CN116744966A - DNA encoded antibodies against SARS-COV-2 - Google Patents

DNA encoded antibodies against SARS-COV-2 Download PDF

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CN116744966A
CN116744966A CN202180056190.7A CN202180056190A CN116744966A CN 116744966 A CN116744966 A CN 116744966A CN 202180056190 A CN202180056190 A CN 202180056190A CN 116744966 A CN116744966 A CN 116744966A
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seq
cov
sars
antibody
nucleic acid
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大卫·韦纳
卡尔·穆图马尼
伊丽莎白·帕尔扎奇
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Da WeiWeina
Ka ErMutumani
Yi LishabaiPaerzhaqi
Wistar Institute of Anatomy and Biology
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Da WeiWeina
Ka ErMutumani
Yi LishabaiPaerzhaqi
Wistar Institute of Anatomy and Biology
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Application filed by Da WeiWeina, Ka ErMutumani, Yi LishabaiPaerzhaqi, Wistar Institute of Anatomy and Biology filed Critical Da WeiWeina
Priority claimed from PCT/US2021/036606 external-priority patent/WO2021252620A2/en
Publication of CN116744966A publication Critical patent/CN116744966A/en
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Abstract

Antibodies to SARS-CoV-2 antigen and recombinant nucleic acid sequences encoding SARS-CoV-2 antibodies are disclosed herein. The application also provides methods of using the compositions and methods to prevent and/or treat SARS-CoV-2 infection or a disease or disorder associated with SARS-CoV-2 infection (e.g., COVID-19) in a subject.

Description

DNA encoded antibodies against SARS-COV-2
RELATED APPLICATIONS
The application requires provisional application number U.S.63/036,809 filed 6/9 in 2020; U.S. provisional application No. 63/036,795, filed 6/9/2020; U.S. provisional application No. 63/068,868, filed 8/21/2020; U.S. provisional application No. 63/083,173, filed on 9/25/2020; and priority of U.S. provisional application number 63/114,271, filed 11/16 in 2020, each of which is hereby incorporated by reference in its entirety.
Technical Field
The present application relates to a composition comprising a recombinant nucleic acid sequence for the in vivo production of one or more synthetic antibodies and functional fragments thereof, and a method of preventing and/or treating a viral infection in a subject by administering said composition.
Background
Coronaviruses (covs) are a family of viruses that are common worldwide and cause a range of diseases in humans from the common cold to Severe Acute Respiratory Syndrome (SARS). Coronaviruses can also cause a variety of diseases in animals. Human coronavirus 229E, OC, NL63 and HKU1 are endemic in the human population.
Covd-19, previously known as 2019-nCoV pneumonia or disease, rapidly emerging as a global public health crisis, adds an increasing number of coronavirus-related diseases to Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS), which jump from animals to humans. There are at least 7 identified coronaviruses that infect humans. SARS-CoV-2 (Zhu et al 2020N Engl J Med,382:727-733; wu et al 2020, nature, 579:265-269) was isolated and sequenced from human airway epithelial cells from an infected patient. Disease symptoms can range from mild influenza-like to severe cases with life-threatening pneumonia (Huang et al 2020, lancet, 395:497-506). The global situation is evolving dynamically and the world health organization announces covd-19 as an international Public Health Emergency (PHEIC) of interest on 1 month 30 and a global pandemic on 3 months 11 of 2020. By month 4 and 1 of 2020, there was 932,605 infections and 46,809 deaths (destination. Org/epilu-applications/global-circuits-covid-19). Infection has spread to many continents. Human-to-human transmission has been observed in a number of countries, and the shortage of personal protective equipment for disposal and the prolonged survival time of coronaviruses on inanimate surfaces (Hulkower et al 2011,Am J Infect Control 39,401-407) have compounded this already fragile situation and increased risk of nosocomial infections. In an effort to protect billions of vulnerable subjects worldwide, advanced research activities must be conducted in parallel to advance the protection model.
Accordingly, there is a need in the art for therapeutic agents that prevent and/or treat covd-19, thereby providing protection against and promoting survival of covd-19 infection. The present invention meets this need.
Disclosure of Invention
In one embodiment, the invention relates to an anti-SARS-CoV-2 antibody or fragment thereof, wherein the antibody is selected from the group consisting of: monoclonal antibodies, polyclonal antibodies, single chain antibodies, immunoconjugates, defucosylated antibodies and bispecific antibodies.
In one embodiment, the immunoconjugate comprises a therapeutic agent or a detection moiety.
In one embodiment, the antibody is selected from the group consisting of: humanized antibodies, chimeric antibodies, fully human antibodies, and antibody mimics.
In one embodiment, the antibody comprises at least one selected from the group consisting of: a) A heavy chain CDR1 sequence selected from the group consisting of SEQ ID NO. 26, SEQ ID NO. 80 and SEQ ID NO. 108; b) A heavy chain CDR2 sequence selected from the group consisting of SEQ ID NO 27, SEQ ID NO 81 and SEQ ID NO 109; c) A heavy chain CDR3 sequence selected from the group consisting of SEQ ID NO. 28, SEQ ID NO. 82 and SEQ ID NO. 110; d) A light chain CDR1 sequence selected from the group consisting of SEQ ID NO. 34, SEQ ID NO. 88 and SEQ ID NO. 116; e) A light chain CDR2 sequence selected from the group consisting of SEQ ID NO. 35, SEQ ID NO. 89 and SEQ ID NO. 117; and f) a light chain CDR3 sequence selected from SEQ ID NO. 36, SEQ ID NO. 90 and SEQ ID NO. 118.
In one embodiment, the antibody comprises at least one selected from the group consisting of: a) An anti-SARS-CoV-2 antibody heavy chain comprising a sequence having at least 80% identity to an amino acid sequence selected from the group consisting of SEQ ID No. 2, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 18, SEQ ID No. 30, SEQ ID No. 42, SEQ ID No. 84, SEQ ID No. 94, SEQ ID No. 100, SEQ ID No. 112, SEQ ID No. 122 and SEQ ID No. 128; b) An anti-SARS-CoV-2 antibody light chain comprising a sequence having at least 80% identity to an amino acid sequence selected from the group consisting of SEQ ID No. 4, SEQ ID No. 8, SEQ ID No. 14, SEQ ID No. 20, SEQ ID No. 38, SEQ ID No. 44, SEQ ID No. 92, SEQ ID No. 96, SEQ ID No. 102, SEQ ID No. 120, SEQ ID No. 124 and SEQ ID No. 130; c) A fragment of the heavy chain of an anti-SARS-CoV-2 antibody comprising at least 80% of the full length sequence selected from the group consisting of SEQ ID No. 2, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 18, SEQ ID No. 30, SEQ ID No. 42, SEQ ID No. 84, SEQ ID No. 94, SEQ ID No. 100, SEQ ID No. 112, SEQ ID No. 122 and SEQ ID No. 128; and d) a fragment of the light chain of an anti-SARS-CoV-2 antibody comprising at least 80% of the full length sequence selected from the group consisting of SEQ ID NO. 4, SEQ ID NO. 8, SEQ ID NO. 14, SEQ ID NO. 20, SEQ ID NO. 38, SEQ ID NO. 44, SEQ ID NO. 92, SEQ ID NO. 96, SEQ ID NO. 102, SEQ ID NO. 120, SEQ ID NO. 124 and SEQ ID NO. 130.
In one embodiment, the antibody comprises at least one selected from the group consisting of: a) Amino acid sequence having at least 80% identity to an amino acid sequence selected from the group consisting of SEQ ID NO 10, SEQ ID NO 16, SEQ ID NO 22, SEQ ID NO 40, SEQ ID NO 46, SEQ ID NO 48, SEQ ID NO 50, SEQ ID NO 52, SEQ ID NO 54, SEQ ID NO 56, SEQ ID NO 58, SEQ ID NO 60, SEQ ID NO 62, SEQ ID NO 64, SEQ ID NO 66, SEQ ID NO 68, SEQ ID NO 70, SEQ ID NO 72, SEQ ID NO 74, SEQ ID NO 76, SEQ ID NO 98, SEQ ID NO 104, SEQ ID NO 126 and SEQ ID NO 132; and b) a fragment comprising at least 80% of the heavy chain of the anti-SARS-CoV-2 antibody selected from the group consisting of SEQ ID NO 10, SEQ ID NO 16, SEQ ID NO 22, SEQ ID NO 40, SEQ ID NO 46, SEQ ID NO 48, SEQ ID NO 50, SEQ ID NO 52, SEQ ID NO 54, SEQ ID NO 56, SEQ ID NO 58, SEQ ID NO 60, SEQ ID NO 62, SEQ ID NO 64, SEQ ID NO 66, SEQ ID NO 68, SEQ ID NO 70, SEQ ID NO 72, SEQ ID NO 74, SEQ ID NO 76, SEQ ID NO 98, SEQ ID NO 104, SEQ ID NO 126 and SEQ ID NO 132.
In one embodiment, the invention relates to a nucleic acid molecule encoding an anti-SARS-CoV-2 antibody or fragment thereof, wherein the antibody is selected from the group consisting of: monoclonal antibodies, polyclonal antibodies, single chain antibodies, immunoconjugates, defucosylated antibodies and bispecific antibodies. In one embodiment, the immunoconjugate comprises a therapeutic agent or a detection moiety.
In one embodiment, the nucleic acid molecule encodes an antibody selected from the group consisting of: humanized antibodies, chimeric antibodies, fully human antibodies, and antibody mimics.
In one embodiment, the nucleic acid molecule encodes an antibody comprising at least one selected from the group consisting of: a) A heavy chain CDR1 sequence selected from the group consisting of SEQ ID NO. 26, SEQ ID NO. 80 and SEQ ID NO. 108; b) A heavy chain CDR2 sequence selected from the group consisting of SEQ ID NO 27, SEQ ID NO 81 and SEQ ID NO 109; c) A heavy chain CDR3 sequence selected from the group consisting of SEQ ID NO. 28, SEQ ID NO. 82 and SEQ ID NO. 110; d) A light chain CDR1 sequence selected from the group consisting of SEQ ID NO. 34, SEQ ID NO. 88 and SEQ ID NO. 116; e) A light chain CDR2 sequence selected from the group consisting of SEQ ID NO. 35, SEQ ID NO. 89 and SEQ ID NO. 117; and f) a light chain CDR3 sequence selected from SEQ ID NO. 36, SEQ ID NO. 90 and SEQ ID NO. 118.
In one embodiment, the nucleic acid molecule encodes an antibody comprising at least one selected from the group consisting of: a) An anti-SARS-CoV-2 antibody heavy chain comprising a sequence having at least 80% identity to an amino acid sequence selected from the group consisting of SEQ ID No. 2, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 18, SEQ ID No. 30, SEQ ID No. 42, SEQ ID No. 84, SEQ ID No. 94, SEQ ID No. 100, SEQ ID No. 112, SEQ ID No. 122 and SEQ ID No. 128; b) An anti-SARS-CoV-2 antibody light chain comprising a sequence having at least 80% identity to an amino acid sequence selected from the group consisting of SEQ ID No. 4, SEQ ID No. 8, SEQ ID No. 14, SEQ ID No. 20, SEQ ID No. 38, SEQ ID No. 44, SEQ ID No. 92, SEQ ID No. 96, SEQ ID No. 102, SEQ ID No. 120, SEQ ID No. 124 and SEQ ID No. 130; c) A fragment of the heavy chain of an anti-SARS-CoV-2 antibody comprising at least 80% of the full length sequence selected from the group consisting of SEQ ID No. 2, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 18, SEQ ID No. 30, SEQ ID No. 42, SEQ ID No. 84, SEQ ID No. 94, SEQ ID No. 100, SEQ ID No. 112, SEQ ID No. 122 and SEQ ID No. 128; and d) a fragment of the light chain of an anti-SARS-CoV-2 antibody comprising at least 80% of the full length sequence selected from the group consisting of SEQ ID NO. 4, SEQ ID NO. 8, SEQ ID NO. 14, SEQ ID NO. 20, SEQ ID NO. 38, SEQ ID NO. 44, SEQ ID NO. 92, SEQ ID NO. 96, SEQ ID NO. 102, SEQ ID NO. 120, SEQ ID NO. 124 and SEQ ID NO. 130.
In one embodiment, the nucleic acid molecule encodes an antibody comprising at least one selected from the group consisting of: a) Amino acid sequence having at least 80% identity to an amino acid sequence selected from the group consisting of SEQ ID NO 10, SEQ ID NO 16, SEQ ID NO 22, SEQ ID NO 40, SEQ ID NO 46, SEQ ID NO 48, SEQ ID NO 50, SEQ ID NO 52, SEQ ID NO 54, SEQ ID NO 56, SEQ ID NO 58, SEQ ID NO 60, SEQ ID NO 62, SEQ ID NO 64, SEQ ID NO 66, SEQ ID NO 68, SEQ ID NO 70, SEQ ID NO 72, SEQ ID NO 74, SEQ ID NO 76, SEQ ID NO 98, SEQ ID NO 104, SEQ ID NO 126 and SEQ ID NO 132; and b) a fragment comprising at least 80% of the heavy chain of the anti-SARS-CoV-2 antibody selected from the group consisting of SEQ ID NO 10, SEQ ID NO 16, SEQ ID NO 22, SEQ ID NO 40, SEQ ID NO 46, SEQ ID NO 48, SEQ ID NO 50, SEQ ID NO 52, SEQ ID NO 54, SEQ ID NO 56, SEQ ID NO 58, SEQ ID NO 60, SEQ ID NO 62, SEQ ID NO 64, SEQ ID NO 66, SEQ ID NO 68, SEQ ID NO 70, SEQ ID NO 72, SEQ ID NO 74, SEQ ID NO 76, SEQ ID NO 98, SEQ ID NO 104, SEQ ID NO 126 and SEQ ID NO 132.
In one embodiment, the nucleic acid molecule further comprises a nucleotide sequence encoding a cleavage domain.
In one embodiment, the nucleic acid molecule comprises at least one selected from the group consisting of: a) A nucleotide sequence selected from the group consisting of SEQ ID NO. 23, SEQ ID NO. 77 and SEQ ID NO. 105 encoding a heavy chain CDR1 sequence; b) A nucleotide sequence selected from the group consisting of SEQ ID NO. 24, SEQ ID NO. 78 and SEQ ID NO. 106 encoding a heavy chain CDR2 sequence; c) A nucleotide sequence selected from the group consisting of SEQ ID NO. 25, SEQ ID NO. 79 and SEQ ID NO. 107 encoding a heavy chain CDR3 sequence; d) A nucleotide sequence selected from the group consisting of SEQ ID NO. 31, SEQ ID NO. 85 and SEQ ID NO. 113 encoding a light chain CDR1 sequence; e) A nucleotide sequence selected from the group consisting of SEQ ID NO. 32, SEQ ID NO. 86 and SEQ ID NO. 114 encoding a light chain CDR2 sequence; and f) a nucleotide sequence selected from the group consisting of SEQ ID NO. 33, SEQ ID NO. 87 and SEQ ID NO. 115 encoding a light chain CDR3 sequence.
In one embodiment, the nucleic acid molecule comprises at least one nucleotide sequence selected from the group consisting of: a) A nucleotide sequence having at least 80% identity to a nucleotide selected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 5, SEQ ID NO. 11, SEQ ID NO. 17, SEQ ID NO. 29, SEQ ID NO. 41, SEQ ID NO. 83, SEQ ID NO. 93, SEQ ID NO. 99, SEQ ID NO. 111, SEQ ID NO. 121 and SEQ ID NO. 127 encoding an anti-SARS-CoV-2 antibody heavy chain; b) A nucleotide sequence having at least 80% identity to a nucleotide selected from the group consisting of SEQ ID NO. 3, SEQ ID NO. 7, SEQ ID NO. 13, SEQ ID NO. 19, SEQ ID NO. 37, SEQ ID NO. 43, SEQ ID NO. 91, SEQ ID NO. 95, SEQ ID NO. 101, SEQ ID NO. 119, SEQ ID NO. 123 and SEQ ID NO. 129 encoding an anti-SARS-CoV-2 antibody light chain; c) A fragment comprising at least 80% of the nucleotide sequence of the full length sequence selected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 5, SEQ ID NO. 11, SEQ ID NO. 17, SEQ ID NO. 29, SEQ ID NO. 41, SEQ ID NO. 83, SEQ ID NO. 93, SEQ ID NO. 99, SEQ ID NO. 111, SEQ ID NO. 121 and SEQ ID NO. 127 encoding an anti-SARS-CoV-2 antibody heavy chain; and d) a fragment comprising at least 80% of the nucleotide sequence selected from the group consisting of SEQ ID NO. 3, SEQ ID NO. 7, SEQ ID NO. 13, SEQ ID NO. 19, SEQ ID NO. 37, SEQ ID NO. 43, SEQ ID NO. 91, SEQ ID NO. 95, SEQ ID NO. 101, SEQ ID NO. 119, SEQ ID NO. 123 and SEQ ID NO. 129, encoding an anti-SARS-CoV-2 antibody light chain.
In one embodiment, the nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of: a) Nucleotide sequence having at least 80% identity with a nucleotide selected from the group consisting of SEQ ID NO 9, SEQ ID NO 15, SEQ ID NO 21, SEQ ID NO 39, SEQ ID NO 45, SEQ ID NO 47, SEQ ID NO 49, SEQ ID NO 51, SEQ ID NO 53, SEQ ID NO 55, SEQ ID NO 57, SEQ ID NO 59, SEQ ID NO 61, SEQ ID NO 63, SEQ ID NO 65, SEQ ID NO 67, SEQ ID NO 69, SEQ ID NO 71, SEQ ID NO 73, SEQ ID NO 75, SEQ ID NO 97, SEQ ID NO 103, SEQ ID NO 125 and SEQ ID NO 131; and b) a fragment comprising at least 80% of the full length sequence of nucleotide sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:15, SEQ ID NO:21, SEQ ID NO:39, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:97, SEQ ID NO:103, SEQ ID NO:125 and SEQ ID NO: 131.
In one embodiment, the nucleotide sequence encodes a leader sequence.
In one embodiment, the nucleic acid molecule comprises an expression vector.
In one embodiment, the invention relates to a composition comprising an anti-SARS-CoV-2 antibody or fragment thereof, wherein the antibody is selected from the group consisting of: monoclonal antibodies, polyclonal antibodies, single chain antibodies, immunoconjugates, defucosylated antibodies and bispecific antibodies.
In one embodiment, the invention relates to a composition comprising at least one nucleic acid molecule encoding an anti-SARS-CoV-2 antibody or fragment thereof, wherein the antibody is selected from the group consisting of: monoclonal antibodies, polyclonal antibodies, single chain antibodies, immunoconjugates, defucosylated antibodies and bispecific antibodies. In one embodiment, the immunoconjugate comprises a therapeutic agent or a detection moiety.
In one embodiment, the composition comprises a first nucleic acid molecule comprising a nucleotide sequence encoding a heavy chain of an anti-SARS-CoV-2 spike antigen synthesis antibody and a second nucleic acid molecule; the second nucleic acid molecule comprises a nucleotide sequence encoding an anti-SARS-CoV-2 spike antigen synthetic antibody light chain.
In one embodiment, the first nucleic acid molecule comprises a nucleotide sequence encoding at least one selected from the group consisting of: a) An anti-SARS-CoV-2 antibody heavy chain comprising a sequence having at least 80% identity to an amino acid sequence selected from the group consisting of SEQ ID No. 2, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 18, SEQ ID No. 30, SEQ ID No. 42, SEQ ID No. 84, SEQ ID No. 94, SEQ ID No. 100, SEQ ID No. 112, SEQ ID No. 122 and SEQ ID No. 128; and b) a fragment of the heavy chain of an anti-SARS-CoV-2 antibody comprising at least 80% of the full length sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:12, SEQ ID NO:18, SEQ ID NO:30, SEQ ID NO:42, SEQ ID NO:84, SEQ ID NO:94, SEQ ID NO:100, SEQ ID NO:112, SEQ ID NO:122 and SEQ ID NO: 128; and the second nucleic acid molecule comprises a nucleotide sequence encoding at least one selected from the group consisting of: c) An anti-SARS-CoV-2 antibody light chain comprising a sequence having at least 80% identity to an amino acid sequence selected from the group consisting of SEQ ID No. 4, SEQ ID No. 8, SEQ ID No. 14, SEQ ID No. 20, SEQ ID No. 38, SEQ ID No. 44, SEQ ID No. 92, SEQ ID No. 96, SEQ ID No. 102, SEQ ID No. 120, SEQ ID No. 124 and SEQ ID No. 130; and d) a fragment of the light chain of an anti-SARS-CoV-2 antibody comprising at least 80% of the full length sequence selected from the group consisting of SEQ ID NO. 4, SEQ ID NO. 8, SEQ ID NO. 14, SEQ ID NO. 20, SEQ ID NO. 38, SEQ ID NO. 44, SEQ ID NO. 92, SEQ ID NO. 96, SEQ ID NO. 102, SEQ ID NO. 120, SEQ ID NO. 124 and SEQ ID NO. 130.
In one embodiment, the first nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of: a) A nucleotide sequence having at least 80% identity to a nucleotide selected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 5, SEQ ID NO. 11, SEQ ID NO. 17, SEQ ID NO. 29, SEQ ID NO. 41, SEQ ID NO. 83, SEQ ID NO. 93, SEQ ID NO. 99, SEQ ID NO. 111, SEQ ID NO. 121 and SEQ ID NO. 127 encoding an anti-SARS-CoV-2 antibody heavy chain; and b) a nucleotide sequence fragment comprising at least 80% of the full length sequence selected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 5, SEQ ID NO. 11, SEQ ID NO. 17, SEQ ID NO. 29, SEQ ID NO. 41, SEQ ID NO. 83, SEQ ID NO. 93, SEQ ID NO. 99, SEQ ID NO. 111, SEQ ID NO. 121 and SEQ ID NO. 127 encoding an anti-SARS-CoV-2 antibody heavy chain; and the second nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of: c) A nucleotide sequence having at least 80% identity to a nucleotide selected from the group consisting of SEQ ID NO. 3, SEQ ID NO. 7, SEQ ID NO. 13, SEQ ID NO. 19, SEQ ID NO. 37, SEQ ID NO. 43, SEQ ID NO. 91, SEQ ID NO. 95, SEQ ID NO. 101, SEQ ID NO. 119, SEQ ID NO. 123 and SEQ ID NO. 129 encoding an anti-SARS-CoV-2 antibody light chain; and d) a fragment comprising at least 80% of the nucleotide sequence selected from the group consisting of SEQ ID NO. 3, SEQ ID NO. 7, SEQ ID NO. 13, SEQ ID NO. 19, SEQ ID NO. 37, SEQ ID NO. 43, SEQ ID NO. 91, SEQ ID NO. 95, SEQ ID NO. 101, SEQ ID NO. 119, SEQ ID NO. 123 and SEQ ID NO. 129, encoding an anti-SARS-CoV-2 antibody light chain.
In one embodiment, the composition further comprises a pharmaceutically acceptable excipient.
In one embodiment, the composition further comprises an adjuvant.
In one embodiment, the invention relates to a method of preventing or treating a disease in a subject, the method comprising administering to the subject an anti-SARS-CoV-2 antibody or fragment thereof, wherein the antibody is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a single chain antibody, an immunoconjugate, a defucosylated antibody, and a bispecific antibody, b) a nucleic acid molecule encoding an anti-SARS-CoV-2 antibody or fragment thereof, wherein the antibody is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a single chain antibody, an immunoconjugate, a defucosylated antibody, and a bispecific antibody, or d) a composition comprising at least one nucleic acid molecule encoding an anti-SARS-CoV-2 antibody or fragment thereof, wherein the antibody is selected from the group consisting of: monoclonal antibodies, polyclonal antibodies, single chain antibodies, immunoconjugates, defucosylated antibodies and bispecific antibodies.
In one embodiment, the disease is COVID-19.
In one embodiment, the method further comprises administering to the subject at least one additional SARS-CoV-2 vaccine or therapeutic agent for the treatment of COVID-19.
In one embodiment, the invention relates to a method of inducing an immune response against SARS-CoV-2 in a subject, the method comprising administering to the subject a) an anti-SARS-CoV-2 antibody or fragment thereof, wherein the antibody is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a single chain antibody, an immunoconjugate, a defucosylated antibody, and a bispecific antibody, b) a nucleic acid molecule encoding an anti-SARS-CoV-2 antibody or fragment thereof, wherein the antibody is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a single chain antibody, an immunoconjugate, a defucosylated antibody, and a bispecific antibody, or d) a composition comprising at least one nucleic acid molecule encoding an anti-SARS-CoV-2 antibody or fragment thereof, wherein the antibody is selected from the group consisting of: monoclonal antibodies, polyclonal antibodies, single chain antibodies, immunoconjugates, defucosylated antibodies and bispecific antibodies.
In one embodiment, the invention relates to a method of inducing an immune response against SARS-CoV-2 in a subject in need thereof, the method comprising administering a combination comprising a first composition comprising a nucleic acid molecule encoding a synthetic anti-SARS-CoV-2 antibody or fragment thereof, wherein the antibody is selected from the group consisting of monoclonal antibodies, polyclonal antibodies, single chain antibodies, immunoconjugates, defucosylated antibodies and bispecific antibodies, and a second composition comprising a nucleic acid molecule encoding a SARS-CoV-2 antigen.
In one embodiment, the nucleic acid molecule encoding a SARS-CoV-2 antigen comprises a nucleotide sequence encoding a peptide comprising an amino acid sequence having at least about 90% identity over the entire length of an amino acid sequence selected from the group consisting of SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:138 and SEQ ID NO: 140.
In one embodiment, the nucleic acid molecule encoding SARS-CoV-2 antigen comprises a nucleotide sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:138 and SEQ ID NO: 140.
In one embodiment, the nucleic acid molecule encoding SARS-CoV-2 antigen comprises a nucleotide sequence that is at least about 90% identical over the entire length of the nucleic acid sequence selected from the group consisting of SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137 and SEQ ID NO: 139.
In one embodiment, the nucleic acid molecule encoding SARS-CoV-2 antigen comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137 and SEQ ID NO: 139.
In one embodiment, the administering includes at least one of electroporation and injection.
In one embodiment, the invention relates to a method of treating or preventing infection of SARS-CoV-2 or a disease or condition associated therewith in a subject in need thereof, the method comprising administering a first composition comprising a nucleic acid molecule encoding a synthetic anti-SARS-CoV-2 antibody or fragment thereof in combination with a second composition, wherein the antibody is selected from the group consisting of monoclonal antibodies, polyclonal antibodies, single chain antibodies, immunoconjugates, defucosylated antibodies and bispecific antibodies, and the second composition comprises a nucleic acid molecule encoding a SARS-CoV-2 antigen.
In one embodiment, the disease is COVID-19.
In one embodiment, the nucleic acid molecule encoding a SARS-CoV-2 antigen comprises a nucleotide sequence encoding a peptide comprising an amino acid sequence having at least about 90% identity over the entire length of an amino acid sequence selected from the group consisting of SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:138 and SEQ ID NO: 140.
In one embodiment, the nucleic acid molecule encoding SARS-CoV-2 antigen comprises a nucleotide sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:138 and SEQ ID NO: 140.
In one embodiment, the nucleic acid molecule encoding SARS-CoV-2 antigen comprises a nucleotide sequence that is at least about 90% identical over the entire length of the nucleic acid sequence selected from the group consisting of SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137 and SEQ ID NO: 139.
In one embodiment, the nucleic acid molecule encoding SARS-CoV-2 antigen comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137 and SEQ ID NO: 139.
In one embodiment, the administering includes at least one of electroporation and injection.
Drawings
FIG. 1 depicts a graph of CR3022 dMAB expression using a two plasmid system.
FIG. 2 depicts exemplary experimental results demonstrating in vitro expression and characterization of CR3022 dMAB.
FIG. 3 depicts exemplary experimental results demonstrating that CR3022 dMAB binds the SARS-CoV-2 Receptor Binding Domain (RBD) in vitro.
FIG. 4 depicts exemplary experimental results demonstrating the in vivo expression kinetics of CR3022 dMAB.
FIG. 5 depicts exemplary experimental results demonstrating in vivo production of CR3022 dMAB binding to the recombinant SARSCOV-2RBD and S1 domains.
FIG. 6 depicts a graph of the development of SARS-CoV-2 clone to express dMAB using either a single plasmid or a dual plasmid system.
Fig. 7 depicts exemplary experimental results demonstrating expression and characterization of S309 dMAB (neutralization).
Fig. 8 depicts exemplary experimental results demonstrating in vitro evaluation of 2130, 2381 and 2196dMAB variants.
Fig. 9 depicts exemplary experimental results demonstrating in vivo expression kinetics of 2130, 2381 and 2196dMAB variants.
Fig. 10 depicts exemplary experimental results demonstrating quantification and binding of 2130, 2381 and 2196dMAB produced in vivo.
FIG. 11 depicts exemplary experimental results demonstrating that serum pools from dMAB-administered mice 2130, 2381 and 2196 demonstrate effective neutralization of SARS-CoV-2 pseudovirus.
Figure 12 depicts exemplary experimental results demonstrating 2196_mod dMAB demonstrates in vivo expression and more efficient neutralization than the parent 2196WT dMAB.
Fig. 13A-13B depict flowcharts of mAb development. FIGS. 13A and 13B show immunization of Balb/c mice with a synthetic consensus sequence for SARS-CoV-2-full length spike DNA as a prime. DNA was injected at 25 μg/mouse doses over a two week interval and boosted with SARS-CoV-2-RBD protein (50 μg/mouse) after the second immunization. Serum from immunized mice was collected and then evaluated by ELISA to detect the presence of anti-SARS-CoV-2 antibody. After confirmation, mouse spleen cells were harvested, B lymphocytes were isolated and used to prepare hybridomas. Finally, positive hybridoma clones were characterized by indirect ELISA, and those selected were further subcloned and amplified for future characterization and analysis.
FIGS. 14A-14E depict the construction and expression of SARS-CoV-2-spike full-length protein. FIG. 14A shows a schematic representation of SARS-CoV-2 full length spike cloned into pCDNA3.1 vector. FIG. 14B depicts the expression of SARS-CoV-2 full length spike protein in a mammalian system having an Fc tag and an Avi tag at the C-terminus, and its size was confirmed in a Bis-TrisPAGE gel. Fig. 14C depicts that it exhibits a purity of >95% as shown by HPLC. FIG. 14D depicts the specificity of the full-length SARS-CoV-2 protein by ELISA using immune serum from SARS-CoV-2-spike DNA vaccinated mice (n=4). In the case of all mouse serum samples, a dose-dependent binding curve was obtained. m1 to m4 represent 4 different mice from which the binding curves were obtained. FIG. 14E depicts Western blot analysis of heavy and light chain expression by SDS-PAGE (12%) for IgG mAb clones (WCoVA 1, WCoVA2, WCoVA3, WCoVA4, WCoVA5, WCoVA6, WCoVA7, WCoVA8, WCoVA9 and WCoVA 10).
FIGS. 15A-15D depict characterization of SARS-CoV-2-spike IgG mAb. FIG. 15A depicts an assessment of the binding potential of IgG clones to SARS-CoV-2-full length spike and RBD. ELISA plates were coated with recombinant SARS-CoV-2-full length spike protein (1. Mu.g/ml) and RBD (1. Mu.g/ml) and tested with mAb clones serially diluted as indicated. FIG. 15B depicts serum IgG endpoint titers of mAb clones against SARS-CoV-2-full length spike protein, and RBD was determined using endpoint titer ELISA. Recombinant Avi-Tag protein was used as a binding control, where no binding of IgG mAb clones was observed. FIG. 15C depicts binding specificity analysis of mAb clones against SARS-CoV-2-RBD protein by Western blotting. Goat anti-mouse IgG-IRDye CW-800 secondary antibody was used to detect the antibody. FIG. 15D depicts the binding kinetics of SARS-CoV-2-spike mAb and its target; SARS-CoV-2-RBD was analyzed by surface plasmon resonance. A sensorgram representing the response relative to IgG mAb clones showing strong binding to SARS-CoV-2-RBD, showing the progress of their interaction.
FIG. 16 shows the analysis of IgG mAb binding to SARS-CoV-2-RBD protein by SPR. The binding horizontal bar graph shows the binding of IgG mabs to SARS-CoV-2-RBD proteins by SPR analysis using the biacore t200SPR system. The Y-axis represents the RU signal subtracted from the reference recorded at the end of the association phase (300 s).
FIGS. 17A-17C depict the competition of SARS-CoV-2-spike mAb with ACE2 receptor for SARS-CoV-2 spike protein binding. FIG. 17A depicts the addition of serial dilutions of SARS-CoV-2-spike IgG mAb to SARS-CoV-2 coated wells prior to the addition of ACE2 protein. The binding of ACE2 to SARS-CoV-2-spike mAb was measured and SARS-CoV-2-spike IgG mAb showed competition for ACE2 receptor binding to SARS-CoV-2-spike protein. FIG. 17B depicts flow cytometry-based receptor binding inhibition; CHO-ACE2 cells stained with SARS-CoV-2 spike and flow cytometry analysis are shown. Cells were analyzed for stokes binding (x-axis). Fig. 17C depicts the percentage of cells that scored negative or positive shown in each quadrant.
FIGS. 18A-18D depict the neutralization efficacy of SARS-CoV-2-spike mAb against a SARS-CoV-2 pseudovirus assay. The function of SARS-CoV-2-spike IgG mAb was assessed using a pseudovirus neutralization assay. Neutralization efficacy of SARS-CoV-2 wild-type and (FIG. 18B) D614G mutated pseudoviruses by SARS-CoV-2IgG mAb clone expressed as% neutralization. FIG. 18C shows the IC50 values of SARS-CoV-2-spike IgG mAb clones represented by the bar graph. Fig. 18D depicts the correlation between ELISA titers and virus neutralization titers. Statistical analysis was performed in GraphPad Prism. The experiment was performed once. IC50, half maximal inhibitory concentration.
FIGS. 19A-19E depict glycogenomics analysis of SARS-CoV-2 antibodies, revealing a spectrum compatible with higher Fc-mediated effector function. Fig. 19A depicts a schematic structure of antibody glycosylation highlighting several monosaccharides and their known effects on Fc-mediated effector function. Figures 19B-19E depict the percentage of total core fucose (figure 19B), terminal sialic acid (figure 19C), bisected GlcNac (figure 19D), and terminal galactose (figure 19E) in the total glycogroup of five SARS-CoV2 antibodies, bulk IgG from three control C57BL/6 mice, and bulk IgG from human control samples (run in triplicate). Median and quartile spacing are shown.
FIGS. 20A-20F depict a computational model of an antibody that interfaces with SARS-CoV-2-spike protein. Fig. 20A and 20D depict the structure of WCoVA7 and WCoVA9 variable regions predicted by AbYmod. FIGS. 20B and 20C depict a docking model of full length SARS-CoV-2 spike structure (PDB: 6 VYB) and WCoVA7 antibody. Left: carton docking model (green: full length spike protein; red: antibody variable region). Right: surface docking model (grey: full length spike protein; red: antibody variable region). FIGS. 20E and 20F depict a docking model of full length SARS-CoV-2 spike structure (PDB: 6 VYB) and WCoVA9 antibody. Animated docking model (green: full length spike protein; red: antibody variable region). Right: surface docking model (grey: full length spike protein; red: antibody variable region).
FIGS. 21A and 21B depict a comparison of SARS-CoV-2, SARS-CoV and MERS-CoV spike glycoproteins. Fig. 21A: amino acid alignment of coronavirus spike proteins, including 11 SARS-CoV-2 sequences with mutations (GISAID). The grey bars represent the same amino acids and the colored bars represent mutations relative to Wuhan-Hu-1. RBD, cleavage site, fusion peptide, and transmembrane domain are indicated in red. Fig. 21B: structural models of SARS-CoV-2, SARS and MERS glycoproteins, with one chain represented by animation and two chains represented by surfaces. The RBD of SARS-CoV-2 is yellow.
FIGS. 22A-22D depict the design and expression of a COVID-19 synthetic DNA vaccine construct. Fig. 22A: schematic representation of the synthetic DNA vaccine constructs of COVID-19, pGX9501 (matched) and pGX9503 (outlier (OL)), containing IgE leader sequences and SARS-CoV-2 spike protein insert. Fig. 22B: RT-PCR assay of RNA extracts from COS-7 cells transfected with pGX9501 and pGX 9503. The extracted RNA was analyzed using RT-PCR using PCR assays designed for each target and for COS-7β -actin mRNA used as an internal expression normalization gene. For each transfection concentration, delta CT (Δ CT ) C calculated as target T Subtracting beta-actin C T And plotted against the log of the quality of pDNA transfection. Fig. 22C: analysis of in vitro expression of spike proteins after transfection of 293T cells with pGX9501, pGX9503 or MOCK plasmid by western blotting. 293T cell lysates were resolved on a gel and probed with polyclonal anti-SARS spike protein. The blots were stripped and then probed with an anti- β -actin loading control. Fig. 22D: in vitro immunofluorescent staining of 293T cells transfected with pGX9501, pGX9503 or pVax (empty control vector) at 3. Mu.g/well. Polyclonal anti-SARS spike protein IgG and anti-IgG secondary (green) were used to measure spike protein expression. Nuclei were counterstained with DAPI (blue). Images were captured using ImageXpress Pico automated cell imaging system.
FIG. 23 shows a panel of IgG binding screens for SARS-CoV-2 and SARS-CoV antigens using serum from INO-4800 treated mice. BALB/c mice were immunized with 25 μg INO-4800 or pVAX-empty vector (control) on day 0 as described in the methods. Protein antigen binding of IgG at 1:50 and 1:250 serum dilutions from mice on day 14. The data shown represent the average OD450nm value (average + SD) for 4 mice per group.
FIGS. 24A-24D depict the humoral response to SARS-CoV-2S1+2 and SRBD protein antigens following a single dose of INO-4800 in BALB/c mice immunized with either a prescribed dose of INO-4800 or pVAX-empty vector on day 0 as described in the methods. (FIG. 4A) SARS-CoV-2S1+2 or (FIG. 4C) SARS-CoV-2RBD protein antigen binding of IgG in serial serum dilutions from mice on day 14. The data shown represent the average OD450nm values (average + SD) for 8 mice (fig. 24A and 24B) and 5 mice (fig. 24C and 24D) per group. Serum IgG binding endpoint titers for SARS-CoV-2S1+2 (FIG. 24B) and SARS-CoV-2RBD proteins (FIG. 24D). Data representing 2 independent experiments.
FIGS. 25A-25C depict serum IgG from INO-4800 immunized mice competing with ACE2 receptor for SARS-CoV-2 spike protein binding. Fig. 25A: soluble ACE2 receptor with EC of 0.025. Mu.g/ml 50 Bind to the full length spike of CoV-2. Fig. 25B: purified serum IgG from BALB/c mice after a second immunization with INO-4800 produced significant competition for ACE2 receptor. Serum IgG samples from animals were run in triplicate. Fig. 25C: igG purified from n=5 mice on day 14 after the second immunization with INO-4800 showed significant competition for ACE2 receptor binding to SARS-CoV-2s1+2 protein. The concentration of soluble ACE2 used in the competition assay was about 0.1. Mu.g/ml.
Figure 26 depicts that IgG purified from n=5 mice on day 14 after the second immunization with INO-4800 showed competition for ACE2 receptor binding to SARS-CoV-2 spike protein compared to pooled native mouse IgG. Initial experimental mice were run in single columnA mice). Vaccinated mice were run in duplicate. If the error bar is not visible, the error is smaller than the data point.
FIGS. 27A and 27B depict the humoral response to SARS-CoV-2 in Hartley guinea pigs after a single dose of INO-4800. Hart1ey guinea pig mice were immunized with 100 μg INO-4800 or pVAX-empty vector as on day 0, as described in these methods. Fig. 27A: on day 0 and day 14, the SARS-CoV-2S protein antigen of IgG in serial serum dilutions was bound. The data shown represent the average OD450nm values (average + SD) for 5 guinea pigs. Fig. 27B: serum IgG binding titers (mean ± SD) to SARS-CoV-2S protein on day 14. P=0.0079, mann-Whitney test.
FIGS. 28A and 28B depict serum mediated inhibition of ACE-2 binding to SARS-CoV-2S protein from INO-4800 immunized guinea pigs. Hart1ey guinea pigs were immunized with 100 μg INO-4800 or pVAX-empty vector on days 0 and 14 as described in the methods. Fig. 28A: serum collected on day 28 (1:20 dilution) was added to SARS-CoV-2 coated wells, followed by serial dilutions of ACE-2 protein. Measurement of detection of ACE-2 binding to SARS-CoV-2S protein. Serum collected from 5 INO-4800 treated and 3 pVAX treated animals was used in this experiment. Fig. 28B: serial dilutions of guinea pig serum collected on day 21 were added to SARS-CoV-2 coated wells prior to the addition of ACE-2 protein. Measurement of detection of ACE-2 binding to SARS-CoV-2S protein. Serum collected from 4 INO-4800 treated and 5 pVAX treated guinea pigs was used in this experiment.
FIGS. 29A-29D depict the detection of SARS-CoV-2S protein reactive antibody in the BAL of INO-4800 immunized animals. BALB/c mice were immunized with INO-4800 or pVAX and BAL collected on day 0 and day 14 (FIGS. 29A and 29B). Hart1ey guinea pigs were immunized with INO-4800 or pVAX and BAL harvested on day 42 on days 0, 14 and 21 (FIGS. 29C and 29D). SARS-CoV-2 spike protein specific antibodies in bronchoalveolar lavage fluid were determined by ELISA. The data are presented as endpoint titers (fig. 29A and 29C) and BAL dilution curves with original OD450nm values (fig. 29B and 29D). The (fig. 29A and 29C) bars represent the average value of each group, and the error bars represent the standard deviation. * P < 0.01, by Mann-Whitney U test. Data represent one experiment for each category of n=5/group
FIGS. 30A-30C depict the rapid induction of T cell responses in BALB/C mice following INO-4800 administration. BALB/c mice (n=5/group) were immunized with 2.5 or 10 μg INO-4800. T cell responses in animals were analyzed on days 4, 7, 10, and 14 of fig. 30A and 30B, and 30C. T cell responses were measured by IFN-. Gamma.ELISPot in spleen cells stimulated for 20 hours with overlapping peptide pools spanning the spike proteins of SARS-CoV-2 (FIG. 30A), SARS-CoV (FIG. 30B) or MERS-CoV (FIG. 30C). Bars represent mean + SD.
FIGS. 31A-31F depict ELISPot images of IFN-gamma+ mouse spleen cells after stimulation with SARS-CoV-2 and SARS antigen. Mice were immunized on day 0 and spleen cells were harvested at the indicated time points. Ifnγ secreting cells in the spleen of immunized animals were counted by ELISpot assay. Representative images show SARS-CoV-2-specific (FIGS. 31A-31C) and SARS-CoV-specific (FIGS. 31D-31F) IFN gamma spot-forming units in the spleen cell population at days 4, 7 and 10 post-immunization. Images were captured by an ImmunoSpot CTL reader.
FIGS. 32A and 32B depict flow cytometry analysis of a T cell population producing IFN-gamma upon stimulation of SARS-CoV-2S protein. Splenocytes harvested from BALB/C and C57BL/6 mice after 14 days of treatment with pVAX or INO-4800 were made into single cell suspensions. Cells were stimulated with SARS-CoV-2 overlapping peptide pool for 6 hours. Fig. 32A: cd4+ and cd8+ T cell gating strategies; gating on (i) followed by gating on lymphocytes (ii) followed by live cd45+ cells (iii). Next, cd3+ cells were gated (iv), and cd4+ (v) and cd8+ (vi) T cells from the population were gated. Ifnγ+ cells were gated from each of the cd4+ (vii) and cd8+ (viii) T cell populations. Fig. 32B: the percentage of cd4+ and cd8+ T cells that produced ifnγ is depicted. Bars represent mean + SD. In this study 4 BALB/C and 4C 57BL/6 mice were used. * p < 0.05,Mann Whitney.
FIGS. 33A and 33B depict T cell epitope mapping after INO-4800 administration to BALB/c mice. Spleen cells were stimulated with SARS-CoV-2 peptide substrate pool for 20 hours. Fig. 33A: the matrix was used to map the T cell response after stimulation of the SARS-CoV-2 peptide pool. Bars represent mean + SD. Fig. 33B: profile of SARS-CoV-2 spike protein and identification of immunodominant peptide in BALB/c mice. Known immunodominant SARS-CoVHLA-A2 epitopes were included for comparison.
Detailed Description
The present invention relates to compositions comprising recombinant nucleic acid sequences encoding antibodies, fragments thereof, variants thereof, or combinations thereof. The composition may be administered to a subject in need thereof to promote expression and formation of synthetic antibodies in vivo.
In particular, heavy and light chain polypeptides expressed from recombinant nucleic acid sequences may be assembled into synthetic antibodies. The heavy and light chain polypeptides can interact with each other such that assembly results in the synthetic antibody being able to bind to an antigen, be more immunogenic than antibodies that are not assembled as described herein, and be able to elicit or induce an immune response against an antigen.
Furthermore, these synthetic antibodies are produced more rapidly in subjects than antibodies produced in response to antigen-induced immune responses. Synthetic antibodies are capable of efficiently binding and neutralizing a range of antigens. Synthetic antibodies can also be effective in protecting against and/or promoting survival of diseases.
1. Definition of the definition
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present invention, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.
As used herein, the terms "comprising," "including," "having," "can," "containing," and variations thereof are intended to be open-ended transitional phrases, terms, or words that do not exclude the possibility of additional acts or structures. The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments "comprising," "consisting of … …," and "consisting essentially of … …," whether or not explicitly stated, of these embodiments or elements presented herein.
"antibody" may refer to antibodies of the IgG, igM, igA, igD or IgE class, or fragments, fragments or derivatives thereof, including Fab, F (ab') 2, fd, and single chain antibodies and derivatives thereof. The antibody may be an antibody isolated from a mammalian serum sample, a polyclonal antibody, an affinity purified antibody or a mixture thereof, which exhibits sufficient binding specificity for a desired epitope or sequence derived therefrom.
An "antibody fragment" or "fragment of an antibody" as used interchangeably herein refers to a portion of an intact antibody that comprises an antigen binding site or variable region. This portion does not include the Fc region of the intact antibody of the constant heavy chain domain (i.e., CH2, CH3, or CH4, depending on the antibody isotype). Examples of antibody fragments include, but are not limited to, fab fragments, fab '-SH fragments, F (ab') 2 fragments, fd fragments, fv fragments, diabodies, single chain Fv (scFv) molecules, single chain polypeptides comprising only one light chain variable domain, single chain polypeptides comprising three CDRs of a light chain variable domain, single chain polypeptides comprising only one heavy chain variable region, and single chain polypeptides comprising three CDRs of a heavy chain variable region.
An "antigen" refers to a protein that has the ability to mount an immune response in a host. The antigen may be recognized and bound by an antibody. The antigen may originate in vivo or from an external environment.
As used herein, "coding sequence" or "coding nucleic acid" may refer to a nucleic acid (RNA or DNA molecule) comprising a nucleotide sequence encoding an antibody as set forth herein. The coding sequence may also comprise a DNA sequence encoding an RNA sequence. The coding sequence may further comprise initiation and termination signals operably linked to regulatory elements including promoters and polyadenylation signals capable of directing expression in cells of the subject or mammal to which the nucleic acid is administered. The coding sequence may further comprise a sequence encoding a signal peptide.
As used herein, "complement" or "complementary" can refer to nucleic acids that can refer to Watson-Crick (e.g., a-T/U and C-G) or Hoogsteen base pairing between nucleotides or nucleotide analogs of a nucleic acid molecule.
As used herein, a "constant current" defines the current received or experienced by a tissue or cells defining the tissue during the duration of an electrical pulse delivered to the same tissue. The electrical pulse is delivered from the electroporation device described herein. This current is maintained at a constant amperage in the tissue during the lifetime of the electrical pulse because the electroporation device provided herein has a feedback element, preferably with instantaneous feedback. The feedback element may measure the resistance of the tissue (or cells) over the duration of the pulse and cause the electroporation device to change its electrical energy output (e.g., increase the voltage) such that the current in the same tissue remains constant throughout the electrical pulse (microsecond) and from pulse to pulse. In some embodiments, the feedback element includes a controller.
As used herein, "current feedback" or "feedback" may be used interchangeably and may refer to an active response of the provided electroporation device that includes measuring current in tissue between electrodes and changing the energy output delivered by the EP device accordingly in order to maintain the current at a constant level. The constant level is preset by the user prior to initiating a pulse sequence or electrical treatment. This feedback may be accomplished by the electroporation component (e.g., controller) of the electroporation device, as the circuitry therein is capable of continuously monitoring the current in the tissue between the electrodes and comparing the monitored current (or current in the tissue) to a preset current and continuously making energy output adjustments to maintain the monitored current at a preset level. The feedback loop may be transient in that it is an analog closed loop feedback.
As used herein, "dispersing current" may refer to patterns of current delivered from different needle electrode arrays of an electroporation device described herein, wherein these patterns minimize or preferably eliminate the occurrence of electroporation-related heat stress on any region of tissue being electroporated.
"electroporation," "electroporation," or "electrokinetic enhancement" ("EP") as used interchangeably herein, can refer to the use of transmembrane electric field pulses to induce microscopic pathways (pores) in a biological membrane; their presence allows biomolecules such as plasmids, oligonucleotides, siRNA, drugs, ions and water to pass from one side of the cell membrane to the other.
As used herein, an "endogenous antibody" may refer to an antibody produced in a subject administered an effective dose of an antigen for inducing a humoral immune response.
As used herein, a "feedback mechanism" may refer to a process performed by software or hardware (or firmware) that receives the impedance of the desired tissue and compares it (before, during, and/or after delivering the energy pulse) to a current value (preferably a current) and adjusts the delivered energy pulse to achieve a preset value. The feedback mechanism may be performed by an analog closed loop circuit.
"fragment" may mean a polypeptide fragment of an antibody that functions, i.e., can bind to a desired target and has the same intended effect as a full-length antibody. Fragments of antibodies may be 100% identical to full length, except for deletion of at least amino acids from the N-and/or C-terminus, in each case with or without a signal peptide and/or methionine at position 1. Fragments may comprise 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more (excluding any heterologous signal peptide added) of the length of a particular full-length antibody. Fragments may comprise fragments of polypeptides having 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to an antibody, and additionally comprise an N-terminal methionine or a heterologous signal peptide that is not included when calculating percent identity. Fragments may further comprise an N-terminal methionine and/or a signal peptide, such as an immunoglobulin signal peptide, e.g. IgE or IgG signal peptide. N-terminal methionine and/or signal peptide may be linked to fragments of the antibody.
Fragments of nucleic acid sequences encoding antibodies may be 100% identical to the full length, except for deletion of at least one nucleotide from the 5 'and/or 3' ends, in each case with or without a sequence encoding a signal peptide and/or methionine at position 1. Fragments may comprise 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more (excluding any heterologous signal peptide added) of the length of a particular full-length coding sequence. Fragments may comprise fragments encoding polypeptides having 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the antibody, and further optionally comprise sequences encoding an N-terminal methionine or a heterologous signal peptide not included when calculating percent identity. Fragments may further comprise coding sequences for N-terminal methionine and/or signal peptides (e.g., immunoglobulin signal peptides, e.g., igE or IgG signal peptides). The coding sequence encoding the N-terminal methionine and/or the signal peptide may be linked to a fragment of the coding sequence.
As used herein, a "genetic construct" refers to a DNA or RNA molecule comprising a nucleotide sequence encoding a protein (e.g., an antibody). Genetic constructs may also refer to DNA molecules that transcribe RNA. The coding sequence includes initiation and termination signals operably linked to regulatory elements including promoters and polyadenylation signals capable of directing expression in cells of a subject to which the nucleic acid molecule is administered. As used herein, the term "expressible form" refers to genetic constructs containing the necessary regulatory elements operably linked to a coding sequence encoding a protein such that when present in a cell of a subject, the coding sequence will be expressed.
As used herein, in the context of two or more nucleic acid or polypeptide sequences, "identical" or "identity" may mean that the sequences have a specified percentage of residues that are identical over a specified region. The percentage can be calculated as follows: optimally aligning the two sequences, comparing the two sequences over a designated region, determining the number of positions in the two sequences where identical residues occur to produce a number of matched positions, dividing the number of matched positions by the total number of positions in the designated region, and multiplying the result by 100 to produce a percentage of sequence identity. In the case where two sequences have different lengths or alignments yielding one or more staggered ends and the designated comparison region comprises only a single sequence, the residues of the single sequence are included in the calculated denominator and not the numerator. Thymine (T) and uracil (U) can be considered equivalent when comparing DNA and RNA. Identity can be performed manually or by using a computer sequence algorithm (e.g., BLAST or BLAST 2.0).
When discussing the feedback mechanism, an "impedance" as used herein may be used and may be converted to a current value according to Ohm's law, so as to be able to compare with a preset current.
As used herein, an "immune response" may mean the activation of the immune system of a host, e.g., the immune system of a mammal, in response to the introduction of one or more nucleic acids and/or peptides. The immune response may be in the form of a cellular or humoral response or both.
As used herein, a "nucleic acid" or "oligonucleotide" or "polynucleotide" may refer to at least two nucleotides that are covalently linked together. The depiction of a single strand also defines the sequence of the complementary strand. Thus, the nucleic acid also includes the depicted single-stranded complementary strand. Many variants of a nucleic acid can be used for the same purpose as a given nucleic acid. Thus, nucleic acids also include substantially identical nucleic acids and complements thereof. The single strand provides a probe that hybridizes to the target sequence under stringent hybridization conditions. Thus, nucleic acids also include probes that hybridize under stringent hybridization conditions.
The nucleic acid may be single-stranded or double-stranded, or may contain portions of both double-stranded and single-stranded sequences. The nucleic acid may be a DNA, RNA or hybrid of genomic and cDNA, wherein the nucleic acid may comprise a combination of deoxyribose and ribonucleotides, as well as combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine. The nucleic acid may be obtained by chemical synthesis methods or by recombinant methods.
As used herein, "operably linked" may refer to expression of a gene under the control of a promoter to which it is spatially linked. Promoters may be located 5 '(upstream) or 3' (downstream) of the gene under their control. The distance between a promoter and a gene may be about the same as the distance between the promoter and the gene it controls in the gene from which the promoter is derived. As known in the art, this change in distance can be accommodated without losing promoter function.
As used herein, "peptide," "protein," or "polypeptide" may mean a linked amino acid sequence and may be natural, synthetic, or a combination of natural and synthetic modifications.
As used herein, a "promoter" may refer to a synthetic or naturally derived molecule capable of conferring, activating, or enhancing expression of a nucleic acid in a cell. Promoters may contain one or more specific transcriptional regulatory sequences to further enhance their expression and/or alter the space and/or timing of their expression. Promoters may also contain distal enhancer or repressor elements, which may be located up to several thousand base pairs from the transcription initiation site. Promoters may be derived from sources including viral, bacterial, fungal, plant, insect and animal sources. Promoters may regulate the expression of a genomic component constitutively or differentially relative to the cell, the tissue or organ in which expression occurs, or relative to the developmental stage in which expression occurs, or in response to an external stimulus such as physiological stress, pathogen, metal ion, or inducer. Representative examples of promoters include phage T7 promoter, phage T3 promoter, SP6 promoter, lac operator promoter, RSV-LTR promoter, tac promoter, SV40 early promoter or SV40 late promoter, and CMVIE promoter.
As used herein, "sample" or "biological sample" refers to biological material isolated from a subject. The biological sample may comprise any biological material suitable for detecting a desired biomarker, and may include cellular and/or non-cellular material obtained from the subject.
"Signal peptide" and "leader sequence" are used interchangeably herein and refer to an amino acid sequence that may be attached to the amino terminus of a protein set forth herein. The signal peptide/leader sequence generally directs the localization of the protein. The signal peptide/leader sequence as used herein preferably facilitates secretion of the protein from the cell in which the signal peptide/leader sequence is produced. The signal peptide/leader sequence is typically cleaved from the remainder of the protein (commonly referred to as the mature protein) when secreted from the cell. The signal peptide/leader sequence is attached at the N-terminus of the protein.
As used herein, "stringent hybridization conditions" may refer to conditions under which a first nucleic acid sequence (e.g., a probe) will hybridize to a second nucleic acid sequence (e.g., a target), such as in a complex mixture of nucleic acids. Stringent conditions are sequence-dependent and will be different in different circumstances. Stringent conditions can be selected to bit sequence thermal melting point (T) m ) About 5 deg.c-10 deg.c lower. T (T) m There may be a temperature (at a defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (since the target sequence is present in excess, at Tm 50% of the probes are occupied at equilibrium). Stringent conditions can be those in which the salt concentration is less than about 1.0M sodium ion, e.g., about 0.01-1.0M sodium ion concentration (or other salt) at ph7.0 to 8.3 and the temperature is at least about 30 ℃ for short probes (e.g., about 10-50 nucleotides) and at least about 60 ℃ for long probes (e.g., greater than about 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, the positive signal may be at least 2 to 10 times that of background hybridization. Exemplary stringent hybridization conditions include the following: 50% formamide, 5 XSSC and 1% SDS, incubated at 42℃or 5 XSSC, 1% SDS, incubated at 65℃and washed at 65℃in 0.2 XSSC and 0.1% SDS.
As used interchangeably herein, "subject" and "patient" refer to any vertebrate, including but not limited to mammals (e.g., cows, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats and mice, non-human primates (e.g., monkeys, such as cynomolgus monkeys or rhesus monkeys, chimpanzees, etc.) and humans. In some embodiments, the subject may be a human or a non-human. The subject or patient may undergo other forms of treatment.
As used herein, "substantially complementary" may refer to a first sequence that is at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a complementary sequence of a second sequence over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more nucleotides or amino acids, or two sequences hybridize under stringent hybridization conditions.
As used herein, "substantially identical" may refer to the first and second sequences being over a region of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or more nucleotides or amino acids, or at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% relative to the nucleic acid if the first sequence is substantially complementary to the second sequence.
As used herein, "synthetic antibody" refers to an antibody encoded by a recombinant nucleic acid sequence described herein and produced in a subject.
As used herein, "treatment" or "treatment" may mean protecting a disease of a subject from the disease by preventing, suppressing, or completely eliminating the disease. Preventing a disease involves administering the vaccine of the invention to a subject prior to the onset of the disease. Inhibiting the disease involves administering the vaccine of the invention to a subject after induction of the disease but before its clinical appearance. Inhibiting the disease involves administering the vaccine of the invention to a subject after clinical occurrence of the disease.
"variant" as used herein with respect to nucleic acids may refer to (i) a portion or fragment of a reference nucleotide sequence; (ii) a complement of a reference nucleotide sequence or a portion thereof; (iii) A nucleic acid substantially identical to a reference nucleic acid or its complement; or (iv) a nucleic acid that hybridizes under stringent conditions to a reference nucleic acid, a complement thereof, or a sequence substantially identical thereto.
A "variant" is a peptide or polypeptide whose amino acid sequence differs by amino acid insertions, deletions, or conservative substitutions, but which retains at least one biological activity. Variant may also mean a protein having substantially the same amino acid sequence as a reference protein having an amino acid sequence that retains at least one biological activity. Conservative substitutions of amino acids, i.e., substitution of an amino acid with a different amino acid having similar properties (e.g., hydrophilicity, degree and distribution of charged regions), are considered in the art to typically involve minor changes. These minor changes can be identified in part by considering the hydropathic index of amino acids, as understood in the art. Kyte et al, J.mol.biol.157:105-132 (1982). The hydropathic index of amino acids is based on their hydrophobicity and charge considerations. It is known in the art that amino acids resembling the hydropathic index can be substituted and still retain protein function. In one aspect, the amino acid having a hydrophilic index of ±2 is substituted. The hydrophilicity of amino acids may also be used to reveal substitutions that will result in the protein retaining biological function. Considering the hydrophilicity of amino acids in the context of a peptide allows the calculation of the maximum local average hydrophilicity of the peptide, which is a useful measure that has been reported to correlate well with antigenicity and immunogenicity. U.S. Pat. No. 4,554,101, which is incorporated by reference in its entirety. As understood in the art, substitution of amino acids with similar hydrophilicity values may result in peptides that retain biological activity, e.g., immunogenicity. Substitutions may be made with amino acids whose hydrophilicity values are within + -2 of each other. Both the hydrophobicity index and the hydrophilicity value of an amino acid are affected by the particular side chain of the amino acid. Consistent with this observation, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of the amino acids, and in particular the side chains of those amino acids, as revealed by hydrophobicity, hydrophilicity, charge, size, and other characteristics.
A variant may be a nucleic acid sequence that is substantially identical over the entire length of the entire gene sequence or fragment thereof. The nucleic acid sequence may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical over the entire length of the gene sequence or fragment thereof. A variant may be an amino acid sequence that is substantially identical over the entire length of the amino acid sequence or fragment thereof. The amino acid sequence may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical over the entire length of the amino acid sequence or fragment thereof.
As used herein, a "vector" may refer to a nucleic acid sequence that contains an origin of replication. The vector may be a plasmid, phage, bacterial artificial chromosome, or yeast artificial chromosome. The vector may be a DNA or RNA vector. The vector may be a self-replicating extra-chromosomal vector or a vector integrated into the host genome.
For recitation of numerical ranges herein, each intermediate number is explicitly contemplated to be of the same degree of accuracy as the other intermediate numbers. For example, for the range of 6-9, the numbers 7 and 8 are considered in addition to 6 and 9, and for the range of 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly considered.
2. Composition and method for producing the same
In some embodiments, the invention provides compositions that bind SARS-CoV-2 antigen, including but not limited to SARS-CoV-2 spike protein. In some embodiments, the composition that binds SARS-CoV-2 spike protein is an antibody.
The present invention relates to the design and development of synthetic DNA plasmid encoding human anti-SARS-CoV-2 monoclonal antibody sequence as a new method of SARS-CoV-2 infection or immunization therapy of COVID-19. A single vaccination with this anti-SARS-CoV-2-dMAB resulted in functional anti-SARS-CoV-2 activity in the serum of vaccinated animals for several weeks. anti-SARS-CoV-2 dMAB can be used as an immunoprophylaxis strategy for SARS-CoV-2 infection or COVID-19.
The present invention relates to compositions comprising recombinant nucleic acid sequences encoding antibodies, fragments thereof, variants thereof, or combinations thereof. When administered to a subject in need thereof, the composition can result in the production of synthetic antibodies in the subject. The synthetic antibodies can bind to a target molecule (i.e., antigen) present in the subject. This binding can neutralize the antigen, block antigen recognition by another molecule, such as a protein or nucleic acid, and elicit or induce an immune response to the antigen.
In one embodiment, the composition comprises a nucleotide sequence encoding a synthetic antibody. In one embodiment, the composition comprises a nucleic acid molecule comprising a first nucleotide sequence encoding a first synthetic antibody and a second nucleotide sequence encoding a second synthetic antibody. In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding a cleavage domain.
In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding an antibody to the SARS-CoV-2 virus (anti-SARS-CoV-2) Receptor Binding Domain (RBD) or spike protein.
In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding a variable heavy chain region of an anti-SARS-CoV-2 antibody and a nucleotide sequence encoding a variable light chain region of an anti-SARS-CoV-2 antibody.
In one embodiment, the invention provides a composition comprising a first nucleic acid molecule comprising a nucleotide sequence encoding a variable heavy chain region of an anti-SARS-CoV-2 antibody and a second nucleic acid molecule comprising a nucleotide sequence encoding a variable light chain region of an anti-SARS-CoV-2 antibody.
In certain embodiments, the antibodies of the invention (including SARS-CoV-2 spike protein fragment) comprise an antibody amino acid sequence disclosed herein that is encoded by any suitable polynucleotide, or any isolated or formulated antibody. Furthermore, the antibodies of the invention comprise antibodies having the structural and/or functional characteristics of the anti-SARS-CoV-2 spike protein antibodies described herein. In one embodiment, the anti-SARS-CoV-2 spike protein antibody binds to SARS-CoV-2 spike protein, thereby partially or completely altering at least one biological activity (e.g., receptor binding activity) of the SARS-CoV-2 spike protein.
In one embodiment, the anti-SARS-CoV-2 spike protein antibody of the invention immunospecifically binds to at least one specific epitope specific for SARS-CoV-2 spike protein and not specifically binds to other polypeptides. The at least one epitope may comprise at least one antibody binding region comprising at least a portion of a SARS-CoV-2 spike protein. As used herein, the term "epitope" refers to a protein determinant capable of binding to an antibody. Epitopes are generally composed of chemically active surface groups of molecules such as amino acids or sugar side chains, and generally have specific three-dimensional structural features as well as specific charge characteristics. Conformational epitopes differ from non-conformational epitopes in that binding to the former is lost in the presence of denaturing solvents rather than the latter.
In some embodiments, the invention includes antibodies that comprise binding specificity to SARS-CoV-2 spike protein (e.g., a binding portion of an antibody). In one embodiment, the anti-SARS-CoV-2 spike protein antibody is a polyclonal antibody. In further embodiments, the anti-SARS-CoV-2 spike protein antibody is a monoclonal antibody. In some embodiments, the anti-SARS-CoV-2 spike protein antibody is a chimeric antibody. In further embodiments, the anti-SARS-CoV-2 spike protein antibody is a humanized antibody.
The binding portion of the antibody comprises one or more fragments of the antibody that retain the ability to specifically bind to a binding partner molecule (e.g., SARS-CoV-2 spike protein). It has been shown that the binding function of antibodies can be performed by fragments of full length antibodies. Examples of binding fragments encompassed within the term "binding moiety" of an antibody include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL and CH1 domains; (ii) A F (ab') 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region; (iii) an Fd fragment consisting of VH and CH1 domains; (iv) Fv fragments consisting of the VL and VH domains of the antibody single arm, (v) dAb fragments consisting of the VH domain (Ward et al, (1989) Nature 341:544-546); and (vi) an isolated Complementarity Determining Region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made into a single protein chain, in which the VL and VH regions pair to form a monovalent molecule (known as a single chain Fv (scFv); see, e.g., bird et al (1988) Science 242:423-426; and Huston et al (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term "binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art and screened for utility in the same manner as the whole antibody. The binding moiety may be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulins.
Antibodies that bind to the SARS-CoV-2 spike protein of the invention are antibodies that inhibit, block, or interfere with at least one SARS-CoV-2 spike protein activity (e.g., receptor binding activity) in vitro, in situ, and/or in vivo.
In one embodiment, the SARS-CoV-2 antibody comprises a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO. 2, SEQ ID NO. 6, SEQ ID NO. 12, SEQ ID NO. 18, SEQ ID NO. 30, SEQ ID NO. 42, SEQ ID NO. 84, SEQ ID NO. 94, SEQ ID NO. 100, SEQ ID NO. 112, SEQ ID NO. 122 or SEQ ID NO. 128. In one embodiment, the SARS-CoV-2 antibody comprises a light chain comprising an amino acid sequence as set forth in SEQ ID NO. 4, SEQ ID NO. 8, SEQ ID NO. 14, SEQ ID NO. 20, SEQ ID NO. 38, SEQ ID NO. 44, SEQ ID NO. 92, SEQ ID NO. 96, SEQ ID NO. 102, SEQ ID NO. 120, SEQ ID NO. 124 or SEQ ID NO. 130.
Whereas certain monoclonal antibodies can bind SARS-CoV-2 spike protein, the VH and VL sequences can be "mixed and matched" to produce other anti-SARS-CoV-2 spike protein binding molecules of the disclosure. Binding of such "mixed and matched" antibodies can be tested using standard binding assays known in the art (e.g., immunoblots, etc.). In some embodiments, when the VH chain and VL chain are mixed and matched, the VH sequence from a particular VH/VL pairing is replaced with a structurally similar VH sequence. Likewise, preferably, the VL sequences from a particular VH/VL pairing are replaced with structurally similar VL sequences.
Accordingly, in one aspect, the present disclosure provides an isolated monoclonal antibody, or binding portion thereof, comprising: (a) A heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 6, SEQ ID NO. 12, SEQ ID NO. 18, SEQ ID NO. 30, SEQ ID NO. 42, SEQ ID NO. 84, SEQ ID NO. 94, SEQ ID NO. 100, SEQ ID NO. 112, SEQ ID NO. 122 or SEQ ID NO. 128; and (b) a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO. 4, SEQ ID NO. 8, SEQ ID NO. 14, SEQ ID NO. 20, SEQ ID NO. 38, SEQ ID NO. 44, SEQ ID NO. 92, SEQ ID NO. 96, SEQ ID NO. 102, SEQ ID NO. 120, SEQ ID NO. 124, or SEQ ID NO. 130, wherein the antibody specifically binds SARS-CoV-2 spike protein.
In some embodiments, the heavy and light chain combination comprises: (a) A heavy chain variable region comprising the amino acid sequence SEQ ID NO. 2, and a light chain variable region comprising the amino acid sequence SEQ ID NO. 4; (b) A heavy chain variable region comprising the amino acid sequence SEQ ID NO. 6, and a light chain variable region comprising the amino acid sequence SEQ ID NO. 8; (c) A heavy chain variable region comprising the amino acid sequence SEQ ID NO. 12 and a light chain variable region comprising the amino acid sequence SEQ ID NO. 14; (d) A heavy chain variable region comprising the amino acid sequence SEQ ID NO. 18, and a light chain variable region comprising the amino acid sequence SEQ ID NO. 20; (e) A heavy chain variable region comprising the amino acid sequence SEQ ID NO. 30; and a light chain variable region comprising the amino acid sequence SEQ ID NO. 38; (f) A heavy chain variable region comprising the amino acid sequence SEQ ID NO. 42 and a light chain variable region comprising the amino acid sequence SEQ ID NO. 44; (g) A heavy chain variable region comprising the amino acid sequence SEQ ID NO. 84; and a light chain variable region comprising the amino acid sequence SEQ ID NO. 92; (h) A heavy chain variable region comprising the amino acid sequence SEQ ID NO. 94; and a light chain variable region comprising the amino acid sequence SEQ ID NO. 96; (i) A heavy chain variable region comprising the amino acid sequence SEQ ID NO. 100 and a light chain variable region comprising the amino acid sequence SEQ ID NO. 102; (j) A heavy chain variable region comprising the amino acid sequence SEQ ID NO 112; and a light chain variable region comprising the amino acid sequence SEQ ID NO. 120; (k) A heavy chain variable region comprising the amino acid sequence SEQ ID NO. 122; and a light chain variable region comprising the amino acid sequence SEQ ID NO. 124; or (l) a heavy chain variable region comprising the amino acid sequence SEQ ID NO:128, and a light chain variable region comprising the amino acid sequence SEQ ID NO:130.
Whereas each of these antibodies can bind SARS-CoV-2 spike protein and the binding specificity is provided primarily by CDR1, CDR2, and CDR3 regions, the VHCDR1, CDR2, and CDR3 sequences and the VLCDR1, CDR2, and CDR3 sequences can be "mixed and matched" to produce other anti-SARS-CoV-2 spike protein binding molecules of the present disclosure. SARS-CoV-2 spike protein binding of such "mixed and matched" antibodies can be tested using standard binding assays known in the art (e.g., immunoblots). Preferably, when the VHCDR sequences are mixed and matched, the CDR1, CDR2 and/or CDR3 sequences from a particular VH sequence are replaced by structurally similar CDR sequences. Likewise, when mixing and matching VLCDR sequences, CDR1, CDR2, and/or CDR3 sequences from a particular VL sequence are preferably replaced with structurally similar CDR sequences. It will be readily apparent to the skilled artisan that new VH and VL sequences can be generated by substituting one or more VH and/or VLCDR region sequences with structurally similar sequences from the CDR sequences disclosed herein.
Thus, in another aspect, the invention provides an isolated monoclonal antibody, or binding portion thereof, comprising at least one selected from the group consisting of: (a) A heavy chain variable region CDRL comprising the amino acid sequence SEQ ID NO. 26, SEQ ID NO. 80 or SEQ ID NO. 108; (b) A heavy chain variable region CDR2 comprising the amino acid sequence SEQ ID NO 27, SEQ ID NO 81 or SEQ ID NO 109; (c) A heavy chain variable region CDR3 comprising the amino acid sequence SEQ ID NO 28, SEQ ID NO 82 or SEQ ID NO 110; (d) A light chain variable region CDRL comprising the amino acid sequence SEQ ID NO. 34, SEQ ID NO. 88 or SEQ ID NO. 116; (e) A light chain variable region CDR2 comprising the amino acid sequence SEQ ID NO. 35, SEQ ID NO. 89 or SEQ ID NO. 117; and (f) a light chain variable region CDR3 comprising the amino acid sequence SEQ ID NO. 36, SEQ ID NO. 90 or SEQ ID NO. 118; wherein the antibody specifically binds SARS-CoV-2 spike protein.
In further embodiments, the antibody comprises (a) a heavy chain variable region CDRL comprising SEQ ID NO. 26; (b) a heavy chain variable region CDR2 comprising SEQ ID NO 27; (c) a heavy chain variable region CDR3 comprising SEQ ID NO. 28; (d) a light chain variable region CDRL comprising SEQ ID NO. 34; (e) A light chain variable region CDR2 comprising SEQ ID NO:35 and (f) a light chain variable region CDR3 comprising SEQ ID NO:36.
In further embodiments, the antibody comprises (a) a heavy chain variable region CDRL comprising SEQ ID NO. 80; (b) a heavy chain variable region CDR2 comprising SEQ ID NO. 81; (c) a heavy chain variable region CDR3 comprising SEQ ID NO 82; (d) a light chain variable region CDRL comprising SEQ ID NO. 88; (e) A light chain variable region CDR2 comprising SEQ ID NO 89 and (f) a light chain variable region CDR3 comprising SEQ ID NO 90.
In further embodiments, the antibody comprises (a) a heavy chain variable region CDRL comprising SEQ ID NO. 109; (b) a heavy chain variable region CDR2 comprising SEQ ID NO. 110; (c) a heavy chain variable region CDR3 comprising SEQ ID NO 111; (d) a light chain variable region CDRL comprising SEQ ID NO. 116; (e) Light chain variable region CDR2 comprising SEQ ID NO. 117 and (f) light chain variable region CDR3 comprising SEQ ID NO. 118.
The foregoing isolated anti-SARS-CoV-2 spike protein antibody CDR sequences establish a novel SARS-CoV-2 spike protein binding protein family that has been isolated according to the present invention and that comprises a polypeptide comprising the listed CDR sequences. In order to generate and select CDRs of the present invention with SARS-CoV-2 spike protein binding and/or SARS-CoV-2 spike protein detection and/or SARS-CoV-2 spike protein neutralizing activity, standard methods known in the art for generating binding proteins of the present invention and assessing the binding and/or detection and/or neutralizing characteristics of these binding proteins, including but not limited to those specifically described herein, can be used.
In one embodiment, the anti-SARS-CoV-2 antibody comprises a sequence identical to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO 8, 10, 12, 14, 16, 18, 20, 22, 30, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 84, 92, 94, 96, 98, 100, 102, 104, 112, 120, 122, 124, or a combination of at least one of the amino acid sequences shown in SEQ ID NO 98, 122, 124, or at least one of the amino acid sequences shown in SEQ ID NO 40, 42, 44, 102, 104, 112, 122, 64, 66, 68, 70, 72, 74, 76, 84, 92, 94, 96, 98, 100, 102, 104, 108, 124, or a combination of the amino acid sequences shown in SEQ ID NO 98, 124, 108, 124, or at least one of the amino acid sequences shown in SEQ ID NO 98, 124, 108, or at least one of the amino acid sequences shown in SEQ ID NO 98, 124, 108, or at least one of the amino acid sequences shown in SEQ ID NO to have the amino acid sequences shown in SEQ ID NO to be at least one of the amino acid sequence of the sequence. In one embodiment, the nucleotide sequence encoding the anti-SARS-CoV-2 antibody comprises a nucleotide sequence encoding SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 12, SEQ ID NO 14, SEQ ID NO 16, SEQ ID NO 18, SEQ ID NO 20, SEQ ID NO 22, SEQ ID NO 30, SEQ ID NO 38, SEQ ID NO 40, SEQ ID NO 42, SEQ ID NO 44, SEQ ID NO 46, SEQ ID NO 48, SEQ ID NO 50, SEQ ID NO 52, SEQ ID NO 54, SEQ ID NO 56, SEQ ID NO 58, SEQ ID NO 60, SEQ ID NO 62, SEQ ID NO 64, SEQ ID NO 66, SEQ ID NO 68, SEQ ID NO 70, SEQ ID NO 72, SEQ ID NO 74, SEQ ID NO 76, SEQ ID NO 84, SEQ ID NO 92, SEQ ID NO 94, SEQ ID NO 96, SEQ ID NO 98, SEQ ID NO 104 or a combination thereof. In one embodiment, the nucleotide sequence encoding the anti-SARS-CoV-2 antibody comprises a codon optimized nucleic acid sequence encoding a fragment comprising at least SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 12, SEQ ID NO 14, SEQ ID NO 16, SEQ ID NO 18, SEQ ID NO 20, SEQ ID NO 22, SEQ ID NO 30, SEQ ID NO 38, SEQ ID NO 40, SEQ ID NO 42, SEQ ID NO 44, SEQ ID NO 46, SEQ ID NO 48, SEQ ID NO 50, SEQ ID NO 52, SEQ ID NO 54, SEQ ID NO 56, SEQ ID NO 58, SEQ ID NO 60, SEQ ID NO 62, SEQ ID NO 64, SEQ ID NO 66, SEQ ID NO 68, SEQ ID NO 70, SEQ ID NO 72, SEQ ID NO 74, SEQ ID NO 76, SEQ ID NO 84, SEQ ID NO 92, SEQ ID NO 48, SEQ ID NO 50, SEQ ID NO 52, SEQ ID NO 54, SEQ ID NO 56, SEQ ID NO 58, SEQ ID NO 60, SEQ ID NO 62, SEQ ID NO 66, SEQ ID NO 68, SEQ ID NO 70, SEQ ID NO 72, SEQ ID NO 74, SEQ ID NO 98, SEQ ID NO 104, or a combination thereof.
In one embodiment, the nucleotide sequence encoding the anti-SARS-CoV-2 antibody comprises an RNA sequence transcribed from a DNA sequence encoding an amino acid sequence having at least one of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:30, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:84, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:98, SEQ ID NO: 108, 96, SEQ ID NO:100, 96, or a combination of at least one of them. In one embodiment, the nucleotide sequence encoding the anti-SARS-CoV-2 antibody comprises an RNA sequence transcribed from a DNA sequence encoding SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 12, SEQ ID NO 14, SEQ ID NO 16, SEQ ID NO 18, SEQ ID NO 20, SEQ ID NO 22, SEQ ID NO 30, SEQ ID NO 38, SEQ ID NO 40, SEQ ID NO 42, SEQ ID NO 44, SEQ ID NO 46, SEQ ID NO 48, SEQ ID NO 50, SEQ ID NO 52, SEQ ID NO 54, SEQ ID NO 56, SEQ ID NO 58, SEQ ID NO 60, SEQ ID NO 62, SEQ ID NO 64, SEQ ID NO 66, SEQ ID NO 68, SEQ ID NO 70, SEQ ID NO 72, SEQ ID NO 74, SEQ ID NO 76, SEQ ID NO 84, SEQ ID NO 92, SEQ ID NO 48, SEQ ID NO 50, SEQ ID NO 60, SEQ ID NO 104. In one embodiment, the nucleotide sequence encoding the anti-SARS-CoV-2 antibody comprises an RNA sequence transcribed from a DNA sequence encoding a polypeptide comprising SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 12, SEQ ID NO 14, SEQ ID NO 16, SEQ ID NO 18, SEQ ID NO 20, SEQ ID NO 22, SEQ ID NO 30, SEQ ID NO 38, SEQ ID NO 40, SEQ ID NO 42, SEQ ID NO 44, SEQ ID NO 46, SEQ ID NO 48, SEQ ID NO 50, SEQ ID NO 52, SEQ ID NO 54, SEQ ID NO 56, SEQ ID NO 58, SEQ ID NO 60, SEQ ID NO 62, SEQ ID NO 64, SEQ ID NO 66, SEQ ID NO 68, SEQ ID NO 70, SEQ ID NO 72, SEQ ID NO 74, SEQ ID NO 76, SEQ ID NO 84, SEQ ID NO 92, SEQ ID NO 94, SEQ ID NO 98, SEQ ID NO 104, at least one of the combination of which is at least one of SEQ ID NO 14, SEQ ID NO 56, SEQ ID NO 58, SEQ ID NO 60, SEQ ID NO 62, SEQ ID NO 64, SEQ ID NO 66, SEQ ID NO 68, SEQ ID NO 72, SEQ ID NO 98, SEQ ID NO 104, or at least one of the combination of them.
In one embodiment, the nucleotide sequence encoding an anti-SARS-CoV-2 antibody comprises a sequence that hybridizes to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, 7, 9, 11, 13, 15, 17, 21, 23, 29, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 83, 91, 93, 95, 97, 99, 101, 103, 111, 123, 121, 125, or a combination of at least one of the sequences shown in SEQ ID NO:17, 51, 53, 55, 67, 61, 63, 65, 67, 69, 71, 73, 75, 83, 75, 95, 125, or a combination of the sequences shown in SEQ ID NO:17, 59, 121, 125, or a combination of at least one of the sequences shown in SEQ ID NO:17, 13, 15, 17, 67, 21, 23, 75, 29, 75, 99, 101, 108, 125, or at least one of the sequences shown in SEQ ID NO:17, 121, 125, 95, 125. In one embodiment, the nucleotide sequence encoding the anti-SARS-CoV-2 antibody comprises SEQ ID NO 1, SEQ ID NO 3, SEQ ID NO 5, SEQ ID NO 7, SEQ ID NO 9, SEQ ID NO 11, SEQ ID NO 13, SEQ ID NO 15, SEQ ID NO 17, SEQ ID NO 21, SEQ ID NO 23, SEQ ID NO 29, SEQ ID NO 37, SEQ ID NO 39, SEQ ID NO 41, SEQ ID NO 43, SEQ ID NO 45, SEQ ID NO 47, SEQ ID NO 49, SEQ ID NO 51, SEQ ID NO 53, SEQ ID NO 55, SEQ ID NO 57, SEQ ID NO 59, SEQ ID NO 61, SEQ ID NO 63, SEQ ID NO 65, SEQ ID NO 67, SEQ ID NO 69, SEQ ID NO 71, SEQ ID NO 73, SEQ ID NO 75, SEQ ID NO 83, SEQ ID NO 91, SEQ ID NO 93, SEQ ID NO 95, SEQ ID NO 101, SEQ ID NO 121, SEQ ID NO 111 or a combination thereof. In one embodiment, the nucleotide sequence encoding the anti-SARS-CoV-2 antibody comprises a nucleic acid sequence comprising SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:29, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:83, SEQ ID NO:91, SEQ ID NO:103, SEQ ID NO:93, SEQ ID NO:95, 125, or a combination of at least one of them, or a portion of them.
In one embodiment, the nucleotide sequence encoding the anti-SARS-CoV-2 antibody comprises an RNA sequence transcribed from a codon optimized nucleotide sequence having a sequence identical to the sequence set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, 7, 9, 11, 13, 15, 17, 21, 23, 29, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 83, 91, 93, 95, 97, 99, 101, 103, 111, 123, 121, 125, or a combination of at least one of the nucleic acid sequences set forth in SEQ ID NO. 17, 59, 61, 63, 65, 67, 69, 71, 73, 75, 83, 95, 125, or a combination of the nucleic acid sequences set forth in at least one of SEQ ID NO. 7, 9, 17, 13, 15, 17, 67, 21, 71, 73, 75, 65, 83, 75, 95, 125, or at least one of the nucleic acid sequences set forth in SEQ ID NO. 101, 103, 111, 121, 125, or at least one of the nucleic acid sequences set forth in the sequence set forth in SEQ ID NO. 7, 51, 53, 55, or 17, or at least one of the sequence set forth in the sequence set forth above. In one embodiment, the nucleotide sequence encoding the anti-SARS-CoV-2 antibody comprises the nucleotide sequence set forth in SEQ ID NO 1, SEQ ID NO 3, SEQ ID NO 5, SEQ ID NO 7, SEQ ID NO 9, SEQ ID NO 11, SEQ ID NO 13, SEQ ID NO 15, SEQ ID NO 17, SEQ ID NO 21, SEQ ID NO 23, SEQ ID NO 29, SEQ ID NO 37, SEQ ID NO 39, SEQ ID NO 41, SEQ ID NO 43, SEQ ID NO 45, SEQ ID NO 47, SEQ ID NO 49, SEQ ID NO 51, SEQ ID NO 53, SEQ ID NO 55, SEQ ID NO 57, SEQ ID NO 59, SEQ ID NO 61, SEQ ID NO 63, SEQ ID NO 65, SEQ ID NO 67, SEQ ID NO 69, SEQ ID NO 71, SEQ ID NO 73, SEQ ID NO 75, SEQ ID NO 83, SEQ ID NO 91, SEQ ID NO 93, SEQ ID NO 95, SEQ ID NO 101, SEQ ID NO 121, SEQ ID NO 17 or a combination thereof. In one embodiment, the nucleotide sequence encoding an anti-SARS-CoV-2 antibody comprises an RNA sequence transcribed from a fragment comprising the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, 7, 9, 11, 13, 15, 17, 21, 23, 29, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 83, 91, 93, 95, 97, 99, 101, 103, 111, 121, 125, or at least one of the combinations of any of the above, or at least one of the above.
In one embodiment, the invention relates to a combination of a first nucleic acid molecule encoding a heavy chain of an anti-SARS-CoV-2 antibody and a second nucleic acid molecule encoding a light chain of an anti-SARS-CoV-2 antibody. In one embodiment, the first nucleic acid molecule is a first plasmid comprising a nucleotide sequence encoding a heavy chain of an anti-SARS-CoV-2 antibody and the second nucleic acid molecule is a second plasmid encoding a light chain of an anti-SARS-CoV-2 antibody.
In one embodiment, the first nucleic acid molecule encoding the heavy chain of an anti-SARS-CoV-2 antibody encodes SEQ ID NO 2, SEQ ID NO 6, SEQ ID NO 12, SEQ ID NO 18, SEQ ID NO 30, SEQ ID NO 42, SEQ ID NO 84, SEQ ID NO 94, SEQ ID NO 100, SEQ ID NO 112, SEQ ID NO 122 or SEQ ID NO 128, or an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO 2, SEQ ID NO 30, SEQ ID NO 42, SEQ ID NO 84, SEQ ID NO 94, SEQ ID NO 100, SEQ ID NO 112, SEQ ID NO 122 or SEQ ID NO 128, or a fragment comprising at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO 2, SEQ ID NO 6, SEQ ID NO 12, 30, SEQ ID NO 42, SEQ ID NO 84, SEQ ID NO 94, SEQ ID NO 80, 100, SEQ ID NO 112, SEQ ID NO 122, or SEQ ID NO 128. In one embodiment, the first nucleic acid molecule encoding the heavy chain of an anti-SARS-CoV-2 antibody comprises a nucleotide sequence of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:17, SEQ ID NO:29, SEQ ID NO:41, SEQ ID NO:83, SEQ ID NO:93, SEQ ID NO:99, SEQ ID NO:111, SEQ ID NO:121 or SEQ ID NO:127 that has at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:17, SEQ ID NO:29, SEQ ID NO:41, SEQ ID NO:83, SEQ ID NO:93, SEQ ID NO:99, SEQ ID NO:111, SEQ ID NO:121 or SEQ ID NO:127, or comprises at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:1, 5, SEQ ID NO:11, SEQ ID NO:17, 29, SEQ ID NO:29, 83, SEQ ID NO:93, SEQ ID NO: 95%, or at least 80% of the full-length of the nucleotide sequence.
In one embodiment, the second nucleic acid molecule encoding the light chain of the anti-SARS-CoV-2 antibody encodes SEQ ID NO 4, SEQ ID NO 8, SEQ ID NO 14, SEQ ID NO 20, SEQ ID NO 38, SEQ ID NO 44, SEQ ID NO 92, SEQ ID NO 96, SEQ ID NO 102, SEQ ID NO 120, SEQ ID NO 124 or SEQ ID NO 130, with the amino acid sequence set forth in SEQ ID NO 4, SEQ ID NO 8, SEQ ID NO 14, SEQ ID NO 20, SEQ ID NO 38, SEQ ID NO 44, SEQ ID NO 92, SEQ ID NO 96, SEQ ID NO 102, SEQ ID NO 120, SEQ ID NO 124 or SEQ ID NO 130 having at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identity or an amino acid sequence comprising at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO 4, SEQ ID NO 8, SEQ ID NO 14, SEQ ID NO 20, 38, SEQ ID NO 14, SEQ ID NO 38, SEQ ID NO 92, 96%, 80%, 90%, 95%, 96% or a fragment of at least 60%, 70%, 80%, 90% or 99% of the amino acid sequence set forth in SEQ ID NO 130. In one embodiment, the second nucleic acid molecule encoding the light chain of an anti-SARS-CoV-2 antibody comprises the nucleotide sequence of SEQ ID NO 3, SEQ ID NO 7, SEQ ID NO 13, SEQ ID NO 19, SEQ ID NO 37, SEQ ID NO 43, SEQ ID NO 91, SEQ ID NO 95, SEQ ID NO 101, SEQ ID NO 119, SEQ ID NO 123 or SEQ ID NO 129 having at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence of SEQ ID NO 3, SEQ ID NO 7, SEQ ID NO 13, SEQ ID NO 19, SEQ ID NO 37, SEQ ID NO 43, SEQ ID NO 91, SEQ ID NO 95, SEQ ID NO 101, SEQ ID NO 119, SEQ ID NO 123 or SEQ ID NO 129, or comprises at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence of SEQ ID NO 3, SEQ ID NO 7, SEQ ID NO 13, SEQ ID NO 19, SEQ ID NO 37, SEQ ID NO 43, SEQ ID NO 95, SEQ ID NO 129, SEQ ID NO 101, SEQ ID NO 119, SEQ ID NO 129, or SEQ ID NO 129.
The compositions of the present invention can treat, prevent and/or protect against any disease, disorder or condition associated with SARS-CoV-2 infection. In certain embodiments, the composition may treat, prevent, and/or protect against viral infection. In certain embodiments, the compositions can treat, prevent and/or protect against SARS-CoV-2 infection-related disease. In certain embodiments, the compositions may treat, prevent, and/or protect against covd-19.
The composition can result in the production of the synthetic antibody in the subject within at least about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 45 hours, 50 hours, or 60 hours of administration of the composition to the subject. The composition can result in the production of the synthetic antibody in the subject within at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days of administration of the composition to the subject. The composition may result in the production of the synthetic antibody in the subject within about 1 hour to about 6 days, about 1 hour to about 5 days, about 1 hour to about 4 days, about 1 hour to about 3 days, about 1 hour to about 2 days, about 1 hour to about 1 day, about 1 hour to about 72 hours, about 1 hour to about 60 hours, about 1 hour to about 48 hours, about 1 hour to about 36 hours, about 1 hour to about 24 hours, about 1 hour to about 12 hours, or about 1 hour to about 6 hours of administration of the composition to the subject.
The composition, when administered to a subject in need thereof, can result in faster production of the synthetic antibody in the subject than an endogenous antibody in the subject administered an antigen to induce a humoral immune response. The composition can result in the production of the synthetic antibody at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days prior to the production of the endogenous antibody in a subject administered an antigen to induce a humoral immune response.
The compositions of the present invention may have the characteristics desired for an effective composition, such as being safe, such that the composition does not cause disease or death; has protective effect on diseases; as well as providing ease of administration, few side effects, biostability, and low cost/dosage.
In some embodiments, the SARS-CoV-2 spike protein binding molecules (e.g., antibodies, etc.) of the invention exhibit a high ability to detect and bind SARS-CoV-2 spike protein in complex mixtures of salts, compounds, and other polypeptides, as assessed by any of several in vitro and in vivo assays known in the art. Those skilled in the art will appreciate that the SARS-CoV-2 spike protein binding molecules (e.g., antibodies, etc.) described herein can be used in methods of diagnosis and treatment and prevention of disease, as well as useful in the procedures and methods of the invention, including but not limited to: immunochromatography assays, immunodeficiency assays, luminex assays, ELISA assays, ELISPOT assays, protein microarray assays, western blot assays, mass spectrometry assays, radioimmunoassays (RIA), radioimmunodiffusion assays, liquid chromatography-tandem mass spectrometry assays, uro-henbane immunodiffusion assays, reversed-phase protein microarrays, rocket immunoelectrophoresis assays, immunohistochemical staining assays, immunoprecipitation assays, complement fixation assays, FACS, protein chip assays, isolation and purification procedures, and affinity chromatography (see, 2007,Van Emon,Immunoassay and Other Bioanalytical Techniques,CRC Press;2005,Wild,Immunoassay Handbook,Gulf Professional Publishing;1996,Diamandis and Christopoulos,Immunoassay,Academic Press;2005,Joos,Microarrays in Clinical Diagnosis,Humana Press;2005,Hamdan and Righetti,Proteomics Today,John Wiley and Sons;2007).
In some embodiments, the SARS-CoV-2 spike protein binding molecules (e.g., antibodies, etc.) of the invention exhibit a high ability to reduce or neutralize SARS-CoV-2 spike protein activity (e.g., receptor binding activity, etc.), as assessed by any of several in vitro and in vivo assays known in the art. For example, these SARS-CoV-2 spike protein binding molecules (e.g., antibodies, etc.) neutralize SARS-CoV-2 associated or SARS-CoV-2 mediated diseases or conditions.
As used herein, a SARS-CoV-2 spike protein binding molecule (e.g., antibody, etc.) that "specifically binds SARS-CoV-2 spike protein" is at 1X10 -6 M or less, more preferably 1x10 -7 M or less, more preferably 1x10 -8 M or less, more preferably 5x10 -9 M or less, more preferably 1x10 -9 M or less or even more preferably 3x10 -10 The KD of M or less binds SARS-CoV-2 spike protein. As used herein, the term "substantially does not bind" to a protein or cell means that it does not bind with high affinity or does not bind with high affinity to a protein or cell, i.e., greater than 1x10 6 M or greater, more preferably 1x10 5 M or greater, more preferably 1x10 4 M or greater, more preferably 1x10 3 M or greater, even more preferably 1x10 2 M or greater KD binds to a protein or cell. As used herein, the term "KD" is intended to refer to the dissociation constant obtained from the ratio of KD to Ka (i.e., KD/Ka) and expressed as molar concentration (M). The KD value of a SARS-CoV-2 spike protein binding molecule (e.g., antibody, etc.) can be determined by methods well known in the art. A preferred method for determining the KD of a binding molecule (e.g., antibody, etc.) is by using surface plasmon resonance, preferably using a biosensor system, such as The system.
As used herein, the term "high affinity" for IgG antibodies refers to having 1x10 for the target binding partner molecule -7 M or less, more preferably 5X10 -8 M or less, even more preferably 1x10 -8 M or less, even more preferably 5x10 -9 M or less and even more preferably 1x10 -9 Antibodies to KD of M or less. However, "high affinity" binding may vary for other antibody isotypes. For example, "high affinity" binding to IgM isotype refers to having 10 -6 M or less, more preferably 10 -7 M or less, even more preferably 10 -8 Antibodies to KD of M or less.
In certain embodiments, the antibody comprises a heavy chain constant region, e.g., an IgG1, igG2, igG3, igG4, igA, igE, igM, or IgD constant region. Preferably, the heavy chain constant region is an IgG1 heavy chain constant region or an IgG4 heavy chain constant region. In addition, antibodies may include a light chain constant region, a kappa light chain constant region, or a lambda light chain constant region. Preferably, the antibody comprises a kappa light chain constant region. Alternatively, the antibody moiety may be, for example, a Fab fragment or a single chain Fv fragment.
Recombinant nucleic acid sequences
As described above, the composition may comprise a recombinant nucleic acid sequence. The recombinant nucleic acid sequence may encode an antibody, a fragment thereof, a variant thereof, or a combination thereof. Antibodies are described in more detail below.
The recombinant nucleic acid sequence may be a heterologous nucleic acid sequence. The recombinant nucleic acid sequence may include one or more heterologous nucleic acid sequences.
The recombinant nucleic acid sequence may be an optimized nucleic acid sequence. Such optimization may increase or alter the immunogenicity of the antibody. Optimization may also improve transcription and/or translation. Optimization may include one or more of the following: increasing transcribed low GC content leader sequences; mRNA stability and codon optimization; adding a kozak sequence for increasing translation; adding an immunoglobulin (Ig) leader sequence encoding a signal peptide; the addition of internal IRES sequences and the elimination of possible cis-acting sequence motifs (i.e., internal TATA boxes).
Recombinant nucleic acid sequence constructs
The recombinant nucleic acid sequence may include one or more recombinant nucleic acid sequence constructs. The recombinant nucleic acid sequence construct may comprise one or more components, which are described in more detail below.
The recombinant nucleic acid sequence construct may comprise a heterologous nucleic acid sequence encoding a heavy chain polypeptide, a fragment thereof, a variant thereof, or a combination thereof. The recombinant nucleic acid sequence construct may comprise a heterologous nucleic acid sequence encoding a light chain polypeptide, a fragment thereof, a variant thereof, or a combination thereof. Recombinant nucleic acid sequence constructs may also include heterologous nucleic acid sequences encoding protease or peptidase cleavage sites. Recombinant nucleic acid sequence constructs may also include heterologous nucleic acid sequences encoding Internal Ribosome Entry Sites (IRES). IRES may be viral IRES or eukaryotic IRES. The recombinant nucleic acid sequence construct may include one or more leader sequences, wherein each leader sequence encodes a signal peptide. The recombinant nucleic acid sequence construct may include one or more promoters, one or more introns, one or more transcription termination regions, one or more start codons, one or more stop or stop codons, and/or one or more polyadenylation signals. The recombinant nucleic acid sequence construct may also include one or more linker or tag sequences. The tag sequence may encode a Hemagglutinin (HA) tag.
(1) Heavy chain polypeptides
The recombinant nucleic acid sequence construct may include a heterologous nucleic acid encoding a heavy chain polypeptide, a fragment thereof, a variant thereof, or a combination thereof. The heavy chain polypeptide may comprise a variable heavy chain (VH) region and/or at least one constant heavy Chain (CH) region. The at least one constant heavy chain region may include one constant heavy chain region 1 (CH 1), one constant heavy chain region 2 (CH 2), and one constant heavy chain region 3 (CH 3), and/or one hinge region.
In some embodiments, the heavy chain polypeptide may include a VH region and a CH1 region. In other embodiments, the heavy chain polypeptide can include a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region.
The heavy chain polypeptide can include a set of complementarity determining regions ("CDRs"). The CDR set may contain three hypervariable regions of the VH region. Starting from the N-terminus of the heavy chain polypeptide, these CDRs are denoted "CDR1", "CDR2" and "CDR3", respectively. CDR1, CDR2, and CDR3 of a heavy chain polypeptide can facilitate binding or recognition of an antigen.
(2) Light chain polypeptides
The recombinant nucleic acid sequence construct may comprise a heterologous nucleic acid sequence encoding the light chain polypeptide, a fragment thereof, a variant thereof, or a combination thereof. The light chain polypeptide may include a variable light chain (VL) region and/or a constant light Chain (CL) region.
The light chain polypeptides may include a set of complementarity determining regions ("CDRs"). The CDR set may comprise three hypervariable regions of the VL region. Starting from the N-terminus of the light chain polypeptide, these CDRs are denoted "CDR1", "CDR2" and "CDR3", respectively. CDR1, CDR2, and CDR3 of a light chain polypeptide can facilitate binding or recognition of an antigen.
(3) Protease cleavage site
The recombinant nucleic acid sequence construct may include a heterologous nucleic acid sequence encoding a protease cleavage site. The protease cleavage site may be recognized by a protease or a peptidase. The protease may be endopeptidase or endoprotease such as, but not limited to, furin, elastase, htrA, calpain, trypsin, chymotrypsin, trypsin and pepsin. The protease may be furin. In other embodiments, the protease may be a serine protease, a threonine protease, a cysteine protease, an aspartic protease, a metalloprotease, a glutamic acid protease, or any protease that cleaves an internal peptide bond (i.e., does not cleave an N-terminal or C-terminal peptide bond).
The protease cleavage site may include one or more amino acid sequences that promote or increase cleavage efficiency. The one or more amino acid sequences may promote or increase the efficiency of forming or producing the discrete polypeptides. The one or more amino acid sequences may comprise a 2A peptide sequence.
(4) Linker sequences
The recombinant nucleic acid sequence construct may comprise one or more linker sequences. The linker sequences may be spatially separated or linked to one or more components described herein. In other embodiments, the linker sequence may encode an amino acid sequence that spatially separates or links two or more polypeptides.
(5) Promoters
The recombinant nucleic acid sequence construct may include one or more promoters. The one or more promoters may be any promoter capable of driving gene expression and regulating gene expression. Such promoters are cis-acting sequence elements required for transcription via DNA-dependent RNA polymerase. The choice of promoter used to direct gene expression depends on the particular application. The promoter may be located in the recombinant nucleic acid sequence construct at about the same distance from the transcription start site as it would be in its natural environment. However, this change in distance can be accommodated without losing promoter function.
The promoter may be operably linked to a heterologous nucleic acid sequence encoding a heavy chain polypeptide and/or a light chain polypeptide. The promoter may be a promoter that exhibits efficient expression in eukaryotic cells. Promoters operably linked to the coding sequence may be a CMV promoter, a promoter from simian virus 40 (SV 40) (e.g., SV40 early promoter and SV40 late promoter), a Mouse Mammary Tumor Virus (MMTV) promoter, a Human Immunodeficiency Virus (HIV) promoter (e.g., bovine Immunodeficiency Virus (BIV) Long Terminal Repeat (LTR) promoter), a moloney virus promoter, an Avian Leukopenia Virus (ALV) promoter, a Cytomegalovirus (CMV) promoter (e.g., CMV immediate early promoter), an Epstein Barr Virus (EBV) promoter, or a Rous Sarcoma Virus (RSV) promoter. The promoter may also be a promoter from a human gene, such as human actin, human myosin, human hemoglobin, human muscle creatine, human polyhedrin, or human metallothionein.
The promoter may be a constitutive or inducible promoter, which initiates transcription only when the host cell is exposed to certain specific external stimuli. In the case of multicellular organisms, the promoter may also be specific for a particular tissue or organ or stage of development. The promoter may also be a tissue specific promoter, such as a natural or synthetic muscle or skin specific promoter. Examples of such promoters are described in U.S. patent application publication no. US20040175727, the entire contents of which are incorporated herein.
The promoter may be associated with an enhancer. Enhancers may be located upstream of the coding sequence. The enhancer may be a human actin, human myosin, human hemoglobin, human muscle creatine or a viral enhancer, such as an enhancer from CMV, FMDV, RSV or EBV. Polynucleotide functional enhancement is described in U.S. patent nos. 5,593,972, 5,962,428 and WO94/016737, each of which is incorporated by reference in its entirety.
(6) Introns
The recombinant nucleic acid sequence construct may include one or more introns. Each intron may include functional splice donor and acceptor sites. The intron may include a spliced enhancer. Introns may include one or more signals required for efficient splicing.
(7) Transcription termination region
The recombinant nucleic acid sequence construct may include one or more transcription termination regions. The transcription termination region may be downstream of the coding sequence to provide efficient termination. The transcription termination region may be obtained from the same gene as the above-described promoter, or may be obtained from one or more different genes.
(8) Initiation codon
The recombinant nucleic acid sequence construct may include one or more initiation codons. The initiation codon may be located upstream of the coding sequence. The initiation codon may be in frame with the coding sequence. The initiation codon can be associated with one or more signals required for efficient translation initiation, such as, but not limited to, a ribosome binding site.
(9) Stop codon
The recombinant nucleic acid sequence construct may include one or more termination or stop codons. The stop codon can be downstream of the coding sequence. The stop codon may be in frame with the coding sequence. The stop codon can be associated with one or more signals required for efficient translation termination.
(10) Polyadenylation signal
The recombinant nucleic acid sequence construct may include one or more polyadenylation signals. Polyadenylation signals may include one or more signals required for effective polyadenylation of a transcript. The polyadenylation signal may be located downstream of the coding sequence. The polyadenylation signal may be an SV40 polyadenylation signal, an LTR polyadenylation signal, a bovine growth hormone (bGH) polyadenylation signal, a human growth hormone (hGH) polyadenylation signal or a human beta-globulin polyadenylation signal. The SV40 polyadenylation signal may be that from the pCEP4 plasmid (Invitrogen, san Diego, calif.).
(11) Leader sequence
The recombinant nucleic acid sequence construct may comprise one or more leader sequences. The leader sequence may encode a signal peptide. The signal peptide may be an immunoglobulin (Ig) signal peptide, such as, but not limited to, an IgG signal peptide and an IgE signal peptide.
Arrangement of recombinant nucleic acid sequence constructs
As described above, the recombinant nucleic acid sequence may comprise one or more recombinant nucleic acid sequence constructs, wherein each recombinant nucleic acid sequence construct may comprise one or more components. One or more of the components are described in detail above. When included in a recombinant nucleic acid sequence construct, one or more components may be arranged in any order relative to one another. In some embodiments, the one or more components may be arranged in a recombinant nucleic acid sequence construct as described below.
(12) Arrangement 1
In one arrangement, the first recombinant nucleic acid sequence construct may comprise a heterologous nucleic acid sequence encoding a heavy chain polypeptide, and the second recombinant nucleic acid sequence construct may comprise a heterologous nucleic acid sequence encoding a light chain polypeptide. The first recombinant nucleic acid sequence construct may be placed in a vector. The second recombinant nucleic acid sequence construct may be placed in a second or separate vector. The placement of the recombinant nucleic acid sequence construct into a vector is described in more detail below.
The first recombinant nucleic acid sequence construct may also include a promoter, an intron, a transcription termination region, a start codon, a stop codon, and/or a polyadenylation signal. The first recombinant nucleic acid sequence construct may further comprise a leader sequence, wherein the leader sequence is upstream (or 5') of the heterologous nucleic acid sequence encoding the heavy chain polypeptide. Thus, the signal peptide encoded by the leader sequence may be linked to the heavy chain polypeptide by a peptide bond.
The second recombinant nucleic acid sequence construct may also include a promoter, an initiation codon, a termination codon, and a polyadenylation signal. The second recombinant nucleic acid sequence construct may further comprise a leader sequence, wherein the leader sequence is located upstream (or 5') of the heterologous nucleic acid sequence encoding the light chain polypeptide. Thus, the signal peptide encoded by the leader sequence may be linked to the light chain polypeptide by a peptide bond.
Thus, one embodiment of arrangement 1 may include a first vector (and thus a first recombinant nucleic acid sequence construct) encoding a heavy chain polypeptide comprising VH and CH1 and a second vector (and thus a second recombinant nucleic acid sequence construct) encoding a light chain polypeptide comprising VL and CL. A second embodiment of arrangement 1 may comprise a first vector (and thus a first recombinant nucleic acid sequence construct) encoding a heavy chain polypeptide comprising VH, CH1, hinge region, CH2 and CH3, and a second vector (and thus a second recombinant nucleic acid sequence construct) encoding a light chain polypeptide comprising VL and CL.
(13) Arrangement 2
In a second arrangement, the recombinant nucleic acid sequence construct may include a heterologous nucleic acid sequence encoding the heavy chain polypeptide and a heterologous nucleic acid sequence encoding the light chain polypeptide. The heterologous nucleic acid sequence encoding a heavy chain polypeptide may be located upstream (or 5') of the heterologous nucleic acid sequence encoding a light chain polypeptide. Alternatively, the heterologous nucleic acid sequence encoding a light chain polypeptide may be located upstream (or 5') of the heterologous nucleic acid sequence encoding a heavy chain polypeptide.
The recombinant nucleic acid sequence construct may be placed in a vector as described in more detail below.
The recombinant nucleic acid sequence construct may include a heterologous nucleic acid sequence encoding a protease cleavage site and/or a linker sequence. If included in the recombinant nucleic acid sequence construct, the heterologous nucleic acid sequence encoding a protease cleavage site can be positioned between the heterologous nucleic acid sequence encoding a heavy chain polypeptide and the heterologous nucleic acid sequence encoding a light chain polypeptide. Thus, protease cleavage sites allow for separation of heavy and light chain polypeptides into different polypeptides upon expression. In other embodiments, if the linker sequence is included in a recombinant nucleic acid sequence construct, the linker sequence may be located between the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the heterologous nucleic acid sequence encoding the light chain polypeptide.
Recombinant nucleic acid sequence constructs can also include promoters, introns, transcription termination regions, start codons, stop codons, and/or polyadenylation signals. The recombinant nucleic acid sequence construct may include one or more promoters. The recombinant nucleic acid sequence construct may comprise two promoters such that one promoter may bind to a heterologous nucleic acid sequence encoding a heavy chain polypeptide and a second promoter may bind to a heterologous nucleic acid sequence encoding a light chain polypeptide. In still other embodiments, the recombinant nucleic acid sequence construct can include a promoter associated with the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the heterologous nucleic acid sequence encoding the light chain polypeptide.
The recombinant nucleic acid sequence construct may further comprise two leader sequences, wherein the first leader sequence is upstream (or 5 ') of the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the second leader sequence is upstream (or 5') of the heterologous nucleic acid sequence encoding the light chain polypeptide. Thus, a first signal peptide encoded by a first leader sequence may be linked to a heavy chain polypeptide by a peptide bond, and a second signal peptide encoded by a second leader sequence may be linked to a light chain polypeptide by a peptide bond.
Thus, one embodiment of arrangement 2 may include a vector (and thus a recombinant nucleic acid sequence construct) encoding a heavy chain polypeptide comprising VH and CH1 and a light chain polypeptide comprising VL and CL, wherein the linker sequence is located between the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the heterologous nucleic acid sequence encoding the light chain polypeptide.
A second embodiment of arrangement 2 may comprise a vector (and thus a recombinant nucleic acid sequence construct) encoding a heavy chain polypeptide comprising VH and CH1 and a light chain polypeptide comprising VL and CL, wherein the heterologous nucleic acid sequence encoding the protease cleavage site is located between the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the heterologous nucleic acid sequence encoding the light chain polypeptide.
A third embodiment of arrangement 2 may include a vector (and thus a recombinant nucleic acid sequence construct) encoding a heavy chain polypeptide including VH, CH1, hinge region, CH2, and CH3 and a light chain polypeptide including VL and CL, wherein the linker sequence is located between the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the heterologous nucleic acid sequence encoding the light chain polypeptide.
A fourth example of arrangement 2 may include a vector (and thus a recombinant nucleic acid sequence construct) encoding a heavy chain polypeptide including VH, CH1, hinge region, CH2, and CH3 and a light chain polypeptide including VL and CL, wherein the heterologous nucleic acid sequence encoding the protease cleavage site is located between the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the heterologous nucleic acid sequence encoding the light chain polypeptide.
Expression from recombinant nucleic acid sequence constructs
As described above, the recombinant nucleic acid sequence construct may include, in one or more components, a heterologous nucleic acid sequence encoding a heavy chain polypeptide and/or a heterologous nucleic acid sequence encoding a light chain polypeptide. Thus, the recombinant nucleic acid sequence construct may facilitate expression of the heavy chain polypeptide and/or the light chain polypeptide.
When using arrangement 1 as described above, the first recombinant nucleic acid sequence construct may facilitate expression of the heavy chain polypeptide and the second recombinant nucleic acid sequence construct may facilitate expression of the light chain polypeptide. When using arrangement 2 as described above, the recombinant nucleic acid sequence construct may facilitate expression of the heavy chain polypeptide and the light chain polypeptide.
When expressed, for example, but not limited to, in a cell, organism, or mammal, the heavy chain polypeptide and the light chain polypeptide can be assembled into the synthetic antibody. In particular, the heavy chain polypeptide and the light chain polypeptide may interact with each other such that assembly produces a synthetic antibody capable of binding an antigen. In other embodiments, the heavy chain polypeptide and the light chain polypeptide can interact with each other such that assembly results in a synthetic antibody that is more immunogenic than an antibody that is not assembled as described herein. In other embodiments, the heavy chain polypeptide and the light chain polypeptide can interact with each other such that assembly produces a synthetic antibody capable of eliciting or inducing an immune response against the antigen.
Carrier body
The recombinant nucleic acid sequence construct described above may be placed in one or more vectors. The one or more vectors may comprise an origin of replication. The one or more vectors may be plasmids, phages, bacterial artificial chromosomes or yeast artificial chromosomes. The one or more vectors may be self-replicating extra-chromosomal vectors or vectors that integrate into the host genome.
Vectors include, but are not limited to, plasmids, expression vectors, recombinant viruses, any form of recombinant "naked DNA" vector, and the like. "vector" includes nucleic acids that can infect, transfect, transiently or permanently transduce a cell. It will be appreciated that the vector may be a naked nucleic acid, or a nucleic acid complexed with a protein or lipid. The vector optionally includes viral or bacterial nucleic acids and/or proteins, and/or membranes (e.g., cell membranes, viral lipid envelopes, etc.). Vectors include, but are not limited to, replicons (e.g., RNA replicons, phage) to which DNA fragments may be attached and become replicated. Thus, vectors include, but are not limited to, RNA, autonomously replicating circular or linear DNA or RNA (e.g., plasmids, viruses, etc., see, e.g., U.S. patent No. 5,217,879, and including both expression plasmids and non-expression plasmids, in some embodiments, the vector includes linear DNA, enzymatic DNA, or synthetic DNA when the recombinant microorganism or cell culture is described as a host "expression vector," this includes extrachromosomal circular and linear DNA as well as DNA that has been incorporated into the host chromosome.
One or more vectors may be heterologous expression constructs, which are typically plasmids used to introduce a particular gene into a target cell. Once the expression vector is within the cell, the heavy and/or light chain polypeptides encoded by the recombinant nucleic acid sequence construct are produced by cellular transcription and translation mechanisms, the ribosomal complex. The one or more vectors can express a large amount of stable messenger RNA and thus express a protein.
(14) Expression vector
The one or more vectors may be circular plasmids or linear nucleic acids. Circular plasmids and linear nucleic acids are capable of directing expression of a particular nucleotide sequence in a suitable subject cell. One or more vectors comprising the recombinant nucleic acid sequence construct may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components.
(15) Plasmid(s)
The one or more vectors may be plasmids. Plasmids can be used to transfect cells with recombinant nucleic acid sequence constructs. Plasmids can be used to introduce a recombinant nucleic acid sequence construct into a subject. The plasmid may also contain a regulatory sequence which may be well suited for gene expression in the cells to which the plasmid is administered.
The plasmid may also contain a mammalian origin of replication in order to maintain the plasmid extrachromosomally and to produce multiple copies of the plasmid in the cell. The plasmid may be pVAX1, pCEP4 or prsp 4 from Invitrogen (Invitrogen) (san diego, california), which may include an epstein barr virus origin of replication and a nuclear antigen EBNA-1 coding region that may produce high copy episomal replication without integration. The backbone of the plasmid may be pAV0242. The plasmid may be a replication defective adenovirus type 5 (Ad 5) plasmid.
The plasmid may be pSE420 (Invitrogen, san Diego, calif.), which may be used for the production of proteins in E.coli (E.coli). The plasmid may also be pYES2 (Invitrogen, san Diego, calif.), which may be used for protein production in the Saccharomyces cerevisiae strain of yeast. The plasmid may also be MAXBAC TM Plasmids of the complete baculovirus expression system (Invitrogen, san Diego, calif.), which can be used for protein production in insect cells. The plasmid may also be pcDNAI or pcDNA3 (Invitrogen, san Diego, calif.), which may be used to produce proteins in mammalian cells such as Chinese Hamster Ovary (CHO) cells.
(16)RNA
In one embodiment, the nucleic acid is an RNA molecule. In one embodiment, the RNA molecule is transcribed from the DNA sequences described herein. Thus, in one embodiment, the invention provides RNA molecules encoding one or more dmabs. The RNA may be positive stranded. Thus, in some embodiments, the RNA molecule can be translated by the cell without any intervening replication steps, such as reverse transcription. RNA molecules useful in the present invention may have a 5' cap (e.g., 7-methylguanosine). This cap can enhance in vivo translation of RNA. The 5 'nucleotides of the RNA molecules useful in the present invention may have 5' triphosphate groups. In the blocked RNA, this can be linked to 7-methylguanosine via a 5 '-to-5' bridge. The RNA molecule may have a 3' poly-A tail. It may also include a poly-A polymerase recognition sequence (e.g., AAUAAA) near its 3' end. RNA molecules useful in the present invention may be single stranded. RNA molecules useful in the present invention may comprise synthetic RNA. In some embodiments, the RNA molecule is a naked RNA molecule. In one embodiment, the RNA molecule is contained within a vector.
In one embodiment, the RNA has 5 'and 3' utrs. In one embodiment, the 5' utr is between 0 and 3000 nucleotides in length. The length of the 5 'and 3' UTR sequences to be added to the coding region may be varied by different methods, including but not limited to designing primers for PCR that anneal to different regions of the UTR. Using this method, one of ordinary skill in the art can modify the 5 'and 3' UTR lengths required to achieve optimal translational efficiency following transfection of transcribed RNA.
The 5 'and 3' UTRs may be naturally occurring, endogenous 5 'and 3' UTRs of the gene of interest. Alternatively, UTR sequences that are not endogenous to the gene of interest may be added by incorporating UTR sequences into the forward and reverse primers or by any other modification of the template. The use of UTR sequences which are not endogenous to the gene of interest can be used to modify the stability and/or translation efficiency of the RNA. For example, AU-rich elements in the 3' utr sequence are known to reduce RNA stability. Thus, based on the characteristics of UTRs well known in the art, a 3' UTR can be selected or designed to increase the stability of transcribed RNA.
In one embodiment, the 5' utr may comprise a Kozak sequence of an endogenous gene. Alternatively, when a 5'utr that is not endogenous to the gene of interest is added by PCR as described above, the consensus Kozak sequence may be redesigned by adding the 5' utr sequence. Kozak sequences may increase the translation efficiency of some RNA transcripts, but it does not seem necessary that all RNAs achieve efficient translation. The requirements of many RNAs for Kozak sequences are known in the art. In other embodiments, the 5' utr may be derived from an RNA virus whose RNA genome is stable in the cell. In other embodiments, various nucleotide analogs can be used in the 3 'or 5' UTR to prevent exonuclease degradation of RNA.
In one embodiment, the RNA has both a cap on the 5 'end and a 3' poly (a) tail, which determines ribosome binding, initiation of translation and stability of the RNA in the cell.
In one embodiment, the RNA is a nucleoside modified RNA. Nucleoside modified RNAs have particular advantages over non-modified RNAs, including, for example, increased stability, low or absent innate immunogenicity, and enhanced translation.
(17) Circular and linear carrier
The one or more vectors may be circular plasmids that can be integrated into the cell genome to transform the target cell or exist extrachromosomally (e.g., autonomously replicating plasmids with origins of replication). The vector may be pVAX, pcdna3.0, or proviax, or any other expression vector capable of expressing the heavy and/or light chain polypeptides encoded by the recombinant nucleic acid sequence construct.
Also provided herein are linear nucleic acids or linear expression cassettes ("LECs") that are capable of being efficiently delivered to a subject via electroporation and express heavy and/or light chain polypeptides encoded by the recombinant nucleic acid sequence constructs. LECs can be any linear DNA that does not contain any phosphate backbone. LECs may not comprise any antibiotic resistance genes and/or phosphate backbones. LECs may not contain other nucleic acid sequences that are not associated with the desired gene expression.
LECs can be derived from any plasmid that can be linearized. The plasmid may be capable of expressing the heavy chain polypeptide and/or the light chain polypeptide encoded by the recombinant nucleic acid sequence construct. The plasmid may be pNP (Puerto Rico/34) or pM2 (New Caledonia/99). The plasmid may be WLV009, pVAX, pcdna3.0 or proviax or any other expression vector capable of expressing the heavy and/or light chain polypeptides encoded by the recombinant nucleic acid sequence construct.
LEC may be PcrM2. LECs can be pcrNP. PcrNP, and pcrMR can be derived from pNP (Puerto Rico/34) and pM2 (New Caledonia/99), respectively.
(18) Viral vectors
In one embodiment, provided herein are viral vectors capable of delivering a nucleic acid of the invention to a cell. The expression vector may be provided to the cell in the form of a viral vector. Viral vector techniques are well known in the art and are described, for example, in Sambrook et al (2001), and Ausubel et al (1997), among other virology and molecular biology manuals. Viruses that may be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. In general, suitable vectors contain an origin of replication functional in at least one organism, a promoter sequence, a convenient restriction endonuclease site and one or more selection markers. (see, e.g., WO01/96584; WO01/29058; and U.S. Pat.No.6,326,193. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian (e.g., human) cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, adeno-associated viruses, etc. see, e.g., U.S. Pat. Nos. 5,350,674 and 5,585,362.
(19) Method for preparing carrier
Provided herein are methods for preparing one or more vectors in which recombinant nucleic acid sequence constructs have been placed. After the final subcloning step, the vector can be used to inoculate cell cultures in a large-scale fermenter using methods known in the art.
In other embodiments, the vector may be used with one or more Electroporation (EP) devices after the final subcloning step. The EP device is described in more detail below.
The one or more vectors may be formulated or manufactured using a combination of known devices and techniques, but are preferably manufactured using plasmid manufacturing techniques described in the authorized, co-pending united states. Provisional application U.S. serial No. 60/939,792 was filed on day 23 of month 5 of 2007. In some embodiments, the DNA plasmids described herein may be formulated at a concentration of greater than or equal to 10 mg/mL. The manufacturing techniques also include or incorporate various devices and schemes commonly known to those of ordinary skill in the art, in addition to those described in U.S. patents. Serial No. 60/939,792, including those described in issued patent us patent No. 7,238,522 issued at 7/3/2007. The above-mentioned applications and patents, U.S. Ser. No. 60/939,792 and U.S. Pat. No. 7,238,522, each of which is incorporated herein by reference in its entirety.
3. Antibodies to
As described above, the recombinant nucleic acid sequence may encode an antibody, a fragment thereof, a variant thereof, or a combination thereof. Antibodies may bind to or react with antigens, as will be described in more detail below.
An antibody may comprise sets of heavy and light chain complementarity determining regions ("CDRs") interposed between sets of heavy and light chain frameworks ("FR"), respectively, that provide support for the CDRs and define the spatial relationship of the CDRs relative to one another. The CDR sets may contain three hypervariable regions of either the heavy or light chain V regions. Starting from the N-terminus of the heavy or light chain, these regions are denoted "CDR1", "CDR2" and "CDR3", respectively. Thus, an antigen binding site may comprise six CDRs, including sets of CDRs from each of the heavy and light chain V regions.
Proteolytic enzyme papain preferentially cleaves IgG molecules to generate several fragments, two of which (F (ab) fragments) each comprise a covalent heterodimer comprising an intact antigen binding site. Pepsin is capable of cleaving IgG molecules to provide several fragments, including F (ab') 2 A fragment comprising two antigen binding sites. Thus, the antibody may be Fab or F (ab') 2 . Fab may include heavy chain polypeptides and light chain polypeptides. The heavy chain polypeptide of a Fab may include a VH region and a CH1 region. The light chain of a Fab may include a VL region and a CL region.
The antibody may be an immunoglobulin (Ig). Ig may be, for example, igA, igM, igD, igE and IgG. Immunoglobulins may include both heavy chain polypeptides and light chain polypeptides. The heavy chain polypeptide of an immunoglobulin may include a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region. The light chain polypeptide of an immunoglobulin may include a VL region and a CL region.
The antibody may be a polyclonal or monoclonal antibody. The antibody may be a chimeric antibody, a single chain antibody, an affinity matured antibody, a human antibody, a humanized antibody or a fully human antibody. The humanized antibody may be an antibody from a non-human species that binds to a desired antigen, the antibody having one or more Complementarity Determining Regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule.
The antibody may be a bispecific antibody as described in more detail below. The antibody may be a bifunctional antibody, as also described in more detail below.
As described above, upon administration of the composition to a subject, antibodies can be produced in the subject. Antibodies may have a half-life in a subject. In some embodiments, the antibody may be modified to extend or shorten its half-life in the subject. Such modifications are described in more detail below.
Antibodies can be defucosylated as described in more detail below.
In one embodiment, the antibody binds to SARS-CoV-2 antigen. In one embodiment, the antibody binds to at least one epitope of SARS-CoV-2 spike protein. In one embodiment, the antibody binds SARS-CoV-2RBD.
Antibodies can be modified to reduce or prevent Antibody Dependent Enhancement (ADE) of antigen-related diseases, as described in more detail below.
Bispecific antibodies
The recombinant nucleic acid sequence may encode a bispecific antibody, a fragment thereof, a variant thereof, or a combination thereof. Bispecific antibodies can bind to or react with two antigens, e.g., two antigens described in more detail below. Bispecific antibodies may be composed of fragments of two of the antibodies described herein, thereby allowing the bispecific antibody to bind to or react with two desired target molecules, which may include antigens, ligands (including ligands for receptors), receptors (including ligand binding sites on receptors), ligand-receptor complexes, and markers, as described in more detail below.
The present invention provides novel bispecific antibodies comprising a first antigen binding site that specifically binds to a first target and a second antigen binding site that specifically binds to a second target, with particularly advantageous properties such as producibility, stability, binding affinity, biological activity, specific targeting of certain T cells, targeting efficiency, and reduced toxicity. In some cases, there is a bispecific antibody, wherein the bispecific antibody binds to the first target with high affinity and binds to the second target with low affinity. In other cases, there is a bispecific antibody, wherein the bispecific antibody binds to the first target with low affinity and binds to the second target with high affinity. In other cases, there is a bispecific antibody, wherein the bispecific antibody binds to the first target with a desired affinity and binds to the second target with a desired affinity.
In one embodiment, the bispecific antibody is a bivalent antibody comprising a) a first light chain and a first heavy chain of an antibody that specifically binds to a first antigen, and b) a second light chain and a second heavy chain of an antibody that specifically binds to a second antigen.
Bispecific antibody molecules according to the invention may have two binding sites with any desired specificity. In some embodiments, one of these binding sites is capable of binding a tumor-associated antigen. In some embodiments, the binding site comprised in the Fab fragment is a binding site specific for the SARS-CoV-2 antigen. In some embodiments, the binding site contained in the single chain Fv fragment is a binding site that is specific for a SARS-CoV-2 antigen (e.g., a SARS-CoV-2 spike antigen).
In some embodiments, one of the binding sites of the bispecific antibody according to the invention is capable of binding to a T cell specific receptor molecule and/or a natural killer cell (NK cell) specific receptor molecule. The T cell specific receptor is a so-called "T cell receptor" (TCR) which allows T cells to bind and, if additional signal is present, activate and respond to an epitope/antigen presented by another cell, known as an antigen presenting cell or APC. T cell receptors are known to resemble Fab fragments of naturally occurring immunoglobulins. It is generally monovalent, comprising alpha-and beta-chains, and in some embodiments, it comprises gamma-and delta-chains. Thus, in some embodiments, the TCR is a TCR (α/β), and in some embodiments, it is a TCR (γ/δ). The T cell receptor forms a complex with the CD 3T-cell co-receptor. CD3 is a protein complex and consists of four distinct chains. In mammals, the complex comprises a CD3 gamma chain, a CD36 chain and two CD3E chains. These chains bind to molecules known as T Cell Receptors (TCRs) and zeta chains to generate activation signals in T lymphocytes. Thus, in some embodiments, the T cell specific receptor is a CD3T cell co-receptor. In some embodiments, the T cell specific receptor is CD28, a protein that is also expressed on T cells. CD28 can provide the costimulatory signal required for T cell activation. CD28 plays an important role in T cell proliferation and survival, cytokine production and T-helper cell-type 2 development. Yet another embodiment of a T cell specific receptor is CD134, also known as Ox40.CD134/OX40 is expressed 24 to 72 hours post activation and can be used to define secondary co-stimulatory molecules. Another example of a T cell receptor is 4-1BB, which is capable of binding to 4-1 BB-ligands on Antigen Presenting Cells (APCs), thereby generating a co-stimulatory signal for the T cell. An additional embodiment of the receptor found mainly on T cells is CD5, CD5 also being found at low levels on B cells. A further embodiment of a receptor that modifies T cell function is CD95, also known as the Fas receptor, which mediates apoptotic signaling through Fas-ligands expressed on the surface of other cells. CD95 has been reported to regulate TCR/CD3 driven signaling pathways in resting T lymphocytes.
An example of an NK cell specific receptor molecule is CD16, a low affinity Fc receptor and NKG2D. Examples of receptor molecules present on the surface of T cells and Natural Killer (NK) cells are CD2 and other members of the CD 2-superfamily. CD2 is able to act as a co-stimulatory molecule on T and NK cells.
In some embodiments, the first binding site of the bispecific antibody molecule binds to a SARS-CoV-2 antigen and the second binding site binds to a T cell specific receptor molecule and/or a Natural Killer (NK) cell specific receptor molecule.
In some embodiments, the first binding site of the antibody molecule binds to a SARS-CoV-2 spike antigen and the second binding site binds to a T cell specific receptor molecule and/or a Natural Killer (NK) cell specific receptor molecule. In some embodiments, the first binding site of the antibody molecule binds to a SARS-CoV-2 spike antigen and the second binding site binds to one of CD3, T Cell Receptor (TCR), CD28, CD16, NKG2D, ox40, 4-1BB, CD2, CD5 and CD 95.
In some embodiments, the first binding site of the antibody molecule binds to a T cell specific receptor molecule and/or a Natural Killer (NK) cell specific receptor molecule and the second binding site binds to a SARS-CoV-2 antigen. In some embodiments, the first binding site of the antibody binds to a T cell specific receptor molecule and/or a Natural Killer (NK) cell specific receptor molecule and the second binding site binds to a SARS-CoV-2 spike antigen. In some embodiments, the first binding site of the antibody binds one of CD3, T Cell Receptor (TCR), CD28, CD16, NKG2D, ox, 4-1BB, CD2, CD5, and CD95, and the second binding site binds SARS-CoV-2 spike antigen.
Bifunctional antibodies
The recombinant nucleic acid sequence may encode a bifunctional antibody, a fragment thereof, a variant thereof, or a combination thereof. The bifunctional antibody may bind to or react with the antigen described below. Bifunctional antibodies may also be modified to confer additional functions to the antibody in addition to recognizing antigen binding. Such modifications may include, but are not limited to, coupling to factor H or a fragment thereof. Factor H is a soluble regulator of complement activation and thus may promote an immune response via complement-mediated lysis (CML).
Extension of antibody half-life
As described above, antibodies can be modified to extend or shorten the half-life of the antibody in a subject. The modification may extend or shorten the half-life of the antibody in the serum of the subject.
Modifications may be present in the constant region of the antibody. The modification may be one or more amino acid substitutions in the constant region of the antibody that extend the half-life of the antibody as compared to the half-life of an antibody that does not comprise the one or more amino acid substitutions. The modification may be one or more amino acid substitutions in the CH2 domain of the antibody that extend the half-life of the antibody compared to the half-life of an antibody that does not comprise the one or more amino acid substitutions.
In some embodiments, one or more amino acid substitutions in the constant region can include replacing a methionine residue in the constant region with a tyrosine residue, replacing a serine residue in the constant region with a threonine residue, replacing a threonine residue in the constant region with a glutamic acid residue, or any combination thereof, thereby extending the half life of the antibody.
In other embodiments, one or more amino acid substitutions in the constant region may include substitution of a tyrosine residue for a methionine residue in the CH2 domain, substitution of a threonine residue for a serine residue in the CH2 domain, substitution of a glutamic acid residue for a threonine residue in the CH2 domain, or any combination thereof, thereby extending the half-life of the antibody.
Desfuction saccharification
The recombinant nucleic acid sequence may encode a nonfucosylated antibody (i.e., a defucosylated antibody or a nonfucosylated antibody), a fragment thereof, a variant thereof, or a combination thereof. Fucosylation involves the addition of the sugar fucose to a molecule, for example linking fucose to N-glycans, O-glycans and glycolipids. Thus, in defucosylated antibodies, fucose is not attached to the carbohydrate chain of the constant region. Furthermore, this lack of fucosylation may improve fcγr11ia binding and antibody directed cytotoxicity (ADCC) activity by antibodies compared to fucosylated antibodies. Thus, in some embodiments, the nonfucosylated antibodies can exhibit increased ADCC activity as compared to the fucosylated antibodies.
Antibodies can be modified to prevent or inhibit fucosylation of the antibody. In some embodiments, such modified antibodies may exhibit increased ADCC activity as compared to unmodified antibodies. The modification may be in the heavy chain, the light chain, or a combination thereof. The modification may be one or more amino acid substitutions in the heavy chain, one or more amino acid substitutions in the light chain, or a combination thereof.
Reduced ADE response
Antibodies can be modified to reduce or prevent Antibody Dependent Enhancement (ADE) of antigen-related diseases, but still neutralize the antigen.
In some embodiments, the antibody may be modified to include one or more amino acid substitutions that reduce or prevent binding of the antibody to fcγr1a. The one or more amino acid substitutions may be in the constant region of the antibody. One or more amino acid substitutions may include substitution of an alanine residue for a leucine residue in the constant region of an antibody, i.e., also referred to herein as LA, LA mutation, or LA substitution. One or more amino acid substitutions may include substitution of two leucine residues with an alanine residue in the constant region of an antibody, each leucine residue also referred to herein as LALA, LALA mutation, or LALA substitution. The presence of LALA substitution may prevent or block binding of the antibody to fcγr1a and, thus, the modified antibody does not enhance or cause ADE of antigen-related diseases, but still neutralizes the antigen.
4. Antigens
The synthetic antibodies are directed against an antigen or fragment or variant thereof. The antigen may be a nucleic acid sequence, an amino acid sequence, a polysaccharide, or a combination thereof. The nucleic acid sequence may be DNA, RNA, cDNA, a variant thereof, a fragment thereof, or a combination thereof. The amino acid sequence may be a protein, a peptide, a variant thereof, a fragment thereof, or a combination thereof. The polysaccharide may be a polysaccharide encoded by a nucleic acid.
The antigen may be from a virus. The antigen may be associated with a viral infection. In one embodiment, the antigen may be associated with SARS-CoV-2 infection or with COVID-19. In one embodiment, the antigen may be a SARS-CoV-2 spike antigen.
In one embodiment, the antigen may be a fragment of SARS-CoV-2 antigen. For example, in one embodiment, the antigen is a fragment of SARS-CoV-2 spike protein. In one embodiment, the antigen is the Receptor Binding Domain (RBD) of SARS-CoV-2 spike protein.
In one embodiment, the synthetic antibodies of the invention target two or more antigens. In one embodiment, the at least one antigen of the bispecific antibody is selected from the antigens described herein. In one embodiment, the two or more antigens are selected from the antigens described herein.
Viral antigens
The viral antigen may be a viral antigen or a fragment or variant thereof. The virus may be a pathogenic virus. The virus may be a coronavirus. The virus may be SARS or SARS-CoV-2 virus.
The antigen may be a SARS-CoV-2 virus antigen or a fragment or variant thereof. The SARS-CoV-2 antigen can be from a factor that allows the virus to replicate, infect or survive. Factors that allow the replication or survival of the SARS-CoV-2 virus include, but are not limited to, structural proteins and non-structural proteins. Such a protein may be a spike protein.
5. Excipients and other components of the composition
The composition may further comprise a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient may be a functional molecule, such as a carrier, carrier or diluent. The pharmaceutically acceptable excipient may be a transfection facilitating agent, which may include surfactants such as Immune Stimulating Complexes (ISCOMS), freunds incomplete adjuvant, LPS analogs (including monophosphoryl lipid a), muramyl peptides, quinone analogs, vesicles (such as squalene and squalene), hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyanions, polycations or nanoparticles, or other known transfection facilitating agents.
Transfection facilitating agents are polyanions, polycations, including poly-L-glutamate (LGS) or lipids. The transfection facilitating agent is poly-L-glutamic acid, and the poly-L-glutamic acid may be present in the composition at a concentration of less than 6 mg/ml. The transfection facilitating agent may also include surfactants such as Immune Stimulating Complexes (ISCOMS), freunds incomplete adjuvant, LPS analogs (including monophosphoryl lipid a), muramyl peptides, quinone analogs, and vesicles (e.g., squalene and squalene), and hyaluronic acid may also be used in combination with the composition. The composition may also include a transfection facilitating agent such as a lipid, liposome (including lecithin liposome or other liposome known in the art) as a DNA-liposome mixture (see, e.g., WO 9324640), calcium ion, viral protein, polyanion, polycation or nanoparticle or other known transfection facilitating agent. Transfection facilitating agents are polyanions, polycations, including poly-L-glutamate (LGS) or lipids. The concentration of transfection agent in the vaccine is less than 4mg/ml, less than 2mg/ml, less than 1mg/ml, less than 0.750mg/ml, less than 0.500mg/ml, less than 0.250mg/ml, less than 0.100mg/ml, less than 0.050mg/ml or less than 0.010mg/ml.
The composition may further comprise a sequence number U.S.021,579, which is fully incorporated by reference, as described in U.S. Pat. No. 4,1, 1994.
The composition may comprise DNA in an amount from about 1 nanogram to 100 milligrams; about 1 microgram to about 10 milligrams; or preferably from about 0.1 micrograms to about 10 milligrams; or more preferably from about 1 mg to about 2 mg. In some preferred embodiments, the compositions according to the invention comprise from about 5 nanograms to about 1000 micrograms of DNA. In some preferred embodiments, the composition may contain about 10 nanograms to about 800 micrograms of DNA. In some preferred embodiments, the composition may comprise about 0.1 to about 500 micrograms of DNA. In some preferred embodiments, the composition may comprise about 1 to about 350 micrograms of DNA. In some preferred embodiments, the composition may comprise about 25 to about 250 micrograms, from about 100 to about 200 micrograms, from about 1 nanogram to 100 milligrams; about 1 microgram to about 10 milligrams; about 0.1 micrograms to about 10 milligrams; about 1 mg to about 2 mg, about 5 nanograms to about 1000 micrograms, about 10 nanograms to about 800 micrograms, about 0.1 to about 500 micrograms, about 1 to about 350 micrograms, about 25 to about 250 micrograms, about 100 to about 200 micrograms of DNA.
The composition may be formulated according to the mode of administration to be used. The injectable pharmaceutical composition may be sterile, pyrogen-free and particle-free. Isotonic formulations or solutions may be used. Additives for isotonicity may include sodium chloride, dextrose, mannitol, sorbitol and lactose. The composition may include a vasoconstrictor. The isotonic solution may include phosphate buffered saline. The composition may further comprise stabilizers, including gelatin and albumin. The stabilizer may allow the formulation to be stable at room or ambient temperature for a long period of time, including LGS or polycations or polyanions.
6. Method for producing synthetic antibodies
The invention also relates to methods of producing synthetic antibodies. The method may comprise administering the composition to a subject in need thereof by using a delivery method described in more detail below. Thus, upon administration of the composition to a subject, a synthetic antibody is produced in or on the subject.
The method may further comprise introducing the composition into one or more cells, and thus, the synthetic antibody may be produced or produced in the one or more cells. The method may further comprise introducing the composition into one or more tissues, such as, but not limited to, skin and muscle, and thus, the synthetic antibody may be produced or produced in the one or more tissues.
7. Methods for identifying or screening antibodies
The invention further relates to methods of identifying or screening for such antibodies, which react with or bind to such antigens. Methods of identifying or screening antibodies can use antigens in methods known to those of skill in the art to identify or screen antibodies. Such methods may include, but are not limited to, selecting antibodies from a library (e.g., phage display) and immunizing an animal, followed by isolation and/or purification of the antibodies.
8. Methods of diagnosing diseases or conditions
The invention further relates to methods of diagnosing a subject as having a disease or disorder using an antibody, fragment thereof, or nucleic acid molecule encoding the same as described herein. In some embodiments, the invention features methods of identifying subjects at risk of transmitting SARS-CoV-2 infection or COVID-19, including those subjects who are asymptomatic or who only display a non-specific indicator of SARS-CoV-2 infection or COVID-19. In some embodiments, the invention may also be used to monitor subjects receiving treatment and therapy for SARS-CoV-2 infection or COVID-19, and to select or alter treatments and therapies effective in subjects having SARS-CoV-2 infection or COVID-19, wherein the selection and use of such treatments and therapies promote immunity to SARS-CoV-2, or prevent infection by SARS-CoV-2.
In one embodiment, the antibodies, fragments thereof, or nucleic acid molecules encoding them can be used in an immunoassay to diagnose a subject with active SARS-CoV-2 infection, COVID-19, or to be immune to SARS-CoV-2 infection, or to monitor a subject receiving treatment and therapy for SARS-CoV-2 infection or COVID-19. Non-limiting exemplary immunoassays include, for example, immunohistochemical assays, immunocytochemical assays, ELISA, capture ELISA, sandwich assays, enzyme immunoassays, radioimmunoassays, fluorescent immunoassays, and the like, all of which are known to those skilled in the art. See, e.g., harlow et al, 1988,Antibodies:A Laboratory Manual,Cold Spring Harbor,New York; harlow et al 1999,Using Antibodies:A Laboratory Manual,Cold Spring Harbor Laboratory Press,NY.
In some embodiments, the methods comprise obtaining a sample from a subject and contacting the sample with an antibody of the invention or a cell expressing an antibody of the invention, and detecting binding of the antibody to an antigen present in the sample.
In some embodiments, the sample may be provided from a subject receiving a therapeutic regimen or therapeutic intervention (e.g., drug therapy, vaccination, etc.) for SARS-CoV-2 infection or COVID-19. Samples may be obtained from the subject at various points in time before, during, or after treatment.
Thus, the SARS-CoV-2 antibodies or nucleic acid molecules encoding antibodies of the invention can be used to generate a risk profile or signature of a subject: (i) A subject who is expected to be immune to a SARS-CoV-2 infection or covd-19 and/or (ii) who is at risk of developing a SARS-CoV-2 infection or covd-19. The antibody profile of the subject can be compared to a predetermined or reference antibody profile to diagnose or identify a subject at risk of developing a SARS-CoV-2 infection or COVID-19, monitor the progression of the disease, and the rate of progression of the disease, and monitor the effectiveness of the SARS-CoV-2 infection or COVID-19 treatment. Data concerning antibodies of the invention may also be combined or correlated with other data or test results of SARS-CoV-2 infection or COVID-19, including but not limited to age, body weight, BMI, imaging data, medical history, smoking status, and any related family history.
The invention also provides methods of identifying agents that treat SARS-CoV-2 infection or COVID-19 that are suitable for or otherwise tailored to a particular subject. In this regard, a test sample of a subject exposed to a therapeutic agent, drug or other treatment regimen may be obtained and the level of one or more SARS-CoV-2 antibodies may be determined. The level of one or more SARS-CoV-2 antibodies can be compared to samples derived from the subject before and after treatment, or the level of one or more SARS-CoV-2 antibodies can be compared to samples derived from one or more subjects exhibiting an improvement in risk factors resulting from such treatment or exposure.
In one embodiment, the invention is a method of diagnosing SARS-CoV-2 infection or COVID-19. In one embodiment, the method comprises determining the immunity of SARS-CoV-2 to infection or reinfection. In some embodiments, these methods can utilize at least one biological sample (e.g., urine, saliva, blood, serum, plasma, amniotic fluid or tears) to detect one or more SARS-CoV-2 antibodies of the invention in the sample. Typically, the sample is a "clinical sample," which is a sample derived from a patient. In one embodiment, the biological sample is a blood sample.
In one embodiment, the method comprises detecting one or more SARS-CoV-2 antigens in at least one biological sample of the subject. In various embodiments, the level of one or more SARS-CoV-2 antigens of the invention in a biological sample from a subject is compared to a comparator. Non-limiting examples of comparators include, but are not limited to, negative controls, positive controls, expected normal background values for a subject, historical normal background values for a subject, expected normal background values for a population of which a subject is a member, or historical normal background values for a population of which a subject is a member.
9. Methods of delivering compositions
The invention also relates to a method of delivering the composition to a subject in need thereof. The delivery method may comprise administering the composition to the subject. Administration may include, but is not limited to, DNA injection with and without in vivo electroporation, liposome-mediated delivery, and nanoparticle-assisted delivery.
The mammal receiving delivery of the composition may be a human, primate, non-human primate, cow, sheep, goat, antelope, bison, buffalo, bison, bovine (bovids), deer, swan, elephant, llama, alpaca, mouse, rat, and chicken.
The composition may be administered by various routes including oral, parenteral, sublingual, transdermal, rectal, transmucosal, topical, via inhalation, via buccal administration, intrathoracic, intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular, intranasal, intrathecal and intra-articular or combinations thereof. For veterinary use, the composition may be administered as a suitably acceptable formulation in accordance with normal veterinary practice. The veterinarian can readily determine the most appropriate dosing regimen and route of administration for a particular animal. The composition may be administered by conventional syringes, needleless injection devices, "microprojectile bombardment vanishing guns" or other physical methods such as electroporation ("EP"), "hydrodynamic methods" or ultrasound.
Electroporation method
Administration of the composition via electroporation may be accomplished using electroporation devices that may be configured to deliver energy pulses to a desired tissue of the mammal effective to cause reversible pores to form in the cell membrane, and preferably the energy pulses are constant currents similar to a preset current input by a user. Electroporation devices may include an electroporation component and an electrode assembly or handle assembly. The electroporation component may include and incorporate one or more of the various elements of the electroporation device, including: a controller, a current waveform generator, an impedance tester, a waveform recorder, an input element, a status reporting element, a communication port, a memory component, a power source, and a power switch. Electroporation can be accomplished using in vivo electroporation devices, such as CELLECTRA EP system (Inovio Pharmaceuticals, plymouth Meeting, pa.) or Elgen electroporation apparatus (Inovio Pharmaceuticals, plymouth Meeting, pa.) to facilitate transfection of cells with plasmids.
The electroporation component may serve as one element of the electroporation device, and the other elements are separate elements (or components) in communication with the electroporation component. The electroporation component may act as more than one element of the electroporation device that may be in communication with other elements of the electroporation device separate from the electroporation component. The elements of the electroporation device that are present as part of one electromechanical or mechanical device may not be limited, as they may function as one device or as separate elements in communication with each other. The electroporation component may be capable of delivering pulses of energy that produce a constant current in the desired tissue, and include a feedback mechanism. The electrode assembly may include an electrode array having a plurality of electrodes arranged in a space, wherein the electrode assembly receives energy pulses from the electroporation component and delivers them to a desired tissue through the electrodes. At least one electrode of the plurality of electrodes is neutral during delivery of the pulse of energy and measures impedance in the desired tissue and transmits the impedance to the electroporation component. The feedback mechanism may receive the measured impedance and may adjust pulses of energy delivered by the electroporation component to maintain a constant current.
Multiple electrodes may deliver energy pulses in a dispersed pattern. The plurality of electrodes may deliver the pulses of energy in a decentralized mode under a programmed sequence through control of the electrodes, and the programmed sequence is input by a user to the electroporation component. The programming sequence may include a plurality of pulses delivered sequentially, wherein each pulse of the plurality of pulses is delivered by at least two active electrodes having a neutral electrode that measures impedance, and wherein a subsequent pulse of the plurality of pulses is delivered by a different active electrode of the at least two active electrodes having a neutral electrode that measures impedance.
The feedback mechanism may be implemented by hardware or software. The feedback mechanism may be performed by an analog closed loop circuit. Feedback occurs every 50, 20, 10, or 1 μs, but is preferably real-time feedback or instantaneous (i.e., substantially instantaneous as determined by available techniques for determining response time). The neutral electrode may measure an impedance in the desired tissue and transmit the impedance to the feedback mechanism, and the feedback mechanism is responsive to the impedance and adjusts the energy pulse to maintain the constant current at a value similar to the preset current. The feedback mechanism may continuously and instantaneously maintain a constant current during the delivery of the energy pulse.
Examples of electroporation devices and methods of electroporation that may facilitate delivery of the compositions of the present invention include those described in U.S. patent No. 7,245,963 to draghia-Akii et al, U.S. patent publication 2005/0052630 to smith et al, the contents of which are incorporated herein by reference in their entirety. Other electroporation devices and methods of electroporation that may be used to facilitate delivery of the compositions include those provided in the co-pending and commonly owned united states. U.S. provisional application sequence No.60/852,149 filed on 10 month 17 of 2007 and No.60/978,982 filed on 10 month 10 of 2007, all of which are incorporated herein in their entirety, are claimed under 35USC 119 (e) for U.S. provisional application sequence No. 11/874072 filed on 10 month 17 of 2007.
U.S. Pat. No. 7,245,963 to Draghia-Akii et al describes a modular electrode system and its use for facilitating the introduction of biomolecules into cells of selected tissues in the body or plants. The modular electrode system may comprise a plurality of needle electrodes; a hypodermic needle; an electrical connector providing a conductive link from the programmable constant current pulse controller to the plurality of needle electrodes; and a power supply. An operator can grasp a plurality of needle electrodes mounted on a support structure and insert them firmly into selected tissue in the body or plant. The biomolecules are then delivered into the selected tissue via a hypodermic needle. The programmable constant current pulse controller is activated and constant current electrical pulses are applied to the plurality of needle electrodes. The applied constant current electrical pulse assists in introducing biomolecules into cells between the plurality of electrodes. U.S. Pat. No. 7,245,963 is incorporated herein by reference in its entirety.
U.S. patent publication 2005/0052630 to Smith et al describes an electroporation device that can be used to effectively promote the introduction of biomolecules into cells of selected tissues in the body or plant. Electroporation devices include motorized devices ("EKD devices") whose operation is specified by software or firmware. The EKD device generates a series of programmable constant current pulse patterns between electrodes in the array based on user control and input of pulse parameters and allows for storage and retrieval of current waveform data. The electroporation device further comprises a replaceable electrode disk having an array of needle electrodes, a central injection channel for the injection needle, and a removable guide disk. U.S. patent publication 2005/0052630 is incorporated herein by reference in its entirety.
The electrode arrays and methods described in U.S. Pat. No. 7,245,963 and U.S. patent publication 2005/0052630 may be suitable for deep penetration into not only tissues (e.g., muscle) but also other tissues or organs. Due to the configuration of the electrode array, the injection needle (delivering the selected biomolecules) is also fully inserted into the target organ and injection is given perpendicular to the target problem in the area predefined by the electrodes. The electrodes are described in U.S. patent No. 7,245,963 and U.S. patent publication 2005/005263, preferably 20mm long and 21 gauge.
In addition, it is contemplated that in some embodiments incorporating electroporation devices and uses thereof, electroporation devices exist, such devices being those described in the following patents: U.S. patent 5,273,525 issued on 12/28/1993, U.S. patent 6,110,161 issued on 8/29/2000, U.S. patent 6,261,281 issued on 17/2001, and U.S. patent 6,939,862 issued on 6,958,060/25/2005 and 6/9/2005. Further, patents covering the subject matter provided in U.S. patent 6,697,669 issued 24-2-2004 (directed to the delivery of DNA using any of a variety of devices) and U.S. patent 7,328,064 issued 5-2-2008 (directed to a method of injecting DNA) are contemplated herein. The entire contents of the above-mentioned patents are incorporated by reference.
10. Therapeutic method
Also provided herein are methods of treating, protecting against, and/or preventing a disease by producing a synthetic antibody in a subject in need thereof. The method may comprise administering the composition to the subject. The compositions may be administered to a subject using the delivery methods described above.
In certain embodiments, the invention provides methods of treating SARS-CoV-2 viral infection and/or preventing SARS-CoV-2 viral infection. In one embodiment, the method treats, protects and/or prevents a disease or disorder associated with SARS-CoV-2 virus infection. In one embodiment, the method treats, prevents, and/or prevents covd-19.
In one embodiment, the subject has or is at risk of a SARS-CoV-2 viral infection.
When a synthetic antibody is produced in a subject, the synthetic antibody may bind to or react with an antigen. Such binding may neutralize an antigen, block recognition of the antigen by another molecule (e.g., a protein or nucleic acid), and elicit or induce an immune response to the antigen, thereby treating, protecting against, and/or preventing a disease associated with the antigen in a subject.
The synthetic antibodies can treat, prevent, and/or protect against disease in a subject to whom the composition is administered. The diseases in the subject to whom the composition is administered may be treated, prevented and/or protected by synthetic antibodies that bind to the antigen. The synthetic antibodies can promote survival of the disease in a subject administered the composition. In one embodiment, the synthetic antibody can provide an increase in survival of the disease in the subject that exceeds the expected survival of a subject with the disease who is not administered the synthetic antibody. In various embodiments, the synthetic antibody can provide an increase in survival of the disease in a subject administered the composition of at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to the expected survival in the absence of the composition. In one embodiment, the synthetic antibody may provide increased protection in a subject against a disease over the expected protection of a subject not administered the synthetic antibody. In various embodiments, the synthetic antibody can protect against disease in at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the subjects beyond the intended protection in the absence of the composition.
The dosage of the composition may be between 1 μg and 10mg active ingredient/kg body weight/time, and may be 20 μg to 10mg ingredient/kg body weight/time. The composition may be administered every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days. The number of doses of the composition for effective treatment may be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
In one embodiment, immunotherapy with the anti-SARS-CoV-2 dMAB of the invention will have a direct therapeutic effect. In one embodiment, immunotherapy using the anti-SARS-CoV-2 dMAB of the invention can be used as an "adjuvant" treatment of immunity to reduce viral protein load in order to provide host immunity and optimize the effect of antiviral drugs.
Combinations of SARS-CoV-2 antigens
In one embodiment, the invention relates to the administration of a SARS-CoV-2 antibody or a nucleic acid encoding a SARS-CoV-2 antibody in combination with a nucleic acid molecule encoding a SARS-CoV-2 antigen. Thus, in some embodiments, the invention relates to immunogenic compositions, e.g., vaccines, comprising a SARS-CoV-2 antibody or nucleic acid encoding a SARS-CoV-2 antibody and a SARS-CoV-2 antigen, fragment, variant thereof. The vaccine can be used to protect against any number of SARS-CoV-2 strains, thereby treating, preventing and/or protecting against SARS-CoV-2 infection or related pathologies, including COVID-19. The vaccine can significantly induce an immune response in a subject administered the vaccine, thereby protecting against and treating SARS-CoV-2 infection or related conditions, including COVID-19.
The vaccine may be a DNA vaccine, a peptide vaccine or a combination of DNA and peptide vaccines. The DNA vaccine may comprise a nucleic acid sequence encoding SARS-CoV-2 antigen. The nucleic acid sequence may be DNA, RNA, cDNA, a variant thereof, a fragment thereof, or a combination thereof. The nucleic acid sequence may also include other sequences encoding linkers, leader sequences, or tag sequences that are linked to the SARS-CoV-2 antigen by peptide bonds. The peptide vaccine may comprise a SARS-CoV-2 antigen peptide, a SARS-CoV-2 antigen protein, a variant thereof, a fragment thereof, or a combination thereof. The DNA and peptide combination vaccine may comprise the above-described nucleic acid sequences encoding SARS-CoV-2 antigen and SARS-CoV-2 antigen peptide or protein, wherein the SARS-CoV-2 antigen peptide or protein and the encoded SARS-CoV-2 antigen have the same amino acid sequence.
In some embodiments, the vaccine can induce a humoral immune response in a subject administered the vaccine. The induced humoral immune response may be specific for the SARS-CoV-2 antigen. The induced humoral immune response may react with SARS-CoV-2 antigen. The humoral immune response may be induced from about 1.5 fold to about 16 fold, from about 2 fold to about 12 fold, or from about 3 fold to about 10 fold in the subject to whom the vaccine is administered. The humoral immune response may induce at least about 1.5 fold, at least about 2.0 fold, at least about 2.5 fold, at least about 3.0 fold, at least about 3.5 fold, at least about 4.0 fold, at least about 4.5 fold, at least about 5.0 fold, at least about 5.5 fold, at least about 6.0 fold, at least about 6.5 fold, at least about 7.0 fold, at least about 7.5 fold, at least about 8.0 fold, at least about 8.5 fold, at least about 9.0 fold, at least about 9.5 fold, at least about 10.0 fold, at least about 10.5 fold, at least about 11.0 fold, at least about 11.5 fold, at least about 12.0 fold, at least about 12.5 fold, at least about 13.0 fold, at least about 13.5 fold, at least about 14.0 fold, at least about 14.5 fold, at least about 15.0 fold, at least about 15.5 fold, or at least about 16.0 fold in the subject to which the vaccine is administered.
The humoral immune response induced by the vaccine may include increased levels of neutralizing antibodies associated with the subject administered the vaccine as compared to the subject not administered the vaccine. The neutralizing antibody may be specific for SARS-CoV-2 antigen. The neutralizing antibody can react with SARS-CoV-2 antigen. Neutralizing antibodies can provide protection against and/or treat a covd-19 infection and its related conditions in a subject administered a vaccine.
The vaccine-induced humoral immune response may include an increase in the level of IgG antibodies associated with a subject administered the vaccine as compared to a subject not administered the vaccine. These IgG antibodies may be specific for SARS-CoV-2 antigen. These IgG antibodies can react with SARS-CoV-2 antigen. Preferably, the humoral response is cross-reactive with two or more strains of covd-19. The level of IgG antibodies associated with a subject administered the vaccine may be increased by about 1.5-fold to about 16-fold, about 2-fold to about 12-fold, or about 3-fold to about 10-fold as compared to a subject not administered the vaccine. The level of IgG antibodies associated with a subject administered a vaccine can be increased by at least about 1.5 fold, at least about 2.0 fold, at least about 2.5 fold, at least about 3.0 fold, at least about 3.5 fold, at least about 4.0 fold, at least about 4.5 fold, at least about 5.0 fold, at least about 5.5 fold, at least about 6.0 fold, at least about 6.5 fold, at least about 7.0 fold, at least about 7.5 fold, at least about 8.0 fold, at least about 8.5 fold, at least about 9.0 fold, at least about 9.5 fold, at least about 10.0 fold, at least about 10.5 fold, at least about 11.0 fold, at least about 11.5 fold, at least about 12.0 fold, at least about 12.5 fold, at least about 13.0 fold, at least about 13.5 fold, at least about 14.0 fold, at least about 14.5 fold, at least about 15.0 fold, as compared to the subject not administered the vaccine.
The vaccine can induce a cellular immune response in a subject administered the vaccine. The induced cellular immune response may be specific for SARS-CoV-2 antigen. The induced cellular immune response can react with SARS-CoV-2 antigen. Preferably, the cell response is cross-reactive to two or more strains of covd-19. The induced cellular immune response may include eliciting CD8 + T cell response. Initiated CD8 + The T cell response can react with SARS-CoV-2 antigen. Outgoing CD8 + The T cell response may be multifunctional. The induced cellular immune response may include eliciting CD8 + T cell response, wherein CD8 + T cells produce interferon-gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), interleukin-2 (IL-2), or a combination of IFN-gamma and TNF-alpha.
The induced cellular immune response may include increased CD8 associated with the subject administered the vaccine as compared to the subject not administered the vaccine + T cell response. CD8 associated with the subject administered the vaccine compared to the subject not administered the vaccine + T cell responses may be increased by about 2-fold to about 30-fold, about 3-fold to about 25-fold, or about 4-fold to about 20-fold. CD8 associated with subjects administered the vaccine + The T cell response may be increased by at least about 1.5 fold, at least about At least about 2.0 fold, at least about 3.0 fold, at least about 4.0 fold, at least about 5.0 fold, at least about 6.0 fold, at least about 6.5 fold, at least about 7.0 fold, at least about 7.5 fold, at least about 8.0 fold, at least about 8.5 fold, at least about 9.0 fold, at least about 9.5 fold, at least about 10.0 fold, at least about 10.5 fold, at least about 11.0 fold, at least about 11.5 fold, at least about 12.0 fold, at least about 12.5 fold, at least about 13.0 fold, at least about 13.5 fold, at least about 14.0 fold, at least about 14.5 fold, at least about 15.0 fold, at least about 16.0 fold, at least about 17.0 fold, at least about 18.0 fold, at least about 19.0 fold, at least about 20.0 fold, at least about 21.0 fold, at least about 22.0 fold, at least about 23.0 fold, at least about 24.0 fold, at least about 25.0 fold, at least about 26.0 fold, at least about 27.0 fold, at least about 29.0 fold, at least about 0.0 fold, or at least about 30.0 fold as compared to the vaccine administered to the subject.
The induced cellular immune response may include IFN-gamma producing CD3 + CD8 + Increased frequency of T cells. CD3 associated with the subject administered the vaccine compared to the subject not administered the vaccine + CD8 + IFN-γ + The frequency of T cells may be increased by at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, or 20-fold.
The induced cellular immune response may include the production of CD3 of TNF-alpha + CD8 + Increased frequency of T cells. CD3 associated with the subject administered the vaccine compared to the subject not administered the vaccine + CD8 + TNF-α + The frequency of T cells may be increased by at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, or 14-fold.
The induced cellular immune response may include production of IL-2 CD3 + CD8 + Increased frequency of T cells. CD3 associated with the subject administered the vaccine compared to the subject not administered the vaccine + CD8 + IL-2 + The frequency of T cells may be increased by at least about 0.5-fold, 1.0-fold, 1.5-fold, 2.0-fold, 2.5-fold, 3.0-fold, 3.5-fold, 4.0-fold, 4.5-fold, or 5.0-fold.
Induced byThe cellular immune response may include production of CD3 for both IFN-gamma and TNF-alpha + CD8 + Increased frequency of T cells. CD3 associated with the subject administered the vaccine compared to the subject not administered the vaccine + CD8 + IFN-γ + TNF-α + The frequency of T cells may be increased by at least about 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, 100-fold, 110-fold, 120-fold, 130-fold, 140-fold, 150-fold, 160-fold, 170-fold, or 180-fold.
The cellular immune response induced by the vaccine may include eliciting CD4 + T cell response. Initiated CD4 + The T cell response can react with SARS-CoV-2 antigen. Initiated CD4 + The T cell response may be multifunctional. The induced cellular immune response may include eliciting CD4 + T cell response, wherein CD4 + T cells produce IFN-gamma, TNF-alpha, IL-2 or a combination of IFN-gamma and TNF-alpha.
The induced cellular immune response may include IFN-gamma producing CD3 + CD4 + Increased frequency of T cells. CD3 associated with the subject administered the vaccine compared to the subject not administered the vaccine + CD4 + IFN-γ + The frequency of T cells may be increased by at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, or 20-fold.
The induced cellular immune response may include the production of CD3 of TNF-alpha + CD4 + Increased frequency of T cells. CD3 associated with the subject administered the vaccine compared to the subject not administered the vaccine + CD4 + TNF-α + The frequency of T cells may be increased by at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, or 22-fold.
The induced cellular immune response may include production of IL-2 CD3 + CD4 + Increased frequency of T cells. CD3 associated with subjects administered the vaccine + CD4 + IL-2 + The frequency of T cells can be increased by at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, 22-fold, 23-fold, 24-fold, 25-fold, 26-fold, 27-fold, 28-fold, 29-fold, 30-fold, 31-fold, 32-fold, 33-fold, 34-fold, 35-fold, 36-fold, 37-fold, 38-fold, 39-fold, 40-fold, 45-fold, 50-fold, 55-fold, or 60-fold as compared to a subject not administered the vaccine.
The induced cellular immune response may include production of IFN-gamma and TNF-alpha CD3 + CD4 + Increased frequency of T cells. CD3 associated with subjects administered the vaccine + CD4 + IFN-γ + TNF-α + The frequency of (c) may be increased by at least about 2-fold, 2.5-fold, 3.0-fold, 3.5-fold, 4.0-fold, 4.5-fold, 5.0-fold, 5.5-fold, 6.0-fold, 6.5-fold, 7.0-fold, 7.5-fold, 8.0-fold, 8.5-fold, 9.0-fold, 9.5-fold, 10.0-fold, 10.5-fold, 11.0-fold, 11.5-fold, 12.0-fold, 12.5-fold, 13.0-fold, 13.5-fold, 14.0-fold, 14.5-fold, 15.0-fold, 15.5-fold, 16.0-fold, 16.5-fold, 17.0-fold, 17.5-fold, 18.0-fold, 18.5-fold, 19.0-fold, 19.5-fold, 20.0-fold, 21-fold, 22-fold, 23-fold, 24-fold, 25-fold, 26-fold, 27-fold, 28-fold, 29-fold, 30-fold, 31-fold as compared to the subject to which the vaccine is not administered.
The vaccine of the present invention may have the characteristics required for an effective vaccine, such as being safe, so that the vaccine itself does not cause disease or death; protective against diseases caused by exposure to living pathogens such as viruses or bacteria; an invention of inducing neutralizing antibodies to prevent cells; inducing protective T cells against intracellular pathogens; and provides ease of administration, few side effects, biostability, and low cost/dosage.
The vaccine may further induce an immune response when administered to different tissues such as muscle or skin. The vaccine may further induce an immune response when administered by electroporation or injection or subcutaneously or intramuscularly.
SARS-CoV-2 antigen
As described above, the vaccine comprises the SARS-CoV-2 antigen, a fragment thereof, a variant thereof, a nucleic acid molecule encoding the same, or a combination thereof. Coronaviruses, including SARS-CoV-2, are enveloped and have a type 1 membrane glycoprotein called spike (S) protein, which forms protruding spikes on the surface of the coronavirus. The spike protein promotes binding of the coronavirus to proteins located on the cell surface (e.g., metalloprotease aminopeptidase N) and mediates cell-virus membrane fusion. Specifically, the spike protein comprises an S1 subunit that promotes binding of the coronavirus to a cell surface protein. Thus, the S1 subunit of the spike protein controls which cells are infected by the coronavirus. The spike protein also contains an S2 subunit, which is a transmembrane subunit that facilitates fusion of the virus and cell membrane. Thus, the SARS-CoV-2 antigen can comprise a SARS-CoV-2 spike protein, the S1 subunit of a SARS-CoV-2 spike protein, or the S2 subunit of a SARS-CoV-2 spike protein.
Upon fusion of the cell surface proteins and membranes, coronaviruses enter the cells and their single stranded RNA genome is released into the cytoplasm of the infected cells. The single stranded RNA genome is a positive strand and can therefore be translated into RNA polymerase that produces additional viral RNAs that are negative strands. Thus, the SARS-CoV-2 antigen can also be a SARS-CoV-2RNA polymerase.
The viral negative RNA strand is transcribed into a smaller subgenomic positive RNA strand, which is used to translate other viral proteins, such as nucleocapsid (N), envelope (E) and matrix (M) proteins. Thus, SARS-CoV-2 antigen can comprise a SARS-CoV-2 nucleocapsid protein, a SARS-CoV-2 envelope protein or a SARS-CoV-2 matrix protein.
Viral negative RNA strands can also be used to replicate viral genomes bound by nucleocapsid proteins. The matrix proteins are integrated into the endoplasmic reticulum of the infected cells along with the spike proteins. The nucleocapsid protein bound to the viral genome, together with the membrane-embedded matrix and spike protein, bud into the lumen of the endoplasmic reticulum, thereby encapsulating the viral genome in the membrane. Viral progeny are then transported to the cell membrane of the infected cell by golgi vesicles and released into the extracellular space by endocytosis.
In some embodiments, the SARS-CoV-2 antigen can be a SARS-CoV-2 spike protein, a SARS-CoV-2RNA polymerase, a SARS-CoV-2 nucleocapsid protein, a SARS-CoV-2 envelope protein, a SARS-CoV-2 matrix protein, a fragment thereof, a variant thereof, or a combination thereof. The SARS-CoV-2 antigen can be a consensus antigen derived from two or more SARS-CoV-2 spike antigens, two or more SARS-CoV-2RNA polymerase, two or more SARS-CoV-2 nucleocapsid proteins, two or more envelope proteins, two or more matrix proteins, or a combination thereof. SARS-CoV-2 consensus antigen can be modified to improve expression. Modifications may include codon optimization, RNA optimization, addition of kozak sequences to increase translation initiation and/or addition of immunoglobulin leader sequences to increase the immunogenicity of the SARS-CoV-2 antigen. In some embodiments, the SARS-CoV-2 antigen comprises an IgE leader sequence that can be the amino acid sequence depicted by SEQ ID NO. 141.
SARS-CoV-2 spike antigen
The SARS-CoV-2 antigen can be a SARS-CoV-2 spike antigen, a fragment thereof, a variant thereof, or a combination thereof. SARS-CoV-2 spike antigen is capable of eliciting an immune response in a mammal against one or more strains of SARS-CoV-2. SARS-CoV-2 spike antigen can comprise epitopes that make it particularly effective as an immunogen against which an immune response is induced against SARS-CoV-2.
The SARS-CoV-2 spike antigen can be a consensus sequence derived from two or more strains of SARS-CoV-2. The SARS-CoV-2 spike antigen can comprise consensus sequences and/or modifications for improved expression. Modifications may include codon optimization, RNA optimization, addition of kozak sequences to increase translation initiation and/or addition of immunoglobulin leader sequences to increase the immunogenicity of SARS-CoV-2 spike antigen. SARS-CoV-2 spike antigen can include a signal peptide, such as an immunoglobulin signal peptide, such as, but not limited to, an immunoglobulin E (IgE) or an immunoglobulin (IgG) signal peptide. In some embodiments, the SARS-CoV-2 spike antigen can comprise a Hemagglutinin (HA) tag. The SARS-CoV-2 spike antigen can be designed to elicit a stronger and wider cellular and/or humoral immune response than the corresponding codon-optimized spike antigen.
The SARS-CoV-2 consensus spike antigen can be the amino acid sequence SEQ ID NO. 134. In some embodiments, the SARS-CoV-2 consensus spike antigen can be an amino acid sequence that has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over the full length of the amino acid sequence set forth in SEQ ID NO. 134.
The SARS-CoV-2 consensus spike antigen can be the nucleic acid sequence SEQ ID NO. 133 encoding SEQ ID NO. 134. In some embodiments, the SARS-CoV-2 consensus spike antigen can be a nucleic acid sequence that has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over the full length of the nucleic acid sequence set forth in SEQ ID NO. 133. In other embodiments, the SARS-CoV-2 consensus spike antigen can be a nucleic acid sequence encoding an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the full length of the amino acid sequence set forth in SEQ ID NO. 134.
SARS-CoV-2 consensus spike antigen can be operably linked to IgE leader. The SARS-CoV-2 consensus spike antigen operatively linked to the IgE leader can be the amino acid sequence SEQ ID NO. 136. In some embodiments, the SARS-CoV-2 consensus spike antigen can be an amino acid sequence that has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over the full length of the amino acid sequence set forth in SEQ ID NO. 136.
The SARS-CoV-2 consensus spike antigen can be the nucleic acid sequence SEQ ID NO:135 encoding SEQ ID NO: 136. In some embodiments, the SARS-CoV-2 consensus spike antigen can be a nucleic acid sequence that has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over the full length of the nucleic acid sequence set forth in SEQ ID NO. 135. In other embodiments, the SARS-CoV-2 consensus spike antigen can be a nucleic acid sequence encoding an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the full length of the amino acid sequence depicted in SEQ ID NO. 136.
Immunogenic fragments of SEQ ID NO. 134 or SEQ ID NO. 136 may be provided. The immunogenic fragment may comprise at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of SEQ ID NO. 134 or SEQ ID NO. 136. In some embodiments, the immunogenic fragment comprises a leader sequence, e.g., an immunoglobulin leader sequence, e.g., an IgE leader sequence. In some embodiments, the immunogenic fragment is free of leader sequences.
An immunogenic fragment of a protein having an amino acid sequence homologous to the immunogenic fragment of SEQ ID NO. 134 or SEQ ID NO. 136 may be provided. Such immunogenic fragments may comprise at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% protein homologous to SEQ ID No. 134 or SEQ ID No. 13695%. Some embodiments relate to immunogenic fragments having 96% homology to immunogenic fragments of the protein sequences herein. Some embodiments relate to immunogenic fragments having 97% homology to immunogenic fragments of the protein sequences herein. Some embodiments relate to immunogenic fragments having 98% homology to immunogenic fragments of the protein sequences herein. Some embodiments relate to immunogenic fragments having 99% homology to immunogenic fragments of the protein sequences herein. In some embodiments, the immunogenic fragment comprises a leader sequence, e.g., an immunoglobulin leader sequence, e.g., an IgE leader sequence. In some embodiments, the immunogenic fragment is free of leader sequences.
Some embodiments relate to immunogenic fragments of SEQ ID NO. 133 or SEQ ID NO. 135. The immunogenic fragment may be at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of SEQ ID NO. 133 or SEQ ID NO. 135. The immunogenic fragment may be at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homologous to the fragment of SEQ ID NO:133 or SEQ ID NO: 135. In some embodiments, the immunogenic fragment comprises a sequence encoding a leader sequence, e.g., an immunoglobulin leader sequence, e.g., an IgE leader sequence. In some embodiments, the fragment does not contain a coding sequence that encodes a leader sequence.
Abnormal SARS-CoV-2 spike antigen
The SARS-CoV-2 antigen can be an aberrant SARS-CoV-2 spike antigen, a fragment thereof, a variant thereof, or a combination thereof. The aberrant SARS-CoV-2 spike antigen is capable of eliciting an immune response in a mammal against one or more strains of SARS-CoV-2. The aberrant SARS-CoV-2 spike antigen can comprise epitopes that render it particularly effective as an immunogen against which an immune response is induced against SARS-CoV-2.
The aberrant SARS-CoV-2 spike antigen can be a consensus sequence derived from two or more strains of SARS-CoV-2. The aberrant SARS-CoV-2 spike antigen can comprise a consensus sequence and/or be modified to improve expression. Modifications may include codon optimization, RNA optimization, addition of kozak sequences to increase translation initiation and/or addition of immunoglobulin leader sequences to increase the immunogenicity of aberrant SARS-CoV-2 spike antigens. The aberrant SARS-CoV-2 spike antigen can include a signal peptide, such as an immunoglobulin signal peptide, such as, but not limited to, an immunoglobulin E (IgE) or an immunoglobulin (IgG) signal peptide. The aberrant SARS-CoV-2 spike antigen can be designed to elicit a stronger and broader cellular and/or humoral immune response than the corresponding spike antigen.
The abnormal SARS-CoV-2 spike antigen can be the amino acid sequence SEQ ID NO. 138. In some embodiments, the aberrant SARS-CoV-2 spike antigen can be an amino acid sequence that has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over the full length of the amino acid sequence set forth in SEQ ID NO. 138.
The aberrant SARS-CoV-2 spike antigen can be the nucleic acid sequence SEQ ID NO:137 encoding SEQ ID NO:138. In some embodiments, the aberrant SARS-CoV-2 spike antigen can be a nucleic acid sequence that has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over the full length of the nucleic acid sequence set forth in SEQ ID NO. 137. In other embodiments, the aberrant SARS-CoV-2 spike antigen can be a nucleic acid sequence encoding an amino acid sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the full length of the amino acid sequence set forth in SEQ ID NO. 138.
The aberrant SARS-CoV-2 spike antigen can be operably linked to the IgE leader. The aberrant SARS-CoV-2 spike antigen operatively linked to the IgE leader may be the amino acid sequence SEQ ID NO. 140. In some embodiments, the aberrant SARS-CoV-2 spike antigen can be an amino acid sequence that has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over the full length of the amino acid sequence set forth in SEQ ID NO. 140.
The aberrant SARS-CoV-2 spike antigen can be the nucleic acid sequence SEQ ID NO. 139 encoding SEQ ID NO. 140. In some embodiments, the aberrant SARS-CoV-2 spike antigen can be a nucleic acid sequence that has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over the full length of the nucleic acid sequence set forth in SEQ ID NO 139. In other embodiments, the aberrant SARS-CoV-2 spike antigen can be a nucleic acid sequence encoding an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the full length of the amino acid sequence set forth in SEQ ID NO. 140.
Immunogenic fragments of SEQ ID NO. 138 or SEQ ID NO. 140 may be provided. The immunogenic fragment may comprise at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of SEQ ID NO. 138 or SEQ ID NO. 140. In some embodiments, the immunogenic fragment comprises a leader sequence, e.g., an immunoglobulin leader sequence, e.g., an IgE leader sequence. In some embodiments, the immunogenic fragment is free of leader sequences.
An immunogenic fragment of a protein having an amino acid sequence homologous to the immunogenic fragment of SEQ ID NO. 138 or SEQ ID NO. 140 may be provided. Such immunogenic fragments may comprise at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% protein that is 95% homologous to SEQ ID NO 138 or SEQ ID NO 140. Some embodiments relate to immunogenic fragments having 96% homology to immunogenic fragments of the protein sequences herein. Some embodiments relate to immunogenic fragments having 97% homology to immunogenic fragments of the protein sequences herein. Some embodiments relate to immunogenic fragments having 98% homology to immunogenic fragments of the protein sequences herein. Some embodiments relate to immunogenic fragments having 99% homology to immunogenic fragments of the protein sequences herein. In some embodiments, the immunogenic fragment comprises a leader sequence, e.g., an immunoglobulin leader sequence, e.g., an IgE leader sequence. In some embodiments, the immunogenic fragment is free of leader sequences.
Some embodiments relate to immunogenic fragments of SEQ ID NO. 137 or SEQ ID NO. 139. The immunogenic fragment may be at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of SEQ ID NO 137 or SEQ ID NO 139. The immunogenic fragment may be at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homologous to the fragment of SEQ ID NO:137 or SEQ ID NO: 139. In some embodiments, the immunogenic fragment comprises a sequence encoding a leader sequence, e.g., an immunoglobulin leader sequence, e.g., an IgE leader sequence. In some embodiments, the fragment does not contain a coding sequence that encodes a leader sequence.
12. Use in combination
The invention also provides a method of treating, protecting against, and/or preventing a disease in a subject in need thereof by administering a combination of a synthetic antibody and a therapeutic agent. In one embodiment, the therapeutic agent is an antiviral agent. In one embodiment, the therapeutic agent is an antibiotic agent. In one embodiment, the therapeutic agent is a SARS-CoV-2 vaccine. In one embodiment, the therapeutic agent is a small molecule drug or biological agent.
The synthetic antibody and therapeutic agent may be administered using any suitable method such that the combination of the synthetic antibody and therapeutic agent is present in the subject. In one embodiment, the method may comprise administering a first composition comprising a synthetic antibody of the invention by any of the methods detailed above, and administering a second composition comprising a therapeutic agent less than 1 day, less than 2 days, less than 3 days, less than 4 days, less than 5 days, less than 6 days, less than 7 days, less than 8 days, less than 9 days, or less than 10 days after administration of the synthetic antibody. In one embodiment, the method may comprise administering a first composition comprising a synthetic antibody of the invention by any of the methods detailed above and administering a second composition comprising a therapeutic agent more than 1 day, more than 2 days, more than 3 days, more than 4 days, more than 5 days, more than 6 days, more than 7 days, more than 8 days, more than 9 days, or more than 10 days after administration of the synthetic antibody. In one embodiment, the method may comprise administering a first composition comprising a therapeutic agent and administering a second composition comprising a synthetic antibody of the invention less than 1 day, less than 2 days, less than 3 days, less than 4 days, less than 5 days, less than 6 days, less than 7 days, less than 8 days, less than 9 days, or less than 10 days after administration of the therapeutic agent. In one embodiment, the method may comprise administering a first composition comprising a therapeutic agent and administering a second composition comprising a synthetic antibody of the invention more than 1 day, more than 2 days, more than 3 days, more than 4 days, more than 5 days, more than 6 days, more than 7 days, more than 8 days, more than 9 days, or more than 10 days after administration of the therapeutic agent. In one embodiment, the method may comprise simultaneously administering a first composition comprising a synthetic antibody of the invention and a second composition comprising a therapeutic agent by any of the methods described in detail above. In one embodiment, the method may comprise simultaneously administering a first composition comprising a synthetic antibody of the invention and a second composition comprising a therapeutic agent by any of the methods described in detail above. In one embodiment, the method may comprise administering a single composition comprising a synthetic antibody of the invention and a therapeutic agent.
Non-limiting examples of antibiotics that may be used in combination with the synthetic antibodies of the invention include aminoglycosides (e.g., gentamicin, amikacin, tobramycin), quinolones (e.g., ciprofloxacin, levofloxacin), cephalosporins (e.g., ceftazidime, cefepime, cefoperazone, cefpirome), anti-pseudomycin classes: carbenicillins (e.g., carbenicillin and ticarcillin) and urea carbapenems (e.g., meloxicam, azlocillin and piperacillin), carbapenems (e.g., meropenem, imipenem, doripenem), polymyxins (e.g., polymyxin B and colistin), and monocyclic lactams (e.g., aztreonam).
13. In vitro and ex vivo production of synthetic antibodies
In one embodiment, the synthetic antibody is produced in vitro or ex vivo. For example, in one embodiment, nucleic acids encoding synthetic antibodies may be introduced and expressed in vitro or in ex vivo cells. Methods for introducing and expressing genes into cells are known in the art. In the context of expression vectors, the vectors may be readily introduced into host cells (e.g., mammalian, bacterial, yeast, or insect cells) by any method in the art. For example, the expression vector may be transferred into the host cell by physical, chemical or biological means.
Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well known in the art. See, e.g., sambrook et al (2012,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory,New York). A preferred method of introducing the polynucleotide into a host cell is calcium phosphate transfection.
Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian (e.g., human) cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, adeno-associated viruses, and the like. See, for example, U.S. patent nos. 5,350,674 and 5,585,362.
Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. Exemplary colloidal systems for use as delivery vehicles in vitro and in vivo are liposomes (e.g., artificial membrane vesicles).
In the case of non-viral delivery systems, an exemplary delivery vehicle is a liposome. Lipid formulations are contemplated for use in introducing nucleic acids into host cells (in vitro, ex vivo, or in vivo). In another aspect, the nucleic acid may be conjugated to a lipid. The nucleic acid associated with the lipid may be encapsulated in the aqueous interior of the liposome, dispersed within the lipid bilayer of the liposome, attached to the liposome via a linking molecule associated with both the liposome and the oligonucleotide, entrapped in the liposome, complexed with the liposome, dispersed in a solution containing the lipid, mixed with the lipid, combined with the lipid, contained in the lipid as a suspension, comprising or complexed with micelles, or otherwise associated with the lipid. The composition associated with the lipid, lipid/DNA or lipid/expression vector is not limited to any particular structure in solution. For example, they may exist as micelles in a bilayer structure, or have a "collapsed" structure. They may also simply be dispersed in solution, possibly forming aggregates of non-uniform size or shape. Lipids are fatty substances that may be naturally occurring or synthetic lipids. For example, lipids include the class of fat droplets naturally occurring in the cytoplasm as well as compounds containing long chain aliphatic hydrocarbons and their derivatives (e.g., fatty acids, alcohols, amines, amino alcohols, and aldehydes).
The invention has a number of aspects, illustrated by the following non-limiting examples.
14. Examples
The invention is further illustrated in the following examples. It should be understood that these embodiments, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Accordingly, various modifications of the present invention, in addition to those shown and described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.
EXAMPLE 1 in vivo production of monoclonal antibodies against SARS-CoV-2/COVID-19 Using synthetic DNA
CR3022 is a cross-reactive monoclonal Ab (mAb) that binds SARS-CoV-2. CR3022 binds to an epitope on the SARS-CoV spike (S) protein within the Receptor Binding Domain (RBD).
Optimized DNA-encoding monoclonal antibodies (dmabs) against SARS-CoV-2 virus have been developed for prophylactic, therapeutic and/or diagnostic use, including treatment or prevention of SARS-CoV-2 mediated diseases (e.g., covd-19). Novel engineering methods were used to develop dmabs for in vivo delivery of anti-SARS-CoV-2/covd-19 mabs with enhanced production (expression, processing, assembly, secretion): 1) Ig gene (heavy chain/HC and light chain/LC) sequences are optimized at the nucleic acid level (DNA and RNA), 2) optimized IgG leader sequences are placed before the genes to ensure secretion into the circulation, and 3) modified pVax expression vectors are used which contain various components to ensure transcript processing (FIG. 1).
Co-transfection of CR3022LC and HC plasmids in vitro resulted in the production and secretion of CR3022IgG1 antibodies, which were detected in the culture supernatant Western blot and quantified by ELISA (FIG. 2).
CR3022 dMAB expressed in vitro showed binding to the appropriate domain of SARS-CoV-2 spike (S) protein (FIG. 3). Consistent with previous reports, it binds to a recombinant version of the Receptor Binding Domain (RBD). RBD is located within the larger subunit 1 (S1) domain of S protein, and therefore CR3022 dMAB also binds recombinant S1 (fig. 3).
Analysis of in vivo expression kinetics showed consistent and durable expression of human CR3022IgG1 dMAB in mice following dual plasmid administration/electroporation (fig. 4).
Serum from mice given CR3022 dMAB via electroporation showed binding to the recombinant SARS-CoV-2RBD and S1 domains by ELISA (fig. 5).
Example 2: in vivo production of monoclonal antibodies against SARS-CoV-2/COVID-19 using synthetic DNA
Optimized DNA-encoded monoclonal antibodies (dmabs) against SARS-CoV-2 virus have been developed for prophylactic, therapeutic and/or diagnostic use (e.g., for the treatment of covd-19 disease). DMAB was developed for in vivo delivery of anti-SARS-CoV-2/covd-19 mAb with enhanced production (expression, processing, assembly, secretion) using single plasmid or dual plasmid systems (fig. 6).
Co-transfection of the S309LC and HC plasmids in vitro resulted in the generation and secretion of neutralizing antibodies that bound the SARS-CoV-2 spike protein Receptor Binding Domain (RBD) (FIG. 7).
In vitro transfection of 2130, 2381 and 2196dMAB plasmids resulted in expression of dMAB and binding to the appropriate domain of SARS-CoV-2 spike (S) protein (FIGS. 8 and 9). Analysis of in vivo expression kinetics showed consistent and durable expression of 2130, 2381 and 2196dMAB after administration/electroporation (FIG. 10). Serum from mice given 2130, 2381 and 2196 dmabs via electroporation demonstrated binding to the recombinant SARS-CoV-2RBD and S1 domains by ELISA (fig. 11). Finally, the modified dMAB (2196_mod dMAB) displayed in vivo expression and had more efficient neutralization than the parent 2196WT dMAB (fig. 12).
Example 3: novel monoclonal antibodies against SARS-CoV-2 spike glycoprotein: potential use as a treatment for patients with covd-19
The spike protein of the SARS-CoV-2 virus is reported to bind to its receptor ACE2. Mabs targeting vulnerable sites on viral surface proteins are increasingly known as a promising class of drugs against infectious diseases and have shown therapeutic efficacy against many viruses (Wang et al 2020 a). In line with this, the identification and cloning of mabs that specifically target surface viral proteins to block viral entry into host cells appears to be a very attractive approach for the prevention and treatment of SARS-CoV-2 (Chen et al 2020). The spike protein of SARS-CoV-2 undergoes major conformational changes and exposes the RBD and important residues of receptor binding to engage host cell receptor ACE2. Thus, binding of RBD to ACE2 receptor protein results in the detachment of S1 from S2, ultimately resulting in S2 mediated virus-host membrane fusion and viral entry. Thus, the role of spike protein in the infection process of SARS-CoV-2 into host cells is highly critical and therefore a highly efficient target for the development of potent mAbs (Chen et al 2020). Antibody responses against SARS-CoV-2 and other enveloped viruses typically include IgM, igG and IgA antibodies against viral envelope glycoproteins and against nucleoproteins. IgG antibodies against the envelope proteins exhibit different functional characteristics, which enable them to provide the most effective systemic antibody response against the virus (French and Moodley, 2020).
Without being bound by theory, it is hypothesized that immunotherapeutic approaches including vaccines and antibodies may provide benefits to patients. In this regard, plasma from patients recovering from disease has been used to treat severe disease patients with covd-19 (Ni et al 2020; zhang et al 2020). This has prompted the development of antibodies against spike proteins for therapeutic use. In this study, 10 SARS-CoV-2-spike protein specific IgG mAbs were developed and successfully cloned. To be effective, antibodies should fulfil several characteristics including specificity, high affinity binding to antigen and the ability to compete with spike proteins binding to the receptor ACE2, thereby blocking infection of cells by the virus. All 10 mAbs were found to have specific and strong binding to SARS-CoV-2-RBD. In addition, they cause a blockade of the interaction between SARS-CoV-2-RBD and ACE2 receptor. Furthermore, the glyco-group spectra of antibodies (especially WCoVA 7) indicate that they have high Fc-mediated effector functions. Therefore, there is a need to further examine the Fc-mediated effector functions of these antibodies. In addition, they cause a blockade of the interaction between SARS-CoV-2-RBD and ACE2 receptor. Furthermore, all 10 IgG clones showed effective neutralization of SARS-CoV-2 protein pseudotyped virus infection with a minimum IC50 value obtained at 2.6ng/ml, mostly less than 10ng/ml. Furthermore, in this study, these IgG clones were shown to effectively neutralize SARS-CoV-2-D614G mutant virus, 4 of which exhibited IC50 values of less than 10ng/ml. Significantly reduced IC50 values strongly indicate the high potential of these spike antibodies for SARS-CoV-2 in terms of neutralizing efficacy. Notably, many different naturally occurring variants in SARS-CoV-2-spike protein have been reported. Initial findings suggest that increased mortality due to covd-19 may be linked to D614G (the most dominant variant). It is speculated that such mutations may lead to conformational changes of the spike protein, ultimately leading to enhanced infectivity (Li et al 2020). Without being bound by theory, it is hypothesized that this varying degree of infectivity and transmissibility may be due to the different reactivities observed in this study between wild-type and D614G mutated SARS-CoV-2 virus for SARS-CoV-2-spike neutralizing antibodies. In summary, these anti-SARS-CoV-2-spike-ACE 2 blocking mabs have significant potential, so these anti-SARS-CoV-2-spike-ACE 2 blocking mabs can be further explored as specific therapeutic options against this severe world pandemic.
The results presented herein emphasize the potential utility of passive immunotherapy for the treatment of covd-19. The identification and characterization of monoclonal antibodies against SARS-CoV-2 spike protein adds a valuable set of agents with therapeutic potential. Of the 10 mabs described, they all exhibited specificity and high affinity for the spike-directed RBD. Thus, monoclonal antibodies have advantages over the use of convalescent polyclonal serum from patients recovered from covd-19. Although these mabs are independent of each other, the epitope recognized by these antibodies is not clear. The functional characteristics of antibodies show their ability to neutralize SARS-CoV-2 spike protein pseudotyped virus, which is an alternative measure of their effect on the virus. Antibodies should be evaluated in a study of SARS-CoV-2 virus infection. Development of unique and specific mabs to spike antigens and epitopes is necessary because they will be able to use a "cocktail" (a mixture of specific bioactive mabs) to simultaneously engage multiple neutralizing epitopes on a viral particle for enhanced therapeutic efficacy. Furthermore, the development of biologically active anti-SARS CoV-2 neutralizing mabs can also provide information about epitopes in antigens that are possible targets for the development of active vaccines for prophylactic purposes. Furthermore, the highly potent SARS-CoV-2-spike mAb has the potential to serve as a candidate biologic from both prophylactic and therapeutic aspects as a tool for developing highly clinically significant SARS-CoV-2-specific diagnostic assays.
The materials and methods used will now be described
Cell culture
HEK293T cells were obtained from ATCC and CHO-ACE2 cell line (stable expression of ACE2 on cell surface) was obtained from Creative Biolabs in the united states. Both cell types were maintained in D10 medium consisting of dulbeck's modified eagle medium (Invitrogen Life Science Technologies, USA) with heat-inactivated fetal bovine serum (10%), glutamine (3 mM), penicillin (100U/ml) and streptomycin (100U/ml). R10 medium containing RPMI1640 (Invitrogen Life Science Technologies, USA), heat-inactivated fetal bovine serum (10%), glutamine (3 mM), penicillin (100U/ml) and streptomycin (100U/ml) was used for mouse spleen cells. All cells were maintained at 37℃and 5% CO 2 。
Construction of SARS-CoV-2-spike synthetic DNA and protein
The SARS-CoV-2-spike plasmid DNA encoding construct is determined by alignment of the available RBD protein sequences in the PubMed database. The sequences corresponding to the immunogen inserts were genetically optimized for enhanced expression in humans, and IgE leader sequences were added to promote expression (Muthumani et al, 2016). Synthetic vaccine constructs were designed and supplied to commercial suppliers (Genscript, NJ, USA) for synthesis. The insert was then subcloned into a modified pMV101 expression vector under the control of the cytomegalovirus immediate early promoter as previously described (muthani et al, 2015;Muthumani et al, 2016).
Generation and evaluation of anti-SARS-CoV-2 hybridoma
On day 0 and day 14, balb/c mice were immunized with the synthetic consensus sequence for SARS-CoV-2-spike antigen at 50 μg/mouse by intramuscular immunization route followed by subcutaneous delivery of recombinant spike-RBD protein (100 μg/mouse). Serum from immunized mice was collected and assessed by ELISA to detect the presence of antibodies targeting SARS-CoV-2 spike. After confirmation, mouse spleen cells were used to produce hybridomas as described previously (Choi et al, 2020). Subsequently, positive hybridoma clones were characterized by indirect ELISA, and those selected were further subcloned and amplified. Antibodies were purified from hybridoma supernatants and used for further study.
ELISA (ELISA)
ELISA assays were performed with mAb clones in order to measure target antigen binding. To measure total IgG in hybridoma supernatants, maxiSorp high binding 96-well ELISA plates (ThermoFisher, USA) were coated overnight at 4 ℃ with 1 μg/mL recombinant SARS-CoV-2-RBD (Sino Biological, USA) and full length stokes. After blocking with 10% fbs in PBS for 1 hour, these plates were incubated with serial dilutions of mAb clones, using PBS with 1% fbs for 2 hours. Samples were then probed with anti-mouse IgG antibody (Sigma Aldrich, USA) conjugated to horseradish peroxidase (HRP) at a dilution of 1:20,000 for 1 hour. Thereafter, tetramethylbenzidine (TMB) substrate (Sigma Aldrich, USA) was added to all wells and incubated for 10 minutes. Then use 2N H 2 SO 4 To stop the reaction. Finally, the optical density was measured at 450nm with the aid of an ELISA plate reader (Biotek, USA). In addition, the average end point titers of SARS-CoV-2-RBD and full length spike-specific antibodies were also determined for all mAb clones. The titer was determined at the highest dilution with S/N (signal/negative) ratio > 2.1. This signal was designated as positive binding to SARS-CoV-2-RBD or full length spike compared to the negative control of antigen binding by an unrelated mAb. OD450 in the negative control is the average of two technical replicates and is designated as antibody endpoint titer of IgG mAb clones.
Western blot analysis
Binding Western blot analysis was performed to assess anti-CoV-2 mAb binding specificity. Briefly, 5 μg of internally generated recombinant human SASR-CoV-2-full-length Stokes (spike) and 2.5 μg of SARS-CoV-2-RBD (Sino Biological Inc, USA) protein were run in a 12% NuPAGE Novex polyacrylamide gel (Invitrogen Life Science Technologies, USA) and transferred onto PVDF membrane (Invitrogen Life Science Technologies, USA). These membranes were blocked using Odyssey blocking buffer (LiCor BioSciences, USA) and then incubated overnight at 4 ℃ with supernatant from mAb clones. After incubation, the membranes were washed with PBS or PBST containing 0.05% tween 20. Subsequently, the membranes were stained with IRDye800 goat anti-mouse secondary antibody (LiCor BioSciences, USA) at RT and then washed with PBST. Finally, these films were scanned using an LI-COR Odyssey CLx imager. Furthermore, to determine the heavy and light chain expression of mAb clones, this assay was performed in which 6ng of each mAb clone was run in a 12% nupagenovex polyacrylamide gel (Invitrogen Life Science Technologies, USA) and subsequently probed with goat anti-mouse IgG secondary antibody (LiCor BioSciences, USA).
SPR (surface plasmon resonance) analysis of SARS-CoV-2-spike monoclonal antibody clone bound to RBD protein
Binding of mAb clones to SARS-CoV-2-spike protein was measured using the Biacore T200SPR system. The SARS-CoV-2-RBD (Sino Biological Inc, USA) protein was immobilized with 10m MH EPES (pH 7.4), 150mM NaCl, 0.05% Tween20 running buffer using standard amine coupling procedure on carboxymethyl dextran sensor chip (CMD 200L, xantec Bioanalytics). Briefly, the chips were first washed with 0.1M sodium borate (pH 9.0), 1M NaCl, and then activated with EDC/NHS for 8min using a running buffer of MilliQ distilled water. After activation, 10 μg/mL of each protein was in 10mM sodium acetate (pH 5) until the desired fixed level was reached. Approximately 2500RUSAR-CoV-RBD protein was immobilized on the flow cell. 5000RU of Bovine Serum Albumin (BSA) was also immobilized to another flow cell that served as a negative control. After immobilization, the remaining activation sites were blocked with 1M ethanolamine at pH 8.5. The running buffer was then switched to 10mM HEPES (pH 7.4), 150mM NaCl, 0.05% Tween20. Each IgG mAb clone was tested in duplicate (0.13 nM, 0.41nM, and 1.2 nM) at three different concentrations. Dilutions were prepared in running buffer. The binding time was 300s and the dissociation time was 600s, with a flow rate of 30. Mu.L/min and a measured temperature of 20 ℃. After each injection, the surface was regenerated by injecting 20mM glycine (pH 2.0) for 60 seconds. Data were collected and analyzed using Biacore evaluation software. In addition, the kinetic parameters of SARS-CoV-2-RBD binding to IgG mAb clones were determined in the Biacore T200SPR system using a protein A/G coated carboxymethyl dextran sensor chip (Xantec Bioanalytics, germany). For each concentration of antigen, approximately 400RU of each mAb clone was captured on the chip surface. SARS-CoV-2-RBD was tested at a concentration ranging from 0-100nM and a flow rate of 30. Mu.L/min. The reference surface was coated with a 400RU mouse IgG isotype control. The association time was 210 seconds and the dissociation time was 900 seconds. After each concentration of antigen, the antibody-antigen complex was removed from the chip using 20mM glycine pH 2.0. The data are the average of two determinations and kinetic parameters are determined using the 1:1 binding model in biacore t200 evaluation software.
Glycan analysis
500 μl of hybridoma supernatant was concentrated using an amicon ultra-0.5 centrifugal filter unit (Millipore Sigma). A large amount of IgG from three C57BL/6 mice and human plasma samples (Innovative Research) was used as a control. Using Pierce TM Total IgG was purified by protein G rotor plates for IgG screening (Thermo Fisher) and further concentrated using an Amicon Ultra-0.5 centrifugal filtration unit (Millipore Sigma). N-glycans were released using peptide-N-glycosidase F (PNGaseF) and labeled with 8-aminopyrene-1, 3, 6-trisulfonic Acid (APTS) using a Glycan AssureAPTS kit (Thermo Fisher) according to the manufacturer's protocol. Labeled N-glycans were analyzed using a 3500 genetic analyzer capillary electrophoresis system. The relative abundance of IgG glycan structures was quantified by calculating the area under the curve for each glycan structure divided by the total glycans.
SARS-CoV-2-alternative virus neutralization assay
SARS-CoV-2-alternative virus neutralization assay kit (Genscript, USA) was used to examine the potential of mAb neutralization of the interaction of SARS-CoV-2-RBD and ACE 2. It is a species and isotype independent blocking ELISA detection tool to determine circulating neutralizing antibodies against SARS-CoV-2, which block protein-protein interactions between RBD and human ACE2 receptor. Briefly, 500ng/ml of each mAb clone and control were pre-incubated with HRP-conjugated RBD (HRP-RBD) for 30 minutes at 37℃to promote binding between mAb clones and HRP-RBD. Subsequently, the mixture was added to a capture plate pre-coated with human ACE2 receptor protein and incubated for 15 minutes at 37 ℃. After washing the plates with wash solution (Genscript, USA), TMB substrate was added to all wells and incubated for 15 minutes. Then, a termination solution (Genscript, USA) was added to each well to terminate the reaction. Finally, absorbance was measured at 450nm using an ELISA microplate reader (Biotek, USA) and inhibition values were determined. Cut-off values of 20 were considered based on a validated set of covd-19 patient serum and healthy control serum validated by Genscript, USA.
Receptor binding inhibition assay based on flow cytometry
Inhibition of SARS-CoV-2-spike protein binding to ACE2 receptor was also assessed by flow cytometry-based assays using a commercially available CHO-ACE2 cell line. For this assay, 2.5ug/ml S1+S2 ECD-his labeled (Sino Biological, USA) was incubated with each of 10 IgG mAb clones (500 ng/ml) for 60 minutes on ice. The mixture was then transferred to CHO-ACE2 cells (150,000 cells/well) that had been plated and then incubated again on ice for 90min. Subsequently, the cells were washed twice with PBS, followed by washing withAPC-conjugated anti-his antibodies (Abcam, USA) were stained. Spike proteins pre-incubated with recombinant human ACE2 (Invitrogen Life Science Technologies, USA) were used as positive controls. All data were obtained from LSR11 flow cytometry (BDbiosciences) and were obtained in FlowJo software (version 10; treeStar, USA).
SARS-CoV-2 pseudovirus production and neutralization assay
SARS-CoV-2 pseudoviruses were generated by co-transfecting HEK293T cells with Gene Jammer (Agilent, USA) in D10 medium at a 1:1 ratio of the DNA plasmid encoding SARS-CoV-2 spike protein (GenScript, USA) and the backbone plasmid pNL4-3.Luc.R-E- (NIHAIDS reagent). The supernatant containing pseudoviruses was harvested 48 hours post-transfection and enriched to 12% total volume with FBS, steri filtered and stored at-80 ℃. Pseudoviruses were titrated using a stable commercially available CHO-ACE2 cell line. For the neutralization assay, 10,000 CHO-ACE2 cells in 100 μld10 medium were plated in 96 well plates and at 37 ℃ and 5% co prior to the neutralization assay 2 Let stand overnight. The following day, serial dilutions of samples were incubated with SARS-CoV-2 pseudovirus for 90 minutes at room temperature, after which the mixture was added to already vaccinated CHO-ACE2 cells. The cells were incubated at 37℃with 5% CO 2 Incubate for 72 hours and then harvest and cleave with brite lite reagent (PerkinElmer, USA). Luminescence from the plates was recorded with a BioTek plate reader and used to calculate the percent neutralization of the samples at each dilution.
Statistical analysis
Statistical analysis was performed by student t-test or nonparametric Spearman (Spearman) correlation test using GraphPadPrism software for calculating statistical significance. Data are expressed as mean ± Standard Error of Mean (SEM). For all tests, p-values < 0.05 were considered significant.
Experimental results will now be described
Generation and characterization of antibodies targeting SARS-CoV-2-spike
There is growing evidence that coronavirus neutralizing antibodies (nabs) exhibit high efficacy in the treatment of a wide range of infectious diseases due to their highly specific antiviral activity and safety. Notably, SARS-CoV and MERS-CoVRBD-vaccine studies revealed strong polyclonal antibody responses in an in vivo environment that lead to inhibition of viral entry, suggesting the high potential of anti-spike antibodies to inhibit SARS-CoV-2 coronavirus entry (Ju et al, 2020; shi et al, 2020).
In this study, balb/c mice were immunized with a synthetic DNA plasmid construct encoding the consensus sequence for the full length SARS-CoV-2-spike antigen (Smith et al 2020) as a prime and the consensus sequence for the SARS-CoV-2-spike-RBD recombinant protein, as described previously (Choi et al 2020). The strategy and steps for immunization and subsequent procedures are summarized in fig. 13A-B. Finally, B lymphocytes were isolated from the spleen of immunized mice and used to generate hybridomas. To analyze serum from immunized animals, full-length spike protein expression constructs were generated as shown in fig. 14A. SARS-CoV-2 spike full-length protein was expressed using a mammalian cell system with Fc and Avi tags at the C-terminus and its size and purity were verified by SDS-PAGE and HPLC techniques (fig. 14B-C). The correct position of the full length spike protein (160 kDa) on SDS-PAGE gels showed clear bands. The protein was also found to have high purity as indicated by the sharp single peak obtained from HPLC. In addition, protein specificity was confirmed by ELISA using immune serum from mice vaccinated with SARS-CoV-2 spike DNA (FIG. 14D). More than 800 hybridomas were generated and then the hybridomas were screened to determine clones capable of producing antibodies with highest affinity for SARS-CoV-2 spike protein. This screening resulted in the identification of the best 10 IgG mabs (WCoVA 1, WCoVA2, WCoVA3, WCoVA4, WCoVA5, WCoVA6, WCoVA7, WCoVA8, WCoVA9 and WCoVA 10). Isotype analysis of the mabs showed clones WCoVA1, WCoVA2, WCoVA3, WCoVA4, WCoVA7 and WCoVA8 with IgG1, while clones WCoVA5, WCoVA6, WCoVA9 and WCoVA10 with IgG2 a. The binding and specificity of individual hybridoma clones was further assessed.
Binding and specificity analysis of SARS-CoV-2 spike monoclonal antibody
Antibody specificity was confirmed by western blot analysis for heavy and light chain expression (fig. 14E). In addition, the ability of these mAbs to bind SARS-CoV-2 full length spike and RBD was studied by indirect ELISA. The results show that all mabs specifically and strongly bind to full length spike and RBD proteins, whereas no binding was observed to the non-specific (NSP) control. Figure 15A shows the dose-dependent binding curve of IgG clones, which is represented by the mean ELISA signal plotted against the different dilutions of mAb clones. All clones showed high endpoint titers without any background (fig. 15B). In addition, the binding specificity of mAb clones was also confirmed by western blot analysis. For this analysis, SARS-CoV-2-RBD protein was loaded and then probed with an equal concentration mAb clone as described in the materials and methods section. All 10 mAb clones were found to bind specifically to SARS-CoV-2-RBD protein (FIG. 15C).
Kinetic analysis of SARS-CoV-2-spike antibodies and their targets by surface plasmon resonance analysis
Surface plasmon resonance binding analysis provides an important tool for mAb antigen binding characterization. mAb clones of SARS-CoV-2 spike and their binding kinetics to the target RBD region were analyzed by SPR. Initially, coV-S-RBD was immobilized on a carboxymethyl dextran sensor chip by amine coupling. The use of recombinant ACE2 to test SARS-CoV-2-RBD is functional after immobilization. All mAb clones were tested at concentrations of 0.13nM, 0.41nM and 1.3nM, and 9 out of 10 clones showed dose-dependent binding to SARS-CoV-2-RBD (FIG. 16). These 9 mAb clones were then further characterized to determine kinetic parameters of interaction. For this experiment, mAb clones were immobilized on a protein a/G sensor chip, which allowed the estimation of active antibodies immobilized on the chip surface. Considering the MW ratio between antibody and SARS-CoV-2-RBD, the target immobilization level of antibody is 400RU, which will yield a theoretical Rmax of 80 RU. The sensorgram of these clones is shown in fig. 15D, and the binding kinetics values for all 9 IgG mAb clones are summarized in table 1. In the case of all 9 IgG mAb clones, very low KD values (< 1 nM) were obtained, which strongly indicate their high affinity interactions with the SARS-CoV-2-RBD protein. WCoVA9 had the highest affinity and slowest dissociation rate. Three of these clones (WCoVA 5, WCoVA8 and WCoVA 9) had Rmax values greater than 100% of the expected Rmax. This suggests that these antibodies can bind 2 SARS-CoV-2-RBD molecules per antibody molecule. Three of these clones (WCoVA 1, WCoVA2 and WCoVA 4) had Rmax values of about 50% of the expected Rmax.
TABLE 1 analysis of IgG mAb clones and their binding kinetics to target SARS-CoV-2-RBD by SPR by immobilization of SARS-CoV-2-RBD using standard amine coupling.
SARS-CoV-2-spike mAb inhibits angiotensin converting enzyme 2 (ACE 2)
Shang and group reported in their studies a crystal structure of the SARS-CoV-2 spike protein RBD complexed with ACE2 and showed that ACE2 binding ridges in SARS-CoV-2RBD have a compact conformation (Shang et al 2020). NAb with the ability to target SARS-CoV-2RBD has the potential to block ACE2 binding by viruses and to prevent viral entry and possibly protect cells from infection therapeutically (Walker et al 2020). Thus, therapeutically active mabs targeting the interaction between SARS-CoV-2 spike protein and ACE2 receptor are of interest for screening hybridomas. Thus, it was assessed with the aid of a blocking ELISA whether these mAbs could block the interaction between SARS-CoV-2-RBD and ACE 2. The results showed that mAb clones were able to effectively block SARS-CoV-2-RBD-ACE2 interaction with variable efficiency (FIG. 17A). In this assay, a total of 5 clones (WCoVA 4, WCoVA5, WCoVA6, WCoVA7 and WCoVA 8) were able to cause more than 70% inhibition of SARS-CoV-2-RBD and ACE2 interactions. The potential of the identified SARS-CoV-2-spike IgG mAb as an inhibitor of viral entry was then assessed. For this purpose, flow cytometry-based assays were used to test the interference of these mAb clones with the binding of SARS-CoV-2 spike protein to CHO-ACE2 cells. Four mAb clones (WCoVA 4, WCoVA5, WCoVA7 and WCoVA 8) blocked SARS-spike binding to ACE2 as shown by flow cytometry (fig. 17B and 17C).
Neutralization of SARS-CoV-2 pseudovirus by SARS-CoV-2-spike antibody
NAb has the ability to inhibit viral infection by blocking the viral replication cycle (to et al 2020). Thus, the function of SARS-CoV-2-spike mAb was assessed using a developed pseudovirus neutralization assay (Smith et al 2020). As expected, pseudovirus neutralization was observed by the positive control ACE2-Ig at an IC50 value of 0.9 μg/mL, but not in the case of the negative control murine antibody TA 99. Antibodies secreted by 10 of the 10 IgG mAb clones were able to neutralize more than 50% of both wild-type (fig. 18A) and D614G mutant viruses (fig. 18B) with minimal sample dilution (10-fold). Furthermore, the IC50 values of these clones were evaluated, indicating that they effectively blocked infection. Cloning mAb; the IC50 values were observed to be 6.9ng/ml, 2.6ng/ml, 4.9ng/ml and 5.0ng/ml for WCoVA4, WCoVA7, WCoVA9 and WCoVA10, respectively, most effective against wild type virus. In addition, WCoVA1, WCoVA2, WCoVA3 and WCoVA4 were found to have higher efficacy against the D614G mutant virus with IC50 values of 8.85ng/ml, 5.05ng/ml, 7.93ng/ml and 6.42ng/ml, respectively (FIG. 18C). Moreover, a significant correlation between the ELISA titres and neutralization titres of these SARS-CoV-2-spike mabs was observed (fig. 18D). These results indicate that increased antiviral activity of these mabs correlates with increased binding affinity to RBD, but their detailed interactions require further investigation.
Sugar component analysis of SARS-CoV-2 antibody
Non-neutralizing Fc-mediated effector functions of antibodies, including antibody-dependent cellular cytotoxicity (ADCC), play an important role in controlling viral infection (Alpert et al, 2012; baum et al, 1996;Bhiman and Lynch,2017;Bruel et al, 2016; chung et al, 2011; halper-Strobng and Nussenzweig,2016;Isitman et al, 2016;Lee and Kent,2018;Madhavi et al, 2015). Antibody glycosylation strongly affects its effector function. The presence of core fucose results in weaker binding to fcγ receptor IIIA and reduced ADCC (masuda et al, 2007). While core fucose has the most significant impact on ADCC, other glycogenic features have also been shown to impact ADCC: terminal sialic acid reduces ADCC, (Naso et al, 2010; raju,2008; raju and scalen, 2007) bisecting GlcNAc induces innate immune function and inflammation (Davies et al, 2001;Takahashi et al, 2009; umana et al, 1999) and terminal galactose induces ADCC (fig. 19A) (Thomann et al, 2016). Glycosylation of WCoVA5, WCoVA7, WCoVA8, WCoVA9 and WCoVA10 was examined. All of these antibodies (especially WCoVA 7) had lower levels of core fucose, lower levels of sialic acid, and higher levels of bisecting GlcNac compared to the bulk of C57BL/6IgG (fig. 19B-D). In addition, WCoVA5, WCoVA7, and WCoVA9 have high levels of terminal galactose (fig. 19B-19E). This glycogen profile (especially for WCoVA 7) suggests that these antibodies will have high Fc mediated effector functions.
Table 2 provides an overview of the characteristics of the downwardly selected antibodies.
Table 2: characterization of downwardly selected antibodies
Computational characterization of antibody-antigen docking
Clones WCoVA7 and WCoVA9 were selected for sequencing and amplified for both heavy and light chains based on overall specificity. These sequences are then used as tools for antibody-antigen docking. The structure of the WCoVA7 and WCoVA9 variable regions was predicted using AbYmod. The SARS-CoV-2 spike structure was obtained from the protein database (PDB: 6 VYB) and the SARS-CoV-2 spike protein was modeled with an antibody complex at the ZDOCK server. ZDOCK performs an exhaustive grid-based search for the docking pattern of two component proteins (Pierce et al 2011). SARS-CoV-2 spike protein is quiescent in the output prediction when the antibody is moving. Antibody-antigen docking revealed that WCoVA7 antibody bound to the interface of RBD domains (fig. 20A-C), while WCoVA9 antibody bound to the interface between the N-terminal domain (NTD) and RBD domains (fig. 20B).
Table 3: IMGT analysis of V (D) J junction of WCoVA7
Table 4: IMGT analysis of V (D) J junction of WCoVA9
Example 4: antibody and dMAB sequences
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Example 5: rapid development of synthetic DNA vaccine for COVID-19
The SARS-CoV-2 spike sequence is most similar to the sequence and structure of the SARS-CoV spike protein (Wrapp et al 2020, science, eabb2507), sharing an overall protein folding structure with the MERS-CoV spike protein (FIG. 21). Unlike HIV and influenza glycoproteins, the pre-fusion form of coronavirus trimer spikes is conformationally dynamic, rarely exposing the receptor binding site completely (Kirchdoerfer et al, 2018,Sci Rep,8:15701). The receptor binding site is a vulnerable target of neutralizing antibodies. Indeed, MERSnAb targeting the Receptor Binding Domain (RBD) tends to have greater neutralizing potency than other epitopes (Wang et al, 2018,J Virol,92:e02002-17). Recent reports indicate that neutralizing anti-SARS antibodies cross-react with the RBD of SARS-CoV-2 (Tian et al, 2020,Emerg Microbes Infect,9:382-385). These data indicate that SARS-CoV-2RBD is an important target for vaccine development. Recent data show that the SARS-CoV-2S protein binds to the same host receptor angiotensin converting enzyme 2 (ACE-2) as the SARS-CoVS protein (Wrapp et al 2020, science, eabb2507).
Here, the design and initial preclinical testing of SARS-CoV-2 synthetic DNA vaccine candidates is described. Expression of SARS-CoV-2S antigen RNA and protein, respectively, was shown after transfection of COS and 293T cells with the candidate vaccine in vitro. Immunization induced by the selected immunogens was followed in mice and guinea pigs, and SARS-CoV-2S protein-specific antibody levels in serum and lung fluids were measured, as well as competitive inhibition of ACE2 binding. The INO-4800 vaccine induces cellular and humoral host immune responses that can be observed within days after a single immunization, including cross-reactive responses against SARS-CoV. In summary, the data show that immunogenicity of SARS-CoV-2 synthetic DNA vaccine candidates targeting SARS-CoV-2S protein supports further translational studies to rapidly accelerate the development of the candidates to address the current global health crisis.
New coronaviruses, SARS-CoV-2, and related COVID-19 diseases spread rapidly and have become a global pandemic. Here, the accelerated preclinical development of INO-4800 against this emerging infectious disease, a synthetic DNA-based SARS-CoV-2 vaccine, is described. Synthetic DNA vaccine design and synthesis began immediately after release of SARS-CoV-2 genomic sequence was published on day 11 of 1/2020. Most of the in vitro and in vivo studies described herein were performed within 6 weeks of SARS-CoV-2 genomic sequence becoming available. The data support the expression and immunogenicity of the INO-4800 synthetic DNA vaccine candidates in a variety of animal models. Humoral and T cell responses were observed in mice after a single dose. In guinea pigs, clinical delivery parameters were employed and antibody titers were observed after a single dose.
Preventing rapidly emerging infectious diseases requires a coordinated response from a global healthy population, and improved strategies are needed to accelerate vaccine development. In response to 2019/2020 coronavirus outbreaks, highly adaptable synthetic DNA drug platforms were rapidly developed. The design and manufacture of synthetic DNA vaccines for neoantigens is a method in which the target antigen sequence is inserted into a highly characterized and clinically tested plasmid vector backbone (pGX 0001). Construct design and engineering parameters were optimized for in vivo gene expression.
SARS-CoV-2S protein was selected as the antigen target. The SARS-CoV-2S protein is a class I membrane fusion protein that is the major envelope protein on the surface of coronaviruses. Preliminary studies have shown that SARS-CoV-2 interaction with its host receptor (ACE-2) can be blocked by antibodies (Zhou et al 2020, nature 579:270-273). In vivo immunogenicity studies in mouse and guinea pig models revealed levels of S protein reactive IgG in serum of INO-4800 immunized animals. In addition to full length S1+S2 and S1, INO-4800 immunity induces RBD binding antibodies, i.e., domains known to be targets of neutralizing antibodies from patients in the SARS-CoV convalescence phase (Zhu et al, 2007,Proc Natl Acad Sci U S A,104:12123-12128; he et al, 2005, virology, 334:74-82). The experiments presented here further demonstrate the functionality of these antibodies by competitively inhibiting the binding of SARS-CoV-2 spike protein to ACE2 receptor in the presence of serum from INO-4800 immunized animals. Importantly, anti-SARS-CoV-2 binding antibodies were detected in lung washes of mice and guinea pigs immunized with INO-4800. The presence of these antibodies in the lung has the potential to protect against these tissues and to prevent LRD, which is associated with severe cases of covd-19. In addition to humoral responses, cellular immune responses have been shown to be associated with more favorable recovery in MERS-CoV infection (Zhao et al, sci Immunol, 2:ean 5393), and may be important for SARS-CoV infection (Oh et al, 2012,Emerg Microbes Infect,1:e23). Here, experiments showed that T cell responses against SARS-CoV-2 were induced as early as day 7 after vaccine delivery. A rapid cellular response has the potential to reduce viral load and may potentially reduce the transmission of SARS-CoV-2 and related covd-19 disease.
In addition to the ability of INO-4800 to rapidly elicit humoral and cellular responses following a single immunization, synthetic DNA drug platforms have several synergistic properties that make them well responsive to disease outbreaks, such as covd-19. As previously mentioned, the ability to design and synthesize candidate vaccine constructs means that in vitro and in vivo tests can potentially begin within days of receiving viral sequences, allowing for an accelerated response to vaccine development. Well-defined and established production methods for DNA plasmid production lead to rapid and scalable production methods with the potential to avoid the complexity of traditional vaccine production in eggs or cell culture. The commercial costs associated with DNA production are also significantly lower than those currently seen for mRNA-based techniques. Recently, reports on stability profiles provided by using optimized DNA formulations have been published (Tebas et al, 2019,J Infect Dis,220:400-410). Stability characteristics mean that the DNA drug product is non-frozen and can be stored for 4.5 years at 2 ℃ -8 ℃, for 1 year and 1 week at room temperature at 37 ℃ while maintaining efficacy at temperatures above 60 ℃. In the case of a pandemic outbreak, the stability profile of the vaccine directly exerts its ability to be deployed and stored in an efficient and executable manner.
While vaccine-induced immunopathology has been proposed as a potential concern for SARS and MERS candidate vaccines, and possibly SARS-CoV-2 vaccines, these concerns may be vaccine platform dependent, and so far no evidence of immune pathogenesis has been reported for MERSDNA vaccines in mouse or non-human primate models (Muthmani et al, 2015,Sci Transl Med,7:301ra13) or SARSDNA vaccines in mice (Yang et al, 2004, nature, 428:561-564). Pulmonary immunopathology characterized by Th 2-associated eosinophilia of whole Inactivated Virus (IV), recombinant proteins, peptides and/or recombinant viral vector vaccines following SARS-CoV challenge has been reported (Tseng et al, 2012,PLoS One,7:e35421;Iwata-Yoshikawa et al, 2014,J Virol,88:8597-8614;Bolles et al, 2011,J Virol,85:12201-12215;Yasui et al, 2008,J Immunol,181:6337-6348; wang et al, 2016,ACS Infect Dis,2:361-376), and recently in the MERS-CoV challenge model (Agrawal et al, 2016,Hum Vaccin Immunother,12:2351-2356). However, the protective efficacy of SARS-CoV and MERS-CoV vaccines against pulmonary immunopathology has also been reported (Yang et al, 2004, nature,428:561-564;Muthmani et al, 2015,Sci Transl Med,7:301ra132;Luoetal, 2018,Virol Sin 33, 201-204; qin et al, 2006, vaccine,24:1028-1034;Roberts et al, 2010,Viral Immunol 23,509-519; deng et al, 2018,Emerg Microbes Infect,7:60;Zhang et al, 2016,Cell Mol Immunol,13:180-190; luke et al, 2016,Sci Transl Med,8:326ra321;Darnell et al, 2007,J Infect Dis,196:1329-1338). It is important to note that most studies demonstrating CoV vaccine-induced immunopathology utilize BALB/c mice, a model known to preferentially develop Th 2-type responses. DNA vaccine platforms induce Th 1-type immune responses and have demonstrated no immunopathological efficacy in respiratory tract infection models, including SARS-CoV (Yang et al, 2004, nature, 428:561-564), MERS-CoV (Muthumb et al, 2015,Sci Transl Med,7:301ra132) and RSV (Smith et al, 2017, vaccine, 35:2840-2847). Future studies will evaluate vaccine-enhanced disease in INO-4800 immunized animals.
Additional preclinical studies are underway to further characterize INO-4800 in small and large animals. The availability of reagents is a major challenge in developing vaccines against emerging infectious diseases, and this limits the ability to report the live virus neutralization activity of antibodies in animal models in this early study. However, it is reported here that INO-4800-induced antibodies block the binding of SARS-CoV-2 spike to host receptor ACE2 using an alternative neutralization assay. Other studies using live virus neutralization assays provide information for studies of antibody functionality and demonstrate the ability of INO-4800 immunity to mediate protection of animals against virus challenge.
In summary, it is promising to describe the results of immunogenicity of the SARS-CoV-2 candidate vaccine INO-4800, measuring antibody and T cell levels at an early time point after a single dose of vaccine is particularly encouraging, supporting further evaluation of the vaccine.
Materials and methods for experiments will now be described
Cell lines
Human Embryonic Kidney (HEK) -293T and COS-7 cell lines were obtained from ATCC (Old Town Manassas, VA). All cell lines were maintained in DMEM supplemented with 10% Fetal Bovine Serum (FBS) and penicillin-streptomycin.
In vitro RNA expression (qRT-PCR)
Plasmid expression in vitro was demonstrated by transfection of COS-7 with serially diluted plasmids, followed by analysis of total RNA extracted from cells using reverse transcription and PCR. UsingTransfection of four concentrations of plasmid was performed with 6 transfection reagents (Promega), which resulted in final masses ranging between 80ng and 10 ng/well. Transfection was performed in duplicate. After 18 to 26 hours of incubation, the cells were lysed with RLT buffer (Qiagen). Total RNA was isolated from each well using the Qiagen RNeasy kit according to the kit instructions. By OD 260/280 The resulting RNA concentration was determined and samples of RNA were diluted to 10 ng/. Mu.L. The kit instructions were then followed using a high capacity cDNA reverse transcription (RevT) kit (Applied Biosystems)One hundred nanograms of RNA was converted into cDNA. RevT reactions containing RNA but not reverse transcriptase (negative RT) served as controls for plasmid DNA or cell genomic DNA sample contamination. Eight μl of sample cDNA was then PCR using primers and probes specific for the target sequence. In a separate reaction, PCR was performed on the same amount of sample cDNA using primers and probes designed for the COS-7 cell line beta-actin sequence. Using the Quant Studio7Flex real-time pcrstatus system (Applied Biosystems), samples were first subjected to 1 minute hold at 95 ℃ and then subjected to 40 cycles of PCR, each cycle consisting of 1 second at 95 ℃ and 20 seconds at 60 ℃. After PCR, the amplification results were analyzed as follows. Negative transfection control, negative RevT control and NTC were checked for each of their respective indications. Threshold period (C) for generating each transfection concentration of INO-4800COVID-19 target mRNA and beta-actin mRNA by Quantum studio software using automatic threshold setting T ). Plasmids are considered active for mRNA expression if expression in any plasmid transfected well is greater than 5CT compared to the negative transfection control.
In vitro protein expression (Western blot)
Human embryonic kidney cells were cultured and transfected as described previously, 293T (Yan et al, 2007, molTher, 15:411-421). 293T cells were transfected with pDNA using TurboFectin8.0 (OriGene) transfection reagent following the manufacturer's protocol. After 48 hours, cell lysates were harvested using modified RIPA cell lysis buffer. Proteins were separated on 4% -12% bis-TRIS gel (Thermo Fisher Scientific), then after transfer, blots were incubated with anti-SARS-CoV spike protein polyclonal antibody (Novus Biologicals) and then visualized with horseradish peroxidase (HRP) -conjugated anti-mouse IgG (GE Amersham).
Immunofluorescence of transfected 293T cells
For in vitro staining of spike protein expression, 293T cells were cultured on 4-well slides (Lab-Tek) and transfected with 3 μg/well of pDNA using Turbo Fectin8.0 (OriGene) transfection reagent following the manufacturer's protocol. After 10 min transfection with 10% neutral buffered formalin (BBC biochemicals, washington State) at Room Temperature (RT) The cells were fixed for 48 hours and then washed with PBS. Prior to staining, chamber slides were blocked with 0.3% (v/v) Triton-X (Sigma), 2% (v/v) donkey serum in PBS for 1 hour at room temperature. Cells were treated with 1% (w/v) BSA (Sigma), 2% (v/v) donkey serum, 0.3% (v/v) Triton-X (Sigma) and 0.025% (v/v) 1gml in PBS -1 A rabbit anti-SARS-CoV spike protein polyclonal antibody (Novus Biologicals) diluted in sodium azide (Sigma) was stained at room temperature for 2 hours. Slides were washed 3 times in PBS for 5 min and then stained with donkey anti-rabbit IgGAF488 (life technology) for 1 hour at RT. Slides were again washed and mounted and covered with DAPI-fluorovent (Southern Biotech).
Animals
Female, 6 week old C57/BL6 and BALB/C mice were purchased from Charles River Laboratories (Malvern, pa.) and The Jackson Laboratory (Bar Harbor, ME). Female 8 week old Hart1ey guinea pigs were purchased from Elm Hill Labs (Chelmsford, mass.). For the mouse study, on day 0, subsequent injection through a needle was followedIn vivo Electroporation (EP) gives a dose of 2.5, 10 or 25 μg pDNA to Tibialis Anterior (TA). />EP delivery consisted of two sets of pulses with a constant current of 0.2 Amp. The second pulse set is delayed by 3 seconds. Within each group there are two 52ms pulses with a 198ms delay between the pulses. Blood was collected on day 0 and day 14. Mice from the parallel groups were serially sacrificed on days 4, 7 and 10 post immunization for analysis of cellular immune responses. For guinea pig studies, on day 0, subsequent injections by Mantoux were performed In vivo EP gives 100 μg pdna to the skin. Blood was collected on day 0 and day 14. Mice and guinea pigs were immunized twice on days 0 and 14 for ACE2 competition ELISA and lung bronchoalveolar lavage. Serum was collected from the mice 14 days after the second immunization, and guinea pigs were collected 28 days after the second immunization.
Antigen binding ELISA
ELISA was performed to determine serum antibody binding titers. At 4℃with 1. Mu.g ml -1 Recombinant protein antigens were coated on NuncELISA plates overnight in Dulbecco Phosphate Buffered Saline (DPBS). Plates were washed three times and then blocked with 3% Bovine Serum Albumin (BSA) in DPBS with 0.05% Tween20 for 2 hours at 37 ℃. The plates were then washed and incubated with serial dilutions of mouse or guinea pig serum and incubated for 2 hours at 37 ℃. Plates were washed again and then incubated with horseradish peroxidase (HRP) -conjugated anti-guinea pig IgG secondary antibody (Sigma-Aldrich, cat) at 1:10,000 dilution. A7289 (HRP) or (HRP) conjugated anti-mouse IgG secondary antibody (Sigma-Aldrich) and incubated for 1 hour at RT. After final washing, sureBlue was used TM TMB1-Component Peroxidase Substrate (KPL, cat.52-00-03) was developed for plates. And the reaction was stopped with TMB stop solution (KPL, cat.50-85-06). Plates were read at 450nm wavelength within 30 minutes using a synergy htx (BioTek Instruments, highland Park, VT). Binding antibody endpoint titers (EPT) were calculated as described in BagarazziM et al. 2012 (Bagarazzi et al 2012, sciTranslMed, 4:155r138). The binding antigens tested included SARS-CoV-2 antigen: s1 spike protein (Sino Biological 40591-V08H), S1+S2ECD spike protein (Sino Biological 40589-V08B 1), RBD (University of Texas, at Austin (McLellan Lab.)); SARS-COV antigen: spike S1 protein (Sino Biological40150-V08B 1), S (1-1190) (Immune Tech IT-002-001P) and spike C-terminus (Meridian Life Science R18572).
ACE-2 competition ELISA
A mouse
ELISA was performed to determine competition of serum IgG antibodies with human ACE2 with a human Fc tag. NuncELISA plates were coated with 1. Mu.g/ml rabbit anti-His 6X in 1XPBS for 4-6 hours at room temperature and washed 4 times with wash buffer (1 XPBS and 0.05% Tween 20). Plates were blocked overnight at 4 ℃ with blocking buffer (1 xpbs,0.05% Tween20,5% evaporated milk and 1% fbs). Plates were washed four times with wash buffer and then incubated with full length (S1+S2) spike protein (Sino Biologics, cat 40589-V08B 1) containing a C-terminal His tag. At room temperature, 10ug/ml was run for 1 hour. Plates were washed and then purified mouse IgG was incubated with serial dilutions of 0.1ug/ml recombinant human ACE2 with human Fc tag (ACE 2-IgHu) for 1-2 hours at room temperature. Plates were washed again and then incubated with a 1:10,000 dilution of horseradish peroxidase (HRP) -conjugated anti-human IgG secondary antibody (Bethyl, cat a 80-304P). And incubated at room temperature for 1 hour. After the final wash, the plates were developed using a 1-StepUltraTMB-ELISA substrate (Thermo, cat.34029). And the reaction was quenched with 1M sulfuric acid. Plates were read at 450nm wavelength over 30 minutes using a Spectra MaxPlus 384 microplate reader (Molecular Devices, sunnyvale, calif.). The competition curves were plotted and the area under the curve (AUC) was calculated using multiple t-tests using Prism8 analysis software to determine statistical significance.
Guinea pig
A96-well half-area assay plate (Costar) was coated overnight at 4℃with 25. Mu.l/well of SARS-CoV-2 spike S1+S2 protein (Sion Biological) diluted in 1xDPBS (Thermofisher). Plates were washed with 1xPBS buffer with 0.05% TWEEN (Sigma). 100 μl/well of 3% (w/v) BSA (Sigma) in 1 xPS with 0.05% TWEEN was added and incubated for 1 hour at 37 ℃. Serum samples were diluted 1:20 in 1% (w/v) BSA in 1xPBS with 0.05% TWEEN. After washing the assay plates, 25 μl/well of diluted serum was added and incubated for 1 hour at 37 ℃. Human recombinant ACE-2-Fc tag (Sino biological) was added directly to diluted serum followed by incubation at 37 ℃ for 1 hour. Plates were washed and 25 μl/well of 1:10,000 dilution goat anti-huFc fragment antibody HRP (bethyl) was added to assay plates. Plates were incubated for 1 hour at room temperature. For development, sureBlue/TMB stop solution (KPL, MD) was used and o.d. was recorded at 450 nm.
Bronchoalveolar lavage collection
Bronchoalveolar lavage (BAL) fluid was collected by washing the lungs of euthanized and exsanguinated mice with 700-1000ul of ice-cold PBS (containing 100 μm edta, 0.05% sodium azide, 0.05% Tween-20, and 1x protease inhibitor (Pierce)) (mucosa preparation solution with blunt tip (MPS)). Guinea pig lungs were washed with 20ml MPS via a 16G catheter inserted into the trachea. The collected BAL liquid was stored at-20C until the time of measurement.
IFN-γELISpot
Spleens from mice were individually collected in RPMI1640 medium supplemented with 10% fbs (R10) and penicillin/streptomycin and processed into single cell suspensions. The cell pellet was resuspended in 5ml ck lysis buffer (Life Technologies, carlsbad, CA), RT for 5 min, and PBS was then added to terminate the reaction. The sample was centrifuged again at 1, 500g for 10min, the cell pellet was resuspended in R10 and then passed through a 45 μm nylon filter before being used in the ELISpot assay. Use of mouse IFN-. Gamma.ELISPot PLUS The ELISPot assay was performed on plates (MABTECH). 96-well ELISpot plates pre-coated with capture antibody were blocked overnight at 4 ℃ with R10 medium. 200,000 mouse spleen cells were plated into each well and stimulated with a 15-mer peptide pool (overlapping with 9 amino acids from SARS-CoV-2, SARS-CoV or MERS-CoV spike protein) for 20 hours (5 peptide pools per protein). In addition, matrix mapping was performed using peptide libraries in the matrices designed to identify immunodominant responses. Cells were stimulated with a final concentration of 5 μl of each peptide per well in rpmi+10% fbs (R10). These spots were developed based on the manufacturer's instructions. R10 and cell stimulation mixtures (Invitrogen) were used for negative and positive controls, respectively. Spots were scanned and quantified by an ImmunoSpot CTL reader. The Spot Formation Units (SFU) per million cells were calculated by subtracting the negative control wells.
Flow cytometry
Spleen cells harvested from BALB/C and C57BL/6 (mouse evaluation with overlapping peptides spanning the SARS-CoV-2S protein) were stained for intracellular cytokines at 37℃and 5% CO2 for 6 hours. Cells were stained with the following antibodies: FITC anti-mouse CD107a, perCP-Cy5.5 anti-mouse CD4 (BD Biosciences), APC anti-mouse CD8a (BD Biosciences), viViViD dyeFixed Viole Dead Cell Stain kit; invitrogen, L34955), APC-Cy7 anti-mouse CD3e (BD Biosciences) and BV605 anti-mouse IFN-gamma (eBiosciences). Phorbol Myristate (PMA) was used as positive control and only complete medium was used as negativeSex control. Cells were washed, fixed, and cell events were acquired using FACSCANTO (BD Biosciences) followed by FlowJo software (FlowJoLLC, ashland, OR) analysis.
Structural modeling
Structural models of SARS-CoV and MERS-CoV were constructed from PDB ID 6acc and 5x59 to assemble a pre-fusion model of all three RBDs with the following conformations. The SARS-CoV-2 structural model was constructed using the SARS-CoV structure (PDB id:6 acc) as a template. A suitable amino acid mutation was prepared using a Rosa tower remodeling simulation and a de novo model of the unstructured SARS-CoV-2 loop in the SARS-CoV structure was constructed (Huang et al 2011, PLoSOne, 6:e24109). Allowing the amino acid positions adjacent to the loops to change the backbone conformation to accommodate the new loops. The structure was prepared using PyMOL.
Statistics
All statistical analyses were performed using GraphPadPrism7 or 8 software (LaJolla, CA). These data are considered significant if p < 0.05. The lines in all graphs represent mean values and the error bars represent standard deviations. No samples or animals were excluded from the analysis. No randomization was performed for animal studies. The samples and animals were not blind until each experiment was performed.
Experimental results will now be described
Design and Synthesis of a synthetic DNA vaccine construct of COVID-19
Four spike protein sequences were retrieved from the first four available SARS-CoV-2 whole genome sequences published by GISAID (Global Initiative on Sharing All Influenza Data). Three spike sequences were 100% matched and one was considered an outlier (98.6% sequence identity with the other sequences). After sequence alignment, SARS-CoV-2 spike glycoprotein sequence was generated and N-terminal IgE leader sequence was added. Highly optimized DNA sequences encoding SARS-CoV-2 IgE-spikes were generated using an Inovio-specific computer gene optimization algorithm to enhance expression and immunogenicity. The optimized DNA sequence was synthesized, digested with BamHI and XhoI, and cloned into the expression vector pGX0001 under the control of the human cytomegalovirus immediate early promoter and the bovine growth hormone polyadenylation signal. The resulting plasmids were designated pGX9501 and pGX9503, designed to encode SARS-CoV-2S protein from 3 matching sequences and outlier sequences, respectively (FIG. 22A).
In vitro characterization of the synthetic DNA vaccine construct of COVID-19
Expression of the encoded SARS-CoV-2 spike transgene was measured at the RNA level in COS-7 cells transfected with pGX9501 and pGX9503. Expression of the ear transgene was confirmed by RT-PCR using total RNA extracted from transfected COS-7 cells (fig. 22B). In vitro spike protein expression in HEK-293T cells was measured by western blot analysis using cross-reactive antibodies against SARS-CoVS protein on cell lysates. Western blotting of lysates of HEK-293T cells transfected with pGX9501 or pGX9503 constructs revealed bands approximating predicted S protein molecular weights (140-142 kDa) (FIG. 22C). In immunofluorescence studies, S protein was detected in 293T cells transfected with pGX9501 or pGX9503 (fig. 22D). In summary, in vitro studies revealed the expression of spike proteins at the RNA and protein levels following transfection of cell lines with candidate vaccine constructs.
Humoral immune response to SARS-CoV-2S protein antigen measured in mice immunized with INO-4800
pGX9501 was chosen as the vaccine construct for immunogenicity studies, pGX9503 due to the wider coverage it would be possible to provide compared to outliers. pGX9501 is subsequently designated INO-4800. Following administration of TA muscle The immunogenicity of INO-4800 was evaluated in BALB/c mice of the delivery device. The reactivity of serum from a group of mice immunized with INO-4800 was measured against a group of SARS-CoV-2 and SARS-CoV antigens (FIG. 23). Analysis showed that IgG binds to the SARS-CoV-2S protein antigen with limited cross-reactivity with the SARS-CoVS protein antigen in the serum of INO-4800 immunized mice. Serum IgG binding endpoint titers against the recombinant SARS-CoV-2 spike protein s1+s2 region (fig. 24A and 24B) and the recombinant SARS-CoV-2 spike protein Receptor Binding Domain (RBD) (fig. 24C and 24D) were measured in mice immunized with pDNA. On day 14 after immunization with a single dose of INO-4800, observations were made in mouse serumTermination titer (fig. 24B-24D).
Inducing antibodies capable of inhibiting the binding of the spike protein host receptor is a key goal of SARS-CoV-2 vaccine development. Thus, experiments were designed to examine the receptor inhibition function of the INO-4800-induced antibody response. Recently, ELISA-based ACE2 inhibition assays were developed as alternatives to neutralization. The assay is in principle similar to other alternative neutralization assays that have been validated against coronaviruses (Rosen et al, 2019, journal of virology Methods 265:77-83). As a control in the assay, ACE2 was shown to be capable of EC at 0.025. Mu.g/ml 50 Binds SARS-CoV-2 spike protein (FIG. 25A). BALB/c mice were immunized with 10 μg of INO-4800 on day 0 and day 14, and serum IgG was purified on day 28 post immunization to ensure inhibition was antibody mediated. Inhibition of spike-ACE 2 interactions using serum IgG from the naive mice and from the INO-4800 vaccinated mice was compared (fig. 25B). The receptor inhibition assay was repeated with a group of five immunized mice and it was shown that the INO-4800-induced antibody competed with ACE2 for binding to SARS-CoV-2 spike protein (fig. 25C and fig. 26). ACE2 is considered to be the primary receptor for SARS-CoV-2 cell entry, blocking this interaction suggests that INO-4800-induced antibodies can prevent host infection.
Detection of humoral immune response to SARS-CoV-2S protein in guinea pigs after intradermal delivery of INO-4800
The immunogenicity of INO-4800 was evaluated in the Hartley guinea pig model, which is an established model for intradermal vaccine delivery (carter et al 2018,SciAdv,4:eaas9930;Schultheis et al, 2017, vaccine, 35:61-70. 100 μg of pDNA was administered to the skin by Mantoux injection followed by day 0 as described in the methods sectionA delivery device. On day 14, anti-spike protein binding of serum antibodies was measured by ELISA. Immunization with INO-4800 revealed an immune response in serum that bound the SARS-CoV-2S1+2 protein to the level of IgG (FIGS. 27A and 27B). Endpoint of day 14 SARS-CoV-2S protein binding titers in guinea pigs treated with 100 μg INO-4800 or pVAX (control), respectively 10, 530 and 21 (fig. 27B). Serum antibody functionality was measured by assessing its ability to inhibit ACE-2 binding to SARS-CoV-2 spike protein. Serum (1:20 dilution) collected from the INO-4800 immunized guinea pigs after the second immunization inhibited SARS-CoV-2 spike protein binding in the ACE-2 concentration range (0.25. Mu.g/ml to 4. Mu.g/ml) (FIG. 28A). In addition, serum dilution curves revealed that serum collected from the INO-4800 immunized guinea pigs blocked ACE-2 binding to SARS-CoV-2 in a dilution-dependent manner (fig. 28B). Serum collected from pVAX-treated animals showed negligible activity in inhibition of ACE-2 binding to viral proteins, and a decrease in OD signal at the highest concentration of serum was considered as a matrix effect in the assay.
In summary, immunogenicity testing in both mice and guinea pigs revealed that the covd-19 candidate vaccine INO-4800 was able to elicit a functional antibody response to SARS-CoV-2 spike protein.
SARS-CoV-2 specific antibody biodistribution of lung in INO-4800 immunized animals
Lower respiratory tract disease (LRD) is associated with severe cases of COVID-19. The presence of antibodies targeting SARS-CoV-2 at the lung mucosa could potentially mediate protection against LRD. Thus, the presence of SARS-CoV-2 specific antibodies was assessed in the lungs of immunized mice and guinea pigs. BALB/c mice and Hartley guinea pigs were immunized with INO-4800 or pVAX control pDNA on days 0 and 14 or days 0, 14 and 28, respectively. Bronchoalveolar lavage (BAL) fluid was collected after sacrifice and subjected to SARS-CoV-2S protein ELISA. In the animals receiving INO-4800, both BALB/c and Hart1ey guinea pigs, statistically significant increases in SARS-CoV-2S protein binding IgG were measured in their BAL fluid compared to animals receiving the pVAX control (FIGS. 29A-29D). Taken together, these data indicate the presence of antibodies specific for anti-SARS-CoV-2 in the lung following immunization with INO-4800.
Early detection of cross-reactive cellular immune responses against SARS-CoV-2 and SARS-CoV in mice immunized with INO-4800
T cell responses against SARS-CoV-2, SARS-CoV and MERS-CoVS antigens were determined by IFN-gamma ELISPot. BALB/c mice groups were sacrificed on day 4, day 7 or day 10 (2.5. Mu.g or 10. Mu.g pDNA) after INO-4800 administration, spleen cells were harvested, andthe single cell suspension was stimulated with 15-mer overlapping peptide pools spanning SARS-CoV-2, SARS-CoV and MERS-CoV spike proteins for 20 hours. On day 7 after INO-4800 dosing, 205 and 552SFU/10 were measured for 2.5 and 10 μg doses, respectively 6 Spleen cells responded to T cells of SARS-CoV-2 (figure 30A). 852 and 2193 SFU/10 were observed on day 10 post INO-4800 dosing 6 Spleen cells respond to the higher orders of magnitude of SARS-CoV-2. In addition, the cross-reactivity of the cellular response elicited by INO-4800 against SARS-CoV was assessed and at day 7 (74 [2.5 μg dose]And 140[10 μg dose ]]SFU/10 6 Splenocytes) and day 10 post-dose (242 [2.5 μg dose)]And 588[10 μg dose ]]SFC/10 6 Splenocytes) a detectable (albeit lower) T cell response was observed (fig. 30B). Interestingly, no cross-reactive T cell responses were observed against MERS-CoV peptides (fig. 30C). Representative images of IFN-. Gamma.ELISPot.plates are provided in FIG. 31. T cell populations producing IFN-gamma were identified. Flow cytometry analysis of splenocytes harvested from BALB/c mice on day 14 after a single INO-4800 immunization revealed that the T cell compartment contained 0.0365% CD4+ and 0.3248% CD8+IFN- γ+ T cells after stimulation with SARS-CoV-2 antigen (FIG. 32).
BALB/c mouse SARS-CoV-2 epitope mapping
Spleen cells from BALB/c mice receiving a dose of 10. Mu.g INO-4800 were epitope mapped. Spleen cells were stimulated for 20 hours using thirty matrix mapping pools and immunodominant responses were detected in multiple peptide pools (fig. 33A). Deconvolving these responses to identify several epitopes in the receptor binding domain and the S2 domain (H2-K d ) Clustering (fig. 33B). Interestingly, a SARS-CoV-2H 2K was observed d Epitope PHGVVFLHV (SEQ ID NO: 142) overlaps and is adjacent to SARS-CoV human HLA-A2 restriction epitope VVFLHVTYV (SEQ ID NO: 143) (Ahmed et al, 2020,Viruses 12:254).
In summary, a rapid T cell response against the SARS-CoV-2S protein epitope was detected in mice immunized with INO-4800.
Example 6: dMAB and DNA vaccine co-delivery
Studies were designed to demonstrate the efficacy of co-delivery of dmab+dna vaccines.
Vaccine sequence:
it is to be understood that the foregoing detailed description and examples are merely illustrative and are not to be considered limiting the scope of the invention, which is defined only by the appended claims and equivalents thereof.
Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications, including but not limited to those involving the chemical structures, substituents, derivatives, intermediates, syntheses, compositions, formulations, or methods of use of the invention, may be made without departing from the spirit and scope of the invention.
Sequence listing
<110> institute of wista anatomy and biology (The Wistar Institute of Anatomy and Biology)
Large Weiwei Na (Weiner, david)
Calf Mu Tuma Ni (Muthumb Kar)
Elizabeth, parzaqi (Parzych, elizabeth)
<120> DNA-encoded antibody against SARS-COV-2
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<150> US 63/036,795
<151> 2020-06-09
<150> US 63/036,809
<151> 2020-06-09
<150> US 63/068,868
<151> 2020-08-21
<150> US 63/083,173
<151> 2020-09-25
<150> US 63/114,271
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atcaactgca agagctccca gagcgtgctg tactctagca tcaacaagaa ttacctggcc 180
tggtatcagc agaagcccgg ccagccccct aagctgctga tctattgggc aagcaccagg 240
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atctcctctc tgcaggccga ggacgtggcc gtgtactatt gccagcagta ctattccacc 360
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Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
145 150 155 160
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
165 170 175
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
180 185 190
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
195 200 205
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
210 215 220
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
225 230 235 240
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
245 250 255
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
260 265 270
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
275 280 285
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
290 295 300
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
305 310 315 320
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
325 330 335
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
340 345 350
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
355 360 365
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
370 375 380
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
385 390 395 400
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
405 410 415
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
420 425 430
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
435 440 445
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
450 455 460
Gly Lys
465
<210> 5
<211> 384
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 5
gaggtgcagc tggtgcagtc aggagccgag gtgaaggagc ccggcagctc cgtgaaggtg 60
tcttgcaagg ccagcggcgg caccttcggc tcttacgccg gcggcacatt tggcagctat 120
gccatcaact gggtgcggca ggcacctgga cagcgcctgg agtggatggg ctggatcgat 180
gccgccaacg gcaataccaa gtacagccag aagttccagg gccgggtgac catcacaggc 240
gacacctctg ccagcacagc ctatatggag ctgtctagcc tgaggtccga ggacaccgcc 300
gtgtactatt gcgcccggga tcgctggatg accacaagag ccttcgacat ctggggccag 360
ggcaccatgg tgacagtgtc ctct 384
<210> 6
<211> 128
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 6
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Glu Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Gly Ser Tyr
20 25 30
Ala Gly Gly Thr Phe Gly Ser Tyr Ala Ile Asn Trp Val Arg Gln Ala
35 40 45
Pro Gly Gln Arg Leu Glu Trp Met Gly Trp Ile Asp Ala Ala Asn Gly
50 55 60
Asn Thr Lys Tyr Ser Gln Lys Phe Gln Gly Arg Val Thr Ile Thr Gly
65 70 75 80
Asp Thr Ser Ala Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser
85 90 95
Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Arg Trp Met Thr Thr
100 105 110
Arg Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser
115 120 125
<210> 7
<211> 333
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 7
cagcctgtgc tgacacagcc acccgcatcc gcctctggaa ccccaggcca gagagtgaca 60
atcagctgtt ccggcagctc ctctaacatc ggcagcaatt acgtgttctg gtatcagcag 120
ctgcccggca tggcccctaa gctgctgatc tcccggaaca atcagcgccc atctggcgtg 180
cccgatcggt tttctggcag caagtccggc acctctgcca gcctggcaat ctccggacca 240
cagtctgagg acgaggccga ttactattgc gcagcatggg acgattccct gaggggacca 300
gtgttcggcg gcggaaccag ggtgacagtg ctg 333
<210> 8
<211> 111
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 8
Gln Pro Val Leu Thr Gln Pro Pro Ala Ser Ala Ser Gly Thr Pro Gly
1 5 10 15
Gln Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser
20 25 30
Asn Tyr Val Phe Trp Tyr Gln Gln Leu Pro Gly Met Ala Pro Lys Leu
35 40 45
Leu Ile Ser Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe
50 55 60
Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Pro
65 70 75 80
Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser
85 90 95
Leu Arg Gly Pro Val Phe Gly Gly Gly Thr Arg Val Thr Val Leu
100 105 110
<210> 9
<211> 2238
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 9
atggattgga cttggaggat tctgtttctg gtcgccgccg ctaccggaac tcacgccgag 60
gtgcagctgg tgcagtcagg agccgaggtg aaggagcccg gcagctccgt gaaggtgtct 120
tgcaaggcca gcggcggcac cttcggctct tacgccggcg gcacatttgg cagctatgcc 180
atcaactggg tgcggcaggc acctggacag cgcctggagt ggatgggctg gatcgatgcc 240
gccaacggca ataccaagta cagccagaag ttccagggcc gggtgaccat cacaggcgac 300
acctctgcca gcacagccta tatggagctg tctagcctga ggtccgagga caccgccgtg 360
tactattgcg cccgggatcg ctggatgacc acaagagcct tcgacatctg gggccagggc 420
accatggtga cagtgtcctc tgcctctaca aagggaccaa gcgtgtttcc actggcaccc 480
agctccaagt ccacctctgg cggcacagcc gccctgggct gtctggtgaa ggattacttc 540
cccgagcctg tgaccgtgtc ctggaactct ggcgccctga ccagcggagt gcacacattt 600
cctgccgtgc tgcagtctag cggcctgtac tccctgtcct ctgtggtgac cgtgccaagc 660
tcctctctgg gcacccagac atatatctgc aacgtgaatc acaagccctc taatacaaag 720
gtggataaga aggtggagcc taagagctgt gacaagaccc acacatgccc tccctgtcca 780
gcaccagagc tgctgggcgg ccctagcgtg ttcctgtttc cacccaagcc aaaggatacc 840
ctgatgatct cccgcacccc agaggtgaca tgcgtggtgg tggacgtgtc tcacgaggac 900
cccgaggtga agttcaactg gtacgtggac ggcgtggagg tgcacaatgc caagacaaag 960
cctagggagg agcagtacaa ctccacctat agagtggtgt ctgtgctgac agtgctgcac 1020
caggactggc tgaacggcaa ggagtataag tgcaaggtga gcaataaggc cctgcctgcc 1080
ccaatcgaga agaccatctc caaggcaaag ggacagcctc gggagccaca ggtgtacaca 1140
ctgcctccaa gccgcgatga gctgaccaag aaccaggtgt ccctgacatg tctggtgaag 1200
ggcttctatc catccgacat cgccgtggag tgggagtcta atggccagcc cgagaacaat 1260
tacaagacca caccccctgt gctggacagc gatggctcct tctttctgta ttctaagctg 1320
accgtggata agagcaggtg gcagcagggc aacgtgttta gctgctccgt gatgcacgag 1380
gccctgcaca atcactacac ccagaagtct ctgagcctgt ccccaggcaa gaggggcaga 1440
aagaggagat ctggcagcgg cgccacaaac ttcagcctgc tgaagcaggc aggcgacgtg 1500
gaggagaatc caggacctat ggtgctgcag acccaggtgt ttatcagcct gctgctgtgg 1560
atctccggag catacggaca gcctgtgctg acacagccac ccgcatccgc ctctggaacc 1620
ccaggccaga gagtgacaat cagctgttcc ggcagctcct ctaacatcgg cagcaattac 1680
gtgttctggt atcagcagct gcccggcatg gcccctaagc tgctgatctc ccggaacaat 1740
cagcgcccat ctggcgtgcc cgatcggttt tctggcagca agtccggcac ctctgccagc 1800
ctggcaatct ccggaccaca gtctgaggac gaggccgatt actattgcgc agcatgggac 1860
gattccctga ggggaccagt gttcggcggc ggaaccaggg tgacagtgct gagaaccgtg 1920
gcagcaccaa gcgtgttcat ctttcctcca tccgatgagc agctgaagag cggcacagcc 1980
tccgtggtgt gcctgctgaa caacttctac cccagggagg ccaaggtgca gtggaaggtg 2040
gacaacgccc tgcagtctgg caatagccag gagtccgtga ccgagcagga ctctaaggat 2100
agcacatatt ccctgagctc caccctgaca ctgtccaagg ccgactacga gaagcacaag 2160
gtgtatgcct gcgaagtcac ccaccagggg ctgaggtctc cagtcactaa atctttcaat 2220
aggggcgaat gctgataa 2238
<210> 10
<211> 744
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 10
Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly
1 5 10 15
Thr His Ala Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Glu
20 25 30
Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe
35 40 45
Gly Ser Tyr Ala Gly Gly Thr Phe Gly Ser Tyr Ala Ile Asn Trp Val
50 55 60
Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met Gly Trp Ile Asp Ala
65 70 75 80
Ala Asn Gly Asn Thr Lys Tyr Ser Gln Lys Phe Gln Gly Arg Val Thr
85 90 95
Ile Thr Gly Asp Thr Ser Ala Ser Thr Ala Tyr Met Glu Leu Ser Ser
100 105 110
Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Arg Trp
115 120 125
Met Thr Thr Arg Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr
130 135 140
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
145 150 155 160
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
165 170 175
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
180 185 190
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
195 200 205
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
210 215 220
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys
225 230 235 240
Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys
245 250 255
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
260 265 270
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
275 280 285
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
290 295 300
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
305 310 315 320
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
325 330 335
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
340 345 350
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
355 360 365
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
370 375 380
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
385 390 395 400
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
405 410 415
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
420 425 430
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
435 440 445
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
450 455 460
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Arg Gly Arg
465 470 475 480
Lys Arg Arg Ser Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln
485 490 495
Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Val Leu Gln Thr Gln
500 505 510
Val Phe Ile Ser Leu Leu Leu Trp Ile Ser Gly Ala Tyr Gly Gln Pro
515 520 525
Val Leu Thr Gln Pro Pro Ala Ser Ala Ser Gly Thr Pro Gly Gln Arg
530 535 540
Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Tyr
545 550 555 560
Val Phe Trp Tyr Gln Gln Leu Pro Gly Met Ala Pro Lys Leu Leu Ile
565 570 575
Ser Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly
580 585 590
Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Pro Gln Ser
595 600 605
Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu Arg
610 615 620
Gly Pro Val Phe Gly Gly Gly Thr Arg Val Thr Val Leu Arg Thr Val
625 630 635 640
Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys
645 650 655
Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg
660 665 670
Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
675 680 685
Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser
690 695 700
Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
705 710 715 720
Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Arg Ser Pro Val Thr
725 730 735
Lys Ser Phe Asn Arg Gly Glu Cys
740
<210> 11
<211> 372
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 11
caggtgcagc tggtgcagtc tggggccgaa gtgaagaagc caggcagctc cgtgaaggtg 60
tcctgcaagg cctctggcgg cacattctct agctacgcca tctcctgggt gaggcaggca 120
ccaggacagg gcctggagtg ggtgggcaga atcatcccta tcttcggcac cgccaactac 180
gcccagtttc agggccgggt gaccatcaca gccgacaagt ccacctctac agcctatatg 240
gagctgtcct ctctgaggag cgaggatacc gccgtgtact attgcgccag agccagctac 300
tgctccacca catcttgtgc aagcggagcc ttcgacatct ggggacaggg caccctggtg 360
acagtgagct cc 372
<210> 12
<211> 124
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 12
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr
20 25 30
Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Val
35 40 45
Gly Arg Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Phe Gln
50 55 60
Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met
65 70 75 80
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Ala Ser Tyr Cys Ser Thr Thr Ser Cys Ala Ser Gly Ala Phe Asp
100 105 110
Ile Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 13
<211> 339
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 13
gacatccaga tgacccagtc tcctgatagc ctggccgtgt ccctgggaga gagagcaaca 60
atcaactgta agtctagcca gagcgtgctg tactcctcta acaataagaa ttacctggcc 120
tggtatcagc agaagccagg ccagccaccc aagctgctga tctattgggc aagcgccagg 180
gagtccggag tgccagacag attcagcggc tccggctctg gaaccgactt caccctgaca 240
atcagctccc tgcagcccga ggacgtggcc atctactatt gccagcagta ctattccgtg 300
cccttcacct ttggccctgg cacaaaggtg gagatcaag 339
<210> 14
<211> 113
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 14
Asp Ile Gln Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser
20 25 30
Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Ala Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Pro Glu Asp Val Ala Ile Tyr Tyr Cys Gln Gln
85 90 95
Tyr Tyr Ser Val Pro Phe Thr Phe Gly Pro Gly Thr Lys Val Glu Ile
100 105 110
Lys
<210> 15
<211> 2232
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 15
atggattgga catggaggat tctgtttctg gtcgccgccg ccacaggaac tcacgctcag 60
gtgcagctgg tgcagtctgg ggccgaagtg aagaagccag gcagctccgt gaaggtgtcc 120
tgcaaggcct ctggcggcac attctctagc tacgccatct cctgggtgag gcaggcacca 180
ggacagggcc tggagtgggt gggcagaatc atccctatct tcggcaccgc caactacgcc 240
cagtttcagg gccgggtgac catcacagcc gacaagtcca cctctacagc ctatatggag 300
ctgtcctctc tgaggagcga ggataccgcc gtgtactatt gcgccagagc cagctactgc 360
tccaccacat cttgtgcaag cggagccttc gacatctggg gacagggcac cctggtgaca 420
gtgagctccg cctccacaaa gggaccaagc gtgttcccac tggcaccctc tagcaagagc 480
acctccggcg gcacagccgc cctgggctgt ctggtgaagg attatttccc cgagcctgtg 540
accgtgtcct ggaactctgg cgccctgacc tccggagtgc acacatttcc cgccgtgctg 600
cagtcctctg gcctgtacag cctgagctcc gtggtgaccg tgccttctag ctccctgggc 660
acccagacat atatctgcaa cgtgaatcac aagcctagca atacaaaggt ggacaagaag 720
gtggagccaa agtcctgtga taagacccac acatgccctc cctgtccagc accagagctg 780
ctgggcggcc caagcgtgtt cctgtttcca cccaagccca aggacacact gatgatctct 840
cgcacccctg aggtgacatg cgtggtggtg gacgtgagcc acgaggaccc cgaggtgaag 900
tttaactggt acgtggatgg cgtggaggtg cacaatgcca agaccaagcc tcgggaggag 960
cagtacaaca gcacctatcg cgtggtgtcc gtgctgacag tgctgcacca ggactggctg 1020
aacggcaagg agtataagtg caaggtgtcc aataaggccc tgcctgcccc aatcgagaag 1080
accatctcta aggcaaaggg acagcctcgg gagccacagg tgtacacact gcctccatcc 1140
cgcgacgagc tgaccaagaa ccaggtgtct ctgacatgtc tggtgaaggg cttctatcca 1200
tctgatatcg ccgtggagtg ggagagcaat ggccagcccg agaacaatta caagaccaca 1260
ccccctgtgc tggacagcga tggctccttc tttctgtatt ccaagctgac cgtggacaag 1320
tctaggtggc agcagggcaa cgtgttttct tgcagcgtga tgcacgaggc cctgcacaat 1380
cactacaccc agaagtccct gtctctgagc ccaggcaaga ggggaaggaa gaggagatcc 1440
ggctctggcg ccacaaactt cagcctgctg aagcaggcag gcgatgtgga ggagaatcca 1500
ggacctatgg tgctgcagac ccaggtgttt atctctctgc tgctgtggat cagcggcgcc 1560
tacggcgaca tccagatgac ccagtctcct gatagcctgg ccgtgtccct gggagagaga 1620
gcaacaatca actgtaagtc tagccagagc gtgctgtact cctctaacaa taagaattac 1680
ctggcctggt atcagcagaa gccaggccag ccacccaagc tgctgatcta ttgggcaagc 1740
gccagggagt ccggagtgcc agacagattc agcggctccg gctctggaac cgacttcacc 1800
ctgacaatca gctccctgca gcccgaggac gtggccatct actattgcca gcagtactat 1860
tccgtgccct tcacctttgg ccctggcaca aaggtggaga tcaagaggac cgtggcagca 1920
cctagcgtgt tcatctttcc tccatccgac gagcagctga agtctggcac agccagcgtg 1980
gtgtgcctgc tgaacaattt ctacccacgc gaggccaagg tgcagtggaa ggtggataac 2040
gccctgcagt ccggcaattc tcaggagagc gtgaccgagc aggactccaa ggattctaca 2100
tatagcctgt ctagcaccct gacactgagc aaggccgatt acgagaagca caaggtgtat 2160
gcctgcgaag tcacccacca ggggctgagg tcaccagtca ccaagtcttt caacagaggg 2220
gaatgttgat aa 2232
<210> 16
<211> 742
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 16
Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly
1 5 10 15
Thr His Ala Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys
20 25 30
Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe
35 40 45
Ser Ser Tyr Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
50 55 60
Glu Trp Val Gly Arg Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala
65 70 75 80
Gln Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr
85 90 95
Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr
100 105 110
Tyr Cys Ala Arg Ala Ser Tyr Cys Ser Thr Thr Ser Cys Ala Ser Gly
115 120 125
Ala Phe Asp Ile Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala
130 135 140
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser
145 150 155 160
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe
165 170 175
Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly
180 185 190
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu
195 200 205
Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr
210 215 220
Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys
225 230 235 240
Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
245 250 255
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
260 265 270
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
275 280 285
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
290 295 300
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
305 310 315 320
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
325 330 335
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
340 345 350
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
355 360 365
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
370 375 380
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
385 390 395 400
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
405 410 415
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
420 425 430
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
435 440 445
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
450 455 460
Lys Ser Leu Ser Leu Ser Pro Gly Lys Arg Gly Arg Lys Arg Arg Ser
465 470 475 480
Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val
485 490 495
Glu Glu Asn Pro Gly Pro Met Val Leu Gln Thr Gln Val Phe Ile Ser
500 505 510
Leu Leu Leu Trp Ile Ser Gly Ala Tyr Gly Asp Ile Gln Met Thr Gln
515 520 525
Ser Pro Asp Ser Leu Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn
530 535 540
Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr
545 550 555 560
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile
565 570 575
Tyr Trp Ala Ser Ala Arg Glu Ser Gly Val Pro Asp Arg Phe Ser Gly
580 585 590
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
595 600 605
Glu Asp Val Ala Ile Tyr Tyr Cys Gln Gln Tyr Tyr Ser Val Pro Phe
610 615 620
Thr Phe Gly Pro Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
625 630 635 640
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
645 650 655
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
660 665 670
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
675 680 685
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
690 695 700
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
705 710 715 720
Ala Cys Glu Val Thr His Gln Gly Leu Arg Ser Pro Val Thr Lys Ser
725 730 735
Phe Asn Arg Gly Glu Cys
740
<210> 17
<211> 375
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 17
caggtgcagc tggtgcagtc tggggctgaa gtgaagaaac caggcagctc cgtcaaggtg 60
tcatgcaaag ccagcggcgg aacattctct agttacgcaa tcagctgggt gcgacaggct 120
ccaggacagg gactggagtg gatgggcggc atcattccta tcttcgggac cgctaactac 180
gcacagaagt ttcagggccg cgtgaccatt acagcagata aatccacttc taccgcctat 240
atggagctgt caagcctgag gagcgaagac actgcagtct actattgcgc cagagtggga 300
tactgctcct ctacctcctg tcacatcggg gccttcgata tttggggaca ggggaccaca 360
gtcaccgtga gttca 375
<210> 18
<211> 125
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 18
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr
20 25 30
Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Val Gly Tyr Cys Ser Ser Thr Ser Cys His Ile Gly Ala Phe
100 105 110
Asp Ile Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
115 120 125
<210> 19
<211> 321
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 19
gaaacaactc tgactcagag ccccggcacc ctgtctctga gtcctggaga gcgagctacc 60
ctgagctgta gggcatcaca gagcgtgtca agctccatcg cctggtatca gcagaagcct 120
ggacaggctc cacgcctgct gatgttcgac tctagtacaa gagctactgg catccccgat 180
cggttctccg ggtctggcag tggaaccgac tttacactga acatttcaag cctggagccc 240
gaagatttcg cagtgtacta ttgccagcag tactcctcta gtccttatac atttgggcag 300
ggcactaagc tggaaatcaa a 321
<210> 20
<211> 107
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 20
Glu Thr Thr Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser
20 25 30
Ile Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Met
35 40 45
Phe Asp Ser Ser Thr Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile Ser Ser Leu Glu Pro
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Ser Ser Ser Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 21
<211> 2216
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 21
atggattgga catggagaat tctgtttctg gtcgccgccg ccactggaac tcacgctcag 60
gtgcagctgg tgcagtctgg ggctgaagtg aagaaaccag gcagctccgt caaggtgtca 120
tgcaaagcca gcggcggaac attctctagt tacgcaatca gctgggtgcg acaggctcca 180
ggacagggac tggagtggat gggcggcatc attcctatct tcgggaccgc taactacgca 240
cagaagtttc agggccgcgt gaccattaca gcagataaat ccacttctac cgcctatatg 300
gagctgtcaa gcctgaggag cgaagacact gcagtctact attgcgccag agtgggatac 360
tgctcctcta cctcctgtca catcggggcc ttcgatattt ggggacaggg gaccacagtc 420
accgtgagtt cagcaagtac aaaggggcct tcagtgtttc ccctggcccc tagctccaaa 480
agtacttcag gagggaccgc cgctctggga tgtctggtga aggactattt ccctgagcca 540
gtcaccgtgt catggaacag cggggccctg acctccggag tccatacatt tccagctgtg 600
ctgcagtcta gtgggctgta ctctctgtca agcgtggtca ctgtcccctc ctctagtctg 660
ggcacacaga cttatatctg caacgtgaat cacaagcctt ccaataccaa agtcgacaag 720
aaagtggaac caaagtcttg tgataaaacc catacatgcc ctccctgtcc agcacctgag 780
ctgctgggcg gaccaagcgt gttcctgttt ccacccaagc ccaaagatac actgatgatt 840
agccggacac ctgaagtcac ttgcgtggtc gtggacgtga gccacgagga ccccgaagtc 900
aagttcaact ggtacgtgga cggcgtcgag gtgcataatg ctaagaccaa accccgcgag 960
gaacagtaca atagcacata tcgagtcgtg tccgtcctga ctgtgctgca ccaggactgg 1020
ctgaacggaa aggagtataa gtgcaaagtg tccaataagg ccctgccagc tcccatcgag 1080
aaaacaattt ctaaggccaa aggccagcca cgggaacccc aggtgtacac tctgcctcca 1140
agccgcgatg agctgactaa gaaccaggtc agcctgacct gtctggtgaa aggcttctat 1200
ccaagtgaca tcgctgtgga gtgggaatct aatggacagc ccgaaaacaa ttacaagact 1260
acccctcccg tgctggactc cgatggctct ttctttctgt attcaaagct gacagtggat 1320
aaaagccggt ggcagcaggg aaacgtcttt agctgctccg tgatgcatga ggccctgcac 1380
aatcattaca cacagaagtc tctgagtctg tcacccggaa agcgaggacg aaaaaggaga 1440
agcggctccg gagctactaa cttctccctg ctgaagcagg caggagacgt ggaggaaaat 1500
cctgggccaa tggtcctgca gacccaggtg tttatctctc tgctgctgtg gattagtggc 1560
gcttacggag aaacaactct gactcagagc cccggcaccc tgtctctgag tcctggagag 1620
cgagctaccc tgagctgtag ggcatcacag agcgtgtcaa gctccatcgc ctggtatcag 1680
cagaagcctg gacaggctcc acgcctgctg atgttcgact ctagtacaag agctactggc 1740
atccccgatc ggttctccgg gtctggcagt ggaaccgact ttacactgaa catttcaagc 1800
ctggagcccg aagatttcgc agtgtactat tgccagcagt actcctctag tccttataca 1860
tttgggcagg gcactaagct ggaaatcaaa aggaccgtcg cagccccttc cgtgttcatt 1920
tttccaccct ctgatgagca gctgaagtcc ggcacagcct ctgtggtgtg cctgctgaac 1980
aacttctacc caagagaagc aaaggtccag tggaaagtgg acaacgccct gcagagtggc 2040
aattcacagg agagcgtgac cgaacaggac tccaaggatt ctacatatag tctgtcaagc 2100
actctgaccc tagcaaagct gactacgaga agcacaaagt gtatgcatgc gaagtgaccc 2160
accaggggct gagaagtcca gtgacaaaat cctttaacag aggggaatgc tgataa 2216
<210> 22
<211> 737
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 22
Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly
1 5 10 15
Thr His Ala Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys
20 25 30
Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe
35 40 45
Ser Ser Tyr Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
50 55 60
Glu Trp Met Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala
65 70 75 80
Gln Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser
85 90 95
Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
100 105 110
Tyr Tyr Cys Ala Arg Val Gly Tyr Cys Ser Ser Thr Ser Cys His Ile
115 120 125
Gly Ala Phe Asp Ile Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
130 135 140
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
145 150 155 160
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
165 170 175
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
180 185 190
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
195 200 205
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
210 215 220
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
225 230 235 240
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
245 250 255
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
260 265 270
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
275 280 285
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
290 295 300
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
305 310 315 320
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
325 330 335
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
340 345 350
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
355 360 365
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
370 375 380
Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
385 390 395 400
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
405 410 415
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
420 425 430
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
435 440 445
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
450 455 460
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Arg Gly Arg Lys Arg Arg
465 470 475 480
Ser Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp
485 490 495
Val Glu Glu Asn Pro Gly Pro Met Val Leu Gln Thr Gln Val Phe Ile
500 505 510
Ser Leu Leu Leu Trp Ile Ser Gly Ala Tyr Gly Glu Thr Thr Leu Thr
515 520 525
Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu
530 535 540
Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser Ile Ala Trp Tyr Gln
545 550 555 560
Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Met Phe Asp Ser Ser Thr
565 570 575
Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr
580 585 590
Asp Phe Thr Leu Asn Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val
595 600 605
Tyr Tyr Cys Gln Gln Tyr Ser Ser Ser Pro Tyr Thr Phe Gly Gln Gly
610 615 620
Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile
625 630 635 640
Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val
645 650 655
Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys
660 665 670
Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu
675 680 685
Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu
690 695 700
Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr
705 710 715 720
His Gln Gly Leu Arg Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu
725 730 735
Cys
<210> 23
<211> 15
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 23
tcctacggaa tgcac 15
<210> 24
<211> 48
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 24
atctcttatg acggcagcaa caagtactat gccgatagcg tgaagggc 48
<210> 25
<211> 60
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 25
gacggatatg gcagcggctc cgattactac tactactact acatggacgt gtggggcaag 60
<210> 26
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 26
Ser Tyr Gly Met His
1 5
<210> 27
<211> 16
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 27
Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys Gly
1 5 10 15
<210> 28
<211> 20
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 28
Asp Gly Tyr Gly Ser Gly Ser Asp Tyr Tyr Tyr Tyr Tyr Tyr Met Asp
1 5 10 15
Val Trp Gly Lys
20
<210> 29
<211> 399
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 29
gaggtgcagc tggtcgagac tggagggggc gtggtgcagc caggccggag cctgagactg 60
agctgcgcag catccggctt cacctttagc tcctacggaa tgcactgggt gaggcaggca 120
ccaggcaagg gcctggagtg ggtggccgtg atctcttatg acggcagcaa caagtactat 180
gccgatagcg tgaagggcag gttcaccatc tcccgcgaca actctaagaa tacactgtac 240
ctgcagatga attccctgag ggccgaggat accgccgtgt actattgcgc ccgcgacgga 300
tatggcagcg gctccgatta ctactactac tactacatgg acgtgtgggg caagggcacc 360
atggtgacag tgtctagctc cgccagcgga gcagagctg 399
<210> 30
<211> 133
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 30
Glu Val Gln Leu Val Glu Thr Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asp Gly Tyr Gly Ser Gly Ser Asp Tyr Tyr Tyr Tyr Tyr Tyr
100 105 110
Met Asp Val Trp Gly Lys Gly Thr Met Val Thr Val Ser Ser Ser Ala
115 120 125
Ser Gly Ala Glu Leu
130
<210> 31
<211> 30
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 31
cagggcgata gcctgagatc ctactatgcc 30
<210> 32
<211> 21
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 32
ggcaagaaca atcggccctc c 21
<210> 33
<211> 30
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 33
aactccaggg attctagcgg caatcaccct 30
<210> 34
<211> 10
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 34
Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala
1 5 10
<210> 35
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 35
Gly Lys Asn Asn Arg Pro Ser
1 5
<210> 36
<211> 10
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 36
Asn Ser Arg Asp Ser Ser Gly Asn His Pro
1 5 10
<210> 37
<211> 324
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 37
acctctagct ccgagctgac acaggaccct gccgtgagcg tggccctggg acagaccgtg 60
cggatcacat gtcagggcga tagcctgaga tcctactatg cctcctggta ccagcagaag 120
ccaggacagg caccagtgcc tgtgatctat ggcaagaaca atcggccctc cggcatccct 180
gacagattct ctggctctag ctccggcaac accgcaagcc tgaccatcac aggagcacag 240
gcagaggacg aggcagatta ctattgcaac tccagggatt ctagcggcaa tcaccctttt 300
ggcaccggca caaaggtgac cctg 324
<210> 38
<211> 108
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 38
Thr Ser Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu
1 5 10 15
Gly Gln Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr
20 25 30
Tyr Ala Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Pro Val
35 40 45
Ile Tyr Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser
50 55 60
Gly Ser Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln
65 70 75 80
Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp Ser Ser Gly
85 90 95
Asn His Pro Phe Gly Thr Gly Thr Lys Val Thr Leu
100 105
<210> 39
<211> 2244
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 39
atggactgga cttggaggat tctgtttctg gtcgccgccg ctactgggac tcacgccgag 60
gtgcagctgg tcgagactgg agggggcgtg gtgcagccag gccggagcct gagactgagc 120
tgcgcagcat ccggcttcac ctttagctcc tacggaatgc actgggtgag gcaggcacca 180
ggcaagggcc tggagtgggt ggccgtgatc tcttatgacg gcagcaacaa gtactatgcc 240
gatagcgtga agggcaggtt caccatctcc cgcgacaact ctaagaatac actgtacctg 300
cagatgaatt ccctgagggc cgaggatacc gccgtgtact attgcgcccg cgacggatat 360
ggcagcggct ccgattacta ctactactac tacatggacg tgtggggcaa gggcaccatg 420
gtgacagtgt ctagctccgc cagcggagca gagctggcat ctacaaaggg accaagcgtg 480
tttccactgg caccctctag caagtctacc agcggcggca cagccgccct gggatgtctg 540
gtgaaggatt acttccctga gccagtgacc gtgtcttgga acagcggcgc cctgaccagc 600
ggagtgcaca catttcccgc cgtgctgcag tcctctggcc tgtactccct gagctccgtg 660
gtgaccgtgc cttctagctc cctgggcacc cagacatata tctgcaacgt gaatcacaag 720
cctagcaata caaaggtgga caagaaggtg gagccaaagt cctgtgataa gacccacaca 780
tgccctccct gtccagcacc tgagctgctg ggcggcccat ccgtgttcct gtttccaccc 840
aagcccaagg acacactgat gatctctcgg accccagagg tgacatgcgt ggtggtggac 900
gtgagccacg aggaccccga ggtgaagttc aactggtacg tggatggcgt ggaggtgcac 960
aatgccaaga ccaagcccag ggaggagcag tacaactcca cctatcgcgt ggtgtctgtg 1020
ctgacagtgc tgcaccagga ctggctgaac ggcaaggagt ataagtgcaa ggtgtccaat 1080
aaggccctgc cagcccccat cgagaagacc atctctaagg caaagggaca gcctcgggag 1140
ccacaggtgt acacactgcc tccatccaga gacgagctga ccaagaacca ggtgtctctg 1200
acatgtctgg tgaagggctt ctatccctct gatatcgccg tggagtggga gagcaatggc 1260
cagcctgaga acaattacaa gaccacaccc cctgtgctgg actccgatgg ctctttcttt 1320
ctgtattcta agctgaccgt ggacaagagc agatggcagc agggcaacgt gttttcctgc 1380
tctgtgatgc acgaggccct gcacaatcac tacacccaga agagcctgtc cctgtctcct 1440
ggcaagaggg gaaggaagcg gagaagcggc tccggagcca caaacttcag cctgctgaag 1500
caggccggcg atgtggagga gaatcctggc ccaatggtgc tgcagaccca ggtgtttatc 1560
agcctgctgc tgtggatctc cggagcatac ggcacctcta gctccgagct gacacaggac 1620
cctgccgtga gcgtggccct gggacagacc gtgcggatca catgtcaggg cgatagcctg 1680
agatcctact atgcctcctg gtaccagcag aagccaggac aggcaccagt gcctgtgatc 1740
tatggcaaga acaatcggcc ctccggcatc cctgacagat tctctggctc tagctccggc 1800
aacaccgcaa gcctgaccat cacaggagca caggcagagg acgaggcaga ttactattgc 1860
aactccaggg attctagcgg caatcaccct tttggcaccg gcacaaaggt gaccctgagg 1920
acagtggcag caccaagcgt gttcatcttt ccaccctccg acgagcagct gaagagcgga 1980
accgcatccg tggtgtgcct gctgaacaac ttctaccccc gggaggccaa ggtgcagtgg 2040
aaggtggata acgccctgca gtctggcaat agccaggagt ccgtgaccga gcaggactct 2100
aaggatagca catattccct gtcctctacc ctgacactga gcaaggccga ctacgagaag 2160
cacaaggtgt atgcctgcga agtcacccac caggggctgc ggtcaccagt cacaaaatcc 2220
tttaatagag gcgaatgttg ataa 2244
<210> 40
<211> 746
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 40
Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly
1 5 10 15
Thr His Ala Glu Val Gln Leu Val Glu Thr Gly Gly Gly Val Val Gln
20 25 30
Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
35 40 45
Ser Ser Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
50 55 60
Glu Trp Val Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala
65 70 75 80
Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
85 90 95
Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
100 105 110
Tyr Tyr Cys Ala Arg Asp Gly Tyr Gly Ser Gly Ser Asp Tyr Tyr Tyr
115 120 125
Tyr Tyr Tyr Met Asp Val Trp Gly Lys Gly Thr Met Val Thr Val Ser
130 135 140
Ser Ser Ala Ser Gly Ala Glu Leu Ala Ser Thr Lys Gly Pro Ser Val
145 150 155 160
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
165 170 175
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
180 185 190
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
195 200 205
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
210 215 220
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
225 230 235 240
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
245 250 255
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
260 265 270
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
275 280 285
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
290 295 300
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
305 310 315 320
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
325 330 335
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
340 345 350
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
355 360 365
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
370 375 380
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
385 390 395 400
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
405 410 415
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
420 425 430
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
435 440 445
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
450 455 460
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
465 470 475 480
Gly Lys Arg Gly Arg Lys Arg Arg Ser Gly Ser Gly Ala Thr Asn Phe
485 490 495
Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met
500 505 510
Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser Gly
515 520 525
Ala Tyr Gly Thr Ser Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser
530 535 540
Val Ala Leu Gly Gln Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu
545 550 555 560
Arg Ser Tyr Tyr Ala Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
565 570 575
Val Pro Val Ile Tyr Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp
580 585 590
Arg Phe Ser Gly Ser Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr
595 600 605
Gly Ala Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp
610 615 620
Ser Ser Gly Asn His Pro Phe Gly Thr Gly Thr Lys Val Thr Leu Arg
625 630 635 640
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
645 650 655
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
660 665 670
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
675 680 685
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
690 695 700
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
705 710 715 720
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Arg Ser Pro
725 730 735
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
740 745
<210> 41
<211> 1425
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 41
atggactgga cctggagaat cctgttcctg gtggcagcag caaccggaac acacgcacag 60
gtgcagctgg tgcagtccgg agcagaggtg aagaagcctg gagcctctgt gaaggtgagc 120
tgcaaggcct ccggctacac ctttacatct tatggaatca gctgggtgag gcaggcacca 180
ggacagggac tggagtggat gggctggatc agcgcctaca acggcaatac aaactatgcc 240
cagaagctgc agggcagagt gaccatgacc acagacacca gcacatccac cgcctacatg 300
gagctgaggt ctctgagaag cgacgataca gccgtgtact attgcgcccg ggactatacc 360
cgcggcgcct ggttcggaga gtctctgatc ggcggatttg ataattgggg ccagggcaca 420
ctggtgaccg tgtctgccag cacaaaggga ccaagcgtgt tcccactggc acccagctcc 480
aagtccacat ctggcggcac cgccgccctg ggatgtctgg tgaaggatta cttcccagag 540
cccgtgaccg tgtcctggaa ttctggcgcc ctgacaagcg gcgtgcacac ctttccagcc 600
gtgctgcagt ctagcggcct gtactccctg tcctctgtgg tgacagtgcc cagctcctct 660
ctgggcacac agacctatat ctgcaatgtg aaccacaagc caagcaacac caaggtggac 720
aagaaggtgg agcccaagtc ctgtgataag acacacacct gccctccctg tcctgcacca 780
gagctgctgg gcggcccatc cgtgttcctg tttccaccca agcctaagga caccctgatg 840
atctctcgga cacccgaggt gacctgcgtg gtggtggacg tgagccacga ggaccccgag 900
gtgaagttta attggtacgt ggatggcgtg gaggtgcaca acgccaagac caagcccagg 960
gaggagcagt acaactccac atatagagtg gtgtctgtgc tgaccgtgct gcaccaggac 1020
tggctgaatg gcaaggagta taagtgcaag gtgtccaaca aggccctgcc cgcccctatc 1080
gagaagacaa tctctaaggc aaagggacag cctcgggagc cacaggtgta caccctgcct 1140
ccatcccgcg acgagctgac aaagaatcag gtgtctctga cctgtctggt gaagggcttc 1200
tatccttctg atatcgcagt ggagtgggag agcaacggac agccagagaa caattacaag 1260
accacacccc ctgtgctgga cagcgatggc tccttctttc tgtatagcaa gctgacagtg 1320
gataagtccc gctggcagca gggcaacgtg ttcagctgtt ccgtgatgca cgaggccctg 1380
cacaaccact acacccagaa gtctctgagc ctgtcccctg gcaag 1425
<210> 42
<211> 475
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 42
Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly
1 5 10 15
Thr His Ala Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys
20 25 30
Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe
35 40 45
Thr Ser Tyr Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
50 55 60
Glu Trp Met Gly Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala
65 70 75 80
Gln Lys Leu Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser
85 90 95
Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val
100 105 110
Tyr Tyr Cys Ala Arg Asp Tyr Thr Arg Gly Ala Trp Phe Gly Glu Ser
115 120 125
Leu Ile Gly Gly Phe Asp Asn Trp Gly Gln Gly Thr Leu Val Thr Val
130 135 140
Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser
145 150 155 160
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
165 170 175
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
180 185 190
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
195 200 205
Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln
210 215 220
Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
225 230 235 240
Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro
245 250 255
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
260 265 270
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
275 280 285
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
290 295 300
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
305 310 315 320
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
325 330 335
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
340 345 350
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
355 360 365
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
370 375 380
Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
385 390 395 400
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
405 410 415
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
420 425 430
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
435 440 445
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
450 455 460
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
465 470 475
<210> 43
<211> 699
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 43
atggtgctgc agacccaggt gttcatctct ctgctgctgt ggatcagcgg agcatacgga 60
gagatcgtgc tgacccagag ccctggcaca ctgagcctgt ccccaggaga gagggccaca 120
ctgtcctgca gagcctctca gagcgtgagc tcctcttacc tggcctggta tcagcagaag 180
ccaggacagg cccccaggct gctgatctat ggagccagct cccgggccac cggcatcccc 240
gaccgcttct ccggctctgg cagcggcaca gacttcaccc tgacaatctc taggctggag 300
cctgaggact tcgccgtgta ctattgccag cagcacgata ccagcctgac atttggccag 360
ggcaccaagg tggagatcaa gagaacagtg gccgccccat ccgtgttcat ctttccccct 420
tctgacgagc agctgaagag cggaaccgca tccgtggtgt gcctgctgaa caatttctac 480
ccccgggagg ccaaggtgca gtggaaggtg gataacgccc tgcagtccgg caattctcag 540
gagagcgtga ccgagcagga caaggattcc acatattctc tgtctagcac cctgacactg 600
tccaaggccg actacgagaa gcacaaggtg tatgcatgcg aggtgaccca ccagggactg 660
tcctctcccg tgacaaagag ctttaaccgc ggcgagtgt 699
<210> 44
<211> 233
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 44
Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser
1 5 10 15
Gly Ala Tyr Gly Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser
20 25 30
Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser
35 40 45
Val Ser Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala
50 55 60
Pro Arg Leu Leu Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro
65 70 75 80
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
85 90 95
Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln His
100 105 110
Asp Thr Ser Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
115 120 125
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
130 135 140
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
145 150 155 160
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
165 170 175
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Lys Asp Ser Thr Tyr
180 185 190
Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His
195 200 205
Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val
210 215 220
Thr Lys Ser Phe Asn Arg Gly Glu Cys
225 230
<210> 45
<211> 1425
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 45
atggactgga cctggagaat cctgttcctg gtggcagcag caaccggaac acacgcacag 60
gtgcagctgg tgcagtccgg agcagaggtg aagaagcctg gagcctctgt gaaggtgagc 120
tgcaaggcct ccggctacac ctttacatct tatggaatca gctgggtgag gcaggcacca 180
ggacagggac tggagtggat gggctggatc agcgcctaca acggcaatac aaactatgcc 240
cagaagctgc agggcagagt gaccatgacc acagacacca gcacatccac cgcctacatg 300
gagctgaggt ctctgagaag cgacgataca gccgtgtact attgcgcccg ggactatacc 360
cgcggcgcct ggttcggaga gtctctgatc ggcggatttg ataattgggg ccagggcaca 420
ctggtgaccg tgtctgccag cacaaaggga ccaagcgtgt tcccactggc acccagctcc 480
aagtccacat ctggcggcac cgccgccctg ggatgtctgg tgaaggatta cttcccagag 540
cccgtgaccg tgtcctggaa ttctggcgcc ctgacaagcg gcgtgcacac ctttccagcc 600
gtgctgcagt ctagcggcct gtactccctg tcctctgtgg tgacagtgcc cagctcctct 660
ctgggcacac agacctatat ctgcaatgtg aaccacaagc caagcaacac caaggtggac 720
aagaaggtgg agcccaagtc ctgtgataag acacacacct gccctccctg tcctgcacca 780
gagctgctgg gcggcccatc cgtgttcctg tttccaccca agcctaagga caccctgatg 840
atctctcgga cacccgaggt gacctgcgtg gtggtggacg tgagccacga ggaccccgag 900
gtgaagttta attggtacgt ggatggcgtg gaggtgcaca acgccaagac caagcccagg 960
gaggagcagt acaactccac atatagagtg gtgtctgtgc tgaccgtgct gcaccaggac 1020
tggctgaatg gcaaggagta taagtgcaag gtgtccaaca aggccctgcc cgcccctatc 1080
gagaagacaa tctctaaggc aaagggacag cctcgggagc cacaggtgta caccctgcct 1140
ccatcccgcg acgagctgac aaagaatcag gtgtctctga cctgtctggt gaagggcttc 1200
tatccttctg atatcgcagt ggagtgggag agcaacggac agccagagaa caattacaag 1260
accacacccc ctgtgctgga cagcgatggc tccttctttc tgtatagcaa gctgacagtg 1320
gataagtccc gctggcagca gggcaacgtg ttcagctgtt ccgtgatgca cgaggccctg 1380
cacaaccact acacccagaa gtctctgagc ctgtcccctg gcaag 1425
<210> 46
<211> 748
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 46
Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly
1 5 10 15
Thr His Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys
20 25 30
Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
35 40 45
Arg Asp Val Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
50 55 60
Glu Trp Val Gly Arg Ile Lys Ser Lys Ile Asp Gly Gly Thr Thr Asp
65 70 75 80
Tyr Ala Ala Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser
85 90 95
Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr
100 105 110
Ala Val Tyr Tyr Cys Thr Thr Ala Gly Ser Tyr Tyr Tyr Asp Thr Val
115 120 125
Gly Pro Gly Leu Pro Glu Gly Lys Phe Asp Tyr Trp Gly Gln Gly Thr
130 135 140
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
145 150 155 160
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
165 170 175
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
180 185 190
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
195 200 205
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
210 215 220
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
225 230 235 240
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
245 250 255
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
260 265 270
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
275 280 285
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
290 295 300
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
305 310 315 320
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
325 330 335
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
340 345 350
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
355 360 365
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
370 375 380
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
385 390 395 400
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
405 410 415
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
420 425 430
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
435 440 445
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
450 455 460
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
465 470 475 480
Arg Gly Arg Lys Arg Arg Ser Gly Ser Gly Ala Thr Asn Phe Ser Leu
485 490 495
Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Val Leu
500 505 510
Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser Gly Ala Tyr
515 520 525
Gly Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu
530 535 540
Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr
545 550 555 560
Ser Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly
565 570 575
Gln Pro Pro Lys Leu Leu Met Tyr Trp Ala Ser Thr Arg Glu Ser Gly
580 585 590
Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Ala Glu Phe Thr Leu
595 600 605
Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Ile Tyr Tyr Cys Gln
610 615 620
Gln Tyr Tyr Ser Thr Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile
625 630 635 640
Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
645 650 655
Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
660 665 670
Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
675 680 685
Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
690 695 700
Ser Thr Tyr Ser Leu Ser Asn Thr Leu Thr Leu Ser Lys Ala Asp Tyr
705 710 715 720
Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
725 730 735
Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
740 745
<210> 47
<211> 2244
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 47
atggactgga catggagaat cctgttcctg gtcgccgccg caactgggac tcacgccgaa 60
gtgcagctgg tcgaatctgg gggaggcctg gtgaagccag gcggcagcct gaggctgtcc 120
tgcgcagcat ctggcttcac ctttagggac gtgtggatgt cctgggtgag acaggcacca 180
ggcaagggcc tggagtgggt gggacgcatc aagagcaaga tcgacggcgg aaccacagat 240
tatgccgccc ccgtgaaggg caggttcacc atcagcagag acgattccaa gaacacactg 300
tacctgcaga tgaattccct gaagaccgag gatacagccg tgtactattg caccacagcc 360
ggctcttact attacgacac agtgggacca ggcctgccag agggcaagtt cgattattgg 420
ggccagggca ccctggtgac agtgagctcc gccagcacca agggcccttc cgtgtttcct 480
ctggccccat ctagcaagtc taccagcggc ggcacagccg ccctgggatg tctggtgaag 540
gactacttcc ccgagcctgt gaccgtgtcc tggaactctg gcgccctgac ctccggagtg 600
cacacatttc ctgccgtgct gcagtcctct ggcctgtata gcctgagctc cgtggtgacc 660
gtgccatcta gctccctggg cacccagaca tacatctgca acgtgaatca caagccttct 720
aatacaaagg tggacaagaa ggtggagcca aagagctgtg ataagaccca cacatgccct 780
ccctgtccag caccagagct gctgggcggc ccatccgtgt tcctgtttcc acccaagccc 840
aaggacacac tgatgatctc ccggacccca gaggtgacat gcgtggtggt ggacgtgtct 900
cacgaggacc ccgaggtgaa gttcaactgg tacgtggatg gcgtggaggt gcacaatgcc 960
aagaccaagc cccgggagga gcagtataac tctacctacc gcgtggtgag cgtgctgaca 1020
gtgctgcacc aggattggct gaacggcaag gagtacaagt gcaaggtgag caataaggcc 1080
ctgcctgccc caatcgagaa gaccatctcc aaggcaaagg gacagcccag ggagcctcag 1140
gtgtatacac tgcctccaag cagagacgag ctgaccaaga accaggtgtc cctgacatgt 1200
ctggtgaagg gcttctaccc ctccgatatc gccgtggagt gggagtctaa tggccagcct 1260
gagaacaatt ataagaccac accccctgtg ctggacagcg atggctcctt ctttctgtac 1320
agcaagctga ccgtggacaa gtcccggtgg cagcagggca acgtgttttc ctgctctgtg 1380
atgcacggcg ccctgcacaa tcactacacc cagaagagcc tgtccctgtc tccaggcaag 1440
aggggaagga agaggagaag cggctccggc gccacaaact tcagcctgct gaagcaggca 1500
ggcgatgtgg aggagaatcc aggacctatg gtgctgcaga cccaggtgtt tatctccctg 1560
ctgctgtgga tctctggcgc ctacggcgac atcgtgatga cccagtctcc tgatagcctg 1620
gccgtgtctc tgggagagag ggcaacaatc aactgtaagt ctagccagag cgtgctgtat 1680
tcctctaaca ataagaatta tctggcatgg taccagcaga agccaggaca gccacccaag 1740
ctgctgatgt actgggcatc tacccgggag agcggagtgc ctgaccgctt ctctggcagc 1800
ggctccggag cagagtttac cctgacaatc agctccctgc aggccgagga tgtggccatc 1860
tattactgcc agcagtatta cagcaccctg accttcggcg gcggcaccaa ggtggagatc 1920
aagaggacag tggccgcccc tagcgtgttc atctttcctc caagcgacga gcagctgaag 1980
tccggcaccg cctctgtggt gtgcctgctg aacaacttct acccaagaga ggccaaggtg 2040
cagtggaagg tggataacgc cctgcagtcc ggcaattctc aggagagcgt gaccgagcag 2100
gactccaagg attctacata ctctctgagc aacaccctga cactgagcaa ggccgactat 2160
gagaagcaca aggtgtacgc ctgcgaagtg acccaccagg ggctgagcag tccagtgacc 2220
aagtctttca atcggggaga atgc 2244
<210> 48
<211> 748
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 48
Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly
1 5 10 15
Thr His Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys
20 25 30
Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
35 40 45
Arg Asp Val Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
50 55 60
Glu Trp Val Gly Arg Ile Lys Ser Lys Ile Asp Gly Gly Thr Thr Asp
65 70 75 80
Tyr Ala Ala Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser
85 90 95
Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr
100 105 110
Ala Val Tyr Tyr Cys Thr Thr Ala Gly Ser Tyr Tyr Tyr Asp Thr Val
115 120 125
Gly Pro Gly Leu Pro Glu Gly Lys Phe Asp Tyr Trp Gly Gln Gly Thr
130 135 140
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
145 150 155 160
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
165 170 175
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
180 185 190
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
195 200 205
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
210 215 220
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
225 230 235 240
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
245 250 255
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
260 265 270
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
275 280 285
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
290 295 300
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
305 310 315 320
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
325 330 335
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
340 345 350
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
355 360 365
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
370 375 380
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
385 390 395 400
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
405 410 415
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
420 425 430
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
435 440 445
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Gly Ala
450 455 460
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
465 470 475 480
Arg Gly Arg Lys Arg Arg Ser Gly Ser Gly Ala Thr Asn Phe Ser Leu
485 490 495
Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Val Leu
500 505 510
Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser Gly Ala Tyr
515 520 525
Gly Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu
530 535 540
Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr
545 550 555 560
Ser Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly
565 570 575
Gln Pro Pro Lys Leu Leu Met Tyr Trp Ala Ser Thr Arg Glu Ser Gly
580 585 590
Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Ala Glu Phe Thr Leu
595 600 605
Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Ile Tyr Tyr Cys Gln
610 615 620
Gln Tyr Tyr Ser Thr Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile
625 630 635 640
Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
645 650 655
Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
660 665 670
Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
675 680 685
Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
690 695 700
Ser Thr Tyr Ser Leu Ser Asn Thr Leu Thr Leu Ser Lys Ala Asp Tyr
705 710 715 720
Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
725 730 735
Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
740 745
<210> 49
<211> 2244
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 49
atggactgga catggagaat cctgttcctg gtcgccgccg caactgggac tcacgccgaa 60
gtgcagctgg tcgaatctgg gggaggcctg gtgaagccag gcggcagcct gaggctgtcc 120
tgcgcagcat ctggcttcac ctttagggac gtgtggatgt cctgggtgag acaggcacca 180
ggcaagggcc tggagtgggt gggacgcatc aagagcaaga tcgacggcgg aaccacagat 240
tatgccgccc ccgtgaaggg caggttcacc atcagcagag acgattccaa gaacacactg 300
tacctgcaga tgaattccct gaagaccgag gatacagccg tgtactattg caccacagcc 360
ggctcttact attacgacac agtgggacca ggcctgccag agggcaagtt cgattattgg 420
ggccagggca ccctggtgac agtgagctcc gccagcacca agggcccttc cgtgtttcct 480
ctggccccat ctagcaagtc taccagcggc ggcacagccg ccctgggatg tctggtgaag 540
gactacttcc ccgagcctgt gaccgtgtcc tggaactctg gcgccctgac ctccggagtg 600
cacacatttc ctgccgtgct gcagtcctct ggcctgtata gcctgagctc cgtggtgacc 660
gtgccatcta gctccctggg cacccagaca tacatctgca acgtgaatca caagccttct 720
aatacaaagg tggacaagaa ggtggagcca aagagctgtg ataagaccca cacatgccct 780
ccctgtccag caccagagct gctgggcggc ccatccgtgt tcctgtttcc acccaagccc 840
aaggacacac tgatgatctc ccggacccca gaggtgacat gcgtggtggt ggacgtgtct 900
cacgaggacc ccgaggtgaa gttcaactgg tacgtggatg gcgtggaggt gcacaatgcc 960
aagaccaagc cccgggagga gcagtataac tctacctacc gcgtggtgag cgtgctgaca 1020
gtgctgcacc aggattggct gaacggcaag gagtacaagt gcaaggtgag caataaggcc 1080
ctgcctgccc caatcgagaa gaccatctcc aaggcaaagg gacagcccag gaagcctcag 1140
gtgtatacac tgcctccaag cagagacgag ctgaccaaga accaggtgtc cctgacatgt 1200
ctggtgaagg gcttctaccc ctccgatatc gccgtggagt gggagtctaa tggccagcct 1260
gagaacaatt ataagaccac accccctgtg ctggacagcg atggctcctt ctttctgtac 1320
agcaagctga ccgtggacaa gtcccggtgg cagcagggca acgtgttttc ctgctctgtg 1380
atgcacggcg ccctgcacaa tcactacacc cagaagagcc tgtccctgtc tccaggcaag 1440
aggggaagga agaggagaag cggctccggc gccacaaact tcagcctgct gaagcaggca 1500
ggcgatgtgg aggagaatcc aggacctatg gtgctgcaga cccaggtgtt tatctccctg 1560
ctgctgtgga tctctggcgc ctacggcgac atcgtgatga cccagtctcc tgatagcctg 1620
gccgtgtctc tgggagagag ggcaacaatc aactgtaagt ctagccagag cgtgctgtat 1680
tcctctaaca ataagaatta tctggcatgg taccagcaga agccaggaca gccacccaag 1740
ctgctgatgt actgggcatc tacccgggag agcggagtgc ctgaccgctt ctctggcagc 1800
ggctccggag cagagtttac cctgacaatc agctccctgc aggccgagga tgtggccatc 1860
tattactgcc agcagtatta cagcaccctg accttcggcg gcggcaccaa ggtggagatc 1920
aagaggacag tggccgcccc tagcgtgttc atctttcctc caagcgacga gcagctgaag 1980
tccggcaccg cctctgtggt gtgcctgctg aacaacttct acccaagaga ggccaaggtg 2040
cagtggaagg tggataacgc cctgcagtcc ggcaattctc aggagagcgt gaccgagcag 2100
gactccaagg attctacata ctctctgagc aacaccctga cactgagcaa ggccgactat 2160
gagaagcaca aggtgtacgc ctgcgaagtg acccaccagg ggctgagcag tccagtgacc 2220
aagtctttca atcggggaga atgc 2244
<210> 50
<211> 748
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 50
Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly
1 5 10 15
Thr His Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys
20 25 30
Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
35 40 45
Arg Asp Val Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
50 55 60
Glu Trp Val Gly Arg Ile Lys Ser Lys Ile Asp Gly Gly Thr Thr Asp
65 70 75 80
Tyr Ala Ala Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser
85 90 95
Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr
100 105 110
Ala Val Tyr Tyr Cys Thr Thr Ala Gly Ser Tyr Tyr Tyr Asp Thr Val
115 120 125
Gly Pro Gly Leu Pro Glu Gly Lys Phe Asp Tyr Trp Gly Gln Gly Thr
130 135 140
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
145 150 155 160
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
165 170 175
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
180 185 190
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
195 200 205
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
210 215 220
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
225 230 235 240
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
245 250 255
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
260 265 270
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
275 280 285
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
290 295 300
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
305 310 315 320
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
325 330 335
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
340 345 350
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
355 360 365
Ile Ser Lys Ala Lys Gly Gln Pro Arg Lys Pro Gln Val Tyr Thr Leu
370 375 380
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
385 390 395 400
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
405 410 415
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
420 425 430
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
435 440 445
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Gly Ala
450 455 460
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
465 470 475 480
Arg Gly Arg Lys Arg Arg Ser Gly Ser Gly Ala Thr Asn Phe Ser Leu
485 490 495
Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Val Leu
500 505 510
Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser Gly Ala Tyr
515 520 525
Gly Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu
530 535 540
Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr
545 550 555 560
Ser Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly
565 570 575
Gln Pro Pro Lys Leu Leu Met Tyr Trp Ala Ser Thr Arg Glu Ser Gly
580 585 590
Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Ala Glu Phe Thr Leu
595 600 605
Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Ile Tyr Tyr Cys Gln
610 615 620
Gln Tyr Tyr Ser Thr Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile
625 630 635 640
Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
645 650 655
Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
660 665 670
Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
675 680 685
Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
690 695 700
Ser Thr Tyr Ser Leu Ser Asn Thr Leu Thr Leu Ser Lys Ala Asp Tyr
705 710 715 720
Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
725 730 735
Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
740 745
<210> 51
<211> 2244
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 51
atggactgga catggagaat cctgttcctg gtcgccgccg caactgggac tcacgccgaa 60
gtgcagctgg tcgaatctgg gggaggcctg gtgaagccag gcggcagcct gaggctgtcc 120
tgcgcagcat ctggcttcac ctttagggac gtgtggatgt cctgggtgag acaggcacca 180
ggcaagggcc tggagtgggt gggacgcatc aagagcaaga tcgacggcgg aaccacagat 240
tatgccgccc ccgtgaaggg caggttcacc atcagcagag acgattccaa gaacacactg 300
tacctgcaga tgaattccct gaagaccgag gatacagccg tgtactattg caccacagcc 360
ggctcttact attacgacac agtgggacca ggcctgccag agggcaagtt cgattattgg 420
ggccagggca ccctggtgac agtgagctcc gccagcacca agggcccttc cgtgtttcct 480
ctggccccat ctagcaagtc taccagcggc ggcacagccg ccctgggatg tctggtgaag 540
gactacttcc ccgagcctgt gaccgtgtcc tggaactctg gcgccctgac ctccggagtg 600
cacacatttc ctgccgtgct gcagtcctct ggcctgtata gcctgagctc cgtggtgacc 660
gtgccatcta gctccctggg cacccagaca tacatctgca acgtgaatca caagccttct 720
aatacaaagg tggacaagaa ggtggagcca aagagctgtg ataagaccca cacatgccct 780
ccctgtccag caccagagtt cgagggcggc ccatccgtgt tcctgtttcc acccaagccc 840
aaggacacac tgatgatctc ccggacccca gaggtgacat gcgtggtggt ggacgtgtct 900
cacgaggacc ccgaggtgaa gttcaactgg tacgtggatg gcgtggaggt gcacaatgcc 960
aagaccaagc cccgggagga gcagtataac tctacctacc gcgtggtgag cgtgctgaca 1020
gtgctgcacc aggattggct gaacggcaag gagtacaagt gcaaggtgag caataaggcc 1080
ctgcctgcca gcatcgagaa gaccatctcc aaggcaaagg gacagcccag ggagcctcag 1140
gtgtatacac tgcctccaag cagagacgag ctgaccaaga accaggtgtc cctgacatgt 1200
ctggtgaagg gcttctaccc ctccgatatc gccgtggagt gggagtctaa tggccagcct 1260
gagaacaatt ataagaccac accccctgtg ctggacagcg atggctcctt ctttctgtac 1320
agcaagctga ccgtggacaa gtcccggtgg cagcagggca acgtgttttc ctgctctgtg 1380
atgcacgagg ccctgcacaa tcactacacc cagaagagcc tgtccctgtc tccaggcaag 1440
aggggaagga agaggagaag cggctccggc gccacaaact tcagcctgct gaagcaggca 1500
ggcgatgtgg aggagaatcc aggacctatg gtgctgcaga cccaggtgtt tatctccctg 1560
ctgctgtgga tctctggcgc ctacggcgac atcgtgatga cccagtctcc tgatagcctg 1620
gccgtgtctc tgggagagag ggcaacaatc aactgtaagt ctagccagag cgtgctgtat 1680
tcctctaaca ataagaatta tctggcatgg taccagcaga agccaggaca gccacccaag 1740
ctgctgatgt actgggcatc tacccgggag agcggagtgc ctgaccgctt ctctggcagc 1800
ggctccggag cagagtttac cctgacaatc agctccctgc aggccgagga tgtggccatc 1860
tattactgcc agcagtatta cagcaccctg accttcggcg gcggcaccaa ggtggagatc 1920
aagaggacag tggccgcccc tagcgtgttc atctttcctc caagcgacga gcagctgaag 1980
tccggcaccg cctctgtggt gtgcctgctg aacaacttct acccaagaga ggccaaggtg 2040
cagtggaagg tggataacgc cctgcagtcc ggcaattctc aggagagcgt gaccgagcag 2100
gactccaagg attctacata ctctctgagc aacaccctga cactgagcaa ggccgactat 2160
gagaagcaca aggtgtacgc ctgcgaagtg acccaccagg ggctgagcag tccagtgacc 2220
aagtctttca atcggggaga atgc 2244
<210> 52
<211> 748
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 52
Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly
1 5 10 15
Thr His Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys
20 25 30
Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
35 40 45
Arg Asp Val Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
50 55 60
Glu Trp Val Gly Arg Ile Lys Ser Lys Ile Asp Gly Gly Thr Thr Asp
65 70 75 80
Tyr Ala Ala Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser
85 90 95
Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr
100 105 110
Ala Val Tyr Tyr Cys Thr Thr Ala Gly Ser Tyr Tyr Tyr Asp Thr Val
115 120 125
Gly Pro Gly Leu Pro Glu Gly Lys Phe Asp Tyr Trp Gly Gln Gly Thr
130 135 140
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
145 150 155 160
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
165 170 175
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
180 185 190
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
195 200 205
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
210 215 220
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
225 230 235 240
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
245 250 255
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser
260 265 270
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
275 280 285
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
290 295 300
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
305 310 315 320
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
325 330 335
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
340 345 350
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Ser Ile Glu Lys Thr
355 360 365
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
370 375 380
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
385 390 395 400
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
405 410 415
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
420 425 430
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
435 440 445
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
450 455 460
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
465 470 475 480
Arg Gly Arg Lys Arg Arg Ser Gly Ser Gly Ala Thr Asn Phe Ser Leu
485 490 495
Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Val Leu
500 505 510
Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser Gly Ala Tyr
515 520 525
Gly Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu
530 535 540
Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr
545 550 555 560
Ser Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly
565 570 575
Gln Pro Pro Lys Leu Leu Met Tyr Trp Ala Ser Thr Arg Glu Ser Gly
580 585 590
Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Ala Glu Phe Thr Leu
595 600 605
Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Ile Tyr Tyr Cys Gln
610 615 620
Gln Tyr Tyr Ser Thr Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile
625 630 635 640
Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
645 650 655
Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
660 665 670
Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
675 680 685
Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
690 695 700
Ser Thr Tyr Ser Leu Ser Asn Thr Leu Thr Leu Ser Lys Ala Asp Tyr
705 710 715 720
Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
725 730 735
Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
740 745
<210> 53
<211> 2211
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 53
atggactgga cttggagaat cctgtttctg gtcgccgccg ctactggaac ccacgctcag 60
atgcagctgg tgcagagtgg acccgaagtg aagaagcccg gcaccagcgt gaaggtgtcc 120
tgtaaggcct ctggcttcac ctttacaagc tccgccgtgc agtgggtgag gcaggccaga 180
ggccagcggc tggagtggat cggatggatc gcagtgggct ccggaaacac caattacgcc 240
cagaagtttc aggagcgcgt gtctatcaca agggacatgt ctaccagcac agcctatatg 300
gagctgtcta gcctgagatc cgaggatacc gccgtgtact attgcgccgc cccttactgt 360
tcccggacat cttgccacga cgcctttgat atctggggcc agggcaccaa ggtgacagtg 420
tcctctgcct ctacaaaggg accaagcgtg ttcccactgg cacctagctc caagtccacc 480
tctggcggca cagccgccct gggctgtctg gtgaaggact actttcctga gccagtgacc 540
gtgagctgga actccggcgc cctgacctcc ggagtgcaca cattccctgc cgtgctgcag 600
tctagcggcc tgtacagcct gtcctctgtg gtgaccgtgc caagctcctc tctgggcacc 660
cagacatata tctgtaacgt gaatcacaag ccttccaata caaaggtgga caagaaggtg 720
gagccaaagt cttgcgataa gacccacaca tgccctccct gtccagcacc tgagctgctg 780
ggcggcccaa gcgtgttcct gtttccaccc aagcccaagg acaccctgat gatcagccgg 840
accccagagg tgacatgcgt ggtggtggac gtgtcccacg aggaccccga ggtgaagttc 900
aactggtacg tggatggcgt ggaggtgcac aatgccaaga ccaagcccag agaggagcag 960
tacaactcca cctatcgggt ggtgtctgtg ctgacagtgc tgcaccagga ctggctgaac 1020
ggcaaggagt acaagtgtaa ggtgtctaat aaggccctgc cagcccccat cgagaagacc 1080
atcagcaagg caaagggaca gccacgcgag ccacaggtgt atacactgcc tccatctagg 1140
gacgagctga ccaagaacca ggtgagcctg acatgcctgg tgaagggctt ttaccccagc 1200
gatatcgccg tggagtggga gtccaatggc cagcctgaga acaattacaa gaccacaccc 1260
cctgtgctgg actctgatgg cagcttcttt ctgtattcta agctgaccgt ggacaagagc 1320
cgctggcagc agggcaacgt gttcagctgt tccgtgatgc acgaggccct gcacaatcac 1380
tatacccaga agtctctgag cctgtcccct ggcaagaggg gaaggaagag gagatctggc 1440
agcggcgcca caaactttag cctgctgaag caggccggcg atgtggagga gaatcctggc 1500
ccaatggtgc tgcagaccca ggtgttcatc agcctgctgc tgtggatctc cggagcatac 1560
ggagaggtgg tgctgaccca gtccccaggc acactgtccc tgtctcctgg agagagagcc 1620
accctgagct gcagggcaag ccagtccgtg agctcctctt acctggcctg gtatcagcag 1680
aagcctggac aggccccacg cctgctgatc tatggagcca gctcccgcgc caccggcatc 1740
cccgacaggt tttctggcag cggctccggc acagatttca ccctgacaat ctccaggctg 1800
gagcctgagg acttcgccgt gtaccactgt cagcactttg gctctagctc ccagtggacc 1860
ttcggccagg gcacaaaggt ggagatcaag agaaccgtgg ccgccccatc cgtgttcatc 1920
tttccaccct ctgacgagca gctgaagagc ggcacagcct ccgtggtgtg cctgctgaac 1980
aatttctatc ccagggaggc caaggtgcag tggaaggtgg ataacgccct gcagtccggc 2040
aattctcagg agagcgtgac cgagcaggac tccaaggatt ctacatactc tctgagcaac 2100
accctgacac tgagcaaggc cgattacgag aagcacaagg tgtatgcctg cgaggtcact 2160
caccaggggc tgtccagtcc cgtcactaag tctttcaata gaggcgaatg t 2211
<210> 54
<211> 737
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 54
Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly
1 5 10 15
Thr His Ala Gln Met Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys
20 25 30
Pro Gly Thr Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe
35 40 45
Thr Ser Ser Ala Val Gln Trp Val Arg Gln Ala Arg Gly Gln Arg Leu
50 55 60
Glu Trp Ile Gly Trp Ile Ala Val Gly Ser Gly Asn Thr Asn Tyr Ala
65 70 75 80
Gln Lys Phe Gln Glu Arg Val Ser Ile Thr Arg Asp Met Ser Thr Ser
85 90 95
Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
100 105 110
Tyr Tyr Cys Ala Ala Pro Tyr Cys Ser Arg Thr Ser Cys His Asp Ala
115 120 125
Phe Asp Ile Trp Gly Gln Gly Thr Lys Val Thr Val Ser Ser Ala Ser
130 135 140
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
145 150 155 160
Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
165 170 175
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
180 185 190
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
195 200 205
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
210 215 220
Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val
225 230 235 240
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
245 250 255
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
260 265 270
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
275 280 285
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
290 295 300
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
305 310 315 320
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
325 330 335
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
340 345 350
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
355 360 365
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
370 375 380
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
385 390 395 400
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
405 410 415
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
420 425 430
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
435 440 445
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
450 455 460
Ser Leu Ser Leu Ser Pro Gly Lys Arg Gly Arg Lys Arg Arg Ser Gly
465 470 475 480
Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu
485 490 495
Glu Asn Pro Gly Pro Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu
500 505 510
Leu Leu Trp Ile Ser Gly Ala Tyr Gly Glu Val Val Leu Thr Gln Ser
515 520 525
Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys
530 535 540
Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala Trp Tyr Gln Gln
545 550 555 560
Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Gly Ala Ser Ser Arg
565 570 575
Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
580 585 590
Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr
595 600 605
His Cys Gln His Phe Gly Ser Ser Ser Gln Trp Thr Phe Gly Gln Gly
610 615 620
Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile
625 630 635 640
Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val
645 650 655
Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys
660 665 670
Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu
675 680 685
Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Asn Thr Leu Thr Leu
690 695 700
Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr
705 710 715 720
His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu
725 730 735
Cys
<210> 55
<211> 2211
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 55
atggactgga cttggagaat cctgtttctg gtcgccgccg ctactggaac ccacgctcag 60
atgcagctgg tgcagagtgg acccgaagtg aagaagcccg gcaccagcgt gaaggtgtcc 120
tgtaaggcct ctggcttcac ctttacaagc tccgccgtgc agtgggtgag gcaggccaga 180
ggccagcggc tggagtggat cggatggatc gcagtgggct ccggaaacac caattacgcc 240
cagaagtttc aggagcgcgt gtctatcaca agggacatgt ctaccagcac agcctatatg 300
gagctgtcta gcctgagatc cgaggatacc gccgtgtact attgcgccgc cccttactgt 360
tcccggacat cttgccacga cgcctttgat atctggggcc agggcaccaa ggtgacagtg 420
tcctctgcct ctacaaaggg accaagcgtg ttcccactgg cacctagctc caagtccacc 480
tctggcggca cagccgccct gggctgtctg gtgaaggact actttcctga gccagtgacc 540
gtgagctgga actccggcgc cctgacctcc ggagtgcaca cattccctgc cgtgctgcag 600
tctagcggcc tgtacagcct gtcctctgtg gtgaccgtgc caagctcctc tctgggcacc 660
cagacatata tctgtaacgt gaatcacaag ccttccaata caaaggtgga caagaaggtg 720
gagccaaagt cttgcgataa gacccacaca tgccctccct gtccagcacc tgagctgctg 780
ggcggcccaa gcgtgttcct gtttccaccc aagcccaagg acaccctgat gatcagccgg 840
accccagagg tgacatgcgt ggtggtggac gtgtcccacg aggaccccga ggtgaagttc 900
aactggtacg tggatggcgt ggaggtgcac aatgccaaga ccaagcccag agaggagcag 960
tacaactcca cctatcgggt ggtgtctgtg ctgacagtgc tgcaccagga ctggctgaac 1020
ggcaaggagt acaagtgtaa ggtgtctaat aaggccctgc cagcccccat cgagaagacc 1080
atcagcaagg caaagggaca gccacgcgag ccacaggtgt atacactgcc tccatctagg 1140
gacgagctga ccaagaacca ggtgagcctg acatgcctgg tgaagggctt ttaccccagc 1200
gatatcgccg tggagtggga gtccaatggc cagcctgaga acaattacaa gaccacaccc 1260
cctgtgctgg actctgatgg cagcttcttt ctgtattcta agctgaccgt ggacaagagc 1320
cgctggcagc agggcaacgt gttcagctgt tccgtgatgc acggcgccct gcacaatcac 1380
tatacccaga agtctctgag cctgtcccct ggcaagaggg gaaggaagag gagatctggc 1440
agcggcgcca caaactttag cctgctgaag caggccggcg atgtggagga gaatcctggc 1500
ccaatggtgc tgcagaccca ggtgttcatc agcctgctgc tgtggatctc cggagcatac 1560
ggagaggtgg tgctgaccca gtccccaggc acactgtccc tgtctcctgg agagagagcc 1620
accctgagct gcagggcaag ccagtccgtg agctcctctt acctggcctg gtatcagcag 1680
aagcctggac aggccccacg cctgctgatc tatggagcca gctcccgcgc caccggcatc 1740
cccgacaggt tttctggcag cggctccggc acagatttca ccctgacaat ctccaggctg 1800
gagcctgagg acttcgccgt gtaccactgt cagcactttg gctctagctc ccagtggacc 1860
ttcggccagg gcacaaaggt ggagatcaag agaaccgtgg ccgccccatc cgtgttcatc 1920
tttccaccct ctgacgagca gctgaagagc ggcacagcct ccgtggtgtg cctgctgaac 1980
aatttctatc ccagggaggc caaggtgcag tggaaggtgg ataacgccct gcagtccggc 2040
aattctcagg agagcgtgac cgagcaggac tccaaggatt ctacatactc tctgagcaac 2100
accctgacac tgagcaaggc cgattacgag aagcacaagg tgtatgcctg cgaggtcact 2160
caccaggggc tgtccagtcc cgtcactaag tctttcaata gaggcgaatg t 2211
<210> 56
<211> 737
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 56
Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly
1 5 10 15
Thr His Ala Gln Met Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys
20 25 30
Pro Gly Thr Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe
35 40 45
Thr Ser Ser Ala Val Gln Trp Val Arg Gln Ala Arg Gly Gln Arg Leu
50 55 60
Glu Trp Ile Gly Trp Ile Ala Val Gly Ser Gly Asn Thr Asn Tyr Ala
65 70 75 80
Gln Lys Phe Gln Glu Arg Val Ser Ile Thr Arg Asp Met Ser Thr Ser
85 90 95
Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
100 105 110
Tyr Tyr Cys Ala Ala Pro Tyr Cys Ser Arg Thr Ser Cys His Asp Ala
115 120 125
Phe Asp Ile Trp Gly Gln Gly Thr Lys Val Thr Val Ser Ser Ala Ser
130 135 140
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
145 150 155 160
Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
165 170 175
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
180 185 190
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
195 200 205
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
210 215 220
Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val
225 230 235 240
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
245 250 255
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
260 265 270
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
275 280 285
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
290 295 300
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
305 310 315 320
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
325 330 335
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
340 345 350
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
355 360 365
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
370 375 380
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
385 390 395 400
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
405 410 415
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
420 425 430
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
435 440 445
Ser Cys Ser Val Met His Gly Ala Leu His Asn His Tyr Thr Gln Lys
450 455 460
Ser Leu Ser Leu Ser Pro Gly Lys Arg Gly Arg Lys Arg Arg Ser Gly
465 470 475 480
Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu
485 490 495
Glu Asn Pro Gly Pro Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu
500 505 510
Leu Leu Trp Ile Ser Gly Ala Tyr Gly Glu Val Val Leu Thr Gln Ser
515 520 525
Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys
530 535 540
Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala Trp Tyr Gln Gln
545 550 555 560
Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Gly Ala Ser Ser Arg
565 570 575
Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
580 585 590
Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr
595 600 605
His Cys Gln His Phe Gly Ser Ser Ser Gln Trp Thr Phe Gly Gln Gly
610 615 620
Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile
625 630 635 640
Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val
645 650 655
Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys
660 665 670
Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu
675 680 685
Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Asn Thr Leu Thr Leu
690 695 700
Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr
705 710 715 720
His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu
725 730 735
Cys
<210> 57
<211> 2211
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 57
atggactgga cttggagaat cctgtttctg gtcgccgccg ctactggaac ccacgctcag 60
atgcagctgg tgcagagtgg acccgaagtg aagaagcccg gcaccagcgt gaaggtgtcc 120
tgtaaggcct ctggcttcac ctttacaagc tccgccgtgc agtgggtgag gcaggccaga 180
ggccagcggc tggagtggat cggatggatc gcagtgggct ccggaaacac caattacgcc 240
cagaagtttc aggagcgcgt gtctatcaca agggacatgt ctaccagcac agcctatatg 300
gagctgtcta gcctgagatc cgaggatacc gccgtgtact attgcgccgc cccttactgt 360
tcccggacat cttgccacga cgcctttgat atctggggcc agggcaccaa ggtgacagtg 420
tcctctgcct ctacaaaggg accaagcgtg ttcccactgg cacctagctc caagtccacc 480
tctggcggca cagccgccct gggctgtctg gtgaaggact actttcctga gccagtgacc 540
gtgagctgga actccggcgc cctgacctcc ggagtgcaca cattccctgc cgtgctgcag 600
tctagcggcc tgtacagcct gtcctctgtg gtgaccgtgc caagctcctc tctgggcacc 660
cagacatata tctgtaacgt gaatcacaag ccttccaata caaaggtgga caagaaggtg 720
gagccaaagt cttgcgataa gacccacaca tgccctccct gtccagcacc tgagctgctg 780
ggcggcccaa gcgtgttcct gtttccaccc aagcccaagg acaccctgat gatcagccgg 840
accccagagg tgacatgcgt ggtggtggac gtgtcccacg aggaccccga ggtgaagttc 900
aactggtacg tggatggcgt ggaggtgcac aatgccaaga ccaagcccag agaggagcag 960
tacaactcca cctatcgggt ggtgtctgtg ctgacagtgc tgcaccagga ctggctgaac 1020
ggcaaggagt acaagtgtaa ggtgtctaat aaggccctgc cagcccccat cgagaagacc 1080
atcagcaagg caaagggaca gccacgcaag ccacaggtgt atacactgcc tccatctagg 1140
gacgagctga ccaagaacca ggtgagcctg acatgcctgg tgaagggctt ttaccccagc 1200
gatatcgccg tggagtggga gtccaatggc cagcctgaga acaattacaa gaccacaccc 1260
cctgtgctgg actctgatgg cagcttcttt ctgtattcta agctgaccgt ggacaagagc 1320
cgctggcagc agggcaacgt gttcagctgt tccgtgatgc acggcgccct gcacaatcac 1380
tatacccaga agtctctgag cctgtcccct ggcaagaggg gaaggaagag gagatctggc 1440
agcggcgcca caaactttag cctgctgaag caggccggcg atgtggagga gaatcctggc 1500
ccaatggtgc tgcagaccca ggtgttcatc agcctgctgc tgtggatctc cggagcatac 1560
ggagaggtgg tgctgaccca gtccccaggc acactgtccc tgtctcctgg agagagagcc 1620
accctgagct gcagggcaag ccagtccgtg agctcctctt acctggcctg gtatcagcag 1680
aagcctggac aggccccacg cctgctgatc tatggagcca gctcccgcgc caccggcatc 1740
cccgacaggt tttctggcag cggctccggc acagatttca ccctgacaat ctccaggctg 1800
gagcctgagg acttcgccgt gtaccactgt cagcactttg gctctagctc ccagtggacc 1860
ttcggccagg gcacaaaggt ggagatcaag agaaccgtgg ccgccccatc cgtgttcatc 1920
tttccaccct ctgacgagca gctgaagagc ggcacagcct ccgtggtgtg cctgctgaac 1980
aatttctatc ccagggaggc caaggtgcag tggaaggtgg ataacgccct gcagtccggc 2040
aattctcagg agagcgtgac cgagcaggac tccaaggatt ctacatactc tctgagcaac 2100
accctgacac tgagcaaggc cgattacgag aagcacaagg tgtatgcctg cgaggtcact 2160
caccaggggc tgtccagtcc cgtcactaag tctttcaata gaggcgaatg t 2211
<210> 58
<211> 737
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 58
Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly
1 5 10 15
Thr His Ala Gln Met Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys
20 25 30
Pro Gly Thr Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe
35 40 45
Thr Ser Ser Ala Val Gln Trp Val Arg Gln Ala Arg Gly Gln Arg Leu
50 55 60
Glu Trp Ile Gly Trp Ile Ala Val Gly Ser Gly Asn Thr Asn Tyr Ala
65 70 75 80
Gln Lys Phe Gln Glu Arg Val Ser Ile Thr Arg Asp Met Ser Thr Ser
85 90 95
Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
100 105 110
Tyr Tyr Cys Ala Ala Pro Tyr Cys Ser Arg Thr Ser Cys His Asp Ala
115 120 125
Phe Asp Ile Trp Gly Gln Gly Thr Lys Val Thr Val Ser Ser Ala Ser
130 135 140
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
145 150 155 160
Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
165 170 175
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
180 185 190
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
195 200 205
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
210 215 220
Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val
225 230 235 240
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
245 250 255
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
260 265 270
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
275 280 285
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
290 295 300
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
305 310 315 320
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
325 330 335
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
340 345 350
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
355 360 365
Arg Lys Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
370 375 380
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
385 390 395 400
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
405 410 415
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
420 425 430
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
435 440 445
Ser Cys Ser Val Met His Gly Ala Leu His Asn His Tyr Thr Gln Lys
450 455 460
Ser Leu Ser Leu Ser Pro Gly Lys Arg Gly Arg Lys Arg Arg Ser Gly
465 470 475 480
Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu
485 490 495
Glu Asn Pro Gly Pro Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu
500 505 510
Leu Leu Trp Ile Ser Gly Ala Tyr Gly Glu Val Val Leu Thr Gln Ser
515 520 525
Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys
530 535 540
Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala Trp Tyr Gln Gln
545 550 555 560
Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Gly Ala Ser Ser Arg
565 570 575
Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
580 585 590
Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr
595 600 605
His Cys Gln His Phe Gly Ser Ser Ser Gln Trp Thr Phe Gly Gln Gly
610 615 620
Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile
625 630 635 640
Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val
645 650 655
Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys
660 665 670
Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu
675 680 685
Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Asn Thr Leu Thr Leu
690 695 700
Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr
705 710 715 720
His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu
725 730 735
Cys
<210> 59
<211> 2211
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 59
atggactgga cttggagaat cctgtttctg gtcgccgccg ctactggaac ccacgctcag 60
atgcagctgg tgcagagtgg acccgaagtg aagaagcccg gcaccagcgt gaaggtgtcc 120
tgtaaggcct ctggcttcac ctttacaagc tccgccgtgc agtgggtgag gcaggccaga 180
ggccagcggc tggagtggat cggatggatc gcagtgggct ccggaaacac caattacgcc 240
cagaagtttc aggagcgcgt gtctatcaca agggacatgt ctaccagcac agcctatatg 300
gagctgtcta gcctgagatc cgaggatacc gccgtgtact attgcgccgc cccttactgt 360
tcccggacat cttgccacga cgcctttgat atctggggcc agggcaccaa ggtgacagtg 420
tcctctgcct ctacaaaggg accaagcgtg ttcccactgg cacctagctc caagtccacc 480
tctggcggca cagccgccct gggctgtctg gtgaaggact actttcctga gccagtgacc 540
gtgagctgga actccggcgc cctgacctcc ggagtgcaca cattccctgc cgtgctgcag 600
tctagcggcc tgtacagcct gtcctctgtg gtgaccgtgc caagctcctc tctgggcacc 660
cagacatata tctgtaacgt gaatcacaag ccttccaata caaaggtgga caagaaggtg 720
gagccaaagt cttgcgataa gacccacaca tgccctccct gtccagcacc tgagttcgag 780
ggcggcccaa gcgtgttcct gtttccaccc aagcccaagg acaccctgat gatcagccgg 840
accccagagg tgacatgcgt ggtggtggac gtgtcccacg aggaccccga ggtgaagttc 900
aactggtacg tggatggcgt ggaggtgcac aatgccaaga ccaagcccag agaggagcag 960
tacaactcca cctatcgggt ggtgtctgtg ctgacagtgc tgcaccagga ctggctgaac 1020
ggcaaggagt acaagtgtaa ggtgtctaat aaggccctgc cagccagcat cgagaagacc 1080
atcagcaagg caaagggaca gccacgcgag ccacaggtgt atacactgcc tccatctagg 1140
gacgagctga ccaagaacca ggtgagcctg acatgcctgg tgaagggctt ttaccccagc 1200
gatatcgccg tggagtggga gtccaatggc cagcctgaga acaattacaa gaccacaccc 1260
cctgtgctgg actctgatgg cagcttcttt ctgtattcta agctgaccgt ggacaagagc 1320
cgctggcagc agggcaacgt gttcagctgt tccgtgatgc acgaggccct gcacaatcac 1380
tatacccaga agtctctgag cctgtcccct ggcaagaggg gaaggaagag gagatctggc 1440
agcggcgcca caaactttag cctgctgaag caggccggcg atgtggagga gaatcctggc 1500
ccaatggtgc tgcagaccca ggtgttcatc agcctgctgc tgtggatctc cggagcatac 1560
ggagaggtgg tgctgaccca gtccccaggc acactgtccc tgtctcctgg agagagagcc 1620
accctgagct gcagggcaag ccagtccgtg agctcctctt acctggcctg gtatcagcag 1680
aagcctggac aggccccacg cctgctgatc tatggagcca gctcccgcgc caccggcatc 1740
cccgacaggt tttctggcag cggctccggc acagatttca ccctgacaat ctccaggctg 1800
gagcctgagg acttcgccgt gtaccactgt cagcactttg gctctagctc ccagtggacc 1860
ttcggccagg gcacaaaggt ggagatcaag agaaccgtgg ccgccccatc cgtgttcatc 1920
tttccaccct ctgacgagca gctgaagagc ggcacagcct ccgtggtgtg cctgctgaac 1980
aatttctatc ccagggaggc caaggtgcag tggaaggtgg ataacgccct gcagtccggc 2040
aattctcagg agagcgtgac cgagcaggac tccaaggatt ctacatactc tctgagcaac 2100
accctgacac tgagcaaggc cgattacgag aagcacaagg tgtatgcctg cgaggtcact 2160
caccaggggc tgtccagtcc cgtcactaag tctttcaata gaggcgaatg t 2211
<210> 60
<211> 737
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 60
Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly
1 5 10 15
Thr His Ala Gln Met Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys
20 25 30
Pro Gly Thr Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe
35 40 45
Thr Ser Ser Ala Val Gln Trp Val Arg Gln Ala Arg Gly Gln Arg Leu
50 55 60
Glu Trp Ile Gly Trp Ile Ala Val Gly Ser Gly Asn Thr Asn Tyr Ala
65 70 75 80
Gln Lys Phe Gln Glu Arg Val Ser Ile Thr Arg Asp Met Ser Thr Ser
85 90 95
Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
100 105 110
Tyr Tyr Cys Ala Ala Pro Tyr Cys Ser Arg Thr Ser Cys His Asp Ala
115 120 125
Phe Asp Ile Trp Gly Gln Gly Thr Lys Val Thr Val Ser Ser Ala Ser
130 135 140
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
145 150 155 160
Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
165 170 175
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
180 185 190
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
195 200 205
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
210 215 220
Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val
225 230 235 240
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
245 250 255
Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
260 265 270
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
275 280 285
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
290 295 300
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
305 310 315 320
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
325 330 335
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
340 345 350
Leu Pro Ala Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
355 360 365
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
370 375 380
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
385 390 395 400
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
405 410 415
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
420 425 430
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
435 440 445
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
450 455 460
Ser Leu Ser Leu Ser Pro Gly Lys Arg Gly Arg Lys Arg Arg Ser Gly
465 470 475 480
Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu
485 490 495
Glu Asn Pro Gly Pro Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu
500 505 510
Leu Leu Trp Ile Ser Gly Ala Tyr Gly Glu Val Val Leu Thr Gln Ser
515 520 525
Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys
530 535 540
Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala Trp Tyr Gln Gln
545 550 555 560
Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Gly Ala Ser Ser Arg
565 570 575
Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
580 585 590
Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr
595 600 605
His Cys Gln His Phe Gly Ser Ser Ser Gln Trp Thr Phe Gly Gln Gly
610 615 620
Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile
625 630 635 640
Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val
645 650 655
Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys
660 665 670
Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu
675 680 685
Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Asn Thr Leu Thr Leu
690 695 700
Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr
705 710 715 720
His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu
725 730 735
Cys
<210> 61
<211> 2211
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 61
atggactgga catggagaat cctgttcctg gtcgccgccg caactggcac tcacgctcag 60
atgcagctgg tgcagtctgg acccgaggtg aagaagcccg gcaccagcgt gaaggtgtcc 120
tgtaaggcct ctggcttcac ctttacaagc tccgccgtgc agtgggtgag gcaggccaga 180
ggccagcggc tggagtggat cggatggatc gcagtgggct ccggaaacac caattacgcc 240
cagaagttcc aggagcgcgt gtctatcaca agggacatgt ccacctctac agcctatatg 300
gagctgtcta gcctgcggtc cgaggataca gccgtgtact attgcgccgc cccttactgt 360
tcctctatca gctgcaacga cggcttcgat atctggggcc agggcaccaa ggtgacagtg 420
agctccgcct ctaccaaggg cccaagcgtg tttcccctgg ccccttctag caagagcacc 480
tccggcggca cagccgccct gggctgtctg gtgaaggact acttccctga gccagtgaca 540
gtgagctgga actccggcgc cctgacctcc ggagtgcaca catttcctgc cgtgctgcag 600
tcctctggcc tgtacagcct gagctccgtg gtgaccgtgc catctagctc cctgggcacc 660
cagacatata tctgtaacgt gaatcacaag ccttccaata caaaggtgga caagaaggtg 720
gagccaaagt cttgcgataa gacccacaca tgccctccct gtccagcacc tgagctgctg 780
ggcggcccaa gcgtgttcct gtttccaccc aagcccaagg acaccctgat gatcagcagg 840
accccagagg tgacatgcgt ggtggtggac gtgtcccacg aggaccccga ggtgaagttt 900
aactggtacg tggatggcgt ggaggtgcac aatgccaaga ccaagcccag agaggagcag 960
tacaattcca cctatcgggt ggtgtctgtg ctgacagtgc tgcaccagga ctggctgaac 1020
ggcaaggagt acaagtgtaa ggtgtctaat aaggccctgc cagcccccat cgagaagacc 1080
atcagcaagg caaagggaca gccacgcgag ccacaggtgt atacactgcc tccatctagg 1140
gacgagctga ccaagaacca ggtgagcctg acatgcctgg tgaagggctt ctaccccagc 1200
gatatcgccg tggagtggga gtccaatggc cagcctgaga acaattacaa gaccacaccc 1260
cctgtgctgg actctgatgg cagcttcttt ctgtattcta agctgaccgt ggacaagagc 1320
agatggcagc agggcaacgt gttttcttgt agcgtgatgc acgaggccct gcacaatcac 1380
tacacccaga agtccctgtc tctgagccct ggcaagaggg gaaggaagag gagatccggc 1440
tctggcgcca caaacttcag cctgctgaag caggccggcg atgtggagga gaatcctggc 1500
ccaatggtgc tgcagaccca ggtgtttatc agcctgctgc tgtggatctc cggagcatat 1560
ggagaggtgg tgctgaccca gtccccaggc acactgagcc tgtcccctgg agagagagcc 1620
accctgagct gcagggcatc tcagagcgtg tctagctcct acctggcctg gtatcagcag 1680
aagcctggcc aggccccaag actgctgatc tacggagcct ctagccgcgc caccggcatc 1740
cccgacaggt tctccggctc tggcagcggc acagacttca ccctgacaat ctcccggctg 1800
gagcctgagg acttcgccgt gtaccactgt cagcactatg gctcctctag ccagtggacc 1860
tttggccagg gcacaaaggt ggagatcaag aggaccgtgg cagcaccatc cgtgttcatc 1920
tttccaccct ctgacgagca gctgaagagc ggcacagcct ccgtggtgtg cctgctgaac 1980
aatttctatc cccgcgaggc caaggtgcag tggaaggtgg ataacgccct gcagtccggc 2040
aattctcagg agagcgtgac cgagcaggac tccaaggatt ctacatactc cctgtctaac 2100
accctgacac tgagcaaggc cgattacgag aagcacaagg tgtatgcctg cgaggtgacc 2160
catcaggggc tgtcttctcc agtgaccaaa tccttcaatc gcggggaatg t 2211
<210> 62
<211> 737
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 62
Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly
1 5 10 15
Thr His Ala Gln Met Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys
20 25 30
Pro Gly Thr Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe
35 40 45
Thr Ser Ser Ala Val Gln Trp Val Arg Gln Ala Arg Gly Gln Arg Leu
50 55 60
Glu Trp Ile Gly Trp Ile Ala Val Gly Ser Gly Asn Thr Asn Tyr Ala
65 70 75 80
Gln Lys Phe Gln Glu Arg Val Ser Ile Thr Arg Asp Met Ser Thr Ser
85 90 95
Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
100 105 110
Tyr Tyr Cys Ala Ala Pro Tyr Cys Ser Ser Ile Ser Cys Asn Asp Gly
115 120 125
Phe Asp Ile Trp Gly Gln Gly Thr Lys Val Thr Val Ser Ser Ala Ser
130 135 140
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
145 150 155 160
Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
165 170 175
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
180 185 190
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
195 200 205
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
210 215 220
Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val
225 230 235 240
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
245 250 255
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
260 265 270
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
275 280 285
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
290 295 300
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
305 310 315 320
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
325 330 335
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
340 345 350
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
355 360 365
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
370 375 380
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
385 390 395 400
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
405 410 415
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
420 425 430
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
435 440 445
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
450 455 460
Ser Leu Ser Leu Ser Pro Gly Lys Arg Gly Arg Lys Arg Arg Ser Gly
465 470 475 480
Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu
485 490 495
Glu Asn Pro Gly Pro Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu
500 505 510
Leu Leu Trp Ile Ser Gly Ala Tyr Gly Glu Val Val Leu Thr Gln Ser
515 520 525
Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys
530 535 540
Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala Trp Tyr Gln Gln
545 550 555 560
Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Gly Ala Ser Ser Arg
565 570 575
Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
580 585 590
Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr
595 600 605
His Cys Gln His Tyr Gly Ser Ser Ser Gln Trp Thr Phe Gly Gln Gly
610 615 620
Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile
625 630 635 640
Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val
645 650 655
Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys
660 665 670
Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu
675 680 685
Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Asn Thr Leu Thr Leu
690 695 700
Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr
705 710 715 720
His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu
725 730 735
Cys
<210> 63
<211> 2211
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 63
atggactgga catggagaat cctgttcctg gtcgccgccg caactggcac tcacgctcag 60
atgcagctgg tgcagtctgg acccgaggtg aagaagcccg gcaccagcgt gaaggtgtcc 120
tgtaaggcct ctggcttcac ctttacaagc tccgccgtgc agtgggtgag gcaggccaga 180
ggccagcggc tggagtggat cggatggatc gcagtgggct ccggaaacac caattacgcc 240
cagaagttcc aggagcgcgt gtctatcaca agggacatgt ccacctctac agcctatatg 300
gagctgtcta gcctgcggtc cgaggataca gccgtgtact attgcgccgc cccttactgt 360
tcctctatca gctgcaacga cggcttcgat atctggggcc agggcaccaa ggtgacagtg 420
agctccgcct ctaccaaggg cccaagcgtg tttcccctgg ccccttctag caagagcacc 480
tccggcggca cagccgccct gggctgtctg gtgaaggact acttccctga gccagtgaca 540
gtgagctgga actccggcgc cctgacctcc ggagtgcaca catttcctgc cgtgctgcag 600
tcctctggcc tgtacagcct gagctccgtg gtgaccgtgc catctagctc cctgggcacc 660
cagacatata tctgtaacgt gaatcacaag ccttccaata caaaggtgga caagaaggtg 720
gagccaaagt cttgcgataa gacccacaca tgccctccct gtccagcacc tgagctgctg 780
ggcggcccaa gcgtgttcct gtttccaccc aagcccaagg acaccctgat gatcagcagg 840
accccagagg tgacatgcgt ggtggtggac gtgtcccacg aggaccccga ggtgaagttt 900
aactggtacg tggatggcgt ggaggtgcac aatgccaaga ccaagcccag agaggagcag 960
tacaattcca cctatcgggt ggtgtctgtg ctgacagtgc tgcaccagga ctggctgaac 1020
ggcaaggagt acaagtgtaa ggtgtctaat aaggccctgc cagcccccat cgagaagacc 1080
atcagcaagg caaagggaca gccacgcgag ccacaggtgt atacactgcc tccatctagg 1140
gacgagctga ccaagaacca ggtgagcctg acatgcctgg tgaagggctt ctaccccagc 1200
gatatcgccg tggagtggga gtccaatggc cagcctgaga acaattacaa gaccacaccc 1260
cctgtgctgg actctgatgg cagcttcttt ctgtattcta agctgaccgt ggacaagagc 1320
agatggcagc agggcaacgt gttttcttgt agcgtgatgc acggcgccct gcacaatcac 1380
tacacccaga agtccctgtc tctgagccct ggcaagaggg gaaggaagag gagatccggc 1440
tctggcgcca caaacttcag cctgctgaag caggccggcg atgtggagga gaatcctggc 1500
ccaatggtgc tgcagaccca ggtgtttatc agcctgctgc tgtggatctc cggagcatat 1560
ggagaggtgg tgctgaccca gtccccaggc acactgagcc tgtcccctgg agagagagcc 1620
accctgagct gcagggcatc tcagagcgtg tctagctcct acctggcctg gtatcagcag 1680
aagcctggcc aggccccaag actgctgatc tacggagcct ctagccgcgc caccggcatc 1740
cccgacaggt tctccggctc tggcagcggc acagacttca ccctgacaat ctcccggctg 1800
gagcctgagg acttcgccgt gtaccactgt cagcactatg gctcctctag ccagtggacc 1860
tttggccagg gcacaaaggt ggagatcaag aggaccgtgg cagcaccatc cgtgttcatc 1920
tttccaccct ctgacgagca gctgaagagc ggcacagcct ccgtggtgtg cctgctgaac 1980
aatttctatc cccgcgaggc caaggtgcag tggaaggtgg ataacgccct gcagtccggc 2040
aattctcagg agagcgtgac cgagcaggac tccaaggatt ctacatactc cctgtctaac 2100
accctgacac tgagcaaggc cgattacgag aagcacaagg tgtatgcctg cgaggtgacc 2160
catcaggggc tgtcttctcc agtgaccaaa tccttcaatc gcggggaatg t 2211
<210> 64
<211> 737
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 64
Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly
1 5 10 15
Thr His Ala Gln Met Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys
20 25 30
Pro Gly Thr Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe
35 40 45
Thr Ser Ser Ala Val Gln Trp Val Arg Gln Ala Arg Gly Gln Arg Leu
50 55 60
Glu Trp Ile Gly Trp Ile Ala Val Gly Ser Gly Asn Thr Asn Tyr Ala
65 70 75 80
Gln Lys Phe Gln Glu Arg Val Ser Ile Thr Arg Asp Met Ser Thr Ser
85 90 95
Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
100 105 110
Tyr Tyr Cys Ala Ala Pro Tyr Cys Ser Ser Ile Ser Cys Asn Asp Gly
115 120 125
Phe Asp Ile Trp Gly Gln Gly Thr Lys Val Thr Val Ser Ser Ala Ser
130 135 140
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
145 150 155 160
Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
165 170 175
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
180 185 190
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
195 200 205
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
210 215 220
Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val
225 230 235 240
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
245 250 255
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
260 265 270
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
275 280 285
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
290 295 300
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
305 310 315 320
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
325 330 335
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
340 345 350
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
355 360 365
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
370 375 380
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
385 390 395 400
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
405 410 415
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
420 425 430
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
435 440 445
Ser Cys Ser Val Met His Gly Ala Leu His Asn His Tyr Thr Gln Lys
450 455 460
Ser Leu Ser Leu Ser Pro Gly Lys Arg Gly Arg Lys Arg Arg Ser Gly
465 470 475 480
Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu
485 490 495
Glu Asn Pro Gly Pro Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu
500 505 510
Leu Leu Trp Ile Ser Gly Ala Tyr Gly Glu Val Val Leu Thr Gln Ser
515 520 525
Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys
530 535 540
Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala Trp Tyr Gln Gln
545 550 555 560
Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Gly Ala Ser Ser Arg
565 570 575
Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
580 585 590
Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr
595 600 605
His Cys Gln His Tyr Gly Ser Ser Ser Gln Trp Thr Phe Gly Gln Gly
610 615 620
Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile
625 630 635 640
Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val
645 650 655
Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys
660 665 670
Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu
675 680 685
Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Asn Thr Leu Thr Leu
690 695 700
Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr
705 710 715 720
His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu
725 730 735
Cys
<210> 65
<211> 2211
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 65
atggactgga catggagaat cctgttcctg gtcgccgccg caactggcac tcacgctcag 60
atgcagctgg tgcagtctgg acccgaggtg aagaagcccg gcaccagcgt gaaggtgtcc 120
tgtaaggcct ctggcttcac ctttacaagc tccgccgtgc agtgggtgag gcaggccaga 180
ggccagcggc tggagtggat cggatggatc gcagtgggct ccggaaacac caattacgcc 240
cagaagttcc aggagcgcgt gtctatcaca agggacatgt ccacctctac agcctatatg 300
gagctgtcta gcctgcggtc cgaggataca gccgtgtact attgcgccgc cccttactgt 360
tcctctatca gctgcaacga cggcttcgat atctggggcc agggcaccaa ggtgacagtg 420
agctccgcct ctaccaaggg cccaagcgtg tttcccctgg ccccttctag caagagcacc 480
tccggcggca cagccgccct gggctgtctg gtgaaggact acttccctga gccagtgaca 540
gtgagctgga actccggcgc cctgacctcc ggagtgcaca catttcctgc cgtgctgcag 600
tcctctggcc tgtacagcct gagctccgtg gtgaccgtgc catctagctc cctgggcacc 660
cagacatata tctgtaacgt gaatcacaag ccttccaata caaaggtgga caagaaggtg 720
gagccaaagt cttgcgataa gacccacaca tgccctccct gtccagcacc tgagctgctg 780
ggcggcccaa gcgtgttcct gtttccaccc aagcccaagg acaccctgat gatcagcagg 840
accccagagg tgacatgcgt ggtggtggac gtgtcccacg aggaccccga ggtgaagttt 900
aactggtacg tggatggcgt ggaggtgcac aatgccaaga ccaagcccag agaggagcag 960
tacaattcca cctatcgggt ggtgtctgtg ctgacagtgc tgcaccagga ctggctgaac 1020
ggcaaggagt acaagtgtaa ggtgtctaat aaggccctgc cagcccccat cgagaagacc 1080
atcagcaagg caaagggaca gccacgcaag ccacaggtgt atacactgcc tccatctagg 1140
gacgagctga ccaagaacca ggtgagcctg acatgcctgg tgaagggctt ctaccccagc 1200
gatatcgccg tggagtggga gtccaatggc cagcctgaga acaattacaa gaccacaccc 1260
cctgtgctgg actctgatgg cagcttcttt ctgtattcta agctgaccgt ggacaagagc 1320
agatggcagc agggcaacgt gttttcttgt agcgtgatgc acggcgccct gcacaatcac 1380
tacacccaga agtccctgtc tctgagccct ggcaagaggg gaaggaagag gagatccggc 1440
tctggcgcca caaacttcag cctgctgaag caggccggcg atgtggagga gaatcctggc 1500
ccaatggtgc tgcagaccca ggtgtttatc agcctgctgc tgtggatctc cggagcatat 1560
ggagaggtgg tgctgaccca gtccccaggc acactgagcc tgtcccctgg agagagagcc 1620
accctgagct gcagggcatc tcagagcgtg tctagctcct acctggcctg gtatcagcag 1680
aagcctggcc aggccccaag actgctgatc tacggagcct ctagccgcgc caccggcatc 1740
cccgacaggt tctccggctc tggcagcggc acagacttca ccctgacaat ctcccggctg 1800
gagcctgagg acttcgccgt gtaccactgt cagcactatg gctcctctag ccagtggacc 1860
tttggccagg gcacaaaggt ggagatcaag aggaccgtgg cagcaccatc cgtgttcatc 1920
tttccaccct ctgacgagca gctgaagagc ggcacagcct ccgtggtgtg cctgctgaac 1980
aatttctatc cccgcgaggc caaggtgcag tggaaggtgg ataacgccct gcagtccggc 2040
aattctcagg agagcgtgac cgagcaggac tccaaggatt ctacatactc cctgtctaac 2100
accctgacac tgagcaaggc cgattacgag aagcacaagg tgtatgcctg cgaggtgacc 2160
catcaggggc tgtcttctcc agtgaccaaa tccttcaatc gcggggaatg t 2211
<210> 66
<211> 737
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 66
Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly
1 5 10 15
Thr His Ala Gln Met Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys
20 25 30
Pro Gly Thr Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe
35 40 45
Thr Ser Ser Ala Val Gln Trp Val Arg Gln Ala Arg Gly Gln Arg Leu
50 55 60
Glu Trp Ile Gly Trp Ile Ala Val Gly Ser Gly Asn Thr Asn Tyr Ala
65 70 75 80
Gln Lys Phe Gln Glu Arg Val Ser Ile Thr Arg Asp Met Ser Thr Ser
85 90 95
Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
100 105 110
Tyr Tyr Cys Ala Ala Pro Tyr Cys Ser Ser Ile Ser Cys Asn Asp Gly
115 120 125
Phe Asp Ile Trp Gly Gln Gly Thr Lys Val Thr Val Ser Ser Ala Ser
130 135 140
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
145 150 155 160
Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
165 170 175
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
180 185 190
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
195 200 205
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
210 215 220
Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val
225 230 235 240
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
245 250 255
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
260 265 270
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
275 280 285
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
290 295 300
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
305 310 315 320
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
325 330 335
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
340 345 350
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
355 360 365
Arg Lys Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
370 375 380
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
385 390 395 400
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
405 410 415
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
420 425 430
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
435 440 445
Ser Cys Ser Val Met His Gly Ala Leu His Asn His Tyr Thr Gln Lys
450 455 460
Ser Leu Ser Leu Ser Pro Gly Lys Arg Gly Arg Lys Arg Arg Ser Gly
465 470 475 480
Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu
485 490 495
Glu Asn Pro Gly Pro Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu
500 505 510
Leu Leu Trp Ile Ser Gly Ala Tyr Gly Glu Val Val Leu Thr Gln Ser
515 520 525
Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys
530 535 540
Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala Trp Tyr Gln Gln
545 550 555 560
Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Gly Ala Ser Ser Arg
565 570 575
Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
580 585 590
Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr
595 600 605
His Cys Gln His Tyr Gly Ser Ser Ser Gln Trp Thr Phe Gly Gln Gly
610 615 620
Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile
625 630 635 640
Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val
645 650 655
Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys
660 665 670
Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu
675 680 685
Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Asn Thr Leu Thr Leu
690 695 700
Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr
705 710 715 720
His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu
725 730 735
Cys
<210> 67
<211> 2211
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 67
atggactgga catggagaat cctgttcctg gtcgccgccg caactggcac tcacgctcag 60
atgcagctgg tgcagtctgg acccgaggtg aagaagcccg gcaccagcgt gaaggtgtcc 120
tgtaaggcct ctggcttcac ctttacaagc tccgccgtgc agtgggtgag gcaggccaga 180
ggccagcggc tggagtggat cggatggatc gcagtgggct ccggaaacac caattacgcc 240
cagaagttcc aggagcgcgt gtctatcaca agggacatgt ccacctctac agcctatatg 300
gagctgtcta gcctgcggtc cgaggataca gccgtgtact attgcgccgc cccttactgt 360
tcctctatca gctgcaacga cggcttcgat atctggggcc agggcaccaa ggtgacagtg 420
agctccgcct ctaccaaggg cccaagcgtg tttcccctgg ccccttctag caagagcacc 480
tccggcggca cagccgccct gggctgtctg gtgaaggact acttccctga gccagtgaca 540
gtgagctgga actccggcgc cctgacctcc ggagtgcaca catttcctgc cgtgctgcag 600
tcctctggcc tgtacagcct gagctccgtg gtgaccgtgc catctagctc cctgggcacc 660
cagacatata tctgtaacgt gaatcacaag ccttccaata caaaggtgga caagaaggtg 720
gagccaaagt cttgcgataa gacccacaca tgccctccct gtccagcacc tgagttcgag 780
ggcggcccaa gcgtgttcct gtttccaccc aagcccaagg acaccctgat gatcagcagg 840
accccagagg tgacatgcgt ggtggtggac gtgtcccacg aggaccccga ggtgaagttt 900
aactggtacg tggatggcgt ggaggtgcac aatgccaaga ccaagcccag agaggagcag 960
tacaattcca cctatcgggt ggtgtctgtg ctgacagtgc tgcaccagga ctggctgaac 1020
ggcaaggagt acaagtgtaa ggtgtctaat aaggccctgc cagccagcat cgagaagacc 1080
atcagcaagg caaagggaca gccacgcgag ccacaggtgt atacactgcc tccatctagg 1140
gacgagctga ccaagaacca ggtgagcctg acatgcctgg tgaagggctt ctaccccagc 1200
gatatcgccg tggagtggga gtccaatggc cagcctgaga acaattacaa gaccacaccc 1260
cctgtgctgg actctgatgg cagcttcttt ctgtattcta agctgaccgt ggacaagagc 1320
agatggcagc agggcaacgt gttttcttgt agcgtgatgc acgaggccct gcacaatcac 1380
tacacccaga agtccctgtc tctgagccct ggcaagaggg gaaggaagag gagatccggc 1440
tctggcgcca caaacttcag cctgctgaag caggccggcg atgtggagga gaatcctggc 1500
ccaatggtgc tgcagaccca ggtgtttatc agcctgctgc tgtggatctc cggagcatat 1560
ggagaggtgg tgctgaccca gtccccaggc acactgagcc tgtcccctgg agagagagcc 1620
accctgagct gcagggcatc tcagagcgtg tctagctcct acctggcctg gtatcagcag 1680
aagcctggcc aggccccaag actgctgatc tacggagcct ctagccgcgc caccggcatc 1740
cccgacaggt tctccggctc tggcagcggc acagacttca ccctgacaat ctcccggctg 1800
gagcctgagg acttcgccgt gtaccactgt cagcactatg gctcctctag ccagtggacc 1860
tttggccagg gcacaaaggt ggagatcaag aggaccgtgg cagcaccatc cgtgttcatc 1920
tttccaccct ctgacgagca gctgaagagc ggcacagcct ccgtggtgtg cctgctgaac 1980
aatttctatc cccgcgaggc caaggtgcag tggaaggtgg ataacgccct gcagtccggc 2040
aattctcagg agagcgtgac cgagcaggac tccaaggatt ctacatactc cctgtctaac 2100
accctgacac tgagcaaggc cgattacgag aagcacaagg tgtatgcctg cgaggtgacc 2160
catcaggggc tgtcttctcc agtgaccaaa tccttcaatc gcggggaatg t 2211
<210> 68
<211> 737
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 68
Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly
1 5 10 15
Thr His Ala Gln Met Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys
20 25 30
Pro Gly Thr Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe
35 40 45
Thr Ser Ser Ala Val Gln Trp Val Arg Gln Ala Arg Gly Gln Arg Leu
50 55 60
Glu Trp Ile Gly Trp Ile Ala Val Gly Ser Gly Asn Thr Asn Tyr Ala
65 70 75 80
Gln Lys Phe Gln Glu Arg Val Ser Ile Thr Arg Asp Met Ser Thr Ser
85 90 95
Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
100 105 110
Tyr Tyr Cys Ala Ala Pro Tyr Cys Ser Ser Ile Ser Cys Asn Asp Gly
115 120 125
Phe Asp Ile Trp Gly Gln Gly Thr Lys Val Thr Val Ser Ser Ala Ser
130 135 140
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
145 150 155 160
Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
165 170 175
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
180 185 190
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
195 200 205
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
210 215 220
Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val
225 230 235 240
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
245 250 255
Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
260 265 270
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
275 280 285
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
290 295 300
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
305 310 315 320
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
325 330 335
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
340 345 350
Leu Pro Ala Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
355 360 365
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
370 375 380
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
385 390 395 400
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
405 410 415
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
420 425 430
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
435 440 445
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
450 455 460
Ser Leu Ser Leu Ser Pro Gly Lys Arg Gly Arg Lys Arg Arg Ser Gly
465 470 475 480
Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu
485 490 495
Glu Asn Pro Gly Pro Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu
500 505 510
Leu Leu Trp Ile Ser Gly Ala Tyr Gly Glu Val Val Leu Thr Gln Ser
515 520 525
Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys
530 535 540
Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala Trp Tyr Gln Gln
545 550 555 560
Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Gly Ala Ser Ser Arg
565 570 575
Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
580 585 590
Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr
595 600 605
His Cys Gln His Tyr Gly Ser Ser Ser Gln Trp Thr Phe Gly Gln Gly
610 615 620
Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile
625 630 635 640
Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val
645 650 655
Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys
660 665 670
Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu
675 680 685
Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Asn Thr Leu Thr Leu
690 695 700
Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr
705 710 715 720
His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu
725 730 735
Cys
<210> 69
<211> 2217
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 69
atggactgga catggagaat cctgttcctg gtcgccgccg caactggcac tcacgctcag 60
atgcagctgg tgcagtctgg acccgaggtg aagaagcccg gcaccagcgt gaaggtgtcc 120
tgtaaggcct ctggcttcac ctttatgagc tccgccgtgc agtgggtgag gcaggccaga 180
ggccagcggc tggagtggat cggatggatc gtgatcggct ccggaaacac caattacgcc 240
cagaagttcc aggagcgcgt gaccatcaca agggacatgt ccacctctac agcctatatg 300
gagctgtcta gcctgcggtc cgaggataca gccgtgtact attgcgccgc cccttactgt 360
tcctctatca gctgcaacga cggcttcgat atctggggcc agggcaccat ggtgacagtg 420
agctccgcct ctaccaaggg cccaagcgtg tttcccctgg ccccttctag caagagcacc 480
tccggcggca cagccgccct gggctgtctg gtgaaggact acttccctga gccagtgaca 540
gtgagctgga actccggcgc cctgacctcc ggagtgcaca catttcctgc cgtgctgcag 600
tcctctggcc tgtacagcct gagctccgtg gtgaccgtgc catctagctc cctgggcacc 660
cagacatata tctgtaacgt gaatcacaag ccttccaata caaaggtgga caagaaggtg 720
gagccaaagt cttgcgataa gacccacaca tgccctccct gtccagcacc tgagctgctg 780
ggcggcccaa gcgtgttcct gtttccaccc aagcccaagg acaccctgat gatcagcagg 840
accccagagg tgacatgcgt ggtggtggac gtgtcccacg aggaccccga ggtgaagttt 900
aactggtacg tggatggcgt ggaggtgcac aatgccaaga ccaagcccag agaggagcag 960
tacaattcca cctatcgggt ggtgtctgtg ctgacagtgc tgcaccagga ctggctgaac 1020
ggcaaggagt acaagtgtaa ggtgtctaat aaggccctgc cagcccccat cgagaagacc 1080
atcagcaagg caaagggaca gccacgcgag ccacaggtgt atacactgcc tccatctagg 1140
gacgagctga ccaagaacca ggtgagcctg acatgcctgg tgaagggctt ctaccccagc 1200
gatatcgccg tggagtggga gtccaatggc cagcctgaga acaattacaa gaccacaccc 1260
cctgtgctgg actctgatgg cagcttcttt ctgtattcta agctgaccgt ggacaagagc 1320
agatggcagc agggcaacgt gttttcttgt agcgtgatgc acgaggccct gcacaatcac 1380
tacacccaga agtccctgtc tctgagccct ggcaagaggg gaaggaagag gagatccggc 1440
tctggcgcca caaacttcag cctgctgaag caggccggcg atgtggagga gaatcctggc 1500
ccaatggtgc tgcagaccca ggtgtttatc agcctgctgc tgtggatctc cggagcatat 1560
ggagagatcg tgctgaccca gtccccaggc acactgagcc tgtcccctgg agagagagcc 1620
accctgagct gcagggcatc tcagagcgtg tctagctcct acctggcctg gtatcagcag 1680
aagcctggcc aggccccaag actgctgatc tacggagcct ctagccgcgc caccggcatc 1740
cccgacaggt tctccggctc tggcagcggc acagacttca ccctgacaat ctcccggctg 1800
gagcctgagg acttcgccgt gtactactgt cagcactatg gctcctctcg gggctggacc 1860
tttggccagg gcacaaaggt ggagatcaag aggaccgtgg cagcaccatc cgtgttcatc 1920
tttccaccct ctgacgagca gctgaagagc ggcacagcct ccgtggtgtg cctgctgaac 1980
aatttctatc cccgcgaggc caaggtgcag tggaaggtgg ataacgccct gcagtccggc 2040
aattctcagg agagcgtgac cgagcaggac tccaaggatt ctacatactc cctgtctaac 2100
accctgacac tgagcaaggc cgattacgag aagcacaagg tgtatgcctg cgaggtgacc 2160
catcaggggc tgtcttctcc agtgaccaaa tccttcaatc gcggggaatg ttgataa 2217
<210> 70
<211> 737
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 70
Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly
1 5 10 15
Thr His Ala Gln Met Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys
20 25 30
Pro Gly Thr Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe
35 40 45
Met Ser Ser Ala Val Gln Trp Val Arg Gln Ala Arg Gly Gln Arg Leu
50 55 60
Glu Trp Ile Gly Trp Ile Val Ile Gly Ser Gly Asn Thr Asn Tyr Ala
65 70 75 80
Gln Lys Phe Gln Glu Arg Val Thr Ile Thr Arg Asp Met Ser Thr Ser
85 90 95
Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
100 105 110
Tyr Tyr Cys Ala Ala Pro Tyr Cys Ser Ser Ile Ser Cys Asn Asp Gly
115 120 125
Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser
130 135 140
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
145 150 155 160
Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
165 170 175
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
180 185 190
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
195 200 205
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
210 215 220
Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val
225 230 235 240
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
245 250 255
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
260 265 270
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
275 280 285
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
290 295 300
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
305 310 315 320
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
325 330 335
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
340 345 350
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
355 360 365
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
370 375 380
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
385 390 395 400
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
405 410 415
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
420 425 430
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
435 440 445
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
450 455 460
Ser Leu Ser Leu Ser Pro Gly Lys Arg Gly Arg Lys Arg Arg Ser Gly
465 470 475 480
Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu
485 490 495
Glu Asn Pro Gly Pro Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu
500 505 510
Leu Leu Trp Ile Ser Gly Ala Tyr Gly Glu Ile Val Leu Thr Gln Ser
515 520 525
Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys
530 535 540
Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala Trp Tyr Gln Gln
545 550 555 560
Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Gly Ala Ser Ser Arg
565 570 575
Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
580 585 590
Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr
595 600 605
Tyr Cys Gln His Tyr Gly Ser Ser Arg Gly Trp Thr Phe Gly Gln Gly
610 615 620
Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile
625 630 635 640
Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val
645 650 655
Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys
660 665 670
Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu
675 680 685
Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Asn Thr Leu Thr Leu
690 695 700
Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr
705 710 715 720
His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu
725 730 735
Cys
<210> 71
<211> 2217
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 71
atggactgga catggagaat cctgttcctg gtcgccgccg caactggcac tcacgctcag 60
atgcagctgg tgcagtctgg acccgaggtg aagaagcccg gcaccagcgt gaaggtgtcc 120
tgtaaggcct ctggcttcac ctttatgagc tccgccgtgc agtgggtgag gcaggccaga 180
ggccagcggc tggagtggat cggatggatc gtgatcggct ccggaaacac caattacgcc 240
cagaagttcc aggagcgcgt gaccatcaca agggacatgt ccacctctac agcctatatg 300
gagctgtcta gcctgcggtc cgaggataca gccgtgtact attgcgccgc cccttactgt 360
tcctctatca gctgcaacga cggcttcgat atctggggcc agggcaccat ggtgacagtg 420
agctccgcct ctaccaaggg cccaagcgtg tttcccctgg ccccttctag caagagcacc 480
tccggcggca cagccgccct gggctgtctg gtgaaggact acttccctga gccagtgaca 540
gtgagctgga actccggcgc cctgacctcc ggagtgcaca catttcctgc cgtgctgcag 600
tcctctggcc tgtacagcct gagctccgtg gtgaccgtgc catctagctc cctgggcacc 660
cagacatata tctgtaacgt gaatcacaag ccttccaata caaaggtgga caagaaggtg 720
gagccaaagt cttgcgataa gacccacaca tgccctccct gtccagcacc tgagctgctg 780
ggcggcccaa gcgtgttcct gtttccaccc aagcccaagg acaccctgat gatcagcagg 840
accccagagg tgacatgcgt ggtggtggac gtgtcccacg aggaccccga ggtgaagttt 900
aactggtacg tggatggcgt ggaggtgcac aatgccaaga ccaagcccag agaggagcag 960
tacaattcca cctatcgggt ggtgtctgtg ctgacagtgc tgcaccagga ctggctgaac 1020
ggcaaggagt acaagtgtaa ggtgtctaat aaggccctgc cagcccccat cgagaagacc 1080
atcagcaagg caaagggaca gccacgcgag ccacaggtgt atacactgcc tccatctagg 1140
gacgagctga ccaagaacca ggtgagcctg acatgcctgg tgaagggctt ctaccccagc 1200
gatatcgccg tggagtggga gtccaatggc cagcctgaga acaattacaa gaccacaccc 1260
cctgtgctgg actctgatgg cagcttcttt ctgtattcta agctgaccgt ggacaagagc 1320
agatggcagc agggcaacgt gttttcttgt agcgtgatgc acggcgccct gcacaatcac 1380
tacacccaga agtccctgtc tctgagccct ggcaagaggg gaaggaagag gagatccggc 1440
tctggcgcca caaacttcag cctgctgaag caggccggcg atgtggagga gaatcctggc 1500
ccaatggtgc tgcagaccca ggtgtttatc agcctgctgc tgtggatctc cggagcatat 1560
ggagagatcg tgctgaccca gtccccaggc acactgagcc tgtcccctgg agagagagcc 1620
accctgagct gcagggcatc tcagagcgtg tctagctcct acctggcctg gtatcagcag 1680
aagcctggcc aggccccaag actgctgatc tacggagcct ctagccgcgc caccggcatc 1740
cccgacaggt tctccggctc tggcagcggc acagacttca ccctgacaat ctcccggctg 1800
gagcctgagg acttcgccgt gtactactgt cagcactatg gctcctctcg gggctggacc 1860
tttggccagg gcacaaaggt ggagatcaag aggaccgtgg cagcaccatc cgtgttcatc 1920
tttccaccct ctgacgagca gctgaagagc ggcacagcct ccgtggtgtg cctgctgaac 1980
aatttctatc cccgcgaggc caaggtgcag tggaaggtgg ataacgccct gcagtccggc 2040
aattctcagg agagcgtgac cgagcaggac tccaaggatt ctacatactc cctgtctaac 2100
accctgacac tgagcaaggc cgattacgag aagcacaagg tgtatgcctg cgaggtgacc 2160
catcaggggc tgtcttctcc agtgaccaaa tccttcaatc gcggggaatg ttgataa 2217
<210> 72
<211> 737
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 72
Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly
1 5 10 15
Thr His Ala Gln Met Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys
20 25 30
Pro Gly Thr Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe
35 40 45
Met Ser Ser Ala Val Gln Trp Val Arg Gln Ala Arg Gly Gln Arg Leu
50 55 60
Glu Trp Ile Gly Trp Ile Val Ile Gly Ser Gly Asn Thr Asn Tyr Ala
65 70 75 80
Gln Lys Phe Gln Glu Arg Val Thr Ile Thr Arg Asp Met Ser Thr Ser
85 90 95
Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
100 105 110
Tyr Tyr Cys Ala Ala Pro Tyr Cys Ser Ser Ile Ser Cys Asn Asp Gly
115 120 125
Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser
130 135 140
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
145 150 155 160
Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
165 170 175
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
180 185 190
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
195 200 205
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
210 215 220
Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val
225 230 235 240
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
245 250 255
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
260 265 270
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
275 280 285
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
290 295 300
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
305 310 315 320
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
325 330 335
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
340 345 350
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
355 360 365
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
370 375 380
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
385 390 395 400
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
405 410 415
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
420 425 430
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
435 440 445
Ser Cys Ser Val Met His Gly Ala Leu His Asn His Tyr Thr Gln Lys
450 455 460
Ser Leu Ser Leu Ser Pro Gly Lys Arg Gly Arg Lys Arg Arg Ser Gly
465 470 475 480
Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu
485 490 495
Glu Asn Pro Gly Pro Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu
500 505 510
Leu Leu Trp Ile Ser Gly Ala Tyr Gly Glu Ile Val Leu Thr Gln Ser
515 520 525
Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys
530 535 540
Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala Trp Tyr Gln Gln
545 550 555 560
Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Gly Ala Ser Ser Arg
565 570 575
Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
580 585 590
Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr
595 600 605
Tyr Cys Gln His Tyr Gly Ser Ser Arg Gly Trp Thr Phe Gly Gln Gly
610 615 620
Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile
625 630 635 640
Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val
645 650 655
Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys
660 665 670
Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu
675 680 685
Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Asn Thr Leu Thr Leu
690 695 700
Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr
705 710 715 720
His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu
725 730 735
Cys
<210> 73
<211> 2217
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 73
atggactgga catggagaat cctgttcctg gtcgccgccg caactggcac tcacgctcag 60
atgcagctgg tgcagtctgg acccgaggtg aagaagcccg gcaccagcgt gaaggtgtcc 120
tgtaaggcct ctggcttcac ctttatgagc tccgccgtgc agtgggtgag gcaggccaga 180
ggccagcggc tggagtggat cggatggatc gtgatcggct ccggaaacac caattacgcc 240
cagaagttcc aggagcgcgt gaccatcaca agggacatgt ccacctctac agcctatatg 300
gagctgtcta gcctgcggtc cgaggataca gccgtgtact attgcgccgc cccttactgt 360
tcctctatca gctgcaacga cggcttcgat atctggggcc agggcaccat ggtgacagtg 420
agctccgcct ctaccaaggg cccaagcgtg tttcccctgg ccccttctag caagagcacc 480
tccggcggca cagccgccct gggctgtctg gtgaaggact acttccctga gccagtgaca 540
gtgagctgga actccggcgc cctgacctcc ggagtgcaca catttcctgc cgtgctgcag 600
tcctctggcc tgtacagcct gagctccgtg gtgaccgtgc catctagctc cctgggcacc 660
cagacatata tctgtaacgt gaatcacaag ccttccaata caaaggtgga caagaaggtg 720
gagccaaagt cttgcgataa gacccacaca tgccctccct gtccagcacc tgagctgctg 780
ggcggcccaa gcgtgttcct gtttccaccc aagcccaagg acaccctgat gatcagcagg 840
accccagagg tgacatgcgt ggtggtggac gtgtcccacg aggaccccga ggtgaagttt 900
aactggtacg tggatggcgt ggaggtgcac aatgccaaga ccaagcccag agaggagcag 960
tacaattcca cctatcgggt ggtgtctgtg ctgacagtgc tgcaccagga ctggctgaac 1020
ggcaaggagt acaagtgtaa ggtgtctaat aaggccctgc cagcccccat cgagaagacc 1080
atcagcaagg caaagggaca gccacgcaag ccacaggtgt atacactgcc tccatctagg 1140
gacgagctga ccaagaacca ggtgagcctg acatgcctgg tgaagggctt ctaccccagc 1200
gatatcgccg tggagtggga gtccaatggc cagcctgaga acaattacaa gaccacaccc 1260
cctgtgctgg actctgatgg cagcttcttt ctgtattcta agctgaccgt ggacaagagc 1320
agatggcagc agggcaacgt gttttcttgt agcgtgatgc acggcgccct gcacaatcac 1380
tacacccaga agtccctgtc tctgagccct ggcaagaggg gaaggaagag gagatccggc 1440
tctggcgcca caaacttcag cctgctgaag caggccggcg atgtggagga gaatcctggc 1500
ccaatggtgc tgcagaccca ggtgtttatc agcctgctgc tgtggatctc cggagcatat 1560
ggagagatcg tgctgaccca gtccccaggc acactgagcc tgtcccctgg agagagagcc 1620
accctgagct gcagggcatc tcagagcgtg tctagctcct acctggcctg gtatcagcag 1680
aagcctggcc aggccccaag actgctgatc tacggagcct ctagccgcgc caccggcatc 1740
cccgacaggt tctccggctc tggcagcggc acagacttca ccctgacaat ctcccggctg 1800
gagcctgagg acttcgccgt gtactactgt cagcactatg gctcctctcg gggctggacc 1860
tttggccagg gcacaaaggt ggagatcaag aggaccgtgg cagcaccatc cgtgttcatc 1920
tttccaccct ctgacgagca gctgaagagc ggcacagcct ccgtggtgtg cctgctgaac 1980
aatttctatc cccgcgaggc caaggtgcag tggaaggtgg ataacgccct gcagtccggc 2040
aattctcagg agagcgtgac cgagcaggac tccaaggatt ctacatactc cctgtctaac 2100
accctgacac tgagcaaggc cgattacgag aagcacaagg tgtatgcctg cgaggtgacc 2160
catcaggggc tgtcttctcc agtgaccaaa tccttcaatc gcggggaatg ttgataa 2217
<210> 74
<211> 737
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 74
Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly
1 5 10 15
Thr His Ala Gln Met Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys
20 25 30
Pro Gly Thr Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe
35 40 45
Met Ser Ser Ala Val Gln Trp Val Arg Gln Ala Arg Gly Gln Arg Leu
50 55 60
Glu Trp Ile Gly Trp Ile Val Ile Gly Ser Gly Asn Thr Asn Tyr Ala
65 70 75 80
Gln Lys Phe Gln Glu Arg Val Thr Ile Thr Arg Asp Met Ser Thr Ser
85 90 95
Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
100 105 110
Tyr Tyr Cys Ala Ala Pro Tyr Cys Ser Ser Ile Ser Cys Asn Asp Gly
115 120 125
Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser
130 135 140
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
145 150 155 160
Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
165 170 175
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
180 185 190
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
195 200 205
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
210 215 220
Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val
225 230 235 240
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
245 250 255
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
260 265 270
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
275 280 285
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
290 295 300
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
305 310 315 320
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
325 330 335
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
340 345 350
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
355 360 365
Arg Lys Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
370 375 380
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
385 390 395 400
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
405 410 415
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
420 425 430
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
435 440 445
Ser Cys Ser Val Met His Gly Ala Leu His Asn His Tyr Thr Gln Lys
450 455 460
Ser Leu Ser Leu Ser Pro Gly Lys Arg Gly Arg Lys Arg Arg Ser Gly
465 470 475 480
Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu
485 490 495
Glu Asn Pro Gly Pro Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu
500 505 510
Leu Leu Trp Ile Ser Gly Ala Tyr Gly Glu Ile Val Leu Thr Gln Ser
515 520 525
Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys
530 535 540
Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala Trp Tyr Gln Gln
545 550 555 560
Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Gly Ala Ser Ser Arg
565 570 575
Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
580 585 590
Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr
595 600 605
Tyr Cys Gln His Tyr Gly Ser Ser Arg Gly Trp Thr Phe Gly Gln Gly
610 615 620
Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile
625 630 635 640
Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val
645 650 655
Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys
660 665 670
Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu
675 680 685
Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Asn Thr Leu Thr Leu
690 695 700
Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr
705 710 715 720
His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu
725 730 735
Cys
<210> 75
<211> 2217
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 75
atggactgga catggagaat cctgttcctg gtcgccgccg caactggcac tcacgctcag 60
atgcagctgg tgcagtctgg acccgaggtg aagaagcccg gcaccagcgt gaaggtgtcc 120
tgtaaggcct ctggcttcac ctttatgagc tccgccgtgc agtgggtgag gcaggccaga 180
ggccagcggc tggagtggat cggatggatc gtgatcggct ccggaaacac caattacgcc 240
cagaagttcc aggagcgcgt gaccatcaca agggacatgt ccacctctac agcctatatg 300
gagctgtcta gcctgcggtc cgaggataca gccgtgtact attgcgccgc cccttactgt 360
tcctctatca gctgcaacga cggcttcgat atctggggcc agggcaccat ggtgacagtg 420
agctccgcct ctaccaaggg cccaagcgtg tttcccctgg ccccttctag caagagcacc 480
tccggcggca cagccgccct gggctgtctg gtgaaggact acttccctga gccagtgaca 540
gtgagctgga actccggcgc cctgacctcc ggagtgcaca catttcctgc cgtgctgcag 600
tcctctggcc tgtacagcct gagctccgtg gtgaccgtgc catctagctc cctgggcacc 660
cagacatata tctgtaacgt gaatcacaag ccttccaata caaaggtgga caagaaggtg 720
gagccaaagt cttgcgataa gacccacaca tgccctccct gtccagcacc tgagttcgag 780
ggcggcccaa gcgtgttcct gtttccaccc aagcccaagg acaccctgat gatcagcagg 840
accccagagg tgacatgcgt ggtggtggac gtgtcccacg aggaccccga ggtgaagttt 900
aactggtacg tggatggcgt ggaggtgcac aatgccaaga ccaagcccag agaggagcag 960
tacaattcca cctatcgggt ggtgtctgtg ctgacagtgc tgcaccagga ctggctgaac 1020
ggcaaggagt acaagtgtaa ggtgtctaat aaggccctgc cagccagcat cgagaagacc 1080
atcagcaagg caaagggaca gccacgcgag ccacaggtgt atacactgcc tccatctagg 1140
gacgagctga ccaagaacca ggtgagcctg acatgcctgg tgaagggctt ctaccccagc 1200
gatatcgccg tggagtggga gtccaatggc cagcctgaga acaattacaa gaccacaccc 1260
cctgtgctgg actctgatgg cagcttcttt ctgtattcta agctgaccgt ggacaagagc 1320
agatggcagc agggcaacgt gttttcttgt agcgtgatgc acgaggccct gcacaatcac 1380
tacacccaga agtccctgtc tctgagccct ggcaagaggg gaaggaagag gagatccggc 1440
tctggcgcca caaacttcag cctgctgaag caggccggcg atgtggagga gaatcctggc 1500
ccaatggtgc tgcagaccca ggtgtttatc agcctgctgc tgtggatctc cggagcatat 1560
ggagagatcg tgctgaccca gtccccaggc acactgagcc tgtcccctgg agagagagcc 1620
accctgagct gcagggcatc tcagagcgtg tctagctcct acctggcctg gtatcagcag 1680
aagcctggcc aggccccaag actgctgatc tacggagcct ctagccgcgc caccggcatc 1740
cccgacaggt tctccggctc tggcagcggc acagacttca ccctgacaat ctcccggctg 1800
gagcctgagg acttcgccgt gtactactgt cagcactatg gctcctctcg gggctggacc 1860
tttggccagg gcacaaaggt ggagatcaag aggaccgtgg cagcaccatc cgtgttcatc 1920
tttccaccct ctgacgagca gctgaagagc ggcacagcct ccgtggtgtg cctgctgaac 1980
aatttctatc cccgcgaggc caaggtgcag tggaaggtgg ataacgccct gcagtccggc 2040
aattctcagg agagcgtgac cgagcaggac tccaaggatt ctacatactc cctgtctaac 2100
accctgacac tgagcaaggc cgattacgag aagcacaagg tgtatgcctg cgaggtgacc 2160
catcaggggc tgtcttctcc agtgaccaaa tccttcaatc gcggggaatg ttgataa 2217
<210> 76
<211> 737
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 76
Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly
1 5 10 15
Thr His Ala Gln Met Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys
20 25 30
Pro Gly Thr Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe
35 40 45
Thr Ser Ser Ala Val Gln Trp Val Arg Gln Ala Arg Gly Gln Arg Leu
50 55 60
Glu Trp Ile Gly Trp Ile Ala Val Gly Ser Gly Asn Thr Asn Tyr Ala
65 70 75 80
Gln Lys Phe Gln Glu Arg Val Ser Ile Thr Arg Asp Met Ser Thr Ser
85 90 95
Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
100 105 110
Tyr Tyr Cys Ala Ala Pro Tyr Cys Ser Ser Ile Ser Cys Asn Asp Gly
115 120 125
Phe Asp Ile Trp Gly Gln Gly Thr Lys Val Thr Val Ser Ser Ala Ser
130 135 140
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
145 150 155 160
Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
165 170 175
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
180 185 190
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
195 200 205
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
210 215 220
Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val
225 230 235 240
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
245 250 255
Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
260 265 270
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
275 280 285
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
290 295 300
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
305 310 315 320
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
325 330 335
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
340 345 350
Leu Pro Ala Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
355 360 365
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
370 375 380
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
385 390 395 400
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
405 410 415
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
420 425 430
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
435 440 445
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
450 455 460
Ser Leu Ser Leu Ser Pro Gly Lys Arg Gly Arg Lys Arg Arg Ser Gly
465 470 475 480
Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu
485 490 495
Glu Asn Pro Gly Pro Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu
500 505 510
Leu Leu Trp Ile Ser Gly Ala Tyr Gly Glu Val Val Leu Thr Gln Ser
515 520 525
Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys
530 535 540
Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala Trp Tyr Gln Gln
545 550 555 560
Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Gly Ala Ser Ser Arg
565 570 575
Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
580 585 590
Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr
595 600 605
His Cys Gln His Tyr Gly Ser Ser Ser Gln Trp Thr Phe Gly Gln Gly
610 615 620
Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile
625 630 635 640
Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val
645 650 655
Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys
660 665 670
Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu
675 680 685
Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Asn Thr Leu Thr Leu
690 695 700
Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr
705 710 715 720
His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu
725 730 735
Cys
<210> 77
<211> 15
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 77
agatactgga tgagt 15
<210> 78
<211> 51
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 78
gaaattaatc cagatagcag tacgataaac tatacgccat ctctaaagga t 51
<210> 79
<211> 33
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 79
atctactatg gttcccctgg tgctatggac tac 33
<210> 80
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 80
Arg Tyr Trp Met Ser
1 5
<210> 81
<211> 17
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 81
Glu Ile Asn Pro Asp Ser Ser Thr Ile Asn Tyr Thr Pro Ser Leu Lys
1 5 10 15
Asp
<210> 82
<211> 11
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 82
Ile Tyr Tyr Gly Ser Pro Gly Ala Met Asp Tyr
1 5 10
<210> 83
<211> 414
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 83
atggattttg ggctgatttt ttttattgtt gctcttttaa aaggggtcca gtgtgaggtg 60
aagcttctcg agtctggagg tggcctggtg cagcctggag gatccctgaa actctcctgt 120
gcagcctcag gattcgattt tagtagatac tggatgagtt gggtccggca ggctccaggg 180
aaagggctag aatggattgg agaaattaat ccagatagca gtacgataaa ctatacgcca 240
tctctaaagg ataaattcat catctccaga gacaacgcca aaaatacgct gtacctgcaa 300
atgagcaaag tgagatctga ggacacagcc ctttattact gtgcaaggat ctactatggt 360
tcccctggtg ctatggacta ctggggtcaa ggaacctcag tcaccgtctc ctca 414
<210> 84
<211> 138
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 84
Met Asp Phe Gly Leu Ile Phe Phe Ile Val Ala Leu Leu Lys Gly Val
1 5 10 15
Gln Cys Glu Val Lys Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
20 25 30
Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe Ser
35 40 45
Arg Tyr Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
50 55 60
Trp Ile Gly Glu Ile Asn Pro Asp Ser Ser Thr Ile Asn Tyr Thr Pro
65 70 75 80
Ser Leu Lys Asp Lys Phe Ile Ile Ser Arg Asp Asn Ala Lys Asn Thr
85 90 95
Leu Tyr Leu Gln Met Ser Lys Val Arg Ser Glu Asp Thr Ala Leu Tyr
100 105 110
Tyr Cys Ala Arg Ile Tyr Tyr Gly Ser Pro Gly Ala Met Asp Tyr Trp
115 120 125
Gly Gln Gly Thr Ser Val Thr Val Ser Ser
130 135
<210> 85
<211> 33
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 85
aaggccagtc agagtgtgag taatgatgta gct 33
<210> 86
<211> 21
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 86
tatgcatcca atcgctacac t 21
<210> 87
<211> 27
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 87
cagcaggatt atttctctcc gtacacg 27
<210> 88
<211> 11
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 88
Lys Ala Ser Gln Ser Val Ser Asn Asp Val Ala
1 5 10
<210> 89
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 89
Tyr Ala Ser Asn Arg Tyr Thr
1 5
<210> 90
<211> 9
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 90
Gln Gln Asp Tyr Phe Ser Pro Tyr Thr
1 5
<210> 91
<211> 381
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 91
atgaagtcac agacccaggt cttcgtattt ctactgctct gtgtgtctgg tgctcatggg 60
agtattgtga tgacccagac tcccaaattc ctgcttgtat cagcaggaga cagggttacc 120
ataacctgca aggccagtca gagtgtgagt aatgatgtag cttggtacca acagaagcca 180
gggcagtctc ctaaactgct gatatactat gcatccaatc gctacactgg agtccctgat 240
cgcttcactg gcagtggata tgggacggat ttcactttca ccatcagcac tgtgcaggct 300
gaagacctgg cagtttattt ctgtcagcag gattatttct ctccgtacac gttcggaggg 360
gggaccaagc tggaaataaa a 381
<210> 92
<211> 127
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 92
Met Lys Ser Gln Thr Gln Val Phe Val Phe Leu Leu Leu Cys Val Ser
1 5 10 15
Gly Ala His Gly Ser Ile Val Met Thr Gln Thr Pro Lys Phe Leu Leu
20 25 30
Val Ser Ala Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser
35 40 45
Val Ser Asn Asp Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro
50 55 60
Lys Leu Leu Ile Tyr Tyr Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp
65 70 75 80
Arg Phe Thr Gly Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser
85 90 95
Thr Val Gln Ala Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln Asp Tyr
100 105 110
Phe Ser Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
115 120 125
<210> 93
<211> 360
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 93
gaggtgaagc tgctggagag cggaggagga ctggtgcagc ctggaggcag cctgaagctg 60
tcctgcgctg cctctggatt cgacttttcc aggtactgga tgtcttgggt gagacaggcc 120
ccaggcaagg gactggagtg gatcggcgag atcaacccag atagctccac aatcaactac 180
accccctccc tgaaggacaa gttcatcatc tctagagata acgctaagaa cacactgtac 240
ctgcagatga gcaaggtgag gtccgaggac accgctctgt actactgcgc cagaatctac 300
tacggcagcc ctggcgccat ggattactgg ggccagggaa catctgtgac cgtgagcagc 360
<210> 94
<211> 120
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 94
Glu Val Lys Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe Ser Arg Tyr
20 25 30
Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Glu Ile Asn Pro Asp Ser Ser Thr Ile Asn Tyr Thr Pro Ser Leu
50 55 60
Lys Asp Lys Phe Ile Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Ser Lys Val Arg Ser Glu Asp Thr Ala Leu Tyr Tyr Cys
85 90 95
Ala Arg Ile Tyr Tyr Gly Ser Pro Gly Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Ser Val Thr Val Ser Ser
115 120
<210> 95
<211> 321
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 95
tccatcgtga tgacacagac ccccaagttt ctgctggtgt ccgccggcga cagggtgaca 60
atcacctgta aggcttccca gtctgtgagc aacgatgtgg cctggtacca gcagaagcct 120
ggacagtccc caaagctgct gatctactac gcctctaacc ggtacacagg cgtgcctgac 180
cgcttcacag gctctggata cggcaccgac ttcaccttca ccatcagcac cgtgcaggct 240
gaggacctgg ccgtgtactt ttgccagcag gattacttca gcccatacac atttggaggc 300
ggaaccaagc tggagatcaa g 321
<210> 96
<211> 107
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 96
Ser Ile Val Met Thr Gln Thr Pro Lys Phe Leu Leu Val Ser Ala Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Ser Asn Asp
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile
35 40 45
Tyr Tyr Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly
50 55 60
Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser Thr Val Gln Ala
65 70 75 80
Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln Asp Tyr Phe Ser Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 97
<211> 2178
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 97
atggactgga cctggagaat cctgttcctg gtggctgctg ctaccggaac acacgctgag 60
gtgaagctgc tggagagcgg aggaggactg gtgcagcctg gaggcagcct gaagctgtcc 120
tgcgctgcct ctggattcga cttttccagg tactggatgt cttgggtgag acaggcccca 180
ggcaagggac tggagtggat cggcgagatc aacccagata gctccacaat caactacacc 240
ccctccctga aggacaagtt catcatctct agagataacg ctaagaacac actgtacctg 300
cagatgagca aggtgaggtc cgaggacacc gctctgtact actgcgccag aatctactac 360
ggcagccctg gcgccatgga ttactggggc cagggaacat ctgtgaccgt gagcagcgcc 420
aagaccacac cacctagcgt gtacccactg gctcctggct ccgctgctca gacaaactct 480
atggtgaccc tgggatgtct ggtgaagggc tacttccctg agccagtgac cgtgacatgg 540
aactctggaa gcctgtcctc tggcgtgcac acctttcccg ccgtgctgca gtccgacctg 600
tacacactga gctcctctgt gaccgtgcct agctccccca ggcctagcga gaccgtgaca 660
tgcaacgtgg ctcaccccgc ctctagcaca aaggtggaca agaagatcgt gcctagagat 720
tgcggctgta agccctgcat ctgtaccgtg cctgaggtga gcagcgtgtt catctttcca 780
cccaagccta aggacgtgct gaccatcaca ctgaccccaa aggtgacctg cgtggtggtg 840
gatatctcta aggacgatcc agaggtgcag ttcagctggt ttgtggacga tgtggaggtg 900
cacacagctc agacccagcc tagggaggag cagttcaaca gcaccttcag gagcgtgagc 960
gagctgccaa tcatgcacca ggattggctg aacggaaagg agttcaagtg ccgggtgaac 1020
tccgctgcct ttccagcccc catcgagaag acaatctcta agaccaaggg acagccacgg 1080
gagccacagg tgtacaccct gcctccaagc cgcgacgagc tgacaaagaa ccaggtgtcc 1140
ctgacctgtc tggtgaaggg attctaccca agcgatatcg ctgtggagtg ggagtccaac 1200
ggccagcccg agaacaacta caagaccaca ccccctgtgc tggactccga tggatctttc 1260
tttctgtaca gcaagctgac cgtggacaag tcccgctggc agcagggcaa cgtgttcagc 1320
tgctccgtga tgcacgaggc cctgcacaac cactacacac agaagtctct gagcctgtcc 1380
ccaggaaaga ggggaaggaa gaggagatct ggcagcggag ctaccaactt ttccctgctg 1440
aagcaggccg gagatgtgga ggagaaccct ggcccaatgg tgctgcagac acaggtgttc 1500
atcagcctgc tgctgtggat cagcggagct tacggctcca tcgtgatgac acagaccccc 1560
aagtttctgc tggtgtccgc cggcgacagg gtgacaatca cctgtaaggc ttcccagtct 1620
gtgagcaacg atgtggcctg gtaccagcag aagcctggac agtccccaaa gctgctgatc 1680
tactacgcct ctaaccggta cacaggcgtg cctgaccgct tcacaggctc tggatacggc 1740
accgacttca ccttcaccat cagcaccgtg caggctgagg acctggccgt gtacttttgc 1800
cagcaggatt acttcagccc atacacattt ggaggcggaa ccaagctgga gatcaagcgg 1860
acagtggctg cccctagcgt gttcatcttc ccacccagcg acgagcagct gaagtctgga 1920
accgccagcg tggtgtgcct gctgaacaac ttctacccac gcgaggctaa ggtgcagtgg 1980
aaggtggata acgccctgca gagcggcaac tcccaggagt ctgtgacaga gcaggacagc 2040
aaggattcca cctactctct gagctccaca ctgaccctgt ccaaggctga ctacgagaag 2100
cacaaggtgt acgcctgcga ggtgacacac cagggactgc ggtctcccgt gaccaagagc 2160
tttaaccgcg gcgagtgt 2178
<210> 98
<211> 726
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 98
Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly
1 5 10 15
Thr His Ala Glu Val Lys Leu Leu Glu Ser Gly Gly Gly Leu Val Gln
20 25 30
Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe
35 40 45
Ser Arg Tyr Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
50 55 60
Glu Trp Ile Gly Glu Ile Asn Pro Asp Ser Ser Thr Ile Asn Tyr Thr
65 70 75 80
Pro Ser Leu Lys Asp Lys Phe Ile Ile Ser Arg Asp Asn Ala Lys Asn
85 90 95
Thr Leu Tyr Leu Gln Met Ser Lys Val Arg Ser Glu Asp Thr Ala Leu
100 105 110
Tyr Tyr Cys Ala Arg Ile Tyr Tyr Gly Ser Pro Gly Ala Met Asp Tyr
115 120 125
Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro
130 135 140
Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser
145 150 155 160
Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val
165 170 175
Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe
180 185 190
Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr
195 200 205
Val Pro Ser Ser Pro Arg Pro Ser Glu Thr Val Thr Cys Asn Val Ala
210 215 220
His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp
225 230 235 240
Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val
245 250 255
Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr
260 265 270
Pro Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu
275 280 285
Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln
290 295 300
Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser
305 310 315 320
Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys
325 330 335
Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile
340 345 350
Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
355 360 365
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
370 375 380
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
385 390 395 400
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
405 410 415
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
420 425 430
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
435 440 445
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Arg
450 455 460
Gly Arg Lys Arg Arg Ser Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu
465 470 475 480
Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Val Leu Gln
485 490 495
Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser Gly Ala Tyr Gly
500 505 510
Ser Ile Val Met Thr Gln Thr Pro Lys Phe Leu Leu Val Ser Ala Gly
515 520 525
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Ser Asn Asp
530 535 540
Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile
545 550 555 560
Tyr Tyr Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly
565 570 575
Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser Thr Val Gln Ala
580 585 590
Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln Asp Tyr Phe Ser Pro Tyr
595 600 605
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
610 615 620
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
625 630 635 640
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
645 650 655
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
660 665 670
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
675 680 685
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
690 695 700
Ala Cys Glu Val Thr His Gln Gly Leu Arg Ser Pro Val Thr Lys Ser
705 710 715 720
Phe Asn Arg Gly Glu Cys
725
<210> 99
<211> 360
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 99
gaggtcaaac tgctggagag tgggggaggg ctggtgcagc ctggcggctc cctgaagctg 60
tcttgcgcag caagcggctt cgacttttct cggtactgga tgagctgggt gagacaggca 120
ccaggcaagg gcctggagtg gatcggcgag atcaaccccg atagctccac catcaattac 180
acaccttctc tgaaggacaa gttcatcatc agcagggata acgccaagaa taccctgtat 240
ctgcagatgt ccaaggtgag gtctgaggac acagccctgt actattgcgc ccgcatctac 300
tatggcagcc caggcgccat ggattactgg ggccagggca cctccgtgac agtgtctagc 360
<210> 100
<211> 120
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 100
Glu Val Lys Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe Ser Arg Tyr
20 25 30
Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Glu Ile Asn Pro Asp Ser Ser Thr Ile Asn Tyr Thr Pro Ser Leu
50 55 60
Lys Asp Lys Phe Ile Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Ser Lys Val Arg Ser Glu Asp Thr Ala Leu Tyr Tyr Cys
85 90 95
Ala Arg Ile Tyr Tyr Gly Ser Pro Gly Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Ser Val Thr Val Ser Ser
115 120
<210> 101
<211> 324
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 101
tccatcgtga tgacccagac acccaagttt ctgctggtga gcgccggcga cagagtgacc 60
atcacctgta aggccagcca gtccgtgtct aacgatgtgg cctggtatca gcagaagcct 120
ggccagagcc caaagctgct gatctactat gcctccaata ggtacaccgg agtgcctgac 180
cgcttcaccg gctccggcta tggcacagat ttcaccttta caatctctac agtgcaggcc 240
gaggacctgg ccgtgtactt ttgccagcag gattacttct ccccctatac cttcggcggc 300
ggcacaaagc tggagatcaa gagg 324
<210> 102
<211> 108
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 102
Ser Ile Val Met Thr Gln Thr Pro Lys Phe Leu Leu Val Ser Ala Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Ser Asn Asp
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile
35 40 45
Tyr Tyr Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly
50 55 60
Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser Thr Val Gln Ala
65 70 75 80
Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln Asp Tyr Phe Ser Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
100 105
<210> 103
<211> 2202
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 103
atggactgga cttggagaat cctgttcctg gtcgctgctg ctaccggcac ccacgctgag 60
gtcaaactgc tggagagtgg gggagggctg gtgcagcctg gcggctccct gaagctgtct 120
tgcgcagcaa gcggcttcga cttttctcgg tactggatga gctgggtgag acaggcacca 180
ggcaagggcc tggagtggat cggcgagatc aaccccgata gctccaccat caattacaca 240
ccttctctga aggacaagtt catcatcagc agggataacg ccaagaatac cctgtatctg 300
cagatgtcca aggtgaggtc tgaggacaca gccctgtact attgcgcccg catctactat 360
ggcagcccag gcgccatgga ttactggggc cagggcacct ccgtgacagt gtctagcgcc 420
tccaccaagg gaccaagcgt gttcccactg gcaccttcct ctaagagcac ctccggcggc 480
acagccgccc tgggctgtct ggtgaaggac tatttccctg agccagtgac cgtgagctgg 540
aactccggcg ccctgaccag cggagtgcac acatttcctg ccgtgctgca gagctccggc 600
ctgtactccc tgtctagcgt ggtgaccgtg ccatcctcta gcctgggcac ccagacatat 660
atctgcaacg tgaatcacaa gccttctaat acaaaggtgg acaagaaggt ggagccaaag 720
agctgtgata agacccacac atgccctccc tgtccagcac ctgagctgct gggcggccca 780
agcgtgttcc tgtttccacc caagcccaag gacaccctga tgatctccag aaccccagag 840
gtgacatgcg tggtggtgga cgtgtctcac gaggaccccg aggtgaagtt caactggtac 900
gtggatggcg tggaggtgca caatgccaag accaagccac gggaggagca gtacaactct 960
acctatagag tggtgagcgt gctgacagtg ctgcaccagg attggctgaa cggcaaggag 1020
tataagtgca aggtgagcaa taaggccctg ccagccccca tcgagaagac catctccaag 1080
gcaaagggac agccacggga gccacaggtg tacacactgc ctccatctag agacgagctg 1140
accaagaacc aggtgagcct gacatgtctg gtgaagggct tttatccctc cgatatcgcc 1200
gtggagtggg agtctaatgg ccagcctgag aacaattaca agaccacacc ccctgtgctg 1260
gactctgatg gcagcttctt tctgtattcc aagctgaccg tggacaagtc tcggtggcag 1320
cagggcaacg tgttctcttg cagcgtgatg cacgaggccc tgcacaatca ctacacccag 1380
aagtccctgt ctctgagccc tggcaagagg ggaaggaagc ggagatccgg ctctggagcc 1440
acaaacttta gcctgctgaa gcaggccggc gatgtggagg agaatcctgg cccaatggtg 1500
ctgcagaccc aggtgttcat ctccctgctg ctgtggatca gcggcgccta cggctccatc 1560
gtgatgaccc agacacccaa gtttctgctg gtgagcgccg gcgacagagt gaccatcacc 1620
tgtaaggcca gccagtccgt gtctaacgat gtggcctggt atcagcagaa gcctggccag 1680
agcccaaagc tgctgatcta ctatgcctcc aataggtaca ccggagtgcc tgaccgcttc 1740
accggctccg gctatggcac agatttcacc tttacaatct ctacagtgca ggccgaggac 1800
ctggccgtgt acttttgcca gcaggattac ttctccccct ataccttcgg cggcggcaca 1860
aagctggaga tcaagaggac cgtggccgcc cctagcgtgt tcatctttcc accctccgac 1920
gagcagctga agagcggcac agcctccgtg gtgtgcctgc tgaacaattt ctacccccgc 1980
gaggccaagg tgcagtggaa ggtggataac gccctgcagt ccggcaattc tcaggagagc 2040
gtgaccgagc aggactccaa ggattctaca tatagcctgt cctctaccct gacactgagc 2100
aaggccgact acgagaagca caaggtgtat gcctgcgagg tcactcacca ggggctgcgc 2160
tcaccagtca caaaatcttt caatagaggg gaatgctgat aa 2202
<210> 104
<211> 732
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 104
Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly
1 5 10 15
Thr His Ala Glu Val Lys Leu Leu Glu Ser Gly Gly Gly Leu Val Gln
20 25 30
Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe
35 40 45
Ser Arg Tyr Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
50 55 60
Glu Trp Ile Gly Glu Ile Asn Pro Asp Ser Ser Thr Ile Asn Tyr Thr
65 70 75 80
Pro Ser Leu Lys Asp Lys Phe Ile Ile Ser Arg Asp Asn Ala Lys Asn
85 90 95
Thr Leu Tyr Leu Gln Met Ser Lys Val Arg Ser Glu Asp Thr Ala Leu
100 105 110
Tyr Tyr Cys Ala Arg Ile Tyr Tyr Gly Ser Pro Gly Ala Met Asp Tyr
115 120 125
Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly
130 135 140
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
145 150 155 160
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
165 170 175
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
180 185 190
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
195 200 205
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
210 215 220
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
225 230 235 240
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
245 250 255
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
260 265 270
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
275 280 285
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
290 295 300
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
305 310 315 320
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
325 330 335
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
340 345 350
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
355 360 365
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
370 375 380
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
385 390 395 400
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
405 410 415
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
420 425 430
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
435 440 445
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
450 455 460
Leu Ser Pro Gly Lys Arg Gly Arg Lys Arg Arg Ser Gly Ser Gly Ala
465 470 475 480
Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro
485 490 495
Gly Pro Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp
500 505 510
Ile Ser Gly Ala Tyr Gly Ser Ile Val Met Thr Gln Thr Pro Lys Phe
515 520 525
Leu Leu Val Ser Ala Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser
530 535 540
Gln Ser Val Ser Asn Asp Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln
545 550 555 560
Ser Pro Lys Leu Leu Ile Tyr Tyr Ala Ser Asn Arg Tyr Thr Gly Val
565 570 575
Pro Asp Arg Phe Thr Gly Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr
580 585 590
Ile Ser Thr Val Gln Ala Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln
595 600 605
Asp Tyr Phe Ser Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
610 615 620
Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
625 630 635 640
Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
645 650 655
Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
660 665 670
Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
675 680 685
Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
690 695 700
Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Arg
705 710 715 720
Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
725 730
<210> 105
<211> 15
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 105
gacacctata tgcac 15
<210> 106
<211> 51
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 106
aggattgatc ctgcgaatgg taatactaaa tatgacccga agttccaggc c 51
<210> 107
<211> 30
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 107
tactacgact acgtgggagc tatggactac 30
<210> 108
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 108
Asp Thr Tyr Met His
1 5
<210> 109
<211> 17
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 109
Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Pro Lys Phe Gln
1 5 10 15
Ala
<210> 110
<211> 10
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 110
Tyr Tyr Asp Tyr Val Gly Ala Met Asp Tyr
1 5 10
<210> 111
<211> 414
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 111
atgaaatgca gctgggttat cttcttcctg atggcagtgg ttacaggggt caattcagag 60
gttcagctgc agcagtctgg ggcagagctt gtgaagccag gggcctcagt caagttgtcc 120
tgcacagctt ctggcttcaa cattaaagac acctatatgc actgggtgaa gcagaggcct 180
gaacagggcc tggagtggat tggaaggatt gatcctgcga atggtaatac taaatatgac 240
ccgaagttcc aggccaaggc cactataaca gcaaacacat cctccaacac agcctacctg 300
cacctcagca gcctgacatc tgaggacact gccgtctatt actgtgcccc atactacgac 360
tacgtgggag ctatggacta ctggggtcaa ggaacctcag tcaccgtctc ctca 414
<210> 112
<211> 138
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 112
Met Lys Cys Ser Trp Val Ile Phe Phe Leu Met Ala Val Val Thr Gly
1 5 10 15
Val Asn Ser Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys
20 25 30
Pro Gly Ala Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile
35 40 45
Lys Asp Thr Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu
50 55 60
Glu Trp Ile Gly Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp
65 70 75 80
Pro Lys Phe Gln Ala Lys Ala Thr Ile Thr Ala Asn Thr Ser Ser Asn
85 90 95
Thr Ala Tyr Leu His Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val
100 105 110
Tyr Tyr Cys Ala Pro Tyr Tyr Asp Tyr Val Gly Ala Met Asp Tyr Trp
115 120 125
Gly Gln Gly Thr Ser Val Thr Val Ser Ser
130 135
<210> 113
<211> 33
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 113
ctggcaagtc agaccattgg tacatggtta gca 33
<210> 114
<211> 21
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 114
gctgcaacca gcttggcaga t 21
<210> 115
<211> 27
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 115
caacaacttt acaatactcc tctgacg 27
<210> 116
<211> 11
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 116
Leu Ala Ser Gln Thr Ile Gly Thr Trp Leu Ala
1 5 10
<210> 117
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 117
Ala Ala Thr Ser Leu Ala Asp
1 5
<210> 118
<211> 9
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 118
Gln Gln Leu Tyr Asn Thr Pro Leu Thr
1 5
<210> 119
<211> 381
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 119
atgaacatgc tcactcagct cctgggatta ctgctgctct ggtttgcagg tggtaaatgt 60
gacattcaga tgacccagtc tcctgcctcc cagtctgcat ctctgggaga aagtgtcacc 120
atcacatgcc tggcaagtca gaccattggt acatggttag catggtatca gcagaaacca 180
gggaaatctc ctcagctcct gatttatgct gcaaccagct tggcagatgg ggtcccatca 240
aggttcagtg gtagtggatc tggcacaaaa ttttctttca agatcagcag cctacaggct 300
gaagattttg taagttatta ctgtcaacaa ctttacaata ctcctctgac gttcggtgga 360
ggcaccaagc tggaaatcaa a 381
<210> 120
<211> 127
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 120
Met Asn Met Leu Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp Phe Ala
1 5 10 15
Gly Gly Lys Cys Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Gln Ser
20 25 30
Ala Ser Leu Gly Glu Ser Val Thr Ile Thr Cys Leu Ala Ser Gln Thr
35 40 45
Ile Gly Thr Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro
50 55 60
Gln Leu Leu Ile Tyr Ala Ala Thr Ser Leu Ala Asp Gly Val Pro Ser
65 70 75 80
Arg Phe Ser Gly Ser Gly Ser Gly Thr Lys Phe Ser Phe Lys Ile Ser
85 90 95
Ser Leu Gln Ala Glu Asp Phe Val Ser Tyr Tyr Cys Gln Gln Leu Tyr
100 105 110
Asn Thr Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
115 120 125
<210> 121
<211> 357
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 121
gaggtgcagc tgcagcagtc cggagctgag ctggtgaagc caggagccag cgtgaagctg 60
tcctgcacag cctctggctt caacatcaag gatacctaca tgcactgggt gaagcagagg 120
ccagagcagg gactggagtg gatcggaaga atcgaccccg ccaacggcaa caccaagtac 180
gatcctaagt tccaggctaa ggccaccatc acagctaaca caagctccaa caccgcctac 240
ctgcacctgt ctagcctgac aagcgaggac accgccgtgt actactgtgc cccttactac 300
gactacgtgg gcgctatgga ttactgggga cagggcacat ctgtgaccgt gtcctct 357
<210> 122
<211> 119
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 122
Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile
35 40 45
Gly Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Pro Lys Phe
50 55 60
Gln Ala Lys Ala Thr Ile Thr Ala Asn Thr Ser Ser Asn Thr Ala Tyr
65 70 75 80
Leu His Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Pro Tyr Tyr Asp Tyr Val Gly Ala Met Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Ser Val Thr Val Ser Ser
115
<210> 123
<211> 330
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 123
aagtgcgata tccagatgac ccagtcccca gcttctcaga gcgcctccct gggagagtct 60
gtgaccatca catgtctggc tagccagaca atcggcacct ggctggcctg gtaccagcag 120
aagccaggaa agagccccca gctgctgatc tacgctgcca caagcctggc tgacggagtg 180
ccctcccggt tttctggaag cggctccgga accaagttct cctttaagat cagctccctg 240
caggccgagg atttcgtgtc ttactactgc cagcagctgt acaacacacc cctgaccttt 300
ggcggaggca ccaagctgga gatcaagcgc 330
<210> 124
<211> 110
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 124
Lys Cys Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Gln Ser Ala Ser
1 5 10 15
Leu Gly Glu Ser Val Thr Ile Thr Cys Leu Ala Ser Gln Thr Ile Gly
20 25 30
Thr Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Gln Leu
35 40 45
Leu Ile Tyr Ala Ala Thr Ser Leu Ala Asp Gly Val Pro Ser Arg Phe
50 55 60
Ser Gly Ser Gly Ser Gly Thr Lys Phe Ser Phe Lys Ile Ser Ser Leu
65 70 75 80
Gln Ala Glu Asp Phe Val Ser Tyr Tyr Cys Gln Gln Leu Tyr Asn Thr
85 90 95
Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
100 105 110
<210> 125
<211> 2214
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 125
atggactgga cctggcgcat cctgttcctg gtggctgctg ctaccggaac acacgctgag 60
gtgcagctgc agcagtccgg agctgagctg gtgaagccag gagccagcgt gaagctgtcc 120
tgcacagcct ctggcttcaa catcaaggat acctacatgc actgggtgaa gcagaggcca 180
gagcagggac tggagtggat cggaagaatc gaccccgcca acggcaacac caagtacgat 240
cctaagttcc aggctaaggc caccatcaca gctaacacaa gctccaacac cgcctacctg 300
cacctgtcta gcctgacaag cgaggacacc gccgtgtact actgtgcccc ttactacgac 360
tacgtgggcg ctatggatta ctggggacag ggcacatctg tgaccgtgtc ctctgctaag 420
accacagctc caagcgtgta cccactggct cccgtgtgcg gaggaaccac aggcagctcc 480
gtgaccctgg gctgtctggt gaagggatac ttcccagagc ccgtgaccct gacatggaac 540
agcggctccc tgtctagcgg agtgcacaca tttcctgccc tgctgcagtc cggcctgtac 600
accctgtcct ctagcgtgac cgtgacatct aacacatggc caagccagac catcacatgc 660
aacgtggctc accccgcctc ctctacaaag gtggacaaga agatcgagcc tcgggtgcca 720
atcacccaga acccatgtcc ccctctgaag gagtgcccac cctgtgctgc ccctaacctg 780
ctgggcggac caagcgtgtt catctttcct ccaaagatca aggatgtgct gatgatctct 840
ctgagcccta tcgtgacctg cgtggtggtg gacgtgtccg aggacgatcc agatgtgcag 900
atctcttggt ttgtgaacaa cgtggaggtg cacacagctc agacccagac acacagggag 960
gactacaaca gcaccctgag agtggtgtcc gccctgccaa tccagcacca ggactggatg 1020
tccggcaagg agttcaagtg caaggtgaac aacaaggatc tgcctgcccc aatcgagcgg 1080
acaatctcta agccaaaggg acctgtgcgc gctccacagg tgtacgtgct gccacctcca 1140
gctgaggaga tgaccaagaa ggagttctct ctgacatgta tgatcaccgg ctttctgcct 1200
gctgagatcg ccgtggattg gacaagcaac ggaagaaccg agcagaacta caagaacacc 1260
gctacagtgc tggactccga tggctcttac ttcatgtact ccaagctgcg ggtgcagaag 1320
tctacctggg agcgcggcag cctgtttgcc tgttctgtgg tgcacgaggt gctgcacaac 1380
cacctgacca caaagacaat ctcccggtct ctgggcaaga ggggaaggaa gaggagaagc 1440
ggcagcggcg ctaccaactt ctccctgctg aagcaggctg gcgacgtgga ggagaaccca 1500
ggacctatgg tgctgcagac acaggtgttt atctctctgc tgctgtggat cagcggcgcc 1560
tacggaaagt gcgatatcca gatgacccag tccccagctt ctcagagcgc ctccctggga 1620
gagtctgtga ccatcacatg tctggctagc cagacaatcg gcacctggct ggcctggtac 1680
cagcagaagc caggaaagag cccccagctg ctgatctacg ctgccacaag cctggctgac 1740
ggagtgccct cccggttttc tggaagcggc tccggaacca agttctcctt taagatcagc 1800
tccctgcagg ccgaggattt cgtgtcttac tactgccagc agctgtacaa cacacccctg 1860
acctttggcg gaggcaccaa gctggagatc aagcgcgctg acgctgcccc tacagtgagc 1920
atcttccccc cttctagcga gcagctgacc agcggaggag cttccgtggt gtgcttcctg 1980
aacaactttt accccaagga catcaacgtg aagtggaaga tcgatggcag cgagaggcag 2040
aacggagtgc tgaactcctg gacagaccag gatagcaagg actccaccta ctctatgtcc 2100
tctaccctga cactgaccaa ggatgagtac gagaggcaca acagctacac atgcgaggcc 2160
acccacaaga catctaccag ccctatcgtg aagtccttca acagaaacga gtgt 2214
<210> 126
<211> 738
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 126
Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly
1 5 10 15
Thr His Ala Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys
20 25 30
Pro Gly Ala Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile
35 40 45
Lys Asp Thr Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu
50 55 60
Glu Trp Ile Gly Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp
65 70 75 80
Pro Lys Phe Gln Ala Lys Ala Thr Ile Thr Ala Asn Thr Ser Ser Asn
85 90 95
Thr Ala Tyr Leu His Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val
100 105 110
Tyr Tyr Cys Ala Pro Tyr Tyr Asp Tyr Val Gly Ala Met Asp Tyr Trp
115 120 125
Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr Ala Pro
130 135 140
Ser Val Tyr Pro Leu Ala Pro Val Cys Gly Gly Thr Thr Gly Ser Ser
145 150 155 160
Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr
165 170 175
Leu Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro
180 185 190
Ala Leu Leu Gln Ser Gly Leu Tyr Thr Leu Ser Ser Ser Val Thr Val
195 200 205
Thr Ser Asn Thr Trp Pro Ser Gln Thr Ile Thr Cys Asn Val Ala His
210 215 220
Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Val Pro
225 230 235 240
Ile Thr Gln Asn Pro Cys Pro Pro Leu Lys Glu Cys Pro Pro Cys Ala
245 250 255
Ala Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys
260 265 270
Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys Val
275 280 285
Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe
290 295 300
Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg Glu
305 310 315 320
Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln His
325 330 335
Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys
340 345 350
Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly Pro
355 360 365
Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Ala Glu Glu Met
370 375 380
Thr Lys Lys Glu Phe Ser Leu Thr Cys Met Ile Thr Gly Phe Leu Pro
385 390 395 400
Ala Glu Ile Ala Val Asp Trp Thr Ser Asn Gly Arg Thr Glu Gln Asn
405 410 415
Tyr Lys Asn Thr Ala Thr Val Leu Asp Ser Asp Gly Ser Tyr Phe Met
420 425 430
Tyr Ser Lys Leu Arg Val Gln Lys Ser Thr Trp Glu Arg Gly Ser Leu
435 440 445
Phe Ala Cys Ser Val Val His Glu Val Leu His Asn His Leu Thr Thr
450 455 460
Lys Thr Ile Ser Arg Ser Leu Gly Lys Arg Gly Arg Lys Arg Arg Ser
465 470 475 480
Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val
485 490 495
Glu Glu Asn Pro Gly Pro Met Val Leu Gln Thr Gln Val Phe Ile Ser
500 505 510
Leu Leu Leu Trp Ile Ser Gly Ala Tyr Gly Lys Cys Asp Ile Gln Met
515 520 525
Thr Gln Ser Pro Ala Ser Gln Ser Ala Ser Leu Gly Glu Ser Val Thr
530 535 540
Ile Thr Cys Leu Ala Ser Gln Thr Ile Gly Thr Trp Leu Ala Trp Tyr
545 550 555 560
Gln Gln Lys Pro Gly Lys Ser Pro Gln Leu Leu Ile Tyr Ala Ala Thr
565 570 575
Ser Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly
580 585 590
Thr Lys Phe Ser Phe Lys Ile Ser Ser Leu Gln Ala Glu Asp Phe Val
595 600 605
Ser Tyr Tyr Cys Gln Gln Leu Tyr Asn Thr Pro Leu Thr Phe Gly Gly
610 615 620
Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala Pro Thr Val Ser
625 630 635 640
Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly Gly Ala Ser Val
645 650 655
Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn Val Lys Trp
660 665 670
Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu Asn Ser Trp Thr
675 680 685
Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser Ser Thr Leu Thr
690 695 700
Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr Thr Cys Glu Ala
705 710 715 720
Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser Phe Asn Arg Asn
725 730 735
Glu Cys
<210> 127
<211> 357
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 127
gaagtgcagc tgcagcagtc tggagccgaa ctggtgaagc caggcgcctc cgtgaagctg 60
tcttgcacag ccagcggctt caacatcaag gacacctaca tgcactgggt gaagcagagg 120
cccgagcagg gcctggagtg gatcggaagg atcgacccag ccaacggcaa taccaagtac 180
gatcccaagt ttcaggccaa ggccaccatc acagccaaca caagctccaa taccgcctat 240
ctgcacctgt ctagcctgac aagcgaggat accgccgtgt actattgtgc cccttactat 300
gactacgtgg gcgccatgga ttattggggc cagggcacat ccgtgaccgt gtcctct 357
<210> 128
<211> 119
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 128
Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile
35 40 45
Gly Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Pro Lys Phe
50 55 60
Gln Ala Lys Ala Thr Ile Thr Ala Asn Thr Ser Ser Asn Thr Ala Tyr
65 70 75 80
Leu His Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Pro Tyr Tyr Asp Tyr Val Gly Ala Met Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Ser Val Thr Val Ser Ser
115
<210> 129
<211> 330
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 129
aagtgcgata tccagatgac ccagagccca gcaagccagt ccgcctctct gggagagtct 60
gtgacaatca cctgtctggc cagccagaca atcggcacct ggctggcctg gtaccagcag 120
aagcctggca agtccccaca gctgctgatc tatgcagcca catctctggc agacggagtg 180
cctagccgct tcagcggctc cggctctgga accaagttct cctttaagat cagctccctg 240
caggccgagg atttcgtgtc ttactattgc cagcagctgt acaacacccc tctgaccttc 300
ggcggcggca caaagctgga gatcaagagg 330
<210> 130
<211> 110
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 130
Lys Cys Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Gln Ser Ala Ser
1 5 10 15
Leu Gly Glu Ser Val Thr Ile Thr Cys Leu Ala Ser Gln Thr Ile Gly
20 25 30
Thr Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Gln Leu
35 40 45
Leu Ile Tyr Ala Ala Thr Ser Leu Ala Asp Gly Val Pro Ser Arg Phe
50 55 60
Ser Gly Ser Gly Ser Gly Thr Lys Phe Ser Phe Lys Ile Ser Ser Leu
65 70 75 80
Gln Ala Glu Asp Phe Val Ser Tyr Tyr Cys Gln Gln Leu Tyr Asn Thr
85 90 95
Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
100 105 110
<210> 131
<211> 2193
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 131
atggattgga cctggaggat tctgtttctg gtcgccgccg ctactggaac tcacgccgaa 60
gtgcagctgc agcagtctgg agccgaactg gtgaagccag gcgcctccgt gaagctgtct 120
tgcacagcca gcggcttcaa catcaaggac acctacatgc actgggtgaa gcagaggccc 180
gagcagggcc tggagtggat cggaaggatc gacccagcca acggcaatac caagtacgat 240
cccaagtttc aggccaaggc caccatcaca gccaacacaa gctccaatac cgcctatctg 300
cacctgtcta gcctgacaag cgaggatacc gccgtgtact attgtgcccc ttactatgac 360
tacgtgggcg ccatggatta ttggggccag ggcacatccg tgaccgtgtc ctctgccagc 420
accaagggac catccgtgtt cccactggca ccttgctccc ggtctacaag cgagtccacc 480
gccgccctgg gatgtctggt gaaggactac ttccccgagc ctgtgacagt gagctggaac 540
tccggcgccc tgacaagcgg agtgcacacc tttccagccg tgctgcagag ctccggcctg 600
tactccctgt ctagcgtggt gaccgtgcca tcctctaatt ttggcaccca gacatatacc 660
tgcaacgtgg accacaagcc cagcaataca aaggtggata agaccgtgga gaggaagtgc 720
tgcgtggagt gccctccctg tccagcccca cccgtggcag gcccttccgt gttcctgttt 780
cctccaaagc ctaaggacac actgatgatc agccgcacac cagaggtgac ctgcgtggtg 840
gtggacgtgt cccacgagga ccccgaggtg cagttcaact ggtacgtgga tggcgtggag 900
gtgcacaatg ccaagaccaa gcctcgggag gagcagttca atagcacctt ccgggtggtg 960
agcgtgctga ccgtggtgca ccaggattgg ctgaacggca aggagtataa gtgcaaggtg 1020
tctaataagg gcctgccagc ccccatcgag aagacaatca gcaagaccaa gggacagcca 1080
cgggagccac aggtgtacac cctgccccct tccagagagg agatgacaaa gaaccaggtg 1140
tctctgacct gtctggtgaa gggcttctat cccagcgaca tcgccgtgga gtgggagtcc 1200
aatggccagc ctgagaacaa ttacaagacc acaccaccca tgctggactc tgatggcagc 1260
ttctttctgt atagcaagct gaccgtggat aagtccaggt ggcagcaggg caacgtgttt 1320
tcttgcagcg tgatgcacga ggccctgcac aatcactaca cacagaagtc cctgtctctg 1380
agcccaggca agaggggaag gaagcggaga tccggctctg gagccaccaa cttcagcctg 1440
ctgaagcagg caggcgacgt ggaggagaat cctggaccaa tggtgctgca gacacaggtg 1500
tttatctccc tgctgctgtg gatctctggc gcctatggca agtgcgatat ccagatgacc 1560
cagagcccag caagccagtc cgcctctctg ggagagtctg tgacaatcac ctgtctggcc 1620
agccagacaa tcggcacctg gctggcctgg taccagcaga agcctggcaa gtccccacag 1680
ctgctgatct atgcagccac atctctggca gacggagtgc ctagccgctt cagcggctcc 1740
ggctctggaa ccaagttctc ctttaagatc agctccctgc aggccgagga tttcgtgtct 1800
tactattgcc agcagctgta caacacccct ctgaccttcg gcggcggcac aaagctggag 1860
atcaagagga ccgtggcagc accatccgtg ttcatctttc ctccatctga cgagcagctg 1920
aagtccggca ccgcctctgt ggtgtgcctg ctgaacaact tctaccctag agaggccaag 1980
gtgcagtgga aggtggataa cgccctgcag tccggcaatt ctcaggagag cgtgacagag 2040
caggactcca aggattctac ctatagcctg tctagcacac tgaccctgag caaggccgac 2100
tacgagaagc acaaggtgta tgcctgtgag gtcacccacc aggggctgag gtcaccagtc 2160
accaagtctt tcaacagggg agaatgttga taa 2193
<210> 132
<211> 729
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 132
Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly
1 5 10 15
Thr His Ala Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys
20 25 30
Pro Gly Ala Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile
35 40 45
Lys Asp Thr Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu
50 55 60
Glu Trp Ile Gly Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp
65 70 75 80
Pro Lys Phe Gln Ala Lys Ala Thr Ile Thr Ala Asn Thr Ser Ser Asn
85 90 95
Thr Ala Tyr Leu His Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val
100 105 110
Tyr Tyr Cys Ala Pro Tyr Tyr Asp Tyr Val Gly Ala Met Asp Tyr Trp
115 120 125
Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
130 135 140
Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr
145 150 155 160
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
165 170 175
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
180 185 190
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
195 200 205
Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp
210 215 220
His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys
225 230 235 240
Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser
245 250 255
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
260 265 270
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
275 280 285
Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
290 295 300
Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val
305 310 315 320
Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
325 330 335
Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr
340 345 350
Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
355 360 365
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
370 375 380
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
385 390 395 400
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp
405 410 415
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
420 425 430
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
435 440 445
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
450 455 460
Arg Gly Arg Lys Arg Arg Ser Gly Ser Gly Ala Thr Asn Phe Ser Leu
465 470 475 480
Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Val Leu
485 490 495
Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser Gly Ala Tyr
500 505 510
Gly Lys Cys Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Gln Ser Ala
515 520 525
Ser Leu Gly Glu Ser Val Thr Ile Thr Cys Leu Ala Ser Gln Thr Ile
530 535 540
Gly Thr Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Gln
545 550 555 560
Leu Leu Ile Tyr Ala Ala Thr Ser Leu Ala Asp Gly Val Pro Ser Arg
565 570 575
Phe Ser Gly Ser Gly Ser Gly Thr Lys Phe Ser Phe Lys Ile Ser Ser
580 585 590
Leu Gln Ala Glu Asp Phe Val Ser Tyr Tyr Cys Gln Gln Leu Tyr Asn
595 600 605
Thr Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr
610 615 620
Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu
625 630 635 640
Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro
645 650 655
Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly
660 665 670
Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr
675 680 685
Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His
690 695 700
Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Arg Ser Pro Val
705 710 715 720
Thr Lys Ser Phe Asn Arg Gly Glu Cys
725
<210> 133
<211> 3783
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 133
agccagtgtg tgaacctgac caccagaaca cagctgcctc ctgcctacac caacagcttc 60
accagaggag tctactaccc agacaaagtc ttcagaagct ctgtgctgca cagcacccag 120
gacctgttcc tgcctttctt cagcaacgtg acctggttcc acgccatcca cgtgtctggc 180
accaacggca ccaagagatt tgacaaccct gttcttcctt tcaatgatgg cgtgtacttt 240
gccagcacag agaagagcaa catcatccga ggctggatct ttggcaccac cctggacagc 300
aaaacccaga gcctgctgat cgtgaacaac gccaccaacg tggtcatcaa ggtgtgtgag 360
ttccagttct gcaatgaccc tttcctgggc gtgtactacc acaagaacaa caagtcctgg 420
atggagtctg agttcagagt ctacagctct gccaacaact gcacatttga atatgtgtcc 480
cagcctttcc tgatggacct ggagggcaag cagggcaact ttaagaacct gagagaattt 540
gtgttcaaga acatcgatgg ctacttcaag atctacagca agcacacacc catcaacctg 600
gtgagagacc tgcctcaggg cttctctgcc ctggagcctc tggtggacct gcccatcggc 660
atcaacatca ccagattcca gaccctgctg gccctgcaca gaagctacct gaccccagga 720
gacagcagca gcggctggac agctggagct gctgcctact acgtgggcta cctgcagccc 780
aggaccttcc tgctgaagta caacgaaaat ggcaccatca cagatgctgt tgactgtgcc 840
ctggaccctc ttagcgagac caagtgcacc ctgaagtcct tcacagtgga gaaaggcatc 900
taccagacca gcaacttccg agtgcagcca acagagagca tcgtgagatt tccaaacatc 960
accaacctgt gcccttttgg agaagtcttc aatgccacca gatttgcttc tgtgtacgcc 1020
tggaacagaa aaagaatcag caactgtgtg gctgactact ctgtgctgta caactctgcc 1080
tccttctcca ccttcaagtg ctatggagtc tctccaacca agctgaatga cctgtgcttc 1140
accaacgtgt atgctgacag ctttgtgatc agaggagatg aagtgcggca gattgctcct 1200
ggccagacag gcaagattgc tgactacaac tacaagctgc ctgatgactt cacaggctgt 1260
gtcatcgcct ggaacagcaa caacctggac agcaaggtgg gcggcaacta caactacctg 1320
tacagacttt tcaggaagag caacctgaag ccttttgaaa gagacatctc cacagagatc 1380
taccaggctg gcagcacacc ctgcaatggt gtggaaggct tcaactgcta cttccctctg 1440
cagagctacg gcttccagcc aacaaatggc gtgggctacc agccttacag agtggtggtg 1500
ctgtcctttg agctgctgca cgcccctgcc acagtgtgtg gccccaagaa gagcaccaac 1560
ctggtgaaga acaaatgtgt gaacttcaat ttcaatggcc tgacaggcac aggagtgctg 1620
acagagagca acaagaagtt tcttcctttc cagcagtttg gaagagacat tgctgacacc 1680
acagatgctg tgagagatcc tcagaccctg gagatcctgg atatcacacc ctgctccttt 1740
ggaggagttt ctgtcatcac acctggcacc aataccagca accaagtggc tgtgctgtac 1800
caagatgtga attgcacaga agtgcctgtg gccatccacg ctgaccagct gacacccacc 1860
tggagagtgt acagcacagg cagcaatgtt ttccagacaa gagctggctg cctgattgga 1920
gcagagcacg tgaacaacag ctatgaatgt gacatcccta ttggagctgg catctgtgcc 1980
agctaccaga cccaaaccaa cagcccaaga agagccagat ctgtggccag ccagagcatc 2040
atcgcctaca ccatgagcct gggagctgag aactctgtgg cctacagcaa caacagcatc 2100
gccatcccca ccaacttcac catctctgtg accacagaga tcctgcctgt gtccatgacc 2160
aagacatctg tggactgcac catgtacatc tgtggagaca gcacagaatg cagcaacctg 2220
ctgctgcagt acggctcctt ctgcacccag ctgaacagag ccctgacagg catcgctgtg 2280
gagcaggaca agaacacaca ggaagtgttt gcccaggtga agcagatcta caaaacacca 2340
cccatcaagg actttggagg cttcaatttc tcccaaatcc tgcctgaccc cagcaagcct 2400
tccaagagaa gcttcattga agacctgctg ttcaacaaag tgaccctggc tgatgctggc 2460
ttcatcaagc agtatggaga ctgcctggga gacattgctg ccagagacct gatctgtgcc 2520
cagaagttta atggcctgac tgtgctgcct cctctgctga cagatgaaat gatcgcccag 2580
tacacatctg ccctgctggc tggcaccatc accagtggct ggacatttgg agctggagct 2640
gccctgcaga tcccttttgc catgcagatg gcctacagat ttaatggcat cggcgtgacc 2700
cagaacgtgc tgtacgagaa ccagaagctg atcgccaacc agttcaactc tgccatcggc 2760
aagatccagg acagcctgag cagcacagcc tctgccctgg gcaagctgca ggatgtggtg 2820
aaccaaaacg cccaggccct gaacaccctg gtgaagcagc tgagcagcaa ctttggagcc 2880
atctcctctg tgctgaatga catcctgagc cggctggaca aggtggaagc agaagtgcag 2940
atcgacagac tcatcacagg ccgcctgcag agcctgcaga cctacgtgac ccagcagctg 3000
atcagagctg ctgagatccg ggcctctgcc aacctggctg ccaccaagat gtcagaatgt 3060
gtgctgggcc agagcaaaag agtggacttc tgtggcaaag gctaccacct gatgtccttc 3120
cctcagtctg ctcctcacgg cgtggtgttc ctgcacgtga cctacgtgcc tgcccaggag 3180
aagaacttca ccacagctcc tgccatctgc cacgatggca aggcccactt cccaagagaa 3240
ggtgtctttg tgtccaatgg cacccactgg ttcgtgaccc agagaaactt ctacgagcct 3300
cagatcatca ccacagacaa cacatttgtg tctggcaact gtgatgtggt catcggcatc 3360
gtgaacaaca cagtttatga ccctctgcag cctgagctgg acagcttcaa agaagagctg 3420
gacaagtact tcaagaacca cacatctcca gatgtggacc tgggagacat ctctggcatc 3480
aatgcctctg tggtgaacat ccagaaggaa attgacaggc tgaacgaagt ggccaagaac 3540
ctgaacgaaa gcctcatcga cctgcaggag ctgggcaagt acgagcagta catcaagtgg 3600
ccttggtaca tctggctggg cttcatcgct ggcctcatcg ccatcgtgat ggtgaccatc 3660
atgctgtgct gcatgaccag ctgctgctct tgcctgaagg gctgctgcag ctgtggcagc 3720
tgctgcaagt ttgatgaaga tgactctgag cctgtgctga agggcgtgaa gctgcactac 3780
aca 3783
<210> 134
<211> 1261
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 134
Ser Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr
1 5 10 15
Thr Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg
20 25 30
Ser Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser
35 40 45
Asn Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr
50 55 60
Lys Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe
65 70 75 80
Ala Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr
85 90 95
Thr Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr
100 105 110
Asn Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe
115 120 125
Leu Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu
130 135 140
Phe Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser
145 150 155 160
Gln Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn
165 170 175
Leu Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr
180 185 190
Ser Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe
195 200 205
Ser Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr
210 215 220
Arg Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly
225 230 235 240
Asp Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly
245 250 255
Tyr Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr
260 265 270
Ile Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys
275 280 285
Cys Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser
290 295 300
Asn Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile
305 310 315 320
Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala
325 330 335
Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp
340 345 350
Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr
355 360 365
Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr
370 375 380
Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro
385 390 395 400
Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp
405 410 415
Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys
420 425 430
Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn
435 440 445
Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly
450 455 460
Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu
465 470 475 480
Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr
485 490 495
Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val
500 505 510
Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn
515 520 525
Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn
530 535 540
Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr
545 550 555 560
Thr Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr
565 570 575
Pro Cys Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr
580 585 590
Ser Asn Gln Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val
595 600 605
Pro Val Ala Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr
610 615 620
Ser Thr Gly Ser Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly
625 630 635 640
Ala Glu His Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala
645 650 655
Gly Ile Cys Ala Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala
660 665 670
Arg Ser Val Ala Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly
675 680 685
Ala Glu Asn Ser Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr
690 695 700
Asn Phe Thr Ile Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr
705 710 715 720
Lys Thr Ser Val Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu
725 730 735
Cys Ser Asn Leu Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn
740 745 750
Arg Ala Leu Thr Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu
755 760 765
Val Phe Ala Gln Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp
770 775 780
Phe Gly Gly Phe Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro
785 790 795 800
Ser Lys Arg Ser Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu
805 810 815
Ala Asp Ala Gly Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile
820 825 830
Ala Ala Arg Asp Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val
835 840 845
Leu Pro Pro Leu Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala
850 855 860
Leu Leu Ala Gly Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala
865 870 875 880
Ala Leu Gln Ile Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly
885 890 895
Ile Gly Val Thr Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala
900 905 910
Asn Gln Phe Asn Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser
915 920 925
Thr Ala Ser Ala Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala
930 935 940
Gln Ala Leu Asn Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala
945 950 955 960
Ile Ser Ser Val Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu
965 970 975
Ala Glu Val Gln Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu
980 985 990
Gln Thr Tyr Val Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala
995 1000 1005
Ser Ala Asn Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly
1010 1015 1020
Gln Ser Lys Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met
1025 1030 1035
Ser Phe Pro Gln Ser Ala Pro His Gly Val Val Phe Leu His Val
1040 1045 1050
Thr Tyr Val Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala
1055 1060 1065
Ile Cys His Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe
1070 1075 1080
Val Ser Asn Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr
1085 1090 1095
Glu Pro Gln Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn
1100 1105 1110
Cys Asp Val Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro
1115 1120 1125
Leu Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr
1130 1135 1140
Phe Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser
1145 1150 1155
Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg
1160 1165 1170
Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu
1175 1180 1185
Gln Glu Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr
1190 1195 1200
Ile Trp Leu Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val
1205 1210 1215
Thr Ile Met Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys
1220 1225 1230
Gly Cys Cys Ser Cys Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp
1235 1240 1245
Ser Glu Pro Val Leu Lys Gly Val Lys Leu His Tyr Thr
1250 1255 1260
<210> 135
<211> 3843
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 135
atggattgga cttggattct ctttctcgtt gctgcagcca cacgcgttca tagcagccag 60
tgtgtgaacc tgaccaccag aacacagctg cctcctgcct acaccaacag cttcaccaga 120
ggagtctact acccagacaa agtcttcaga agctctgtgc tgcacagcac ccaggacctg 180
ttcctgcctt tcttcagcaa cgtgacctgg ttccacgcca tccacgtgtc tggcaccaac 240
ggcaccaaga gatttgacaa ccctgttctt cctttcaatg atggcgtgta ctttgccagc 300
acagagaaga gcaacatcat ccgaggctgg atctttggca ccaccctgga cagcaaaacc 360
cagagcctgc tgatcgtgaa caacgccacc aacgtggtca tcaaggtgtg tgagttccag 420
ttctgcaatg accctttcct gggcgtgtac taccacaaga acaacaagtc ctggatggag 480
tctgagttca gagtctacag ctctgccaac aactgcacat ttgaatatgt gtcccagcct 540
ttcctgatgg acctggaggg caagcagggc aactttaaga acctgagaga atttgtgttc 600
aagaacatcg atggctactt caagatctac agcaagcaca cacccatcaa cctggtgaga 660
gacctgcctc agggcttctc tgccctggag cctctggtgg acctgcccat cggcatcaac 720
atcaccagat tccagaccct gctggccctg cacagaagct acctgacccc aggagacagc 780
agcagcggct ggacagctgg agctgctgcc tactacgtgg gctacctgca gcccaggacc 840
ttcctgctga agtacaacga aaatggcacc atcacagatg ctgttgactg tgccctggac 900
cctcttagcg agaccaagtg caccctgaag tccttcacag tggagaaagg catctaccag 960
accagcaact tccgagtgca gccaacagag agcatcgtga gatttccaaa catcaccaac 1020
ctgtgccctt ttggagaagt cttcaatgcc accagatttg cttctgtgta cgcctggaac 1080
agaaaaagaa tcagcaactg tgtggctgac tactctgtgc tgtacaactc tgcctccttc 1140
tccaccttca agtgctatgg agtctctcca accaagctga atgacctgtg cttcaccaac 1200
gtgtatgctg acagctttgt gatcagagga gatgaagtgc ggcagattgc tcctggccag 1260
acaggcaaga ttgctgacta caactacaag ctgcctgatg acttcacagg ctgtgtcatc 1320
gcctggaaca gcaacaacct ggacagcaag gtgggcggca actacaacta cctgtacaga 1380
cttttcagga agagcaacct gaagcctttt gaaagagaca tctccacaga gatctaccag 1440
gctggcagca caccctgcaa tggtgtggaa ggcttcaact gctacttccc tctgcagagc 1500
tacggcttcc agccaacaaa tggcgtgggc taccagcctt acagagtggt ggtgctgtcc 1560
tttgagctgc tgcacgcccc tgccacagtg tgtggcccca agaagagcac caacctggtg 1620
aagaacaaat gtgtgaactt caatttcaat ggcctgacag gcacaggagt gctgacagag 1680
agcaacaaga agtttcttcc tttccagcag tttggaagag acattgctga caccacagat 1740
gctgtgagag atcctcagac cctggagatc ctggatatca caccctgctc ctttggagga 1800
gtttctgtca tcacacctgg caccaatacc agcaaccaag tggctgtgct gtaccaagat 1860
gtgaattgca cagaagtgcc tgtggccatc cacgctgacc agctgacacc cacctggaga 1920
gtgtacagca caggcagcaa tgttttccag acaagagctg gctgcctgat tggagcagag 1980
cacgtgaaca acagctatga atgtgacatc cctattggag ctggcatctg tgccagctac 2040
cagacccaaa ccaacagccc aagaagagcc agatctgtgg ccagccagag catcatcgcc 2100
tacaccatga gcctgggagc tgagaactct gtggcctaca gcaacaacag catcgccatc 2160
cccaccaact tcaccatctc tgtgaccaca gagatcctgc ctgtgtccat gaccaagaca 2220
tctgtggact gcaccatgta catctgtgga gacagcacag aatgcagcaa cctgctgctg 2280
cagtacggct ccttctgcac ccagctgaac agagccctga caggcatcgc tgtggagcag 2340
gacaagaaca cacaggaagt gtttgcccag gtgaagcaga tctacaaaac accacccatc 2400
aaggactttg gaggcttcaa tttctcccaa atcctgcctg accccagcaa gccttccaag 2460
agaagcttca ttgaagacct gctgttcaac aaagtgaccc tggctgatgc tggcttcatc 2520
aagcagtatg gagactgcct gggagacatt gctgccagag acctgatctg tgcccagaag 2580
tttaatggcc tgactgtgct gcctcctctg ctgacagatg aaatgatcgc ccagtacaca 2640
tctgccctgc tggctggcac catcaccagt ggctggacat ttggagctgg agctgccctg 2700
cagatccctt ttgccatgca gatggcctac agatttaatg gcatcggcgt gacccagaac 2760
gtgctgtacg agaaccagaa gctgatcgcc aaccagttca actctgccat cggcaagatc 2820
caggacagcc tgagcagcac agcctctgcc ctgggcaagc tgcaggatgt ggtgaaccaa 2880
aacgcccagg ccctgaacac cctggtgaag cagctgagca gcaactttgg agccatctcc 2940
tctgtgctga atgacatcct gagccggctg gacaaggtgg aagcagaagt gcagatcgac 3000
agactcatca caggccgcct gcagagcctg cagacctacg tgacccagca gctgatcaga 3060
gctgctgaga tccgggcctc tgccaacctg gctgccacca agatgtcaga atgtgtgctg 3120
ggccagagca aaagagtgga cttctgtggc aaaggctacc acctgatgtc cttccctcag 3180
tctgctcctc acggcgtggt gttcctgcac gtgacctacg tgcctgccca ggagaagaac 3240
ttcaccacag ctcctgccat ctgccacgat ggcaaggccc acttcccaag agaaggtgtc 3300
tttgtgtcca atggcaccca ctggttcgtg acccagagaa acttctacga gcctcagatc 3360
atcaccacag acaacacatt tgtgtctggc aactgtgatg tggtcatcgg catcgtgaac 3420
aacacagttt atgaccctct gcagcctgag ctggacagct tcaaagaaga gctggacaag 3480
tacttcaaga accacacatc tccagatgtg gacctgggag acatctctgg catcaatgcc 3540
tctgtggtga acatccagaa ggaaattgac aggctgaacg aagtggccaa gaacctgaac 3600
gaaagcctca tcgacctgca ggagctgggc aagtacgagc agtacatcaa gtggccttgg 3660
tacatctggc tgggcttcat cgctggcctc atcgccatcg tgatggtgac catcatgctg 3720
tgctgcatga ccagctgctg ctcttgcctg aagggctgct gcagctgtgg cagctgctgc 3780
aagtttgatg aagatgactc tgagcctgtg ctgaagggcg tgaagctgca ctacacataa 3840
tga 3843
<210> 136
<211> 1279
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 136
Met Asp Trp Thr Trp Ile Leu Phe Leu Val Ala Ala Ala Thr Arg Val
1 5 10 15
His Ser Ser Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro
20 25 30
Ala Tyr Thr Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val
35 40 45
Phe Arg Ser Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe
50 55 60
Phe Ser Asn Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn
65 70 75 80
Gly Thr Lys Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val
85 90 95
Tyr Phe Ala Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe
100 105 110
Gly Thr Thr Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn
115 120 125
Ala Thr Asn Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp
130 135 140
Pro Phe Leu Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu
145 150 155 160
Ser Glu Phe Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr
165 170 175
Val Ser Gln Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe
180 185 190
Lys Asn Leu Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys
195 200 205
Ile Tyr Ser Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln
210 215 220
Gly Phe Ser Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn
225 230 235 240
Ile Thr Arg Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr
245 250 255
Pro Gly Asp Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr
260 265 270
Val Gly Tyr Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn
275 280 285
Gly Thr Ile Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu
290 295 300
Thr Lys Cys Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln
305 310 315 320
Thr Ser Asn Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro
325 330 335
Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg
340 345 350
Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val
355 360 365
Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys
370 375 380
Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn
385 390 395 400
Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile
405 410 415
Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro
420 425 430
Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp
435 440 445
Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys
450 455 460
Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln
465 470 475 480
Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe
485 490 495
Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln
500 505 510
Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala
515 520 525
Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys
530 535 540
Val Asn Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu
545 550 555 560
Ser Asn Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala
565 570 575
Asp Thr Thr Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp
580 585 590
Ile Thr Pro Cys Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr
595 600 605
Asn Thr Ser Asn Gln Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr
610 615 620
Glu Val Pro Val Ala Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg
625 630 635 640
Val Tyr Ser Thr Gly Ser Asn Val Phe Gln Thr Arg Ala Gly Cys Leu
645 650 655
Ile Gly Ala Glu His Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile
660 665 670
Gly Ala Gly Ile Cys Ala Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg
675 680 685
Arg Ala Arg Ser Val Ala Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser
690 695 700
Leu Gly Ala Glu Asn Ser Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile
705 710 715 720
Pro Thr Asn Phe Thr Ile Ser Val Thr Thr Glu Ile Leu Pro Val Ser
725 730 735
Met Thr Lys Thr Ser Val Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser
740 745 750
Thr Glu Cys Ser Asn Leu Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln
755 760 765
Leu Asn Arg Ala Leu Thr Gly Ile Ala Val Glu Gln Asp Lys Asn Thr
770 775 780
Gln Glu Val Phe Ala Gln Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile
785 790 795 800
Lys Asp Phe Gly Gly Phe Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser
805 810 815
Lys Pro Ser Lys Arg Ser Phe Ile Glu Asp Leu Leu Phe Asn Lys Val
820 825 830
Thr Leu Ala Asp Ala Gly Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly
835 840 845
Asp Ile Ala Ala Arg Asp Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu
850 855 860
Thr Val Leu Pro Pro Leu Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr
865 870 875 880
Ser Ala Leu Leu Ala Gly Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala
885 890 895
Gly Ala Ala Leu Gln Ile Pro Phe Ala Met Gln Met Ala Tyr Arg Phe
900 905 910
Asn Gly Ile Gly Val Thr Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu
915 920 925
Ile Ala Asn Gln Phe Asn Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu
930 935 940
Ser Ser Thr Ala Ser Ala Leu Gly Lys Leu Gln Asp Val Val Asn Gln
945 950 955 960
Asn Ala Gln Ala Leu Asn Thr Leu Val Lys Gln Leu Ser Ser Asn Phe
965 970 975
Gly Ala Ile Ser Ser Val Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys
980 985 990
Val Glu Ala Glu Val Gln Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln
995 1000 1005
Ser Leu Gln Thr Tyr Val Thr Gln Gln Leu Ile Arg Ala Ala Glu
1010 1015 1020
Ile Arg Ala Ser Ala Asn Leu Ala Ala Thr Lys Met Ser Glu Cys
1025 1030 1035
Val Leu Gly Gln Ser Lys Arg Val Asp Phe Cys Gly Lys Gly Tyr
1040 1045 1050
His Leu Met Ser Phe Pro Gln Ser Ala Pro His Gly Val Val Phe
1055 1060 1065
Leu His Val Thr Tyr Val Pro Ala Gln Glu Lys Asn Phe Thr Thr
1070 1075 1080
Ala Pro Ala Ile Cys His Asp Gly Lys Ala His Phe Pro Arg Glu
1085 1090 1095
Gly Val Phe Val Ser Asn Gly Thr His Trp Phe Val Thr Gln Arg
1100 1105 1110
Asn Phe Tyr Glu Pro Gln Ile Ile Thr Thr Asp Asn Thr Phe Val
1115 1120 1125
Ser Gly Asn Cys Asp Val Val Ile Gly Ile Val Asn Asn Thr Val
1130 1135 1140
Tyr Asp Pro Leu Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu
1145 1150 1155
Asp Lys Tyr Phe Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly
1160 1165 1170
Asp Ile Ser Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu
1175 1180 1185
Ile Asp Arg Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu
1190 1195 1200
Ile Asp Leu Gln Glu Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp
1205 1210 1215
Pro Trp Tyr Ile Trp Leu Gly Phe Ile Ala Gly Leu Ile Ala Ile
1220 1225 1230
Val Met Val Thr Ile Met Leu Cys Cys Met Thr Ser Cys Cys Ser
1235 1240 1245
Cys Leu Lys Gly Cys Cys Ser Cys Gly Ser Cys Cys Lys Phe Asp
1250 1255 1260
Glu Asp Asp Ser Glu Pro Val Leu Lys Gly Val Lys Leu His Tyr
1265 1270 1275
Thr
<210> 137
<211> 3783
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 137
agccagtgtg tgaacctgac caccagaaca cagctgcctc ctgcctacac caacagcttc 60
accagaggag tctactaccc agacaaggtg ttcagaagct ctgtgctgca cagcacccag 120
gacctcttcc tgcctttctt cagcaacgtg acctggttcc acgccatcca cgtgtctggc 180
accaacggca ccaagagatt tgacaaccct gtgctgcctt tcaatgatgg tgtgtacttt 240
gccagcacag agaagagcaa catcatccga ggctggatct ttggcaccac cctggacagc 300
aaaacacaga gcctgctgat cgtgaataat gccaccaacg tggtcatcaa ggtgtgtgag 360
ttccagttct gcaatgaccc tttcctgggc gtgtactacc acaagaacaa caagtcctgg 420
atggagtctg agttccgagt gtacagctct gccaacaact gcacatttga atatgtgtcc 480
cagcctttcc tgatggacct ggagggcaag cagggcaatt tcaagaacct gagagaattt 540
gtgttcaaga acatcgatgg ctacttcaag atctacagca agcacacacc catcaacctg 600
gtgagagatc ttcctcaggg cttctctgcc ctggagcctc tggtggacct gcccatcggc 660
atcaacatca cccgctttca gaccctgctg gccctgcaca gaagctacct gaccccagga 720
gacagcagca gcggctggac agctggagct gctgcctact acgtgggcta cctgcagcca 780
agaaccttcc tgctgaagta caacgaaaat ggcaccatca ctgtggctgt ggcctgtgcc 840
ctggaccctc tttctgagac caagtgcacc ctgaagtcct tcacagtgga gaaaggcatc 900
taccagacca gcaacttcag agttcagcca acagagagca tcgtgagatt tccaaacatc 960
accaacctgt gtccttttgg agaagtcttc aatgccacca gatttgcttc tgtgtacgcc 1020
tggaacagaa aaagaatcag caactgtgtg gctgactact ctgtgctgta caactctgcc 1080
tccttctcca ccttcaagtg ctacggtgtg tctcctacca agctgaatga cctgtgcttc 1140
accaacgtgt atgctgacag ctttgtcatc agaggagatg aagtgcggca gatcgcccct 1200
ggccagacag gcaagattgc tgactacaac tacaagctgc ctgatgactt cacaggctgt 1260
gtcatcgcct ggaacagcaa caacctggac agcaaggtgg gcggcaacta caactacctg 1320
tacagacttt tcaggaagag caacctgaag ccttttgaaa gagacatctc cacagagatc 1380
taccaggctg gcagcacacc ctgcaatgga gtggaaggct tcaactgcta cttccctctg 1440
cagagctacg gcttccagcc caccaatggc gtgggctacc agccttacag agtggtggtg 1500
ctgtcctttg agctgctgca cgcccctgcc acagtgtgtg gccccaagaa gagcaccaac 1560
ctggtgaaga acaaatgtgt gaacttcaat ttcaatggcc tgacaggcac aggagtgctg 1620
acagagagca acaagaagtt cctgcctttc cagcagtttg gaagagacat tgctgacacc 1680
acagatgctg tgagagatcc tcagaccctg gagatcctgg acatcacacc ctgctccttt 1740
ggaggagttt ctgtcatcac acctggagcc aacaccagca accaagtgac agtgctgtac 1800
caagatgtga actgcacaga agttcctgtg gccatccacg ctgaccagct gaccccaacc 1860
tggagagtct acagcacagg cagcaacgtg tttaaaacaa gagctggctg cctgattgga 1920
gcagagcacg tgaacaacag ctatgaatgt gacatcccta ttggagctgg catctgtgcc 1980
agctaccaga cccaaaccaa cagcccaaga agagccagga gcacagccag ccagagcatc 2040
atcgcctaca ccatgagcct gggagcagag aactctgtgg cctacagcaa caacagcatc 2100
gtcatcccca ccaacttcac catctctgtg accacagaga tcctgcctgt gtccatgacc 2160
aagacatctg tggactgcac catgtacatc tgcagtgaca gcacagaatg cagcaaccct 2220
ctgctgcagt acggctcctt ctgcacccag ctgaacagag ccctgacagg catcgctgtg 2280
gagcaggaca agaacacaca ggaagtgttt gcccaggtga agcagatcta caaaacacca 2340
cccatcaagg actttggagg cttcaacttc tcccagatcc tgcctgaccc cagcaagccc 2400
agcaagagaa gcttcattga agacctgctg ttcaacaaag tgaccctggc tgatgctggc 2460
ttcatcaaac aatatggaga ctgcctggga gacattgctg ccagagacct gatctgtgcc 2520
cagaagttta atggcctgac tgtgctgcct cctctgctga cagatgaaat gatcgcccag 2580
tacacatctg ccctgctggc tggcaccatc acatctggct ggacatttgg agctggagct 2640
gccctgcaga tcccttttgc catgcagatg gcctacagat ttaatggcat cagagtgacc 2700
cagaacgtgc tgtatgaaaa ccagaagctg atcgccaacc agttcaactc tgccatcggc 2760
aagatccagg acagcctgag cagcacagcc tctgccctgg gcaagctgca ggatgtggtg 2820
aaccaaaatg cccaggccct gaacaccctg gtgaagcagc tgagcagcac cttctccacc 2880
atctccagcg tgctgaatga catcctgagc cggctggaca aggtggaagc tgaggtgcag 2940
atcgacagac tcatcacagg ccggctgcag agcctgcaga cctacgtgac ccagcagctg 3000
atcagagctg ctgagatcag agcttctgcc aacctgaagg ccaccaagat gtcagaatgt 3060
gtgctgggcc agagcaagag agtggacttc tgtggcaaag gctaccacct gatgtccttc 3120
cctcagtctg ctcctcacgg cgtggtgttc ctgcacgtga cctacgtgcc tgcccaggag 3180
aagaacttca ccacagctcc tgccacctgc cacgatggca aagcccactt cccaagagaa 3240
ggcgtctttg tgtccaatgg cacccactgg ttcgtgaccc agagaaactt tgatgagcct 3300
cagatcatca ccacagacaa cacatttgtt tctggcaact gtgatgtggt catcggcatc 3360
gtgaacaaca cagtttatga ccctctgcag cctgagctgg acagcttcaa agaagagctg 3420
gacaagtact tcaagaacca cacatctcca gatgtggacc tgggagacat ctctggcatc 3480
aatgcctctg tggtgaacat ccagaaggaa attgacaggc tgaacgaagt ggccaagaac 3540
ctgaacgaaa gcctcatcga cctgcaggag ctgggcaagt acgagcagta catcaagtgg 3600
ccttggtaca tctggctggg cttcattgct ggcctcatcg ccatcgtgat ggtgaccatc 3660
atgctgtgct gcatgaccag ctgctgctct tgcctgaagg gctgctgcag ctgtggcagc 3720
tgctgcaagt ttgatgaaga tgactctgag cctgtgctga agggcgtgaa gctgcactac 3780
aca 3783
<210> 138
<211> 1261
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 138
Ser Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr
1 5 10 15
Thr Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg
20 25 30
Ser Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser
35 40 45
Asn Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr
50 55 60
Lys Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe
65 70 75 80
Ala Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr
85 90 95
Thr Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr
100 105 110
Asn Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe
115 120 125
Leu Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu
130 135 140
Phe Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser
145 150 155 160
Gln Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn
165 170 175
Leu Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr
180 185 190
Ser Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe
195 200 205
Ser Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr
210 215 220
Arg Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly
225 230 235 240
Asp Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly
245 250 255
Tyr Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr
260 265 270
Ile Thr Val Ala Val Ala Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys
275 280 285
Cys Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser
290 295 300
Asn Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile
305 310 315 320
Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala
325 330 335
Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp
340 345 350
Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr
355 360 365
Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr
370 375 380
Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro
385 390 395 400
Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp
405 410 415
Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys
420 425 430
Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn
435 440 445
Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly
450 455 460
Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu
465 470 475 480
Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr
485 490 495
Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val
500 505 510
Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn
515 520 525
Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn
530 535 540
Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr
545 550 555 560
Thr Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr
565 570 575
Pro Cys Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Ala Asn Thr
580 585 590
Ser Asn Gln Val Thr Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val
595 600 605
Pro Val Ala Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr
610 615 620
Ser Thr Gly Ser Asn Val Phe Lys Thr Arg Ala Gly Cys Leu Ile Gly
625 630 635 640
Ala Glu His Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala
645 650 655
Gly Ile Cys Ala Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala
660 665 670
Arg Ser Thr Ala Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly
675 680 685
Ala Glu Asn Ser Val Ala Tyr Ser Asn Asn Ser Ile Val Ile Pro Thr
690 695 700
Asn Phe Thr Ile Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr
705 710 715 720
Lys Thr Ser Val Asp Cys Thr Met Tyr Ile Cys Ser Asp Ser Thr Glu
725 730 735
Cys Ser Asn Pro Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn
740 745 750
Arg Ala Leu Thr Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu
755 760 765
Val Phe Ala Gln Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp
770 775 780
Phe Gly Gly Phe Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro
785 790 795 800
Ser Lys Arg Ser Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu
805 810 815
Ala Asp Ala Gly Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile
820 825 830
Ala Ala Arg Asp Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val
835 840 845
Leu Pro Pro Leu Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala
850 855 860
Leu Leu Ala Gly Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala
865 870 875 880
Ala Leu Gln Ile Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly
885 890 895
Ile Arg Val Thr Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala
900 905 910
Asn Gln Phe Asn Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser
915 920 925
Thr Ala Ser Ala Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala
930 935 940
Gln Ala Leu Asn Thr Leu Val Lys Gln Leu Ser Ser Thr Phe Ser Thr
945 950 955 960
Ile Ser Ser Val Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu
965 970 975
Ala Glu Val Gln Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu
980 985 990
Gln Thr Tyr Val Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala
995 1000 1005
Ser Ala Asn Leu Lys Ala Thr Lys Met Ser Glu Cys Val Leu Gly
1010 1015 1020
Gln Ser Lys Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met
1025 1030 1035
Ser Phe Pro Gln Ser Ala Pro His Gly Val Val Phe Leu His Val
1040 1045 1050
Thr Tyr Val Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala
1055 1060 1065
Thr Cys His Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe
1070 1075 1080
Val Ser Asn Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Asp
1085 1090 1095
Glu Pro Gln Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn
1100 1105 1110
Cys Asp Val Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro
1115 1120 1125
Leu Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr
1130 1135 1140
Phe Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser
1145 1150 1155
Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg
1160 1165 1170
Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu
1175 1180 1185
Gln Glu Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr
1190 1195 1200
Ile Trp Leu Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val
1205 1210 1215
Thr Ile Met Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys
1220 1225 1230
Gly Cys Cys Ser Cys Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp
1235 1240 1245
Ser Glu Pro Val Leu Lys Gly Val Lys Leu His Tyr Thr
1250 1255 1260
<210> 139
<211> 3843
<212> DNA
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 139
atggattgga cctggattct ttttctcgtt gcagctgcta cacgcgttca tagcagccag 60
tgtgtgaacc tgaccaccag aacacagctg cctcctgcct acaccaacag cttcaccaga 120
ggagtctact acccagacaa ggtgttcaga agctctgtgc tgcacagcac ccaggacctc 180
ttcctgcctt tcttcagcaa cgtgacctgg ttccacgcca tccacgtgtc tggcaccaac 240
ggcaccaaga gatttgacaa ccctgtgctg cctttcaatg atggtgtgta ctttgccagc 300
acagagaaga gcaacatcat ccgaggctgg atctttggca ccaccctgga cagcaaaaca 360
cagagcctgc tgatcgtgaa taatgccacc aacgtggtca tcaaggtgtg tgagttccag 420
ttctgcaatg accctttcct gggcgtgtac taccacaaga acaacaagtc ctggatggag 480
tctgagttcc gagtgtacag ctctgccaac aactgcacat ttgaatatgt gtcccagcct 540
ttcctgatgg acctggaggg caagcagggc aatttcaaga acctgagaga atttgtgttc 600
aagaacatcg atggctactt caagatctac agcaagcaca cacccatcaa cctggtgaga 660
gatcttcctc agggcttctc tgccctggag cctctggtgg acctgcccat cggcatcaac 720
atcacccgct ttcagaccct gctggccctg cacagaagct acctgacccc aggagacagc 780
agcagcggct ggacagctgg agctgctgcc tactacgtgg gctacctgca gccaagaacc 840
ttcctgctga agtacaacga aaatggcacc atcactgtgg ctgtggcctg tgccctggac 900
cctctttctg agaccaagtg caccctgaag tccttcacag tggagaaagg catctaccag 960
accagcaact tcagagttca gccaacagag agcatcgtga gatttccaaa catcaccaac 1020
ctgtgtcctt ttggagaagt cttcaatgcc accagatttg cttctgtgta cgcctggaac 1080
agaaaaagaa tcagcaactg tgtggctgac tactctgtgc tgtacaactc tgcctccttc 1140
tccaccttca agtgctacgg tgtgtctcct accaagctga atgacctgtg cttcaccaac 1200
gtgtatgctg acagctttgt catcagagga gatgaagtgc ggcagatcgc ccctggccag 1260
acaggcaaga ttgctgacta caactacaag ctgcctgatg acttcacagg ctgtgtcatc 1320
gcctggaaca gcaacaacct ggacagcaag gtgggcggca actacaacta cctgtacaga 1380
cttttcagga agagcaacct gaagcctttt gaaagagaca tctccacaga gatctaccag 1440
gctggcagca caccctgcaa tggagtggaa ggcttcaact gctacttccc tctgcagagc 1500
tacggcttcc agcccaccaa tggcgtgggc taccagcctt acagagtggt ggtgctgtcc 1560
tttgagctgc tgcacgcccc tgccacagtg tgtggcccca agaagagcac caacctggtg 1620
aagaacaaat gtgtgaactt caatttcaat ggcctgacag gcacaggagt gctgacagag 1680
agcaacaaga agttcctgcc tttccagcag tttggaagag acattgctga caccacagat 1740
gctgtgagag atcctcagac cctggagatc ctggacatca caccctgctc ctttggagga 1800
gtttctgtca tcacacctgg agccaacacc agcaaccaag tgacagtgct gtaccaagat 1860
gtgaactgca cagaagttcc tgtggccatc cacgctgacc agctgacccc aacctggaga 1920
gtctacagca caggcagcaa cgtgtttaaa acaagagctg gctgcctgat tggagcagag 1980
cacgtgaaca acagctatga atgtgacatc cctattggag ctggcatctg tgccagctac 2040
cagacccaaa ccaacagccc aagaagagcc aggagcacag ccagccagag catcatcgcc 2100
tacaccatga gcctgggagc agagaactct gtggcctaca gcaacaacag catcgtcatc 2160
cccaccaact tcaccatctc tgtgaccaca gagatcctgc ctgtgtccat gaccaagaca 2220
tctgtggact gcaccatgta catctgcagt gacagcacag aatgcagcaa ccctctgctg 2280
cagtacggct ccttctgcac ccagctgaac agagccctga caggcatcgc tgtggagcag 2340
gacaagaaca cacaggaagt gtttgcccag gtgaagcaga tctacaaaac accacccatc 2400
aaggactttg gaggcttcaa cttctcccag atcctgcctg accccagcaa gcccagcaag 2460
agaagcttca ttgaagacct gctgttcaac aaagtgaccc tggctgatgc tggcttcatc 2520
aaacaatatg gagactgcct gggagacatt gctgccagag acctgatctg tgcccagaag 2580
tttaatggcc tgactgtgct gcctcctctg ctgacagatg aaatgatcgc ccagtacaca 2640
tctgccctgc tggctggcac catcacatct ggctggacat ttggagctgg agctgccctg 2700
cagatccctt ttgccatgca gatggcctac agatttaatg gcatcagagt gacccagaac 2760
gtgctgtatg aaaaccagaa gctgatcgcc aaccagttca actctgccat cggcaagatc 2820
caggacagcc tgagcagcac agcctctgcc ctgggcaagc tgcaggatgt ggtgaaccaa 2880
aatgcccagg ccctgaacac cctggtgaag cagctgagca gcaccttctc caccatctcc 2940
agcgtgctga atgacatcct gagccggctg gacaaggtgg aagctgaggt gcagatcgac 3000
agactcatca caggccggct gcagagcctg cagacctacg tgacccagca gctgatcaga 3060
gctgctgaga tcagagcttc tgccaacctg aaggccacca agatgtcaga atgtgtgctg 3120
ggccagagca agagagtgga cttctgtggc aaaggctacc acctgatgtc cttccctcag 3180
tctgctcctc acggcgtggt gttcctgcac gtgacctacg tgcctgccca ggagaagaac 3240
ttcaccacag ctcctgccac ctgccacgat ggcaaagccc acttcccaag agaaggcgtc 3300
tttgtgtcca atggcaccca ctggttcgtg acccagagaa actttgatga gcctcagatc 3360
atcaccacag acaacacatt tgtttctggc aactgtgatg tggtcatcgg catcgtgaac 3420
aacacagttt atgaccctct gcagcctgag ctggacagct tcaaagaaga gctggacaag 3480
tacttcaaga accacacatc tccagatgtg gacctgggag acatctctgg catcaatgcc 3540
tctgtggtga acatccagaa ggaaattgac aggctgaacg aagtggccaa gaacctgaac 3600
gaaagcctca tcgacctgca ggagctgggc aagtacgagc agtacatcaa gtggccttgg 3660
tacatctggc tgggcttcat tgctggcctc atcgccatcg tgatggtgac catcatgctg 3720
tgctgcatga ccagctgctg ctcttgcctg aagggctgct gcagctgtgg cagctgctgc 3780
aagtttgatg aagatgactc tgagcctgtg ctgaagggcg tgaagctgca ctacacataa 3840
tga 3843
<210> 140
<211> 1279
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 140
Met Asp Trp Thr Trp Ile Leu Phe Leu Val Ala Ala Ala Thr Arg Val
1 5 10 15
His Ser Ser Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro
20 25 30
Ala Tyr Thr Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val
35 40 45
Phe Arg Ser Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe
50 55 60
Phe Ser Asn Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn
65 70 75 80
Gly Thr Lys Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val
85 90 95
Tyr Phe Ala Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe
100 105 110
Gly Thr Thr Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn
115 120 125
Ala Thr Asn Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp
130 135 140
Pro Phe Leu Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu
145 150 155 160
Ser Glu Phe Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr
165 170 175
Val Ser Gln Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe
180 185 190
Lys Asn Leu Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys
195 200 205
Ile Tyr Ser Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln
210 215 220
Gly Phe Ser Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn
225 230 235 240
Ile Thr Arg Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr
245 250 255
Pro Gly Asp Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr
260 265 270
Val Gly Tyr Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn
275 280 285
Gly Thr Ile Thr Val Ala Val Ala Cys Ala Leu Asp Pro Leu Ser Glu
290 295 300
Thr Lys Cys Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln
305 310 315 320
Thr Ser Asn Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro
325 330 335
Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg
340 345 350
Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val
355 360 365
Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys
370 375 380
Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn
385 390 395 400
Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile
405 410 415
Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro
420 425 430
Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp
435 440 445
Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys
450 455 460
Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln
465 470 475 480
Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe
485 490 495
Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln
500 505 510
Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala
515 520 525
Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys
530 535 540
Val Asn Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu
545 550 555 560
Ser Asn Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala
565 570 575
Asp Thr Thr Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp
580 585 590
Ile Thr Pro Cys Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Ala
595 600 605
Asn Thr Ser Asn Gln Val Thr Val Leu Tyr Gln Asp Val Asn Cys Thr
610 615 620
Glu Val Pro Val Ala Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg
625 630 635 640
Val Tyr Ser Thr Gly Ser Asn Val Phe Lys Thr Arg Ala Gly Cys Leu
645 650 655
Ile Gly Ala Glu His Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile
660 665 670
Gly Ala Gly Ile Cys Ala Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg
675 680 685
Arg Ala Arg Ser Thr Ala Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser
690 695 700
Leu Gly Ala Glu Asn Ser Val Ala Tyr Ser Asn Asn Ser Ile Val Ile
705 710 715 720
Pro Thr Asn Phe Thr Ile Ser Val Thr Thr Glu Ile Leu Pro Val Ser
725 730 735
Met Thr Lys Thr Ser Val Asp Cys Thr Met Tyr Ile Cys Ser Asp Ser
740 745 750
Thr Glu Cys Ser Asn Pro Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln
755 760 765
Leu Asn Arg Ala Leu Thr Gly Ile Ala Val Glu Gln Asp Lys Asn Thr
770 775 780
Gln Glu Val Phe Ala Gln Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile
785 790 795 800
Lys Asp Phe Gly Gly Phe Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser
805 810 815
Lys Pro Ser Lys Arg Ser Phe Ile Glu Asp Leu Leu Phe Asn Lys Val
820 825 830
Thr Leu Ala Asp Ala Gly Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly
835 840 845
Asp Ile Ala Ala Arg Asp Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu
850 855 860
Thr Val Leu Pro Pro Leu Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr
865 870 875 880
Ser Ala Leu Leu Ala Gly Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala
885 890 895
Gly Ala Ala Leu Gln Ile Pro Phe Ala Met Gln Met Ala Tyr Arg Phe
900 905 910
Asn Gly Ile Arg Val Thr Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu
915 920 925
Ile Ala Asn Gln Phe Asn Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu
930 935 940
Ser Ser Thr Ala Ser Ala Leu Gly Lys Leu Gln Asp Val Val Asn Gln
945 950 955 960
Asn Ala Gln Ala Leu Asn Thr Leu Val Lys Gln Leu Ser Ser Thr Phe
965 970 975
Ser Thr Ile Ser Ser Val Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys
980 985 990
Val Glu Ala Glu Val Gln Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln
995 1000 1005
Ser Leu Gln Thr Tyr Val Thr Gln Gln Leu Ile Arg Ala Ala Glu
1010 1015 1020
Ile Arg Ala Ser Ala Asn Leu Lys Ala Thr Lys Met Ser Glu Cys
1025 1030 1035
Val Leu Gly Gln Ser Lys Arg Val Asp Phe Cys Gly Lys Gly Tyr
1040 1045 1050
His Leu Met Ser Phe Pro Gln Ser Ala Pro His Gly Val Val Phe
1055 1060 1065
Leu His Val Thr Tyr Val Pro Ala Gln Glu Lys Asn Phe Thr Thr
1070 1075 1080
Ala Pro Ala Thr Cys His Asp Gly Lys Ala His Phe Pro Arg Glu
1085 1090 1095
Gly Val Phe Val Ser Asn Gly Thr His Trp Phe Val Thr Gln Arg
1100 1105 1110
Asn Phe Asp Glu Pro Gln Ile Ile Thr Thr Asp Asn Thr Phe Val
1115 1120 1125
Ser Gly Asn Cys Asp Val Val Ile Gly Ile Val Asn Asn Thr Val
1130 1135 1140
Tyr Asp Pro Leu Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu
1145 1150 1155
Asp Lys Tyr Phe Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly
1160 1165 1170
Asp Ile Ser Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu
1175 1180 1185
Ile Asp Arg Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu
1190 1195 1200
Ile Asp Leu Gln Glu Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp
1205 1210 1215
Pro Trp Tyr Ile Trp Leu Gly Phe Ile Ala Gly Leu Ile Ala Ile
1220 1225 1230
Val Met Val Thr Ile Met Leu Cys Cys Met Thr Ser Cys Cys Ser
1235 1240 1245
Cys Leu Lys Gly Cys Cys Ser Cys Gly Ser Cys Cys Lys Phe Asp
1250 1255 1260
Glu Asp Asp Ser Glu Pro Val Leu Lys Gly Val Lys Leu His Tyr
1265 1270 1275
Thr
<210> 141
<211> 18
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 141
Met Asp Trp Thr Trp Ile Leu Phe Leu Val Ala Ala Ala Thr Arg Val
1 5 10 15
His Ser
<210> 142
<211> 9
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 142
Pro His Gly Val Val Phe Leu His Val
1 5
<210> 143
<211> 9
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 143
Val Val Phe Leu His Val Thr Tyr Val
1 5
<210> 144
<211> 15
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 144
Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys
1 5 10 15
<210> 145
<211> 15
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 145
Lys Asn Lys Cys Val Asn Phe Asn Phe Asn Gly Leu Thr Gly Thr
1 5 10 15
<210> 146
<211> 15
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 146
Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr
1 5 10 15
<210> 147
<211> 15
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 147
Ser Phe Pro Gln Ser Ala Pro His Gly Val Val Phe Leu His Val
1 5 10 15
<210> 148
<211> 15
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 148
Phe Thr Thr Ala Pro Ala Ile Cys His Asp Gly Lys Ala His Phe
1 5 10 15
<210> 149
<211> 15
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 149
Pro His Gly Val Val Phe Leu His Val Thr Tyr Val Pro Ala Gln
1 5 10 15
<210> 150
<211> 15
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 150
Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala
1 5 10 15
<210> 151
<211> 15
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 151
Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser
1 5 10 15
<210> 152
<211> 15
<212> PRT
<213> artificial sequence
<220>
<223> chemical Synthesis
<400> 152
Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu
1 5 10 15

Claims (37)

1. An anti-SARS-CoV-2 antibody or fragment thereof, wherein said antibody is selected from the group consisting of: monoclonal antibodies, polyclonal antibodies, single chain antibodies, immunoconjugates, defucosylated antibodies and bispecific antibodies.
2. The anti-SARS-CoV-2 antibody or fragment thereof according to claim 1, wherein said immunoconjugate comprises a therapeutic agent or a detection moiety.
3. The anti-SARS-CoV-2 antibody or fragment thereof according to claim 1, wherein said antibody is selected from the group consisting of: humanized antibodies, chimeric antibodies, fully human antibodies, and antibody mimics.
4. The anti-SARS-CoV-2 antibody or fragment thereof according to claim 1, wherein said antibody comprises at least one selected from the group consisting of:
a) A heavy chain CDR1 sequence selected from the group consisting of SEQ ID NO. 26, SEQ ID NO. 80 and SEQ ID NO. 108;
b) A heavy chain CDR2 sequence selected from the group consisting of SEQ ID NO 27, SEQ ID NO 81 and SEQ ID NO 109;
c) A heavy chain CDR3 sequence selected from the group consisting of SEQ ID NO. 28, SEQ ID NO. 82 and SEQ ID NO. 110;
d) A light chain CDR1 sequence selected from the group consisting of SEQ ID NO. 34, SEQ ID NO. 88 and SEQ ID NO. 116;
e) A light chain CDR2 sequence selected from the group consisting of SEQ ID NO. 35, SEQ ID NO. 89 and SEQ ID NO. 117; and
f) A light chain CDR3 sequence selected from SEQ ID NO. 36, SEQ ID NO. 90 and SEQ ID NO. 118.
5. The anti-SARS-CoV-2 antibody or fragment thereof according to claim 1, wherein said antibody comprises at least one selected from the group consisting of:
a) An anti-SARS-CoV-2 antibody heavy chain comprising a sequence having at least 80% identity to an amino acid sequence selected from the group consisting of SEQ ID No. 2, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 18, SEQ ID No. 30, SEQ ID No. 42, SEQ ID No. 84, SEQ ID No. 94, SEQ ID No. 100, SEQ ID No. 112, SEQ ID No. 122 and SEQ ID No. 128;
b) An anti-SARS-CoV-2 antibody light chain comprising a sequence having at least 80% identity to an amino acid sequence selected from the group consisting of SEQ ID No. 4, SEQ ID No. 8, SEQ ID No. 14, SEQ ID No. 20, SEQ ID No. 38, SEQ ID No. 44, SEQ ID No. 92, SEQ ID No. 96, SEQ ID No. 102, SEQ ID No. 120, SEQ ID No. 124 and SEQ ID No. 130;
c) A fragment of the heavy chain of an anti-SARS-CoV-2 antibody comprising at least 80% of the full length sequence selected from the group consisting of SEQ ID No. 2, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 18, SEQ ID No. 30, SEQ ID No. 42, SEQ ID No. 84, SEQ ID No. 94, SEQ ID No. 100, SEQ ID No. 112, SEQ ID No. 122 and SEQ ID No. 128; and
d) A fragment of the light chain of an anti-SARS-CoV-2 antibody comprising at least 80% of the full length sequence selected from the group consisting of SEQ ID No. 4, SEQ ID No. 8, SEQ ID No. 14, SEQ ID No. 20, SEQ ID No. 38, SEQ ID No. 44, SEQ ID No. 92, SEQ ID No. 96, SEQ ID No. 102, SEQ ID No. 120, SEQ ID No. 124 and SEQ ID No. 130.
6. The anti-SARS-CoV-2 antibody or fragment thereof according to claim 1, wherein said antibody comprises at least one selected from the group consisting of:
a) An amino acid sequence having at least 80% identity to an amino acid sequence selected from the group consisting of: SEQ ID NO 10, SEQ ID NO 16, SEQ ID NO 22, SEQ ID NO 40, SEQ ID NO 46, SEQ ID NO 48, SEQ ID NO 50, SEQ ID NO 52, SEQ ID NO 54, SEQ ID NO 56, SEQ ID NO 58, SEQ ID NO 60, SEQ ID NO 62, SEQ ID NO 64, SEQ ID NO 66, SEQ ID NO 68, SEQ ID NO 70, SEQ ID NO 72, SEQ ID NO 74, SEQ ID NO 76, SEQ ID NO 98, SEQ ID NO 104, SEQ ID NO 126 and SEQ ID NO 132. And
b) An antibody comprising at least 80% of the full length sequence selected from the group consisting of
Fragment of SARS-CoV-2 antibody heavy chain: SEQ ID NO 10, SEQ ID NO 16, SEQ ID NO 22, SEQ ID NO 40, SEQ ID NO 46, SEQ ID NO 48, SEQ ID NO 50, SEQ ID NO 52, SEQ ID NO 54, SEQ ID NO 56, SEQ ID NO 58, SEQ ID NO 60, SEQ ID NO 62, SEQ ID NO 64, SEQ ID NO 66, SEQ ID NO 68, SEQ ID NO 70, SEQ ID NO 72, SEQ ID NO 74, SEQ ID NO 76, SEQ ID NO 98, SEQ ID NO 104, SEQ ID NO 126 and SEQ ID NO 132.
7. A nucleic acid molecule encoding the anti-SARS-CoV-2 antibody or fragment thereof of any one of claims 1 to 6.
8. The nucleic acid molecule of claim 7, further comprising a nucleotide sequence encoding a cleavage domain.
9. The nucleic acid molecule of claim 7, wherein the nucleic acid molecule comprises at least one selected from the group consisting of:
a) A nucleotide sequence selected from the group consisting of SEQ ID NO. 23, SEQ ID NO. 77 and SEQ ID NO. 105 encoding said heavy chain CDR1 sequence;
b) A nucleotide sequence selected from the group consisting of SEQ ID NO. 24, SEQ ID NO. 78 and SEQ ID NO. 106 encoding said heavy chain CDR2 sequence;
c) A nucleotide sequence selected from the group consisting of SEQ ID NO. 25, SEQ ID NO. 79 and SEQ ID NO. 107 encoding said heavy chain CDR3 sequence;
d) A nucleotide sequence selected from the group consisting of SEQ ID NO. 31, SEQ ID NO. 85 and SEQ ID NO. 113 encoding said light chain CDR1 sequence;
e) A nucleotide sequence selected from the group consisting of SEQ ID NO. 32, SEQ ID NO. 86 and SEQ ID NO. 114 encoding said light chain CDR2 sequence; and
f) A nucleotide sequence selected from the group consisting of SEQ ID NO. 33, SEQ ID NO. 87 and SEQ ID NO. 115 encoding the light chain CDR3 sequence.
10. The nucleic acid molecule of claim 7, wherein the nucleic acid molecule comprises at least one nucleotide sequence selected from the group consisting of:
a) A nucleotide sequence having at least 80% identity to a nucleotide selected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 5, SEQ ID NO. 11, SEQ ID NO. 17, SEQ ID NO. 29, SEQ ID NO. 41, SEQ ID NO. 83, SEQ ID NO. 93, SEQ ID NO. 99, SEQ ID NO. 111, SEQ ID NO. 121 and SEQ ID NO. 127 encoding an anti-SARS-CoV-2 antibody heavy chain;
b) A nucleotide sequence having at least 80% identity to a nucleotide selected from the group consisting of SEQ ID NO. 3, SEQ ID NO. 7, SEQ ID NO. 13, SEQ ID NO. 19, SEQ ID NO. 37, SEQ ID NO. 43, SEQ ID NO. 91, SEQ ID NO. 95, SEQ ID NO. 101, SEQ ID NO. 119, SEQ ID NO. 123 and SEQ ID NO. 129 encoding an anti-SARS-CoV-2 antibody light chain;
c) A fragment comprising at least 80% of the nucleotide sequence of the full length sequence selected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 5, SEQ ID NO. 11, SEQ ID NO. 17, SEQ ID NO. 29, SEQ ID NO. 41, SEQ ID NO. 83, SEQ ID NO. 93, SEQ ID NO. 99, SEQ ID NO. 111, SEQ ID NO. 121 and SEQ ID NO. 127 encoding an anti-SARS-CoV-2 antibody heavy chain; and
d) A fragment comprising at least 80% of the nucleotide sequence selected from the group consisting of SEQ ID No. 3, SEQ ID No. 7, SEQ ID No. 13, SEQ ID No. 19, SEQ ID No. 37, SEQ ID No. 43, SEQ ID No. 91, SEQ ID No. 95, SEQ ID No. 101, SEQ ID No. 119, SEQ ID No. 123 and SEQ ID No. 129, which encodes an anti-SARS-CoV-2 antibody light chain.
11. The nucleic acid molecule of claim 7, wherein the nucleic acid molecule comprises a nucleotide sequence in the group consisting of:
a) Nucleotide sequence having at least 80% identity with a nucleotide selected from the group consisting of SEQ ID NO 9, SEQ ID NO 15, SEQ ID NO 21, SEQ ID NO 39, SEQ ID NO 45, SEQ ID NO 47, SEQ ID NO 49, SEQ ID NO 51, SEQ ID NO 53, SEQ ID NO 55, SEQ ID NO 57, SEQ ID NO 59, SEQ ID NO 61, SEQ ID NO 63, SEQ ID NO 65, SEQ ID NO 67, SEQ ID NO 69, SEQ ID NO 71, SEQ ID NO 73, SEQ ID NO 75, SEQ ID NO 97, SEQ ID NO 103, SEQ ID NO 125 and SEQ ID NO 131; and
b) A fragment comprising at least 80% of the nucleotide sequence of the full length sequence selected from the group consisting of SEQ ID NO 9, SEQ ID NO 15, SEQ ID NO 21, SEQ ID NO 39, SEQ ID NO 45, SEQ ID NO 47, SEQ ID NO 49, SEQ ID NO 51, SEQ ID NO 53, SEQ ID NO 55, SEQ ID NO 57, SEQ ID NO 59, SEQ ID NO 61, SEQ ID NO 63, SEQ ID NO 65, SEQ ID NO 67, SEQ ID NO 69, SEQ ID NO 71, SEQ ID NO 73, SEQ ID NO 75, SEQ ID NO 97, SEQ ID NO 103, SEQ ID NO 125 and SEQ ID NO 131.
12. The nucleic acid molecule of any one of claims 7 to 11, wherein the nucleotide sequence encodes a leader sequence.
13. The nucleic acid molecule of any one of claims 7 to 11, wherein the nucleic acid molecule comprises an expression vector.
14. A composition comprising at least one anti-SARS-CoV-2 antibody or fragment thereof as claimed in any one of claims 1 to 6.
15. A composition comprising at least one nucleic acid molecule according to any one of claims 7 to 11.
16. The composition of claim 15, comprising a first nucleic acid molecule comprising a nucleotide sequence encoding a heavy chain of an anti-SARS-CoV-2 spike antigen synthesis antibody and a second nucleic acid molecule; the second nucleic acid molecule comprises a nucleotide sequence encoding a light chain of an anti-SARS-CoV-2 spike antigen synthesis antibody.
17. The composition of claim 16, wherein the first nucleic acid molecule comprises a nucleotide sequence encoding at least one selected from the group consisting of:
a) An anti-SARS-CoV-2 antibody heavy chain comprising a sequence having at least 80% identity to an amino acid sequence selected from the group consisting of SEQ ID No. 2, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 18, SEQ ID No. 30, SEQ ID No. 42, SEQ ID No. 84, SEQ ID No. 94, SEQ ID No. 100, SEQ ID No. 112, SEQ ID No. 122 and SEQ ID No. 128; and
b) A fragment of the heavy chain of an anti-SARS-CoV-2 antibody comprising at least 80% of the full length sequence selected from the group consisting of SEQ ID No. 2, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 18, SEQ ID No. 30, SEQ ID No. 42, SEQ ID No. 84, SEQ ID No. 94, SEQ ID No. 100, SEQ ID No. 112, SEQ ID No. 122 and SEQ ID No. 128; and
wherein the second nucleic acid molecule comprises a nucleotide sequence encoding at least one selected from the group consisting of:
c) An anti-SARS-CoV-2 antibody light chain comprising a sequence having at least 80% identity to an amino acid sequence selected from the group consisting of SEQ ID No. 4, SEQ ID No. 8, SEQ ID No. 14, SEQ ID No. 20, SEQ ID No. 38, SEQ ID No. 44, SEQ ID No. 92, SEQ ID No. 96, SEQ ID No. 102, SEQ ID No. 120, SEQ ID No. 124 and SEQ ID No. 130; and
d) A fragment of the light chain of an anti-SARS-CoV-2 antibody comprising at least 80% of the full length sequence selected from the group consisting of SEQ ID No. 4, SEQ ID No. 8, SEQ ID No. 14, SEQ ID No. 20, SEQ ID No. 38, SEQ ID No. 44, SEQ ID No. 92, SEQ ID No. 96, SEQ ID No. 102, SEQ ID No. 120, SEQ ID No. 124 and SEQ ID No. 130.
18. The composition of claim 16, wherein the first nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of:
a) A nucleotide sequence having at least 80% identity to a nucleotide selected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 5, SEQ ID NO. 11, SEQ ID NO. 17, SEQ ID NO. 29, SEQ ID NO. 41, SEQ ID NO. 83, SEQ ID NO. 93, SEQ ID NO. 99, SEQ ID NO. 111, SEQ ID NO. 121 and SEQ ID NO. 127 encoding an anti-SARS-CoV-2 antibody heavy chain; and
b) A fragment comprising at least 80% of the nucleotide sequence of the full length sequence selected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 5, SEQ ID NO. 11, SEQ ID NO. 17, SEQ ID NO. 29, SEQ ID NO. 41, SEQ ID NO. 83, SEQ ID NO. 93, SEQ ID NO. 99, SEQ ID NO. 111, SEQ ID NO. 121 and SEQ ID NO. 127 encoding an anti-SARS-CoV-2 antibody heavy chain; and is also provided with
Wherein the second nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of:
c) A nucleotide sequence having at least 80% identity to a nucleotide selected from the group consisting of SEQ ID NO. 3, SEQ ID NO. 7, SEQ ID NO. 13, SEQ ID NO. 19, SEQ ID NO. 37, SEQ ID NO. 43, SEQ ID NO. 91, SEQ ID NO. 95, SEQ ID NO. 101, SEQ ID NO. 119, SEQ ID NO. 123 and SEQ ID NO. 129 encoding an anti-SARS-CoV-2 antibody light chain; and
d) A fragment comprising at least 80% of the nucleotide sequence selected from the group consisting of SEQ ID No. 3, SEQ ID No. 7, SEQ ID No. 13, SEQ ID No. 19, SEQ ID No. 37, SEQ ID No. 43, SEQ ID No. 91, SEQ ID No. 95, SEQ ID No. 101, SEQ ID No. 119, SEQ ID No. 123 and SEQ ID No. 129, which encodes an anti-SARS-CoV-2 antibody light chain.
19. The composition of claim 14, further comprising a pharmaceutically acceptable excipient.
20. The composition of any one of claims 15 to 18, further comprising a pharmaceutically acceptable excipient.
21. A method of preventing or treating a disease in a subject, the method comprising administering to the subject the antibody or antibody fragment of any one of claims 1 to 6, the nucleic acid molecule of any one of claims 7 to 13, or the composition of any one of claims 14 to 20.
22. The method of claim 21, wherein the disease is covd-19.
23. The method of claim 22, further comprising administering to the subject at least one additional SARS-CoV-2 vaccine or therapeutic agent for the treatment of covd-19.
24. A method of inducing an immune response against SARS-CoV-2 in a subject, said method comprising administering to the subject the antibody or antibody fragment of any one of claims 1 to 6, the nucleic acid molecule of any one of claims 7 to 13, or the composition of any one of claims 14 to 20.
25. A method of inducing an immune response against SARS-CoV-2 in a subject in need thereof, the method comprising administering to the subject a combination of a first composition comprising the nucleic acid molecule encoding the synthetic anti-SARS-CoV-2 antibody or fragment thereof of any one of claims 7 to 13 and a second composition comprising the nucleic acid molecule encoding the SARS-CoV-2 antigen.
26. The method of claim 25, wherein the nucleic acid molecule encoding a SARS-CoV-2 antigen comprises a nucleotide sequence encoding a peptide comprising an amino acid sequence having at least about 90% identity over the entire length of an amino acid sequence selected from the group consisting of SEQ ID No. 134, SEQ ID No. 136, SEQ ID No. 138, and SEQ ID No. 140.
27. The method of claim 26, wherein the nucleic acid molecule encoding a SARS-CoV-2 antigen comprises a nucleotide sequence encoding an amino acid sequence selected from the group consisting of SEQ ID No. 134, SEQ ID No. 136, SEQ ID No. 138, and SEQ ID No. 140.
28. The method of claim 25, wherein the nucleic acid molecule encoding a SARS-CoV-2 antigen comprises a nucleotide sequence that is at least about 90% identical over the entire length of a nucleic acid sequence selected from the group consisting of SEQ ID No. 133, SEQ ID No. 135, SEQ ID No. 137, and SEQ ID No. 139.
29. The method of claim 28, wherein the nucleic acid molecule encoding a SARS-CoV-2 antigen comprises a nucleotide sequence selected from the group consisting of SEQ ID No. 133, SEQ ID No. 135, SEQ ID No. 137, and SEQ ID No. 139.
30. The method of claim 25, wherein administering comprises at least one of electroporation and injection.
31. A method of treating or protecting a subject in need thereof from infection by SARS-CoV-2 or a disease or disorder associated therewith, the method comprising administering to the subject a combination of a first composition comprising a nucleic acid molecule encoding a synthetic anti-SARS-CoV-2 antibody or fragment thereof of any one of claims 7 to 13 and a second composition comprising a nucleic acid molecule encoding a SARS-CoV-2 antigen.
32. The method of claim 31, wherein the disease is covd-19.
33. The method of claim 31, wherein the nucleic acid molecule encoding a SARS-CoV-2 antigen comprises a nucleotide sequence encoding a peptide comprising an amino acid sequence having at least about 90% identity over the entire length of an amino acid sequence selected from the group consisting of SEQ ID No. 134, SEQ ID No. 136, SEQ ID No. 138, and SEQ ID No. 140.
34. The method of claim 33, wherein the nucleic acid molecule encoding a SARS-CoV-2 antigen comprises a nucleotide sequence encoding an amino acid sequence selected from the group consisting of SEQ ID No. 134, SEQ ID No. 136, SEQ ID No. 138, and SEQ ID No. 140.
35. The method of claim 31, wherein the nucleic acid molecule encoding a SARS-CoV-2 antigen comprises a nucleotide sequence that is at least about 90% identical over the entire length of a nucleic acid sequence selected from the group consisting of SEQ ID No. 133, SEQ ID No. 135, SEQ ID No. 137, and SEQ ID No. 139.
36. The method of claim 35, wherein the nucleic acid molecule encoding the SARS-CoV-2 antigen comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137 and SEQ ID NO: 139.
37. The method of claim 31, wherein administering comprises at least one of electroporation and injection.
CN202180056190.7A 2020-06-09 2021-06-09 DNA encoded antibodies against SARS-COV-2 Pending CN116744966A (en)

Applications Claiming Priority (7)

Application Number Priority Date Filing Date Title
US63/036,795 2020-06-09
US63/036,809 2020-06-09
US63/068,868 2020-08-21
US63/083,173 2020-09-25
US202063114271P 2020-11-16 2020-11-16
US63/114,271 2020-11-16
PCT/US2021/036606 WO2021252620A2 (en) 2020-06-09 2021-06-09 Dna encoded antibodies for use against sars-cov-2

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