CN113480617A - Rotavirus strains and their use as vaccines - Google Patents

Rotavirus strains and their use as vaccines Download PDF

Info

Publication number
CN113480617A
CN113480617A CN202110780463.4A CN202110780463A CN113480617A CN 113480617 A CN113480617 A CN 113480617A CN 202110780463 A CN202110780463 A CN 202110780463A CN 113480617 A CN113480617 A CN 113480617A
Authority
CN
China
Prior art keywords
amino acid
leu
seq
acid sequence
ile
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202110780463.4A
Other languages
Chinese (zh)
Inventor
倪世利
邢苗苗
李慧娟
于菲
邹水燕
曲衍洪
孙萌
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shandong Weigao Litong Biological Products Co Ltd
Original Assignee
Shandong Weigao Litong Biological Products Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shandong Weigao Litong Biological Products Co Ltd filed Critical Shandong Weigao Litong Biological Products Co Ltd
Priority to CN202110780463.4A priority Critical patent/CN113480617A/en
Publication of CN113480617A publication Critical patent/CN113480617A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/12—Antidiarrhoeals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5252—Virus inactivated (killed)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2720/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsRNA viruses
    • C12N2720/00011—Details
    • C12N2720/12011—Reoviridae
    • C12N2720/12311—Rotavirus, e.g. rotavirus A
    • C12N2720/12321—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2720/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsRNA viruses
    • C12N2720/00011—Details
    • C12N2720/12011—Reoviridae
    • C12N2720/12311—Rotavirus, e.g. rotavirus A
    • C12N2720/12322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2720/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsRNA viruses
    • C12N2720/00011—Details
    • C12N2720/12011—Reoviridae
    • C12N2720/12311—Rotavirus, e.g. rotavirus A
    • C12N2720/12334—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/14—Reoviridae, e.g. rotavirus, bluetongue virus, Colorado tick fever virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/10—Detection of antigens from microorganism in sample from host
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention relates to the field of virus, in particular to separation, culture and identification of group A rotavirus G9P [8] strain and application as a vaccine. The invention relates to a group A rotavirus LSR-8 strain, which has the full amino acid sequence shown in SEQ ID No.1 to SEQ ID No.11 and the full nucleic acid sequence shown in SEQ ID No.12 to SEQ ID No. 22. The invention also relates to the use of the LSR-8 strain in the production of inactivated human rotavirus vaccine, attenuated live human rotavirus vaccine and combined vaccine, and can be used for preventing, alleviating or treating rotavirus infection and diseases caused by rotavirus infection, such as rotavirus gastroenteritis, diarrhea and the like.

Description

Rotavirus strains and their use as vaccines
Technical Field
The present invention relates to the field of viruses, in particular to rotavirus strains and their use as vaccines.
Background
Group a rotavirus causes more than 90% of human rotavirus gastroenteritis, 2016, children less than 5 years of global age die from about 128500 people infected with rotavirus, and children less than 5 years of global age are infected with rotavirus by about 2.6 billion people. Group a rotaviruses are highly contagious zoonotic pathogens via the fecal-oral route and can also be transmitted via the respiratory tract in aerosol form. As for the infection rate and incidence rate of rotavirus, the developed countries and the developing countries have no obvious difference, and the difference of substances and living environment is not an important factor for inhibiting rotavirus infection. Rotavirus infection has no specific treatment medicine, the vaccine is the first means for preventing and controlling rotavirus infection, the world health organization WHO actively promotes the research and development of rotavirus vaccines by governments, and suggests that all countries in the world should incorporate the rotavirus vaccines into the national immune program.
The rotavirus belongs to Reoviridae (Reoviridae), and mature virus particles are icosahedral viruses in smooth cartwheel-shaped axial-radial form under an electron microscopeThe diameter is about 65-75nm, and there are 3 layers of protein capsids. The genome consists of 11 double-stranded RNA fragments, each of which is a gene encoding a protein. The 11 genes encode 6 structural proteins of the virus: VP1-VP4, VP6, VP7 and 6 non-structural proteins NSP1-NSP 6. Rotaviruses were divided into groups A-G7 based on their antigenicity of VP6 protein. All rotaviruses cause disease in other animals, but only A, B and C groups infect humans, and infection with group A rotaviruses accounts for 90% of all cases where rotaviruses infect humans. The VP4 and VP7 protein as neutralizing antigen of rotavirus can stimulate organism to produce neutralizing antibody, so as to inhibit rotavirus infection. The VP4 protein can be changed into VP5 and VP8 under the action of trypsin in intestinal tract, the VP8 protein can be combined with sialic acid receptor on the cell surface, so as to mediate the adsorption process of rotavirus, the VP5 membrane fusion site mediates the cell-entering process of rotavirus, and the VP4 protein determines the toxicity and P-type serum type of rotavirus. The VP7 protein is glycosylated protein and has Ca2+Dependent, it constitutes the outermost surface of the virus and determines the P-serotype of rotavirus. According to VP4, rotavirus is divided into 35 genotypes, and the rotavirus serotype which is currently prevalent worldwide is mainly P [4 ]]And P [8]](ii) a According to VP7, rotavirus is divided into 27 serotypes, and the serotype of rotavirus strains currently circulating worldwide is mainly G1, G2, G3, G4 and G9.
The inactivated rotavirus vaccine is a vaccine for safely and effectively preventing diseases of children caused by rotavirus infection. However, no inactivated rotavirus vaccine product is on the market so far. The protection efficiency of all currently used rotavirus attenuated live vaccines is about 75 percent, and the protection effect of the vaccines is yet to be improved; secondly, the rotavirus attenuated live vaccine can survive in the environment for a long time after preparation, use and detoxification after immunization, and has potential safety hazards including toxicity reversion, intussusception risks and the like; the attenuated live vaccine has the defects of foreign factor pollution risk, incapability of being used for people with immunodeficiency or immunosuppression treatment and the like in the preparation process, so that the development of the inactivated rotavirus vaccine has important significance and application prospect.
Disclosure of Invention
In view of this, the present invention provides rotavirus strains and their use as vaccines. The LSR-8 strain can be used for producing inactivated human rotavirus vaccines, human rotavirus attenuated live vaccines, subunit vaccines, genetically engineered recombinant protein vaccines, conjugate vaccines and combination vaccines, and can be used for preventing, relieving or treating rotavirus infection and diseases caused by the rotavirus infection, such as rotavirus gastroenteritis, diarrhea and the like.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a polypeptide or a combination of polypeptides, the polypeptide comprising the following sequence:
(I) an amino acid sequence as shown in any of SEQ ID Nos. 1-11;
or
(II) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence of (I), and the amino acid sequence has the same or similar functions with the amino acid sequence of (I);
or
(III) an amino acid sequence having at least 99.4% identity to an amino acid sequence as set forth in any of SEQ ID Nos. 1 to 11;
the number of the plurality is 5.
In some embodiments of the invention, the polypeptide comprises the sequence:
an amino acid sequence having more than 99.4% identity with the amino acid sequence shown as SEQ ID No. 4;
an amino acid sequence having more than 99.4% identity with the amino acid sequence shown as SEQ ID No. 6; and/or
An amino acid sequence having more than 99.7% identity with the amino acid sequence shown as SEQ ID No. 8; and/or
An amino acid sequence having more than 99.7% identity with the amino acid sequence shown as SEQ ID No. 9; and/or
An amino acid sequence with more than 99.5 percent of identity with the amino acid sequence shown as SEQ ID No. 11.
In some embodiments of the invention, the polypeptide comprises the sequence:
a polypeptide comprising an amino acid sequence which is at least 99.4% identical to the amino acid sequence shown in SEQ ID No. 4; and/or
A polypeptide comprising an amino acid sequence which is at least 99.4% identical to the amino acid sequence shown in SEQ ID No. 6; and/or
A polypeptide comprising an amino acid sequence which is at least 99.5% identical to the amino acid sequence shown in SEQ ID No. 11.
In some embodiments of the invention, the polypeptide comprises the sequence:
an amino acid sequence shown as SEQ ID No. 4; and/or
An amino acid sequence shown as SEQ ID No. 6; and/or
The amino acid sequence shown as SEQ ID No. 11.
In some embodiments of the invention, the polypeptide comprises the sequence:
a polypeptide comprising the amino acid sequence shown as SEQ ID No 2; and
a polypeptide comprising the amino acid sequence shown as SEQ ID No 5;
further comprising one or more of the following sequences:
a polypeptide comprising an amino acid sequence which is at least 99.4% identical to the amino acid sequence shown as SEQ ID No. 6; and/or
A polypeptide comprising an amino acid sequence corresponding to that shown as SEQ ID No. 7; and/or
A polypeptide comprising an amino acid sequence having at least 99.7% identity to the amino acid sequence set forth in SEQ ID No. 8; and/or
A polypeptide comprising an amino acid sequence which is at least 99.7% identical to the amino acid sequence shown as SEQ ID No. 9; and/or
A polypeptide comprising the amino acid sequence shown as SEQ ID No. 10; and/or
A polypeptide comprising an amino acid sequence which is at least 99.5% identical to the amino acid sequence shown as SEQ ID No. 11; and/or
A polypeptide comprising the amino acid sequence shown as SEQ ID No. 1; and/or
A polypeptide comprising the amino acid sequence shown as SEQ ID No. 3; and/or
A polypeptide comprising an amino acid sequence which is at least 99.4% identical to the amino acid sequence shown as SEQ ID No. 4.
In some embodiments of the invention, the polypeptide comprises the sequence:
a polypeptide comprising an amino acid sequence as set forth in SEQ ID NO 2;
a polypeptide comprising the amino acid sequence shown as SEQ ID No. 5; and
a polypeptide comprising an amino acid sequence which is at least 99.4% identical to the amino acid sequence shown as SEQ ID No. 6;
further comprising one or more of the following sequences:
a polypeptide comprising an amino acid sequence corresponding to that shown as SEQ ID No. 7; and/or
A polypeptide comprising an amino acid sequence having at least 99.7% identity to the amino acid sequence set forth in SEQ ID No. 8; and/or
A polypeptide comprising an amino acid sequence which is at least 99.7% identical to the amino acid sequence shown in SEQ ID No. 9; and/or
A polypeptide comprising the amino acid sequence shown in SEQ ID No. 10; and/or
A polypeptide comprising an amino acid sequence which is at least 99.5% identical to the amino acid sequence shown in SEQ ID No. 11; and/or
A polypeptide comprising the amino acid sequence shown in SEQ ID No. 1; and/or
A polypeptide comprising the amino acid sequence shown in SEQ ID No. 3; and/or
A polypeptide comprising an amino acid sequence which is at least 99.4% identical to the amino acid sequence shown in SEQ ID No. 4.
In some embodiments of the invention, the polypeptide comprises the sequence:
the 75 th and 76 th nucleotides of the nucleic acid molecule of the polypeptide for encoding the amino acid sequence shown as SEQ ID No.4 are GA, and the corresponding 22 th and 23 th amino acids are MK; and/or
A nucleic acid molecule encoding a polypeptide having the amino acid sequence shown in SEQ ID No.4 having nucleotide 499 as G and corresponding amino acid 175 as a; and/or
The 772 th nucleotide of the nucleic acid molecule encoding the polypeptide with the amino acid sequence as shown in SEQ ID No.4 is C, and the corresponding 266 th amino acid is H; and/or
The 1129 th nucleotide of a nucleic acid molecule of the polypeptide for encoding the amino acid sequence shown as SEQ ID No.4 is C, and the corresponding 385 th amino acid is H; and/or
The 404 th nucleotide of a nucleic acid molecule of the polypeptide for encoding the amino acid sequence shown as SEQ ID No.6 is G, and the corresponding 119 th amino acid is R; and/or
The 656 nucleotide of the nucleic acid molecule, which encodes a polypeptide having the amino acid sequence shown in SEQ ID No.6, is C, which corresponds to amino acid S at position 203.
The invention also provides nucleic acid molecules encoding the polypeptides or combinations of polypeptides.
In some embodiments of the invention, the nucleic acid molecule comprises the following sequence:
(IV) a nucleotide sequence encoding an amino acid sequence as shown in any of SEQ ID Nos. 1 to 11; and/or
(V) an antisense complementary sequence of a nucleotide sequence encoding an amino acid sequence as shown in any of SEQ ID Nos. 1 to 11;
(VI) a nucleotide sequence which hybridizes with a nucleotide sequence encoding an amino acid sequence as shown in any of SEQ ID Nos. 1 to 11 under a high stringency condition;
(VII) a nucleotide sequence obtained by substituting, deleting or adding one or more bases in the nucleotide sequence shown In (IV), (V) or (VI), and the nucleotide sequence has the same or similar functions with the nucleotide sequence shown In (IV);
(VIII) a nucleotide sequence which has at least 99.4% identity with the nucleotide sequence shown In (IV), (V) or (VI).
The plurality is 2, 3, 4 or 5.
In some embodiments of the invention, the nucleic acid molecule comprises a nucleotide sequence as set forth in any of SEQ ID Nos. 12 to 22.
In some embodiments of the invention, the nucleic acid molecule comprises the following sequence:
a nucleic acid molecule encoding the amino acid sequence shown as SEQ ID No.1, said nucleic acid molecule having the nucleotide sequence shown as SEQ ID No. 12; and/or
A nucleic acid molecule encoding the amino acid sequence shown as SEQ ID No.2, said nucleic acid molecule having the nucleotide sequence shown as SEQ ID No. 13; and/or
A nucleic acid molecule encoding the amino acid sequence shown as SEQ ID No.3, said nucleic acid molecule having the nucleotide sequence shown as SEQ ID No. 14; and/or
A nucleic acid molecule encoding the amino acid sequence shown as SEQ ID No.4, said nucleic acid molecule having the nucleotide sequence shown as SEQ ID No. 15; and/or
A nucleic acid molecule encoding the amino acid sequence shown as SEQ ID No.5, said nucleic acid molecule having the nucleotide sequence shown as SEQ ID No. 16; and/or
A nucleic acid molecule encoding the amino acid sequence shown as SEQ ID No.6, said nucleic acid molecule having the nucleotide sequence shown as SEQ ID No. 17; and/or
A nucleic acid molecule encoding the amino acid sequence shown as SEQ ID No.7, said nucleic acid molecule having the nucleotide sequence shown as SEQ ID No. 18; and/or
A nucleic acid molecule encoding the amino acid sequence shown as SEQ ID No.8, said nucleic acid molecule having the nucleotide sequence shown as SEQ ID No. 19; and/or
A nucleic acid molecule encoding the amino acid sequence shown as SEQ ID No.9, said nucleic acid molecule having the nucleotide sequence shown as SEQ ID No. 20; and/or
A nucleic acid molecule encoding the amino acid sequence shown as SEQ ID No.10, said nucleic acid molecule having the nucleotide sequence shown as SEQ ID No. 21; and/or
A nucleic acid molecule encoding the amino acid sequence shown as SEQ ID No.11, said nucleic acid molecule having the nucleotide sequence shown as SEQ ID No. 22.
The invention also provides an expression vector comprising the nucleic acid molecule.
In addition, the invention also provides a host cell for transforming or transfecting the expression vector.
More importantly, the invention also provides rotavirus which is characterized by comprising the polypeptide or the polypeptide combination or the nucleic acid molecule.
In some embodiments of the invention, the rotavirus has a accession number of CGMCC No. 20292.
More importantly, the invention also provides the application of the polypeptide or the polypeptide combination, the nucleic acid molecule or the rotavirus in preparing vaccines and/or medicines for preventing and/or treating diseases;
including, but not limited to, rotavirus-induced disease, diphtheria, tetanus, pertussis, Haemophilus influenzae type b-induced disease, Hepatitis B (HBV) -induced disease, polio and/or pneumococcal-induced disease
The invention also provides a medicament which comprises the polypeptide or the polypeptide combination, the nucleic acid molecule or the rotavirus and pharmaceutically acceptable auxiliary materials.
The invention also provides a pharmaceutical composition comprising the polypeptide or the polypeptide combination, the nucleic acid molecule or the rotavirus and a medicament for treating other diseases.
The invention also provides a vaccine comprising the polypeptide or the polypeptide combination, the nucleic acid molecule or the rotavirus and an acceptable adjuvant.
The invention also provides a method for preventing and/or treating diseases, and the medicine, the pharmaceutical composition or the vaccine is administered.
The invention also provides a kit comprising a nucleic acid molecule capable of amplifying said polypeptide or combination of polypeptides or said rotavirus.
In addition, the invention also provides a kit comprising a polypeptide or a combination of polypeptides, a nucleic acid molecule or a rotavirus capable of specifically binding to the polypeptide or the combination of polypeptides.
The invention also provides a disease diagnosis method, which uses the kit to detect virus expression and judges whether the disease is caused according to the expression quantity of the virus.
The invention also provides a preparation method of the rotavirus inactivated vaccine, which is characterized by comprising the following steps:
step 1: the polypeptide or the polypeptide combination, the nucleic acid molecule or the rotavirus are inoculated in Vero cells of enlarged subculture for culture after adaptive culture and plaque purification;
step 2: harvesting virus culture solution, filtering to remove impurities, concentrating, purifying and inactivating to obtain virus stock solution;
and step 3: and (3) filtering and sterilizing the virus stock solution prepared in the step (2), and adding an adjuvant to obtain the rotavirus inactivated vaccine.
In some embodiments of the present invention, the culturing in step 1 is carried out at 35-37 ℃ for 72-120 hours.
In some embodiments of the invention, the inactivation of step 2 is inactivation with formalin or B-propiolactone or heat.
In some embodiments of the invention, the purification in step 2 is anion exchange chromatography, molecular sieve gel filtration chromatography.
In some embodiments of the invention, the adjuvant of step 3 is an aluminum hydroxide solution.
In summary, the present invention provides G9P [8] rotavirus, specifically the LSR-8 strain of group A rotavirus. In some embodiments, the isolation and characterization of strains, and adaptation of strains on MA104 cells and Vero cells, respectively, is disclosed.
In some embodiments, pharmaceutical compositions are disclosed that may include an adjuvant and/or inactivated G9P [8] rotavirus.
The inactivated rotavirus may also be included in a pharmaceutical composition to induce a response against other pathogens, such as, but not limited to, diphtheria, tetanus, pertussis, haemophilus influenzae type b, Hepatitis B (HBV), polio, and/or pneumococcal disease.
The immune response may be a protective immune response. The subject may be a child, e.g., a human child less than 5 years of age or 1 year of age; the subject may also be an adult. The method may comprise one priming and at least one boosting.
The invention discloses an isolated nucleic acid molecule from LSR-8 rotavirus and an isolated polypeptide sequence from LSR-8 rotavirus. These molecules can be included in pharmaceutical compositions and can be used to induce an immune response.
Biological preservation Instructions
Biological material: LSR-8, class name: rotavirus is preserved in China general microbiological culture Collection center (CGMCC) at 31.8.2020, with the address of No.3 Siro-1 of Beijing, Chaoyang, and the preservation number of CGMCC No. 20292.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 shows VP4 and VP7 sequences in relation to other rotaviruses, wherein FIG. 1A is NC1LSR-8VP4, representing the VP4 nucleic acid sequence of LSR-8, and FIG. 1B is NC1LSR-8VP7, representing the VP7 nucleic acid sequence of LSR-8;
FIG. 2 shows an image of RNA electrophoresis type;
FIG. 3 shows a plaque purification picture;
FIG. 4 shows the proliferation profile of LSR-8 rotavirus on MA104 cells;
FIG. 5 shows a photograph of a colloidal gold reagent strip for LSR-8 rotavirus;
FIG. 6 shows immunofluorescence pictures of LSR-8 rotavirus on MA104 cells.
Detailed Description
The invention discloses rotavirus strains and application thereof as vaccines, and can be realized by appropriately improving process parameters by taking the contents as reference by the technical personnel in the field. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
In the rotavirus strain and the application thereof as the vaccine, the used raw materials and reagents can be purchased from the market.
The invention is further illustrated by the following examples:
example 1 isolation and characterization of LSR-8 Strain
Obtaining a viral sample
Collecting diarrhea excrement specimen of children with pediatric diarrhea patient in hospital, suspending in 0.01M PBS solution with pH 7.2, centrifuging at 8000rpm and 4 deg.C for 20 min, collecting supernatant, detecting rotavirus positive by colloidal gold kit, sterilizing and filtering with 0.22 μ M filter, and subpackaging in 1.5ml sterile centrifuge tube, and storing at-80 deg.C for use.
Plaque purification of LSR-8 strain virus morphology
Adding pancreatin into the rotavirus harvest liquid, activating in 37 deg.C constant temperature water bath for 1 hr, diluting with 10 times of maintenance liquid to 10-1、10-2、10-3、10-4、10-5、10-6、10-7、10-8。
MA104 cells were seeded on 6-well plates, and when the cells grew into a dense monolayer, the growth medium was discarded, and the cell surface was washed 3 times with maintenance medium. And (3) sucking out residual maintenance liquid, adding 500 mu l of activated rotavirus liquid into each hole, adsorbing for 60-120 minutes at 37 ℃, then sucking out the virus liquid, and washing the cell surface for 1 time by using the maintenance liquid. 3ml of a maintenance solution containing 0.3% agar was added to each well, and each dilution was graded into 2 wells, and transferred to an inverted culture in an incubator after the agar was solidified. After 72 hours, a layer of maintenance medium containing 0.006% neutral red and 0.3% agar was placed over the original agar layer in the incubator protected from light. Cytopathic conditions were observed daily and appropriate dilutions of the virulent seed of individual plaques were picked and cultured.
Adaptation of LSR-8 strain virus on MA104 cells
The rotavirus harvest was added to pancreatin at a final concentration of 20. mu.g/ml and activated in a thermostatic water bath at 37 ℃ for 1 hour.
MA104 cells grown as a dense monolayer in a T25 flask were removed from the growth medium and the cell surface was washed 3 times with the maintenance medium. Sucking out residual maintenance liquid, adding activated rotavirus liquid 500 μ l, adsorbing at 37 deg.C for 60-120 min, adding maintenance liquid 10ml containing pancreatin with final concentration of 4 μ g/ml, and adsorbing at 37 deg.C with 5% CO2Medium culture, observation of cytopathic effect CPE.
When 75% CPE appeared in the cells above, the virus was harvested. The harvested virus culture solution was frozen and thawed alternately at-20 ℃ and 25 ℃ for 2 times. Centrifuging at 8000rpm and 4 deg.C for 20 min, collecting supernatant, inoculating MA104 cell by the same method, and passaging or storing at-80 deg.C.
The rotavirus antigen is detected by a colloidal gold kit, and the titer of the LSR-8 virus is detected by a plaque method.
LSR-8 strain genome nucleic acid banding pattern
Nucleic acid extraction of LSR-8 strain virus genome was performed according to the instruction manual of the GeneJET Viral DNA and RNA Purification Kit virus genome RNA extraction Kit. The nucleic acid banding pattern is observed by non-denaturing polyacrylamide gel electrophoresis, after 90V and 6h, silver staining and color development.
LSR-8 strain genomic sequence analysis
Extracted RNA was sequenced according to clonal sequencing. Whole genotyping was performed using the Rotac v2.0 online tool. The phylogenetic tree was generated using the adjacency method with Mega 10.1.8 software. The LSR-8VP4 gene is highly similar (99.7%) to the VP4 gene of the japanese strain isolated in 2018 and the wuhan strain isolated in 2019. The LSR-8VP7 gene was closely related to the G9 strain from Tokyo, Japan, which was collected in 2018.
LSR-8 can be grown in Vero cell to 106-107Pfu/ml and shows strong stability.
LSR-8 strain virus morphology
Dripping 10 μ l of the virus suspension purified by the sucrose pad on a copper mesh, standing for 5 minutes, then sucking the redundant suspension with filter paper, and standing for tens of seconds to dry the copper mesh; and (3) dripping 10 mu l of 2% phosphotungstic acid dye solution on a copper mesh, standing for 5 minutes, sucking the redundant dye solution by using filter paper, standing for dozens of seconds, and directly observing the morphological characteristics of the virus under a transmission electron microscope after the copper mesh is dried.
Example 2 Adaptation and passaging of the LSR-8 Strain
Adaptation of LSR-8 strain virus on VERO cells
Adding pancreatin into the rotavirus harvest liquid, and activating in a constant temperature water bath at 37 ℃ for 1 hour.
The VERO cells grown in a dense monolayer in a T25 flask were removed from the growth medium and the cell surface was washed with maintenance medium. Sucking out residual maintenance liquid, adding activated rotavirus liquid 500ul, adsorbing at 37 deg.C for 60-120 min, adding maintenance liquid 10ml containing pancreatin with final concentration of 4 μ lg/ml, and adsorbing at 37 deg.C with 5% CO2Medium culture, observation of cytopathic effect CPE.
When 75% CPE appeared in the cells above, the virus was harvested. Freezing and thawing the harvested virus culture solution at-20 deg.C and 25 deg.C alternately for 2 times, centrifuging at 8000rpm and 4 deg.C for 20 min, collecting supernatant, inoculating VERO cells by the same method, and passaging or storing at-80 deg.C for use.
Example 3 identification method of LSR-8 Strain
Virus titer assay for LSR-8 strain:
adding pancreatin into the rotavirus harvest solution, activating in 37 deg.C constant temperature water bath for 1 hr, diluting with 10 times of maintenance solution to 10-1、10-2、10-3、10-4、10-5、10-6、10-7、10-8。
MA104 cells were seeded on 6-well plates, and when the cells grew into a dense monolayer, the growth medium was discarded, and the cell surface was washed 3 times with maintenance medium. Sucking out residual maintaining liquid, adding activated rotavirus liquid 500 μ l, adsorbing at 37 deg.C for 60-120 min, removing the virus liquid, adding maintaining liquid 3ml containing 0.3% agar, each dilution gradient is 2 holes, and transferring into incubator after agar is solidified. After 72 hours, a layer of maintenance medium containing 0.006% neutral red and 0.3% agar was placed over the original agar layer in the incubator protected from light. Continuing inversion at 37 DEG CAfter 4 days of culture, the cell lesions were observed every day, and the titer was calculated. The LSR-8 strain can grow to 10 in Vero cells5.5-107Pfu/ml and shows strong stability.
Neutralization discrimination test
MA104 cell line 104The cells/well were seeded in 96-well plates, and when the cells grew into a dense monolayer, the growth medium was discarded, and the cell surface was washed with maintenance medium. Sucking out residual maintenance liquid, diluting the virus antiserum of the separated strain according to 2-fold gradient, adding the virus antiserum into a 96-hole plate according to 100 ul/hole, adding virus activating liquid of 100Pfu/100 mu l/hole, uniformly mixing, and then carrying out CO at 37 DEG C2Neutralizing in incubator for 2 hr, discarding antiserum, adding maintenance liquid, and adding CO at 37 deg.C2And culturing in an incubator for 8-10 days, and observing the result.
Example 4 Generation and purification of LSR-8 Strain vaccine
Production of rotaviruses is achieved by using large-scale production roller bottles. Confluent monolayers of Vero cells in roller bottles were infected with specific rotavirus strains with multiplicity of infection MOI of 0.05-0.5. Filtering the virus liquid obtained by repeated freeze thawing, removing impurities, concentrating, purifying and inactivating to obtain virus stock solution, diluting according to a certain antigen content, and adding 0-1.6mg/ml aluminum hydroxide adjuvant.
Example 5 adjuvant
The aluminum hydroxide adjuvant has obvious effect on improving the immunogenicity of the rotavirus inactivated vaccine. Injecting vaccines with different aluminum hydroxide adjuvant contents into the muscle of the antibody-negative mouse, immunizing for 2 times at intervals of 28 days, collecting blood after immunization for 0, 14, 28 and 56 days, and detecting the titer of neutralizing antibodies in the serum of the mouse by a plaque reduction neutralization test. The experiment for the influence of aluminum hydroxide on the immunogenicity of the vaccine comprises two parts: the comparison of the first part of adjuvants with different concentrations on the antigen with the same vaccine concentration and the comparison of the second part of adjuvants with the same adjuvant concentration (0.8mg/ml) on the antigen with different concentrations show that the lead hydroxide adjuvant has obvious effect on improving the immunogenicity of the rotavirus inactivated vaccine.
Example 6 method for evaluating the immunopotency of LSR-8 Strain
(1) Analysis of immunogenicity of rotavirus vaccine samples
Before immunization, blood is collected from mice, serum is separated, and whether the mother mice contain rotavirus specific antibodies or not is detected through ELSA, so that positive mice are excluded. The virus harvest is purified and inactivated to prepare vaccine samples for immunization, 10 mice are injected intramuscularly in each dose of 0EU/100 mul, 40EU/100 mul, 80EU/100 mul and 160EU/100 mul 4 doses, 10 negative controls are injected, 100ul PBS is injected, and one-needle boosting immunization is carried out after 28 days. The microneutralization assay measures neutralizing antibody titers by measuring antibodies in blood samples obtained at different times 2, 4, 6, 8 weeks after dosing.
(2) Evaluation experiment of neutralizing antibody titer of immune serum rotavirus
1) MA104 cells were seeded in 96-well plates at 37 ℃ CO2Culturing in incubator until compact monolayer is formed.
2) The activated rotavirus strain is diluted to 1000Pfu/100 mul, and serum after the test animal is immunized is diluted by 4 times and diluted by 2 times of equal ratio gradient.
3) Mu.l of activated rotavirus strain was mixed with diluted serum 1:1 and neutralized for 1 hour at 37 ℃ in a CO2 incubator.
4) 100. mu.l/well of the neutralization solution was inoculated into 96-well culture plates which had been washed with a maintenance solution, and treated with CO2 at 37 ℃ for 1 hour.
5) The non-adsorbed neutralizing solution was discarded, and the cells were washed once with MEM maintenance solution, and then cultured for 7 days with the addition of the maintenance solution.
6) Goat anti-rotavirus immune polyclonal antibody (Millipore corporation) was treated with carbonate buffer (pH 9.6) at a ratio of 1: 1000 dilutions, 100. mu.l per well, were coated overnight at 4 ℃ in 96-well ELISA plates;
7) removing coating solution, washing the plate with PBS solution (PBST solution) containing 0.5% Tween-20 for 3 times (3 min each time); blocking with PBST solution containing 3% bovine serum albumin, 100. mu.l wells, at 37 ℃ for 2 hours;
8) removing the blocking solution, washing the plate 3 times by using the PBST solution, completely transferring the liquid in the neutralized 96-well cell plate to an ELISA plate, and incubating for 1h at 37 ℃;
9) after washing the plates 3 times with PBST, add buffer 1: HRP-labeled goat anti-rotavirus polyclonal antibody diluted 2000, 100 mul/hole, combined for 1h at 37 ℃;
10) after PBST washing for 3 times, adding TMB color development solution with 100 mul/hole, developing for 5min at room temperature, adding 2M H2SO4 with 100 mul/hole, and stopping developing;
11) the absorbance was measured at a wavelength of 450nm, 630nm (the test wavelength) using a microplate reader.
Cutoff value: the Cutoff threshold is equal to the negative hole mean value multiplied by 2.1, and if the negative mean value is less than or equal to 0.050, the Cutoff threshold is calculated according to 0.050. When the detection value of the sample to be detected is higher than the threshold value, the sample to be detected is considered as a positive hole, and the reading value of the antigen amount (EU/mL) is the positive highest dilution multiplied by 10.
Sequence listing
<110> Shandongwei Gaertong biological products Ltd
<120> rotavirus strain and use thereof as vaccine
<130> MP2028672
<160> 22
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1088
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Met Gly Lys Tyr Asn Leu Ile Leu Ser Glu Tyr Leu Ser Phe Val Tyr
1 5 10 15
Asn Ser Gln Ser Ala Val Gln Ile Pro Ile Tyr Tyr Ser Ser Asn Pro
20 25 30
Glu Leu Glu Lys Arg Cys Ile Glu Phe His Ala Lys Cys Val Asp Asn
35 40 45
Ser Lys Lys Gly Ile Ser Leu Lys Pro Val Phe Glu Glu Tyr Lys Asp
50 55 60
Val Ile Asp Asn Ala Thr Leu Leu Ser Ile Leu Ser Tyr Ser Tyr Asp
65 70 75 80
Lys Tyr Asn Ala Val Glu Arg Lys Leu Val Asn Tyr Ala Lys Gly Lys
85 90 95
Pro Leu Glu Ala Asp Leu Thr Ala Asn Glu Leu Asp Tyr Glu Asn Asn
100 105 110
Lys Ile Thr Ser Glu Leu Phe Gln Ser Ala Glu Glu Tyr Thr Asp Ser
115 120 125
Leu Met Asp Pro Ala Ile Leu Thr Ser Leu Ser Ser Asn Leu Asn Ala
130 135 140
Val Met Phe Trp Leu Glu Arg His Ser Asn Asp Val Ala Asp Ala Asn
145 150 155 160
Lys Ile Tyr Lys Arg Arg Leu Asp Leu Phe Thr Ile Val Ala Ser Thr
165 170 175
Ile Asn Lys Tyr Gly Val Pro Arg His Asn Glu Lys Tyr Arg Tyr Glu
180 185 190
Tyr Glu Val Met Lys Asp Lys Pro Tyr Tyr Leu Val Thr Trp Ala Asn
195 200 205
Ser Ser Ile Glu Met Leu Met Ser Val Phe Ser His Glu Asp Tyr Leu
210 215 220
Ile Ala Lys Glu Leu Ile Ile Leu Ser Tyr Ser Asn Arg Ser Thr Leu
225 230 235 240
Ala Lys Leu Val Ser Ser Pro Met Ser Ile Leu Val Ala Leu Ile Asp
245 250 255
Ile Asn Gly Thr Phe Ile Thr Asn Glu Glu Leu Glu Leu Glu Phe Ser
260 265 270
Asp Lys Tyr Val Lys Ala Ile Val Pro Asp Gln Ile Phe Asp Glu Leu
275 280 285
Gln Glu Met Ile Asp Asn Met Lys Lys Val Gly Leu Val Asp Ile Pro
290 295 300
Arg Met Ile Gln Glu Trp Leu Val Asp Cys Ser Leu Glu Lys Phe Thr
305 310 315 320
Leu Met Ser Lys Ile Tyr Ser Trp Ser Phe His Val Gly Phe Arg Lys
325 330 335
Gln Lys Met Ile Asp Ala Ala Leu Asp Gln Leu Lys Thr Glu Tyr Thr
340 345 350
Glu Asp Val Asp Gly Glu Met Tyr Asn Glu Tyr Thr Met Leu Ile Arg
355 360 365
Asp Glu Ile Val Lys Met Leu Glu Val Pro Val Lys His Asp Asp His
370 375 380
Leu Leu Arg Asp Ser Glu Leu Ala Gly Leu Leu Ser Met Ser Ser Ala
385 390 395 400
Ser Asn Gly Glu Ser Arg Gln Leu Lys Phe Gly Arg Lys Thr Ile Phe
405 410 415
Ser Thr Lys Lys Asn Met His Val Met Asp Asp Ile Ala His Gly Lys
420 425 430
Tyr Thr Pro Gly Val Ile Pro Pro Val Asn Val Asp Arg Pro Ile Pro
435 440 445
Leu Gly Arg Arg Asp Val Pro Gly Arg Arg Thr Arg Ile Ile Phe Ile
450 455 460
Leu Pro Tyr Glu Tyr Phe Ile Ala Gln His Ala Val Val Glu Lys Met
465 470 475 480
Leu Ser Tyr Ala Lys His Thr Arg Glu Tyr Ala Glu Phe Tyr Ser Gln
485 490 495
Ser Asn Gln Leu Leu Ser Tyr Gly Asp Val Thr Arg Phe Leu Ser Ser
500 505 510
Asn Ser Met Val Leu Tyr Thr Asp Val Ser Gln Trp Asp Ser Ser Gln
515 520 525
His Asn Thr Gln Pro Phe Arg Lys Gly Ile Ile Met Gly Leu Asp Met
530 535 540
Leu Ser Asn Met Thr Asn Asp Pro Lys Val Ile Gln Thr Leu Asn Leu
545 550 555 560
Tyr Lys Gln Thr Gln Val Asn Leu Met Asp Ser Tyr Val Gln Ile Pro
565 570 575
Asp Gly Asn Val Ile Lys Lys Ile Gln Tyr Gly Ala Val Ala Ser Gly
580 585 590
Glu Lys Gln Thr Lys Ala Ala Asn Ser Ile Ala Asn Leu Ala Leu Ile
595 600 605
Lys Thr Val Leu Ser Arg Ile Ala Asn Lys Tyr Ser Phe Ile Thr Lys
610 615 620
Ile Ile Arg Val Asp Gly Asp Asp Asn Tyr Ala Val Leu Gln Phe Asn
625 630 635 640
Thr Asp Val Thr Lys Gln Met Val Gln Asp Val Ser Asn Asp Val Arg
645 650 655
His Ile Tyr Ser Arg Met Asn Ala Lys Val Lys Ala Leu Val Ser Thr
660 665 670
Val Gly Ile Glu Ile Ala Lys Arg Tyr Ile Ala Gly Gly Lys Ile Phe
675 680 685
Phe Arg Ala Gly Ile Asn Leu Leu Asn Asn Glu Lys Arg Gly Gln Ser
690 695 700
Thr Gln Trp Asp Gln Ala Ala Ile Leu Tyr Ser Asn Tyr Ile Val Asn
705 710 715 720
Lys Leu Arg Gly Phe Glu Thr Asp Arg Glu Phe Ile Leu Thr Lys Ile
725 730 735
Ile Gln Met Thr Ser Val Ala Ile Thr Gly Ser Leu Arg Leu Phe Pro
740 745 750
Ser Glu Arg Val Leu Thr Thr Asn Ser Thr Phe Lys Val Phe Asp Ser
755 760 765
Glu Asp Phe Ile Ile Glu Tyr Gly Thr Thr Asp Asp Glu Val Tyr Ile
770 775 780
Gln Arg Ala Phe Met Ser Leu Ser Ser Gln Lys Ser Gly Ile Ala Asp
785 790 795 800
Glu Ile Ala Ser Ser Gln Thr Phe Lys Asn Tyr Val Asn Lys Leu Ser
805 810 815
Asp Gln Leu Leu Ile Ser Lys Asn Val Ile Val Ser Lys Gly Ile Ala
820 825 830
Val Thr Glu Lys Ala Lys Leu Asn Ser Tyr Ala Pro Val Tyr Leu Glu
835 840 845
Lys Arg Arg Ala Gln Ile Ser Ala Leu Leu Thr Met Leu Gln Lys Pro
850 855 860
Val Ser Phe Lys Ser Asn Lys Ile Thr Ile Asn Asp Ile Leu Arg Asp
865 870 875 880
Ile Lys Pro Phe Phe Val Thr Ser Glu Ala Asn Leu Ser Ile Gln Tyr
885 890 895
Arg Lys Phe Met Pro Thr Leu Pro Asp Asn Val Gln Tyr Val Ile Gln
900 905 910
Cys Ile Gly Ser Arg Thr Tyr Gln Ile Glu Asp Ser Gly Ser Lys Ser
915 920 925
Ser Ile Ser Lys Leu Ile Ser Lys Tyr Ser Val Tyr Lys Pro Ser Ile
930 935 940
Glu Glu Leu Tyr Lys Val Ile Ser Leu Arg Glu Gln Glu Ile Gln Leu
945 950 955 960
Tyr Leu Val Ser Leu Gly Val Pro Pro Val Asp Ala Gly Thr Tyr Val
965 970 975
Gly Ser Arg Ile Tyr Ser Gln Asp Lys Tyr Lys Ile Leu Glu Ser Tyr
980 985 990
Val Tyr Asn Leu Leu Ser Ile Asn Tyr Gly Cys Tyr Gln Leu Phe Asp
995 1000 1005
Phe Asn Ser Pro Asp Leu Glu Lys Leu Ile Arg Ile Pro Phe Lys Gly
1010 1015 1020
Lys Ile Pro Ala Val Thr Phe Ile Leu His Leu Tyr Ala Lys Leu Glu
1025 1030 1035 1040
Ile Ile Asn His Ala Ile Lys Asn Gly Ala Trp Ile Ser Leu Phe Cys
1045 1050 1055
Asn Tyr Pro Lys Ser Glu Met Ile Lys Leu Trp Lys Lys Met Trp Asn
1060 1065 1070
Ile Thr Ala Leu Arg Ser Pro Tyr Thr Ser Ala Asn Phe Phe Gln Asp
1075 1080 1085
<210> 2
<211> 894
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Met Ala Tyr Arg Lys Arg Gly Ala Lys Arg Glu Asp Leu Pro Gln Gln
1 5 10 15
Asn Glu Arg Leu Gln Glu Lys Glu Ile Glu Asn Asn Thr Asp Val Thr
20 25 30
Met Glu Asn Lys Asn Lys Asn Arg Asn Asn Asn Arg Lys Gln Gln Leu
35 40 45
Ser Asp Lys Val Leu Ser Gln Lys Glu Glu Ile Ile Thr Asp Val Gln
50 55 60
Asp Asp Ile Lys Ile Ala Asp Glu Val Lys Lys Ser Ser Lys Glu Glu
65 70 75 80
Ser Lys Gln Leu Leu Glu Ile Leu Lys Thr Lys Glu Asp His Gln Lys
85 90 95
Glu Val Gln Tyr Glu Ile Leu Gln Lys Thr Ile Pro Thr Phe Glu Pro
100 105 110
Lys Glu Ser Ile Leu Lys Lys Leu Glu Asp Ile Lys Pro Glu Gln Ala
115 120 125
Lys Lys Gln Met Lys Leu Phe Arg Ile Phe Glu Pro Arg Gln Leu Pro
130 135 140
Ile Tyr Arg Ala Asn Gly Glu Lys Glu Leu Arg Asn Arg Trp Tyr Trp
145 150 155 160
Lys Leu Lys Lys Asp Thr Leu Pro Asp Gly Asp Tyr Asp Val Arg Glu
165 170 175
Tyr Phe Leu Asn Leu Tyr Asp Gln Ile Leu Ile Glu Met Pro Asp Tyr
180 185 190
Leu Leu Leu Lys Asp Met Ala Val Glu Asn Lys Asn Ser Arg Asp Ala
195 200 205
Gly Lys Val Val Asp Ser Glu Thr Ala Ser Ile Cys Asp Ala Ile Phe
210 215 220
Gln Asp Glu Glu Thr Glu Gly Val Ile Arg Arg Phe Ile Ala Asp Met
225 230 235 240
Arg Gln Gln Val Gln Ala Asp Arg Asn Ile Val Asn Tyr Pro Ser Ile
245 250 255
Leu His Pro Ile Asp His Ala Phe Asn Glu Tyr Phe Leu Asn His Gln
260 265 270
Leu Val Glu Pro Leu Asn Asn Asp Ile Ile Phe Asn Tyr Ile Pro Glu
275 280 285
Arg Ile Arg Asn Asp Val Asn Tyr Ile Leu Asn Met Asp Met Asn Leu
290 295 300
Pro Ser Thr Ala Arg Tyr Ile Arg Pro Asn Leu Leu Gln Asp Arg Leu
305 310 315 320
Asn Leu His Asp Asn Phe Glu Ser Leu Trp Asp Thr Ile Thr Thr Ser
325 330 335
Asn Tyr Ile Leu Ala Arg Ser Val Val Pro Asp Leu Lys Glu Lys Glu
340 345 350
Leu Val Ser Thr Glu Ala Gln Ile Gln Lys Met Ser Gln Asp Leu Gln
355 360 365
Leu Glu Ala Leu Thr Ile Gln Ser Glu Thr Gln Phe Leu Ala Gly Ile
370 375 380
Asn Ser Gln Ala Ala Asn Asp Cys Phe Lys Thr Leu Ile Ala Ala Met
385 390 395 400
Leu Ser Gln Arg Thr Met Ser Leu Asp Phe Val Thr Thr Asn Tyr Met
405 410 415
Ser Leu Ile Ser Gly Met Trp Leu Leu Thr Val Ile Pro Asn Asp Met
420 425 430
Phe Leu Arg Glu Ser Leu Val Ala Cys Glu Leu Ala Ile Ile Asn Thr
435 440 445
Ile Val Tyr Pro Ala Phe Gly Met Gln Arg Met His Tyr Arg Asn Gly
450 455 460
Asp Pro Gln Thr Pro Phe Gln Ile Ala Glu Gln Gln Ile Gln Asn Phe
465 470 475 480
Gln Val Ala Asn Trp Leu His Phe Ile Asn Asn Asn Arg Phe Arg Gln
485 490 495
Val Val Ile Asp Gly Val Leu Asn Gln Thr Leu Asn Asp Asn Ile Arg
500 505 510
Asn Gly Gln Val Ile Asn Gln Leu Met Glu Ala Leu Met Gln Leu Ser
515 520 525
Arg Gln Gln Phe Pro Thr Met Pro Val Asp Tyr Lys Arg Ser Ile Gln
530 535 540
Arg Gly Ile Leu Leu Leu Ser Asn Arg Leu Gly Gln Leu Val Asp Leu
545 550 555 560
Thr Arg Leu Leu Ser Tyr Asn Tyr Glu Thr Leu Met Ala Cys Ile Thr
565 570 575
Met Asn Met Gln His Val Gln Thr Leu Thr Thr Glu Arg Leu Gln Leu
580 585 590
Thr Ser Val Thr Ser Leu Cys Met Leu Ile Gly Asn Thr Thr Val Ile
595 600 605
Pro Ser Pro Gln Thr Leu Phe His Tyr Tyr Asn Val Asn Val Asn Phe
610 615 620
His Ser Asn Tyr Asn Glu Arg Ile Asn Asp Ala Val Ala Ile Ile Thr
625 630 635 640
Ala Ala Asn Arg Leu Asn Leu Tyr Gln Lys Lys Met Lys Ser Ile Val
645 650 655
Glu Asp Phe Leu Lys Arg Leu Gln Ile Phe Asp Val Pro Arg Val Pro
660 665 670
Asp Asp Gln Met Tyr Arg Leu Arg Asp Arg Leu Arg Leu Leu Pro Val
675 680 685
Glu Arg Arg Arg Leu Asp Ile Phe Asn Leu Ile Leu Met Asn Met Glu
690 695 700
Gln Ile Glu Arg Ala Ser Asp Lys Ile Ala Gln Gly Val Ile Ile Ala
705 710 715 720
Tyr Arg Asp Met Gln Leu Glu Arg Asp Glu Met Tyr Gly Phe Val Asn
725 730 735
Ile Ala Arg Asn Leu Asp Gly Tyr Gln Gln Ile Asn Leu Glu Glu Leu
740 745 750
Met Arg Thr Gly Asp Tyr Gly Gln Ile Thr Asn Met Leu Leu Asn Asn
755 760 765
Gln Pro Val Ala Leu Val Gly Ala Leu Pro Phe Val Thr Asp Ser Ser
770 775 780
Val Ile Ser Leu Ile Ala Lys Leu Asp Ala Thr Val Phe Ala Gln Ile
785 790 795 800
Val Lys Leu Arg Lys Val Asp Thr Leu Lys Pro Ile Leu Tyr Lys Ile
805 810 815
Asn Ser Asp Ser Asn Asp Phe Tyr Leu Val Ala Asn Tyr Asp Trp Ile
820 825 830
Pro Thr Ser Thr Thr Lys Val Tyr Lys Gln Val Pro Gln Pro Phe Asp
835 840 845
Phe Arg Ala Ser Met His Met Leu Thr Ser Asn Leu Thr Phe Thr Val
850 855 860
Tyr Ser Asp Leu Leu Ser Phe Val Ser Ala Asp Thr Val Glu Pro Ile
865 870 875 880
Asn Ala Ile Ala Phe Asp Asn Met Arg Ile Met Asn Glu Leu
885 890
<210> 3
<211> 835
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 3
Met Lys Val Leu Ala Leu Arg His Ser Val Ala Gln Val Tyr Ala Asp
1 5 10 15
Thr Gln Thr Tyr Leu His Asp Asp Ser Lys Asp Glu Tyr Glu Asn Ala
20 25 30
Phe Leu Ile Ser Asn Leu Thr Thr His Asn Ile Leu Tyr Leu Asn Tyr
35 40 45
Ser Leu Lys Thr Leu Lys Ile Leu Asn Lys Ser Gly Ile Ala Ala Val
50 55 60
Glu Val Gln Ser Pro Asp Glu Leu Phe Ala Leu Ile Arg Cys Asn Phe
65 70 75 80
Thr Tyr Asp Tyr Glu Asn Asn Ile Ile Tyr Leu His Asp Tyr Ser Tyr
85 90 95
Tyr Thr Asn Asn Glu Ile Arg Thr Asp Gln His Trp Ile Thr Lys Thr
100 105 110
Asn Ile Thr Glu Tyr Leu Leu Pro Gly Trp Lys Leu Thr Tyr Val Gly
115 120 125
Tyr Asn Gly Lys Asn Thr Arg Gly His Tyr Asn Phe Ser Phe Leu Cys
130 135 140
Gln Asn Ala Ala Thr Asp Asp Asp Ile Ile Ile Glu Tyr Ile Tyr Ser
145 150 155 160
Asn Glu Leu Asp Phe Gln Asn Phe Leu Leu Arg Lys Ile Lys Glu Arg
165 170 175
Met Thr Thr Ser Leu Pro Ile Ala Arg Leu Ser Asn Arg Val Phe Arg
180 185 190
Asp Lys Leu Phe Pro Ser Ile Val Asn Met Tyr Lys Lys Val Ile Asn
195 200 205
Val Gly Pro Arg Asn Glu Ser Met Phe Thr Phe Leu Asn Phe Pro Thr
210 215 220
Ile Lys Gln Phe Ser Asn Gly Ala Tyr Ile Val Lys His Thr Ile Lys
225 230 235 240
Leu Lys Gln Glu Lys Trp Leu Gly Lys Arg Val Ser Gln Phe Asp Ile
245 250 255
Gly Gln Tyr Lys Asn Met Leu Asn Val Val Thr Thr Ile Tyr Tyr Tyr
260 265 270
Tyr Asn Leu Tyr His Ser Lys Pro Ile Ile Tyr Met Leu Gly Ser Ala
275 280 285
Pro Ser Tyr Trp Ile Tyr Asp Ile Lys Gln Tyr Ser Asp Phe Thr Phe
290 295 300
Glu Thr Trp Asp Pro Leu Asp Thr Pro Tyr Ser Thr Ile His His Lys
305 310 315 320
Glu Leu Phe Phe Asp Lys Asp Val Asn Lys Leu Lys Asp Asn Ser Val
325 330 335
Leu Tyr Ile Asp Ile Arg Thr Asp Arg Gly Asn Met Asp Trp Lys Glu
340 345 350
Trp Arg Lys Val Val Glu Gln Gln Thr Ile Asn Asn Leu Asn Ile Ala
355 360 365
Tyr Lys Tyr Leu Ala Thr Gly Lys Ala Lys Val Cys Cys Val Lys Leu
370 375 380
Thr Ala Met Asp Leu Glu Leu Pro Ile Thr Ala Lys Leu Leu His His
385 390 395 400
Pro Thr Thr Glu Val Arg Ser Glu Phe Tyr Ala Ile Leu Asp Val Trp
405 410 415
Asp Ile Ile Thr Ile Lys Arg Phe Ile Pro Lys Gly Val Phe Tyr Ala
420 425 430
Phe Ile Asn Asn Ile Ile Thr Glu Asn Val Phe Ile Gln Pro Ser Phe
435 440 445
Lys Leu Lys Val Ser Pro Thr Asp Tyr Ile Val Ala Leu Tyr Ala Leu
450 455 460
Ser Asn Asp Phe Asn Ser Arg Gln Asp Val Ile Asn Leu Ile Asn Lys
465 470 475 480
Gln Lys Gln Ser Leu Ile Thr Val Arg Met Asn Asn Thr Phe Lys Asp
485 490 495
Glu Pro Lys Val Asn Phe Lys Asn Ile Tyr Asp Trp Thr Phe Leu Pro
500 505 510
Thr Asp Phe Glu Leu Lys Asp Ser Ile Ile Thr Ser Tyr Asp Gly Cys
515 520 525
Leu Gly Met Phe Gly Leu Ser Ile Ser Leu Ser Ser Lys Pro Thr Gly
530 535 540
Asn Asn His Leu Phe Ile Ile Asn Gly Thr Asp Lys Tyr Asp Lys Leu
545 550 555 560
Asp Gln Tyr Ala Asn His Met Gly Ile Ser Arg Arg Ser His Gln Ile
565 570 575
Arg Phe Ser Glu Ser Ala Thr Ser Tyr Ser Gly Tyr Ile Phe Arg Asp
580 585 590
Leu Ser Asn Asn Asn Phe Asn Leu Ile Gly Thr Asn Val Glu Asn Ser
595 600 605
Val Ser Gly His Val Tyr Asn Ala Leu Ile Tyr Tyr Arg Tyr Asn Tyr
610 615 620
Ala Phe Asp Leu Lys Arg Trp Ile Tyr Leu His Ser Ile Gly Lys Ala
625 630 635 640
Ala Val Glu Gly Gly Arg Tyr Tyr Glu His Ala Pro Ile Glu Leu Ile
645 650 655
Tyr Ala Cys Arg Ser Ala Lys Glu Phe Ala Ile Leu Gln Asp Asp Leu
660 665 670
Thr Val Leu Arg Tyr Ala Asn Glu Ile Glu Glu Tyr Ile Asn Lys Val
675 680 685
Tyr Ser Ile Thr Tyr Ala Asp Asp Pro Asn Tyr Phe Ile Gly Ile Lys
690 695 700
Phe Asn Ser Ile Pro Tyr Glu Tyr Asp Val Lys Val Pro His Leu Thr
705 710 715 720
Leu Gly Val Leu Phe Ile Ser Asp Asn Met Ile His Asp Val Val Thr
725 730 735
Val Leu Lys Lys Met Lys Thr Glu Leu Phe Lys Thr Glu Ile Ser Thr
740 745 750
Ser Tyr Thr Tyr Met Leu Ser Asp Asn Ile Tyr Val Ala Asn Ala Ser
755 760 765
Gly Val Leu Ser Thr Tyr Phe Lys Leu Tyr Asn Met Phe Tyr Arg Asn
770 775 780
His Ile Thr Phe Gly Gln Ser Arg Met Phe Ile Pro His Ile Thr Leu
785 790 795 800
Ser Phe Ser Asn Lys Gln Thr Val Arg Ile Glu Ser Thr Arg Leu Lys
805 810 815
Ile Asn Ser Ile Tyr Leu Arg Lys Ile Lys Gly Glu Thr Val Phe Asp
820 825 830
Met Ser Glu
835
<210> 4
<211> 775
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 4
Met Ala Ser Leu Ile Tyr Arg Gln Leu Leu Thr Asn Ser Tyr Ser Val
1 5 10 15
Asp Leu Tyr Asp Glu Met Lys Gln Ile Gly Ser Glu Lys Thr Gln Asn
20 25 30
Val Thr Ile Asn Pro Gly Pro Phe Ala Gln Thr Arg Tyr Ala Pro Val
35 40 45
Asn Trp Gly His Gly Glu Ile Asn Asp Ser Thr Thr Val Glu Pro Ile
50 55 60
Leu Asp Gly Pro Tyr Gln Pro Thr Thr Phe Thr Pro Pro Thr Asp Tyr
65 70 75 80
Trp Ile Leu Ile Asn Ser Asn Thr Asn Gly Val Val Tyr Glu Ser Thr
85 90 95
Asn Asn Ser Asp Phe Trp Thr Ala Val Ile Ala Val Glu Pro His Val
100 105 110
Asp Pro Val Asp Arg Gln Tyr Asn Val Phe Gly Glu Asn Lys Gln Phe
115 120 125
Asn Val Arg Asn Asp Ser Asp Lys Trp Lys Phe Leu Glu Met Phe Arg
130 135 140
Gly Ser Ser Gln Ser Asp Phe Tyr Asn Arg Arg Thr Leu Thr Ser Asp
145 150 155 160
Thr Lys Leu Val Gly Ile Leu Lys Tyr Gly Gly Arg Ile Trp Ala Phe
165 170 175
His Gly Glu Thr Pro Arg Ala Thr Thr Asp Ser Ser Asn Thr Ala Asn
180 185 190
Leu Asn Gly Ile Ser Ile Thr Ile His Ser Glu Phe Tyr Ile Ile Pro
195 200 205
Arg Ser Gln Glu Ser Lys Cys Asn Glu Tyr Ile Asn Asn Gly Leu Pro
210 215 220
Pro Ile Gln Asn Thr Arg Asn Val Val Pro Leu Ser Leu Ser Ser Arg
225 230 235 240
Ser Ile Gln Tyr Thr Arg Ala Gln Val Asn Glu Asp Ile Thr Ile Ser
245 250 255
Lys Thr Ser Leu Trp Lys Glu Met Gln His Asn Arg Asp Ile Ile Ile
260 265 270
Arg Phe Lys Phe Gly Asn Ser Ile Ile Lys Leu Gly Gly Leu Gly Tyr
275 280 285
Lys Trp Ser Glu Ile Ser Tyr Lys Ala Ala Asn Tyr Gln Tyr Asn Tyr
290 295 300
Leu Arg Asp Gly Glu Gln Val Thr Ala His Thr Thr Cys Ser Val Asn
305 310 315 320
Gly Val Asn Asn Phe Ser Tyr Asn Gly Gly Ser Leu Pro Thr Asp Phe
325 330 335
Ser Ile Ser Arg Tyr Glu Val Ile Lys Glu Asn Ser Tyr Val Tyr Val
340 345 350
Asp Tyr Trp Asp Asp Ser Lys Ala Phe Arg Asn Met Val Tyr Val Arg
355 360 365
Ser Leu Ala Ala Asn Leu Asn Ser Val Lys Cys Thr Gly Gly Ser Tyr
370 375 380
His Phe Ser Ile Pro Val Gly Ala Trp Pro Val Met Asn Gly Gly Ala
385 390 395 400
Val Ser Leu His Phe Ala Gly Val Thr Leu Ser Thr Gln Phe Thr Asp
405 410 415
Phe Val Ser Leu Asn Ser Leu Arg Phe Arg Phe Ser Leu Thr Val Asp
420 425 430
Glu Pro Ser Phe Ser Ile Leu Arg Thr Arg Thr Val Asn Leu Tyr Gly
435 440 445
Leu Pro Ala Ala Asn Pro Asn Asn Gly Asn Glu Tyr Tyr Glu Ile Ser
450 455 460
Gly Arg Phe Ser Leu Ile Ser Leu Val Pro Thr Asn Asp Asp Tyr Gln
465 470 475 480
Thr Pro Ile Met Asn Ser Val Thr Val Arg Gln Asp Leu Glu Arg Gln
485 490 495
Leu Thr Asp Leu Arg Glu Glu Phe Asn Ser Leu Ser Gln Glu Ile Ala
500 505 510
Met Ser Gln Leu Ile Asp Leu Ala Leu Leu Pro Leu Asp Met Phe Ser
515 520 525
Met Phe Ser Gly Ile Lys Ser Thr Ile Asp Leu Thr Lys Ser Met Ala
530 535 540
Thr Ser Val Met Lys Lys Phe Arg Lys Ser Lys Leu Ala Thr Ser Val
545 550 555 560
Ser Glu Met Thr Asn Ser Leu Ser Asp Ala Ala Ser Ser Ala Ser Arg
565 570 575
Ser Val Ser Val Arg Ser Asn Ile Ser Ala Phe Ser Asn Trp Thr Asn
580 585 590
Val Ser Asn Asp Val Ser Asn Val Thr Asn Ser Val Asn Asp Ile Ser
595 600 605
Thr Gln Thr Ser Thr Ile Ser Lys Asn Leu Arg Leu Arg Glu Met Ile
610 615 620
Thr Gln Thr Glu Gly Met Ser Phe Asp Asp Ile Ser Ala Ala Val Leu
625 630 635 640
Lys Thr Lys Ile Asp Met Ser Thr Gln Ile Gly Lys Asn Thr Leu Pro
645 650 655
Asp Ile Val Thr Glu Ala Ser Glu Lys Phe Ile Pro Lys Arg Ser Tyr
660 665 670
Arg Ile Leu Lys Asp Asp Glu Val Met Glu Ile Asn Thr Glu Gly Lys
675 680 685
Phe Phe Ala Tyr Lys Ile Asp Thr Leu Asn Glu Val Pro Phe Asp Val
690 695 700
Asn Lys Phe Ala Glu Leu Val Thr Asn Ser Pro Val Ile Ser Ala Ile
705 710 715 720
Ile Asp Phe Lys Thr Leu Lys Asn Leu Asn Asp Asn Tyr Gly Ile Thr
725 730 735
Arg Thr Glu Ala Leu Asn Leu Ile Lys Ser Asn Pro Asn Val Leu Arg
740 745 750
Asn Phe Ile Asn Gln Asn Asn Pro Ile Ile Arg Asn Arg Ile Glu Gln
755 760 765
Leu Ile Leu Gln Cys Lys Leu
770 775
<210> 5
<211> 397
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 5
Met Glu Val Leu Tyr Ser Leu Ser Lys Thr Leu Lys Asp Ala Arg Asp
1 5 10 15
Lys Ile Val Glu Gly Thr Leu Tyr Ser Asn Val Ser Asp Leu Ile Gln
20 25 30
Gln Phe Asn Gln Met Ile Val Thr Met Asn Gly Asn Asp Phe Gln Thr
35 40 45
Gly Gly Ile Gly Asn Leu Pro Ile Arg Asn Trp Thr Phe Asp Phe Gly
50 55 60
Leu Leu Gly Thr Thr Leu Leu Asn Leu Asp Ala Asn Tyr Val Glu Thr
65 70 75 80
Ala Arg Thr Thr Ile Glu Tyr Phe Ile Asp Phe Ile Asp Asn Val Cys
85 90 95
Met Asp Glu Met Ala Arg Glu Ser Gln Arg Asn Gly Val Ala Pro Gln
100 105 110
Ser Glu Ala Leu Arg Lys Leu Ala Gly Ile Lys Phe Lys Arg Ile Asn
115 120 125
Phe Asn Asn Ser Ser Glu Tyr Ile Glu Asn Trp Asn Leu Gln Asn Arg
130 135 140
Arg Gln Arg Thr Gly Phe Val Phe His Lys Pro Asn Ile Phe Pro Tyr
145 150 155 160
Ser Ala Ser Phe Thr Leu Asn Arg Ser Gln Pro Met His Asp Asn Leu
165 170 175
Met Gly Thr Met Trp Leu Asn Ala Gly Ser Glu Ile Gln Val Ala Gly
180 185 190
Phe Asp Tyr Ser Cys Ala Leu Asn Ala Pro Ala Asn Ile Gln Gln Phe
195 200 205
Glu His Ile Val Gln Leu Arg Arg Ala Leu Thr Thr Ala Thr Ile Thr
210 215 220
Leu Leu Pro Asp Ala Glu Arg Phe Ser Phe Pro Arg Val Ile Asn Ser
225 230 235 240
Ala Asp Gly Ala Thr Thr Trp Phe Phe Asn Pro Ile Ile Leu Arg Pro
245 250 255
Asn Asn Val Glu Val Glu Phe Leu Leu Asn Gly Gln Ile Ile Asn Thr
260 265 270
Tyr Gln Ala Arg Phe Gly Thr Ile Val Ala Arg Asn Phe Asp Thr Ile
275 280 285
Arg Leu Ser Phe Gln Leu Met Arg Pro Pro Asn Met Thr Pro Ala Val
290 295 300
Asn Ala Leu Phe Pro Gln Ala Gln Pro Phe Gln His His Ala Thr Val
305 310 315 320
Gly Leu Thr Leu Arg Ile Glu Ser Ala Val Cys Glu Ser Val Leu Ala
325 330 335
Asp Ala Asn Glu Thr Leu Leu Ala Asn Val Thr Ala Val Arg Gln Glu
340 345 350
Tyr Ala Ile Pro Val Gly Pro Val Phe Pro Pro Gly Met Asn Trp Thr
355 360 365
Glu Leu Ile Thr Asn Tyr Ser Pro Ser Arg Glu Asp Asn Leu Gln Arg
370 375 380
Val Phe Thr Val Ala Ser Ile Arg Ser Met Leu Ile Lys
385 390 395
<210> 6
<211> 326
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 6
Met Tyr Gly Ile Glu Tyr Thr Thr Val Leu Thr Phe Leu Ile Ser Ile
1 5 10 15
Val Leu Leu Asn Tyr Ile Leu Lys Ser Leu Thr Ser Ala Met Asp Phe
20 25 30
Ile Ile Tyr Arg Phe Leu Leu Leu Ile Val Ile Leu Ser Pro Phe Val
35 40 45
Lys Thr Gln Asn Tyr Gly Ile Asn Leu Pro Ile Thr Gly Ser Met Asp
50 55 60
Thr Ala Tyr Ala Asn Ser Ser Gln Gln Glu Thr Phe Leu Thr Ser Thr
65 70 75 80
Leu Cys Leu Tyr Tyr Pro Thr Glu Ala Ser Thr Gln Ile Gly Asp Thr
85 90 95
Glu Trp Lys Asn Thr Leu Ser Gln Leu Phe Leu Thr Lys Gly Trp Pro
100 105 110
Thr Gly Ser Val Tyr Phe Arg Glu Tyr Thr Asp Ile Ala Ser Phe Ser
115 120 125
Ile Asp Pro Gln Leu Tyr Cys Asp Tyr Asn Val Val Leu Met Lys His
130 135 140
Asp Ser Thr Leu Glu Leu Asp Met Ser Glu Leu Ala Asp Leu Ile Leu
145 150 155 160
Asn Glu Trp Leu Cys Asn Pro Met Asp Ile Thr Leu Tyr Tyr Tyr Gln
165 170 175
Gln Thr Asp Glu Ser Asn Lys Trp Ile Ser Met Gly Gln Ser Cys Thr
180 185 190
Ile Lys Val Cys Pro Leu Asn Thr Gln Thr Ser Gly Ile Gly Cys Thr
195 200 205
Thr Thr Asn Thr Ala Thr Phe Glu Glu Val Ala Thr Ser Glu Lys Leu
210 215 220
Val Ile Thr Asp Val Val Asp Gly Val Asn His Lys Leu Asp Val Thr
225 230 235 240
Thr Asn Thr Cys Thr Ile Arg Asn Cys Lys Lys Leu Gly Pro Arg Glu
245 250 255
Asn Val Ala Ile Ile Gln Val Gly Gly Ser Glu Val Leu Asp Ile Thr
260 265 270
Ala Asp Pro Thr Thr Thr Pro Gln Thr Glu Arg Met Met Arg Val Asn
275 280 285
Trp Lys Lys Trp Trp Gln Val Phe Tyr Thr Val Val Asp Tyr Ile Asn
290 295 300
Gln Ile Val Gln Val Met Ser Lys Arg Ser Arg Ser Leu Asn Ser Ala
305 310 315 320
Ala Phe Tyr Tyr Arg Val
325
<210> 7
<211> 486
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 7
Met Ala Thr Phe Lys Asp Ala Cys Tyr Tyr Tyr Lys Arg Ile Asn Lys
1 5 10 15
Leu Asn His Ala Val Leu Lys Leu Gly Val Asn Asp Thr Trp Arg Thr
20 25 30
Ser Pro Pro Thr Lys Tyr Lys Gly Trp Cys Leu Asp Cys Cys Gln His
35 40 45
Thr Asp Leu Thr Tyr Cys Arg Gly Cys Thr Met Tyr His Val Cys Gln
50 55 60
Trp Cys Ser Gln Tyr Asp Arg Cys Phe Leu Asp Asn Gln Pro His Leu
65 70 75 80
Leu Arg Met Arg Thr Phe Lys Asn Glu Val Thr Lys Asn Asp Leu Met
85 90 95
Asn Leu Ile Asp Met Tyr Asn Val Leu Phe Pro Ile Asn Gln Arg Ile
100 105 110
Val Asp Lys Phe Ile Asn Ser Thr Arg Gln His Lys Cys Arg Asn Glu
115 120 125
Cys Met Thr Gln Trp Tyr Asn His Leu Leu Met Pro Ile Thr Leu Gln
130 135 140
Ser Leu Ser Ile Glu Leu Asp Gly Asp Val Tyr Tyr Val Phe Gly Tyr
145 150 155 160
Tyr Asp Ser Met Ser Asp Ile Asn Gln Thr Pro Phe Ser Phe Thr Asn
165 170 175
Leu Ile Gly Met Tyr Asp Lys Leu Leu Leu Asp Asn Val Asn Phe Asn
180 185 190
Arg Met Ser Phe Leu Pro Val Ala Leu Gln Gln Glu Tyr Ala Leu Arg
195 200 205
Tyr Phe Ser Lys Ser Arg Phe Ile Ser Glu Lys Arg Lys Cys Ile Ser
210 215 220
Asp Leu His Phe Ser Thr Asn Val Ile Glu Asn Leu His Asn Pro Ser
225 230 235 240
Phe Lys Ile Gln Ile Thr Arg Asn Cys Ser Glu Leu Ser Ser Asp Trp
245 250 255
Asn Gly Ala Cys Lys Leu Val Lys Asp Met Ser Thr Tyr Phe Asn Val
260 265 270
Leu Lys Thr Ser His Ile Glu Phe Tyr Ser Ile Ser Thr Arg Cys Arg
275 280 285
Ile Phe Thr Gln His Lys Leu Lys Ile Ala Ser Lys His Ile Lys Pro
290 295 300
Asn Tyr Val Thr Ser Asn His Lys Thr Ser Ala Thr Glu Val His Asn
305 310 315 320
Cys Lys Trp Cys Ser Ile Asn Asn Gly Tyr Thr Val Trp Asn Asp Phe
325 330 335
Arg Ile Lys Lys Ile Tyr Asp Asn Ile Phe Asn Phe Leu Arg Ala Leu
340 345 350
Val Lys Ser Asn Ser Asn Ile Gly His Cys Ser Ser Gln Glu Lys Ile
355 360 365
Tyr Glu His Ile Lys Gly Val Leu Asp Ile Cys Asp Asp Glu Lys Trp
370 375 380
Lys Met Ala Val Ala Glu Ile Phe Asn Cys Leu Glu Pro Val Glu Leu
385 390 395 400
Asp Thr Val Lys Tyr Val Leu Phe Asn His Glu Val Asn Trp Asp Val
405 410 415
Ile Asn Leu Leu Val Gln Ser Val Gly Lys Val Pro Gln Ile Leu Thr
420 425 430
Leu Asn Asp Ile Val Ile Ile Met Lys Ser Ile Ile Tyr Asp Trp Phe
435 440 445
Asp Ile Arg Tyr Met Arg Asn Thr Pro Met Thr Thr Phe Thr Val Asp
450 455 460
Lys Leu Arg Gln Leu Cys Thr Gly Val Lys Thr Val Asp Tyr Asp Ser
465 470 475 480
Gly Ile Ser Asp Val Glu
485
<210> 8
<211> 317
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 8
Met Ala Glu Leu Ala Cys Phe Cys Tyr Pro His Leu Glu Asn Asp Ser
1 5 10 15
Tyr Lys Phe Ile Pro Phe Asn Asn Leu Ala Ile Lys Cys Met Leu Thr
20 25 30
Ala Lys Ile Asp Lys Lys Asp Gln Asp Lys Phe Tyr Asn Ser Ile Val
35 40 45
Tyr Gly Ile Ala Pro Pro Pro Gln Phe Arg Lys Arg Tyr Asn Thr Ser
50 55 60
Asp Asn Ser Arg Gly Met Asn Tyr Glu Thr Ile Met Phe Asn Lys Val
65 70 75 80
Ala Ile Leu Ile Cys Glu Ala Leu Asn Ser Ile Lys Val Thr Gln Ser
85 90 95
Glu Val Ala Asn Val Leu Ser Arg Val Val Ser Val Arg His Leu Glu
100 105 110
Asn Leu Val Leu Arg Lys Glu Asn His Gln Asp Val Leu Phe His Ser
115 120 125
Lys Glu Leu Leu Leu Lys Ser Val Leu Ile Ala Ile Gly Gln Ser Lys
130 135 140
Glu Ile Glu Thr Thr Ala Thr Ala Glu Gly Gly Glu Ile Val Phe Gln
145 150 155 160
Asn Ala Ala Phe Thr Met Trp Lys Leu Thr Tyr Leu Asp His Lys Leu
165 170 175
Met Pro Ile Leu Asp Gln Asn Phe Ile Glu Tyr Lys Ile Thr Leu Asn
180 185 190
Glu Asp Lys Pro Ile Ser Asp Val Cys Val Lys Glu Leu Val Ala Glu
195 200 205
Leu Arg Trp Gln Tyr Asn Arg Phe Ala Ile Ile Thr His Gly Lys Gly
210 215 220
His Tyr Arg Val Val Lys Tyr Ser Ser Val Ala Asn His Ala Asp Arg
225 230 235 240
Val Phe Ala Thr Tyr Lys Asn Asn Ala Lys Ser Gly Asn Thr Thr Asp
245 250 255
Phe Asn Leu Leu Asp Gln Arg Ile Ile Trp Gln Asn Trp Tyr Ala Phe
260 265 270
Thr Ser Ser Met Lys Gln Gly Asn Thr Ile Asp Val Cys Lys Lys Leu
275 280 285
Leu Phe Gln Lys Met Lys Gln Glu Lys Asn Pro Phe Lys Gly Leu Ser
290 295 300
Thr Asp Arg Lys Met Asp Glu Val Ser His Val Gly Ile
305 310 315
<210> 9
<211> 310
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 9
Met Glu Ser Thr Gln Gln Met Val Ser Ser Ile Ile Asn Thr Ser Phe
1 5 10 15
Glu Ala Ala Val Val Ala Ala Thr Ser Thr Leu Glu Leu Met Gly Ile
20 25 30
Gln Tyr Asp Tyr Asn Glu Val Phe Thr Arg Val Lys Ser Lys Phe Asp
35 40 45
Tyr Val Met Asp Asp Ser Gly Val Lys Asn Asn Leu Leu Gly Lys Ala
50 55 60
Ile Thr Ile Asp Gln Ala Leu Asn Gly Lys Phe Gly Ser Ala Ile Arg
65 70 75 80
Asn Arg Asn Trp Met Thr Asp Ser Lys Thr Val Ala Lys Leu Asp Glu
85 90 95
Asp Val Asn Lys Leu Arg Met Thr Leu Ser Ser Lys Gly Ile Asp Gln
100 105 110
Lys Met Arg Val Leu Asn Ala Cys Phe Ser Val Lys Arg Ile Pro Gly
115 120 125
Lys Ser Ser Ser Ile Ile Lys Cys Thr Lys Leu Met Lys Asp Lys Ile
130 135 140
Glu Arg Gly Glu Val Glu Val Asn Asp Ser Tyr Ile Asp Glu Lys Met
145 150 155 160
Glu Ile Asp Thr Ile Asp Trp Lys Ser Arg Tyr Asp Gln Leu Glu Lys
165 170 175
Arg Phe Glu Ser Leu Lys Gln Arg Val Thr Glu Lys Tyr Asn Thr Trp
180 185 190
Val Gln Lys Ala Lys Lys Val Asn Glu Asn Met Tyr Ser Leu Gln Asn
195 200 205
Val Ile Ser Gln Gln Gln Asn Gln Ile Ala Asp Leu Gln Gln Tyr Cys
210 215 220
Asn Lys Leu Glu Thr Asp Leu Gln Gly Lys Phe Ser Ser Leu Val Ser
225 230 235 240
Ser Val Glu Trp Tyr Leu Arg Ser Met Glu Leu Pro Asp Asp Val Lys
245 250 255
Asn Asp Ile Glu Gln Gln Leu Asn Ser Ile Asp Leu Ile Asn Pro Ile
260 265 270
Asn Ala Ile Asp Asp Ile Glu Ser Leu Val Arg Asn Leu Val Gln Asp
275 280 285
Tyr Asp Arg Thr Phe Leu Met Leu Lys Gly Leu Leu Lys Gln Cys Asn
290 295 300
Tyr Glu Tyr Val Tyr Glu
305 310
<210> 10
<211> 175
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 10
Met Glu Lys Leu Thr Asp Leu Asn Tyr Thr Leu Ser Val Ile Thr Leu
1 5 10 15
Met Asn Ser Thr Leu His Thr Ile Leu Glu Asp Pro Gly Met Ala Tyr
20 25 30
Phe Pro Tyr Ile Ala Ser Val Leu Thr Val Leu Phe Thr Leu His Lys
35 40 45
Ala Ser Ile Pro Thr Met Lys Ile Ala Leu Lys Thr Ser Lys Cys Ser
50 55 60
Tyr Lys Val Val Lys Tyr Cys Ile Val Thr Ile Phe Asn Thr Leu Leu
65 70 75 80
Lys Leu Ala Gly Tyr Lys Glu Gln Ile Thr Thr Lys Asp Glu Ile Glu
85 90 95
Lys Gln Met Asp Arg Val Val Lys Glu Met Arg Arg Gln Leu Glu Met
100 105 110
Ile Asp Lys Leu Thr Thr Arg Glu Ile Glu Gln Val Glu Leu Leu Lys
115 120 125
Arg Ile Tyr Asp Lys Leu Met Val Gln Ser Val Gly Glu Ile Asp Met
130 135 140
Thr Lys Glu Ile Asn Gln Lys Asn Val Lys Thr Leu Glu Glu Trp Glu
145 150 155 160
Ser Gly Arg Asn Pro Tyr Glu Pro Lys Glu Val Thr Ala Ala Met
165 170 175
<210> 11
<211> 197
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 11
Met Ser Leu Ser Ile Asp Val Thr Ser Leu Pro Ser Ile Ser Ser Ser
1 5 10 15
Ile Phe Lys Asn Glu Ser Ser Ser Thr Thr Ser Thr Leu Ser Gly Lys
20 25 30
Ser Ile Gly Arg Ser Glu Gln Tyr Ile Ser Pro Asp Val Glu Ala Phe
35 40 45
Asn Lys Tyr Met Leu Ser Lys Ser Pro Glu Asp Ile Gly Pro Ser Asp
50 55 60
Ser Ala Ser Asn Asp Pro Leu Thr Asn Phe Ser Ile Arg Ser Asn Ala
65 70 75 80
Val Lys Thr Asn Ala Asp Ala Gly Val Ser Met Asp Ser Ser Thr Gln
85 90 95
Ser Arg Pro Ser Ser Asn Val Gly Cys Asp Gln Leu Asp Phe Ser Leu
100 105 110
Thr Lys Gly Ile Asn Val Ser Ala Asn Leu Asp Ser Cys Ile Ser Ile
115 120 125
Ser Thr Asp His Lys Lys Glu Lys Ser Lys Lys Asp Lys Ser Arg Lys
130 135 140
His Tyr Pro Arg Ile Glu Ala Asp Ser Asp Ser Glu Asp Tyr Val Leu
145 150 155 160
Asp Asp Ser Asp Ser Asp Asp Gly Lys Cys Lys Asn Cys Lys Tyr Lys
165 170 175
Lys Lys Tyr Phe Ala Leu Arg Met Arg Met Lys Arg Val Ala Met Gln
180 185 190
Leu Ile Glu Asp Leu
195
<210> 12
<211> 3302
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
ggctattaaa gctgtacaat ggggaagtac aatctaatct tgtcagaata tttatcattt 60
gtttacaatt cacaatctgc agttcaaata ccaatttatt attcttccaa tccagaatta 120
gaaaaaagat gtattgaatt tcatgctaaa tgtgttgaca attctaaaaa aggtatatca 180
ttaaaacctg tatttgaaga gtataaagat gtaatagata atgcaacgtt actatctata 240
ttatcatatt cttatgataa atacaacgct gtggaacgga aactagtcaa ttatgctaaa 300
ggtaaacctt tagaggctga tttaacggca aacgagcttg attatgaaaa taacaaaata 360
acttctgaac tgtttcagtc agctgaagaa tataccgatt cattaatgga tcctgctata 420
ctaacttcgt tatcttcaaa tttaaatgca gttatgtttt ggttagaacg acactcaaat 480
gacgttgctg atgcaaataa aatttataag cgtagactag atttatttac catagtggca 540
tctacaataa ataaatacgg agtacccaga cataatgaaa aatacagata tgaatacgaa 600
gtgatgaagg ataaaccgta ttacttagta acttgggcta actcatctat agaaatgctt 660
atgtcagtgt tttcacatga ggattattta atagcaaaag agttaataat cttatcatat 720
tcaaatagat caacgttagc aaaactagtt tcatctccaa tgtcaatatt agttgcatta 780
atagatatca atggtacgtt tattacgaat gaagaattgg aacttgagtt ttcggataaa 840
tatgttaagg caattgtacc tgatcaaatt ttcgacgaat tacaggaaat gattgacaat 900
atgaagaaag ttggtttagt agacatacca agaatgattc aagaatggtt agttgattgt 960
tcattagaga aatttacact gatgtcaaaa atttattctt ggtcatttca tgttggtttt 1020
agaaaacaaa aaatgattga tgcagcatta gaccaattga agacagagta cactgaagat 1080
gtagatggtg agatgtacaa tgagtacaca atgttaatta gagatgaaat agttaaaatg 1140
ttagaagtac cagttaaaca tgacgatcat ctacttcgtg attcagaatt agctggattg 1200
ttatcaatgt catcagcttc aaatggtgaa tcaaggcaac ttaaatttgg tcgtaaaaca 1260
atattttcaa ctaagaaaaa tatgcatgtt atggatgata ttgcacatgg aaagtatact 1320
cctggtgtca ttcctccagt aaacgtagat agaccaattc cactgggtcg tagagatgtt 1380
cctggacgaa gaacaagaat tatattcata ttgccatacg aatactttat tgcgcagcac 1440
gctgtcgtag aaaaaatgtt atcatacgca aagcatacta gagagtatgc ggaattttac 1500
tcacagtcaa atcaattgct atcatacggt gatgttacaa gattcttatc cagtaattct 1560
atggtgttat acacagacgt ttcacaatgg gattcgtcac aacataacac acagccattt 1620
agaaaaggaa taattatggg tttagatatg ttatctaata tgactaatga tccaaaagta 1680
atacaaacgc taaatttata taaacaaaca caagttaatc tcatggattc atatgttcaa 1740
atacctgatg gtaatgtaat caaaaagatt cagtatggtg ctgttgcttc aggagaaaaa 1800
caaactaagg cagccaattc tatagctaat ttggcactca ttaaaacagt attgtcaagg 1860
attgcaaata aatattcttt tataaccaaa ataatcagag ttgatggtga tgataattat 1920
gcagtactac aatttaacac cgatgttact aaacaaatgg tccaagatgt gtcaaacgat 1980
gtgagacata tatattctag aatgaatgct aaagttaaag cattggtatc tacagtcggt 2040
attgaaatag caaaaagata tatagctgga ggaaaaatat tttttagagc tggtataaac 2100
ttattaaata atgagaagcg tggacaaagt acacaatggg atcaagcagc tattttatat 2160
tcgaactaca ttgttaacaa attacgagga tttgagactg atagagaatt tatattaact 2220
aaaattatac aaatgacatc tgtagccatt actggatcac taaggctatt tccgtcagaa 2280
cgagtgttaa ctactaattc tacattcaaa gtatttgact cagaagattt cattatagag 2340
tatggaacaa ctgacgatga agtatatata cagagagcat ttatgtcatt gtctagtcaa 2400
aagtcaggaa tagctgatga aattgcctct tcacaaacat tcaaaaatta tgttaataaa 2460
ttgtctgacc aactactaat atcaaaaaac gtaattgtat ccaaaggtat agctgtaaca 2520
gaaaaagcaa aattgaattc atatgcacca gtttatttag aaaaacgccg tgcacaaata 2580
tcagcgctat taactatgtt acagaagcca gtgtcattta aatcaaataa aattactatt 2640
aatgacattt tgcgtgacat aaaaccattt tttgttactt ctgaagccaa tttgtcaatt 2700
caatacagaa aatttatgcc aacattacct gataacgtcc aatatgttat acaatgtata 2760
ggatcaagga cgtatcagat agaagatagt gggtcgaaat catcgatttc gaagttaata 2820
tcaaaatact cagtttacaa accatcaatt gaggaactat ataaagttat atctttacga 2880
gaacaagaaa tacagttgta tttagtttca ctaggagttc caccagttga tgcaggcaca 2940
tacgtcggat caagaatata ttctcaagac aaatataaaa tattagaatc ttacgtatac 3000
aatttattat caattaatta tggatgttat caattattcg attttaattc tccagattta 3060
gagaaactta tacgaattcc ttttaaaggt aagataccag ccgtaacatt tatattacat 3120
ctttacgcaa aacttgaaat aataaatcat gccattaaga atggagcatg gattagtttg 3180
ttctgtaatt atccaaaatc tgagatgatt aaactatgga agaaaatgtg gaatataaca 3240
gcattacggt ctccctatac tagtgcgaat ttctttcaag attagagtgc ttagatgtga 3300
cc 3302
<210> 13
<211> 2729
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
ggctattaaa ggctcaatgg cgtacaggaa gcgcggagct aaacgtgaag acttaccaca 60
acaaaatgaa cgtctgcaag aaaaagaaat tgaaaataat acagacgtaa ctatggaaaa 120
taaaaataaa aatagaaata ataatagaaa gcagcaatta tctgacaaag tgctatcaca 180
aaaagaagaa ataataactg atgtacaaga tgatattaaa atagctgatg aggttaaaaa 240
atcatcaaag gaggagtcga aacagttact tgaaatatta aaaacgaaag aagatcatca 300
gaaagaagta caatacgaaa ttctacaaaa aacaataccg acttttgaac caaaagaatc 360
aattttgaaa aaattagagg atataaaacc agagcaagct aagaagcaaa tgaaattatt 420
tagaatcttt gaaccaaggc aattaccaat ataccgagca aatggtgaaa aagaattaag 480
gaatagatgg tattggaaat tgaaaaagga tacgctacca gacggagatt atgacgtacg 540
cgagtatttt ttaaatttat atgatcaaat actgatagaa atgccagatt atttattgct 600
aaaagatatg gctgtagaaa ataaaaactc tagggatgct ggtaaagttg tagattctga 660
gactgcaagt atttgtgatg ctatatttca ggatgaggaa acagagggag ttatcagaag 720
atttatcgca gatatgagac aacaagttca ggccgacaga aatattgtca attatccatc 780
aattttacat ccaattgatc atgcgtttaa cgaatatttt ctgaaccatc aattagttga 840
accattaaat aatgacatta tttttaatta tataccagaa agaataagaa atgacgtcaa 900
ctatattttg aatatggata tgaatttgcc atcaacagca agatatatta gaccaaatct 960
attgcaagat agactaaatt tacatgataa ttttgaatca ttatgggata caataactac 1020
gtcaaattac atattagcaa gatcagtcgt gcctgatttg aaggaaaaag aattagtttc 1080
aactgaagct cagatacaga aaatgtctca agatttgcag cttgaagctt taacaataca 1140
atctgaaacg cagtttctgg ccggcataaa ttcacaagca gcaaatgatt gttttaaaac 1200
actgatagca gctatgttaa gccaacgtac aatgtcatta gattttgtaa ctacgaatta 1260
tatgtcactt atatctggca tgtggctatt gactgttata ccaaatgata tgtttcttcg 1320
tgagtcgcta gttgcatgcg aattggctat aataaacact atagtttacc cagcatttgg 1380
aatgcaaaga atgcattata gaaatggcga tcctcagact ccatttcaaa tagcagagca 1440
acaaatacaa aattttcaag tagctaattg gttacatttt attaataata atagatttag 1500
acaagttgtt attgatggag tgttaaatca aacactaaat gacaacatta gaaatggaca 1560
agttattaat caattaatgg aagcattaat gcagttatcc agacaacaat ttccgactat 1620
gccagttgat tataaaagat caatccaaag aggaatattg ttattatcta atagattggg 1680
tcagttagtt gatttaacaa gattattatc gtataattat gaaactctaa tggcttgtat 1740
aactatgaac atgcaacatg ttcaaactct cactaccgaa agattacagt taacttctgt 1800
cacatcctta tgtatgttaa ttggaaatac cacagtaatt ccaagtccac aaacgctatt 1860
tcactattat aacgtaaatg taaattttca ttcaaattat aatgaacgaa ttaacgatgc 1920
tgtggctatt attacggctg ctaatagact aaacttatat cagaaaaaaa tgaaatcaat 1980
agtcgaggac tttttaaaaa gattgcaaat ttttgatgta cctcgagtac cagatgatca 2040
aatgtatagg ttgagagaca gacttagatt attgccagtt gaaagacgaa gacttgatat 2100
atttaactta atattaatga acatggagca gatcgaacga gcttcagata aaattgctca 2160
aggagtaata attgcttaca gagatatgca actagaaaga gatgagatgt atggatttgt 2220
taacatcgct aggaacctcg atggatatca acaaatcaat ttagaagagt taatgagaac 2280
tggagactat ggtcaaatta ctaatatgct attaaacaac cagcctgtag ctttggtagg 2340
agcattgcca tttgtgaccg attcatcagt tatatcactc attgcaaaat tggatgccac 2400
agtttttgct caaatagtta aacttagaaa agtggacact ttaaaaccga tattatataa 2460
aataaattct gattctaatg atttctactt agttgcaaat tatgattgga taccgacttc 2520
aaccacaaaa gtttacaagc aagtaccaca accttttgat ttcagagcgt caatgcacat 2580
gttaacgtct aatttaactt ttaccgttta ttctgattta ctatctttcg tttctgcaga 2640
cacggttgaa cctattaacg caattgcttt tgacaatatg cgcattatga acgaactgta 2700
aacgccaacc ccactgtgga gatatgacc 2729
<210> 14
<211> 2591
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
ggctattaaa gcaatactag tagtgcgttt tacctctgat ggtgtaagca tgaaagtatt 60
agctttaaga cacagtgtgg ctcaggtgta tgcagatacg caaacttact tgcatgacga 120
ttcgaaagat gagtatgaaa atgcatttct catttctaat ctaactacac ataacatatt 180
atatttaaat tatagtctta aaacattaaa aatattaaat aaatcaggca tagcggcagt 240
tgaagtacaa tctccagatg aattatttgc tttaataaga tgtaatttta catatgatta 300
tgagaataac ataatttatt tacatgatta ttcatattat actaacaatg agataagaac 360
agatcaacat tggataacta aaaccaacat aactgaatat ctactacctg gatggaaatt 420
aacatatgta ggatataatg gaaaaaatac acgtggtcac tataatttct catttttatg 480
tcaaaatgca gctacagatg acgatataat aattgaatat atatattcca atgaattaga 540
ttttcaaaat tttctgttaa gaaaaattaa agagagaatg actacatctc ttccaattgc 600
tagattgtca aatcgggtgt ttagagataa attatttcca tctattgtga acatgtataa 660
aaaagtgata aatgttggac caagaaacga atccatgttc acattcttaa atttcccaac 720
tattaagcaa ttttcaaacg gtgcatacat cgtaaaacat actattaaat taaaacaaga 780
gaaatggctg ggtaaaagag tgtcacaatt tgacattgga caatataaaa atatgttaaa 840
tgtagttact accatctatt actactataa tttataccat tcgaaaccaa taatatacat 900
gctcggttca gctccatctt attggattta tgatattaaa cagtattctg attttacatt 960
tgaaacatgg gatccattag acactccata ttctacaata catcataagg aattattttt 1020
tgataaggac gtcaataaac ttaaagataa ttcagttcta tatattgata tacgaactga 1080
taggggaaat atggattgga aagagtggag aaaagtagtg gaacagcaaa ctatcaacaa 1140
tctaaatatt gcatataaat atctagcaac aggaaaagcc aaagtgtgtt gtgttaaatt 1200
aacagctatg gatctagaat tgccaatcac agcaaaatta cttcatcatc caactactga 1260
agttagatca gaattttatg caatcttaga tgtatgggat attattacta ttaaaagatt 1320
tataccaaag ggtgtatttt atgcttttat aaataatata atcacagaga atgtgtttat 1380
acaaccttct tttaaattga aagtatcacc tactgattat atagtggcat tgtatgcttt 1440
atctaacgat tttaattcaa gacaagatgt tattaattta attaataaac aaaaacaatc 1500
gcttattact gttcgtatga ataacacctt taaagacgaa cctaaagtaa attttaaaaa 1560
tatttatgat tggacgtttc tgccaacaga tttcgaactt aaagattcga taattacatc 1620
gtatgatggt tgtttaggca tgtttggatt atcgatatct ctatcttcta aacctactgg 1680
taataatcat ttatttataa taaatggaac tgacaaatat gataaactag atcagtatgc 1740
aaatcatatg ggtatttcta ggagatcaca tcaaattaga ttttcagagt ctgcgacatc 1800
atattcaggg tatattttta gagatctttc aaataataat ttcaacttga taggtactaa 1860
tgtagaaaat tcagtttcag gacatgttta taatgcattg atttactaca ggtataatta 1920
tgcatttgat cttaaaagat ggatttattt acactcaatt ggaaaagccg ctgtggaagg 1980
tggaagatat tatgaacatg caccaataga attgatatat gcatgccggt cagctaaaga 2040
atttgctata ttgcaagatg acttaacggt attgagatac gctaatgaaa ttgaagagta 2100
cataaacaaa gtatatagta taacttatgc tgatgatcct aattacttta taggaattaa 2160
atttaacagt ataccttatg aatatgatgt taaagttcca catttgacgt taggagtact 2220
atttatatct gataatatga tacatgatgt agttacagta ttgaagaaaa tgaagacaga 2280
actatttaag acggaaatta gtactagtta cacttatatg ctatctgata atatatatgt 2340
agcaaatgcc agcggcgtgt tatcaacgta ttttaaatta tataatatgt tttacagaaa 2400
tcacatcacg tttggacaat cgcgaatgtt tattccacat attacactga gttttagtaa 2460
taaacaaaca gttagaatag aaagcacaag actcaagatt aattcaattt acttaagaaa 2520
gattaaaggg gagaccgtat ttgatatgtc tgagtgagct aaaaacttaa cacactagtc 2580
atgatgtgac c 2591
<210> 15
<211> 2359
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
ggctataaaa tggcttcgct catttataga cagcttctca ctaattcata ttcagtagat 60
ttatatgatg aaatgaagca aattggatca gaaaaaactc aaaatgtaac tataaaccca 120
ggtccatttg ctcaaacaag atatgctcca gtcaattggg gtcatggaga aataaatgat 180
tcaactacag tggaaccaat tttagatggt ccttatcaac ctactacatt tacaccacct 240
actgattact ggatacttat taactcaaat acaaatggag tagtatacga gagtacaaat 300
aatagtgact tttggactgc agtcattgct gttgaaccgc acgtcgatcc agtagataga 360
caatataatg tatttggtga aaataaacaa tttaatgtaa ggaatgattc agataaatgg 420
aagtttttag aaatgtttag aggcagtagt caaagtgact tttataatag acgtacacta 480
acttctgata ctaaactcgt aggaatatta aaatatggtg gaagaatatg ggcatttcat 540
ggtgaaacac caagggctac taccgatagc tcaaacactg caaatttgaa cggtatatca 600
attacaattc attcagaatt ttatattatt ccaagatccc aagagtctaa gtgtaatgaa 660
tatattaata acggtctacc accaattcaa aatactagaa atgtagtacc attatcatta 720
tcatctagat ctatacagta tacgagagca caagttaatg aagacattac aatttcaaag 780
acttcattat ggaaagaaat gcaacataac agagatatta taattaggtt taaatttggc 840
aatagtatta ttaaattagg gggattgggt tataaatggt ctgaaatatc atataaagca 900
gcgaactatc aatataatta tttacgtgat ggtgaacaag taactgcaca tactacctgt 960
tcagtaaacg gagtaaataa ttttagctac aacggaggat ctttgcccac tgattttagt 1020
atttcaagat atgaagttat taaagaaaat tcctatgtat acgtagatta ttgggacgat 1080
tcaaaagcat ttcgaaatat ggtgtatgtt agatcattag cagctaattt aaactcagtg 1140
aaatgtacag gtggaagtta ccattttagt atacctgtgg gtgcatggcc agttatgaat 1200
ggaggtgccg tttcgttgca tttcgctggt gttacattat ctacgcaatt cacagatttc 1260
gtatcattaa attcattacg gtttagattt agtttgacag tagatgaacc atctttttca 1320
atattgagaa cacgtacggt aaatttatat ggattaccag ctgctaatcc aaataatgga 1380
aatgaatatt acgaaatatc aggaaggttt tcgcttattt ctctagttcc aaccaacgat 1440
gattatcaaa ctccaattat gaattcagta acagtaagac aagatttaga acgtcaacta 1500
actgatttac gagaggaatt caattcatta tcacaagaaa tagctatgtc acaattaatt 1560
gatttagcgt tattaccttt agacatgttt tctatgtttt cagggattaa aagcacaatt 1620
gatctaacca aatcaatggc gactagcgta atgaaaaaat ttagaaaatc aaagttagct 1680
acatcagttt cagaaatgac taactcattg tcagacgcag catcatcagc atcaagaagc 1740
gtttctgtta gatcgaatat atccgcattt tcaaactgga ctaatgtttc aaatgatgta 1800
tcaaatgtga ctaattcagt aaatgatatt tcaacgcaaa catctacgat cagtaaaaac 1860
cttagattaa gagaaatgat cactcaaact gaaggaatga gttttgatga tatttcagca 1920
gcagtactaa aaacgaaaat agatatgtct actcaaattg ggaaaaatac tttacctgac 1980
atagtcacag aggcatctga gaaatttatt ccaaaacgct cgtatcgaat actaaaagat 2040
gatgaagtaa tggaaattaa tactgaagga aaattctttg catataaaat tgatacactt 2100
aatgaagtgc catttgatgt aaataaattt gctgaacttg taacaaattc cccagttata 2160
tcagcaataa tcgattttaa aacattgaag aatttgaatg ataattatgg aattacccga 2220
actgaagcgt taaatttaat taaatcaaat ccaaatgtat tacgtaattt tattaaccag 2280
aataatccaa ttataaggaa tagaattgaa cagttaattc tacaatgtaa gttgtgagag 2340
cgctattgag gatgtgacc 2359
<210> 16
<211> 1356
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
ggcttttaaa cgaagtcttc gacatggagg ttctgtattc attgtcaaaa actcttaaag 60
atgctaggga taagattgtt gaaggtacat tatattctaa tgttagtgat cttattcagc 120
aatttaatca aatgatagta accatgaacg gaaatgactt tcaaactgga ggaattggca 180
atttacccat tagaaattgg acattcgact ttggtctact aggaactacg cttttaaatc 240
ttgatgctaa ttatgttgag accgccagaa ctacgattga gtattttatt gatttcattg 300
ataatgtatg tatggatgaa atggcaagag agtctcaaag aaatggagta gctccacaat 360
ctgaggcatt gagaaagcta gccggtatta aatttaaaag aataaacttt aataattcat 420
cagaatatat agaaaattgg aatttacaaa atagaagaca acgtactgga tttgtttttc 480
ataaacctaa tatatttcca tactcagcat catttacttt aaatagatct caaccaatgc 540
atgacaattt aatgggaact atgtggctta atgctggatc agaaattcaa gtggctggat 600
ttgactattc gtgtgctcta aacgctccag caaatattca gcaatttgaa catatcgtcc 660
agctaaggcg tgcgctaact acagctacta taactttgtt acctgatgca gaaagattta 720
gttttccaag agttattaat tcagcagatg gcgcaactac atggttcttt aatccaatca 780
tcctaagacc aaacaacgta gaagtagaat ttttactgaa tggacaaatt attaatacat 840
atcaagctag atttggcact attgtcgcaa gaaatttcga tacaattcgt ctatcattcc 900
aattaatgcg tccaccaaac atgacgccag ccgtaaatgc attatttccg caagcacaac 960
cttttcaaca tcatgcaaca gttggactta cgttacgtat tgagtctgca gtttgtgaat 1020
cagtgcttgc ggatgcaaat gaaactttat tggcgaatgt taccgcagta cgtcaagagt 1080
atgctatacc agttggacca gtatttccac caggcatgaa ttggactgag ctgattacta 1140
actattcacc atctagggaa gataatttgc aacgtgtctt tacagtagcc tctatcagaa 1200
gcatgttgat taagtgagga ccagactaac catctggtat ctaatcttag ttagcatgta 1260
gctatatcaa gtcattcaga ctctataagt aaggacatgg ctccatgttc gctacgtaga 1320
gtaactgtat gaatgatata gtgagaggat gtgacc 1356
<210> 17
<211> 1061
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
ggctttaaaa gagagaattt ccgtttggct agcggttagc tccttttaat gtatggtatt 60
gaatatacca cagttctaac ctttctgata tcaatagttt tattgaacta catattaaaa 120
tcactaacta gtgcgatgga ctttataatt tatagatttc ttttacttat tgttattttg 180
tcgccatttg tcaaaacaca aaattatggg ataaatttac caattactgg ctccatggac 240
acagcatatg caaattcgtc acagcaagaa acattcttaa cttcaacgct atgtttatat 300
tatcctactg aagcatcaac tcaaattgga gatactgaat ggaagaatac tctatctcaa 360
ttattcttga ctaaggggtg gccaactgga tcagtctatt ttagagaata tacagatatc 420
gcttctttct caattgatcc acaactttat tgtgattata atgttgtgct aatgaagcat 480
gattcaacgt tagagctaga tatgtctgaa ttagctgatt tgattctaaa tgaatggtta 540
tgcaatccaa tggatataac attatattat tatcagcaaa cagatgaatc gaataaatgg 600
atatcgatgg gacaatcttg taccataaaa gtgtgcccat taaatacaca aacttcagga 660
ataggttgta ctactacaaa tacagcgaca tttgaagaag tagctactag tgagaaatta 720
gtgataactg atgttgttga tggcgtgaat cataaacttg atgtaactac aaatacctgt 780
acaattagaa attgtaagaa gttaggaccg agagaaaatg tagcaattat acaagtcggt 840
ggctcagaag tgttagatat tacagcggat ccaactacca caccacaaac tgagcgtatg 900
atgcgagtaa attggaagaa atggtggcaa gttttctata cagtagtaga ttacattaat 960
cagattgtgc aagttatgtc caaaagatca cggtcattaa attcagcagc tttttattat 1020
agagtctgat atatcttagg ttagaattgg tcgatgtgac c 1061
<210> 18
<211> 1061
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
ggctttaaaa gagagaattt ccgtttggct agcggttagc tccttttaat gtatggtatt 60
gaatatacca cagttctaac ctttctgata tcaatagttt tattgaacta catattaaaa 120
tcactaacta gtgcgatgga ctttataatt tatagatttc ttttacttat tgttattttg 180
tcgccatttg tcaaaacaca aaattatggg ataaatttac caattactgg ctccatggac 240
acagcatatg caaattcgtc acagcaagaa acattcttaa cttcaacgct atgtttatat 300
tatcctactg aagcatcaac tcaaattgga gatactgaat ggaagaatac tctatctcaa 360
ttattcttga ctaaggggtg gccaactgga tcagtctatt ttagagaata tacagatatc 420
gcttctttct caattgatcc acaactttat tgtgattata atgttgtgct aatgaagcat 480
gattcaacgt tagagctaga tatgtctgaa ttagctgatt tgattctaaa tgaatggtta 540
tgcaatccaa tggatataac attatattat tatcagcaaa cagatgaatc gaataaatgg 600
atatcgatgg gacaatcttg taccataaaa gtgtgcccat taaatacaca aacttcagga 660
ataggttgta ctactacaaa tacagcgaca tttgaagaag tagctactag tgagaaatta 720
gtgataactg atgttgttga tggcgtgaat cataaacttg atgtaactac aaatacctgt 780
acaattagaa attgtaagaa gttaggaccg agagaaaatg tagcaattat acaagtcggt 840
ggctcagaag tgttagatat tacagcggat ccaactacca caccacaaac tgagcgtatg 900
atgcgagtaa attggaagaa atggtggcaa gttttctata cagtagtaga ttacattaat 960
cagattgtgc aagttatgtc caaaagatca cggtcattaa attcagcagc tttttattat 1020
agagtctgat atatcttagg ttagaattgg tcgatgtgac c 1061
<210> 19
<211> 1059
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
ggcttttaaa gcgtctcagt cgccgtttga gccttgcggt gtagccatgg ctgagctagc 60
ttgcttttgt tatcctcatt tggagaacga tagctataaa tttattcctt ttaataattt 120
agcaataaaa tgcatgttga cagcaaaaat agataaaaaa gatcaagata aattttacaa 180
ttctattgtt tatgggattg cgccaccacc acaatttaga aaacgttata atactagtga 240
taattcgaga ggaatgaatt acgagacaat tatgtttaat aaggtggcta tcttaatttg 300
tgaagcactt aattcgatta aagttacaca atctgaagtt gcaaatgttc tttcaagagt 360
agtttccgtt agacatttgg aaaatctagt actaaggaaa gaaaatcatc aagacgtact 420
tttccattcg aaagaactac tcttaaaatc tgtgttgata gctattggtc agtcaaaaga 480
aatcgaaact accgctactg ccgaaggagg agaaatagta tttcagaatg cagcttttac 540
tatgtggaaa ttgacgtatt tagatcataa attaatgcct attttggatc agaatttcat 600
tgaatataaa attacattga atgaagataa accaatttca gatgtatgtg ttaaagaact 660
tgttgctgaa ttaagatggc agtataacag atttgctata ataacgcatg gtaaaggtca 720
ctatagagtt gttaaatatt catcagttgc taaccatgca gatagagtat ttgctacata 780
taagaataat gctaagagtg gtaatactac tgatttcaat ttactagacc aaagaattat 840
ttggcaaaat tggtatgcat ttacatcttc aatgaaacaa ggtaatacaa ttgatgtatg 900
taagaaacta ctctttcaaa agatgaaaca agagaaaaat ccgttcaaag gattgtcaac 960
tgatagaaaa atggatgaag tctcacatgt tggaatttaa ttcgctttcg attcaagaat 1020
gatgatgacg aagcaagaat agaaagcgct tatgtgacc 1059
<210> 20
<211> 1074
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
ggcttttaat gcttttcagt ggttgctgct caagatggag tctactcagc agatggtaag 60
ctctattatt aacacttctt ttgaagctgc agttgttgct gccacttcaa cattagaatt 120
aatgggtatt caatatgatt acaatgaagt atttactaga gttaaaagta aatttgatta 180
tgtaatggat gactctggtg ttaaaaataa tcttttgggt aaagctataa ctattgacca 240
ggcgttaaat ggaaagtttg gctcagctat tagaaataga aattggatga ctgactctaa 300
aacggttgct aaattggatg aagacgtgaa taaacttaga atgacattgt cttctaaagg 360
aatcgatcaa aagatgagag tgcttaatgc ttgttttagt gtgaaaagaa taccaggaaa 420
atcatcatca ataattaagt gcactaaact tatgaaggat aaaatagaac gtggggaagt 480
tgaggttaat gattcatata ttgatgagaa aatggaaatt gataccattg attggaaatc 540
tcgttatgac caattagaaa aaagatttga gtcactaaaa caaagagtta ctgagaaata 600
caatacttgg gtacaaaaag cgaagaaagt aaatgagaat atgtactctc ttcagaatgt 660
catttcacaa cagcaaaatc aaatagcaga tcttcaacaa tattgtaata aattggaaac 720
tgacttgcaa ggcaaattta gttcattagt gtcatcagtt gagtggtatt taagatctat 780
ggaattacca gatgatgtaa agaatgatat tgaacagcag ttaaattcaa ttgatttaat 840
taatcccatt aatgctatag atgatatcga atcattggtt agaaatttag ttcaggatta 900
tgatagaaca tttttaatgt taaagggact gttgaagcaa tgcaactatg aatatgtata 960
tgaatagtca cataattaaa atcattaacc atctacacat gaccctctat gagcacaata 1020
gttaaaagct aacactgtca aaaacctaaa tggctatagg ggcgttatgt gacc 1074
<210> 21
<211> 751
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
ggcttttaaa agttctgttc cgagagagcg cgtgcggaaa gatggaaaag cttaccgacc 60
tcaactacac attgagtgta atcactctaa tgaacagtac actacataca atactagagg 120
atccaggaat ggcgtatttt ccctatattg catctgtcct aacggttttg ttcacattgc 180
ataaagcatc aattccaacg atgaaaatag cattgaaaac gtcaaaatgt tcgtataaag 240
tagtaaagta ttgtattgta acaattttta atacgttact gaaactagca ggttataaag 300
agcaaataac tactaaagat gaaatagaaa aacaaatgga tagagtcgtt aaagaaatga 360
gacgtcagct ggaaatgatt gataaactga ctacacgtga aattgagcaa gtagagctac 420
ttaaacgtat atatgataaa ttgatggtgc aatcagttgg cgagatagac atgacgaaag 480
aaattaatca gaagaacgtg aaaacgctag aagaatggga gagtggaaga aatccttatg 540
aaccgaaaga agtgactgca gcaatgtaag aggttgagct gccgtcgact gtcttcggaa 600
gcggcggagt tctttacagt aagccccatc ggacctgatg gctggctgag aagccacagt 660
cagtcatatc gcgtgtggct caagccttaa tcccgtttaa ccaatccggt cagcaccgga 720
cgttaatgga aggaacggtc ttaatgtgac c 751
<210> 22
<211> 664
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
ggctttaaaa gcgctacagt gatgtctctc agcattgacg taacaagtct tccctcaatt 60
tcttctagta tctttaaaaa tgaatcgtct tctacaacgt caactctttc tggaaaatct 120
attggtagga gtgaacagta catttcacca gatgtagaag cattcaataa atacatgttg 180
tcgaagtctc cagaggatat tggaccatct gattctgctt caaacgatcc actcaccaac 240
ttttcgatta gatcgaatgc agttaagaca aatgcagacg ctggcgtgtc tatggattca 300
tcgacgcaat cacgaccttc aagcaacgtt ggatgtgatc aattggattt ctccttaact 360
aaaggtatta atgttagtgc taatcttgat tcatgtatat caatttcaac tgatcataag 420
aaggagaaat ctaagaaaga taaaagtagg aaacactacc caagaattga agcagattct 480
gattctgaag attatgtttt ggatgattca gatagtgatg acggtaaatg taagaattgt 540
aaatacaaga aaaaatattt cgcactaaga atgaggatga agcgagtcgc aatgcaattg 600
atcgaagatt tgtaatgtta acctgagagc acactaggga gctccccact cccgttttgt 660
gacc 664

Claims (21)

1. A polypeptide or combination of polypeptides, wherein the polypeptide comprises the sequence:
(I) an amino acid sequence as shown in any of SEQ ID Nos. 1-11;
or
(II) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence of (I), and the amino acid sequence has the same or similar functions with the amino acid sequence of (I);
or
(III) an amino acid sequence having at least 99.4% identity to an amino acid sequence as set forth in any of SEQ ID Nos. 1 to 11;
the number of the plurality is 5.
2. The polypeptide or combination of polypeptides of claim 1, wherein the polypeptide comprises the sequence:
an amino acid sequence having more than 99.4% identity with the amino acid sequence shown as SEQ ID No. 4;
an amino acid sequence having more than 99.4% identity with the amino acid sequence shown as SEQ ID No. 6; and/or
An amino acid sequence having more than 99.7% identity with the amino acid sequence shown as SEQ ID No. 8; and/or
An amino acid sequence having more than 99.7% identity with the amino acid sequence shown as SEQ ID No. 9; and/or
An amino acid sequence with more than 99.5 percent of identity with the amino acid sequence shown as SEQ ID No. 11.
3. The polypeptide or polypeptide combination of claim 1 or 2, wherein the polypeptide comprises the sequence:
a polypeptide comprising the amino acid sequence shown as SEQ ID No 2; and
a polypeptide comprising the amino acid sequence shown as SEQ ID No 5;
further comprising one or more of the following sequences:
a polypeptide comprising an amino acid sequence which is at least 99.4% identical to the amino acid sequence shown as SEQ ID No. 6; and/or
A polypeptide comprising an amino acid sequence corresponding to that shown as SEQ ID No. 7; and/or
A polypeptide comprising an amino acid sequence having at least 99.7% identity to the amino acid sequence set forth in SEQ ID No. 8; and/or
A polypeptide comprising an amino acid sequence which is at least 99.7% identical to the amino acid sequence shown as SEQ ID No. 9; and/or
A polypeptide comprising the amino acid sequence shown as SEQ ID No. 10; and/or
A polypeptide comprising an amino acid sequence which is at least 99.5% identical to the amino acid sequence shown as SEQ ID No. 11; and/or
A polypeptide comprising the amino acid sequence shown as SEQ ID No. 1; and/or
A polypeptide comprising the amino acid sequence shown as SEQ ID No. 3; and/or
A polypeptide comprising an amino acid sequence which is at least 99.4% identical to the amino acid sequence shown as SEQ ID No. 4.
4. The polypeptide or polypeptide combination of claim 1 or 2, wherein the polypeptide comprises the sequence:
a polypeptide comprising an amino acid sequence as set forth in SEQ ID No. 2;
a polypeptide comprising the amino acid sequence shown as SEQ ID No. 5; and
a polypeptide comprising an amino acid sequence which is at least 99.4% identical to the amino acid sequence shown as SEQ ID No. 6;
further comprising one or more of the following sequences:
a polypeptide comprising an amino acid sequence corresponding to that shown as SEQ ID No. 7; and/or
A polypeptide comprising an amino acid sequence having at least 99.7% identity to the amino acid sequence set forth in SEQ ID No. 8; and/or
A polypeptide comprising an amino acid sequence which is at least 99.7% identical to the amino acid sequence shown in SEQ ID No. 9; and/or
A polypeptide comprising the amino acid sequence shown in SEQ ID No. 10; and/or
A polypeptide comprising an amino acid sequence which is at least 99.5% identical to the amino acid sequence shown in SEQ ID No. 11; and/or
A polypeptide comprising the amino acid sequence shown in SEQ ID No. 1; and/or
A polypeptide comprising the amino acid sequence shown in SEQ ID No. 3; and/or
A polypeptide comprising an amino acid sequence which is at least 99.4% identical to the amino acid sequence shown in SEQ ID No. 4.
5. A nucleic acid molecule encoding a polypeptide or combination of polypeptides according to any one of claims 1 to 4.
6. The nucleic acid molecule of claim 5, comprising the sequence:
(IV) a nucleotide sequence encoding an amino acid sequence as shown in any of SEQ ID Nos. 1 to 11; and/or
(V) an antisense complementary sequence of a nucleotide sequence encoding an amino acid sequence as shown in any of SEQ ID Nos. 1 to 11;
(VI) a nucleotide sequence which hybridizes with a nucleotide sequence encoding an amino acid sequence as shown in any of SEQ ID Nos. 1 to 11 under a high stringency condition;
(VII) a nucleotide sequence obtained by substituting, deleting or adding one or more bases to the nucleotide sequence shown In (IV), (V) or (VI), and the nucleotide sequence has the same or similar functions with the nucleotide sequence shown In (IV);
(VIII) a nucleotide sequence which is at least 99.4% identical to the nucleotide sequence shown In (IV), (V) or (VI);
the number of the plurality is 5.
7. The nucleic acid molecule of claim 5 or 6, comprising a nucleotide sequence as set forth in any of SEQ ID Nos. 12 to 22.
8. The nucleic acid molecule of claim 7, comprising the sequence:
a nucleic acid molecule encoding the amino acid sequence shown as SEQ ID No.1, said nucleic acid molecule having the nucleotide sequence shown as SEQ ID No. 12; and/or
A nucleic acid molecule encoding the amino acid sequence shown as SEQ ID No.2, said nucleic acid molecule having the nucleotide sequence shown as SEQ ID No. 13; and/or
A nucleic acid molecule encoding the amino acid sequence shown as SEQ ID No.3, said nucleic acid molecule having the nucleotide sequence shown as SEQ ID No. 14; and/or
A nucleic acid molecule encoding the amino acid sequence shown as SEQ ID No.4, said nucleic acid molecule having the nucleotide sequence shown as SEQ ID No. 15; and/or
A nucleic acid molecule encoding the amino acid sequence shown as SEQ ID No.5, said nucleic acid molecule having the nucleotide sequence shown as SEQ ID No. 16; and/or
A nucleic acid molecule encoding the amino acid sequence shown as SEQ ID No.6, said nucleic acid molecule having the nucleotide sequence shown as SEQ ID No. 17; and/or
A nucleic acid molecule encoding the amino acid sequence shown as SEQ ID No.7, said nucleic acid molecule having the nucleotide sequence shown as SEQ ID No. 18; and/or
A nucleic acid molecule encoding the amino acid sequence shown as SEQ ID No.8, said nucleic acid molecule having the nucleotide sequence shown as SEQ ID No. 19; and/or
A nucleic acid molecule encoding the amino acid sequence shown as SEQ ID No.9, said nucleic acid molecule having the nucleotide sequence shown as SEQ ID No. 20; and/or
A nucleic acid molecule encoding the amino acid sequence shown as SEQ ID No.10, said nucleic acid molecule having the nucleotide sequence shown as SEQ ID No. 21; and/or
A nucleic acid molecule encoding the amino acid sequence shown as SEQ ID No.11, said nucleic acid molecule having the nucleotide sequence shown as SEQ ID No. 22.
9. Rotavirus which comprises a polypeptide or a combination of polypeptides according to any one of claims 1 to 4 or a nucleic acid molecule according to any one of claims 5 to 8.
10. The rotavirus of claim 9 which has a accession number of CGMCC No. 20292.
11. Use of a polypeptide or combination of polypeptides according to any one of claims 1 to 4, a nucleic acid molecule according to any one of claims 5 to 8 or a rotavirus according to any one of claims 9 to 10 in the manufacture of a vaccine and/or medicament for the prevention and/or treatment of a disease;
such diseases include, but are not limited to, rotavirus-induced disease, diphtheria, tetanus, pertussis, haemophilus influenzae type b-induced disease, Hepatitis B (HBV) -induced disease, polio and/or pneumococcal-induced disease.
12. Medicament comprising a polypeptide or a combination of polypeptides according to any one of claims 1 to 4, a nucleic acid molecule according to any one of claims 5 to 8 or a rotavirus according to any one of claims 9 to 10 and a pharmaceutically acceptable excipient.
13. Pharmaceutical composition comprising a polypeptide or a combination of polypeptides according to any one of claims 1 to 4, a nucleic acid molecule according to any one of claims 5 to 8 or a rotavirus according to any one of claims 9 to 10 and a drug for the treatment of other diseases.
14. Vaccine comprising a polypeptide or a combination of polypeptides according to any one of claims 1 to 4, a nucleic acid molecule according to any one of claims 5 to 8 or a rotavirus according to any one of claims 9 to 10 and an acceptable adjuvant.
15. A kit comprising a nucleic acid molecule capable of amplifying a polypeptide or combination of polypeptides as claimed in any one of claims 1 to 4, a nucleic acid molecule as claimed in any one of claims 5 to 8 or a rotavirus as claimed in any one of claims 9 to 10.
16. A kit comprising a polypeptide or combination of polypeptides capable of specifically binding to a polypeptide as claimed in any one of claims 1 to 4, a nucleic acid molecule as claimed in any one of claims 5 to 8 or a rotavirus as claimed in any one of claims 9 to 10.
17. The preparation method of the rotavirus inactivated vaccine is characterized by comprising the following steps:
step 1: the polypeptide or the polypeptide combination according to any one of claims 1 to 4, the nucleic acid molecule according to any one of claims 5 to 8 or the rotavirus according to any one of claims 9 to 10 is subjected to adaptive culture and plaque purification, and inoculated in Vero cells of expanded subculture for culture;
step 2: harvesting virus culture solution, filtering to remove impurities, concentrating, purifying and inactivating to obtain virus stock solution;
and step 3: and (3) filtering and sterilizing the virus stock solution prepared in the step (2), and adding an adjuvant to obtain the rotavirus inactivated vaccine.
18. The method according to claim 17, wherein the culturing in step 1 is carried out at 35 to 37 ℃ for 72 to 120 hours.
19. The method of claim 17 or 18, wherein the inactivation of step 2 is inactivation by formaldehyde solution or β -propiolactone or heat.
20. The method of any one of claims 17 to 19, wherein the purification of step 2 is anion exchange chromatography, molecular sieve gel filtration chromatography or adsorption chromatography.
21. The method of any one of claims 17 to 20, wherein the adjuvant of step 3 is aluminum hydroxide.
CN202110780463.4A 2021-07-09 2021-07-09 Rotavirus strains and their use as vaccines Pending CN113480617A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202110780463.4A CN113480617A (en) 2021-07-09 2021-07-09 Rotavirus strains and their use as vaccines

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202110780463.4A CN113480617A (en) 2021-07-09 2021-07-09 Rotavirus strains and their use as vaccines

Publications (1)

Publication Number Publication Date
CN113480617A true CN113480617A (en) 2021-10-08

Family

ID=77938437

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202110780463.4A Pending CN113480617A (en) 2021-07-09 2021-07-09 Rotavirus strains and their use as vaccines

Country Status (1)

Country Link
CN (1) CN113480617A (en)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102695522A (en) * 2009-05-12 2012-09-26 美国政府(由卫生和人类服务部、疾病控制和预防中心的部长所代表) New human rotavirus strains and vaccines
CN104258387A (en) * 2014-07-25 2015-01-07 武汉博沃生物科技有限公司 Human rotavirus vaccine and preparation method thereof
CN108431214A (en) * 2015-10-05 2018-08-21 美国政府(由卫生和人类服务部的部长所代表) Human rotavirus G9P [6] strains and the purposes as vaccine

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102695522A (en) * 2009-05-12 2012-09-26 美国政府(由卫生和人类服务部、疾病控制和预防中心的部长所代表) New human rotavirus strains and vaccines
CN104258387A (en) * 2014-07-25 2015-01-07 武汉博沃生物科技有限公司 Human rotavirus vaccine and preparation method thereof
CN108431214A (en) * 2015-10-05 2018-08-21 美国政府(由卫生和人类服务部的部长所代表) Human rotavirus G9P [6] strains and the purposes as vaccine

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ESONA, M.等: "outer capside spike protein VP4 [Human rotavirus A]", GENBANK登录号：QHX59681.1, pages 1 - 2 *
FUJII,Y.等, GENBANK登录号：BBJ70207.1, pages 1 - 2 *
XUAN ZHOU等: "Surveillance of human rotavirus in Wuhan, China (2011-2019): predominance of G9P[8] and emergence of G12", PATHOGENS, vol. 9, pages 1 - 18 *
马鑫 等: "2009-2011年河北省卢龙地区G9P[8]型人A组轮状病毒全基因组序列分析", 病毒学报, vol. 30, no. 2, pages 119 - 127 *

Similar Documents

Publication Publication Date Title
CN101514334B (en) Chicken infectivity bronchitis virus attenuated vaccine strain and application thereof
CN113337478B (en) Cat parvovirus strain and application thereof
CN109868262B (en) Canine distemper attenuated strain and application thereof
CN107184969A (en) A kind of A types Sai Nika paddy viral inactivation vaccines and its preparation method and application
CN114854697B (en) Trivalent inactivated vaccine of porcine rotavirus G4-G5-G9 and preparation method and application thereof
CN107287218B (en) Avian infectious bronchitis virulent strain S1 gene and virulent strain and application thereof
WO2023246621A1 (en) Coxsackievirus a10 strain and use thereof
CN114525261B (en) Porcine epidemic diarrhea and porcine rotavirus bivalent inactivated vaccine and preparation method thereof
CN103087994B (en) Coxsackievirus A16-type virus strain and use thereof
CN112500458B (en) Novel variant subunit vaccine of chicken infectious bursal disease virus, preparation method and application thereof
CN102559606B (en) A16 type strain of Coxsackie virus and application of the strain
CN111073862B (en) Bovine viral diarrhea type2 attenuated strain and application thereof
CN106854647B (en) Duck viral hepatitis bivalent yolk antibody and preparation method and application thereof
CN108431214B (en) Human rotavirus G9P [6] strain and use as vaccine
WO2000020565A1 (en) Enhanced immunogen for inactivated vaccine for infection with japanese encephalitis viruses and process for producing the same
CN116042540A (en) Separation and application of cat rhinotracheitis virus FHV-1WH-2017 strain
CN113480617A (en) Rotavirus strains and their use as vaccines
Barros et al. Genetic variation of foot-and-mouth disease virus isolates recovered from persistently infected water buffalo (Bubalus bubalis)
CN102363770A (en) Recombinant baculovirus capable of expressing porcine circovirus type 2 Cap protein and somatostatin in fusion manner, and subunit vaccine thereof
RU2563522C1 (en) STRAIN O №2102/Zabaikalsky/2010 FMD VIRUS Aphtae epizooticae OF TYPE O FOR CONTROL OF ANTIGENIC AND IMMUNOGENIC ACTIVITY OF FMD VACCINES AND FOR PRODUCTION OF BIOLOGICAL PRODUCTS FOR DIAGNOSTICS AND SPECIFIC PROPHYLAXIS OF FMD OF TYPE O
CN111073863A (en) Porcine epidemic diarrhea and porcine delta coronavirus bivalent attenuated vaccine and preparation method thereof
CN108653726A (en) RSV-PCV combined vaccines and preparation method thereof
RU2140452C1 (en) Foot and mouth virus disease type a strain n 1707 &#34;armenia-98&#34; used for preparing diagnostic and vaccine preparations
CN114958780B (en) Bovine Aichivirus D virus isolate and application thereof
CN112725288B (en) Canine adenovirus type 2 attenuated vaccine strain and application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination