CN112375748B - Novel coronavirus chimeric recombinant vaccine based on vesicular stomatitis virus vector, and preparation method and application thereof - Google Patents

Novel coronavirus chimeric recombinant vaccine based on vesicular stomatitis virus vector, and preparation method and application thereof Download PDF

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CN112375748B
CN112375748B CN202110027659.6A CN202110027659A CN112375748B CN 112375748 B CN112375748 B CN 112375748B CN 202110027659 A CN202110027659 A CN 202110027659A CN 112375748 B CN112375748 B CN 112375748B
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郑爱华
李虹悦
张毓航
温丹
袁菲
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Institute of Zoology of CAS
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Abstract

The invention discloses a novel coronavirus chimeric recombinant vaccine based on a vesicular stomatitis virus vector, and a preparation method and application thereof. The active component of the recombinant vaccine is recombinant virus rVSV-SARS-CoV/2-RBD, which is obtained by replacing glycoprotein G of vesicular stomatitis virus with chimeric envelope protein S; the chimeric envelope protein S is obtained by replacing the RBD of the SARS-CoV envelope protein S with the RBD of the SARS-CoV-2 envelope protein S; the amino acid sequence of the RBD of the SARS-CoV envelope protein S is 315-536 of the amino acid sequence of the SARS-CoV envelope protein S; the amino acid sequence of the RBD of the SARS-CoV-2 cyst membrane protein S is 319-541 th position of the amino acid sequence of the SARS-CoV-2 cyst membrane protein S. The recombinant virus has important significance for the development of vaccines of new coronavirus.

Description

Novel coronavirus chimeric recombinant vaccine based on vesicular stomatitis virus vector, and preparation method and application thereof
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a novel coronavirus chimeric recombinant vaccine based on a vesicular stomatitis virus vector, and a preparation method and application thereof.
Background
The novel coronavirus (SARS-CoV-2) is a newly emerged coronavirus of the beta genus. The virus genome analysis shows that the virus is very close to SARS-CoV (Severe acid metabolism syndrome-coronavirus) of the same genus (89.1% nucleotide similarity), and can cause symptoms such as human pneumonia after infection. The novel coronavirus is a single-stranded positive-strand RNA virus with an envelope. The virus particles are circular or elliptical and have radial protrusions when observed under an electron microscope. This radial bulge is a characteristic structure of coronaviruses and is composed of the envelope protein spike (S). The S protein is an important structural protein of coronavirus, and plays a key role in the process of virus invasion into host cells. The S protein is also a target of a coronavirus neutralizing antibody and is an important target protein for vaccine research.
A novel coronavirus pneumonia (COVID-19) caused by a novel coronavirus (SARS-CoV-2) has been incorporated into a "type B" infectious disease prescribed by the infectious disease prevention and control Law of the people's republic of China, and a prevention and control measure for the "type A" infectious disease has been taken. The COVID-19 epidemiological characteristics are as follows: the main transmission route is transmission through respiratory droplets and can also be transmission through contact; the disease is more serious after the infection of the old and the patients with basic diseases, and the children and the infants also have the disease. Based on current epidemiological investigations, the patient latency is typically 3-7 days, up to 14 days. The clinical manifestations are fever, hypodynamia and dry cough. A few patients have nasal obstruction, watery nasal discharge, diarrhea, etc. Severe patients manifest as dyspnea, acute respiratory distress syndrome, septic shock, uncorrectable metabolic acidosis, and hemorrhagic clotting dysfunction.
Since no specific medicine for the coronavirus pneumonia exists at present, symptomatic treatment and supportive treatment are mainly carried out on patients. The patient lies in bed for rest, and sufficient heat is ensured; the balance of water and electrolyte is noticed, and the internal environment is maintained to be stable; vital signs, finger oxygen saturation, etc. are closely monitored. The antiviral treatment method comprises: the interferon-alpha is atomized and inhaled and lopinavir/ritonavir is taken. In addition, the pneumonia infected by the novel coronavirus belongs to the category of epidemic diseases in the traditional Chinese medicine, the cause of the pneumonia is the qi of epidemic infection crime, and the dialectical treatment can be carried out by using a traditional Chinese medicine scheme.
At present, vaccines and medicines for the novel coronavirus (SARS-CoV-2) are still in clinical trials or earlier stages, and comprehensive evaluation and further optimization are urgently needed. The inventor uses Vesicular Stomatitis Virus (VSV) as a vector in the earlier stage, replaces glycoprotein G of the VSV Virus with envelope protein S of a novel coronavirus (SARS-CoV-2), and constructs a replication type recombinant Virus rVSV-SARS-CoV-2. The recombinant virus surface expression of the novel coronavirus envelope protein S can simulate the process of new coronavirus invading cells, can stimulate an organism to generate an immune response aiming at SARS-CoV-2, can generate high-titer new coronavirus neutralizing antibodies in cynomolgus monkeys through nasal spray immunity, and can generate protection aiming at the new coronavirus. There are also two recent documents: replication-component Vector preservation viral Vaccine against SARS-CoV-2-Mediated Pathogenesis in Rice (Cell Host Microbe. 2020 Sep 9;28(3): 465-474) and A single dose of recombinant VSV- Δ G-spike Vaccine preservation against SARS-CoV-2 change (bioRxiv 2020.06.18.160655) reported that expression of the envelope protein S of SARS-CoV-2 as antigen produced an immune response and protection against SARS-CoV-2 in a small animal model using VSV as the Vector. The replication-competent recombinant viruses used in these two documents are similar to rVSV-SARS-CoV-2 constructed in the previous invention by the present inventors, and the immune response and protective effect induced by the virus are similar, and both of them need to be further improved.
Disclosure of Invention
In order to develop a novel coronavirus vaccine with better immune protection effect, the invention provides a novel coronavirus chimeric recombinant vaccine based on a vesicular stomatitis virus vector, and a preparation method and application thereof.
In a first aspect, the present invention protects a recombinant virus.
The recombinant virus protected by the invention is 1) or 2):
1) replacing glycoprotein G of vesicular stomatitis virus with chimeric envelope protein S to obtain virus; the chimeric envelope protein S is obtained by replacing RBD (Receptor binding domain) of SARS-CoV envelope protein S with RBD of SARS-CoV-2 envelope protein S;
2) replacing glycoprotein G of vesicular stomatitis virus with chimeric envelope protein S to obtain virus; the chimeric envelope protein S is obtained by replacing the RBD of the SARS-CoV envelope protein S with the RBD of the SARS-CoV-2 envelope protein S and deleting the 1261-1279 th position of the SARS-CoV envelope protein S amino acid sequence;
the amino acid sequence of the RBD of the SARS-CoV envelope protein S is 315-536 of the amino acid sequence of the SARS-CoV envelope protein S;
the amino acid sequence of the RBD of the SARS-CoV-2 cyst membrane protein S is 319-541 th position of the amino acid sequence of the SARS-CoV-2 cyst membrane protein S.
In the recombinant protein, the SARS-CoV-2 cyst membrane protein S amino acid sequence is the sequence 4 in the sequence table.
The SARS-CoV cyst membrane protein S amino acid sequence is sequence 6 in the sequence table.
Further, the amino acid sequence of the chimeric envelope protein S is a) or b) or c):
a) the amino acid sequence is a protein shown in a sequence 2;
b) the protein with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 2;
c) and (b) a protein having 85% or more homology with the amino acid sequence shown in the sequence 2 and having the same function.
Furthermore, the recombinant virus is obtained by replacing the coding gene sequence of glycoprotein G in the genome sequence of the vesicular stomatitis virus with the coding gene sequence of the chimeric envelope protein S. The coding gene sequence of the chimeric envelope protein S is a sequence 1 in a sequence table.
In a second aspect, the invention features a recombinant virus.
The recombinant virus protected by the invention is obtained by transfecting a recombinant virus vector into a virus packaging cell and then carrying out cell culture; the recombinant viral vector is A) or B) as follows:
A) replacing the coding gene sequence of glycoprotein G in the genome sequence of the vesicular stomatitis virus in the vesicular stomatitis virus vector with the coding gene sequence of the chimeric envelope protein S to obtain a vector;
B) a vector obtained by inserting a reporter gene sequence into the recombinant viral vector of A).
Further, the coding gene sequence of the chimeric envelope protein S is sequence 1 in the sequence table.
The reporter gene can be a reporter gene commonly used in the prior art, such as an eGFP gene (shown as a sequence 14 in a sequence table) and a Luciferase gene.
In the A), the recombinant virus vector is obtained by inserting the DNA molecule shown in the sequence 1 between the restriction enzyme sites MluI and NotI of the rVSV delta G vector.
The rVSV delta G vector comprises a T7 promoter sequence, a whole genome sequence of a vesicular stomatitis virus Indiana strain with a deletion glycoprotein G coding gene sequence and an HDV terminator sequence, wherein the nucleotide sequence is a sequence 7 in a sequence table.
The virus packaging cell can be a cell line commonly used in the prior art for virus packaging, such as 293T cell, Vero cell and BHK cell, and can be Vero cell.
The recombinant virus is obtained by transfecting the virus packaging cell with the recombinant virus vector, a plasmid for expressing the N protein of VSV, a plasmid for expressing the P protein of VSV, a plasmid for expressing the L protein of VSV, a plasmid for expressing the M protein of VSV, a plasmid for expressing the G protein of VSV and a plasmid for expressing T7RNA polymerase together, and then carrying out cell culture.
The plasmid for expressing the N protein of the VSV is specifically a plasmid obtained by cloning a N protein coding gene sequence (sequence 8 in a sequence table) in a VSV genome into a eukaryotic expression plasmid pCDNA3.1(+) through BamHI and EcoRI enzyme cutting sites. The plasmid for expressing the P protein of the VSV is specifically a plasmid obtained by cloning a P protein coding gene sequence (sequence 9 in a sequence table) in a VSV genome into a eukaryotic expression plasmid pCDNA3.1(+) through BamHI and EcoRI enzyme cutting sites. The plasmid for expressing the VSV L protein is specifically a plasmid obtained by cloning an L protein coding gene sequence (sequence 10 in a sequence table) in a VSV genome into a eukaryotic expression plasmid pCDNA3.1(+) through BamHI and EcoRI enzyme cutting sites. The plasmid for expressing the M protein of the VSV is specifically a plasmid obtained by cloning an M protein coding gene sequence (a sequence 11 in a sequence table) in a VSV genome into a eukaryotic expression plasmid pCDNA3.1(+) through BamHI and EcoRI enzyme cutting sites. The plasmid for expressing the G protein of the VSV is specifically a plasmid obtained by cloning a G protein coding gene sequence (sequence 12 in a sequence table) in a VSV genome into a eukaryotic expression plasmid pCDNA3.1(+) through BamHI and EcoRI enzyme cutting sites. The plasmid for expressing the T7RNA polymerase is specifically a plasmid obtained by cloning a T7RNA polymerase coding gene sequence (sequence 13 in a sequence table) into a eukaryotic expression plasmid pCDNA3.1(+) through BamHI and EcoRI enzyme cutting sites.
The SARS-CoV-2 is SARS-CoV-2Wuhan-Hu-1 strain (GenBank: NC-045512.2);
the SARS-CoV is SARS-CoV BJ01 strain (GenBank: AY 278488.2);
the vesicular stomatitis virus is specifically vesicular stomatitis virus Indiana strain (GenBank: KF 935251.1).
The recombinant viral vectors described above also fall within the scope of the present invention.
In a third aspect, the present invention provides a novel use of the above recombinant virus or the above recombinant viral vector.
The present invention protects the use of the above recombinant virus or the above recombinant viral vector in any one of the following X1) -X3):
x1) preparing a novel coronavirus vaccine;
x2) for the preparation of a product for the prevention and/or treatment of diseases caused by novel coronaviruses;
x3) screening for novel coronavirus invasion inhibitors.
In the above application, the screening of the novel coronavirus invasion inhibitor is embodied in the detection of the titer of the neutralizing antibody induced by the novel coronavirus vaccine.
In a fourth aspect, the invention protects a product for the prevention and/or treatment of diseases caused by a novel coronavirus.
The active ingredient of the product for preventing and/or treating diseases caused by the novel coronavirus is the recombinant virus or the recombinant virus vector.
In any of the uses or products described above, the product is a novel coronavirus vaccine.
In any of the above applications or products, the novel coronavirus is specifically SARS-CoV-2.
The disease caused by the novel coronavirus is particularly COVID-19 caused by the novel coronavirus.
The concept involved in the present invention is as follows:
the vaccine is an automatic immune preparation for preventing infectious diseases, which is prepared by artificially attenuating and inactivating pathogenic microorganisms (such as bacteria, rickettsia, viruses and the like) and metabolites thereof or by utilizing genetic engineering and other methods. The vaccine retains the property of pathogenic bacteria to stimulate the immune system of an animal. When an animal body is contacted with the pathogen without harm, the immune system can generate certain protective substances, such as antibodies and the like; when the animal is exposed to the pathogenic bacteria again, the immune system of the animal will follow its original memory and produce more protective substances to prevent the pathogenic bacteria from harming.
Vesicular Stomatitis Virus (VSV) is a single negative strand non-segmented RNA Virus with a genome length of about 11Kb and a simple structure. 5 mRNAs are transcribed sequentially from 3 'to 5', encoding 5 proteins: nucleocapsid protein N (nucleocapsid protein), phosphoprotein P (phosphoprotein), matrix protein M (matrix protein), glycoprotein G (glycoprotein), large polymerase L (large protein). The G protein is an integral membrane protein of type I, exists in a trimer form on the surface of virion and performs the functions of binding with a target cell receptor and membrane fusion. In animals infected with VSV, the majority of the antibodies produced are directed against the G protein. VSV is a representative model virus species of the rhabdoviridae family and is widely used to study the mechanisms of entry, replication, and assembly of enveloped viruses into cells. Vesicular stomatitis caused by VSV virus is a benign disease of contact infection, primarily infecting rodents, cattle, pigs and horses, and can also infect humans and other animals. Humans are only infected by chance, but often with few symptoms or only mild fever. The rate of VSV antibodies in the population is very low, and only those who are exposed to VSV frequently have a higher seroprevalence, e.g., some researchers, farmers who are veterinarians who are exposed to sick animals. The neutralizing antibody target of VSV is the G protein, and once the G protein is replaced with the envelope protein of the foreign virus, the recombinant virus will not be affected by pre-existing immunity (pre-existing immunity) in the human body. Absence of preexisting immunity and lack of apparent pathogenicity are prerequisites for VSV as a vaccine vector.
Among the existing live Virus vaccine vectors, Vesicular Stomatitis Virus (VSV) has a low seroprevalence among the population; can induce mucosal immunity; the virus vaccine vector has the outstanding advantages of simple genome, easy large-scale production and the like, and is considered to be one of the virus vaccine vectors with development potential. Successful recovery of infectious VSV virus from DNA makes genetic manipulation of VSV possible. Exogenous viral functional envelope proteins with appropriate cytoplasmic tails can be efficiently packaged into the envelope of the virus to form various chimeric recombinant VSVs packaging heterologous envelope proteins. The non-coding region between each transcription unit of the recombinant VSV can tolerate the insertion of foreign genes with the length of 4.5kb and obtain high-efficiency expression. These properties confer potential for VSV applications as live virus vaccine vectors.
As an emerging live vector vaccine, VSV has the following advantages: (1) easy culture: VSV is available in very high titers on most mammalian cells and is easily prepared in large quantities. (2) High efficiency: in a mouse model of VSV-HA infection, 10 infectious viral particles induced an immune response and 105The immune response elicited by each infectious particle was equally significant. (3) The use is easy: immunization can be carried out by various vaccination routes, and often one vaccination can cause a strong immune response. (4) And (3) generating stronger immune response: can stimulate organism to generate strong cellular immune response and humoral immune response, can also cause stronger mucosal immune response, and is particularly suitable for the development of vaccines of respiratory pathogens infected through mucosa. (5) The safety is good: since VSV virus replicates completely in the cytoplasm, only from RNA → RNA, it does not integrate into the DNA of the host cell and is eventually cleared by the host immune system. Moreover, genome mutation and/or modification can be carried out by reverse genetic manipulation, so that the VSV virus can be weakened appropriately, and becomes a safer recombinant vaccine vector.
The VSV recombinant virus prepared by the invention is different from a VSV pseudovirus, and the most difference is that the VSV recombinant virus prepared by the invention can be replicated after infecting cells, and the VSV pseudovirus only can infect the cells but cannot replicate. In addition, the preparation method and the application range of the two are also different. Although both can be used for screening of viral inhibitors and for basic study of viral infection mechanism, the VSV recombinant virus produced by the present invention can also be used for producing vaccines, whereas VSV pseudoviruses cannot be used for producing vaccines.
SARS coronavirus (SARS-CoV) and novel coronavirus (SARS-CoV-2) have high similarity, belong to the genus beta coronavirus, and the Receptor Binding Domain (RBD) of the S protein is the main epitope using the same cell receptor ACE 2. However, the research finds that the level of the neutralizing antibody induced by the VSV recombinant SARS coronavirus rVSV-SARS-CoV is far higher than that of the VSV recombinant novel coronavirus rVSV-SARS-CoV-2. In order to further improve the immune protection effect of the VSV recombinant novel coronavirus vector vaccine, the RBD of the envelope protein S of the SARS coronavirus is replaced by the RBD of the novel coronavirus to construct a replication type recombinant virus rVSV-SARS-CoV/2-RBD. The recombinant virus surface expresses the chimeric protein of the novel coronavirus and the SARS coronavirus envelope protein S, wherein the main epitope is RBD of the novel coronavirus (SARS-CoV-2), so that the chimeric protein S stimulates the immune response mainly aiming at the novel coronavirus. Moreover, after the main epitope RBD of the novel coronavirus is embedded on the S protein of the SARS coronavirus with stronger immunogenicity, a neutralizing antibody with higher titer than that of the wild novel coronavirus S protein can be induced in mouse and macaque animal models, thereby having better protection effect. The recombinant virus has important significance for the development of vaccines of novel coronavirus.
Drawings
FIG. 1 is a schematic illustration of the chimerization of chimeric envelope protein S.
FIG. 2 is a schematic diagram showing construction of VSV-SARS-CoV/2-RBD.
FIG. 3 shows the expression level detection of S protein in recombinant virus rVSV-SARS-CoV/2-RBD.
FIG. 4 is a graph showing the growth curve of rVSV-SARS-CoV/2-RBD.
FIG. 5 shows the neutralizing antibodies generated by recombinant viruses rVSV-SARS-CoV-2 and rVSV-SARS-CoV inoculated human ACE2 transgenic mouse hACE 2-KI/NIFDC.
FIG. 6 shows the neutralizing antibodies generated by the inoculation of Balb/c mice with rVSV-SARS-CoV/2-RBD and the control rVSV-SARS-CoV-2.
FIG. 7 shows neutralizing antibodies generated by inoculation of human ACE2 transgenic mice hACE2-KI/NIFDC with recombinant virus rVSV-SARS-CoV/2-RBD and control recombinant virus rVSV-SARS-CoV-2.
FIG. 8 shows neutralizing antibodies produced by cynomolgus monkey inoculated with rVSV-SARS-CoV/2-RBD recombinant virus and rVSV-SARS-CoV-2 control recombinant virus.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The test methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
The rVSV Δ G vectors in the examples described below are described in the documents "Single dose of a rVSV-based vaccine experiments complete protection against virus vector live viewer with a thrombocytopathogenic syndrome virus, NPJ vitamins, 2019 Jan 25, 4: 5", publicly available from animal research in the national academy of sciences, the biomaterials being used only for the repetition of experiments related to the present invention and not for other uses. The nucleotide sequence of the rVSV delta G vector is a sequence 7 in a sequence table, and comprises a T7 promoter sequence, a vesicular stomatitis virus Indiana strain whole genome sequence deleting a glycoprotein G coding gene sequence and an HDV terminator sequence.
The plasmid for expressing the N protein of VSV in the following examples is obtained by cloning the gene sequence encoding the N protein in the VSV genome (sequence 8 in the sequence table) into the eukaryotic expression plasmid pCDNA3.1(+) (Beijing Shengyue Gegen Biotech Co., Ltd.) through BamHI and EcoRI cleavage sites.
The plasmid expressing the P protein of VSV in the following examples is obtained by cloning the gene sequence encoding the P protein in the VSV genome (sequence 9 in the sequence table) into the eukaryotic expression plasmid pCDNA3.1(+) (Beijing Shengyue Gegen Biotech Co., Ltd.) through BamHI and EcoRI cleavage sites.
The plasmid for expressing the L protein of VSV in the following examples is obtained by cloning the gene sequence encoding the L protein in the VSV genome (sequence 10 in the sequence table) into the eukaryotic expression plasmid pCDNA3.1(+) (Beijing Shengyue Gegen Biotech Co., Ltd.) through BamHI and EcoRI cleavage sites.
The plasmid expressing the M protein of VSV in the following examples is obtained by cloning the M protein coding gene sequence (sequence 11 in the sequence table) in the VSV genome into the eukaryotic expression plasmid pCDNA3.1(+) (Beijing Shengyue Gegen Biotech Co., Ltd.) through BamHI and EcoRI cleavage sites.
The plasmid expressing the G protein of VSV in the following examples is obtained by cloning the G protein coding gene sequence (sequence 12 in the sequence table) in the VSV genome into the eukaryotic expression plasmid pCDNA3.1(+) (Beijing Shengyue Gegen Biotech Co., Ltd.) through BamHI and EcoRI cleavage sites.
The plasmid expressing T7RNA polymerase in the following examples was obtained by cloning a gene sequence encoding T7RNA polymerase (SEQ ID NO: 13 in the sequence Listing) into a eukaryotic expression plasmid pCDNA3.1(+) (Beijing Shengyue Gegen Biotech Co., Ltd.) through BamHI and EcoRI cleavage sites.
The Vero cells in the following examples are ATCC (American type culture collection) products, and the product number is CCL-81.
Example 1 preparation of recombinant Virus rVSV-SARS-CoV/2-RBD
Preparation of recombinant vector VSV-SARS-CoV/2-RBD
1. Optimization of chimeric envelope protein S sequences
In order to develop a novel coronavirus vaccine, the coding gene sequences of 1-314 th and 537 1259 th positions of the envelope protein S amino acid sequence (gene bank accession number AAP 30030.1) of SARS-CoV BJ01 strain (gene bank accession number AY 278488.2) and the coding gene sequence of 319-541 th position of the envelope protein S amino acid sequence (gene bank accession number YP _ 009724390.1) of SARS-CoV-2Wuhan-Hu-1 strain (gene bank accession number NC _ 045512.2) are humanized, optimized and chimeric to obtain the coding gene sequence of the chimeric envelope protein S, and the nucleotide sequence of the coding gene sequence is shown as sequence 1 in the sequence table.
The chimeric envelope protein S is a protein obtained by replacing the RBD (position 315 and 536 of the amino acid sequence of SARS coronavirus (SARS-CoV) envelope protein S) of SARS coronavirus (SARS-CoV-2) envelope protein S with the RBD (position 319 and 541) of the novel coronavirus (SARS-CoV-2 envelope protein S), deleting the 1261 and 1279 (19 amino acids at the C-terminal of the intracellular region of SARS-CoV envelope protein S) of the amino acid sequence of SARS coronavirus (SARS-CoV) envelope protein S, and keeping the other amino acid sequences of SARS coronavirus (SARS-CoV) envelope protein S unchanged (FIG. 1), wherein the amino acid sequence is shown as sequence 2 in the sequence table.
2. Construction of recombinant vector VSV-SARS-CoV/2-RBD
Inserting the coding gene sequence (sequence 1) of the chimeric envelope protein S in the step 1 between the restriction enzyme sites MluI and NotI of the rVSV delta G vector to obtain a recombinant vector, and naming the recombinant vector as VSV-SARS-CoV/2-RBD.
Preparation of recombinant virus rVSV-SARS-CoV/2-RBD
And (2) co-transfecting the recombinant vector VSV-SARS-CoV/2-RBD in the step one and auxiliary plasmids (a plasmid for expressing the N protein of VSV, a plasmid for expressing the P protein of VSV, a plasmid for expressing the L protein of VSV, a plasmid for expressing the M protein of VSV, a plasmid for expressing the G protein of VSV and a plasmid for expressing T7RNA polymerase) into Vero cells to prepare the recombinant virus rVSV-SARS-CoV/2-RBD. The structure schematic diagram of the recombinant virus rVSV-SARS-CoV/2-RBD is shown in FIG. 2. The method comprises the following specific steps:
1. and (3) subculturing the Vero cells into a culture dish, and when the cell density reaches 70-80% the next day, replacing the complete culture medium with a DMEM (Thermo Fisher, the product number is SH30243.01B) medium containing 2% (volume fraction) fetal bovine serum (FBS, Thermo Fisher, the product number is 10091) to obtain a Vero cell culture system.
2. After completion of step 1, 36. mu.L of FuGENE 6 (Promega, cat # E2692) was incubated with Opti-MEM medium (Thermo Fisher, cat # 51985091) at room temperature for 5 minutes, and then incubated with the recombinant vector plasmid VSV-SARS-CoV/2-RBD (1.59. mu.g), plasmid expressing N protein of VSV (1.286. mu.g), plasmid expressing P protein of VSV (639 ng), plasmid expressing L protein of VSV (159.9 ng), plasmid expressing M protein of VSV (159.9 ng), plasmid expressing G protein of VSV (159.9 ng), and plasmid expressing T7RNA polymerase (8.1. mu.g) in step one, followed by incubation at room temperature for 15 minutes to obtain a post-incubation solution (total volume of 600. mu.L).
3. And (3) after the step 2 is finished, adding the solution incubated in the step 2 into the Vero cell culture system in the step 1, and replacing the fresh culture medium after 6 hours. Cell supernatants were harvested 3 days after transfection and contained the recombinant virus rVSV-SARS-CoV/2-RBD. The obtained supernatant is used to infect new Vero cell to realize the amplification of recombinant virus rVSV-SARS-CoV/2-RBD. The recombinant virus rVSV-SARS-CoV/2-RBD is obtained by replacing the coding gene sequence of glycoprotein G in the genome sequence of vesicular stomatitis virus with the coding gene sequence of chimeric envelope protein S.
Preparation of control recombinant virus rVSV-SARS-CoV-2
1. Preparation of recombinant vector VSV-SARS-CoV-2
Inserting the coding gene sequence of the envelope protein S of SARS-CoV-2 shown in the sequence 3 between the restriction enzyme cutting sites MluI and NotI of the rVSV delta G vector to obtain a recombinant vector, and naming the recombinant vector as VSV-SARS-CoV-2. The amino acid sequence of the envelope protein S of SARS-CoV-2 is shown as sequence 4 in the sequence table.
2. Preparation of recombinant Virus rVSV-SARS-CoV-2
The recombinant vector VSV-SARS-CoV-2 in step 1 and helper plasmids (plasmid expressing N protein of VSV, plasmid expressing P protein of VSV, plasmid expressing L protein of VSV, plasmid expressing M protein of VSV, plasmid expressing G protein of VSV, and plasmid expressing T7RNA polymerase) were co-transfected into Vero cells according to the method in step two to prepare recombinant virus rVSV-SARS-CoV-2. The recombinant virus rVSV-SARS-CoV-2 is obtained by replacing the coding gene sequence of glycoprotein G in the genome sequence of vesicular stomatitis virus with the coding gene sequence of envelope protein S of SARS-CoV-2.
Preparation of control recombinant Virus rVSV-SARS-CoV
1. Preparation of recombinant vector VSV-SARS-CoV
Inserting the coding gene sequence of the envelope protein S of SARS-CoV shown in the sequence 5 between the restriction enzyme cutting sites MluI and NotI of rVSV delta G vector to obtain recombinant vector, and naming the recombinant vector as VSV-SARS-CoV. The amino acid sequence of the envelope protein S of SARS-CoV is shown as sequence 6 in the sequence table.
2. Preparation of recombinant Virus rVSV-SARS-CoV
The recombinant vector VSV-SARS-CoV in step 1 and helper plasmids (plasmid expressing N protein of VSV, plasmid expressing P protein of VSV, plasmid expressing L protein of VSV, plasmid expressing M protein of VSV, plasmid expressing G protein of VSV, and plasmid expressing T7RNA polymerase) were co-transfected into Vero cells according to the method in step two to prepare recombinant virus rVSV-SARS-CoV. The recombinant virus rVSV-SARS-CoV is obtained by replacing the coding gene sequence of glycoprotein G in the genome sequence of vesicular stomatitis virus with the coding gene sequence of envelope protein S of SARS-CoV.
Example 2 identification of antigen expression of recombinant Virus rVSV-SARS-CoV/2-RBD
To identify the expression of chimeric S protein by rVSV-SARS-CoV/2-RBD, Vero cell supernatants infected with rVSV-SARS-CoV/2-RBD prepared in example 1 and control recombinant virus rVSV-SARS-CoV-2 were collected, centrifuged at 39,000 rpm and 4 ℃ for 3 hours (Beckman SW41 rpm) by ultracentrifugation to obtain virus pellet, and the virus pellet was resuspended to obtain concentrated virus. Then, the expression of the S protein in the supernatant of the purified virus particles and infected Vero cells was detected by Western Blot (antibody against RBD protein, Beijing Yiqiao Shenzhou science and technology Co., Ltd., rabbit anti-SARS-CoV-2 RBD polyclonal antibody, cat No. 40592-T62). Uninfected Vero cells were used as controls.
The results are shown in FIG. 3. The results show that: expression of the S protein was detected on both rVSV-SARS-CoV/2-RBD infected Vero cell supernatant and purified virus particles, with a band size of about 180kD, indicating successful packaging of the chimeric S protein into recombinant virus. The control virus rVSV-SARS-CoV-2 has a 110KD band corresponding to the S and S1 proteins, respectively, in addition to the 180KD band.
Example 3 growth Curve of recombinant Virus rVSV-SARS-CoV/2-RBD
Vero cells were passaged at a ratio of 1:3 in 10cm cell culture dishes. The recombinant virus rVSV-SARS-CoV/2-RBD prepared in example 1 and the control recombinant virus rVSV-SARS-CoV-2, M.O.I. =0.01 were added when the cells grew to a density of about 80% on the next day, and the medium was changed to DMEM medium containing 2% (volume fraction) FBS 2 hours after infection. Samples were taken every 12 hours post-infection, up to 108 hours post-infection. The virus titer in the supernatants at different infection times was determined by immunofluorescence.
The operation method for measuring the virus titer by the immunofluorescence method comprises the following steps: vero cells were passaged in 96-well plates at 1.0 ten thousand cells per well. And adding the supernatant of the virus to be detected when the cells grow to about 80-90% of the density on the next day. And (3) performing 10-time gradient dilution on the virus supernatant, namely uniformly mixing 30 mul of virus stock solution and 270 mul of virus diluent, and performing analogy to dilute 6-7 gradients. The virus dilution was DMEM medium containing 2% (volume fraction) FBS. 100 mul of virus solution was added to each well, and 3 wells were repeated for each dilution. The cells were placed in a 37 ℃ cell incubator and 2 hours after infection, the medium was changed to contain 20mM NH4DMEM medium of Cl and 2% (volume fraction) FBS, the cells were placed in a cell culture chamber at 28 ℃. Viral titers (in units of FFU/ml) were detected 24 hours after infection by staining with anti-RBD protein antibody. Selection of fluorescence number in 96-well plates at 102Counting the left and right holes, wherein the calculation method comprises the following steps: mean fluorescence number in three wells x dilution factor x 10. For example, a dilution factor of 104The fluorescence in the wells of (1) was 21, and the virus titer was 21X 104×10=2.1×106 FFU/ml。
The results are shown in FIG. 4. The results show that: comparing the growth curves of the two recombinant viruses, rVSV-SARS-CoV/2-RBD amplification was faster, reaching 10 hours after infection6 FFU/ml. While rVSV-SARS-CoV-2 reached 10 hours after infection6 FFU/ml。
Example 4 immunization experiment
Human ACE2 transgenic mouse immunization experiment
Test animals: human ACE2 transgenic mouse hACE2-KI/NIFDC model (humanized animal model obtained by inserting human hACE2 gene cDNAs under mouse mACE2 promoter based on CRISPR/Cas9 technology on C57BL/6 mouse background). This Model is described in the document "A Mouse Model of SARS-CoV-2 Infection and pathogenesis. Cell Host Microbe. 2020;28(1):124-133. e4.".
The test method comprises the following steps: the recombinant viruses rVSV-SARS-CoV-2 and rVSV-SARS-CoV prepared in example 1 were inoculated into hACE2-KI/NIFDC mice by intramuscular injection (at a dose of 8X 10)5FFU), 4 mice per group. Blood was collected 14, 28 days after inoculation, and serum neutralizing antibody titers were then measured using rVSV-eGFP-SARS-CoV-2 and rVSV-eGFP-SARS-CoV, respectively. rVSV-eGFP-SARS-CoV-2 is described in the literature "assessment of reproducibility-compliance storage of virus-based recombinant virus sustitutable for SARS-CoV-2 entry and neutralization assays. emery Microbes Infect. 2020 Dec;9(1): 2269-. The preparation method of rVSV-eGFP-SARS-CoV is as follows: 1) the eGFP gene sequence shown in sequence No. 14 was inserted between nucleotides 62 to 63 of the nucleotide sequence of the VSV vector to obtain plasmid rVSV-eGFP-G. 2) Inserting the coding gene sequence of the envelope protein S of SARS-CoV-2 shown in the sequence 5 between the restriction enzyme cutting sites MluI and NotI of the rVSV-eGFP-G plasmid, replacing the coding gene sequence of the VSV envelope protein G in the original plasmid, and obtaining the recombinant vector VSV-eGFP-SARS-CoV. 3) The recombinant virus rVSV-eGFP-SARS-CoV was prepared according to the method described in step two of example 1.
The specific steps of the method for detecting the titer of the neutralizing antibody are as follows: vero cells were passaged in 96-well plates at 1.0 ten thousand cells per well. The next day the cells grew to around 80% -90% and were ready for testing. The serum is first diluted by a 5-fold dilution method, e.g., 1:10, 1:50, 1:250, 1: 1250. Simultaneously diluting rVSV-eGFP-SARS-CoV-2 virus to 2X 103FFU/ml, resulting in a final number of 100FFU per well. After adding the same amount of the diluted virus solution to each empty 96-well plate, the serum was diluted in the same amount according to the corresponding dilution ratioAdding the components one by one, then mixing uniformly, and the dilution times of the serum are changed to be twice of the original dilution times correspondingly after mixing uniformly. After incubation at room temperature for 30min, 100. mu.l of the mixture was applied to the Vero cell layer. After 2 hours, the reaction solution was replaced with 20mM NH4Cl medium, after 24 hours the number of GFP positive cells in each well was read under a fluorescent microscope. Calculation of neutralizing antibody Titers (FRNT) according to Reed Muench method50). Calculating the formula: FRNT50=【1/10】(less than 50% cell infection number dilution logarithm + distance ratio x dilution factor logarithm). Distance ratio = (50% -less than 50% cell infection number positive rate)/(greater than 50% cell infection number positive rate-less than 50% cell infection number positive rate). The dilution coefficient is the multiple dilution gradient.
The results are shown in FIG. 5. The results show that: the level of neutralizing antibody induced by rVSV-SARS-CoV immunized mice is much higher than that induced by rVSV-SARS-CoV-2, and is about 5 times higher than that induced by rVSV-SARS-CoV-2.
Second, mouse immunization experiment
Test animals: BALB/c mice (Sibefu, Beijing Biotechnology, Inc., female, 6-8 weeks old).
The test method comprises the following steps: the recombinant virus rVSV-SARS-CoV/2-RBD prepared in example 1 and the control recombinant virus rVSV-SARS-CoV-2 were inoculated into BALB/c mice by intramuscular injection and nasal drip route (intramuscular injection 3X 105FFU, nose 7X 104FFU), 6 mice per group. Blood was collected at 14 and 28 days after inoculation, and the neutralizing antibody titer in the serum was measured using rVSV-eGFP-SARS-CoV-2.
The results are shown in FIG. 6. The results show that: no neutralizing antibody was detected in rVSV-SARS-CoV-2 immunized mice on either day 14 or day 28. After immunization with rVSV-SARS-CoV/2-RBD, neutralizing antibody against SARS-CoV-2 can be generated by either intramuscular injection or nasal drop route. Wherein the average neutralizing antibody titer of the intramuscular injection group at 28 days after immunization is 121.7, and the average neutralizing antibody titer of the nasal drip group is 112.
Third, human ACE2 transgenic mouse immunity experiment
Test animals: human ACE2 transgenic mouse hACE2-KI/NIFDC model.
The test method comprises the following steps: the recombinant virus rVSV-SARS-CoV/2-RBD prepared in example 1 and the control recombinant virus rVSV-SARS-CoV-2 were inoculated into hACE2-KI/NIFDC mice by intramuscular injection and nasal drip route (intramuscular injection 8X 10)5FFU, nose drop 2X 105FFU), 4 mice per group. Blood was collected 14, 28 days after inoculation, and serum neutralizing antibody titers were then measured using rVSV-eGFP-SARS-CoV-2.
The results are shown in FIG. 7. The results show that: in rVSV-SARS-CoV-2 immunized mice, neutralizing antibodies were detected only in the intramuscular injection group, and the average neutralizing antibody titer at 28 days was 301.2. After immunization with rVSV-SARS-CoV/2-RBD, neutralizing antibody against SARS-CoV-2 can be produced by intramuscular injection or nasal drip, wherein the average neutralizing antibody titer of 28 days after immunization in the intramuscular injection group is 717.1, and the average neutralizing antibody titer of the nasal drip group is 339.9. Therefore, the level of the neutralizing antibody induced by the rVSV-SARS-CoV/2-RBD is obviously superior to that of the rVSV-SARS-CoV-2.
Immunization experiment of macaque
Test animals: 3 aged 20 years old macaques (equivalent to 80 years old man) (female, beijing zhongkongling biotechnology corporation).
The test method comprises the following steps: the recombinant virus rVSV-SARS-CoV/2-RBD prepared in example 1 and the control recombinant virus rVSV-SARS-CoV-2 were inoculated into 3 20-year-old macaques by nasal drip route at a dose of 1X 107And (4) FFU. Blood was collected at 14, 21 and 28 days after the inoculation, and the neutralizing antibody titer of the serum was measured using rVSV-eGFP-SARS-CoV-2.
The results are shown in FIG. 8. The results show that: one of the rVSV-SARS-CoV/2-RBD immune groups produced very high titer neutralizing antibody, the titer was 2174.6. While the highest neutralizing antibody titer produced in the rVSV-SARS-CoV-2 immunization group was 1075.7. Overall, the immune effect of the rVSV-SARS-CoV/2-RBD immune group is better than that of the rVSV-SARS-CoV-2 immune group.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the technical principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> institute of animal research of Chinese academy of sciences
<120> novel coronavirus chimeric recombinant vaccine based on vesicular stomatitis virus vector, and preparation method and application thereof
<160> 14
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3783
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<213> Artificial Sequence
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atggatgcaa tgaagagagg gctctgctgt gtgctgctgc tgtgtggagc agtcttcgtt 60
tcggctagcg acctggacag gtgcaccacc ttcgacgacg tgcaggcccc caactacacc 120
cagcacacca gcagcatgag gggcgtgtac taccccgacg agatcttcag gagcgacacc 180
ctgtacctga cccaggacct gttcctgccc ttctacagca acgtgaccgg cttccacacc 240
atcaaccaca ccttcgacaa ccccgtgatc cccttcaagg acggcatcta cttcgccgcc 300
accgagaaga gcaacgtggt gaggggctgg gtgttcggca gcaccatgaa caacaagagc 360
cagagcgtga tcatcatcaa caacagcacc aacgtggtga tcagggcctg caacttcgag 420
ctgtgcgaca accccttctt cgccgtgagc aagcccatgg gcacccagac ccacaccatg 480
atcttcgaca acgccttcaa ctgcaccttc gagtacatca gcgacgcctt cagcctggac 540
gtgagcgaga agagcggcaa cttcaagcac ctgagggagt tcgtgttcaa gaacaaggac 600
ggcttcctgt acgtgtacaa gggctaccag cccatcgacg tggtgaggga cctgcccagc 660
ggcttcaaca ccctgaagcc catcttcaag ctgcccctgg gcatcaacat caccaacttc 720
agggccatcc tgaccgcctt cagccccgcc caggacacct ggggcaccag cgctgcagcc 780
tacttcgtgg gctacctgaa gcccaccacc ttcatgctga agtacgacga gaacggcacc 840
atcaccgacg ccgtggactg cagccagaac cccctggccg agctgaagtg cagcgtgaag 900
agcttcgaga tcgacaaggg catctaccag accagcaact tccgggtgca gcctacagag 960
tctattgtgc ggttcccaaa catcacaaac ctgtgccctt tcggcgaggt gttcaacgcc 1020
acccggttcg cctctgtgta cgcctggaac cggaagcgga tctctaactg cgtggccgac 1080
tactccgtgc tgtacaactc cgcctctttc tctacattca agtgctacgg cgtgtcccct 1140
acaaagctga acgacctgtg cttcaccaac gtgtacgccg actctttcgt gattagaggc 1200
gacgaggtga ggcagattgc ccccggccag acaggcaaga tcgccgacta caactacaag 1260
ctgcccgacg acttcacagg ctgcgtgatc gcctggaact ctaacaacct ggactctaag 1320
gtgggcggca actacaacta cctgtacaga ctgttccgga agtctaacct gaagccattc 1380
gagagggaca ttagcaccga gatttaccag gccggctcta ccccatgcaa cggcgtggag 1440
ggcttcaact gctacttccc actgcagtcc tacggcttcc agcctacaaa cggcgtgggc 1500
taccagcctt accgggtggt ggtgctgtct ttcgagctgc tccacgcccc cgccacagtg 1560
tgcggcccaa agaagagcac aaacctcgtg aagaacaagt gcgtgaactt caacttcaac 1620
ggcctgaccg gcaccggcgt gctgacacct agcagcaaga ggttccagcc cttccagcag 1680
ttcggcaggg acgtgagcga cttcaccgac agcgtgaggg accccaagac cagcgagatc 1740
ctggacatca gcccttgcag cttcggcggc gtgagcgtga tcacacctgg caccaacgcc 1800
agcagcgagg tggccgtgct gtaccaggac gtgaactgca ccgacgtgag caccgccatc 1860
cacgccgacc agctgacacc tgcctggagg atctacagca ccggcaacaa cgtgttccag 1920
acccaggccg gctgcctgat cggcgccgag cacgtggaca ccagctacga gtgcgacatc 1980
cctatcggcg ccggcatctg cgccagctac cacaccgtga gcctgctgag gagcaccagc 2040
cagaagagca tcgtggccta caccatgagc ctgggcgccg acagcagcat cgcctacagc 2100
aacaacacca tcgccattcc taccaacttc agcatcagca tcaccaccga ggtgatgccc 2160
gtgagcatgg ccaagaccag cgtggactgc aacatgtaca tctgcggcga cagcaccgag 2220
tgcgccaacc tgctgctcca gtacggcagc ttctgcaccc agctgaacag ggccctgagc 2280
ggcatcgccg ccgagcagga caggaacacc agggaggtgt tcgcccaggt gaagcagatg 2340
tacaagaccc caaccctgaa gtacttcggc ggcttcaact tcagccagat cctgcccgac 2400
cccctgaagc ccaccaagag gagcttcatc gaggacctgc tgttcaacaa ggtgaccctg 2460
gccgacgccg gcttcatgaa gcagtacggc gagtgcctgg gcgacatcaa cgccagggac 2520
ctgatctgcg cccagaagtt caacggcctg accgtgctgc cacctctgct gaccgacgac 2580
atgatcgccg cctacaccgc cgccctggtg agcggcaccg ccaccgccgg ctggaccttc 2640
ggcgccggcg ccgccctcca gatccccttc gccatgcaga tggcctacag gttcaacggc 2700
atcggcgtga cccagaacgt gctgtacgag aaccagaagc agatcgccaa ccagttcaac 2760
aaggccatca gccagatcca ggagagcctg accaccacca gcaccgccct gggcaagctc 2820
caggacgtgg tgaaccagaa cgcccaggcc ctgaacaccc tggtgaagca gctgagcagc 2880
aacttcggcg ccatcagcag cgtgctgaac gacatcctgt ctagactgga caaggtggag 2940
gccgaggtgc agatcgacag gctgatcacc ggcaggctgc agagcctgca gacctacgtg 3000
acccagcagc tgatcagggc cgccgagatc agggccagcg ccaacctggc cgccaccaag 3060
atgagcgagt gcgtgctggg ccagagcaag agggtggact tctgcggcaa gggctaccac 3120
ctgatgagct tcccccaggc cgccccccac ggcgtggtgt tcctgcacgt gacctacgtg 3180
cccagccagg agaggaactt caccaccgcc cccgccatct gccacgaggg caaggcctac 3240
ttccccaggg agggcgtgtt cgtgttcaac ggcaccagct ggttcatcac ccagaggaac 3300
ttcttcagcc cccagatcat caccaccgac aacaccttcg tgagcggcaa ctgcgacgtg 3360
gtgatcggca tcatcaacaa caccgtgtac gaccccctgc agcccgagct ggacagcttc 3420
aaggaggagc tggacaagta cttcaagaac cacaccagcc ccgacgtgga cctgggcgac 3480
atcagcggca tcaacgccag cgtggtgaac atccagaagg agatcgacag gctgaacgag 3540
gtggccaaga acctgaacga gagcctgatc gacctgcagg agctgggcaa gtacgagcag 3600
tacatcaagt ggccctggta cgtgtggctg ggcttcatcg ccggcctgat cgccatcgtg 3660
atggtgacca tcctgctgtg ctgcatgacc agctgctgca gctgcctgaa gagactcaaa 3720
aggtcaatgc taatgggtaa tccagatgac cgtataccga gggacacata tacattagag 3780
tga 3783
<210> 2
<211> 1260
<212> PRT
<213> Artificial Sequence
<400> 2
Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly
1 5 10 15
Ala Val Phe Val Ser Ala Ser Asp Leu Asp Arg Cys Thr Thr Phe Asp
20 25 30
Asp Val Gln Ala Pro Asn Tyr Thr Gln His Thr Ser Ser Met Arg Gly
35 40 45
Val Tyr Tyr Pro Asp Glu Ile Phe Arg Ser Asp Thr Leu Tyr Leu Thr
50 55 60
Gln Asp Leu Phe Leu Pro Phe Tyr Ser Asn Val Thr Gly Phe His Thr
65 70 75 80
Ile Asn His Thr Phe Asp Asn Pro Val Ile Pro Phe Lys Asp Gly Ile
85 90 95
Tyr Phe Ala Ala Thr Glu Lys Ser Asn Val Val Arg Gly Trp Val Phe
100 105 110
Gly Ser Thr Met Asn Asn Lys Ser Gln Ser Val Ile Ile Ile Asn Asn
115 120 125
Ser Thr Asn Val Val Ile Arg Ala Cys Asn Phe Glu Leu Cys Asp Asn
130 135 140
Pro Phe Phe Ala Val Ser Lys Pro Met Gly Thr Gln Thr His Thr Met
145 150 155 160
Ile Phe Asp Asn Ala Phe Asn Cys Thr Phe Glu Tyr Ile Ser Asp Ala
165 170 175
Phe Ser Leu Asp Val Ser Glu Lys Ser Gly Asn Phe Lys His Leu Arg
180 185 190
Glu Phe Val Phe Lys Asn Lys Asp Gly Phe Leu Tyr Val Tyr Lys Gly
195 200 205
Tyr Gln Pro Ile Asp Val Val Arg Asp Leu Pro Ser Gly Phe Asn Thr
210 215 220
Leu Lys Pro Ile Phe Lys Leu Pro Leu Gly Ile Asn Ile Thr Asn Phe
225 230 235 240
Arg Ala Ile Leu Thr Ala Phe Ser Pro Ala Gln Asp Thr Trp Gly Thr
245 250 255
Ser Ala Ala Ala Tyr Phe Val Gly Tyr Leu Lys Pro Thr Thr Phe Met
260 265 270
Leu Lys Tyr Asp Glu Asn Gly Thr Ile Thr Asp Ala Val Asp Cys Ser
275 280 285
Gln Asn Pro Leu Ala Glu Leu Lys Cys Ser Val Lys Ser Phe Glu Ile
290 295 300
Asp Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val Gln Pro Thr Glu
305 310 315 320
Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu
325 330 335
Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys
340 345 350
Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala
355 360 365
Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn
370 375 380
Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly
385 390 395 400
Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp
405 410 415
Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp
420 425 430
Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu
435 440 445
Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile
450 455 460
Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu
465 470 475 480
Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr
485 490 495
Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu
500 505 510
Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn
515 520 525
Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn Gly Leu Thr Gly
530 535 540
Thr Gly Val Leu Thr Pro Ser Ser Lys Arg Phe Gln Pro Phe Gln Gln
545 550 555 560
Phe Gly Arg Asp Val Ser Asp Phe Thr Asp Ser Val Arg Asp Pro Lys
565 570 575
Thr Ser Glu Ile Leu Asp Ile Ser Pro Cys Ser Phe Gly Gly Val Ser
580 585 590
Val Ile Thr Pro Gly Thr Asn Ala Ser Ser Glu Val Ala Val Leu Tyr
595 600 605
Gln Asp Val Asn Cys Thr Asp Val Ser Thr Ala Ile His Ala Asp Gln
610 615 620
Leu Thr Pro Ala Trp Arg Ile Tyr Ser Thr Gly Asn Asn Val Phe Gln
625 630 635 640
Thr Gln Ala Gly Cys Leu Ile Gly Ala Glu His Val Asp Thr Ser Tyr
645 650 655
Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala Ser Tyr His Thr
660 665 670
Val Ser Leu Leu Arg Ser Thr Ser Gln Lys Ser Ile Val Ala Tyr Thr
675 680 685
Met Ser Leu Gly Ala Asp Ser Ser Ile Ala Tyr Ser Asn Asn Thr Ile
690 695 700
Ala Ile Pro Thr Asn Phe Ser Ile Ser Ile Thr Thr Glu Val Met Pro
705 710 715 720
Val Ser Met Ala Lys Thr Ser Val Asp Cys Asn Met Tyr Ile Cys Gly
725 730 735
Asp Ser Thr Glu Cys Ala Asn Leu Leu Leu Gln Tyr Gly Ser Phe Cys
740 745 750
Thr Gln Leu Asn Arg Ala Leu Ser Gly Ile Ala Ala Glu Gln Asp Arg
755 760 765
Asn Thr Arg Glu Val Phe Ala Gln Val Lys Gln Met Tyr Lys Thr Pro
770 775 780
Thr Leu Lys Tyr Phe Gly Gly Phe Asn Phe Ser Gln Ile Leu Pro Asp
785 790 795 800
Pro Leu Lys Pro Thr Lys Arg Ser Phe Ile Glu Asp Leu Leu Phe Asn
805 810 815
Lys Val Thr Leu Ala Asp Ala Gly Phe Met Lys Gln Tyr Gly Glu Cys
820 825 830
Leu Gly Asp Ile Asn Ala Arg Asp Leu Ile Cys Ala Gln Lys Phe Asn
835 840 845
Gly Leu Thr Val Leu Pro Pro Leu Leu Thr Asp Asp Met Ile Ala Ala
850 855 860
Tyr Thr Ala Ala Leu Val Ser Gly Thr Ala Thr Ala Gly Trp Thr Phe
865 870 875 880
Gly Ala Gly Ala Ala Leu Gln Ile Pro Phe Ala Met Gln Met Ala Tyr
885 890 895
Arg Phe Asn Gly Ile Gly Val Thr Gln Asn Val Leu Tyr Glu Asn Gln
900 905 910
Lys Gln Ile Ala Asn Gln Phe Asn Lys Ala Ile Ser Gln Ile Gln Glu
915 920 925
Ser Leu Thr Thr Thr Ser Thr Ala Leu Gly Lys Leu Gln Asp Val Val
930 935 940
Asn Gln Asn Ala Gln Ala Leu Asn Thr Leu Val Lys Gln Leu Ser Ser
945 950 955 960
Asn Phe Gly Ala Ile Ser Ser Val Leu Asn Asp Ile Leu Ser Arg Leu
965 970 975
Asp Lys Val Glu Ala Glu Val Gln Ile Asp Arg Leu Ile Thr Gly Arg
980 985 990
Leu Gln Ser Leu Gln Thr Tyr Val Thr Gln Gln Leu Ile Arg Ala Ala
995 1000 1005
Glu Ile Arg Ala Ser Ala Asn Leu Ala Ala Thr Lys Met Ser Glu Cys
1010 1015 1020
Val Leu Gly Gln Ser Lys Arg Val Asp Phe Cys Gly Lys Gly Tyr His
1025 1030 1035 1040
Leu Met Ser Phe Pro Gln Ala Ala Pro His Gly Val Val Phe Leu His
1045 1050 1055
Val Thr Tyr Val Pro Ser Gln Glu Arg Asn Phe Thr Thr Ala Pro Ala
1060 1065 1070
Ile Cys His Glu Gly Lys Ala Tyr Phe Pro Arg Glu Gly Val Phe Val
1075 1080 1085
Phe Asn Gly Thr Ser Trp Phe Ile Thr Gln Arg Asn Phe Phe Ser Pro
1090 1095 1100
Gln Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val
1105 1110 1115 1120
Val Ile Gly Ile Ile Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro Glu
1125 1130 1135
Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn His Thr
1140 1145 1150
Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn Ala Ser Val
1155 1160 1165
Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu Val Ala Lys Asn
1170 1175 1180
Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu Gly Lys Tyr Glu Gln
1185 1190 1195 1200
Tyr Ile Lys Trp Pro Trp Tyr Val Trp Leu Gly Phe Ile Ala Gly Leu
1205 1210 1215
Ile Ala Ile Val Met Val Thr Ile Leu Leu Cys Cys Met Thr Ser Cys
1220 1225 1230
Cys Ser Cys Leu Lys Arg Leu Lys Arg Ser Met Leu Met Gly Asn Pro
1235 1240 1245
Asp Asp Arg Ile Pro Arg Asp Thr Tyr Thr Leu Glu
1250 1255 1260
<210> 3
<211> 3822
<212> DNA
<213> Artificial Sequence
<400> 3
atgttcgtgt tcctcgtgct cctgcctctg gtgtctagcc agtgcgtgaa cctgaccaca 60
cggacccagc tccctcccgc ctacacaaac tctttcaccc ggggcgtgta ctaccccgac 120
aaggtgttcc ggtctagcgt gctccactct acacaggacc tgttcctccc tttcttcagc 180
aacgtgacat ggttccacgc catccacgtg tctggcacaa acggcacaaa gcggttcgac 240
aaccccgtgc tccctttcaa cgacggcgtg tacttcgcca gcaccgagaa gtctaacatt 300
atccggggct ggattttcgg caccacactc gactctaaga cacagtccct cctgattgtg 360
aacaacgcca caaacgtggt gattaaggtg tgcgagttcc agttctgcaa cgaccctttc 420
ctgggcgtgt actaccacaa gaacaacaag tcttggatgg agtctgagtt cagagtgtac 480
tctagcgcca acaactgcac cttcgagtac gtgtcccagc ctttcctcat ggacctggag 540
ggcaagcagg gcaacttcaa gaacctgaga gagttcgtgt tcaagaacat tgacggctac 600
ttcaagattt actctaagca caccccaatt aacctcgtga gggacctccc tcagggcttc 660
tccgccttag aaccactggt ggacctccct attggcatta acatcacacg cttccagaca 720
ctgctcgccc tccaccggtc ttacctgacc ccaggcgact ctagctctgg ctggacagcc 780
ggcgccgccg cctactacgt gggctacctg cagcctagga ccttcctcct gaagtacaac 840
gagaacggca caattaccga cgccgtggac tgcgccctgg acccactgtc cgagacaaag 900
tgcacactga agtccttcac agtggagaag ggcatttacc agacatctaa cttccgggtg 960
cagcctacag agtctattgt gcggttccca aacatcacaa acctgtgccc tttcggcgag 1020
gtgttcaacg ccacccggtt cgcctctgtg tacgcctgga accggaagcg gatctctaac 1080
tgcgtggccg actactccgt gctgtacaac tccgcctctt tctctacatt caagtgctac 1140
ggcgtgtccc ctacaaagct gaacgacctg tgcttcacca acgtgtacgc cgactctttc 1200
gtgattagag gcgacgaggt gaggcagatt gcccccggcc agacaggcaa gatcgccgac 1260
tacaactaca agctgcccga cgacttcaca ggctgcgtga tcgcctggaa ctctaacaac 1320
ctggactcta aggtgggcgg caactacaac tacctgtaca gactgttccg gaagtctaac 1380
ctgaagccat tcgagaggga cattagcacc gagatttacc aggccggctc taccccatgc 1440
aacggcgtgg agggcttcaa ctgctacttc ccactgcagt cctacggctt ccagcctaca 1500
aacggcgtgg gctaccagcc ttaccgggtg gtggtgctgt ctttcgagct gctccacgcc 1560
cccgccacag tgtgcggccc aaagaagagc acaaacctcg tgaagaacaa gtgcgtgaac 1620
ttcaacttca acggcctcac aggcacaggc gtgctcaccg agtctaacaa gaagttcctc 1680
cctttccagc agttcggccg cgacattgcc gacaccaccg acgccgtgcg ggaccctcag 1740
acactggaaa ttctcgacat caccccttgc agcttcggcg gcgtgtccgt gatcacccca 1800
ggcacaaaca catctaacca ggtggccgtg ctgtaccagg acgtgaactg caccgaggtg 1860
ccagtggcca tccacgccga ccagctcacc ccaacatgga gggtgtacag cacaggctct 1920
aacgtgttcc agacccgggc cggctgcctc attggcgccg agcacgtgaa caactcttac 1980
gagtgcgaca tccctattgg cgccggcatt tgcgcctctt accagaccca gacaaactct 2040
ccacggagag cccggtctgt ggcctctcag agcattattg cctacaccat gtctctgggc 2100
gccgagaact ctgtggccta ctctaacaac tctattgcca tccctacaaa cttcacaatt 2160
tctgtgacca ccgagattct cccagtgtct atgaccaaga catctgtgga ctgcaccatg 2220
tacatttgcg gcgactccac cgagtgctct aacctcctgc tccagtacgg ctctttctgc 2280
acccagctca accgcgccct gacaggcatc gccgtggagc aggacaagaa cacccaggag 2340
gtgttcgccc aggtgaagca gatttacaag acccccccaa ttaaggactt cggcggcttc 2400
aacttctctc agattctccc cgacccatcc aagcctagca agcggtcctt cattgaggac 2460
ctcctgttca acaaggtgac actggccgac gccggcttca ttaagcagta cggcgactgc 2520
ctgggcgaca ttgccgcccg ggacctgatt tgcgcccaga agttcaacgg cctcacagtg 2580
ctccccccac tgctcaccga cgagatgatt gcccagtaca catctgccct cctggccggc 2640
acaattacat ctggctggac cttcggcgcc ggcgccgccc tgcagatccc tttcgccatg 2700
cagatggcct accgcttcaa cggcatcggc gtgacacaga acgtgctgta cgagaaccag 2760
aagctgatcg ccaaccagtt caacagcgcc attggcaaga ttcaggactc tctgagcagc 2820
acagccagcg ccctgggcaa gctgcaggac gtggtgaacc agaacgccca ggccctgaac 2880
acactggtga agcagctgtc ttctaacttc ggcgccattt ctagcgtgct gaacgacatt 2940
ctgtcgcggc tggacaaggt ggaggccgag gtgcagattg acaggctcat cacaggcaga 3000
ctgcagtctc tgcagacata cgtgacccag cagctgatta gagccgccga gattagagcc 3060
tccgccaacc tggccgccac caagatgagc gagtgcgtgc tcggccagtc taagcgggtg 3120
gacttctgcg gcaagggcta ccacctcatg tctttccctc agtccgcccc tcacggcgtg 3180
gtgttcctcc acgtgacata cgtgcccgcc caggagaaga acttcaccac agcccccgcc 3240
atttgccacg acggcaaggc ccacttccct agggagggcg tgttcgtgtc taacggcacc 3300
cactggttcg tgacccagcg gaacttctac gagcctcaga ttattaccac agacaacaca 3360
ttcgtgagcg gcaactgcga cgtggtgatt ggcattgtga acaacacagt gtacgaccca 3420
ctgcagcctg agttggactc tttcaaggag gaactcgaca agtacttcaa gaaccacaca 3480
tctcctgacg tggacctggg cgacattagc ggcattaacg cctctgtggt gaacattcag 3540
aaggagattg acagactgaa cgaggtggcc aagaacctga acgagtctct cattgacctg 3600
caggagctgg gcaagtacga gcagtacatt aagtggcctt ggtacatttg gctgggcttc 3660
attgccggcc tgatcgccat tgtgatggtg accatcatgc tgtgctgcat gacatcttgc 3720
tgcagctgcc tgaagggctg ctgctcttgc ggctcttgct gcaagttcga cgaggacgac 3780
tctgagcccg tgctgaaggg cgtgaagctc cactacacct ga 3822
<210> 4
<211> 1273
<212> PRT
<213> Artificial Sequence
<400> 4
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val
1 5 10 15
Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe
20 25 30
Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu
35 40 45
His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp
50 55 60
Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp
65 70 75 80
Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu
85 90 95
Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser
100 105 110
Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile
115 120 125
Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr
130 135 140
Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr
145 150 155 160
Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu
165 170 175
Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe
180 185 190
Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr
195 200 205
Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu
210 215 220
Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr
225 230 235 240
Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser
245 250 255
Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro
260 265 270
Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala
275 280 285
Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys
290 295 300
Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val
305 310 315 320
Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys
325 330 335
Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala
340 345 350
Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu
355 360 365
Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro
370 375 380
Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe
385 390 395 400
Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly
405 410 415
Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys
420 425 430
Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn
435 440 445
Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe
450 455 460
Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys
465 470 475 480
Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly
485 490 495
Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val
500 505 510
Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys
515 520 525
Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn
530 535 540
Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu
545 550 555 560
Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val
565 570 575
Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe
580 585 590
Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val
595 600 605
Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile
610 615 620
His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser
625 630 635 640
Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val
645 650 655
Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala
660 665 670
Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg Ser Val Ala
675 680 685
Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser
690 695 700
Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile
705 710 715 720
Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val
725 730 735
Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu
740 745 750
Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr
755 760 765
Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln
770 775 780
Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe
785 790 795 800
Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser
805 810 815
Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly
820 825 830
Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp
835 840 845
Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu
850 855 860
Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly
865 870 875 880
Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile
885 890 895
Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr
900 905 910
Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn
915 920 925
Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala
930 935 940
Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn
945 950 955 960
Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val
965 970 975
Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln
980 985 990
Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val
995 1000 1005
Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu
1010 1015 1020
Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg Val
1025 1030 1035 1040
Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln Ser Ala
1045 1050 1055
Pro His Gly Val Val Phe Leu His Val Thr Tyr Val Pro Ala Gln Glu
1060 1065 1070
Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His Asp Gly Lys Ala His
1075 1080 1085
Phe Pro Arg Glu Gly Val Phe Val Ser Asn Gly Thr His Trp Phe Val
1090 1095 1100
Thr Gln Arg Asn Phe Tyr Glu Pro Gln Ile Ile Thr Thr Asp Asn Thr
1105 1110 1115 1120
Phe Val Ser Gly Asn Cys Asp Val Val Ile Gly Ile Val Asn Asn Thr
1125 1130 1135
Val Tyr Asp Pro Leu Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu
1140 1145 1150
Asp Lys Tyr Phe Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp
1155 1160 1165
Ile Ser Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp
1170 1175 1180
Arg Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu
1185 1190 1195 1200
Gln Glu Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile
1205 1210 1215
Trp Leu Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile
1220 1225 1230
Met Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys
1235 1240 1245
Ser Cys Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro Val
1250 1255 1260
Leu Lys Gly Val Lys Leu His Tyr Thr
1265 1270
<210> 5
<211> 3837
<212> DNA
<213> Artificial Sequence
<400> 5
atggatgcaa tgaagagagg gctctgctgt gtgctgctgc tgtgtggagc agtcttcgtt 60
tcggctagcg acctggacag gtgcaccacc ttcgacgacg tgcaggcccc caactacacc 120
cagcacacca gcagcatgag gggcgtgtac taccccgacg agatcttcag gagcgacacc 180
ctgtacctga cccaggacct gttcctgccc ttctacagca acgtgaccgg cttccacacc 240
atcaaccaca ccttcgacaa ccccgtgatc cccttcaagg acggcatcta cttcgccgcc 300
accgagaaga gcaacgtggt gaggggctgg gtgttcggca gcaccatgaa caacaagagc 360
cagagcgtga tcatcatcaa caacagcacc aacgtggtga tcagggcctg caacttcgag 420
ctgtgcgaca accccttctt cgccgtgagc aagcccatgg gcacccagac ccacaccatg 480
atcttcgaca acgccttcaa ctgcaccttc gagtacatca gcgacgcctt cagcctggac 540
gtgagcgaga agagcggcaa cttcaagcac ctgagggagt tcgtgttcaa gaacaaggac 600
ggcttcctgt acgtgtacaa gggctaccag cccatcgacg tggtgaggga cctgcccagc 660
ggcttcaaca ccctgaagcc catcttcaag ctgcccctgg gcatcaacat caccaacttc 720
agggccatcc tgaccgcctt cagccccgcc caggacacct ggggcaccag cgctgcagcc 780
tacttcgtgg gctacctgaa gcccaccacc ttcatgctga agtacgacga gaacggcacc 840
atcaccgacg ccgtggactg cagccagaac cccctggccg agctgaagtg cagcgtgaag 900
agcttcgaga tcgacaaggg catctaccag accagcaact tcagggtggt gcccagcggc 960
gacgtggtga ggttccccaa catcaccaac ctgtgcccct tcggcgaggt gttcaacgcc 1020
accaagttcc ccagcgtata cgcctgggag aggaagaaga tcagcaactg cgtggccgac 1080
tacagcgtgc tgtacaacag caccttcttc agcaccttca agtgctacgg cgtgagcgcc 1140
accaagctga acgacctgtg cttcagcaac gtgtacgccg acagcttcgt ggtgaagggc 1200
gacgacgtga ggcagatcgc tcctggacag accggcgtga tcgccgacta caactacaag 1260
ctgcccgacg acttcatggg ctgcgtgctg gcctggaaca ccaggaacat cgacgccacc 1320
agcaccggca actacaacta caagtacagg tacctgaggc acggcaagct gaggcccttc 1380
gagagggaca tcagcaacgt gccattcagc cctgacggca agccctgcac accacctgcc 1440
ctgaactgct actggccact gaacgactac ggcttctaca ccaccaccgg catcggctac 1500
cagccctaca gggtggtggt gctgagcttc gagctgctga acgctcctgc caccgtgtgc 1560
ggccctaagc tgagcaccga cctgatcaag aaccagtgcg tgaacttcaa cttcaacggc 1620
ctgaccggca ccggcgtgct gacacctagc agcaagaggt tccagccctt ccagcagttc 1680
ggcagggacg tgagcgactt caccgacagc gtgagggacc ccaagaccag cgagatcctg 1740
gacatcagcc cttgcagctt cggcggcgtg agcgtgatca cacctggcac caacgccagc 1800
agcgaggtgg ccgtgctgta ccaggacgtg aactgcaccg acgtgagcac cgccatccac 1860
gccgaccagc tgacacctgc ctggaggatc tacagcaccg gcaacaacgt gttccagacc 1920
caggccggct gcctgatcgg cgccgagcac gtggacacca gctacgagtg cgacatccct 1980
atcggcgccg gcatctgcgc cagctaccac accgtgagcc tgctgaggag caccagccag 2040
aagagcatcg tggcctacac catgagcctg ggcgccgaca gcagcatcgc ctacagcaac 2100
aacaccatcg ccattcctac caacttcagc atcagcatca ccaccgaggt gatgcccgtg 2160
agcatggcca agaccagcgt ggactgcaac atgtacatct gcggcgacag caccgagtgc 2220
gccaacctgc tgctccagta cggcagcttc tgcacccagc tgaacagggc cctgagcggc 2280
atcgccgccg agcaggacag gaacaccagg gaggtgttcg cccaggtgaa gcagatgtac 2340
aagaccccaa ccctgaagta cttcggcggc ttcaacttca gccagatcct gcccgacccc 2400
ctgaagccca ccaagaggag cttcatcgag gacctgctgt tcaacaaggt gaccctggcc 2460
gacgccggct tcatgaagca gtacggcgag tgcctgggcg acatcaacgc cagggacctg 2520
atctgcgccc agaagttcaa cggcctgacc gtgctgccac ctctgctgac cgacgacatg 2580
atcgccgcct acaccgccgc cctggtgagc ggcaccgcca ccgccggctg gaccttcggc 2640
gccggcgccg ccctccagat ccccttcgcc atgcagatgg cctacaggtt caacggcatc 2700
ggcgtgaccc agaacgtgct gtacgagaac cagaagcaga tcgccaacca gttcaacaag 2760
gccatcagcc agatccagga gagcctgacc accaccagca ccgccctggg caagctccag 2820
gacgtggtga accagaacgc ccaggccctg aacaccctgg tgaagcagct gagcagcaac 2880
ttcggcgcca tcagcagcgt gctgaacgac atcctgtcta gactggacaa ggtggaggcc 2940
gaggtgcaga tcgacaggct gatcaccggc aggctgcaga gcctgcagac ctacgtgacc 3000
cagcagctga tcagggccgc cgagatcagg gccagcgcca acctggccgc caccaagatg 3060
agcgagtgcg tgctgggcca gagcaagagg gtggacttct gcggcaaggg ctaccacctg 3120
atgagcttcc cccaggccgc cccccacggc gtggtgttcc tgcacgtgac ctacgtgccc 3180
agccaggaga ggaacttcac caccgccccc gccatctgcc acgagggcaa ggcctacttc 3240
cccagggagg gcgtgttcgt gttcaacggc accagctggt tcatcaccca gaggaacttc 3300
ttcagccccc agatcatcac caccgacaac accttcgtga gcggcaactg cgacgtggtg 3360
atcggcatca tcaacaacac cgtgtacgac cccctgcagc ccgagctgga cagcttcaag 3420
gaggagctgg acaagtactt caagaaccac accagccccg acgtggacct gggcgacatc 3480
agcggcatca acgccagcgt ggtgaacatc cagaaggaga tcgacaggct gaacgaggtg 3540
gccaagaacc tgaacgagag cctgatcgac ctgcaggagc tgggcaagta cgagcagtac 3600
atcaagtggc cctggtacgt gtggctgggc ttcatcgccg gcctgatcgc catcgtgatg 3660
gtgaccatcc tgctgtgctg catgaccagc tgctgcagct gcctgaagag actcaaaagg 3720
tcaatgctaa tgggtaatcc agatgaccgt ataccgaggg acacatatac attagagccg 3780
aagatcagac atatgtacac aaacggtggg tttgatgcga tggctgagaa aagatga 3837
<210> 6
<211> 1278
<212> PRT
<213> Artificial Sequence
<400> 6
Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly
1 5 10 15
Ala Val Phe Val Ser Ala Ser Asp Leu Asp Arg Cys Thr Thr Phe Asp
20 25 30
Asp Val Gln Ala Pro Asn Tyr Thr Gln His Thr Ser Ser Met Arg Gly
35 40 45
Val Tyr Tyr Pro Asp Glu Ile Phe Arg Ser Asp Thr Leu Tyr Leu Thr
50 55 60
Gln Asp Leu Phe Leu Pro Phe Tyr Ser Asn Val Thr Gly Phe His Thr
65 70 75 80
Ile Asn His Thr Phe Asp Asn Pro Val Ile Pro Phe Lys Asp Gly Ile
85 90 95
Tyr Phe Ala Ala Thr Glu Lys Ser Asn Val Val Arg Gly Trp Val Phe
100 105 110
Gly Ser Thr Met Asn Asn Lys Ser Gln Ser Val Ile Ile Ile Asn Asn
115 120 125
Ser Thr Asn Val Val Ile Arg Ala Cys Asn Phe Glu Leu Cys Asp Asn
130 135 140
Pro Phe Phe Ala Val Ser Lys Pro Met Gly Thr Gln Thr His Thr Met
145 150 155 160
Ile Phe Asp Asn Ala Phe Asn Cys Thr Phe Glu Tyr Ile Ser Asp Ala
165 170 175
Phe Ser Leu Asp Val Ser Glu Lys Ser Gly Asn Phe Lys His Leu Arg
180 185 190
Glu Phe Val Phe Lys Asn Lys Asp Gly Phe Leu Tyr Val Tyr Lys Gly
195 200 205
Tyr Gln Pro Ile Asp Val Val Arg Asp Leu Pro Ser Gly Phe Asn Thr
210 215 220
Leu Lys Pro Ile Phe Lys Leu Pro Leu Gly Ile Asn Ile Thr Asn Phe
225 230 235 240
Arg Ala Ile Leu Thr Ala Phe Ser Pro Ala Gln Asp Thr Trp Gly Thr
245 250 255
Ser Ala Ala Ala Tyr Phe Val Gly Tyr Leu Lys Pro Thr Thr Phe Met
260 265 270
Leu Lys Tyr Asp Glu Asn Gly Thr Ile Thr Asp Ala Val Asp Cys Ser
275 280 285
Gln Asn Pro Leu Ala Glu Leu Lys Cys Ser Val Lys Ser Phe Glu Ile
290 295 300
Asp Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val Val Pro Ser Gly
305 310 315 320
Asp Val Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu
325 330 335
Val Phe Asn Ala Thr Lys Phe Pro Ser Val Tyr Ala Trp Glu Arg Lys
340 345 350
Lys Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Thr
355 360 365
Phe Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Ala Thr Lys Leu Asn
370 375 380
Asp Leu Cys Phe Ser Asn Val Tyr Ala Asp Ser Phe Val Val Lys Gly
385 390 395 400
Asp Asp Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Val Ile Ala Asp
405 410 415
Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Met Gly Cys Val Leu Ala Trp
420 425 430
Asn Thr Arg Asn Ile Asp Ala Thr Ser Thr Gly Asn Tyr Asn Tyr Lys
435 440 445
Tyr Arg Tyr Leu Arg His Gly Lys Leu Arg Pro Phe Glu Arg Asp Ile
450 455 460
Ser Asn Val Pro Phe Ser Pro Asp Gly Lys Pro Cys Thr Pro Pro Ala
465 470 475 480
Leu Asn Cys Tyr Trp Pro Leu Asn Asp Tyr Gly Phe Tyr Thr Thr Thr
485 490 495
Gly Ile Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu
500 505 510
Leu Asn Ala Pro Ala Thr Val Cys Gly Pro Lys Leu Ser Thr Asp Leu
515 520 525
Ile Lys Asn Gln Cys Val Asn Phe Asn Phe Asn Gly Leu Thr Gly Thr
530 535 540
Gly Val Leu Thr Pro Ser Ser Lys Arg Phe Gln Pro Phe Gln Gln Phe
545 550 555 560
Gly Arg Asp Val Ser Asp Phe Thr Asp Ser Val Arg Asp Pro Lys Thr
565 570 575
Ser Glu Ile Leu Asp Ile Ser Pro Cys Ser Phe Gly Gly Val Ser Val
580 585 590
Ile Thr Pro Gly Thr Asn Ala Ser Ser Glu Val Ala Val Leu Tyr Gln
595 600 605
Asp Val Asn Cys Thr Asp Val Ser Thr Ala Ile His Ala Asp Gln Leu
610 615 620
Thr Pro Ala Trp Arg Ile Tyr Ser Thr Gly Asn Asn Val Phe Gln Thr
625 630 635 640
Gln Ala Gly Cys Leu Ile Gly Ala Glu His Val Asp Thr Ser Tyr Glu
645 650 655
Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala Ser Tyr His Thr Val
660 665 670
Ser Leu Leu Arg Ser Thr Ser Gln Lys Ser Ile Val Ala Tyr Thr Met
675 680 685
Ser Leu Gly Ala Asp Ser Ser Ile Ala Tyr Ser Asn Asn Thr Ile Ala
690 695 700
Ile Pro Thr Asn Phe Ser Ile Ser Ile Thr Thr Glu Val Met Pro Val
705 710 715 720
Ser Met Ala Lys Thr Ser Val Asp Cys Asn Met Tyr Ile Cys Gly Asp
725 730 735
Ser Thr Glu Cys Ala Asn Leu Leu Leu Gln Tyr Gly Ser Phe Cys Thr
740 745 750
Gln Leu Asn Arg Ala Leu Ser Gly Ile Ala Ala Glu Gln Asp Arg Asn
755 760 765
Thr Arg Glu Val Phe Ala Gln Val Lys Gln Met Tyr Lys Thr Pro Thr
770 775 780
Leu Lys Tyr Phe Gly Gly Phe Asn Phe Ser Gln Ile Leu Pro Asp Pro
785 790 795 800
Leu Lys Pro Thr Lys Arg Ser Phe Ile Glu Asp Leu Leu Phe Asn Lys
805 810 815
Val Thr Leu Ala Asp Ala Gly Phe Met Lys Gln Tyr Gly Glu Cys Leu
820 825 830
Gly Asp Ile Asn Ala Arg Asp Leu Ile Cys Ala Gln Lys Phe Asn Gly
835 840 845
Leu Thr Val Leu Pro Pro Leu Leu Thr Asp Asp Met Ile Ala Ala Tyr
850 855 860
Thr Ala Ala Leu Val Ser Gly Thr Ala Thr Ala Gly Trp Thr Phe Gly
865 870 875 880
Ala Gly Ala Ala Leu Gln Ile Pro Phe Ala Met Gln Met Ala Tyr Arg
885 890 895
Phe Asn Gly Ile Gly Val Thr Gln Asn Val Leu Tyr Glu Asn Gln Lys
900 905 910
Gln Ile Ala Asn Gln Phe Asn Lys Ala Ile Ser Gln Ile Gln Glu Ser
915 920 925
Leu Thr Thr Thr Ser Thr Ala Leu Gly Lys Leu Gln Asp Val Val Asn
930 935 940
Gln Asn Ala Gln Ala Leu Asn Thr Leu Val Lys Gln Leu Ser Ser Asn
945 950 955 960
Phe Gly Ala Ile Ser Ser Val Leu Asn Asp Ile Leu Ser Arg Leu Asp
965 970 975
Lys Val Glu Ala Glu Val Gln Ile Asp Arg Leu Ile Thr Gly Arg Leu
980 985 990
Gln Ser Leu Gln Thr Tyr Val Thr Gln Gln Leu Ile Arg Ala Ala Glu
995 1000 1005
Ile Arg Ala Ser Ala Asn Leu Ala Ala Thr Lys Met Ser Glu Cys Val
1010 1015 1020
Leu Gly Gln Ser Lys Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu
1025 1030 1035 1040
Met Ser Phe Pro Gln Ala Ala Pro His Gly Val Val Phe Leu His Val
1045 1050 1055
Thr Tyr Val Pro Ser Gln Glu Arg Asn Phe Thr Thr Ala Pro Ala Ile
1060 1065 1070
Cys His Glu Gly Lys Ala Tyr Phe Pro Arg Glu Gly Val Phe Val Phe
1075 1080 1085
Asn Gly Thr Ser Trp Phe Ile Thr Gln Arg Asn Phe Phe Ser Pro Gln
1090 1095 1100
Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val Val
1105 1110 1115 1120
Ile Gly Ile Ile Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro Glu Leu
1125 1130 1135
Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn His Thr Ser
1140 1145 1150
Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn Ala Ser Val Val
1155 1160 1165
Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu Val Ala Lys Asn Leu
1170 1175 1180
Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu Gly Lys Tyr Glu Gln Tyr
1185 1190 1195 1200
Ile Lys Trp Pro Trp Tyr Val Trp Leu Gly Phe Ile Ala Gly Leu Ile
1205 1210 1215
Ala Ile Val Met Val Thr Ile Leu Leu Cys Cys Met Thr Ser Cys Cys
1220 1225 1230
Ser Cys Leu Lys Arg Leu Lys Arg Ser Met Leu Met Gly Asn Pro Asp
1235 1240 1245
Asp Arg Ile Pro Arg Asp Thr Tyr Thr Leu Glu Pro Lys Ile Arg His
1250 1255 1260
Met Tyr Thr Asn Gly Gly Phe Asp Ala Met Ala Glu Lys Arg
1265 1270 1275
<210> 7
<211> 12687
<212> DNA
<213> Artificial Sequence
<400> 7
acgaagacaa acaaaccatt attatcatta aaaggctcag gagaaacttt aacagtaatc 60
aaaatgtctg ttacagtcaa gagaatcatt gacaacacag tcatagttcc aaaacttcct 120
gcaaatgagg atccagtgga atacccggca gattacttca gaaaatcaaa ggagattcct 180
ctttacatca atactacaaa aagtttgtca gatctaagag gatatgtcta ccaaggcctc 240
aaatccggaa atgtatcaat catacatgtc aacagctact tgtatggagc attaaaggac 300
atccggggta agttggataa agattggtca agtttcggaa taaacatcgg gaaagcaggg 360
gatacaatcg gaatatttga ccttgtatcc ttgaaagccc tggacggcgt acttccagat 420
ggagtatcgg atgcttccag aaccagcgca gatgacaaat ggttgccttt gtatctactt 480
ggcttataca gagtgggcag aacacaaatg cctgaataca gaaaaaagct catggatggg 540
ctgacaaatc aatgcaaaat gatcaatgaa cagtttgaac ctcttgtgcc agaaggtcgt 600
gacatttttg atgtgtgggg aaatgacagt aattacacaa aaattgtcgc tgcagtggac 660
atgttcttcc acatgttcaa aaaacatgaa tgtgcctcgt tcagatacgg aactattgtt 720
tccagattca aagattgtgc tgcattggca acatttggac acctctgcaa aataaccgga 780
atgtctacag aagatgtaac gacctggatc ttgaaccgag aagttgcaga tgaaatggtc 840
caaatgatgc ttccaggcca agaaattgac aaggccgatt catacatgcc ttatttgatc 900
gactttggat tgtcttctaa gtctccatat tcttccgtca aaaaccctgc cttccacttc 960
tgggggcaat tgacagctct tctgctcaga tccaccagag caaggaatgc ccgacagcct 1020
gatgacattg agtatacatc tcttactaca gcaggtttgt tgtacgctta tgcagtagga 1080
tcctctgccg acttggcaca acagttttgt gttggagata acaaatacac tccagatgat 1140
agtaccggag gattgacgac taatgcaccg ccacaaggca gagatgtggt cgaatggctc 1200
ggatggtttg aagatcaaaa cagaaaaccg actcctgata tgatgcagta tgcgaaaaga 1260
gcagtcatgt cactgcaagg cctaagagag aagacaattg gcaagtatgc taagtcagaa 1320
tttgacaaat gaccctataa ttctcagatc acctattata tattatgcta catatgaaaa 1380
aaactaacag atatcatgga taatctcaca aaagttcgtg agtatctcaa gtcctactct 1440
cgtctagatc aggcggtagg agagatagat gagatcgaag cacaacgagc tgaaaagtcc 1500
aattatgagt tgttccaaga ggacggagtg gaagagcata ctaggccctc ttattttcag 1560
gcagcagatg attctgacac agaatctgaa ccagaaattg aagacaatca aggcttgtat 1620
gtaccagatc cggaagctga gcaagttgaa ggctttatac aggggccttt agatgactat 1680
gcagatgagg acgtggatgt tgtattcact tcggactgga aacagcctga gcttgaatcc 1740
gacgagcatg gaaagacctt acggttgaca ttgccagagg gtttaagtgg agagcagaaa 1800
tcccagtggc ttttgacgat taaagcagtc gttcaaagtg ccaaacactg gaatctggca 1860
gagtgcacat ttgaagcatc gggagaaggg gtcatcataa aaaagcgcca gataactccg 1920
gatgtatata aggtcactcc agtgatgaac acacatccgt accaatcaga agccgtatca 1980
gatgtttggt ctctctcaaa gacatccatg actttccaac ccaagaaagc aagtcttcag 2040
cctctcacca tatccttgga tgaattgttc tcatctagag gagaattcat ctctgtcgga 2100
ggtaacggac gaatgtctca taaagaggcc atcctgctcg gtctgaggta caaaaagttg 2160
tacaatcagg cgagagtcaa atattctctg tagactatga aaaaaagtaa cagatatcac 2220
aatctaagtg ttatcccaat ccattcatca tgagttcctt aaagaagatt ctcggtctga 2280
aggggaaagg taagaaatct aagaaattag ggatcgcacc acccccttat gaagaggaca 2340
ctagcatgga gtatgctccg agcgctccaa ttgacaaatc ctattttgga gttgacgaga 2400
tggacaccta tgatccgaat caattaagat atgagaaatt cttctttaca gtgaaaatga 2460
cggttagatc taatcgtccg ttcagaacat actcagatgt ggcagccgct gtatcccatt 2520
gggatcacat gtacatcgga atggcaggga aacgtccctt ctacaaaatc ttggcttttt 2580
tgggttcttc taatctaaag gccactccag cggtattggc agatcaaggt caaccagagt 2640
atcacgctca ctgcgaaggc agggcttatt tgccacatag gatggggaag acccctccca 2700
tgctcaatgt accagagcac ttcagaagac cattcaatat aggtctttac aagggaacga 2760
ttgagctcac aatgaccatc tacgatgatg agtcactgga agcagctcct atgatctggg 2820
atcatttcaa ttcttccaaa ttttctgatt tcagagagaa ggccttaatg tttggcctga 2880
ttgtcgagaa aaaggcatct ggagcgtggg tcctggattc tatcagccac ttcaaatgag 2940
ctagtctagc ttccagcttc tgaacaatcc ccggtttact cagtctctcc taattccagc 3000
ctttcgaaca actaatatcc tgtcttttct atccctatga aaaaaactaa cagagatcga 3060
tctgtttcct tgaccctgca caacagattc ttcatgtttg aaccaaatca acttgtgata 3120
tcatgctcaa agaggcctta attatatttt aatttttaat ttttatgaaa aaaactaaca 3180
gcaatcatgg aagtccacga ttttgagacc gacgagttca atgatttcaa tgaagatgac 3240
tatgccacaa gagaattcct gaatcccgat gagcgcatga cgtacttgaa tcatgctgat 3300
tacaatttga attctcctct aattagtgat gatattgaca atttgatcag gaaattcaat 3360
tctcttccga ttccctcgat gtgggatagt aagaactggg atggagttct tgagatgtta 3420
acatcatgtc aagccaatcc catctcaaca tctcagatgc ataaatggat gggaagttgg 3480
ttaatgtctg ataatcatga tgccagtcaa gggtatagtt ttttacatga agtggacaaa 3540
gaggcagaaa taacatttga cgtggtggag accttcatcc gcggctgggg caacaaacca 3600
attgaataca tcaaaaagga aagatggact gactcattca aaattctcgc ttatttgtgt 3660
caaaagtttt tggacttaca caagttgaca ttaatcttaa atgctgtctc tgaggtggaa 3720
ttgctcaact tggcgaggac tttcaaaggc aaagtcagaa gaagttctca tggaacgaac 3780
atatgcagga ttagggttcc cagcttgggt cctactttta tttcagaagg atgggcttac 3840
ttcaagaaac ttgatattct aatggaccga aactttctgt taatggtcaa agatgtgatt 3900
atagggagga tgcaaacggt gctatccatg gtatgtagaa tagacaacct gttctcagag 3960
caagacatct tctcccttct aaatatctac agaattggag ataaaattgt ggagaggcag 4020
ggaaattttt cttatgactt gattaaaatg gtggaaccga tatgcaactt gaagctgatg 4080
aaattagcaa gagaatcaag gcctttagtc ccacaattcc ctcattttga aaatcatatc 4140
aagacttctg ttgatgaagg ggcaaaaatt gaccgaggta taagattcct ccatgatcag 4200
ataatgagtg tgaaaacagt ggatctcaca ctggtgattt atggatcgtt cagacattgg 4260
ggtcatcctt ttatagatta ttacactgga ctagaaaaat tacattccca agtaaccatg 4320
aagaaagata ttgatgtgtc atatgcaaaa gcacttgcaa gtgatttagc tcggattgtt 4380
ctatttcaac agttcaatga tcataaaaag tggttcgtga atggagactt gctccctcat 4440
gatcatccct ttaaaagtca tgttaaagaa aatacatggc ccacagctgc tcaagttcaa 4500
gattttggag ataaatggca tgaacttccg ctgattaaat gttttgaaat acccgactta 4560
ctagacccat cgataatata ctctgacaaa agtcattcaa tgaataggtc agaggtgttg 4620
aaacatgtcc gaatgaatcc gaacactcct atccctagta aaaaggtgtt gcagactatg 4680
ttggacacaa aggctaccaa ttggaaagaa tttcttaaag agattgatga gaagggctta 4740
gatgatgatg atctaattat tggtcttaaa ggaaaggaga gggaactgaa gttggcaggt 4800
agatttttct ccctaatgtc ttggaaattg cgagaatact ttgtaattac cgaatatttg 4860
ataaagactc atttcgtccc tatgtttaaa ggcctgacaa tggcggacga tctaactgca 4920
gtcattaaaa agatgttaga ttcctcatcc ggccaaggat tgaagtcata tgaggcaatt 4980
tgcatagcca atcacattga ttacgaaaaa tggaataacc accaaaggaa gttatcaaac 5040
ggcccagtgt tccgagttat gggccagttc ttaggttatc catccttaat cgagagaact 5100
catgaatttt ttgagaaaag tcttatatac tacaatggaa gaccagactt gatgcgtgtt 5160
cacaacaaca cactgatcaa ttcaacctcc caacgagttt gttggcaagg acaagagggt 5220
ggactggaag gtctacggca aaaaggatgg agtatcctca atctactggt tattcaaaga 5280
gaggctaaaa tcagaaacac tgctgtcaaa gtcttggcac aaggtgataa tcaagttatt 5340
tgcacacagt ataaaacgaa gaaatcgaga aacgttgtag aattacaggg tgctctcaat 5400
caaatggttt ctaataatga gaaaattatg actgcaatca aaatagggac agggaagtta 5460
ggacttttga taaatgacga tgagactatg caatctgcag attacttgaa ttatggaaaa 5520
ataccgattt tccgtggagt gattagaggg ttagagacca agagatggtc acgagtgact 5580
tgtgtcacca atgaccaaat acccacttgt gctaatataa tgagctcagt ttccacaaat 5640
gctctcaccg tagctcattt tgctgagaac ccaatcaatg ccatgataca gtacaattat 5700
tttgggacat ttgctagact cttgttgatg atgcatgatc ctgctcttcg tcaatcattg 5760
tatgaagttc aagataagat accgggcttg cacagttcta ctttcaaata cgccatgttg 5820
tatttggacc cttccattgg aggagtgtcg ggcatgtctt tgtccaggtt tttgattaga 5880
gccttcccag atcccgtaac agaaagtctc tcattctgga gattcatcca tgtacatgct 5940
cgaagtgagc atctgaagga gatgagtgca gtatttggaa accccgagat agccaagttt 6000
cgaataactc acatagacaa gctagtagaa gatccaacct ctctgaacat cgctatggga 6060
atgagtccag cgaacttgtt aaagactgag gttaaaaaat gcttaatcga atcaagacaa 6120
accatcagga accaggtgat taaggatgca accatatatt tgtatcatga agaggatcgg 6180
ctcagaagtt tcttatggtc aataaatcct ctgttcccta gatttttaag tgaattcaaa 6240
tcaggcactt ttttgggagt cgcagacggg ctcatcagtc tatttcaaaa ttctcgtact 6300
attcggaact cctttaagaa aaagtatcat agggaattgg atgatttgat tgtgaggagt 6360
gaggtatcct ctttgacaca tttagggaaa cttcatttga gaaggggatc atgtaaaatg 6420
tggacatgtt cagctactca tgctgacaca ttaagataca aatcctgggg ccgtacagtt 6480
attgggacaa ctgtacccca tccattagaa atgttgggtc cacaacatcg aaaagagact 6540
ccttgtgcac catgtaacac atcagggttc aattatgttt ctgtgcattg tccagacggg 6600
atccatgacg tctttagttc acggggacca ttgcctgctt atctagggtc taaaacatct 6660
gaatctacat ctattttgca gccttgggaa agggaaagca aagtcccact gattaaaaga 6720
gctacacgtc ttagagatgc tatctcttgg tttgttgaac ccgactctaa actagcaatg 6780
actatacttt ctaacatcca ctctttaaca ggcgaagaat ggaccaaaag gcagcatggg 6840
ttcaaaagaa cagggtctgc ccttcatagg ttttcgacat ctcggatgag ccatggtggg 6900
ttcgcatctc agagcactgc agcattgacc aggttgatgg caactacaga caccatgagg 6960
gatctgggag atcagaattt cgacttttta ttccaagcaa cgttgctcta tgctcaaatt 7020
accaccactg ttgcaagaga cggatggatc accagttgta cagatcatta tcatattgcc 7080
tgtaagtcct gtttgagacc catagaagag atcaccctgg actcaagtat ggactacacg 7140
cccccagatg tatcccatgt gctgaagaca tggaggaatg gggaaggttc gtggggacaa 7200
gagataaaac agatctatcc tttagaaggg aattggaaga atttagcacc tgctgagcaa 7260
tcctatcaag tcggcagatg tataggtttt ctatatggag acttggcgta tagaaaatct 7320
actcatgccg aggacagttc tctatttcct ctatctatac aaggtcgtat tagaggtcga 7380
ggtttcttaa aagggttgct agacggatta atgagagcaa gttgctgcca agtaatacac 7440
cggagaagtc tggctcattt gaagaggccg gccaacgcag tgtacggagg tttgatttac 7500
ttgattgata aattgagtgt atcacctcca ttcctttctc ttactagatc aggacctatt 7560
agagacgaat tagaaacgat tccccacaag atcccaacct cctatccgac aagcaaccgt 7620
gatatggggg tgattgtcag aaattacttc aaataccaat gccgtctaat tgaaaaggga 7680
aaatacagat cacattattc acaattatgg ttattctcag atgtcttatc catagacttc 7740
attggaccat tctctatttc caccaccctc ttgcaaatcc tatacaagcc atttttatct 7800
gggaaagata agaatgagtt gagagagctg gcaaatcttt cttcattgct aagatcagga 7860
gaggggtggg aagacataca tgtgaaattc ttcaccaagg acatattatt gtgtccagag 7920
gaaatcagac atgcttgcaa gttcgggatt gctaaggata ataataaaga catgagctat 7980
cccccttggg gaagggaatc cagagggaca attacaacaa tccctgttta ttatacgacc 8040
accccttacc caaagatgct agagatgcct ccaagaatcc aaaatcccct gctgtccgga 8100
atcaggttgg gccaattacc aactggcgct cattataaaa ttcggagtat attacatgga 8160
atgggaatcc attacaggga cttcttgagt tgtggagacg gctccggagg gatgactgct 8220
gcattactac gagaaaatgt gcatagcaga ggaatattca atagtctgtt agaattatca 8280
gggtcagtca tgcgaggcgc ctctcctgag ccccccagtg ccctagaaac tttaggagga 8340
gataaatcga gatgtgtaaa tggtgaaaca tgttgggaat atccatctga cttatgtgac 8400
ccaaggactt gggactattt cctccgactc aaagcaggct tggggcttca aattgattta 8460
attgtaatgg atatggaagt tcgggattct tctactagcc tgaaaattga gacgaatgtt 8520
agaaattatg tgcaccggat tttggatgag caaggagttt taatctacaa gacttatgga 8580
acatatattt gtgagagcga aaagaatgca gtaacaatcc ttggtcccat gttcaagacg 8640
gtcgacttag ttcaaacaga atttagtagt tctcaaacgt ctgaagtata tatggtatgt 8700
aaaggtttga agaaattaat cgatgaaccc aatcccgatt ggtcttccat caatgaatcc 8760
tggaaaaacc tgtacgcatt ccagtcatca gaacaggaat ttgccagagc aaagaaggtt 8820
agtacatact ttaccttgac aggtattccc tcccaattca ttcctgatcc ttttgtaaac 8880
attgagacta tgctacaaat attcggagta cccacgggtg tgtctcatgc ggctgcctta 8940
aaatcatctg atagacctgc agatttattg accattagcc ttttttatat ggcgattata 9000
tcgtattata acatcaatca tatcagagta ggaccgatac ctccgaaccc cccatcagat 9060
ggaattgcac aaaatgtggg gatcgctata actggtataa gcttttggct gagtttgatg 9120
gagaaagaca ttccactata tcaacagtgt ttggcagtta tccagcaatc atttccgatt 9180
aggtgggagg ctatttcagt aaaaggagga tacaagcaga agtggagtac tagaggtgat 9240
gggctcccaa aagatacccg aatttcagac tccttggccc caatcgggaa ctggatcaga 9300
tctttggaat tggtccgaaa ccaagttcgt ctaaatccat tcaataagat cttgttcaat 9360
cagctatgtc gtacagtgga taatcatttg aagtggtcaa atttgcgaaa aaacacagga 9420
atgattgaat ggatcaatgg gcgaatttca aaagaagacc ggtctatact gatgttgaag 9480
agtgacctac atgaggaaaa ctcttggaga gattaaaaaa tcaggaggag actccaaact 9540
ttaagtatga aaaaaacttt gatccttaag accctcttgt ggtttttatt tttttatctg 9600
gttttgtggt cttcgtgggt cggcatggca tctccacctc ctcgcggtcc gacctgggca 9660
tccgaaggag gacgtcgtcc actcggatgg ctaagggaga gctcggatcc ggctgctaac 9720
aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc 9780
cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga 9840
tcgagatcct ctagagtcga cctgcaggca tgcaagcttg tattctatag tgtcacctaa 9900
atcgtatgtg tatgatacat aaggttatgt attaattgta gccgcgttct aacgacaata 9960
tgtacaagcc taattgtgta gcatctggct tactgaagca gaccctatca tctctctcgt 10020
aaactgccgt cagagtcggt ttggttggac gaaccttctg agtttctggt aacgccgtcc 10080
cgcacccgga aatggtcagc gaaccaatca gcagggtcat cgctagccag atcctctacg 10140
ccggacgcat cgtggccggc atcaccggcg ccacaggtgc ggttgctggc gcctatatcg 10200
ccgacatcac cgatggggaa gatcgggctc gccacttcgg gctcatgagc gcttgtttcg 10260
gcgtgggtat ggtggcaggc cccgtggccg ggggactgtt gggcgccatc tccttgcacc 10320
attccttgcg gcggcggtgc tcaacggcct caacctacta ctgggctgct tcctaatgca 10380
ggagtcgcat aagggagagc gtcgaatggt gcactctcag tacaatctgc tctgatgccg 10440
catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc 10500
tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga 10560
ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt 10620
tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa 10680
atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca 10740
tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc 10800
aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc 10860
acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt 10920
acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt 10980
ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg 11040
ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact 11100
caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg 11160
ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga 11220
aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg 11280
aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa 11340
tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac 11400
aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc 11460
cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca 11520
ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga 11580
gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta 11640
agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc 11700
atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc 11760
cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt 11820
cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac 11880
cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct 11940
tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact 12000
tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg 12060
ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata 12120
aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga 12180
cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag 12240
ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg 12300
agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac 12360
ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca 12420
acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg 12480
cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc 12540
gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa 12600
tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcaggggg atctcgatcc 12660
cgcgaaatta atacgactca ctatagg 12687
<210> 8
<211> 1269
<212> DNA
<213> Artificial Sequence
<400> 8
atgtctgtta cagtcaagag aatcattgac aacacagtca tagttccaaa acttcctgca 60
aatgaggatc cagtggaata cccggcagat tacttcagaa aatcaaagga gattcctctt 120
tacatcaata ctacaaaaag tttgtcagat ctaagaggat atgtctacca aggcctcaaa 180
tccggaaatg tatcaatcat acatgtcaac agctacttgt atggagcatt aaaggacatc 240
cggggtaagt tggataaaga ttggtcaagt ttcggaataa acatcgggaa agcaggggat 300
acaatcggaa tatttgacct tgtatccttg aaagccctgg acggcgtact tccagatgga 360
gtatcggatg cttccagaac cagcgcagat gacaaatggt tgcctttgta tctacttggc 420
ttatacagag tgggcagaac acaaatgcct gaatacagaa aaaagctcat ggatgggctg 480
acaaatcaat gcaaaatgat caatgaacag tttgaacctc ttgtgccaga aggtcgtgac 540
atttttgatg tgtggggaaa tgacagtaat tacacaaaaa ttgtcgctgc agtggacatg 600
ttcttccaca tgttcaaaaa acatgaatgt gcctcgttca gatacggaac tattgtttcc 660
agattcaaag attgtgctgc attggcaaca tttggacacc tctgcaaaat aaccggaatg 720
tctacagaag atgtaacgac ctggatcttg aaccgagaag ttgcagatga aatggtccaa 780
atgatgcttc caggccaaga aattgacaag gccgattcat acatgcctta tttgatcgac 840
tttggattgt cttctaagtc tccatattct tccgtcaaaa accctgcctt ccacttctgg 900
gggcaattga cagctcttct gctcagatcc accagagcaa ggaatgcccg acagcctgat 960
gacattgagt atacatctct tactacagca ggtttgttgt acgcttatgc agtaggatcc 1020
tctgccgact tggcacaaca gttttgtgtt ggagataaca aatacactcc agatgatagt 1080
accggaggat tgacgactaa tgcaccgcca caaggcagag atgtggtcga atggctcgga 1140
tggtttgaag atcaaaacag aaaaccgact cctgatatga tgcagtatgc gaaaagagca 1200
gtcatgtcac tgcaaggcct aagagagaag acaattggca agtatgctaa gtcagaattt 1260
gacaaatga 1269
<210> 9
<211> 798
<212> DNA
<213> Artificial Sequence
<400> 9
atggataatc tcacaaaagt tcgtgagtat ctcaagtcct actctcgtct agatcaggcg 60
gtaggagaga tagatgagat cgaagcacaa cgagctgaaa agtccaatta tgagttgttc 120
caagaggacg gagtggaaga gcatactagg ccctcttatt ttcaggcagc agatgattct 180
gacacagaat ctgaaccaga aattgaagac aatcaaggct tgtatgtacc agatccggaa 240
gctgagcaag ttgaaggctt tatacagggg cctttagatg actatgcaga tgaggacgtg 300
gatgttgtat tcacttcgga ctggaaacag cctgagcttg aatccgacga gcatggaaag 360
accttacggt tgacattgcc agagggttta agtggagagc agaaatccca gtggcttttg 420
acgattaaag cagtcgttca aagtgccaaa cactggaatc tggcagagtg cacatttgaa 480
gcatcgggag aaggggtcat cataaaaaag cgccagataa ctccggatgt atataaggtc 540
actccagtga tgaacacaca tccgtaccaa tcagaagccg tatcagatgt ttggtctctc 600
tcaaagacat ccatgacttt ccaacccaag aaagcaagtc ttcagcctct caccatatcc 660
ttggatgaat tgttctcatc tagaggagaa ttcatctctg tcggaggtaa cggacgaatg 720
tctcataaag aggccatcct gctcggtctg aggtacaaaa agttgtacaa tcaggcgaga 780
gtcaaatatt ctctgtag 798
<210> 10
<211> 6330
<212> DNA
<213> Artificial Sequence
<400> 10
atggaagtcc acgattttga gaccgacgag ttcaatgatt tcaatgaaga tgactatgcc 60
acaagagaat tcctgaatcc cgatgagcgc atgacgtact tgaatcatgc tgattacaat 120
ttgaattctc ctctaattag tgatgatatt gacaatttga tcaggaaatt caattctctt 180
ccgattccct cgatgtggga tagtaagaac tgggatggag ttcttgagat gttaacatca 240
tgtcaagcca atcccatctc aacatctcag atgcataaat ggatgggaag ttggttaatg 300
tctgataatc atgatgccag tcaagggtat agttttttac atgaagtgga caaagaggca 360
gaaataacat ttgacgtggt ggagaccttc atccgcggct ggggcaacaa accaattgaa 420
tacatcaaaa aggaaagatg gactgactca ttcaaaattc tcgcttattt gtgtcaaaag 480
tttttggact tacacaagtt gacattaatc ttaaatgctg tctctgaggt ggaattgctc 540
aacttggcga ggactttcaa aggcaaagtc agaagaagtt ctcatggaac gaacatatgc 600
aggattaggg ttcccagctt gggtcctact tttatttcag aaggatgggc ttacttcaag 660
aaacttgata ttctaatgga ccgaaacttt ctgttaatgg tcaaagatgt gattataggg 720
aggatgcaaa cggtgctatc catggtatgt agaatagaca acctgttctc agagcaagac 780
atcttctccc ttctaaatat ctacagaatt ggagataaaa ttgtggagag gcagggaaat 840
ttttcttatg acttgattaa aatggtggaa ccgatatgca acttgaagct gatgaaatta 900
gcaagagaat caaggccttt agtcccacaa ttccctcatt ttgaaaatca tatcaagact 960
tctgttgatg aaggggcaaa aattgaccga ggtataagat tcctccatga tcagataatg 1020
agtgtgaaaa cagtggatct cacactggtg atttatggat cgttcagaca ttggggtcat 1080
ccttttatag attattacac tggactagaa aaattacatt cccaagtaac catgaagaaa 1140
gatattgatg tgtcatatgc aaaagcactt gcaagtgatt tagctcggat tgttctattt 1200
caacagttca atgatcataa aaagtggttc gtgaatggag acttgctccc tcatgatcat 1260
ccctttaaaa gtcatgttaa agaaaataca tggcccacag ctgctcaagt tcaagatttt 1320
ggagataaat ggcatgaact tccgctgatt aaatgttttg aaatacccga cttactagac 1380
ccatcgataa tatactctga caaaagtcat tcaatgaata ggtcagaggt gttgaaacat 1440
gtccgaatga atccgaacac tcctatccct agtaaaaagg tgttgcagac tatgttggac 1500
acaaaggcta ccaattggaa agaatttctt aaagagattg atgagaaggg cttagatgat 1560
gatgatctaa ttattggtct taaaggaaag gagagggaac tgaagttggc aggtagattt 1620
ttctccctaa tgtcttggaa attgcgagaa tactttgtaa ttaccgaata tttgataaag 1680
actcatttcg tccctatgtt taaaggcctg acaatggcgg acgatctaac tgcagtcatt 1740
aaaaagatgt tagattcctc atccggccaa ggattgaagt catatgaggc aatttgcata 1800
gccaatcaca ttgattacga aaaatggaat aaccaccaaa ggaagttatc aaacggccca 1860
gtgttccgag ttatgggcca gttcttaggt tatccatcct taatcgagag aactcatgaa 1920
ttttttgaga aaagtcttat atactacaat ggaagaccag acttgatgcg tgttcacaac 1980
aacacactga tcaattcaac ctcccaacga gtttgttggc aaggacaaga gggtggactg 2040
gaaggtctac ggcaaaaagg atggagtatc ctcaatctac tggttattca aagagaggct 2100
aaaatcagaa acactgctgt caaagtcttg gcacaaggtg ataatcaagt tatttgcaca 2160
cagtataaaa cgaagaaatc gagaaacgtt gtagaattac agggtgctct caatcaaatg 2220
gtttctaata atgagaaaat tatgactgca atcaaaatag ggacagggaa gttaggactt 2280
ttgataaatg acgatgagac tatgcaatct gcagattact tgaattatgg aaaaataccg 2340
attttccgtg gagtgattag agggttagag accaagagat ggtcacgagt gacttgtgtc 2400
accaatgacc aaatacccac ttgtgctaat ataatgagct cagtttccac aaatgctctc 2460
accgtagctc attttgctga gaacccaatc aatgccatga tacagtacaa ttattttggg 2520
acatttgcta gactcttgtt gatgatgcat gatcctgctc ttcgtcaatc attgtatgaa 2580
gttcaagata agataccggg cttgcacagt tctactttca aatacgccat gttgtatttg 2640
gacccttcca ttggaggagt gtcgggcatg tctttgtcca ggtttttgat tagagccttc 2700
ccagatcccg taacagaaag tctctcattc tggagattca tccatgtaca tgctcgaagt 2760
gagcatctga aggagatgag tgcagtattt ggaaaccccg agatagccaa gtttcgaata 2820
actcacatag acaagctagt agaagatcca acctctctga acatcgctat gggaatgagt 2880
ccagcgaact tgttaaagac tgaggttaaa aaatgcttaa tcgaatcaag acaaaccatc 2940
aggaaccagg tgattaagga tgcaaccata tatttgtatc atgaagagga tcggctcaga 3000
agtttcttat ggtcaataaa tcctctgttc cctagatttt taagtgaatt caaatcaggc 3060
acttttttgg gagtcgcaga cgggctcatc agtctatttc aaaattctcg tactattcgg 3120
aactccttta agaaaaagta tcatagggaa ttggatgatt tgattgtgag gagtgaggta 3180
tcctctttga cacatttagg gaaacttcat ttgagaaggg gatcatgtaa aatgtggaca 3240
tgttcagcta ctcatgctga cacattaaga tacaaatcct ggggccgtac agttattggg 3300
acaactgtac cccatccatt agaaatgttg ggtccacaac atcgaaaaga gactccttgt 3360
gcaccatgta acacatcagg gttcaattat gtttctgtgc attgtccaga cgggatccat 3420
gacgtcttta gttcacgggg accattgcct gcttatctag ggtctaaaac atctgaatct 3480
acatctattt tgcagccttg ggaaagggaa agcaaagtcc cactgattaa aagagctaca 3540
cgtcttagag atgctatctc ttggtttgtt gaacccgact ctaaactagc aatgactata 3600
ctttctaaca tccactcttt aacaggcgaa gaatggacca aaaggcagca tgggttcaaa 3660
agaacagggt ctgcccttca taggttttcg acatctcgga tgagccatgg tgggttcgca 3720
tctcagagca ctgcagcatt gaccaggttg atggcaacta cagacaccat gagggatctg 3780
ggagatcaga atttcgactt tttattccaa gcaacgttgc tctatgctca aattaccacc 3840
actgttgcaa gagacggatg gatcaccagt tgtacagatc attatcatat tgcctgtaag 3900
tcctgtttga gacccataga agagatcacc ctggactcaa gtatggacta cacgccccca 3960
gatgtatccc atgtgctgaa gacatggagg aatggggaag gttcgtgggg acaagagata 4020
aaacagatct atcctttaga agggaattgg aagaatttag cacctgctga gcaatcctat 4080
caagtcggca gatgtatagg ttttctatat ggagacttgg cgtatagaaa atctactcat 4140
gccgaggaca gttctctatt tcctctatct atacaaggtc gtattagagg tcgaggtttc 4200
ttaaaagggt tgctagacgg attaatgaga gcaagttgct gccaagtaat acaccggaga 4260
agtctggctc atttgaagag gccggccaac gcagtgtacg gaggtttgat ttacttgatt 4320
gataaattga gtgtatcacc tccattcctt tctcttacta gatcaggacc tattagagac 4380
gaattagaaa cgattcccca caagatccca acctcctatc cgacaagcaa ccgtgatatg 4440
ggggtgattg tcagaaatta cttcaaatac caatgccgtc taattgaaaa gggaaaatac 4500
agatcacatt attcacaatt atggttattc tcagatgtct tatccataga cttcattgga 4560
ccattctcta tttccaccac cctcttgcaa atcctataca agccattttt atctgggaaa 4620
gataagaatg agttgagaga gctggcaaat ctttcttcat tgctaagatc aggagagggg 4680
tgggaagaca tacatgtgaa attcttcacc aaggacatat tattgtgtcc agaggaaatc 4740
agacatgctt gcaagttcgg gattgctaag gataataata aagacatgag ctatccccct 4800
tggggaaggg aatccagagg gacaattaca acaatccctg tttattatac gaccacccct 4860
tacccaaaga tgctagagat gcctccaaga atccaaaatc ccctgctgtc cggaatcagg 4920
ttgggccaat taccaactgg cgctcattat aaaattcgga gtatattaca tggaatggga 4980
atccattaca gggacttctt gagttgtgga gacggctccg gagggatgac tgctgcatta 5040
ctacgagaaa atgtgcatag cagaggaata ttcaatagtc tgttagaatt atcagggtca 5100
gtcatgcgag gcgcctctcc tgagcccccc agtgccctag aaactttagg aggagataaa 5160
tcgagatgtg taaatggtga aacatgttgg gaatatccat ctgacttatg tgacccaagg 5220
acttgggact atttcctccg actcaaagca ggcttggggc ttcaaattga tttaattgta 5280
atggatatgg aagttcggga ttcttctact agcctgaaaa ttgagacgaa tgttagaaat 5340
tatgtgcacc ggattttgga tgagcaagga gttttaatct acaagactta tggaacatat 5400
atttgtgaga gcgaaaagaa tgcagtaaca atccttggtc ccatgttcaa gacggtcgac 5460
ttagttcaaa cagaatttag tagttctcaa acgtctgaag tatatatggt atgtaaaggt 5520
ttgaagaaat taatcgatga acccaatccc gattggtctt ccatcaatga atcctggaaa 5580
aacctgtacg cattccagtc atcagaacag gaatttgcca gagcaaagaa ggttagtaca 5640
tactttacct tgacaggtat tccctcccaa ttcattcctg atccttttgt aaacattgag 5700
actatgctac aaatattcgg agtacccacg ggtgtgtctc atgcggctgc cttaaaatca 5760
tctgatagac ctgcagattt attgaccatt agcctttttt atatggcgat tatatcgtat 5820
tataacatca atcatatcag agtaggaccg atacctccga accccccatc agatggaatt 5880
gcacaaaatg tggggatcgc tataactggt ataagctttt ggctgagttt gatggagaaa 5940
gacattccac tatatcaaca gtgtttggca gttatccagc aatcatttcc gattaggtgg 6000
gaggctattt cagtaaaagg aggatacaag cagaagtgga gtactagagg tgatgggctc 6060
ccaaaagata cccgaatttc agactccttg gccccaatcg ggaactggat cagatctttg 6120
gaattggtcc gaaaccaagt tcgtctaaat ccattcaata agatcttgtt caatcagcta 6180
tgtcgtacag tggataatca tttgaagtgg tcaaatttgc gaaaaaacac aggaatgatt 6240
gaatggatca atgggcgaat ttcaaaagaa gaccggtcta tactgatgtt gaagagtgac 6300
ctacatgagg aaaactcttg gagagattaa 6330
<210> 11
<211> 690
<212> DNA
<213> Artificial Sequence
<400> 11
atgagttcct taaagaagat tctcggtctg aaggggaaag gtaagaaatc taagaaatta 60
gggatcgcac caccccctta tgaagaggac actagcatgg agtatgctcc gagcgctcca 120
attgacaaat cctattttgg agttgacgag atggacacct atgatccgaa tcaattaaga 180
tatgagaaat tcttctttac agtgaaaatg acggttagat ctaatcgtcc gttcagaaca 240
tactcagatg tggcagccgc tgtatcccat tgggatcaca tgtacatcgg aatggcaggg 300
aaacgtccct tctacaaaat cttggctttt ttgggttctt ctaatctaaa ggccactcca 360
gcggtattgg cagatcaagg tcaaccagag tatcacgctc actgcgaagg cagggcttat 420
ttgccacata ggatggggaa gacccctccc atgctcaatg taccagagca cttcagaaga 480
ccattcaata taggtcttta caagggaacg attgagctca caatgaccat ctacgatgat 540
gagtcactgg aagcagctcc tatgatctgg gatcatttca attcttccaa attttctgat 600
ttcagagaga aggccttaat gtttggcctg attgtcgaga aaaaggcatc tggagcgtgg 660
gtcctggatt ctatcagcca cttcaaatga 690
<210> 12
<211> 1536
<212> DNA
<213> Artificial Sequence
<400> 12
atgaagtgcc ttttgtactt agctttttta ttcatcgggg tgaattgcaa gttcaccata 60
gtttttccac acaaccgaaa aggaaactgg aaaaatgttc cttccaatta ccattattgc 120
ccgtcaagct cagatttaaa ttggcataat gacttaatag gcacagcctt acaagtcaaa 180
atgcccaaga gtcacaaggc tattcaagca gacggttgga tgtgtcatgc ttccaaatgg 240
gtcactactt gtgatttccg ctggtacgga ccggagtata taacacattc catccgatcc 300
ttcactccat ctgtagaaca atgcaaggaa agcattgaac aaacgaaaca aggaacttgg 360
ctgaatccag gcttccctcc tcaaagttgt ggatatgcaa ctgtgacgga tgctgaagca 420
gcgattgtcc aggtgactcc tcaccatgtg cttgttgatg aatacacagg agaatgggtt 480
gattcacagt tcatcaacgg aaaatgcagc aatgacatat gccccactgt ccataactcc 540
acaacctggc attccgacta taaggtcaaa gggctatgtg attctaacct catttccatg 600
gacatcacct tcttctcaga ggacggagag ctatcatccc taggaaagga gggcacaggg 660
ttcagaagta actactttgc ttatgaaact ggagacaagg cctgcaaaat gcagtactgc 720
aagcattggg gagtcagact cccatcaggt gtctggttcg agatggctga taaggatctc 780
tttgctgcag ccagattccc tgaatgccca gaagggtcaa gtatctctgc tccatctcag 840
acctcagtgg atgtaagtct cattcaggac gttgagagga tcttggatta ttccctctgc 900
caagaaacct ggagcaaaat cagagcgggt cttcccatct ctccagtgga tctcagctat 960
cttgctccta aaaacccagg aaccggtcct gtctttacca taatcaatgg taccctaaaa 1020
tactttgaga ccagatacat cagagtcgat attgctgctc caatcctctc aagaatggtc 1080
ggaatgatca gtggaactac cacagaaagg gaactgtggg atgactgggc tccatatgaa 1140
gacgtggaaa ttggacccaa tggagttctg aggaccagtt caggatataa gtttccttta 1200
tatatgattg gacatggtat gttggactcc gatcttcatc ttagctcaaa ggctcaggtg 1260
tttgaacatc ctcacattca agacgctgct tcgcagcttc ctgatgatga gactttattt 1320
tttggtgata ctgggctatc caaaaatcca atcgagtttg tagaaggttg gttcagtagt 1380
tggaagagct ctattgcctc tttttgcttt atcatagggt taatcattgg actattcttg 1440
gttctccgag ttggtattta tctttgcatt aaattaaagc acaccaagaa aagacagatt 1500
tatacagaca tagagatgaa ccgacttgga aagtaa 1536
<210> 13
<211> 2652
<212> DNA
<213> Artificial Sequence
<400> 13
atgaacacga ttaacatcgc taagaacgac ttctctgaca tcgaactggc tgctatcccg 60
ttcaacactc tggctgacca ttacggtgag cgtttagctc gcgaacagtt ggcccttgag 120
catgagtctt acgagatggg tgaagcacgc ttccgcaaga tgtttgagcg tcaacttaaa 180
gctggtgagg ttgcggataa cgctgccgcc aagcctctca tcactaccct actccctaag 240
atgattgcac gcatcaacga ctggtttgag gaagtgaaag ctaagcgcgg caagcgcccg 300
acagccttcc agttcctgca agaaatcaag ccggaagccg tagcgtacat caccattaag 360
accactctgg cttgcctaac cagtgctgac aatacaaccg ttcaggctgt agcaagcgca 420
atcggtcggg ccattgagga cgaggctcgc ttcggtcgta tccgtgacct tgaagctaag 480
cacttcaaga aaaacgttga ggaacaactc aacaagcgcg tagggcacgt ctacaagaaa 540
gcatttatgc aagttgtcga ggctgacatg ctctctaagg gtctactcgg tggcgaggcg 600
tggtcttcgt ggcataagga agactctatt catgtaggag tacgctgcat cgagatgctc 660
attgagtcaa ccggaatggt tagcttacac cgccaaaatg ctggcgtagt aggtcaagac 720
tctgagacta tcgaactcgc acctgaatac gctgaggcta tcgcaacccg tgcaggtgcg 780
ctggctggca tctctccgat gttccaacct tgcgtagttc ctcctaagcc gtggactggc 840
attactggtg gtggctattg ggctaacggt cgtcgtcctc tggcgctggt gcgtactcac 900
agtaagaaag cactgatgcg ctacgaagac gtttacatgc ctgaggtgta caaagcgatt 960
aacattgcgc aaaacaccgc atggaaaatc aacaagaaag tcctagcggt cgccaacgta 1020
atcaccaagt ggaagcattg tccggtcgag gacatccctg cgattgagcg tgaagaactc 1080
ccgatgaaac cggaagacat cgacatgaat cctgaggctc tcaccgcgtg gaaacgtgct 1140
gccgctgctg tgtaccgcaa ggacaaggct cgcaagtctc gccgtatcag ccttgagttc 1200
atgcttgagc aagccaataa gtttgctaac cataaggcca tctggttccc ttacaacatg 1260
gactggcgcg gtcgtgttta cgctgtgtca atgttcaacc cgcaaggtaa cgatatgacc 1320
aaaggactgc ttacgctggc gaaaggtaaa ccaatcggta aggaaggtta ctactggctg 1380
aaaatccacg gtgcaaactg tgcgggtgtc gataaggttc cgttccctga gcgcatcaag 1440
ttcattgagg aaaaccacga gaacatcatg gcttgcgcta agtctccact ggagaacact 1500
tggtgggctg agcaagattc tccgttctgc ttccttgcgt tctgctttga gtacgctggg 1560
gtacagcacc acggcctgag ctataactgc tcccttccgc tggcgtttga cgggtcttgc 1620
tctggcatcc agcacttctc cgcgatgctc cgagatgagg taggtggtcg cgcggttaac 1680
ttgcttccta gtgaaaccgt tcaggacatc tacgggattg ttgctaagaa agtcaacgag 1740
attctacaag cagacgcaat caatgggacc gataacgaag tagttaccgt gaccgatgag 1800
aacactggtg aaatctctga gaaagtcaag ctgggcacta aggcactggc tggtcaatgg 1860
ctggcttacg gtgttactcg cagtgtgact aagcgttcag tcatgacgct ggcttacggg 1920
tccaaagagt tcggcttccg tcaacaagtg ctggaagata ccattcagcc agctattgat 1980
tccggcaagg gtctgatgtt cactcagccg aatcaggctg ctggatacat ggctaagctg 2040
atttgggaat ctgtgagcgt gacggtggta gctgcggttg aagcaatgaa ctggcttaag 2100
tctgctgcta agctgctggc tgctgaggtc aaagataaga agactggaga gattcttcgc 2160
aagcgttgcg ctgtgcattg ggtaactcct gatggtttcc ctgtgtggca ggaatacaag 2220
aagcctattc agacgcgctt gaacctgatg ttcctcggtc agttccgctt acagcctacc 2280
attaacacca acaaagatag cgagattgat gcacacaaac aggagtctgg tatcgctcct 2340
aactttgtac acagccaaga cggtagccac cttcgtaaga ctgtagtgtg ggcacacgag 2400
aagtacggaa tcgaatcttt tgcactgatt cacgactcct tcggtaccat tccggctgac 2460
gctgcgaacc tgttcaaagc agtgcgcgaa actatggttg acacatatga gtcttgtgat 2520
gtactggctg atttctacga ccagttcgct gaccagttgc acgagtctca attggacaaa 2580
atgccagcac ttccggctaa aggtaacttg aacctccgtg acatcttaga gtcggacttc 2640
gcgttcgcgt aa 2652
<210> 14
<211> 720
<212> DNA
<213> Artificial Sequence
<400> 14
atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60
ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120
ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180
ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240
cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300
ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360
gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420
aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480
ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540
gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600
tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660
ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtga 720

Claims (9)

1. A recombinant virus which is 1) or 2) below:
1) replacing glycoprotein G of vesicular stomatitis virus with chimeric envelope protein S to obtain virus; the chimeric envelope protein S is obtained by replacing the RBD of the SARS-CoV envelope protein S with the RBD of the SARS-CoV-2 envelope protein S;
2) replacing glycoprotein G of vesicular stomatitis virus with chimeric envelope protein S to obtain virus; the chimeric envelope protein S is obtained by replacing the RBD of the SARS-CoV envelope protein S with the RBD of the SARS-CoV-2 envelope protein S and deleting the 1261-1279 th site of the SARS-CoV envelope protein S;
the amino acid sequence of the RBD of the SARS-CoV envelope protein S is 315-536 of the amino acid sequence of the SARS-CoV envelope protein S;
the amino acid sequence of the RBD of the SARS-CoV-2 cyst membrane protein S is 319-541 th position of the amino acid sequence of the SARS-CoV-2 cyst membrane protein S.
2. The recombinant virus of claim 1, wherein: the amino acid sequence of the chimeric envelope protein S is shown as a sequence 2.
3. The recombinant virus of claim 1, wherein: the recombinant virus is obtained by replacing the coding gene sequence of glycoprotein G in the genome sequence of the vesicular stomatitis virus with the coding gene sequence of chimeric envelope protein S; the coding gene sequence of the chimeric envelope protein S is a sequence 1 in a sequence table.
4. A recombinant virus is obtained by transfecting a recombinant virus vector into a virus packaging cell and then carrying out cell culture; the recombinant viral vector is A) or B) as follows:
A) replacing the coding gene sequence of glycoprotein G in the genome sequence of the vesicular stomatitis virus in the vesicular stomatitis virus vector with the coding gene sequence of the chimeric envelope protein S to obtain a vector;
B) a vector obtained by inserting a reporter gene sequence into the recombinant virus vector of A);
the amino acid sequence of the chimeric envelope protein S is shown as a sequence 2.
5. The recombinant virus of claim 4, wherein: the coding gene sequence of the chimeric envelope protein S is a sequence 1 in a sequence table.
6. The recombinant virus of any one of claims 1-5, wherein: the SARS-CoV-2 is SARS-CoV-2Wuhan-Hu-1 strain;
or, the SARS-CoV is SARS-CoV BJ01 strain;
or, the vesicular stomatitis virus is a vesicular stomatitis virus Indiana strain.
7. A recombinant viral vector as described in claim 4.
8. Use of the recombinant virus of any one of claims 1 to 6 or the recombinant viral vector of claim 7 in any one of the following X1) -X3):
x1) preparing a novel coronavirus vaccine;
x2) for the preparation of a product for the prevention and/or treatment of diseases caused by novel coronaviruses;
x3) screening for novel coronavirus invasion inhibitors;
the novel coronavirus is SARS-CoV-2;
the disease caused by the novel coronavirus is COVID-19 caused by the novel coronavirus.
9. A product for preventing and/or treating diseases caused by a novel coronavirus, which comprises the recombinant virus according to any one of claims 1 to 6 or the recombinant viral vector according to claim 7 as an active ingredient; the novel coronavirus is SARS-CoV-2;
or, the disease caused by the novel coronavirus is COVID-19 caused by the novel coronavirus.
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