CN1475571A - Expression and application of a recombinant SARS virus gene in Hansenula polymorpha - Google Patents

Abstract

The present invention belongs to the field of protein expression genetic engineering, specifically using Hansenula as a cell factory to express the product of exogenous genes. Since SARS virus and Hansenula are far in evolutionary relationship, SARS viruses S, S1, S2 and The codon usage of the main antigenic epitope gene in the process of protein translation is very different from that of Hansenula, which leads to an abnormal pause in translation. In the industrial production process of SARS virus S, S1, S2 genes and main antigen epitope gene expression products, the present invention redesigns SARS virus S, S1, S2 genes and main antigens according to the codon usage of Hansenula highly expressed genes epitope gene. The highly expressed product can be used as a vaccine to prevent atypical pneumonia caused by SARS virus. The gene designed in the present invention can also be used for fusion expression with the cholera toxin B subunit gene (patent application number: 03110441.X) for mucosal immunity in Hansenula yeast, and the expression product can be used as an oral vaccine.

Description

Expression and application of a recombinant SARS virus gene in Hansenula polymorpha

technical field

The invention belongs to the field of protein expression genetic engineering, and in particular relates to the use of SARS virus recombinant S, S1, S2 and major epitope genes and the use of Hansenula polymorpha containing these genes as a cell factory for high-efficiency expression As well as the application of the expression product in the development of a vaccine for preventing SARS virus.

Background technique

The SARS virus is a newly emerged coronavirus that causes severe acute respiratory syndrome (Severe Acute Respiratory Syndrome, SARS) in humans and is the culprit of atypical pneumonia. The viral genome is single-stranded positive-sense RNA. The RNA is about 30Kb in full length, the largest among all RNA viruses, and is infectious. The pathogenesis of SARS virus is still unclear. It has a wide range of natural hosts, can survive in the air and feces for a long time, and is mainly transmitted through the respiratory system. Anaphylaxis may occur with chronic infection; after acute infection, persistent infection may also occur. There is currently no effective vaccine to prevent it. Like other coronaviruses, SARS virus is a typical enveloped virus, and many glycosylated Spike proteins, namely S protein, are exposed outside its envelope. The S protein is encoded by the S gene, which is composed of two proteins of approximately the same size, S1 and S2, which directly bind to the host cell receptors, cause cell fusion through the conformational changes of S1 and S2, and induce the production of neutralizing antibodies and cells immunity. In other coronaviruses, the S protein is the main site of virus neutralizing antigens, contains many antigenic epitopes, and induces neutralizing antibodies and fusion membrane reactions. This suggests that we can use the S gene to design a SARS virus vaccine.

The inventors used DNAStar, VectorNTI and other software and their accompanying online analysis tools to predict and analyze SARS virus isolates in the United States, Canada, Hong Kong, Beijing, Guangzhou and other places from nucleotide sequence to protein structure. It is found that the Spike protein difference of all SARS virus isolates is only pure on S1, and S2 is highly conserved, and the difference of all SARS virus isolate proteins is only pure on S1, S2 is highly conserved, and the S2 of all current SARS virus isolates The genes are all the same. The full length of the S gene is 3768bp, encoding 1255 amino acids (AA). Among them, 1-16AA is the signal peptide, 66-609AA is the S1 protein region, and 641-1247AA is the S2 protein region. In the S2 protein, the potential glycosylation sites are mainly concentrated in the 787-1221AA region, which is very similar to the fusion protein (Fusion) of paramyxovirus. At the 1148-1236AA position of the S2 protein, there is a special coiled coil structure (1149-1188AA), a leucine zipper (1148-1182AA) and a Cys-rich structure (1217-1236AA). The inventors speculate that this region may be in The SARS virus plays a very critical role when it fuses with the host cell. For this reason, the inventor designed a series of S genes of different lengths according to the codon usage table of highly expressed genes of Hansenula polymorpha (Patent Application No.: 03110441.X) to express efficiently in Hansenula polymorpha, according to these The blocking effect of the antibody produced by the gene fragment on the SARS virus will determine the best plan for the development of therapeutic and preventive vaccines for atypical pneumonia.

Contents of the invention

Hansenula polymorpha (Hansenula polymorpha) belongs to methanol yeast, also known as Pichiaaugusta. Its optimum growth temperature is high, and its growth rate is fast, which is conducive to large-scale fermentation production, and can efficiently express many genes that are difficult to express efficiently in other systems. Because Hansenula polymorpha can step-by-step integrate multiple genes according to a certain gene dosage ratio, the recombinant bacteria can express the required genes according to the optimal ratio, which has not been reported in other methanolic yeasts. In addition, Hansenula polymorpha has the advantages of simple genetic manipulation, high copy number of exogenous gene, high yield of exogenous protein, and easy industrial production. Yeast, etc.) exogenous gene expression system has been widely concerned. Especially in secretory expression, exogenous proteins can complete post-translational processes such as proteolytic maturation, glycosylation modification, and disulfide bond formation through the secretory pathway, making the expressed protein closer to the natural protein form with biological activity , and avoid over-glycosylation in brewer's yeast. However, the SARS virus has a distant evolutionary relationship with yeast, and its codon usage is very different from that of Hansenula during protein translation, resulting in an abnormal pause in translation. So far, there has been no report of using the Hansenula system to express any gene of the SARS virus. When we use Hansenula polymorpha AS 2.2412 (provided by CGMCC, General Microorganism Center of China Microbiological Culture Collection Management Committee) to express SARS virus S, S1, S2 genes and their main epitope genes, according to the high expression gene of Hansenula polymorpha The codon usage of these genes was redesigned, and the expression was greatly improved.

The present invention has been accomplished based on such a discovery. Therefore, the object of the present invention is to provide a kind of recombinant SARS virus S, S1, S2 gene and main epitope gene thereof and utilize the Hansenula polymorpha that contains this gene to carry out efficient expression as cell factory, and utilize these expression products in Application in preparation of SARS virus genetic engineering vaccine. Hansenula polymorpha recombinant SARS virus S, S1, S1-1, S1-2, S1-3, S2, S2-1, S2-2, S2-3, S2-4, S2-5 gene of the present invention The nucleotide sequences correspond to the nucleotide sequences shown in SEQ ID NO: 1-11, respectively, and the encoded amino acid sequences correspond to the amino acid sequences shown in SEQ ID NO: 12-22. To achieve, the technical route of the present invention is to utilize the codon usage table of highly expressed genes of Hansenula polymorpha (as shown in Figure 1, Chinese patent application number: 03110441.X). According to the nucleotide sequence (as shown in Figure 2, full-length 3768bp) of the S gene (accession number: AY278554, CUHK-W1 strain) of SARS virus coding reported on Genbank, the aminoacid sequence of its coding is as SEQ ID NO: 12 shown (full length 1255 amino acids). According to the Vector NTI 8.0 software and its accompanying online programs such as SMART (Simple Modular Architecture Research Tool), the structural domain composition, protein secondary structure, transmembrane helix, glycosylation site, and antigenic epitope were analyzed. According to the analysis results, the inventor divided the encoded protein of SARS virus S gene into S (1-1255AA), S1 (17-640AA), S1-1 (66-609AA), S1-2 (17-232AA), S1-3 (258-572AA), S2(641-1247AA), S2-1(641-856AA), S2-2(883-1197AA), S2-3(1149-1183AA), S2-4(1149-1236AA), S2 -5 (787-1188AA) has a total of 11 areas. S(1-1255AA), S1(17-640AA), S1-1(66-609AA), S1-2(17-232AA), S1-3(258- 572AA), S2(641-1247AA), S2-1(641-856AA), S2-2(883-1197AA), S2-3(1149-1183AA), S2-4(1149-1236AA), S2-5( 787-1188AA) region corresponding to the amino acid sequence is converted into a nucleotide sequence, that is, the coding gene designed according to the codon usage of the highly expressed gene of Hansenula yeast is obtained, corresponding to the nucleotides shown in SEQ ID NO: 1-11 respectively sequence. These genes (SEQ ID NO: 1-11) are 100% homologous to the amino acids encoded by the corresponding encoding genes of SARS virus, and the amino acid sequences encoded by them are shown in (SEQ ID NO: 12-22) respectively, as for the nucleoside The acid sequence identity is at least 75%. Considering the swing of individual synonymous codon preferences of highly expressed genes of Hansenula spp. There are 5 amino acid differences in the encoded protein, and the S2 gene is completely consistent. Therefore, the nucleotides of optimized recombinant SARS virus S, S1, S1-1, S1-2, S1-3, S2, S2-1, S2-2, S2-3, S2-4, S2-5 genes Sequences, except for the nucleotide sequences shown in SEQ ID NO: 1-11, due to the deletion and increase of nucleotide sequence mutations, nucleotide sequences encoding proteins with the same function are still produced. It should be pointed out that these sequences correspond to the above-mentioned The nucleotide sequences shown in SEQ ID NO: 1-11 have at least 75% homology. The above-mentioned nucleotide sequences having at least 75% homology also relate to one of the objectives of the present invention. The SARS virus S, S1, S2 genes and their main antigen epitope genes designed according to the codon usage of Hansenula highly expressed genes obtained by the present invention overcome the incompatibility in the Hansenula expression system, thereby making these genes Highly expressed in Hansenula. The SARS virus S, S1, S2 genes and their main epitope genes expressed in Hansenula are safer and more reliable, and have no potential tumorigenicity compared with mammalian cell expression products. After purification, it can be directly used clinically to prevent "atypical pneumonia" caused by SARS virus, or it can be fused with the coding gene of cholera toxin B subunit (Chinese patent application number: 03110441.X) to improve vaccine immunity through mucosal immunity. Effect.

Another object of the present invention is to provide a method for preparing SARS virus S, S1, S1-1, S1-2, S1-3, S2, S2-1, S2-2, S2 using the codon usage of Hansenula highly expressed genes -3. A method for encoding proteins by S2-4 and S2-5 genes, the method involving the following steps:

1. According to the codon usage (Fig. 1) of the highly expressed gene of Hansenula polymorpha, optimize the design of SARS virus S, S1, S1-1, S1-2, S1-3, S2, S2-1, S2-2, S2-3, S2-4, S2-5 genes;

2. Artificially synthesize the newly designed coding gene through the gene synthesizer;

3. Using the Hansenula polymorpha expression vectors pHMOXZ-A (intracellular expression), pHFMDHZ-A (intracellular expression), pHMOXZα-A (secretory expression), pHFMDHZα in the Chinese patent (application number: 03110441.X) -A (secretory expression) or any other eukaryotic expression vector that can be integrated and expressed in Hansenula, construct the optimized SARS virus S, S1, S1-1, S1-2, S1-3, S2, S2 -1, recombinant expression vectors of S2-2, S2-3, S2-4, S2-5 genes;

4. Hansenula polymorpha recombinant SARS virus S, S1, S1-1, S1-2, S1-3, S2, S2-1, S2-2, S2-3, S2-4, S2-5 gene encoded protein Induced expression;

5. Expression product identification and biological activity identification.

Obtain Hansenula polymorpha high-efficiency expression containing SARS virus S, S1, S1-1, S1-2, S1-3, S2, S2-1, S2-2, S2-3, S2-4 according to the above method steps , the recombinant of S2-5 gene, prepare SARS virus S, S1, S1-1, S1-2, S1-3, S2, S2-1, S2-2, S2-3, S2-4, S2-5 gene Encoding proteins is within the reach of those skilled in the art.

So far, there are no reports about the expression of SARS virus S, S1, S2 genes and their main epitope genes in Hansenula at home and abroad. The SARS virus S, S1, S1-1, S1-2, S1-3, S2, S2-1, S2-2, S2-3, S2-4, S2-5 gene of the optimized design of the present invention in Hansenula The expression is superior to other expression systems regardless of the expression biomass and extraction preparation steps, so that the recombinant SARS virus S, S1, S1-1, S1-2, S1-3, S2, S2-1, S2-2, S2-3 , S2-4, S2-5 gene-encoded protein industrial production and application has become a reality.

Description of drawings

为本发明中优化设计S、S1、S1-1、S1-2、S1-3、S2、S2-1、S2-2、S2-3、S2-4、S2-5基因时选用的密码子Figure 1: Comparison of synonymous codon usage between highly expressed genes and low expressed genes in Hansenula. High: Highly expressed genes; Low: Low expressed genes. N is the number of codons for each amino acid when the Codon W program classifies high and low expression genes. RSCU (Relative synonymous codon usage, relative synonymous codon usage), RSCU reflects the usage of each synonymous codon in a gene, and its value is equal to the actual observed value of a certain codon in the gene, which is the same as that of each codon The ratio between the expected values when the frequency of the frequency appears; the introduction of the RSCU value can make the codon usage comparison between databases composed of different amino acids. ^*@ indicates the superior codon corresponding to the highly expressed gene, where ^* means the difference is extremely significant after the chi-square test; ^@ means the difference is significant after the chi-square test.

Codons selected for optimal design of S, S1, S1-1, S1-2, S1-3, S2, S2-1, S2-2, S2-3, S2-4, S2-5 genes in the present invention

Figure 2: The nucleotide sequence of the encoded S gene (accession number: AY278554, CUHK-W1 strain) of the SARS virus reported on Genbank

Detailed ways

The following examples can make those skilled in the technical field understand the present invention more comprehensively, but do not limit the present invention in any way. The design of embodiment 1S, S1, S1-1, S1-2, S1-3, S2, S2-1, S2-2, S2-3, S2-4, S2-5 gene

1. Embed the H. polymorpha highly expressed gene codon usage table (Chinese patent application number: 03110441.X) described in Figure 1 into the DNAStar program

2. According to the positions corresponding to the encoded amino acids of the SARS virus S gene (accession number: AY278554, CUHK-W1 strain) reported on Genbank, S, S1, S1-1, S1-2, S1-3, S2, S2-1, S2-2, S2-3, S2-4, S2-5 gene encoding corresponding amino acid sequence S(1-1255AA), S1(17-640AA), S1-1(66-609AA), S1- 2(17-232AA), S1-3(258-572AA), S2(641-1247AA), S2-1(641-856AA), S2-2(883-1197AA), S2-3(1149-1183AA), S2-4(1149-1236AA), S2-5(787-1188AA); that is, SEQ ID NO: 12-22 were converted into corresponding nucleotide sequences respectively, which were designed according to the codon usage of highly expressed genes of Hansenula yeast The coding genes are shown in SEQ ID NO: 1-11 respectively.

3. Artificially synthesize the newly designed coding gene through the gene synthesizer

Construction, transformation and screening of embodiment 2 recombinant expression vector

1. Construction of recombinant expression vectors: using Hansenula polymorpha expression vectors pHMOXZ-A (intracellular expression), pHFMDHZ-A (intracellular expression), pHMOXZα-A (secretory expression), pHFMDHZα-A (secretory expression ) or any other eukaryotic expression vectors that can be integrated and expressed in Hansenula, construct optimized SARS virus S, S1, S1-1, S1-2, S1-3, S2, S2-1, S2-2 , S2-3, S2-4, S2-5 gene recombinant expression vector. Any of the above recombinant expression vectors can be highly expressed in Hansenula polymorpha.

2. Transformation and screening of recombinant expression vectors: Transformation of Hansenula polymorpha by electroporation transformation method. Pick a single clone of Hansenula polymorpha NCYC495 in 5ml YPD liquid medium, culture overnight at 37°C

3. Take 2ml of the bacterial liquid and add it to 200ml of pre-warmed YPD, and cultivate it at 37°C until OD600=1.2-1.5 (about 6h)

4. Centrifuge at 6000 rpm for 5 minutes at room temperature, discard the supernatant

5. Suspend cells with 500ml TED (100mM Tris-HCl; 50mM EDTA; 25mM DTT; pH=8.0)

6. Shaker at 37°C, shake at 200rpm for 15min

Centrifuge at 6000rpm×5min at 7.4°C, discard the supernatant

8. Gently suspend the cells with 200ml pre-cooled 270mM sucrose

Centrifuge at 6000rpm×5min at 9.4°C, discard the supernatant

10. Gently suspend the cells with 100ml pre-cooled 270mM sucrose

Centrifuge at 6000rpm×5min at 11.4°C, discard the supernatant

12. Gently suspend the cells with 1ml of pre-cooled 270mM sucrose, aliquot into 60ul/tube, and store in liquid nitrogen or in a refrigerator below -80°C for later use.

13. Add 5ul of plasmid DNA (about 100-500ng) to 60ul of competent cells, mix gently and add to an electric shock cup with a pore size of 2mm

14. Electric shock parameters: 50uF, 100Ω, 1.5KV

15. Immediately after the electric shock, add 940ul of YPDTM (1% yeast extract; 1% peptone; 2% glucose; 1mM Tris-HCl; 1mM MgCl ₂ ), suck it into a small tube of 2-5ml, shake it at 37°C at 200rpm 1h

16. Take 100ul of YPD plates coated with Zeocin antibiotics (final concentration 100ug/ml), and culture them at 37°C for 2 days. Colonies of large, medium and small forms appear on the plates. Pick clones and identify recombinants by PCR, and use Southern hybridization to identify copies number

17. Transformants were grown and screened on YPD (1% yeast extract, 1% peptone, 2% glucose) plates coated with Zeocin antibiotics. If other vectors that can be integrated and expressed in Hansenula are used, please refer to the manual of the vector. Extract the DNA of large colony yeast and use PCR method to detect whether there is recombinant insertion. Pick the correct clones identified by PCR for fermentation

Example 3 Fermentation and induced expression

After the positive recombinant yeast clones containing multiple copies of the gene were cultured in YPD medium at 37°C for 12 hours, they were inoculated into fresh YPD fermentation medium (containing 1.5% glycerol) at a ratio of 1:20, and the fermentation temperature was maintained at 30-37°C. ℃, pH3-5, dissolved oxygen 20%, air flow rate 5-10L/min. After 24 hours, when the _O2 pressure increased sharply (prompting that the glycerol in the medium had been consumed), start feeding (YPD containing 50% glycerol), strictly control the feed rate, so that the final concentration of glycerol in the fermentation broth was maintained at 0.05 %-0.4%. After 24-48 hours of fermentation, it can be harvested. For the fermentation broth with intracellular expression, the cells were harvested by centrifugation at 12,000 rpm for 2 min at 4°C; for the fermentation broth with secretory expression, the supernatant was harvested by centrifugation at 4°C. Perform SDS-PAGE and Western-blotting analysis. In addition, in addition to using glycerol to induce fermentation, the Hansenula polymorpha expression system can also use 1% methanol and 1% glucose to induce fermentation.

The biological activity identification of embodiment 4 expression product

After purifying the expression products of S, S1, S1-1, S1-2, S1-3, S2, S2-1, S2-2, S2-3, S2-4, S2-5 genes, immunize mice, Separate serum. Virus blocking tests are carried out in laboratories above P3 level. In Vero cells infected with SARS virus, the vaccine corresponding to the serum that can block or inhibit virus replication is an effective vaccine.

Sequence Listing <110> Institute of Microbiology, Chinese Academy of Sciences <120> A kind of Hansenula polymorpha expressing Severe Acute Respiratory Syndrome (SARS) virus recombinant S, S1, S2 genes and main epitope genes and its application <141 >2003-05-20<160>22<170>PatentIn version 3.2<210>1<211>3768<212>DNA<213>Artificial Sequence<220><223>according to Hansenula Polymorpha high表达基因密码子用法设计<400>1atgttcatct tcttgttgtt cttgaccttg acctccggtt ccgacttgga cagatgtacc     60accttcgacg acgtccaagc tccaaactac acccaacaca cctcctccat gagaggtgtc    120tactacccag acgagatctt cagatccgac accttgtact tgacccaaga cttgttcttg    180ccattctact ccaacgtcac cggtttccac accatcaacc acaccttcga caacccagtc    240atcccattca aggacggtat ctacttcgct gctaccgaga agtccaacgt cgtcagaggt    300tgggtcttcg gttccaccat gaacaacaag tcccaatccg tcatcatcat caacaactcc    360accaacgtcg tcatcagagc ttgtaacttc gagttgtgtg acaacccatt 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gctgaggtcc aaatcgacag attgatcacc   2940ggtagattgc aatccttgca aacctacgtc acccaacaat tgatcagagc tgctgagatc   3000agagcttccg ctaacttggc tgctaccaag atgtccgagt gtgtcttggg tcaatccaag   3060agagtcgact tctgtggtaa gggttaccac ttgatgtcct tcccacaagc tgctccacac   3120ggtgtcgtct tcttgcacgt cacctacgtc ccatcccaag agagaaactt caccaccgct   3180ccagctatct gtcacgaggg taaggcttac ttcccaagag agggtgtctt cgtcttcaac   3240ggtacctcct ggttcatcac ccaaagaaac ttcttctccc cacaaatcat caccaccgac   3300aacaccttcg tctccggtaa ctgtgacgtc gtcatcggta tcatcaacaa caccgtctac   3360gacccattgc aaccagagtt ggactccttc aaggaggagt tggacaagta cttcaagaac   3420cacacctccc cagacgtcga cttgggtgac atctccggta tcaacgcttc cgtcgtcaac   3480atccaaaagg agatcgacag attgaacgag gtcgctaaga acttgaacga gtccttgatc   3540gacttgcaag agttgggtaa gtacgagcaa tacatcaagt ggccatggta cgtctggttg   3600ggtttcatcg ctggtttgat cgctatcgtc atggtcacca tcttgttgtg ttgtatgacc   3660tcctgttgtt cctgtttgaa gggtgcttgt tcctgtggtt cctgttgtaa gttcgacgag   3720gacgactccg agccagtctt gaagggtgtc aagttgcact acacctga                3768<210>2<211>1872<212>DNA< 213>Artificial Sequence<220><223>根据多形汉逊酵母(Hansenula Polymorpha)高表达基因密码子用法设计<400>2gacagatgta ccaccttcga cgacgtccaa gctccaaact acacccaaca cacctcctcc     60atgagaggtg tctactaccc agacgagatt ttcagatccg acaccttgta cttgacccaa    120gacttgttct tgccattcta ctccaacgtc accggtttcc acaccatcaa ccacaccttc    180gacaacccag tcatcccatt caaggacggt atctacttcg ctgctaccga gaagtccaac    240gtcgtcagag gttgggtctt cggttccacc atgaacaaca agtcccaatc cgtcatcatc    300atcaacaact ccaccaacgt cgtcatcaga gcttgtaact tcgagttgtg tgacaaccca    360ttcttcgctg tctccaagcc aatgggtacc caaacccaca ccatgatctt cgacaacgct    420ttcaactgta ccttcgagta catctccgac gctttctcct tggacgtctc cgagaagtcc    480ggtaacttca agcacttgag agagttcgtc ttcaagaaca aggacggttt cttgtacgtc    540tacaagggtt accaaccaat cgacgtcgtc agagacttgc catccggttt caacaccttg    600aagccaatct tcaagttgcc attgggtatc aacatcacca acttcagagc tatcttgacc    660gctttctccc cagctcaaga cacctggggt acctccgctg ctgcttactt cgtcggttac    720ttgaagccaa ccaccttcat gttgaagtac gacgagaacg gtaccatcac cgacgctgtc    780gactgttccc aaaacccatt ggctgagttg aagtgttccg tcaagtcctt cgagatcgac    840aagggtatct accaaacctc caacttcaga gtcgtcccat ccggtgacgt cgtcagattc    900ccaaacatca ccaacttgtg tccattgggt gaggtcttca acgctaccaa gttcccatcc    960gtctacgctt gggagagaaa gaagatctcc aactgtgtcg ctgactactc cgtcttgtac   1020aactccacct tcttctccac cttcaagtgt tacggtgtct ccgctaccaa gttgaacgac   1080ttgtgtttct ccaacgtcta cgctgactcc ttcgtcgtca agggtgacga cgtcagacaa   1140atcgctccag gtccaaccgg tgtcatcgct gactacaact acaagttgcc agacgacttc 1200atgggttgtg tcttggcttg gaacaccaga aacatcgacg ctacctccac cggtaactac   1260aactacaagt acagatactt gagacacggt aagttgagac cattcgagag agacatctcc   1320aacgtcccat tctccccaga cggtaagcca tgtaccccac cagctttgaa ctgttactgg   1380ccattgaacg actacggttt ctacaccacc accggtatcg gttaccaacc atacagagtc   1440gtcgtcttgt ccttcgagtt gttgaacgct ccagctaccg tctgtggtcc aaagttgtcc   1500accgacttga tcaagaacca atgtgtcaac ttcaacttca acggtttgac cggtaccggt   1560gtcttgaccc catcctccaa gagattccaa ccattccaac aattcggtag agacgtctcc   1620gacttcaccg actccgtcag agacccaaag acctccgaga ttttggacat ctccccatgt   1680tccttcggtg gtgtctccgt catcacccca ggtaccaacg cttcctccga ggtcgctgtc   1740ttgtaccaag acgtcaactg taccgacgtc tccaccgcta tccacgctga ccaattgacc   1800ccagcttgga gaatctactc caccggtaac aacgtcttcc aaacccaagc tggttgtttg   1860atcggtgctg ag                                                       1872<210>3<211>1632<212>DNA<213>Artificial Sequence<220><223>根据多形汉逊酵母(Hansenula Polymorpha)高表达基因密码子用法设计<400>3gtcaccggtt tccacaccat caaccacacc ttcgacaacc cagtcatccc attcaaggac     60ggtatctact tcgctgctac cgagaagtcc aacgtcgtca gaggttgggt cttcggttcc    120accatgaaca acaagtccca atccgtcatc atcatcaaca actccaccaa cgtcgtcatc    180agagcttgta acttcgagtt gtgtgacaac ccattcttcg ctgtctccaa gccaatgggt    240acccaaaccc acaccatgat cttcgacaac gctttcaact gtaccttcga gtacatctcc    300gacgctttct ccttggacgt ctccgagaag tccggtaact tcaagcactt gagagagttc    360gtcttcaaga acaaggacgg tttcttgtac gtctacaagg gttaccaacc aatcgacgtc    420gtcagagact tgccatccgg tttcaacacc ttgaagccaa tcttcaagtt gccattgggt    480atcaacatca ccaacttcag agctatcttg accgctttct ccccagctca agacacctgg    540ggtacctccg ctgctgctta cttcgtcggt tacttgaagc caaccacctt catgttgaag    600tacgacgaga acggtaccat caccgacgct gtcgactgtt cccaaaaccc attggctgag    660ttgaagtgtt ccgtcaagtc cttcgagatc gacaagggta tctaccaaac ctccaacttc    720agagtcgtcc catccggtga cgtcgtcaga ttcccaaaca tcaccaactt gtgtccattg    780ggtgaggtct tcaacgctac caagttccca tccgtctacg cttgggagag aaagaagatc    840tccaactgtg tcgctgacta ctccgtcttg tacaactcca ccttcttctc caccttcaag    900tgttacggtg tctccgctac caagttgaac gacttgtgtt tctccaacgt ctacgctgac    960tccttcgtcg tcaagggtga cgacgtcaga caaatcgctc caggtccaac cggtgtcatc   1020gctgactaca actacaagtt gccagacgac ttcatgggtt gtgtcttggc ttggaacacc   1080agaaacatcg acgctacctc caccggtaac tacaactaca agtacagata cttgagacac   1140ggtaagttga gaccattcga gagagacatc tccaacgtcc cattctcccc agacggtaag   1200ccatgtaccc caccagcttt gaactgttac tggccattga acgactacgg tttctacacc   1260accaccggta tcggttacca accatacaga gtcgtcgtct tgtccttcga gttgttgaac   1320gctccagcta ccgtctgtgg tccaaagttg tccaccgact tgatcaagaa ccaatgtgtc 1380aacttcaact tcaacggttt gaccggtacc ggtgtcttga ccccatcctc caagagattc   1440caaccattcc aacaattcgg tagagacgtc tccgacttca ccgactccgt cagagaccca   1500aagacctccg agattttgga catctcccca tgttccttcg gtggtgtctc cgtcatcacc   1560ccaggtacca acgcttcctc cgaggtcgct gtcttgtacc aagacgtcaa ctgtaccgac   1620gtctccaccg ct                                                       1632<210>4<211>648<212>DNA<213>Artificial Sequence<220><223>根据多形汉逊酵母(Hansenula Polymorpha)高表达基因密码子用法设计<400>4gacagatgta ccaccttcga cgacgtccaa gctccaaact acacccaaca cacctcctcc     60atgagaggtg tctactaccc agacgagatt ttcagatccg acaccttgta cttgacccaa    120gacttgttct tgccattcta ctccaacgtc accggtttcc acaccatcaa ccacaccttc    180gacaacccag tcatcccatt caaggacggt atctacttcg ctgctaccga gaagtccaac    240gtcgtcagag gttgggtctt cggttccacc atgaacaaca agtcccaatc cgtcatcatc 300atcaacaact ccaccaacgt cgtcatcaga gcttgtaact tcgagttgtg tgacaaccca    360ttcttcgctg tctccaagcc aatgggtacc caaacccaca ccatgatctt cgacaacgct    420ttcaactgta ccttcgagta catctccgac gctttctcct tggacgtctc cgagaagtcc    480ggtaacttca agcacttgag agagttcgtc ttcaagaaca aggacggttt cttgtacgtc    540tacaagggtt accaaccaat cgacgtcgtc agagacttgc catccggttt caacaccttg    600aagccaatct tcaagttgcc attgggtatc aacatcacca acttcaga                 648<210>5<211>945<212>DNA<213 >Artificial Sequence<220><223>根据多形汉逊酵母(Hansenula Polymorpha)高表达基因密码子用法设计<400>5aagccaacca ccttcatgtt gaagtacgac gagaacggta ccatcaccga cgctgtcgac     60tgttcccaaa acccattggc tgagttgaag tgttccgtca agtccttcga gatcgacaag    120ggtatctacc aaacctccaa cttcagagtc gtcccatccg gtgacgtcgt cagattccca    180aacatcacca acttgtgtcc attgggtgag gtcttcaacg ctaccaagtt cccatccgtc    240tacgcttggg agagaaagaa gatctccaac tgtgtcgctg actactccgt cttgtacaac    300tccaccttct tctccacctt caagtgttac ggtgtctccg ctaccaagtt gaacgacttg    360tgtttctcca acgtctacgc tgactccttc gtcgtcaagg gtgacgacgt cagacaaatc    420gctccaggtc caaccggtgt catcgctgac tacaactaca agttgccaga cgacttcatg    480ggttgtgtct tggcttggaa caccagaaac atcgacgcta cctccaccgg taactacaac    540tacaagtaca gatacttgag acacggtaag ttgagaccat tcgagagaga catctccaac    600gtcccattct ccccagacgg taagccatgt accccaccag ctttgaactg ttactggcca    660ttgaacgact acggtttcta caccaccacc ggtatcggtt accaaccata cagagtcgtc    720gtcttgtcct tcgagttgtt gaacgctcca gctaccgtct gtggtccaaa gttgtccacc    780gacttgatca agaaccaatg tgtcaacttc aacttcaacg gtttgaccgg taccggtgtc    840ttgaccccat cctccaagag attccaacca ttccaacaat tcggtagaga cgtctccgac    900ttcaccgact ccgtcagaga cccaaagacc tccgagattt tggac                    945<210>6<211>1821<212>DNA<213>Artificial Sequence<220><223>根据多形汉逊酵母(Hansenula Polymorpha)高表达基因密码子用法设计<400>6cacgtcgaca cctcctacga gtgtgacatc ccaatcggtg ctggtatctg tgcttcctac     60cacaccgtct ccttgttgag atccacctcc caaaagtcca tcgtcgctta caccatgtcc    120ttgggtgctg actcctccat cgcttactcc aacaacacca tcgctatccc aaccaacttc    180tccatctcca tcaccaccga ggtcatgcca gtctccatgg ctaagacctc cgtcgactgt    240aacatgtaca tctgtggtga ctccaccgag tgtgctaact tgttgttgca atacggttcc    300ttctgtaccc aattgaacag agctttgtcc ggtatcgctg ctgagcaaga cagaaacacc    360agagaggtct tcgctcaagt caagcaaatg tacaagaccc caaccttgaa gtacttcggt    420ggtttcaact tctcccaaat cttgccagac ccattgaagc caaccaagag atccttcatc    480gaggacttgt tgttcaacaa ggtcaccttg gctgacgctg gtttcatgaa gcaatacggt    540gagtgtttgg gtgacatcaa cgctagagac ttgatctgtg ctcaaaagtt caacggtttg    600accgtcttgc caccattgtt gaccgacgac atgatcgctg cttacaccgc tgctttggtc    660tccggtaccg ctaccgctgg ttggaccttc ggtgctggtg ctgctttgca aatcccattc    720gctatgcaaa tggcttacag attcaacggt atcggtgtca cccaaaacgt cttgtacgag    780aaccaaaagc aaatcgctaa ccaattcaac aaggctatct cccaaatcca agagtccttg    840accaccacct ccaccgcttt gggtaagttg caagacgtcg tcaaccaaaa cgctcaagct    900ttgaacacct tggtcaagca attgtcctcc aacttcggtg ctatctcctc cgtcttgaac    960gacatcttgt ccagattgga caaggtcgag gctgaggtcc aaatcgacag attgatcacc   1020ggtagattgc aatccttgca aacctacgtc acccaacaat tgatcagagc tgctgagatc   1080agagcttccg ctaacttggc tgctaccaag atgtccgagt gtgtcttggg tcaatccaag   1140agagtcgact tctgtggtaa gggttaccac ttgatgtcct tcccacaagc tgctccacac   1200ggtgtcgtct tcttgcacgt cacctacgtc ccatcccaag agagaaactt caccaccgct   1260ccagctatct gtcacgaggg taaggcttac ttcccaagag agggtgtctt cgtcttcaac 1320ggtacctcct ggttcatcac ccaaagaaac ttcttctccc cacaaatcat caccaccgac   1380aacaccttcg tctccggtaa ctgtgacgtc gtcatcggta tcatcaacaa caccgtctac   1440gacccattgc aaccagagtt ggactccttc aaggaggagt tggacaagta cttcaagaac   1500cacacctccc cagacgtcga cttgggtgac atctccggta tcaacgcttc cgtcgtcaac   1560atccaaaagg agatcgacag attgaacgag gtcgctaaga acttgaacga gtccttgatc   1620gacttgcaag agttgggtaa gtacgagcaa tacatcaagt ggccatggta cgtctggttg   1680ggtttcatcg ctggtttgat cgctatcgtc atggtcacca tcttgttgtg ttgtatgacc   1740tcctgttgtt cctgtttgaa gggtgcttgt tcctgtggtt cctgttgtaa gttcgacgag   1800gacgactccg agccagtctt g                                             1821<210>7<211>648<212>DNA<213>Artificial Sequence<220><223>根据多形汉逊酵母(Hansenula Polymorpha)高表达基因密码子用法设计<400>7cacgtcgaca cctcctacga gtgtgacatc ccaatcggtg ctggtatctg tgcttcctac     60cacaccgtct ccttgttgag atccacctcc caaaagtcca tcgtcgctta caccatgtcc    120ttgggtgctg actcctccat cgcttactcc aacaacacca tcgctatccc aaccaacttc    180tccatctcca tcaccaccga ggtcatgcca gtctccatgg ctaagacctc cgtcgactgt    240aacatgtaca tctgtggtga ctccaccgag tgtgctaact tgttgttgca atacggttcc    300ttctgtaccc aattgaacag agctttgtcc ggtatcgctg ctgagcaaga cagaaacacc    360agagaggtct tcgctcaagt caagcaaatg tacaagaccc caaccttgaa gtacttcggt    420ggtttcaact tctcccaaat cttgccagac ccattgaagc caaccaagag atccttcatc    480gaggacttgt tgttcaacaa ggtcaccttg gctgacgctg gtttcatgaa gcaatacggt    540gagtgtttgg gtgacatcaa cgctagagac ttgatctgtg ctcaaaagtt caacggtttg    600accgtcttgc caccattgtt gaccgacgac atgatcgctg cttacacc                 648<210>8<211>945<212>DNA<213>Artificial Sequence<220><223>根据多形汉逊酵母(Hansenula Polymorpha)高表达基因密码子用法设计<400>8caaatggctt acagattcaa cggtatcggt gtcacccaaa acgtcttgta cgagaaccaa     60aagcaaatcg ctaaccaatt caacaaggct atctcccaaa tccaagagtc cttgaccacc    120acctccaccg ctttgggtaa gttgcaagac gtcgtcaacc aaaacgctca agctttgaac    180accttggtca agcaattgtc ctccaacttc ggtgctatct cctccgtctt gaacgacatc    240ttgtccagat tggacaaggt cgaggctgag gtccaaatcg acagattgat caccggtaga    300ttgcaatcct tgcaaaccta cgtcacccaa caattgatca gagctgctga gatcagagct    360tccgctaact tggctgctac caagatgtcc gagtgtgtct tgggtcaatc caagagagtc    420gacttctgtg gtaagggtta ccacttgatg tccttcccac aagctgctcc acacggtgtc    480gtcttcttgc acgtcaccta cgtcccatcc caagagagaa acttcaccac cgctccagct    540atctgtcacg agggtaaggc ttacttccca agagagggtg tcttcgtctt caacggtacc    600tcctggttca tcacccaaag aaacttcttc tccccacaaa tcatcaccac cgacaacacc    660ttcgtctccg gtaactgtga cgtcgtcatc ggtatcatca acaacaccgt ctacgaccca    720ttgcaaccag agttggactc cttcaaggag gagttggaca agtacttcaa gaaccacacc    780tccccagacg tcgacttggg tgacatctcc ggtatcaacg cttccgtcgt caacatccaa    840aaggagatcg acagattgaa cgaggtcgct aagaacttga acgagtcctt gatcgacttg    900caagagttgg gtaagtacga gcaatacatc aagtggccat ggtac                    945 <210>9<211>120<212>DNA<213>Artificial Sequence<220><223>Designed according to the codon usage of highly expressed genes in Hansenula Polymorpha ctaagaactt gaacgagtcc ttgatcgact tgcaagagtt gggtaagtac 120<210>10<211>264<212>DNA<213>Artificial Sequence<220><223>Designed according to the codon usage of highly expressed genes in Hansenula Polymorpha <400>10ggtgacatct ccggtatcaa cgcttccgtc gtcaacatcc aaaaggagat cgacagattg     60aacgaggtcg ctaagaactt gaacgagtcc ttgatcgact tgcaagagtt gggtaagtac    120gagcaataca tcaagtggcc atggtacgtc tggttgggtt tcatcgctgg tttgatcgct    180atcgtcatgg tcaccatctt gttgtgttgt atgacctcct gttgttcctg tttgaagggt    240gcttgttcct gtggttcctg ttgt                                           264<210>11<211>1206<212>DNA<213>Artificial Sequence<220><223>根据多形汉逊酵母(Hansenula Polymorpha)高表达基因密码子用法设计<400>11atcttgccag acccattgaa gccaaccaag agatccttca tcgaggactt gttgttcaac     60aaggtcacct tggctgacgc tggtttcatg aagcaatacg gtgagtgttt gggtgacatc    120aacgctagag acttgatctg tgctcaaaag ttcaacggtt tgaccgtctt gccaccattg    180ttgaccgacg acatgatcgc tgcttacacc gctgctttgg tctccggtac cgctaccgct    240ggttggacct tcggtgctgg tgctgctttg caaatcccat tcgctatgca aatggcttac 300agattcaacg gtatcggtgt cacccaaaac gtcttgtacg agaaccaaaa gcaaatcgct    360aaccaattca acaaggctat ctcccaaatc caagagtcct tgaccaccac ctccaccgct    420ttgggtaagt tgcaagacgt cgtcaaccaa aacgctcaag ctttgaacac cttggtcaag    480caattgtcct ccaacttcgg tgctatctcc tccgtcttga acgacatctt gtccagattg    540gacaaggtcg aggctgaggt ccaaatcgac agattgatca ccggtagatt gcaatccttg    600caaacctacg tcacccaaca attgatcaga gctgctgaga tcagagcttc cgctaacttg    660gctgctacca agatgtccga gtgtgtcttg ggtcaatcca agagagtcga cttctgtggt    720aagggttacc acttgatgtc cttcccacaa gctgctccac acggtgtcgt cttcttgcac    780gtcacctacg tcccatccca agagagaaac ttcaccaccg ctccagctat ctgtcacgag    840ggtaaggctt acttcccaag agagggtgtc ttcgtcttca acggtacctc ctggttcatc    900acccaaagaa acttcttctc cccacaaatc atcaccaccg acaacacctt cgtctccggt    960aactgtgacg tcgtcatcgg tatcatcaac aacaccgtct acgacccatt gcaaccagag   1020ttggactcct tcaaggagga gttggacaag tacttcaaga accacacctc cccagacgtc   1080gacttgggtg acatctccgg tatcaacgct tccgtcgtca acatccaaaa ggagatcgac   1140agattgaacg aggtcgctaa gaacttgaac gagtccttga tcgacttgca agagttgggt   1200aagtac                                                              1206<210>12<211>1255 <212> PRT <213> CORONAVIRUS <400> 1MET PHE ILE PHE Leu Phe Leu Thr Leu ThR Sergr Seru1 5 10 15ASP ARG CYS THR ThRs Ala Pro Asn Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr THRn THR's THR -PLN -thr THR -S that

20 25 30His Thr Ser Ser Met Arg Gly Val Tyr Tyr Pro Asp Glu Ile Phe Arg

35 40 45Ser Asp Thr Leu Tyr Leu Thr Gln Asp Leu Phe Leu Pro Phe Tyr Ser

50 55 60asn Val THR GLY PHE HIS THR ILE Asn His Thr PHE ASN Pro Val65 70 75 80ile Pro PHE LYS Asp Gly Ile Tyr Ala THR GLU LYS Sernrs Sernrner Asn

85 90 95Val Val Arg Gly Trp Val Phe Gly Ser Thr Met Asn Asn Lys Ser Gln

100 105 110Ser Val Ile Ile Ile Asn Asn Ser Thr Asn Val Val Ile Arg Ala Cys

115 120 125Asn Phe Glu Leu Cys Asp Asn Pro Phe Phe Ala Val Ser Lys Pro Met

130 135 140gly Thr Gln Thr His Thr Met Ile PHE ASN Ala Phe asn Cys THR145 150 155 160phe GLU Tyr Ile Serle Seru Seru ASP Val Glu LYS Ser.

165 170 175Gly Asn Phe Lys His Leu Arg Glu Phe Val Phe Lys Asn Lys Asp Gly

180 185 190Phe Leu Tyr Val Tyr Lys Gly Tyr Gln Pro Ile Asp Val Val Arg Asp

195 200 205Leu Pro Ser Gly Phe Asn Thr Leu Lys Pro Ile Phe Lys Leu Pro Leu

210 215 220Gly Ile asn Ile Thr Asn PHE Ala Ile Leu ThR Ala PHE Ser PRO2225 230 235 240ALA GLN ASP THR THR Sera Ala Ala Ala Tyr Phe Val Gly Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr

245 250 255Leu Lys Pro Thr Thr Phe Met Leu Lys Tyr Asp Glu Asn Gly Thr Ile

260 265 270Thr Asp Ala Val Asp Cys Ser Gln Asn Pro Leu Ala Glu Leu Lys Cys

275 280 285Ser Val Lys Ser Phe Glu Ile Asp Lys Gly Ile Tyr Gln Thr Ser Asn

290 295 300phe ARG Val Val Val Pro Serg Val Val ARG PHE PRO Asn Ile ThR30 310 320asn Leu Cys Pro PHLU VAL PHE Ala ThR LYS PHE Pro Ser

325 330 335Val Tyr Ala Trp Glu Arg Lys Lys Ile Ser Asn Cys Val Ala Asp Tyr

340 345 350Ser Val Leu Tyr Asn Ser Thr Phe Phe Ser Thr Phe Lys Cys Tyr Gly

355 360 365Val Ser Ala Thr Lys Leu Asn Asp Leu Cys Phe Ser Asn Val Tyr Ala

370 375 380ASP Ser PHE VAL VAL LYS GLY ASP Val ARG Gln Ile Ala Pro Gly385 395 400GLN Thr Gly Val Ile Ala ASN Tyr Lysp Asp Asp asp

405 410 415 Met Gly Cys Val Leu Ala Trp Asn Thr Arg Asn Ile Asp Ala Thr Ser

420 425 430Thr Gly Asn Tyr Asn Tyr Lys Tyr Arg Tyr Leu Arg His Gly Lys Leu

435 440 445Arg Pro Phe Glu Arg Asp Ile Ser Asn Val Pro Phe Ser Pro Asp Gly

450 455 460LYS Pro CYS THR Pro Pro Ala Leu Asn Cys Tyr Tyr Tyr Leu asn ASN ASN ASN ASN ASN ASN ASN ASN ASN ASN ASN ASN ASN

485 490 495Val Val Leu Ser Phe Glu Leu Leu Asn Ala Pro Ala Thr Val Cys Gly

500 505 510Pro Lys Leu Ser Thr Asp Leu Ile Lys Asn Gln Cys Val Asn Phe Asn

515 520 525Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Pro Ser Ser Lys Arg

530 535 540phe Gln Pro PHE GLN GLN GLN PHE GLY ASP Val Ser ASP PHE ThR ASP545 550 560SER Val ARG ARG ARS THR Serle Leu ASP Ile Serle Serle Serle Cyser

565 570 575Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Ala Ser Ser

580 585 590Glu Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Asp Val Ser Thr

595 600 605Ala Ile His Ala Asp Gln Leu Thr Pro Ala Trp Arg Ile Tyr Ser Thr

610 615 620Gly Asn Val Phe Gln Thr Gln Ala Gly Cys Leu Ile GLY Ala Glu625 635 640his Val ASP TYR GLU CYS ASLA GLY Ala Gly Ala Gly Ile

645 650 655Cys Ala Ser Tyr His Thr Val Ser Leu Leu Arg Ser Thr Ser Gln Lys

660 665 670Ser Ile Val Ala Tyr Thr Met Ser Leu Gly Ala Asp Ser Ser Ile Ala

675 680 685Tyr Ser Asn Asn Thr Ile Ala Ile Pro Thr Asn Phe Ser Ile Ser Ile

690 695 700thr Thr Glu Val Met Pro Val Serite Ala Lys Thr Ser Val ASP CYS7070 715 720asn Met Tyr Ile Cys GLU CYS ALA Asn Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu's M's TEU Leu'sR -

725 730 735Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Ser Gly Ile

740 745 750Ala Ala Glu Gln Asp Arg Asn Thr Arg Glu Val Phe Ala Gln Val Lys

755 760 765Gln Met Tyr Lys Thr Pro Thr Leu Lys Tyr Phe Gly Gly Phe Asn Phe

770 775 780ser Gln Ile Leu Pro ASP Pro Leu Lys Pro ThR LYS ARG Serg Serg 785 795 800GLU ASN LEU PHE ASP ALA GLY PHE MET MET

805 810 815Lys Gln Tyr Gly Glu Cys Leu Gly Asp Ile Asn Ala Arg Asp Leu Ile

820 825 830Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu Leu Thr

835 840 845Asp Asp Met Ile Ala Ala Tyr Thr Ala Ala Leu Val Ser Gly Thr Ala

850                 855                 860Thr Ala Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile Pro Phe865                 870                 875                 880Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr Gln Asn

885 890 895Val Leu Tyr Glu Asn Gln Lys Gln Ile Ala Asn Gln Phe Asn Lys Ala

900 905 910Ile Ser Gln Ile Gln Glu Ser Leu Thr Thr Thr Ser Ser Thr Ala Leu Gly

915 920 925Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn Thr Leu

930 935 940val LYS GLN Leu Sero Sern Phe Gly Ala Ile Ser Val Leu asn945 955 960asp Ile Leu Serg Leu AS Val Val Glu Val Gln Ile ASP ASP

965 970 975Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val Thr Gln

980 985 990Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu Ala Ala

995 1000 1005Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg Val Asp

1010 1015 1020Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln Ala Ala

1025 1030 1035Pro His Gly Val Val Phe Leu His Val Thr Tyr Val Pro Ser Gln

1040 1045 1050Glu Arg Asn Phe Thr Thr Ala Pro Ala Ile Cys His Glu Gly Lys

1055 1060 1065Ala Tyr Phe Pro Arg Glu Gly Val Phe Val Phe Asn Gly Thr Ser

1070 1075 1080Trp Phe Ile Thr Gln Arg Asn Phe Phe Ser Pro Gln Ile Ile Thr

1085 1090 1095Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val Val Ile Gly

1100 1105 1110Ile Ile Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro Glu Leu Asp

1115 1120 1125Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn His Thr Ser

1130 1135 1140Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn Ala Ser Val

1145 1150 1155Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu Val Ala Lys

1160 1165 1170Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu Gly Lys Tyr

1175 1180 1185Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Val Trp Leu Gly Phe Ile

1190 1195 1200Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Leu Leu Cys Cys

1205 1210 1215 Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Ala Cys Ser Cys Gly

1220 1225 1230Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro Val Leu Lys

1235 1240 1245Gly Val Lys Leu His Tyr Thr

1250 1255 <210> 1311> 624 <212> PRT <213> Coronavirus <400> 13ASP ARG CYS ThR ThR PHE ASP Val Gln Ala Pro Asn Tyr Tyr Gln1 5 10 15HIS SEHIS Serg Gly Val Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr's Asp Glu Ile Phe Arg

20 25 25 30Ser Asp Thr Leu Tyr Leu Thr Gln Asp Leu Phe Leu Pro Phe Tyr Ser

35 40 45Asn Val Thr Gly Phe His Thr Ile Asn His Thr Phe Asp Asn Pro Val

50 55 60ILE PRO PHE LYS Asp Gly Ile Tyr Phe Ala Ala THR GLU LYS Sern65 75 80VAL VAL ARG GLE VAL PHE GLY Ser THR MET Asn Lysr Gln Gln Gln Gln Gln Gln

85 90 95Ser Val Ile Ile Ile Asn Asn Ser Thr Asn Val Val Ile Arg Ala Cys

100 105 110Asn Phe Glu Leu Cys Asp Asn Pro Phe Phe Ala Val Ser Lys Pro Met

115 120 125Gly Thr Gln Thr His Thr Met Ile Phe Asn Ala Phe Asn Cys Thr

130 135 140phe Glu Tyr Ile Serite Ala Phe Ser Leu ASP Val Sergs Ser145 150 155 160160Gly As His Leu Phe Val Phe Lysn Lysn Lysn Lysn Lysn Lysn Lysn Lysn Lysn Lysn Lysn Lysn Lysp Gly Gly Gly Gly

165 170 175Phe Leu Tyr Val Tyr Lys Gly Tyr Gln Pro Ile Asp Val Val Arg Asp

180 185 190Leu Pro Ser Gly Phe Asn Thr Leu Lys Pro Ile Phe Lys Leu Pro Leu

195 200 205Gly Ile Asn Ile Thr Asn Phe Arg Ala Ile Leu Thr Ala Phe Ser Pro

210 215 220ALA GLN ASP THR TRP GLY THR Sera Ala Ala Tyr PHE Val Gly Gly Tyr2225 235 240leu LYS Pro ThR Thr PHE MET Leu LYS TYR ASN GLY Thr Ile

245 250 255Thr Asp Ala Val Asp Cys Ser Gln Asn Pro Leu Ala Glu Leu Lys Cys

260 265 270Ser Val Lys Ser Phe Glu Ile Asp Lys Gly Ile Tyr Gln Thr Ser Asn

275 280 285Phe Arg Val Val Pro Ser Gly Asp Val Val Arg Phe Pro Asn Ile Thr

290                 295                 300Asn Leu Cys Pro Leu Gly Glu Val Phe Asn Ala Thr Lys Phe Pro Ser305                 310                 315                 320Val Tyr Ala Trp Glu Arg Lys Lys Ile Ser Asn Cys Val Ala Asp Tyr

325 330 335Ser Val Leu Tyr Asn Ser Thr Phe Phe Ser Thr Phe Lys Cys Tyr Gly

340 345 350Val Ser Ala Thr Lys Leu Asn Asp Leu Cys Phe Ser Asn Val Tyr Ala

355 360 365Asp Ser Phe Val Val Lys Gly Asp Asp Val Arg Gln Ile Ala Pro Gly

370 375 380pro Thr Gly Val Ile Ala Ala asn Tyr Lys Leu Prou ASP PHE385 395 400MET GLY CYS VAL Leu Ala THR ARG Asn Ile Ala Thr Ser

405 410 415Thr Gly Asn Tyr Asn Tyr Lys Tyr Arg Tyr Leu Arg His Gly Lys Leu

420 425 430Arg Pro Phe Glu Arg Asp Ile Ser Asn Val Pro Phe Ser Pro Asp Gly

435 440 445Lys Pro Cys Thr Pro Pro Ala Leu Asn Cys Tyr Trp Pro Leu Asn Asp

450 455 460TYR GLY PHE TYR THR THR THR GLY ILE GLY TYR GLN Pro Tyr ARG Val465 475 480VAL VAL Leu Seru Leu Leu Ala Ala ThR Val Cys Gly

485 490 495Pro Lys Leu Ser Thr Asp Leu Ile Lys Asn Gln Cys Val Asn Phe Asn

500 505 510Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Pro Ser Ser Lys Arg

515 520 525Phe Gln Pro Phe Gln Gln Phe Gly Arg Asp Val Ser Asp Phe Thr Asp

530 535 540ser Val ARG ASP Pro LYS THR SERU Ileu ASP Ile Serle Serle Serle Cys545 550 560SER PHE GLY GLY VAL VR Pro Gly Thr Asn Ala Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Seer

565 570 575Glu Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Asp Val Ser Thr

580 585 590Ala Ile His Ala Asp Gln Leu Thr Pro Ala Trp Arg Ile Tyr Ser Thr

595 600 605Gly Asn Asn Val Phe Gln Thr Gln Ala Gly Cys Leu Ile Gly Ala Glu

610 615 620 <210 <211> 544 <212> PRT <213> Coronavirus <400> 14VAL THR GLY PHE HIS THR ILE Asn His THR PHE ASN Pro Val Ile1 5 PHE LYS Asp Gly Ile Tyr PHER ALA ALA ALA ALA ALA ALA ALA ALA ALA ALA ALA ALA ALA ALA Thr Glu Lys Ser Asn Val

20 25 30Val Arg Gly Trp Val Phe Gly Ser Thr Met Asn Asn Lys Ser Gln Ser

35 40 45Val Ile Ile Ile Asn Asn Ser Thr Asn Val Val Ile Arg Ala Cys Asn

50 55 60phe Glu Leu Cys ASP Asn Pro PHE ALA VAL Ser Lys Pro Met Gly65 70 75 80thr Gln Thr His Thr Met iLe Phe Ala Phe asn Cys THR PHE

85 90 95Glu Tyr Ile Ser Asp Ala Phe Ser Leu Asp Val Ser Glu Lys Ser Gly

100 105 110Asn Phe Lys His Leu Arg Glu Phe Val Phe Lys Asn Lys Asp Gly Phe

115 120 Leu Tyr Val Tyr Lys Gly Tyr Gln Pro Ile Asp Val Val Arg Asp Leu

130 135 140Pro Ser Gly Phe asn Thr Leu Lys Pro Ile PHE LYS Leu Prou Leu Gly145 150 155 160le THR Asn Phe Ala Ile Leu ThR Ala Pro Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala

165 170 175Gln Asp Thr Trp Gly Thr Ser Ala Ala Ala Tyr Phe Val Gly Tyr Leu

180 185 190Lys Pro Thr Thr Phe Met Leu Lys Tyr Asp Glu Asn Gly Thr Ile Thr

195 200 205Asp Ala Val Asp Cys Ser Gln Asn Pro Leu Ala Glu Leu Lys Cys Ser

210 215 220val LYS Serle Asp Lys Gly Ile Tyr Gln Thr Serte 2225 235 240ARG VAL VAL VAL VAL VAL VAL ARG PRO Pro Asn Ile THR ASN

245 250 255Leu Cys Pro Leu Gly Glu Val Phe Asn Ala Thr Lys Phe Pro Ser Val

260 265 270Tyr Ala Trp Glu Arg Lys Lys Ile Ser Asn Cys Val Ala Asp Tyr Ser

275 280 285Val Leu Tyr Asn Ser Thr Phe Phe Ser Thr Phe Lys Cys Tyr Gly Val

290 295 300ser Ala ThR LYS Leu asn Asp Leu Cys PHE Ser Asn Val Tyr Ala Ala Ala Ala Ala Ala asp305 320r PHE Val Val Val ASP Val ARG Gln Ile Ala GLY Pro GLY Pro GLY Pro GLY Pro

325 330 335Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Met

340 345 350Gly Cys Val Leu Ala Trp Asn Thr Arg Asn Ile Asp Ala Thr Ser Thr

355 360 365Gly Asn Tyr Asn Tyr Lys Tyr Arg Tyr Leu Arg His Gly Lys Leu Arg

370 375 380Pro PHLU ARG ARG Asp Ile Sern Val Pro PHE Ser Pro ASP GLY LYS385 395 400Pro CYS THR Pro Ala Leu Asn Cysn Asn Asn Asn Asn Asn Asn Asn Asn Asn

405 410 415Gly Phe Tyr Thr Thr Thr Gly Ile Gly Tyr Gln Pro Tyr Arg Val Val

420 425 430Val Leu Ser Phe Glu Leu Leu Asn Ala Pro Ala Thr Val Cys Gly Pro

435 440 445Lys Leu Ser Thr Asp Leu Ile Lys Asn Gln Cys Val Asn Phe Asn Phe

450 455 460ASN GLY Leu Thr Gly Thr Gly Val Leu Thr Pro Serg PHE465 475 480GLN PHE GLN GLN GLN GLN GLY ARG ASP Val Ser ASP Ser Ap Ser ASP Ser ASP Thr Asr ASP VHR ThR -R that Ge

485 490 495Val Arg Asp Pro Lys Thr Ser Glu Ile Leu Asp Ile Ser Pro Cys Ser

500 505 510Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Ala Ser Ser Glu

515 520 525Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Asp Val Ser Thr Ala

530 535 540 <210 <211> 216 <212> PRT <213> Coronavirus <400> 15ASP ARG CYS ThR ThR PHE ASP Val Gln Ala Pro Asn Tyr Tyr GLN1 5 10 15HIS Serg Gly Val Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Pro Asp Glu Ile Phe Arg

20 25 25 30Ser Asp Thr Leu Tyr Leu Thr Gln Asp Leu Phe Leu Pro Phe Tyr Ser

35 40 45Asn Val Thr Gly Phe His Thr Ile Asn His Thr Phe Asp Asn Pro Val

50 55 60ILE Pro PHE LYS ASP GLY Ile Tyr Phe Ala Ala Thr Glu Lysn65 70 75 80VAL VAL TRP Valphe Gly Ser THR MET Asn Lysr Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln

85 90 95Ser Val Ile Ile Ile Asn Asn Ser Thr Asn Val Val Ile Arg Ala Cys

100 105 110Asn Phe Glu Leu Cys Asp Asn Pro Phe Phe Ala Val Ser Lys Pro Met

115 120 125Gly Thr Gln Thr His Thr Met Ile Phe Asn Ala Phe Asn Cys Thr

130 135 140phe Glu Tyr Ile Serite Ala Phe Ser Leu ASP Val Sergs Ser145 150 155 160160Gly As His Leu Phe Val Phe Lysn Lysn Lysn Lysn Lysn Lysn Lysn Lysn Lysn Lysn Lysn Lysn Lysp Gly Gly Gly Gly

165 170 175Phe Leu Tyr Val Tyr Lys Gly Tyr Gln Pro Ile Asp Val Val Arg Asp

180 185 190Leu Pro Ser Gly Phe Asn Thr Leu Lys Pro Ile Phe Lys Leu Pro Leu

195 200 205Gly Ile Asn Ile Thr Asn Phe Arg

210 215 <210> 1615 <212> PRT <213> Coronavirus <400> 16lys Pro ThR ThR PHR PHE Leu Lys Tyr ASP GLU ASN GLY THR ILE THR1 5 10 15ASP CYS Sern ASN PRO Leu Ala Glu Leu Lys Cys Ser

20 25 30Val Lys Ser Phe Glu Ile Asp Lys Gly Ile Tyr Gln Thr Ser Asn Phe

35 40 45Arg Val Val Pro Ser Gly Asp Val Val Arg Phe Pro Asn Ile Thr Asn

50 55 60leu Cys Pro Leu GLY GLU VAL PHE Asn Ala THR LYS PRO PRO Ser Val65 70 75 80thr Ala Tru ARG LYS LYS Ile Ser ALA Ala asr Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Seer

85 90 95Val Leu Tyr Asn Ser Thr Phe Phe Ser Thr Phe Lys Cys Tyr Gly Val

100 105 110Ser Ala Thr Lys Leu Asn Asp Leu Cys Phe Ser Asn Val Tyr Ala Asp

115 120 125Ser Phe Val Val Lys Gly Asp Asp Val Arg Gln Ile Ala Pro Gly Pro

130 135 140thr GLY VAL ILE ALA ASP Tyr Asn Tyr Lys Leu Pro ASP ASP PHE MET145 150 155 160GLY CYS Val Leu Ala THR ARG Asn Ile Ala THR Serte Thr Thr Thr Thr Thr Thr Thr Thr Thr Thr Thr Thr Thr Thr Thr Thr Thr Thr THR Serte Thr

165 170 175Gly Asn Tyr Asn Tyr Lys Tyr Arg Tyr Leu Arg His Gly Lys Leu Arg

180 185 190Pro Phe Glu Arg Asp Ile Ser Asn Val Pro Phe Ser Pro Asp Gly Lys

195 200 205Pro Cys Thr Pro Pro Ala Leu Asn Cys Tyr Trp Pro Leu Asn Asp Tyr

210 215 220Gly PHR THR THR THR THR GLY TYR GLN Pro Tyr ARG Val Val 2225 235 240VAL Leu Seru Leu Leu Ala THR Val Cys Gly Pro

245 250 255Lys Leu Ser Thr Asp Leu Ile Lys Asn Gln Cys Val Asn Phe Asn Phe

260 265 270Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Pro Ser Ser Lys Arg Phe

275 280 285Gln Pro Phe Gln Gln Phe Gly Arg Asp Val Ser Asp Phe Thr Asp Ser

290 295 300VAL ASP Pro LYS THR Series Glu Ile Leu ASP305 315 <210> 17 <211> 607 <212> PRT <213> Coronavirus <400> 17HIS Val ASP TYR GLU CYS Asp Ile Gly GLY GLY GLY GLY ILE1 5 10 15Cys Ala Ser Tyr His Thr Val Ser Leu Leu Arg Ser Thr Ser Gln Lys

20 25 30Ser Ile Val Ala Tyr Thr Met Ser Leu Gly Ala Asp Ser Ser Ile Ala

35 40 45Tyr Ser Asn Asn Thr Ile Ala Ile Pro Thr Asn Phe Ser Ile Ser Ile

50 55 60thr Thr Glu Val Met Pro Val Serite Ala Lys Thr Val ASP CYS65 75 80asn Met Tyr Ile Cys GLY ASN Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu's's's TEU -S's

85 90 95Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Ser Gly Ile

100 105 110Ala Ala Glu Gln Asp Arg Asn Thr Arg Glu Val Phe Ala Gln Val Lys

115 120 125Gln Met Tyr Lys Thr Pro Thr Leu Lys Tyr Phe Gly Gly Phe Asn Phe

130 135 140ser Gln Ile Leu Pro ASP PRO Leu Lys Pro Thr Lys ARG Serg Serg Serite Ile145 150 155 160GLU ASN LEU PHE As Val Val THR Leu Ala Gly PHE MET MET MET MET MET MET MET MET MET MET MET MET MET MET MET MET MET MET MET MET MET MET MET MET MET MET MET MET MET MET MET MET MET MET MET MET MET

165 170 175Lys Gln Tyr Gly Glu Cys Leu Gly Asp Ile Asn Ala Arg Asp Leu Ile

180 185 190Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu Leu Thr

195 200 205Asp Asp Met Ile Ala Ala Tyr Thr Ala Ala Leu Val Ser Gly Thr Ala

210                 215                 220Thr Ala Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile Pro Phe225                 230                 235                 240Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr Gln Asn

245 250 255Val Leu Tyr Glu Asn Gln Lys Gln Ile Ala Asn Gln Phe Asn Lys Ala

260 265 270Ile Ser Gln Ile Gln Glu Ser Leu Thr Thr Thr Ser Ser Thr Ala Leu Gly

275 280 285Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn Thr Leu

290 295 300Val LYS GLN Leu Sero Sern Phe Gly Ala iLe Ser Val Leu asn305 315 320asp Ile Leu Serg Leu as Val Glu Val Gln Ile ASP

325 330 335Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val Thr Gln

340 345 350Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu Ala Ala

355 360 365Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg Val Asp Phe

370                 375                 380Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln Ala Ala Pro His385                 390                 395                 400Gly Val Val Phe Leu His Val Thr Tyr Val Pro Ser Gln Glu Arg Asn

405 410 415Phe Thr Ala Thr Ala Pro Ala Ile Cys His Glu Gly Lys Ala Tyr Phe Pro

420 425 430Arg Glu Gly Val Phe Val Phe Asn Gly Thr Ser Trp Phe Ile Thr Gln

435 440 445Arg Asn Phe Phe Ser Pro Gln Ile Ile Thr Thr Asp Asn Thr Phe Val

450 455 460r Gly Asn Cys ASP Val Val Ile Gly ILE ILE Asn Thr Val Tyr465 475 480ASP Pro Leu Gln Pro Glu Leu Leu Leu Leu Leu ASP LYS

485 490 495Tyr Phe Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser

500 505 510Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu

515 520 525Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu

530 535 540leu Gly LYS Tyr Gln Tyr Ile Lys TRP Pro Tr Val Trp Leu545 550 560Gly PHE Ile Ala Gly Leu Ile Val Met Val THR ILE Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu's Mary Metal that thedeter

565 570 575Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Ala Cys Ser Cys

580 585 590Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro Val Leu

595 600 605 <210> 18 <211> 216 <212> PRT <213> Coronavirus <400> 18HIS Val ASP THR SER GLU CYS ASP ILE GLY ALA GLY ALA GLY Ile1 5 10 15CYR His ThR Val Seruuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuu Arg Ser Thr Ser Gln Lys

20 25 30Ser Ile Val Ala Tyr Thr Met Ser Leu Gly Ala Asp Ser Ser Ile Ala

35 40 45Tyr Ser Asn Asn Thr Ile Ala Ile Pro Thr Asn Phe Ser Ile Ser Ile

50 55 60thr Thr Glu Val Met Pro Val Serite Ala Lys Thr Val ASP CYS65 75 80asn Met Tyr Ile Cys GLY ASN Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu's's's TEU -S's

85 90 95Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Ser Gly Ile

100 105 110Ala Ala Glu Gln Asp Arg Asn Thr Arg Glu Val Phe Ala Gln Val Lys

115 120 125Gln Met Tyr Lys Thr Pro Thr Leu Lys Tyr Phe Gly Gly Phe Asn Phe

130 135 140ser Gln Ile Leu Pro ASP PRO Leu Lys Pro Thr Lys ARG Serg Serg Serite Ile145 150 155 160GLU ASN LEU PHE As Val Val THR Leu Ala Gly PHE MET MET MET MET MET MET MET MET MET MET MET MET MET MET MET MET MET MET MET MET MET MET MET MET MET MET MET MET MET MET MET MET MET MET MET MET MET

165 170 175Lys Gln Tyr Gly Glu Cys Leu Gly Asp Ile Asn Ala Arg Asp Leu Ile

180 185 190Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu Leu Thr

195 200 205Asp Asp Met Ile Ala Ala Tyr Thr

210 215 <210> 19 <2111> 315 <212> PRT <213> Coronavirus <400> 19GLN MET ALA TYR ARG PHE Asn Gly Val THR GLN Val Leu1 5 10 15tyr Gln LYS Gln Gln PHE Asn Lys Ala Ile Ser

20 25 30Gln Ile Gln Glu Ser Leu Thr Thr Thr Ser Ser Thr Ala Leu Gly Lys Leu

35 40 45Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn Thr Leu Val Lys

50 55 60GLN Leu Ser Sern Phe Gly Ala iLe Ser Val Leu asn ASP ILE65 70 80leu Serg Leu ASP LYS Val Glu Val Gln Ile ARG Leuuuuu

85 90 95Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val Thr Gln Gln Leu

100 105 110Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu Ala Ala Thr Lys

115 120 125 Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg Val Asp Phe Cys Gly

130 135140lys Gly Tyr His Leu Met Ser, Pro Gln Ala Ala Ala His Gly Val145 150 160VAL PHE Leu His Val Pro Val Gln Gln Phe Thr

165 170 175Thr Ala Pro Ala Ile Cys His Glu Gly Lys Ala Tyr Phe Pro Arg Glu

180 185 190Gly Val Phe Val Phe Asn Gly Thr Ser Trp Phe Ile Thr Gln Arg Asn

195 200 205Phe Phe Ser Pro Gln Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly

210                 215                 220Asn Cys Asp Val Val Ile Gly Ile Ile Asn Asn Thr Val Tyr Asp Pro225                 230                 235                 240Leu Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe

245 250 255Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile

260 265 270Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu

275 280 285Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu Gly

290 295 300lys Ty Gln Tyr Ile Lys TRP Pro TRP TYR305 315 <210> 20 <211> 40 <212> PRT <213> Coronavirus <400> 20gly Asr Gly Ile Asr Val Val Val Ale Gln LYS GLU11111111 5 10 15Ile Asp Arg Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile

20 25 30Asp Leu Gln Glu Leu Gly Lys Tyr

35 40 <210> 21 <211> 88 <212> PRT <213> Coronavirus <400> 21gly Asp Ile Serle Asn Ala Ser Val Val Val Ale Gln LYS GLU1 5 10 15ILE ARG Leu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile

20 25 25 30Asp Leu Gln Glu Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp

35 40 45Tyr Val Trp Leu Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val

50 55 60thr Ile Leu Leu Cys Met THR Ser Cys Cys Seru Lys Gly65 70 80ALA CYS Ser Cys Cys Cys Cys Cys Cys Cys Cys Cys Cys Cys Cys

85 <20> 22 <211 402 <212> PRT <213> Coronavirus <400> 22ILE Leu Pro ASP Pro Leu Lys Pro Thr Lys ARG Serge Ile Glu ASP1 5LEU PHE ASN LEU ALA ALA ALA ALA GLE Phe Met Lys Gln

20 25 25 30Tyr Gly Glu Cys Leu Gly Asp Ile Asn Ala Arg Asp Leu Ile Cys Ala

35 40 45Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu Leu Thr Asp Asp

50 55 60MET ILE Ala Ala Tyr Tyr Ala Leu Val Gly ThR Ala Thr 60 75 80GLY THR PHE GLY ALA Ala Ala Ala Pro PHE Ala Met

85 90 95Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr Gln Asn Val Leu

100 105 110Tyr Glu Asn Gln Lys Gln Ile Ala Asn Gln Phe Asn Lys Ala Ile Ser

115 120 125Gln Ile Gln Glu Ser Leu Thr Thr Thr Ser Ser Thr Ala Leu Gly Lys Leu

130                 135                 140Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn Thr Leu Val Lys145                 150                 155                 160Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val Leu Asn Asp Ile

165 170 175Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln Ile Asp Arg Leu

180 185 190Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val Thr Gln Gln Leu

195 200 205Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu Ala Ala Thr Lys

210 215 220MET Ser Glu Cys Val Val Leu Gly Gln Serg Val ASP PHE CYS GLY225 230 235LYS GLY TYR His Leu Met Sern Ala Ala Pro His Gly Val Val Val Val Val Val Val Val Val Val Val

245 250 255Val Phe Leu His Val Thr Tyr Val Pro Ser Gln Glu Arg Asn Phe Thr

260 265 270Thr Ala Pro Ala Ile Cys His Glu Gly Lys Ala Tyr Phe Pro Arg Glu

275 280 285Gly Val Phe Val Phe Asn Gly Thr Ser Trp Phe Ile Thr Gln Arg Asn

290 295 300phe Phe Ser Pro Gln Ile Ile THR THR ASN Thr Phe Val Ser Gly305 315 320asn CYS Asp Val Val Ile Ile Asn Thr Val Tyr ASP PRO

325 330 335Leu Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe

340 345 350Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile

355 360 Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu

370 375 380val Ala Lysn Leu asn Glu Serle ASP Leu Gln Glu Leu Gly385 395 400lys Tyr

Claims (16)

1. Recombinant SARS virus S, S1, S1-1, S1-2, S1-3, S2, S2-1, S2-2, S2-3, S2- derived from Hansenula polymorpha (Hansenula polymorpha) expression 4. S2-5 gene, a gene with similar functions formed by modifying, extending or shortening the nucleotide sequence of the above-mentioned gene, and its nucleotide sequence is the corresponding nucleoside shown in SEQ ID NO: 1-11 The acid sequences share at least 75% homology. 2. the recombinant SARS virus S, S1, S1-1, S1-2, S1-3, S2, S2-1, S2-2, S1-3, S2, S2-1, S2-2 derived from the recombinant SARS virus expressed by Hansenula polymorpha (Hansenulapolymorpha) according to claim 1 S2-3, S2-4, and S2-5 genes respectively have nucleotide sequences corresponding to SEQ ID NO: 1-11. 3. gene according to claim 2, wherein the recombinant SARS virus S gene expressed by Hansenula polymorpha (Hansenula polymorpha), it has the nucleotide sequence shown in SEQ ID NO: 1, and the protein of its coding has as SEQ ID NO: ID NO: amino acid sequence shown in 12. 4. gene according to claim 2, wherein the recombinant SARS virus S1 gene of Hansenula polymorpha (Hansenulapolymorpha) expression, it has the nucleotide sequence shown in SEQ ID NO: 2, and the protein of its coding has as SEQ ID NO: ID NO: amino acid sequence shown in 13. 5. the gene according to claim 2, wherein the recombinant SARS virus S1-1 gene expressed by Hansenula polymorpha (Hansenula polymorpha), it has the nucleotide sequence shown in SEQ ID NO: 3, and the protein encoded by it has Amino acid sequence as shown in SEQ ID NO: 14. 6. gene according to claim 2, wherein the recombinant SARS virus S1-2 gene of Hansenula polymorpha (Hansenula polymorpha) expression, it has the nucleotide sequence shown in SEQ ID NO: 4, and the protein of its coding has Amino acid sequence as shown in SEQ ID NO:15. 7. A recombinant SARS virus S1-3 gene derived from Hansenula polymorpha (Hansenula polymorpha) expression, it has the nucleotide sequence shown in SEQ ID NO: 5, and the protein encoded by it has such as SEQ ID NO: The amino acid sequence shown in 16. 8. The gene according to claim 2, wherein the recombinant SARS virus S2 gene expressed by Hansenula polymorpha (Hansenulapolymorpha), it has the nucleotide sequence shown in SEQ ID NO: 6, the protein encoded by it It has the amino acid sequence shown in SEQ ID NO: 17. 9. The gene according to claim 2, wherein the recombinant SARS virus S2-1 gene expressed by Hansenula polymorpha (Hansenula polymorpha), it has the nucleotide sequence shown in SEQ ID NO: 7, and the protein encoded by it has Amino acid sequence as shown in SEQ ID NO: 18. 10. The gene according to claim 2, wherein the recombinant SARS virus S2-2 gene expressed by Hansenula polymorpha (Hansenula polymorpha), it has the nucleotide sequence shown in SEQ ID NO: 8, and the protein encoded by it has Amino acid sequence as shown in SEQ ID NO: 19. 11. The gene according to claim 2, wherein the recombinant SARS virus S2-3 gene expressed by Hansenula polymorpha (Hansenula polymorpha), it has the nucleotide sequence shown in SEQ ID NO: 9, and the protein encoded by it has Amino acid sequence as shown in SEQ ID NO:20. 12. The gene according to claim 2, wherein the recombinant SARS virus S2-4 gene expressed by Hansenula polymorpha (Hansenulapolymorpha) has the nucleotide sequence shown in SEQ ID NO: 10, which encodes The protein has the amino acid sequence shown in SEQ ID NO: 21. 13. The gene according to claim 2, wherein the recombinant SARS virus S2-5 gene expressed by Hansenula polymorpha (Hansenulapolymorpha), it has the nucleotide sequence shown in SEQ ID NO: 11, and the protein encoded by it has Amino acid sequence as shown in SEQ ID NO:22. 14. The gene according to claim 1 or 2, they are expressed in the recombinant SARS virus S, S1, S1-1, S1-2, S1-3, S2, The coding protein of S2-1, S2-2, S2-3, S2-4, S2-5 genes and functionally similar genes, the preparation method thereof comprises the following steps: a) Optimal design of SARS virus S, S1, S1-1, S1-2, S1-3, S2, S2-1, S2-2, S2-3, S2- according to the codon usage of highly expressed genes of Hansenula polymorpha 4. S2-5 gene; b) Artificially synthesize the newly designed S, S1, S1-1, S1-2, S1-3, S2, S2-1, S2-2, S2-3, S2-4, S2-5 genes through a gene synthesizer ; c) Utilize the Hansenula polymorpha expression vector or any other eukaryotic expression vector that can be integrated and expressed in Hansenula to construct the optimized SARS virus S, S1, S1-1, S1-2, S1-3, Recombinant expression vectors of S2, S2-1, S2-2, S2-3, S2-4, S2-5 genes; d) Hansenula polymorpha recombinant SARS virus S, S1, S1-1, S1-2, S1-3, S2, S2-1, S2-2, S2-3, S2-4, S2-5 gene encoded protein Induced expression; e) Expression product identification and biological activity identification. 15. according to the recombinant SARS virus S, S1, S1-1, S1-2, S1-3, S2, S2-1, S2-2 of expression of Hansenula polymorpha (Hansenulapolymorpha) described in claim 1-13, The application of S2-3, S2-4, S2-5 genes and genes with similar functions, optionally one of them in the preparation of therapeutic drugs and preventive vaccines for SARS virus disease. 16. according to the recombinant SARS virus S, S1, S1-1, S1-2, S1-3, S2, S2-1, S2-2, S2-1, S2-2 expressed by Hansenula polymorpha (Hansenula polymorpha) described in claim 1-14, S2-3, S2-4, S2-5 genes and the proteins encoded by genes with similar functions, and the application of one of the proteins in the preparation of therapeutic drugs and preventive vaccines for SARS virus disease.

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