CN1244698C - Expression of recombination SARS virus gene in pleiomorphic Hansen yeast and its use - Google Patents

Expression of recombination SARS virus gene in pleiomorphic Hansen yeast and its use Download PDF

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CN1244698C
CN1244698C CNB031411584A CN03141158A CN1244698C CN 1244698 C CN1244698 C CN 1244698C CN B031411584 A CNB031411584 A CN B031411584A CN 03141158 A CN03141158 A CN 03141158A CN 1244698 C CN1244698 C CN 1244698C
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CN1475571A (en
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邱并生
宋厚辉
李勇
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Institute of Microbiology of CAS
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Abstract

The present invention belongs to the field of protein expression gene engineering, more specifically a product for expressing foreign genes by using a Hansen yeast as a cell factory. Because the relationship of SARS viruses is relatively far from the Hansen yeast on an evolutionary relationship, and a codon using method of the SARS viruses S, S1 and S2 and main antigen epiposition genes in a protein translation process makes a great difference from the Hansen yeast, so the translation can be stopped anomaly. In the product, S, S1 and S2 genes of the SARS viruses and the main antigen expression genes are redesigned according to the codon using method in a Hansen yeast high expression gene during the industrial production process of an expression product of the S, S1 and S2 genes of the SARS viruses and the main antigen epiposition genes, and the high expression product can be used as a vaccine for preventing atypical pneumonitis resulted from the SARS viruses. The genes designed in the product can also be used for fusion expression in the Hansen yeast with cholera toxin B subunit genes (patent application number: 03110441. X) of mucosal immunization, and an expression product can be used as an oral vaccine.

Description

A kind of expression and the purposes of SARS virus gene in multiple-shaped nuohan inferior yeast of recombinating
Technical field
The invention belongs to protein expression genetically engineered field, the multiple-shaped nuohan inferior yeast (Hansenula polymorpha) that relates to specifically utilize SARS virus reorganization S, S1, S2 and major antigen epitope gene and utilization to contain these genes efficiently expresses and utilizes its expression product in the application that is used for developing the vaccine that prevents SARS virus as cell factory.
Background technology
SARS virus is a kind of emerging coronavirus, causes that (Severe Acute Respiratory Syndrome SARS), is the arch-criminal of severe acute respiratory syndrome to human SARS (Severe Acute Respiratory Syndrome).This viral genome is the sub-thread positive chain RNA.The about 30Kb of this RNA total length is maximum in all RNA viruses, and has infectivity.The pathogenesis of SARS virus it be unclear that, and its natural reservoir (of bird flu viruses) scope is wider, can survive for a long time in air, ight soil, mainly propagates through respiratory system.Anaphylaxis may take place during chronic infection; After the acute infection, also persistent infection may take place.Still not having effective vaccine at present can prevent.The same with other coronavirus, SARS virus is typical togavirus, and exposing in its cyst membrane outside has many glycosylated fibres dash forward (Spike) albumen, i.e. S albumen.S albumen is by the S genes encoding, and it is formed near onesize Protein S 1 and S2 by two, directly combines with host cell receptor, and the conformational change by S1 and S2 causes cytogamy, and induces generation neutralizing antibody and cellular immunization.In other coronavirus, S albumen is the antigenic main position of virus neutralization, contains many epitopes, and induces the generation neutrality antibody and melt film reaction.This points out us can utilize the S gene to carry out the design of SARS virus vaccine.
The contriver utilizes software and incidental on-line analysis instruments thereof such as DNAStar, VectorNTI, and the SARS virus strain isolated on ground such as the U.S., Canada, Hong Kong, Beijing, Guangzhou is predicted from the nucleotide sequence to the protein structure and analyzed.Find that the proteic difference of all SARS virus strain isolated Spike is only pure on S1 at present, the S2 high conservative, and the proteic difference of all SARS virus strain isolateds is only pure on S1 at present, the S2 high conservative, and also the S2 gene of all SARS virus strain isolateds is all consistent at present.S full length gene 3768bp, 1255 amino acid (AA) of encoding.Wherein 1-16AA is a signal peptide, and 66-609AA is the S1 protein region, and 641-1247AA is the S2 protein region.In S2 albumen, the potential glycosylation site mainly concentrates on the 787-1221AA zone, the fusion rotein of very similar paramyxovirus (Fusion).In the proteic 1148-1236AA of S2 position, have a special Coiled coil structure (1149-1188AA) and leucine zipper (1148-1182AA) and be rich in the structure (1217-1236AA) of Cys, the contriver infers that this zone may play a part when SARS virus and host cell merge very crucial.For this reason, the contriver is according to debaryomyces hansenii cance high-expression gene codon usage table (number of patent application: 03110441.X) designed the S gene of a series of different lengthss, in debaryomyces hansenii (Hansenula polymorpha), to efficiently express, the antibody that produces according to these gene fragments is determined the preferred plan of severe acute respiratory syndrome therapeutic and preventative vaccine development to the barrier effect of SARS virus.
Summary of the invention
Multiple-shaped nuohan inferior yeast (Hansenula polymorpha) belongs to methanol yeast, is called Pichiaaugusta again.Its optimum growth temperature height, growth velocity is fast, is beneficial to large scale fermentation production, can efficiently express many genes of efficiently expressing of being difficult in other systems.Because multiple-shaped nuohan inferior yeast can be integrated a plurality of genes than substep by certain gene dosage, the reorganization bacterium can be by the required gene of the ratio expression of the best, and this does not appear in the newspapers in other methanol yeast.In addition, multiple-shaped nuohan inferior yeast has that genetic manipulation is simple, copy number of foreign gene is high, foreign protein output height, be easy to advantages such as suitability for industrialized production, be a heterologous gene expression system that is better than intestinal bacteria and other yeast (as pichia pastoris phaff and cereuisiae fermentum etc.), obtained extensive concern.Especially in secretion type expression, foreign protein can be finished translation post-treatment processes such as proteolysis maturation, glycosylation modified and disulfide linkage formation by Secretory Pathway, make the more approaching native protein form of expressed albumen, avoided the mistake glycosylation phenomenon in the cereuisiae fermentum again with biologic activity.But SARS virus and yeast relationship on evolutionary relationship is far away, and its codon usage and debaryomyces hansenii difference are very big in the protein translation process, thereby causes translating unusual pause.So far do not see the report that utilizes any gene of debaryomyces hansenii system expression SARS virus as yet.When we utilize multiple-shaped nuohan inferior yeast AS 2.2412 (the common micro-organisms center C GMCC of China Committee for Culture Collection of Microorganisms provides) to express SARS virus S, S1, S2 gene and major antigen epitope gene thereof, codon usage according to the multiple-shaped nuohan inferior yeast cance high-expression gene has redesigned these encoding genes, and its expression amount is improved greatly.
The present invention is based on a kind of like this discovery and finishes.Therefore, the object of the present invention is to provide the multiple-shaped nuohan inferior yeast that a kind of recombinate SARS virus S, S1, S2 gene and major antigen epitope gene thereof and utilization contain this gene to efficiently express, and utilize the application of these expression products in preparation SARS virus recombinant vaccine as cell factory.The nucleotide sequence of multiple-shaped nuohan inferior yeast reorganization SARS virus S of the present invention, S1, S1-1, S1-2, S1-3, S2, S2-1, S2-2, S2-3, S2-4, S2-5 gene corresponds respectively to the nucleotide sequence shown in the SEQ ID NO:1-11, and its amino acid sequence coded corresponds respectively to the aminoacid sequence shown in the SEQ ID NO:12-22.For reaching, technological line of the present invention is to utilize multiple-shaped nuohan inferior yeast cance high-expression gene codon usage table (Chinese patent application number as shown in Figure 1: 03110441.X).Go up the nucleotide sequence (as shown in Figure 2, total length 3768bp) of S gene (accession number: AY278554, CUHK-W1 strain) of the SARS virus coding of report according to Genbank, its amino acid sequence coded is (total length 1255 amino acid) shown in SEQ ID NO:12.Carry out structural domain composition, albumen secondary structure, transbilayer helix, glycosylation site, Characterization of antigenic epitopes according to Vector NTI 8.0 softwares and incidental SMART (SimpleModular Architecture Research Tool) etc. thereof at sequence of threads.According to analytical results, the contriver is divided 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) totally 11 zones with the proteins encoded of SARS virus S gene.According to the codon usage of debaryomyces hansenii cance high-expression gene with 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), the regional corresponding amino acid sequence of S2-5 (787-1188AA) is transformed into nucleotide sequence, promptly obtain encoding gene, correspond respectively to as the nucleotide sequence shown in the SEQ IDNO:1-11 according to debaryomyces hansenii cance high-expression gene codon usage design.The amino acid identity of these genes (SEQ ID NO:1-11) and SARS virus respective coding genes encoding is 100%, its amino acid sequence coded respectively shown in (SEQ ID NO:12-22), as for nucleotide sequence homology at least 75%.Consider swing and SARS different isolates the difference on S1 gene coded protein of debaryomyces hansenii cance high-expression gene to indivedual synonym preferences, the albumen of the S1 genes encoding of the SARS virus strain isolated on ground such as the U.S., Canada, Hong Kong, Beijing, Guangzhou has 5 amino acid differences, and the S2 gene is in full accord.Therefore, the nucleotide sequence of the coding reorganization SARS virus S that optimizes, S1, S1-1, S1-2, S1-3, S2, S2-1, S2-2, S2-3, S2-4, S2-5 gene, except that the nucleotide sequence shown in the SEQ IDNO:1-11, because the disappearance of nucleotide sequence sudden change, increase still produce the proteic nucleotide sequence of coding identical function, should be pointed out that these sequences correspond respectively to the nucleotides sequence shown in the above-mentioned SEQ ID NO:1-11 and have at least 75% homology.The above-described nucleotide sequence that these have at least 75% homology also relates to one of purpose that the present invention will reach.SARS virus S, S1, S2 gene and the major antigen epitope gene thereof that obtain by the present invention according to debaryomyces hansenii cance high-expression gene codon usage design, overcome the inadaptability in the expressed by Hansenula yeast system, thereby these genes are efficiently expressed in debaryomyces hansenii.The SARS virus S that expresses in debaryomyces hansenii, S1, S2 gene and major antigen epitope gene safety and reliability thereof are compared with the mammalian cell expression product, no potential tumorigenicity." severe acute respiratory syndrome " that can directly be used to clinically after purified to prevent SARS virus to cause, perhaps with the encoding gene of choleratoxin B subunit (Chinese patent application number: 03110441.X) amalgamation and expression, improve vaccine effect by mucosal immunity.
Another object of the present invention is to provide a kind of method of utilizing debaryomyces hansenii cance high-expression gene codon usage to prepare SARS virus S, S1, S1-1, S1-2, S1-3, S2, S2-1, S2-2, S2-3, S2-4, S2-5 gene coded protein, and this method relates to following steps:
1. according to the codon usage (Fig. 1) of multiple-shaped nuohan inferior yeast cance high-expression gene, optimization design SARS virus S, S1, S1-1, S1-2, S1-3, S2, S2-1, S2-2, S2-3, S2-4, S2-5 gene;
2. pass through gene machine, the newly-designed encoding gene of synthetic;
3. utilize Chinese patent (application number: the multiple-shaped nuohan inferior yeast expression vector pHMOXZ-A 03110441.X) (born of the same parents in express), pHFMDHZ-A (expressing in the born of the same parents), pHMOXZ α-A (secretion type expression), pHFMDHZ α-A (secretion type expression) or other any can be in debaryomyces hansenii the carrier for expression of eukaryon of integrative gene expression, make up the recombinant expression vector that contains the SARS virus S, the S1 that have optimized, S1-1, S1-2, S1-3, S2, S2-1, S2-2, S2-3, S2-4, S2-5 gene;
4. the abduction delivering of multiple-shaped nuohan inferior yeast reorganization SARS virus S, S1, S1-1, S1-2, S1-3, S2, S2-1, S2-2, S2-3, S2-4, S2-5 gene coded protein;
5. expression product is identified and the biologic activity evaluation.
Step obtains debaryomyces hansenii (Hansenula polymorpha) and efficiently expresses the recon that contains SARS virus S, S1, S1-1, S1-2, S1-3, S2, S2-1, S2-2, S2-3, S2-4, S2-5 gene according to the method described above, and preparation SARS virus S, S1, S1-1, S1-2, S1-3, S2, S2-1, S2-2, S2-3, S2-4, S2-5 gene coded protein are that those skilled in the art can realize.
Up to now, relevant SARS virus S, S1, S2 gene and the expression of major antigen epitope gene in debaryomyces hansenii thereof yet there are no report both at home and abroad.The SARS virus S of optimization design of the present invention, S1, S1-1, S1-2, S1-3, S2, S2-1, S2-2, S2-3, S2-4, the expression of S2-5 gene in debaryomyces hansenii are expressed biomass and are extracted preparation process and all be better than other expression system, and the industrialization production and the application of reorganization SARS virus S, S1, S1-1, S1-2, S1-3, S2, S2-1, S2-2, S2-3, S2-4, S2-5 gene coded protein are become a reality.
Description of drawings
Fig. 1: debaryomyces hansenii cance high-expression gene and low expressing gene synonym usage are relatively.High: the expression cance high-expression gene; Low: the low expressing gene of expression.N is that Codon W program is carried out the branch time-like to high and low expressing gene, each amino acid whose codon number.RSCU (Relative synonymouscodon usage, relative synonym usage), RSCU reflection be the service condition of each synonym in the gene, the ratio between the expected value when its numerical value equals the actual observed value of certain codon in gene and occurs with identical frequency with each codon; Carry out codon usage relatively between the database that introducing RSCU value can make different aminoacids form. * @The superior codon of expression cance high-expression gene correspondence, wherein *For difference after chi square test extremely remarkable; @Be significant difference after chi square test.Be the codon of selecting for use when optimization design S, S1, S1-1, S1-2, S1-3, S2, S2-1, S2-2, S2-3, S2-4, S2-5 gene among the present invention
The nucleotide sequence of the S gene of the coding of the SARS virus of the last report of Fig. 2: Genbank (accession number: AY278554, CUHK-W1 strain)
Embodiment
Following embodiment can make the technician of this professional skill field more fully understand the present invention, but does not limit the present invention in any way.
Embodiment 1
The design of S, S1, S1-1, S1-2, S1-3, S2, S2-1, S2-2, S2-3, S2-4, S2-5 gene
1. with the described multiple-shaped nuohan inferior yeast cance high-expression gene of Fig. 1 codon usage table (Chinese patent application number: 03110441.X) intercalation of DNA Star program
2. go up the SARS virus S gene (accession number: AY278554 of report according to Genbank, the CUHK-W1 strain) position of coded amino acid correspondence, by the DNAStar program with S, S1, S1-1, S1-2, S1-3, S2, S2-1, S2-2, S2-3, S2-4, S2-5 genes 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); Be that SEQ ID NO:12-22 is transformed into corresponding nucleotide sequence respectively, promptly obtain encoding gene, respectively shown in SEQ ID NO:1-11 according to the codon usage design of debaryomyces hansenii cance high-expression gene.
3. pass through gene machine, the newly-designed encoding gene of synthetic
Embodiment 2
The structure of recombinant expression vector, conversion and screening
1. the structure of recombinant expression vector: utilize multiple-shaped nuohan inferior yeast expression vector pHMOXZ-A (expressing in the born of the same parents), pHFMDHZ-A (expressing in the born of the same parents), pHMOXZ α-A (secretion type expression), pHFMDHZ α-A (secretion type expression) or other any can be in debaryomyces hansenii the carrier for expression of eukaryon of integrative gene expression, make up the recombinant expression vector that contains the SARS virus S, the S1 that have optimized, S1-1, S1-2, S1-3, S2, S2-1, S2-2, S2-3, S2-4, S2-5 gene.Above-mentioned any recombinant expression vector all can efficiently express in multiple-shaped nuohan inferior yeast.
2. the conversion of recombinant expression vector and screening: adopt the electroporation conversion method to transform multiple-shaped nuohan inferior yeast.Choose multiple-shaped nuohan inferior yeast NCYC495 mono-clonal in the 5mlYPD liquid nutrient medium, 37 ℃ of incubated overnight
3. get 2ml bacterium liquid and add among the pre-warm YPD of 200ml, 37 ℃ are cultured to (about 6h) between the OD600=1.2-1.5
4. the centrifugal 5min of room temperature 6000rpm abandons supernatant
5. with 500mlTED (100mM Tris-HCl; 50mM EDTA; 25mM DTT; PH=8.0) suspension cell
6. 37 ℃ of shaking tables, 200rpm shakes 15min
7. 4 ℃ of centrifugal 6000rpm * 5min abandon supernatant
8. with the sucrose of the 270mM of 200ml precooling suspension cell gently
9. 4 ℃ of centrifugal 6000rpm * 5min abandon supernatant
10. with the sucrose of the 270mM of 100ml precooling suspension cell gently
11. 4 ℃ of centrifugal 6000rpm * 5min abandon supernatant
12. with the sucrose of the 270mM of 1ml precooling suspension cell gently, be distributed into the 60ul/ pipe, liquid nitrogen or refrigerator below-80 ℃ are preserved standby.
13. in the competent cell of 60ul, add 5ul plasmid DNA (about 100-500ng), add behind the mixing in the electric shock cup in 2mm aperture gently
14. shock parameters: 50uF, 100 Ω, 1.5KV
15. add YPDTM (1% yeast extract of 940ul after the electric shock immediately; 1% peptone; 2% glucose; 1mM Tris-HCl; 1mM MgCl 2), be drawn in the tubule of 2-5ml, 37 ℃ of shaking tables, 200rpm shakes 1h
Be coated with the YPD flat board that contains Zeocin microbiotic (final concentration 100ug/ml) 16. get 100ul, cultivate after 2 days for 37 ℃, occur the bacterium colony of large, medium and small three kinds of forms on the flat board, choose clone PCR and identify recon, identify copy number with Southern hybridization
17. transformant is scribbling growth screening on the antibiotic YPD of Zeocin (1% yeast extract, 1% peptone, the 2% glucose) flat board.If adopt other can be in debaryomyces hansenii the carrier of integrative gene expression, can be with reference to the service manual of this carrier.Whether extract macrocolony zymic DNA utilizes the PCR method detection to have recon to insert.Picking ferments through the correct clone of PCR evaluation
Embodiment 3
Fermentation and abduction delivering
The positive recombinant yeast clone that will contain multi-copy gene in the YPD substratum 37 ℃ cultivate after 12 hours, inoculate in the fresh YPD fermention medium (containing 1.5% glycerine) according to 1: 20 ratio, keep leavening temperature 30-37 ℃, pH3-5, dissolved oxygen amount 20%, air velocity 5-10L/min.Work as O after 24 hours 2When pressure sharply raises (glycerine of prompting in the substratum is exhausted), begin to feed in raw material (YPD that contains 50% glycerine), strict control feed rate makes that the final concentration of glycerine maintains between the 0.05%-0.4% in the fermented liquid.Ferment after 24-48 hour, can gather in the crops.For the fermented liquid of expressing in the born of the same parents, 4 ℃ of centrifugal results thalline of 12000rpm * 2min; For the fermented liquid of secretion type expression, 4 ℃ of centrifugal results supernatants.Carrying out SDS-PAGE and Western-blotting analyzes.In addition, except that inducing the fermentation with glycerine, the multiple-shaped nuohan inferior yeast expression system also can ferment with 1% methyl alcohol and 1% glucose induction.
Embodiment 4
The biologic activity of expression product is identified
Behind S, S1, S1-1, S1-2, S1-3, S2, S2-1, S2-2, S2-3, S2-4, each expression of gene product purification of S2-5, immune mouse, separation of serum.Carry out viral blocking test in the above rank of P3 laboratory.In the Vero cell that infects SARS virus, the vaccine that can block or suppress the serum correspondence of virus replication is an effective vaccine.
Sequence table
＜110〉Institute of Microorganism, Academia Sinica
＜120〉a kind of multiple-shaped nuohan inferior yeast is expressed the reorganization of serious disease acute respiratory syndrome (SARS) virus S, S1, S2 gene and major antigen epitope gene and uses thereof
<141>2003-05-20
<160>22
<170>PatentIn?version?3.2
<210>1
<211>3768
<212>DNA
<213>Artificial?Sequence
<220>
＜223〉design according to multiple-shaped nuohan inferior yeast (Hansenula Polymorpha) cance high-expression gene codon usage
<400>1
atgttcatct?tcttgttgtt?cttgaccttg?acctccggtt?ccgacttgga?cagatgtacc 60
accttcgacg?acgtccaagc?tccaaactac?acccaacaca?cctcctccat?gagaggtgtc 120
tactacccag?acgagatctt?cagatccgac?accttgtact?tgacccaaga?cttgttcttg 180
ccattctact?ccaacgtcac?cggtttccac?accatcaacc?acaccttcga?caacccagtc 240
atcccattca?aggacggtat?ctacttcgct?gctaccgaga?agtccaacgt?cgtcagaggt 300
tgggtcttcg?gttccaccat?gaacaacaag?tcccaatccg?tcatcatcat?caacaactcc 360
accaacgtcg?tcatcagagc?ttgtaacttc?gagttgtgtg?acaacccatt?cttcgctgtc 420
tccaagccaa?tgggtaccca?aacccacacc?atgatcttcg?acaacgcttt?caactgtacc 480
ttcgagtaca?tctccgacgc?tttctccttg?gacgtctccg?agaagtccgg?taacttcaag 540
cacttgagag?agttcgtctt?caagaacaag?gacggtttct?tgtacgtcta?caagggttac 600
caaccaatcg?acgtcgtcag?agacttgcca?tccggtttca?acaccttgaa?gccaatcttc 660
aagttgccat?tgggtatcaa?catcaccaac?ttcagagcta?tcttgaccgc?tttctcccca 720
gctcaagaca?cctggggtac?ctccgctgct?gcttacttcg?tcggttactt?gaagccaacc 780
accttcatgt?tgaagtacga?cgagaacggt?accatcaccg?acgctgtcga?ctgttcccaa 840
aacccattgg?ctgagttgaa?gtgttccgtc?aagtccttcg?agatcgacaa?gggtatctac 900
caaacctcca?acttcagagt?cgtcccatcc?ggtgacgtcg?tcagattccc?aaacatcacc 960
aacttgtgtc?cattcggtga?ggtcttcaac?gctaccaagt?tcccatccgt?ctacgcttgg 1020
gagagaaaga?agatctccaa?ctgtgtcgct?gactactccg?tcttgtacaa?ctccaccttc 1080
ttctccacct?tcaagtgtta?cggtgtctcc?gctaccaagt?tgaacgactt?gtgtttctcc 1140
aacgtctacg?ctgactcctt?cgtcgtcaag?ggtgacgacg?tcagacaaat?cgctccaggt 1200
caaaccggtg?tcatcgctga?ctacaactac?aagttgccag?acgacttcat?gggttgtgtc 1260
ttggcttgga?acaccagaaa?catcgacgct?acctccaccg?gtaactacaa?ctacaagtac 1320
agatacttga?gacacggtaa?gttgagacca?ttcgagagag?acatctccaa?cgtcccattc 1380
tccccagacg?gtaagccatg?taccccacca?gctttgaact?gttactggcc?attgaacgac 1440
tacggtttct?acaccaccac?cggtatcggt?taccaaccat?acagagtcgt?cgtcttgtcc 1500
ttcgagttgt?tgaacgctcc?agctaccgtc?tgtggtccaa?agttgtccac?cgacttgatc 1560
aagaaccaat?gtgtcaactt?caacttcaac?ggtttgaccg?gtaccggtgt?cttgacccca 1620
tcctccaaga?gattccaacc?attccaacaa?ttcggtagag?acgtctccga?cttcaccgac 1680
tccgtcagag?acccaaagac?ctccgagatc?ttggacatct?ccccatgttc?cttcggtggt 1740
gtctccgtca?tcaccccagg?taccaacgct?tcctccgagg?tcgctgtctt?gtaccaagac 1800
gtcaactgta?ccgacgtctc?caccgctatc?cacgctgacc?aattgacccc?agcttggaga 1860
atctactcca?ccggtaacaa?cgtcttccaa?acccaagctg?gttgtttgat?cggtgctgag 1920
cacgtcgaca?cctcctacga?gtgtgacatc?ccaatcggtg?ctggtatctg?tgcttcctac 1980
cacaccgtct?ccttgttgag?atccacctcc?caaaagtcca?tcgtcgctta?caccatgtcc 2040
ttgggtgctg?actcctccat?cgcttactcc?aacaacacca?tcgctatccc?aaccaacttc 2100
tccatctcca?tcaccaccga?ggtcatgcca?gtctccatgg?ctaagacctc?cgtcgactgt 2160
aacatgtaca?tctgtggtga?ctccaccgag?tgtgctaact?tgttgttgca?atacggttcc 2220
ttctgtaccc?aattgaacag?agctttgtcc?ggtatcgctg?ctgagcaaga?cagaaacacc 2280
agagaggtct?tcgctcaagt?caagcaaatg?tacaagaccc?caaccttgaa?gtacttcggt 2340
ggtttcaact?tctcccaaat?cttgccagac?ccattgaagc?caaccaagag?atccttcatc 2400
gaggacttgt?tgttcaacaa?ggtcaccttg?gctgacgctg?gtttcatgaa?gcaatacggt 2460
gagtgtttgg?gtgacatcaa?cgctagagac?ttgatctgtg?ctcaaaagtt?caacggtttg 2520
accgtcttgc?caccattgtt?gaccgacgac?atgatcgctg?cttacaccgc?tgctttggtc 2580
tccggtaccg?ctaccgctgg?ttggaccttc?ggtgctggtg?ctgctttgca?aatcccattc 2640
gctatgcaaa?tggcttacag?attcaacggt?atcggtgtca?cccaaaacgt?cttgtacgag 2700
aaccaaaagc?aaatcgctaa?ccaattcaac?aaggctatct?cccaaatcca?agagtccttg 2760
accaccacct?ccaccgcttt?gggtaagttg?caagacgtcg?tcaaccaaaa?cgctcaagct 2820
ttgaacacct?tggtcaagca?attgtcctcc?aacttcggtg?ctatctcctc?cgtcttgaac 2880
gacatcttgt?ccagattgga?caaggtcgag?gctgaggtcc?aaatcgacag?attgatcacc 2940
ggtagattgc?aatccttgca?aacctacgtc?acccaacaat?tgatcagagc?tgctgagatc 3000
agagcttccg?ctaacttggc?tgctaccaag?atgtccgagt?gtgtcttggg?tcaatccaag 3060
agagtcgact?tctgtggtaa?gggttaccac?ttgatgtcct?tcccacaagc?tgctccacac 3120
ggtgtcgtct?tcttgcacgt?cacctacgtc?ccatcccaag?agagaaactt?caccaccgct 3180
ccagctatct?gtcacgaggg?taaggcttac?ttcccaagag?agggtgtctt?cgtcttcaac 3240
ggtacctcct?ggttcatcac?ccaaagaaac?ttcttctccc?cacaaatcat?caccaccgac 3300
aacaccttcg?tctccggtaa?ctgtgacgtc?gtcatcggta?tcatcaacaa?caccgtctac 3360
gacccattgc?aaccagagtt?ggactccttc?aaggaggagt?tggacaagta?cttcaagaac 3420
cacacctccc?cagacgtcga?cttgggtgac?atctccggta?tcaacgcttc?cgtcgtcaac 3480
atccaaaagg?agatcgacag?attgaacgag?gtcgctaaga?acttgaacga?gtccttgatc 3540
gacttgcaag?agttgggtaa?gtacgagcaa?tacatcaagt?ggccatggta?cgtctggttg 3600
ggtttcatcg?ctggtttgat?cgctatcgtc?atggtcacca?tcttgttgtg?ttgtatgacc 3660
tcctgttgtt?cctgtttgaa?gggtgcttgt?tcctgtggtt?cctgttgtaa?gttcgacgag 3720
gacgactccg?agccagtctt?gaagggtgtc?aagttgcact?acacctga 3768
<210>2
<211>1872
<212>DNA
<213>Artificial?Sequence
<220>
＜223〉design according to multiple-shaped nuohan inferior yeast (Hansenula Polymorpha) cance high-expression gene codon usage
<400>2
gacagatgta?ccaccttcga?cgacgtccaa?gctccaaact?acacccaaca?cacctcctcc 60
atgagaggtg?tctactaccc?agacgagatt?ttcagatccg?acaccttgta?cttgacccaa 120
gacttgttct?tgccattcta?ctccaacgtc?accggtttcc?acaccatcaa?ccacaccttc 180
gacaacccag?tcatcccatt?caaggacggt?atctacttcg?ctgctaccga?gaagtccaac 240
gtcgtcagag?gttgggtctt?cggttccacc?atgaacaaca?agtcccaatc?cgtcatcatc 300
atcaacaact?ccaccaacgt?cgtcatcaga?gcttgtaact?tcgagttgtg?tgacaaccca 360
ttcttcgctg?tctccaagcc?aatgggtacc?caaacccaca?ccatgatctt?cgacaacgct 420
ttcaactgta?ccttcgagta?catctccgac?gctttctcct?tggacgtctc?cgagaagtcc 480
ggtaacttca?agcacttgag?agagttcgtc?ttcaagaaca?aggacggttt?cttgtacgtc 540
tacaagggtt?accaaccaat?cgacgtcgtc?agagacttgc?catccggttt?caacaccttg 600
aagccaatct?tcaagttgcc?attgggtatc?aacatcacca?acttcagagc?tatcttgacc 660
gctttctccc?cagctcaaga?cacctggggt?acctccgctg?ctgcttactt?cgtcggttac 720
ttgaagccaa?ccaccttcat?gttgaagtac?gacgagaacg?gtaccatcac?cgacgctgtc 780
gactgttccc?aaaacccatt?ggctgagttg?aagtgttccg?tcaagtcctt?cgagatcgac 840
aagggtatct?accaaacctc?caacttcaga?gtcgtcccat?ccggtgacgt?cgtcagattc 900
ccaaacatca?ccaacttgtg?tccattgggt?gaggtcttca?acgctaccaa?gttcccatcc 960
gtctacgctt?gggagagaaa?gaagatctcc?aactgtgtcg?ctgactactc?cgtcttgtac 1020
aactccacct?tcttctccac?cttcaagtgt?tacggtgtct?ccgctaccaa?gttgaacgac 1080
ttgtgtttct?ccaacgtcta?cgctgactcc?ttcgtcgtca?agggtgacga?cgtcagacaa 1140
atcgctccag?gtccaaccgg?tgtcatcgct?gactacaact?acaagttgcc?agacgacttc 1200
atgggttgtg?tcttggcttg?gaacaccaga?aacatcgacg?ctacctccac?cggtaactac 1260
aactacaagt?acagatactt?gagacacggt?aagttgagac?cattcgagag?agacatctcc 1320
aacgtcccat?tctccccaga?cggtaagcca?tgtaccccac?cagctttgaa?ctgttactgg 1380
ccattgaacg?actacggttt?ctacaccacc?accggtatcg?gttaccaacc?atacagagtc 1440
gtcgtcttgt?ccttcgagtt?gttgaacgct?ccagctaccg?tctgtggtcc?aaagttgtcc 1500
accgacttga?tcaagaacca?atgtgtcaac?ttcaacttca?acggtttgac?cggtaccggt 1560
gtcttgaccc?catcctccaa?gagattccaa?ccattccaac?aattcggtag?agacgtctcc 1620
gacttcaccg?actccgtcag?agacccaaag?acctccgaga?ttttggacat?ctccccatgt 1680
tccttcggtg?gtgtctccgt?catcacccca?ggtaccaacg?cttcctccga?ggtcgctgtc 1740
ttgtaccaag?acgtcaactg?taccgacgtc?tccaccgcta?tccacgctga?ccaattgacc 1800
ccagcttgga?gaatctactc?caccggtaac?aacgtcttcc?aaacccaagc?tggttgtttg 1860
atcggtgctg?ag 1872
<210>3
<211>1632
<212>DNA
<213>Artificial?Sequence
<220>
＜223〉design according to multiple-shaped nuohan inferior yeast (Hansenula Polymorpha) cance high-expression gene codon usage
<400>3
gtcaccggtt?tccacaccat?caaccacacc?ttcgacaacc?cagtcatccc?attcaaggac 60
ggtatctact?tcgctgctac?cgagaagtcc?aacgtcgtca?gaggttgggt?cttcggttcc 120
accatgaaca?acaagtccca?atccgtcatc?atcatcaaca?actccaccaa?cgtcgtcatc 180
agagcttgta?acttcgagtt?gtgtgacaac?ccattcttcg?ctgtctccaa?gccaatgggt 240
acccaaaccc?acaccatgat?cttcgacaac?gctttcaact?gtaccttcga?gtacatctcc 300
gacgctttct?ccttggacgt?ctccgagaag?tccggtaact?tcaagcactt?gagagagttc 360
gtcttcaaga?acaaggacgg?tttcttgtac?gtctacaagg?gttaccaacc?aatcgacgtc 420
gtcagagact?tgccatccgg?tttcaacacc?ttgaagccaa?tcttcaagtt?gccattgggt 480
atcaacatca?ccaacttcag?agctatcttg?accgctttct?ccccagctca?agacacctgg 540
ggtacctccg?ctgctgctta?cttcgtcggt?tacttgaagc?caaccacctt?catgttgaag 600
tacgacgaga?acggtaccat?caccgacgct?gtcgactgtt?cccaaaaccc?attggctgag 660
ttgaagtgtt?ccgtcaagtc?cttcgagatc?gacaagggta?tctaccaaac?ctccaacttc 720
agagtcgtcc?catccggtga?cgtcgtcaga?ttcccaaaca?tcaccaactt?gtgtccattg 780
ggtgaggtct?tcaacgctac?caagttccca?tccgtctacg?cttgggagag?aaagaagatc 840
tccaactgtg?tcgctgacta?ctccgtcttg?tacaactcca?ccttcttctc?caccttcaag 900
tgttacggtg?tctccgctac?caagttgaac?gacttgtgtt?tctccaacgt?ctacgctgac 960
tccttcgtcg?tcaagggtga?cgacgtcaga?caaatcgctc?caggtccaac?cggtgtcatc 1020
gctgactaca?actacaagtt?gccagacgac?ttcatgggtt?gtgtcttggc?ttggaacacc 1080
agaaacatcg?acgctacctc?caccggtaac?tacaactaca?agtacagata?cttgagacac 1140
ggtaagttga?gaccattcga?gagagacatc?tccaacgtcc?cattctcccc?agacggtaag 1200
ccatgtaccc?caccagcttt?gaactgttac?tggccattga?acgactacgg?tttctacacc 1260
accaccggta?tcggttacca?accatacaga?gtcgtcgtct?tgtccttcga?gttgttgaac 1320
gctccagcta?ccgtctgtgg?tccaaagttg?tccaccgact?tgatcaagaa?ccaatgtgtc 1380
aacttcaact?tcaacggttt?gaccggtacc?ggtgtcttga?ccccatcctc?caagagattc 1440
caaccattcc?aacaattcgg?tagagacgtc?tccgacttca?ccgactccgt?cagagaccca 1500
aagacctccg?agattttgga?catctcccca?tgttccttcg?gtggtgtctc?cgtcatcacc 1560
ccaggtacca?acgcttcctc?cgaggtcgct?gtcttgtacc?aagacgtcaa?ctgtaccgac 1620
gtctccaccg?ct 1632
<210>4
<211>648
<212>DNA
<213>Artificial?Seauence
<220>
＜223〉design according to multiple-shaped nuohan inferior yeast (Hansenula Polymorpha) cance high-expression gene codon usage
<400>4
gacagatgta?ccaccttcga?cgacgtccaa?gctccaaact?acacccaaca?cacctcctcc 60
atgagaggtg?tctactaccc?agacgagatt?ttcagatccg?acaccttgta?cttgacccaa 120
gacttgttct?tgccattcta?ctccaacgtc?accggtttcc?acaccatcaa?ccacaccttc 180
gacaacccag?tcatcccatt?caaggacggt?atctacttcg?ctgctaccga?gaagtccaac 240
gtcgtcagag?gttgggtctt?cggttccacc?atgaacaaca?agtcccaatc?cgtcatcatc 300
atcaacaact?ccaccaacgt?cgtcatcaga?gcttgtaact?tcgagttgtg?tgacaaccca 360
ttcttcgctg?tctccaagcc?aatgggtacc?caaacccaca?ccatgatctt?cgacaacgct 420
ttcaactgta?ccttcgagta?catctccgac?gctttctcct?tggacgtctc?cgagaagtcc 480
ggtaacttca?agcacttgag?agagttcgtc?ttcaagaaca?aggacggttt?cttgtacgtc 540
tacaagggtt?accaaccaat?cgacgtcgtc?agagacttgc?catccggttt?caacaccttg 600
aagccaatct?tcaagttgcc?attgggtatc?aacatcacca?acttcaga 648
<210>5
<211>945
<212>DNA
<213>Artificial?Sequence
<220>
＜223〉design according to multiple-shaped nuohan inferior yeast (Hansenula Polymorpha) cance high-expression gene codon usage
<400>5
aagccaacca?ccttcatgtt?gaagtacgac?gagaacggta?ccatcaccga?cgctgtcgac 60
tgttcccaaa?acccattggc?tgagttgaag?tgttccgtca?agtccttcga?gatcgacaag 120
ggtatctacc?aaacctccaa?cttcagagtc?gtcccatccg?gtgacgtcgt?cagattccca 180
aacatcacca?acttgtgtcc?attgggtgag?gtcttcaacg?ctaccaagtt?cccatccgtc 240
tacgcttggg?agagaaagaa?gatctccaac?tgtgtcgctg?actactccgt?cttgtacaac 300
tccaccttct?tctccacctt?caagtgttac?ggtgtctccg?ctaccaagtt?gaacgacttg 360
tgtttctcca?acgtctacgc?tgactccttc?gtcgtcaagg?gtgacgacgt?cagacaaatc 420
gctccaggtc?caaccggtgt?catcgctgac?tacaactaca?agttgccaga?cgacttcatg 480
ggttgtgtct?tggcttggaa?caccagaaac?atcgacgcta?cctccaccgg?taactacaac 540
tacaagtaca?gatacttgag?acacggtaag?ttgagaccat?tcgagagaga?catctccaac 600
gtcccattct?ccccagacgg?taagccatgt?accccaccag?ctttgaactg?ttactggcca 660
ttgaacgact?acggtttcta?caccaccacc?ggtatcggtt?accaaccata?cagagtcgtc 720
gtcttgtcct?tcgagttgtt?gaacgctcca?gctaccgtct?gtggtccaaa?gttgtccacc 780
gacttgatca?agaaccaatg?tgtcaacttc?aacttcaacg?gtttgaccgg?taccggtgtc 840
ttgaccccat?cctccaagag?attccaacca?ttccaacaat?tcggtagaga?cgtctccgac 900
ttcaccgact?ccgtcagaga?cccaaagacc?tccgagattt?tggac 945
<210>6
<211>1821
<212>DNA
<213>Artificial?Sequence
<220>
＜223〉design according to multiple-shaped nuohan inferior yeast (Hansenula Polymorpha) cance high-expression gene codon usage
<400>6
cacgtcgaca?cctcctacga?gtgtgacatc?ccaatcggtg?ctggtatctg?tgcttcctac 60
cacaccgtct?ccttgttgag?atccacctcc?caaaagtcca?tcgtcgctta?caccatgtcc 120
ttgggtgctg?actcctccat?cgcttactcc?aacaacacca?tcgctatccc?aaccaacttc 180
tccatctcca?tcaccaccga?ggtcatgcca?gtctccatgg?ctaagacctc?cgtcgactgt 240
aacatgtaca?tctgtggtga?ctccaccgag?tgtgctaact?tgttgttgca?atacggttcc 300
ttctgtaccc?aattgaacag?agctttgtcc?ggtatcgctg?ctgagcaaga?cagaaacacc 360
agagaggtct?tcgctcaagt?caagcaaatg?tacaagaccc?caaccttgaa?gtacttcggt 420
ggtttcaact?tctcccaaat?cttgccagac?ccattgaagc?caaccaagag?atccttcatc 480
gaggacttgt?tgttcaacaa?ggtcaccttg?gctgacgctg?gtttcatgaa?gcaatacggt 540
gagtgtttgg?gtgacatcaa?cgctagagac?ttgatctgtg?ctcaaaagtt?caacggtttg 600
accgtcttgc?caccattgtt?gaccgacgac?atgatcgctg?cttacaccgc?tgctttggtc 660
tccggtaccg?ctaccgctgg?ttggaccttc?ggtgctggtg?ctgctttgca?aatcccattc 720
gctatgcaaa?tggcttacag?attcaacggt?atcggtgtca?cccaaaacgt?cttgtacgag 780
aaccaaaagc?aaatcgctaa?ccaattcaac?aaggctatct?cccaaatcca?agagtccttg 840
accaccacct?ccaccgcttt?gggtaagttg?caagacgtcg?tcaaccaaaa?cgctcaagct 900
ttgaacacct?tggtcaagca?attgtcctcc?aacttcggtg?ctatctcctc?cgtcttgaac 960
gacatcttgt?ccagattgga?caaggtcgag?gctgaggtcc?aaatcgacag?attgatcacc 1020
ggtagattgc?aatccttgca?aacctacgtc?acccaacaat?tgatcagagc?tgctgagatc 1080
agagcttccg?ctaacttggc?tgctaccaag?atgtccgagt?gtgtcttggg?tcaatccaag 1140
agagtcgact?tctgtggtaa?gggttaccac?ttgatgtcct?tcccacaagc?tgctccacac 1200
ggtgtcgtct?tcttgcacgt?cacctacgtc?ccatcccaag?agagaaactt?caccaccgct 1260
ccagctatct?gtcacgaggg?taaggcttac?ttcccaagag?agggtgtctt?cgtcttcaac 1320
ggtacctcct?ggttcatcac?ccaaagaaac?ttcttctccc?cacaaatcat?caccaccgac 1380
aacaccttcg?tctccggtaa?ctgtgacgtc?gtcatcggta?tcatcaacaa?caccgtctac 1440
gacccattgc?aaccagagtt?ggactccttc?aaggaggagt?tggacaagta?cttcaagaac 1500
cacacctccc?cagacgtcga?cttgggtgac?atctccggta?tcaacgcttc?cgtcgtcaac 1560
atccaaaagg?agatcgacag?attgaacgag?gtcgctaaga?acttgaacga?gtccttgatc 1620
gacttgcaag?agttgggtaa?gtacgagcaa?tacatcaagt?ggccatggta?cgtctggttg 1680
ggtttcatcg?ctggtttgat?cgctatcgtc?atggtcacca?tcttgttgtg?ttgtatgacc 1740
tcctgttgtt?cctgtttgaa?gggtgcttgt?tcctgtggtt?cctgttgtaa?gttcgacgag 1800
gacgactccg?agccagtctt?g 1821
<210>7
<211>648
<212>DNA
<213>Artificial?Sequence
<220>
＜223〉design according to multiple-shaped nuohan inferior yeast (Hansenula Polymorpha) cance high-expression gene codon usage
<400>7
cacgtcgaca?cctcctacga?gtgtgacatc?ccaatcggtg?ctggtatctg?tgcttcctac 60
cacaccgtct?ccttgttgag?atccacctcc?caaaagtcca?tcgtcgctta?caccatgtcc 120
ttgggtgctg?actcctccat?cgcttactcc?aacaacacca?tcgctatccc?aaccaacttc 180
tccatctcca?tcaccaccga?ggtcatgcca?gtctccatgg?ctaagacctc?cgtcgactgt 240
aacatgtaca?tctgtggtga?ctccaccgag?tgtgctaact?tgttgttgca?atacggttcc 300
ttctgtaccc?aattgaacag?agctttgtcc?ggtatcgctg?ctgagcaaga?cagaaacacc 360
agagaggtct?tcgctcaagt?caagcaaatg?tacaagaccc?caaccttgaa?gtacttcggt 420
ggtttcaact?tctcccaaat?cttgccagac?ccattgaagc?caaccaagag?atccttcatc 480
gaggacttgt?tgttcaacaa?ggtcaccttg?gctgacgctg?gtttcatgaa?gcaatacggt 540
gagtgtttgg?gtgacatcaa?cgctagagac?ttgatctgtg?ctcaaaagtt?caacggtttg 600
accgtcttgc?caccattgtt?gaccgacgac?atgatcgctg?cttacacc 648
<210>8
<211>945
<212>DNA
<213>Artificial?Sequence
<220>
＜223〉design according to multiple-shaped nuohan inferior yeast (Hansenula Polymorpha) cance high-expression gene codon usage
<400>8
caaatggctt?acagattcaa?cggtatcggt?gtcacccaaa?acgtcttgta?cgagaaccaa 60
aagcaaatcg?ctaaccaatt?caacaaggct?atctcccaaa?tccaagagtc?cttgaccacc 120
acctccaccg?ctttgggtaa?gttgcaagac?gtcgtcaacc?aaaacgctca?agctttgaac 180
accttggtca?agcaattgtc?ctccaacttc?ggtgctatct?cctccgtctt?gaacgacatc 240
ttgtccagat?tggacaaggt?cgaggctgag?gtccaaatcg?acagattgat?caccggtaga 300
ttgcaatcct?tgcaaaccta?cgtcacccaa?caattgatca?gagctgctga?gatcagagct 360
tccgctaact?tggctgctac?caagatgtcc?gagtgtgtct?tgggtcaatc?caagagagtc 420
gacttctgtg?gtaagggtta?ccacttgatg?tccttcccac?aagctgctcc?acacggtgtc 480
gtcttcttgc?acgtcaccta?cgtcccatcc?caagagagaa?acttcaccac?cgctccagct 540
atctgtcacg?agggtaaggc?ttacttccca?agagagggtg?tcttcgtctt?caacggtacc 600
tcctggttca?tcacccaaag?aaacttcttc?tccccacaaa?tcatcaccac?cgacaacacc 660
ttcgtctccg?gtaactgtga?cgtcgtcatc?ggtatcatca?acaacaccgt?ctacgaccca 720
ttgcaaccag?agttggactc?cttcaaggag?gagttggaca?agtacttcaa?gaaccacacc 780
tccccagacg?tcgacttggg?tgacatctcc?ggtatcaacg?cttccgtcgt?caacatccaa 840
aaggagatcg?acagattgaa?cgaggtcgct?aagaacttga?acgagtcctt?gatcgacttg 900
caagagttgg?gtaagtacga?gcaatacatc?aagtggccat?ggtac 945
<210>9
<211>120
<212>DNA
<213>Artificial?Sequence
<220>
＜223〉design according to multiple-shaped nuohan inferior yeast (Hansenula Polymorpha) cance high-expression gene codon usage
<400>9
ggtgacatct?ccggtatcaa?cgcttccgtc?gtcaacatcc?aaaaggagat?cgacagattg 60
aacgaggtcg?ctaagaactt?gaacgagtcc?ttgatcgact?tgcaagagtt?gggtaagtac 120
<210>10
<211>264
<212>DNA
<213>Artificial?Sequence
<220>
＜223〉design according to multiple-shaped nuohan inferior yeast (Hansenula Polymorpha) cance high-expression gene codon usage
<400>10
ggtgacatct?ccggtatcaa?cgcttccgtc?gtcaacatcc?aaaaggagat?cgacagattg 60
aacgaggtcg?ctaagaactt?gaacgagtcc?ttgatcgact?tgcaagagtt?gggtaagtac 120
gagcaataca?tcaagtggcc?atggtacgtc?tggttgggtt?tcatcgctgg?tttgatcgct 180
atcgtcatgg?tcaccatctt?gttgtgttgt?atgacctcct?gttgttcctg?tttgaagggt 240
gcttgttcct?gtggttcctg?ttgt 264
<210>11
<211>1206
<212>DNA
<213>Artificial?Sequence
<220>
＜223〉design according to multiple-shaped nuohan inferior yeast (Hansenula Polymorpha) cance high-expression gene codon usage
<400>11
atcttgccag?acccattgaa?gccaaccaag?agatccttca?tcgaggactt?gttgttcaac 60
aaggtcacct?tggctgacgc?tggtttcatg?aagcaatacg?gtgagtgttt?gggtgacatc 120
aacgctagag?acttgatctg?tgctcaaaag?ttcaacggtt?tgaccgtctt?gccaccattg 180
ttgaccgacg?acatgatcgc?tgcttacacc?gctgctttgg?tctccggtac?cgctaccgct 240
ggttggacct?tcggtgctgg?tgctgctttg?caaatcccat?tcgctatgca?aatggcttac 300
agattcaacg?gtatcggtgt?cacccaaaac?gtcttgtacg?agaaccaaaa?gcaaatcgct 360
aaccaattca?acaaggctat?ctcccaaatc?caagagtcct?tgaccaccac?ctccaccgct 420
ttgggtaagt?tgcaagacgt?cgtcaaccaa?aacgctcaag?ctttgaacac?cttggtcaag 480
caattgtcct?ccaacttcgg?tgctatctcc?tccgtcttga?acgacatctt?gtccagattg 540
gacaaggtcg?aggctgaggt?ccaaatcgac?agattgatca?ccggtagatt?gcaatccttg 600
caaacctacg?tcacccaaca?attgatcaga?gctgctgaga?tcagagcttc?cgctaacttg 660
gctgctacca?agatgtccga?gtgtgtcttg?ggtcaatcca?agagagtcga?cttctgtggt 720
aagggttacc?acttgatgtc?cttcccacaa?gctgctccac?acggtgtcgt?cttcttgcac 780
gtcacctacg?tcccatccca?agagagaaac ttcaccaccg?ctccagctat?ctgtcacgag 840
ggtaaggctt?acttcccaag?agagggtgtc?ttcgtcttca?acggtacctc?ctggttcatc 900
acccaaagaa?acttcttctc?cccacaaatc atcaccaccg?acaacacctt?cgtctccggt 960
aactgtgacg?tcgtcatcgg?tatcatcaac?aacaccgtct?acgacccatt?gcaaccagag 1020
ttggactcct?tcaaggagga?gttggacaag?tacttcaaga?accacacctc?cccagacgtc 1080
gacttgggtg?acatctccgg?tatcaacgct?tccgtcgtca?acatccaaaa?ggagatcgac 1140
agattgaacg?aggtcgctaa?gaacttgaac?gagtccttga?tcgacttgca?agagttgggt 1200
aagtac 1206
<210>12
<211>1255
<212>PRT
<213>Coronavirus
<400>12
Met?Phe?Ile?Phe?Leu?Leu?Phe?Leu?Thr?Leu?Thr?Ser?Gly?Ser?Asp?Leu
1 5 10 15
Asp?Arg?Cys?Thr?Thr?Phe?Asp?Asp?Val?Gln?Ala?Pro?Asn?Tyr?Thr?Gln
20 25 30
His?Thr?Ser?Ser?Met?Arg?Gly?Val?Tyr?Tyr?Pro?Asp?Glu?Ile?Phe?Arg
35 40 45
Ser?Asp?Thr?Leu?Tyr?Leu?Thr?Gln?Asp?Leu?Phe?Leu?Pro?Phe?Tyr?Ser
50 55 60
Asn?Val?Thr?Gly?Phe?His?Thr?Ile?Asn?His?Thr?Phe?Asp?Asn?Pro?Val
65 70 75 80
Ile?Pro?Phe?Lys?Asp?Gly?Ile?Tyr?Phe?Ala?Ala?Thr?Glu?Lys?Ser?Asn
85 90 95
Val?Val?Arg?Gly?Trp?Val?Phe?Gly?Ser?Thr?Met?Asn?Asn?Lys?Ser?Gln
100 105 110
Ser?Val?Ile?Ile?Ile?Asn?Asn?Ser?Thr?Asn?Val?Val?Ile?Arg?Ala?Cys
115 120 125
Asn?Phe?Glu?Leu?Cys?Asp?Asn?Pro?Phe?Phe?Ala?Val?Ser?Lys?Pro?Met
130 135 140
Gly?Thr?Gln?Thr?His?Thr?Met?Ile?Phe?Asp?Asn?Ala?Phe?Asn?Cys?Thr
145 150 155 160
Phe?Glu?Tyr?Ile?Ser?Asp?Ala?Phe?Ser?Leu?Asp?Val?Ser?Glu?Lys?Ser
165 170 175
Gly?Asn?Phe?Lys?His?Leu?Arg?Glu?Phe?Val?Phe?Lys?Asn?Lys?Asp?Gly
180 185 190
Phe?Leu?Tyr?Val?Tyr?Lys?Gly?Tyr?Gln?Pro?Ile?Asp?Val?Val?Arg?Asp
195 200 205
Leu?Pro?Ser?Gly?Phe?Asn?Thr?Leu?Lys?Pro?Ile?Phe?Lys?Leu?Pro?Leu
210 215 220
Gly?Ile?Asn?Ile?Thr?Asn?Phe?Arg?Ala?Ile?Leu?Thr?Ala?Phe?Ser?Pro
225 230 235 240
Ala?Gln?Asp?Thr?Trp?Gly?Thr?Ser?Ala?Ala?Ala?Tyr?Phe?Val?Gly?Tyr
245 250 255
Leu?Lys?Pro?Thr?Thr?Phe?Met?Leu?Lys?Tyr?Asp?Glu?Asn?Gly?Thr?Ile
260 265 270
Thr?Asp?Ala?Val?Asp?Cys?Ser?Gln?Asn?Pro?Leu?Ala?Glu?Leu?Lys?Cys
275 280 285
Ser?Val?Lys?Ser?Phe?Glu?Ile?Asp?Lys?Gly?Ile?Tyr?Gln?Thr?Ser?Asn
290 295 300
Phe?Arg?Val?Val?Pro?Ser?Gly?Asp?Val?Val?Arg?Phe?Pro?Asn?Ile?Thr
305 310 315 320
Asn?Leu?Cys?Pro?Phe?Gly?Glu?Val?Phe?Asn?Ala?Thr?Lys?Phe?Pro?Ser
325 330 335
Val?Tyr?Ala?Trp?Glu?Arg?Lys?Lys?Ile?Ser?Asn?Cys?Val?Ala?Asp?Tyr
340 345 350
Ser?Val?Leu?Tyr?Asn?Ser?Thr?Phe?Phe?Ser?Thr?Phe?Lys?Cys?Tyr?Gly
355 360 365
Val?Ser?Ala?Thr?Lys?Leu?Asn?Asp?Leu?Cys?Phe?Ser?Asn?Val?Tyr?Ala
370 375 380
Asp?Ser?Phe?Val?Val?Lys?Gly?Asp?Asp?Val?Arg?Gln?Ile?Ala?Pro?Gly
385 390 395 400
Gln?Thr?Gly?Val?Ile?Ala?Asp?Tyr?Asn?Tyr?Lys?Leu?Pro?Asp?Asp?Phe
405 410 415
Met?Gly?Cys?Val?Leu?Ala?Trp?Asn?Thr?Arg?Asn?Ile?Asp?Ala?Thr?Ser
420 425 430
Thr?Gly?Asn?Tyr?Asn?Tyr?Lys?Tyr?Arg?Tyr?Leu?Arg?His?Gly?Lys?Leu
435 440 445
Arg?Pro?Phe?Glu?Arg?Asp?Ile?Ser?Asn?Val?Pro?Phe?Ser?Pro?Asp?Gly
450 455 460
Lys?Pro?Cys?Thr?Pro?Pro?Ala?Leu?Asn?Cys?Tyr?Trp?Pro?Leu?Asn?Asp
465 470 475 480
Tyr?Gly?Phe?Tyr?Thr?Thr?Thr?Gly?Ile?Gly?Tyr?Gln?Pro?Tyr?Arg?Val
485 490 495
Val?Val?Leu?Ser?Phe?Glu?Leu?Leu?Asn?Ala?Pro?Ala?Thr?Val?Cys?Gly
500 505 510
Pro?Lys?Leu?Ser?Thr?Asp?Leu?Ile?Lys?Asn?Gln?Cys?Val?Asn?Phe?Asn
515 520 525
Phe?Asn?Gly?Leu?Thr?Gly?Thr?Gly?Val?Leu?Thr?Pro?Ser?Ser?Lys?Arg
530 535 540
Phe?Gln?Pro?Phe?Gln?Gln?Phe?Gly?Arg?Asp?Val?Ser?Asp?Phe?Thr?Asp
545 550 555 560
Ser?Val?Arg?Asp?Pro?Lys?Thr?Ser?Glu?Ile?Leu?Asp?Ile?Ser?Pro?Cys
565 570 575
Ser?Phe?Gly?Gly?Val?Ser?Val?Ile?Thr?Pro?Gly?Thr?Asn?Ala?Ser?Ser
580 585 590
Glu?Val?Ala?Val?Leu?Tyr?Gln?Asp?Val?Asn?Cys?Thr?Asp?Val?Ser?Thr
595 600 605
Ala?Ile?His?Ala?Asp?Gln?Leu?Thr?Pro?Ala?Trp?Arg?Ile?Tyr?Ser?Thr
610 615 620
Gly?Asn?Asn?Val?Phe?Gln?Thr?Gln?Ala?Gly?Cys?Leu?Ile?Gly?Ala?Glu
625 630 635 640
His?Val?Asp?Thr?Ser?Tyr?Glu?Cys?Asp?Ile?Pro?Ile?Gly?Ala?Gly?Ile
645 650 655
Cys?Ala?Ser?Tyr?His?Thr?Val?Ser?Leu?Leu?Arg?Ser?Thr?Ser?Gln?Lys
660 665 670
Ser?Ile?Val?Ala?Tyr?Thr?Met?Ser?Leu?Gly?Ala?Asp?Ser?Ser?Ile?Ala
675 680 685
Tyr?Ser?Asn?Asn?Thr?Ile?Ala?Ile?Pro?Thr?Asn?Phe?Ser?Ile?Ser?Ile
690 695 700
Thr?Thr?Glu?Val?Met?Pro?Val?Ser?Met?Ala?Lys?Thr?Ser?Val?Asp?Cys
705 710 715 720
Asn?Met?Tyr?Ile?Cys?Gly?Asp?Ser?Thr?Glu?Cys?Ala?Asn?Leu?Leu?Leu
725 730 735
Gln?Tyr?Gly?Ser?Phe?Cys?Thr?Gln?Leu?Asn?Arg?Ala?Leu?Ser?Gly?Ile
740 745 750
Ala?Ala?Glu?Gln?Asp?Arg?Asn?Thr?Arg?Glu?Val?Phe?Ala?Gln?Val?Lys
755 760 765
Gln?Met?Tyr?Lys?Thr?Pro?Thr?Leu?Lys?Tyr?Phe?Gly?Gly?Phe?Asn?Phe
770 775 780
Ser?Gln?Ile?Leu?Pro?Asp?Pro?Leu?Lys?Pro?Thr?Lys?Arg?Ser?Phe?Ile
785 790 795 800
Glu?Asp?Leu?Leu?Phe?Asn?Lys?Val?Thr?Leu?Ala?Asp?Ala?Gly?Phe?Met
805 810 815
Lys?Gln?Tyr?Gly?Glu?Cys?Leu?Gly?Asp?Ile?Asn?Ala?Arg?Asp?Leu?Ile
820 825 830
Cys?Ala?Gln?Lys?Phe?Asn?Gly?Leu?Thr?Val?Leu?Pro?Pro?Leu?Leu?Thr
835 840 845
Asp?Asp?Met?Ile?Ala?Ala?Tyr?Thr?Ala?Ala?Leu?Val?Ser?Gly?Thr?Ala
850 855 860
Thr?Ala?Gly?Trp?Thr?Phe?Gly?Ala?Gly?Ala?Ala?Leu?Gln?Ile?Pro?Phe
865 870 875 880
Ala?Met?Gln?Met?Ala?Tyr?Arg?Phe?Asn?Gly?Ile?Gly?Val?Thr?Gln?Asn
885 890 895
Val?Leu?Tyr?Glu?Asn?Gln?Lys?Gln?Ile?Ala?Asn?Gln?Phe?Asn?Lys?Ala
900 905 910
Ile?Ser?Gln?Ile?Gln?Glu?Ser?Leu?Thr?Thr?Thr?Ser?Thr?Ala?Leu?Gly
915 920 925
Lys?Leu?Gln?Asp?Val?Val?Asn?Gln?Asn?Ala?Gln?Ala?Leu?Asn?Thr?Leu
930 935 940
Val?Lys?Gln?Leu?Ser?Ser?Asn?Phe?Gly?Ala?Ile?Ser?Ser?Val?Leu?Asn
945 950 955 960
Asp?Ile?Leu?Ser?Arg?Leu?Asp?Lys?Val?Glu?Ala?Glu?Val?Gln?Ile?Asp
965 970 975
Arg?Leu?Ile?Thr?Gly?Arg?Leu?Gln?Ser?Leu?Gln?Thr?Tyr?Val?Thr?Gln
980 985 990
Gln?Leu?Ile?Arg?Ala?Ala?Glu?Ile?Arg?Ala?Ser?Ala?Asn?Leu?Ala?Ala
995 1000 1005
Thr?Lys?Met?Ser?Glu?Cys?Val?Leu?Gly?Gln?Ser?Lys?Arg?Val?Asp
1010 1015 1020
Phe?Cys?Gly?Lys?Gly?Tyr?His?Leu?Met?Ser?Phe?Pro?Gln?Ala?Ala
1025 1030 1035
Pro?His?Gly?Val?Val?Phe?Leu?His?Val?Thr?Tyr?Val?Pro?Ser?Gln
1040 1045 1050
Glu?Arg?Asn?Phe?Thr?Thr?Ala?Pro?Ala?Ile?Cys?His?Glu?Gly?Lys
1055 1060 1065
Ala?Tyr?Phe?Pro?Arg?Glu?Gly?Val?Phe?Val?Phe?Asn?Gly?Thr?Ser
1070 1075 1080
Trp?Phe?Ile?Thr?Gln?Arg?Asn?Phe?Phe?Ser?Pro?Gln?Ile?Ile?Thr
1085 1090 1095
Thr?Asp?Asn?Thr?Phe?Val?Ser?Gly?Asn?Cys?Asp?Val?Val?Ile?Gly
1100 1105 1110
Ile?Ile?Asn?Asn?Thr?Val?Tyr?Asp?Pro?Leu?Gln?Pro?Glu?Leu?Asp
1115 1120 1125
Ser?Phe?Lys?Glu?Glu?Leu?Asp?Lys?Tyr?Phe?Lys?Asn?His?Thr?Ser
1130 1135 1140
Pro?Asp?Val?Asp?Leu?Gly?Asp?Ile?Ser?Gly?Ile?Asn?Ala?Ser?Val
1145 1150 1155
Val?Asn?Ile?Gln?Lys?Glu?Ile?Asp?Arg?Leu?Asn?Glu?Val?Ala?Lys
1160 1165 1170
Asn?Leu?Asn?Glu?Ser?Leu?Ile?Asp?Leu?Gln?Glu?Leu?Gly?Lys?Tyr
1175 1180 1185
Glu?Gln?Tyr?Ile?Lys?Trp?Pro?Trp?Tyr?Val?Trp?Leu?Gly?Phe?Ile
1190 1195 1200
Ala?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 1230
Ser?Cys?Cys?Lys?Phe?Asp?Glu?Asp?Asp?Ser?Glu?Pro?Val?Leu?Lys
1235 1240 1245
Gly?Val?Lys?Leu?His?Tyr?Thr
1250 1255
<210>13
<211>624
<212>PRT
<213>Coronavirus
<400>13
Asp?Arg?Cys?Thr?Thr?Phe?Asp?Asp?Val?Gln?Ala?Pro?Asn?Tyr?Thr?Gln
1 5 10 15
His?Thr?Ser?Ser?Met?Arg?Gly?Val?Tyr?Tyr?Pro?Asp?Glu?Ile?Phe?Arg
20 25 30
Ser?Asp?Thr?Leu?Tyr?Leu?Thr?Gln?Asp?Leu?Phe?Leu?Pro?Phe?Tyr?Ser
35 40 45
Asn?Val?Thr?Gly?Phe?His?Thr?Ile?Asn?His?Thr?Phe?Asp?Asn?Pro?Val
50 55 60
Ile?Pro?Phe?Lys?Asp?Gly?Ile?Tyr?Phe?Ala?Ala?Thr?Glu?Lys?Ser?Asn
65 70 75 80
Val?Val?Arg?Gly?Trp?Val?Phe?Gly?Ser?Thr?Met?Asn?Asn?Lys?Ser?Gln
85 90 95
Ser?Val?Ile?Ile?Ile?Asn?Asn?Ser?Thr?Asn?Val?Val?Ile?Arg?Ala?Cys
100 105 110
Asn?Phe?Glu?Leu?Cys?Asp?Asn?Pro?Phe?Phe?Ala?Val?Ser?Lys?Pro?Met
115 120 125
Gly?Thr?Gln?Thr?His?Thr?Met?Ile?Phe?Asp?Asn?Ala?Phe?Asn?Cys?Thr
130 135 140
Phe?Glu?Tyr?Ile?Ser?Asp?Ala?Phe?Ser?Leu?Asp?Val?Ser?Glu?Lys?Ser
145 150 155 160
Gly?Asn?Phe?Lys?His?Leu?Arg?Glu?Phe?Val?Phe?Lys?Asn?Lys?Asp?Gly
165 170 175
Phe?Leu?Tyr?Val?Tyr?Lys?Gly?Tyr?Gln?Pro?Ile?Asp?Val?Val?Arg?Asp
180 185 190
Leu?Pro?Ser?Gly?Phe?Asn?Thr?Leu?Lys?Pro?Ile?Phe?Lys?Leu?Pro?Leu
195 200 205
Gly?Ile?Asn?Ile?Thr?Asn?Phe?Arg?Ala?Ile?Leu?Thr?Ala?Phe?Ser?Pro
210 215 220
Ala?Gln?Asp?Thr?Trp?Gly?Thr?Ser?Ala?Ala?Ala?Tyr?Phe?Val?Gly?Tyr
225 230 235 240
Leu?Lys?Pro?Thr?Thr?Phe?Met?Leu?Lys?Tyr?Asp?Glu?Asn?Gly?Thr?Ile
245 250 255
Thr?Asp?Ala?Val?Asp?Cys?Ser?Gln?Asn?Pro?Leu?Ala?Glu?Leu?Lys?Cys
260 265 270
Ser?Val?Lys?Ser?Phe?Glu?Ile?Asp?Lys?Gly?Ile?Tyr?Gln?Thr?Ser?Asn
275 280 285
Phe?Arg?Val?Val?Pro?Ser?Gly?Asp?Val?Val?Arg?Phe?Pro?Asn?Ile?Thr
290 295 300
Asn?Leu?Cys?Pro?Leu?Gly?Glu?Val?Phe?Asn?Ala?Thr?Lys?Phe?Pro?Ser
305 310 315 320
Val?Tyr?Ala?Trp?Glu?Arg?Lys?Lys?Ile?Ser?Asn?Cys?Val?Ala?Asp?Tyr
325 330 335
Ser?Val?Leu?Tyr?Asn?Ser?Thr?Phe?Phe?Ser?Thr?Phe?Lys?Cys?Tyr?Gly
340 345 350
Val?Ser?Ala?Thr?Lys?Leu?Asn?Asp?Leu?Cys?Phe?Ser?Asn?Val?Tyr?Ala
355 360 365
Asp?Ser?Phe?Val?Val?Lys?Gly?Asp?Asp?Val?Arg?Gln?Ile?Ala?Pro?Gly
370 375 380
Pro?Thr?Gly?Val?Ile?Ala?Asp?Tyr?Asn?Tyr?Lys?Leu?Pro?Asp?Asp?Phe
385 390 395 400
Met?Gly?Cys?Val?Leu?Ala?Trp?Asn?Thr?Arg?Asn?Ile?Asp?Ala?Thr?Ser
405 410 415
Thr?Gly?Asn?Tyr?Asn?Tyr?Lys?Tyr?Arg?Tyr?Leu?Arg?His?Gly?Lys?Leu
420 425 430
Arg?Pro?Phe?Glu?Arg?Asp?Ile?Ser?Asn?Val?Pro?Phe?Ser?Pro?Asp?Gly
435 440 445
Lys?Pro?Cys?Thr?Pro?Pro?Ala?Leu?Asn?Cys?Tyr?Trp?Pro?Leu?Asn?Asp
450 455 460
Tyr?Gly?Phe?Tyr?Thr?Thr?Thr?Gly?Ile?Gly?Tyr?Gln?Pro?Tyr?Arg?Val
465 470 475 480
Val?Val?Leu?Ser?Phe?Glu?Leu?Leu?Asn?Ala?Pro?Ala?Thr?Val?Cys?Gly
485 490 495
Pro?Lys?Leu?Ser?Thr?Asp?Leu?Ile?Lys?Asn?Gln?Cys?Val?Asn?Phe?Asn
500 505 510
Phe?Asn?Gly?Leu?Thr?Gly?Thr?Gly?Val?Leu?Thr?Pro?Ser?Ser?Lys?Arg
515 520 525
Phe?Gln?Pro?Phe?Gln?Gln?Phe?Gly?Arg?Asp?Val?Ser?Asp?Phe?Thr?Asp
530 535 540
Ser?Val?Arg?Asp?Pro?Lys?Thr?Ser?Glu?Ile?Leu?Asp?Ile?Ser?Pro?Cys
545 550 555 560
Ser?Phe?Gly?Gly?Val?Ser?Val?Ile?Thr?Pro?Gly?Thr?Asn?Ala?Ser?Ser
565 570 575
Glu?Val?Ala?Val?Leu?Tyr?Gln?Asp?Val?Asn?Cys?Thr?Asp?Val?Ser?Thr
580 585 590
Ala?Ile?His?Ala?Asp?Gln?Leu?Thr?Pro?Ala?Trp?Arg?Ile?Tyr?Ser?Thr
595 600 605
Gly?Asn?Asn?Val?Phe?Gln?Thr?Gln?Ala?Gly?Cys?Leu?Ile?Gly?Ala?Glu
610 615 620
<210>14
<211>544
<212>PRT
<213>Coronavirus
<400>14
Val?Thr?Gly?Phe?His?Thr?Ile?Asn?His?Thr?Phe?Asp?Asn?Pro?Val?Ile
1 5 10 15
Pro?Phe?Lys?Asp?Gly?Ile?Tyr?Phe?Ala?Ala?Thr?Glu?Lys?Ser?Asn?Val
20 25 30
Val?Arg?Gly?Trp?Val?Phe?Gly?Ser?Thr?Met?Asn?Asn?Lys?Ser?Gln?Ser
35 40 45
Val?Ile?Ile?Ile?Asn?Asn?Ser?Thr?Asn?Val?Val?Ile?Arg?Ala?Cys?Asn
50 55 60
Phe?Glu?Leu?Cys?Asp?Asn?Pro?Phe?Phe?Ala?Val?Ser?Lys?Pro?Met?Gly
65 70 75 80
Thr?Gln?Thr?His?Thr?Met?Ile?Phe?Asp?Asn?Ala?Phe?Asn?Cys?Thr?Phe
85 90 95
Glu?Tyr?Ile?Ser?Asp?Ala?Phe?Ser?Leu?Asp?Val?Ser?Glu?Lys?Ser?Gly
100 105 110
Asn?Phe?Lys?His?Leu?Arg?Glu?Phe?Val?Phe?Lys?Asn?Lys?Asp?Gly?Phe
115 120 125
Leu?Tyr?Val?Tyr?Lys?Gly?Tyr?Gln?Pro?Ile?Asp?Val?Val?Arg?Asp?Leu
130 135 140
Pro?Ser?Gly?Phe?Asn?Thr?Leu?Lys?Pro?Ile?Phe?Lys?Leu?Pro?Leu?Gly
145 150 155 160
Ile?Asn?Ile?Thr?Asn?Phe?Arg?Ala?Ile?Leu?Thr?Ala?Phe?Ser?Pro?Ala
165 170 175
Gln?Asp?Thr?Trp?Gly?Thr?Ser?Ala?Ala?Ala?Tyr?Phe?Val?Gly?Tyr?Leu
180 185 190
Lys?Pro?Thr?Thr?Phe?Met?Leu?Lys?Tyr?Asp?Glu?Asn?Gly?Thr?Ile?Thr
195 200 205
Asp?Ala?Val?Asp?Cys?Ser?Gln?Asn?Pro?Leu?Ala?Glu?Leu?Lys?Cys?Ser
210 215 220
Val?Lys?Ser?Phe?Glu?Ile?Asp?Lys?Gly?Ile?Tyr?Gln?Thr?Ser?Asn?Phe
225 230 235 240
Arg?Val?Val?Pro?Ser?Gly?Asp?Val?Val?Arg?Phe?Pro?Asn?Ile?Thr?Asn
245 250 255
Leu?Cys?Pro?Leu?Gly?Glu?Val?Phe?Asn?Ala?Thr?Lys?Phe?Pro?Ser?Val
260 265 270
Tyr?Ala?Trp?Glu?Arg?Lys?Lys?Ile?Ser?Asn?Cys?Val?Ala?Asp?Tyr?Ser
275 280 285
Val?Leu?Tyr?Asn?Ser?Thr?Phe?Phe?Ser?Thr?Phe?Lys?Cys?Tyr?Gly?Val
290 295 300
Ser?Ala?Thr?Lys?Leu?Asn?Asp?Leu?Cys?Phe?Ser?Asn?Val?Tyr?Ala?Asp
305 310 315 320
Ser?Phe?Val?Val?Lys?Gly?Asp?Asp?Val?Arg?Gln?Ile?Ala?Pro?Gly?Pro
325 330 335
Thr?Gly?Val?Ile?Ala?Asp?Tyr?Asn?Tyr?Lys?Leu?Pro?Asp?Asp?Phe?Met
340 345 350
Gly?Cys?Val?Leu?Ala?Trp?Asn?Thr?Arg?Asn?Ile?Asp?Ala?Thr?Ser?Thr
355 360 365
Gly?Asn?Tyr?Asn?Tyr?Lys?Tyr?Arg?Tyr?Leu?Arg?His?Gly?Lys?Leu?Arg
370 375 380
Pro?Phe?Glu?Arg?Asp?Ile?Ser?Asn?Val?Pro?Phe?Ser?Pro?Asp?Gly?Lys
385 390 395 400
Pro?Cys?Thr?Pro?Pro?Ala?Leu?Asn?Cys?Tyr?Trp?Pro?Leu?Asn?Asp?Tyr
405 410 415
Gly?Phe?Tyr?Thr?Thr?Thr?Gly?Ile?Gly?Tyr?Gln?Pro?Tyr?Arg?Val?Val
420 425 430
Val?Leu?Ser?Phe?Glu?Leu?Leu?Asn?Ala?Pro?Ala?Thr?Val?Cys?Gly?Pro
435 440 445
Lys?Leu?Ser?Thr?Asp?Leu?Ile?Lys?Asn?Gln?Cys?Val?Asn?Phe?Asn?Phe
450 455 460
Asn?Gly?Leu?Thr?Gly?Thr?Gly?Val?Leu?Thr?Pro?Ser?Ser?Lys?Arg?Phe
465 470 475 480
Gln?Pro?Phe?Gln?Gln?Phe?Gly?Arg?Asp?Val?Ser?Asp?Phe?Thr?Asp?Ser
485 490 495
Val?Arg?Asp?Pro?Lys?Thr?Ser?Glu?Ile?Leu?Asp?Ile?Ser?Pro?Cys?Ser
500 505 510
Phe?Gly?Gly?Val?Ser?Val?Ile?Thr?Pro?Gly?Thr?Asn?Ala?Ser?Ser?Glu
515 520 525
Val?Ala?Val?Leu?Tyr?Gln?Asp?Val?Asn?Cys?Thr?Asp?Val?Ser?Thr?Ala
530 535 540
<210>15
<211>216
<212>PRT
<213>Coronavirus
<400>15
Asp?Arg?Cys?Thr?Thr?Phe?Asp?Asp?Val?Gln?Ala?Pro?Asn?Tyr?Thr?Gln
1 5 10 15
His?Thr?Ser?Ser?Met?Arg?Gly?Val?Tyr?Tyr?Pro?Asp?Glu?Ile?Phe?Arg
20 25 30
Ser?Asp?Thr?Leu?Tyr?Leu?Thr?Gln?Asp?Leu?Phe?Leu?Pro?Phe?Tyr?Ser
35 40 45
Asn?Val?Thr?Gly?Phe?His?Thr?Ile?Asn?His?Thr?Phe?Asp?Asn?Pro?Val
50 55 60
Ile?Pro?Phe?Lys?Asp?Gly?Ile?Tyr?Phe?Ala?Ala?Thr?Glu?Lys?Ser?Asn
65 70 75 80
Val?Val?Arg?Gly?Trp?Val?Phe?Gly?Ser?Thr?Met?Asn?Asn?Lys?Ser?Gln
85 90 95
Ser?Val?Ile?Ile?Ile?Asn?Asn?Ser?Thr?Asn?Val?Val?Ile?Arg?Ala?Cys
100 105 110
Asn?Phe?Glu?Leu?Cys?Asp?Asn?Pro?Phe?Phe?Ala?Val?Ser?Lys?Pro?Met
115 120 125
Gly?Thr?Gln?Thr?His?Thr?Met?Ile?Phe?Asp?Asn?Ala?Phe?Asn?Cys?Thr
130 135 140
Phe?Glu?Tyr?Ile?Ser?Asp?Ala?Phe?Ser?Leu?Asp?Val?Ser?Glu?Lys?Ser
145 150 155 160
Gly?Asn?Phe?Lys?His?Leu?Arg?Glu?Phe?Val?Phe?Lys?Asn?Lys?Asp?Gly
165 170 175
Phe?Leu?Tyr?Val?Tyr?Lys?Gly?Tyr?Gln?Pro?Ile?Asp?Val?Val?Arg?Asp
180 185 190
Leu?Pro?Ser?Gly?Phe?Asn?Thr?Leu?Lys?Pro?Ile?Phe?Lys?Leu?Pro?Leu
195 200 205
Gly?Ile?Asn?Ile?Thr?Asn?Phe?Arg
210 215
<210>16
<211>315
<212>PRT
<213>Coronavirus
<400>16
Lys?Pro?Thr?Thr?Phe?Met?Leu?Lys?Tyr?Asp?Glu?Asn?Gly?Thr?Ile?Thr
1 5 10 15
Asp?Ala?Val?Asp?Cys?Ser?Gln?Asn?Pro?Leu?Ala?Glu?Leu?Lys?Cys?Ser
20 25 30
Val?Lys?Ser?Phe?Glu?Ile?Asp?Lys?Gly?Ile?Tyr?Gln?Thr?Ser?Asn?Phe
35 40 45
Arg?Val?Val?Pro?Ser?Gly?Asp?Val?Val?Arg?Phe?Pro?Asn?Ile?Thr?Asn
50 55 60
Leu?Cys?Pro?Leu?Gly?Glu?Val?Phe?Asn?Ala?Thr?Lys?Phe?Pro?Ser?Val
65 70 75 80
Tyr?Ala?Trp?Glu?Arg?Lys?Lys?Ile?Ser?Asn?Cys?Val?Ala?Asp?Tyr?Ser
85 90 95
Val?Leu?Tyr?Asn?Ser?Thr?Phe?Phe?Ser?Thr?Phe?Lys?Cys?Tyr?Gly?Val
100 105 110
Ser?Ala?Thr?Lys?Leu?Asn?Asp?Leu?Cys?Phe?Ser?Asn?Val?Tyr?Ala?Asp
115 120 125
Ser?Phe?Val?Val?Lys?Gly?Asp?Asp?Val?Arg?Gln?Ile?Ala?Pro?Gly?Pro
130 135 140
Thr?Gly?Val?Ile?Ala?Asp?Tyr?Asn?Tyr?Lys?Leu?Pro?Asp?Asp?Phe?Met
145 150 155 160
Gly?Cys?Val?Leu?Ala?Trp?Asn?Thr?Arg?Asn?Ile?Asp?Ala?Thr?Ser?Thr
165 170 175
Gly?Asn?Tyr?Asn?Tyr?Lys?Tyr?Arg?Tyr?Leu?Arg?His?Gly?Lys?Leu?Arg
180 185 190
Pro?Phe?Glu?Arg?Asp?Ile?Ser?Asn?Val?Pro?Phe?Ser?Pro?Asp?Gly?Lys
195 200 205
Pro?Cys?Thr?Pro?Pro?Ala?Leu?Asn?Cys?Tyr?Trp?Pro?Leu?Asn?Asp?Tyr
210 215 220
Gly?Phe?Tyr?Thr?Thr?Thr?Gly?Ile?Gly?Tyr?Gln?Pro?Tyr?Arg?Val?Val
225 230 235 240
Val?Leu?Ser?Phe?Glu?Leu?Leu?Asn?Ala?pro?Ala?Thr?Val?Cys?Gly?Pro
245 250 255
Lys?Leu?Ser?Thr?Asp?Leu?Ile?Lys?Asn?Gln?Cys?Val?Asn?Phe?Asn?Phe
260 265 270
Asn?Gly?Leu?Thr?Gly?Thr?Gly?Val?Leu?Thr?Pro?Ser?Ser?Lys?Arg?Phe
275 280 285
Gln?Pro?Phe?Gln?Gln?Phe?Gly?Arg?Asp?Val?Ser?Asp?Phe?Thr?Asp?Ser
290 295 300
Val?Arg?Asp?Pro?Lys?Thr?Ser?Glu?Ile?Leu?Asp
305 310 315
<210>17
<211>607
<212>PRT
<213>Coronavirus
<400>17
His?Val?Asp?Thr?Ser?Tyr?Glu?Cys?Asp?Ile?Pro?Ile?Gly?Ala?Gly?Ile
1 5 10 15
Cys?Ala?Ser?Tyr?His?Thr?Val?Ser?Leu?Leu?Arg?Ser?Thr?Ser?Gln?Lys
20 25 30
Ser?Ile?Val?Ala?Tyr?Thr?Met?Ser?Leu?Gly?Ala?Asp?Ser?Ser?Ile?Ala
35 40 45
Tyr?Ser?Asn?Asn?Thr?Ile?Ala?Ile?Pro?Thr?Asn?Phe?Ser?Ile?Ser?Ile
50 55 60
Thr?Thr?Glu?Val?Met?Pro?Val?Ser?Met?Ala?Lys?Thr?Ser?Val?Asp?Cys
65 70 75 80
Asn?Met?Tyr?Ile?Cys?Gly?Asp?Ser?Thr?Glu?Cys?Ala?Asn?Leu?Leu?Leu
85 90 95
Gln?Tyr?Gly?Ser?Phe?Cys?Thr?Gln?Leu?Asn?Arg?Ala?Leu?Ser?Gly?Ile
100 105 110
Ala?Ala?Glu?Gln?Asp?Arg?Asn?Thr?Arg?Glu?Val?Phe?Ala?Gln?Val?Lys
115 120 125
Gln?Met?Tyr?Lys?Thr?Pro?Thr?Leu?Lys?Tyr?Phe?Gly?Gly?Phe?Asn?Phe
130 135 140
Ser?Gln?Ile?Leu?Pro?Asp?Pro?Leu?Lys?Pro?Thr?Lys?Arg?Ser?Phe?Ile
145 150 155 160
Glu?Asp?Leu?Leu?Phe?Asn?Lys?Val?Thr?Leu?Ala?Asp?Ala?Gly?Phe?Met
165 170 175
Lys?Gln?Tyr?Gly?Glu?Cys?Leu?Gly?Asp?Ile?Asn?Ala?Arg?Asp?Leu?Ile
180 185 190
Cys?Ala?Gln?Lys?Phe?Asn?Gly?Leu?Thr?Val?Leu?Pro?Pro?Leu?Leu?Thr
195 200 205
Asp?Asp?Met?Ile?Ala?Ala?Tyr?Thr?Ala?Ala?Leu?Val?Ser?Gly?Thr?Ala
210 215 220
Thr?Ala?Gly?Trp?Thr?Phe?Gly?Ala?Gly?Ala?Ala?Leu?Gln?Ile?Pro?Phe
225 230 235 240
Ala?Met?Gln?Met?Ala?Tyr?Arg?Phe?Asn?Gly?Ile?Gly?Val?Thr?Gln?Asn
245 250 255
Val?Leu?Tyr?Glu?Asn?Gln?Lys?Gln?Ile?Ala?Asn?Gln?Phe?Asn?Lys?Ala
260 265 270
Ile?Ser?Gln?Ile?Gln?Glu?Ser?Leu?Thr?Thr?Thr?Ser?Thr?Ala?Leu?Gly
275 280 285
Lys?Leu?Gln?Asp?Val?Val?Asn?Gln?Asn?Ala?Gln?Ala?Leu?Asn?Thr?Leu
290 295 300
Val?Lys?Gln?Leu?Ser?Ser?Asn?Phe?Gly?Ala?Ile?Ser?Ser?Val?Leu?Asn
305 310 315 320
Asp?Ile?Leu?Ser?Arg?Leu?Asp?Lys?Val?Glu?Ala?Glu?Val?Gln?Ile?Asp
325 330 335
Arg?Leu?Ile?Thr?Gly?Arg?Leu?Gln?Ser?Leu?Gln?Thr?Tyr?Val?Thr?Gln
340 345 350
Gln?Leu?Ile?Arg?Ala?Ala?Glu?Ile?Arg?Ala?Ser?Ala?Asn?Leu?Ala?Ala
355 360 365
Thr?Lys?Met?Ser?Glu?Cys?Val?Leu?Gly?Gln?Ser?Lys?Arg?Val?Asp?Phe
370 375 380
Cys?Gly?Lys?Gly?Tyr?His?Leu?Met?Ser?Phe?Pro?Gln?Ala?Ala?Pro?His
385 390 395 400
Gly?Val?Val?Phe?Leu?His?Val?Thr?Tyr?Val?Pro?Ser?Gln?Glu?Arg?Asn
405 410 415
Phe?Thr?Thr?Ala?Pro?Ala?Ile?Cys?His?Glu?Gly?Lys?Ala?Tyr?Phe?Pro
420 425 430
Arg?Glu?Gly?Val?Phe?Val?Phe?Asn?Gly?Thr?Ser?Trp?Phe?Ile?Thr?Gln
435 440 445
Arg?Asn?Phe?Phe?Ser?Pro?Gln?Ile?Ile?Thr?Thr?Asp?Asn?Thr?Phe?Val
450 455 460
Ser?Gly?Asn?Cys?Asp?Val?Val?Ile?Gly?Ile?Ile?Asn?Asn?Thr?Val?Tyr
465 470 475 480
Asp?Pro?Leu?Gln?Pro?Glu?Leu?Asp?Ser?Phe?Lys?Glu?Glu?Leu?Asp?Lys
485 490 495
Tyr?Phe?Lys?Asn?His?Thr?Ser?Pro?Asp?Val?Asp?Leu?Gly?Asp?Ile?Ser
500 505 510
Gly?Ile?Asn?Ala?Ser?Val?Val?Asn?Ile?Gln?Lys?Glu?Ile?Asp?Arg?Leu
515 520 525
Asn?Glu?Val?Ala?Lys?Asn?Leu?Asn?Glu?Ser?Leu?Ile?Asp?Leu?Gln?Glu
530 535 540
Leu?Gly?Lys?Tyr?Glu?Gln?Tyr?Ile?Lys?Trp?Pro?Trp?Tyr?Val?Trp?Leu
545 550 555 560
Gly?Phe?Ile?Ala?Gly?Leu?Ile?Ala?Ile?Val?Met?Val?Thr?Ile?Leu?Leu
565 570 575
Cys?Cys?Met?Thr?Ser?Cys?Cys?Ser?Cys?Leu?Lys?Gly?Ala?Cys?Ser?Cys
580 585 590
Gly?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>18
His?Val?Asp?Thr?Ser?Tyr?Glu?Cys?Asp?Ile?Pro?Ile?Gly?Ala?Gly?Ile
1 5 10 15
Cys?Ala?Ser?Tyr?His?Thr?Val?Ser?Leu?Leu?Arg?Ser?Thr?Ser?Gln?Lys
20 25 30
Ser?Ile?Val?Ala?Tyr?Thr?Met?Ser?Leu?Gly?Ala?Asp?Ser?Ser?Ile?Ala
35 40 45
Tyr?Ser?Asn?Asn?Thr?Ile?Ala?Ile?Pro?Thr?Asn?Phe?Ser?Ile?Ser?Ile
50 55 60
Thr?Thr?Glu?Val?Met?Pro?Val?Ser?Met?Ala?Lys?Thr?Ser?Val?Asp?Cys
65 70 75 80
Asn?Met?Tyr?Ile?Cys?Gly?Asp?Ser?Thr?Glu?Cys?Ala?Asn?Leu?Leu?Leu
85 90 95
Gln?Tyr?Gly?Ser?Phe?Cys?Thr?Gln?Leu?Asn?Arg?Ala?Leu?Ser?Gly?Ile
100 105 110
Ala?Ala?Glu?Gln?Asp?Arg?Asn?Thr?Arg?Glu?Val?Phe?Ala?Gln?Val?Lys
115 120 125
Gln?Met?Tyr?Lys?Thr?Pro?Thr?Leu?Lys?Tyr?Phe?Gly?Gly?Phe?Asn?Phe
130 135 140
Ser?Gln?Ile?Leu?Pro?Asp?Pro?Leu?Lys?Pro?Thr?Lys?Arg?Ser?Phe?Ile
145 150 155 160
Glu?Asp?Leu?Leu?Phe?Asn?Lys?Val?Thr?Leu?Ala?Asp?Ala?Gly?Phe?Met
165 170 175
Lys?Gln?Tyr?Gly?Glu?Cys?Leu?Gly?Asp?Ile?Asn?Ala?Arg?Asp?Leu?Ile
180 185 190
Cys?Ala?Gln?Lys?Phe?Asn?Gly?Leu?Thr?Val?Leu?Pro?Pro?Leu?Leu?Thr
195 200 205
Asp?Asp?Met?Ile?Ala?Ala?Tyr?Thr
210 215
<210>19
<211>315
<212>PRT
<213>Coronavirus
<400>19
Gln?Met?Ala?Tyr?Arg?Phe?Asn?Gly?Ile?Gly?Val?Thr?Gln?Asn?Val?Leu
1 5 10 15
Tyr?Glu?Asn?Gln?Lys?Gln?Ile?Ala?Asn?Gln?Phe?Asn?Lys?Ala?Ile?Ser
20 25 30
Gln?Ile?Gln?Glu?Ser?Leu?Thr?Thr?Thr?Ser?Thr?Ala?Leu?Gly?Lys?Leu
35 40 45
Gln?Asp?Val?Val?Asn?Gln?Asn?Ala?Gln?Ala?Leu?Asn?Thr?Leu?Val?Lys
50 55 60
Gln?Leu?Ser?Ser?Asn?Phe?Gly?Ala?Ile?Ser?Ser?Val?Leu?Asn?Asp?Ile
65 70 75 80
Leu?Ser?Arg?Leu?Asp?Lys?Val?Glu?Ala?Glu?Val?Gln?Ile?Asp?Arg?Leu
85 90 95
Ile?Thr?Gly?Arg?Leu?Gln?Ser?Leu?Gln?Thr?Tyr?Val?Thr?Gln?Gln?Leu
100 105 110
Ile?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
130 135 140
Lys?Gly?Tyr?His?Leu?Met?Ser?Phe?Pro?Gln?Ala?Ala?Pro?His?Gly?Val
145 150 155 160
Val?Phe?Leu?His?Val?Thr?Tyr?Val?Pro?Ser?Gln?Glu?Arg?Asn?Phe?Thr
165 170 175
Thr?Ala?Pro?Ala?Ile?Cys?His?Glu?Gly?Lys?Ala?Tyr?Phe?Pro?Arg?Glu
180 185 190
Gly?Val?Phe?Val?Phe?Asn?Gly?Thr?Ser?Trp?Phe?Ile?Thr?Gln?Arg?Asn
195 200 205
Phe?Phe?Ser?Pro?Gln?Ile?Ile?Thr?Thr?Asp?Asn?Thr?Phe?Val?Ser?Gly
210 215 220
Asn?Cys?Asp?Val?Val?Ile?Gly?Ile?Ile?Asn?Asn?Thr?Val?Tyr?Asp?Pro
225 230 235 240
Leu?Gln?Pro?Glu?Leu?Asp?Ser?Phe?Lys?Glu?Glu?Leu?Asp?Lys?Tyr?Phe
245 250 255
Lys?Asn?His?Thr?Ser?Pro?Asp?Val?Asp?Leu?Gly?Asp?Ile?Ser?Gly?Ile
260 265 270
Asn?Ala?Ser?Val?Val?Asn?Ile?Gln?Lys?Glu?Ile?Asp?Arg?Leu?Asn?Glu
275 280 285
Val?Ala?Lys?Asn?Leu?Asn?Glu?Ser?Leu?Ile?Asp?Leu?Gln?Glu?Leu?Gly
290 295 300
Lys?Tyr?Glu?Gln?Tyr?Ile?Lys?Trp?Pro?Trp?Tyr
305 310 315
<210>20
<211>40
<212>PRT
<213>Coronavirus
<400>20
Gly?Asp?Ile?Ser?Gly?Ile?Asn?Ala?Ser?Val?Val?Asn?Ile?Gln?Lys?Glu
1 5 10 15
Ile?Asp?Arg?Leu?Asn?Glu?Val?Ala?Lys?Asn?Leu?Asn?Glu?Ser?Leu?Ile
20 25 30
Asp?Leu?Gln?Glu?Leu?Gly?Lys?Tyr
35 40
<210>21
<211>88
<212>PRT
<213>Coronavirus
<4O0>21
Gly?Asp?Ile?Ser?Gly?Ile?Asn?Ala?Ser?Val?Val?Asn?Ile?Gln?Lys?Glu
1 5 10 15
Ile?Asp?Arg?Leu?Asn?Glu?Val?Ala?Lys?Asn?Leu?Asn?Glu?Ser?Leu?Ile
20 25 30
Asp?Leu?Gln?Glu?Leu?Gly?Lys?Tyr?Glu?Gln?Tyr?Ile?Lys?Trp?Pro?Trp
35 40 45
Tyr?Val?Trp?Leu?Gly?Phe?Ile?Ala?Gly?Leu?Ile?Ala?Ile?Val?Met?Val
50 55 60
Thr?Ile?Leu?Leu?Cys?Cys?Met?Thr?Ser?Cys?Cys?Ser?Cys?Leu?Lys?Gly
65 70 75 80
Ala?Cys?Ser?Cys?Gly?Ser?Cys?Cys
85
<210>22
<211>402
<212>PRT
<213>Coronavirus
<400>22
Ile?Leu?Pro?Asp?Pro?Leu?Lys?Pro?Thr?Lys?Arg?Ser?Phe?Ile?Glu?Asp
1 5 10 15
Leu?Leu?Phe?Asn?Lys?Val?Thr?Leu?Ala?Asp?Ala?Gly?Phe?Met?Lys?Gln
20 25 30
Tyr?Gly?Glu?Cys?Leu?Gly?Asp?Ile?Asn?Ala?Arg?Asp?Leu?Ile?Cys?Ala
35 40 45
Gln?Lys?Phe?Asn?Gly?Leu?Thr?Val?Leu?Pro?Pro?Leu?Leu?Thr?Asp?Asp
50 55 60
Met?Ile?Ala?Ala?Tyr?Thr?Ala?Ala?Leu?Val?Ser?Gly?Thr?Ala?Thr?Ala
65 70 75 80
Gly?Trp?Thr?Phe?Gly?Ala?Gly?Ala?Ala?Leu?Gln?Ile?Pro?Phe?Ala?Met
85 90 95
Gln?Met?Ala?Tyr?Arg?Phe?Asn?Gly?Ile?Gly?Val?Thr?Gln?Asn?Val?Leu
100 105 110
Tyr?Glu?Asn?Gln?Lys?Gln?Ile?Ala?Asn?Gln?Phe?Asn?Lys?Ala?Ile?Ser
115 120 125
Gln?Ile?Gln?Glu?Ser?Leu?Thr?Thr?Thr?Ser?Thr?Ala?Leu?Gly?Lys?Leu
130 135 140
Gln?Asp?Val?Val?Asn?Gln?Asn?Ala?Gln?Ala?Leu?Asn?Thr?Leu?Val?Lys
145 150 155 160
Gln?Leu?Ser?Ser?Asn?Phe?Gly?Ala?Ile?Ser?Ser?Val?Leu?Asn?Asp?Ile
165 170 175
Leu?Ser?Arg?Leu?Asp?Lys?Val?Glu?Ala?Glu?Val?Gln?Ile?Asp?Arg?Leu
180 185 190
Ile?Thr?Gly?Arg?Leu?Gln?Ser?Leu?Gln?Thr?Tyr?Val?Thr?Gln?Gln?Leu
195 200 205
Ile?Arg?Ala?Ala?Glu?Ile?Arg?Ala?Ser?Ala?Asn?Leu?Ala?Ala?Thr?Lys
210 215 220
Met?Ser?Glu?Cys?Val?Leu?Gly?Gln?Ser?Lys?Arg?Val?Asp?Phe?Cys?Gly
225 230 235 240
Lys?Gly?Tyr?His?Leu?Met?Ser?Phe?Pro?Gln?Ala?Ala?Pro?His?Gly?Val
245 250 255
Val?Phe?Leu?His?Val?Thr?Tyr?Val?Pro?Ser?Gln?Glu?Arg?Asn?Phe?Thr
260 265 270
Thr?Ala?Pro?Ala?Ile?Cys?His?Glu?Gly?Lys?Ala?Tyr?Phe?Pro?Arg?Glu
275 280 285
Gly?Val?Phe?Val?Phe?Asn?Gly?Thr?Ser?Trp?Phe?Ile?Thr?Gln?Arg?Asn
290 295 300
Phe?Phe?Ser?Pro?Gln?Ile?Ile?Thr?Thr?Asp?Asn?Thr?Phe?Val?Ser?Gly
305 3l0 315 320
Asn?Cys?Asp?Val?Val?Ile?Gly?Ile?Ile?Asn?Asn?Thr?Val?Tyr?Asp?Pro
325 330 335
Leu?Gln?Pro?Glu?Leu?Asp?Ser?Phe?Lys?Glu?Glu?Leu?Asp?Lys?Tyr?Phe
340 345 350
Lys?Asn?His?Thr?Ser?Pro?Asp?Val?Asp?Leu?Gly?Asp?Ile?Ser?Gly?Ile
355 360 365
Asn?Ala?Ser?Val?Val?Asn?Ile?Gln?Lys?Glu?Ile?Asp?Arg?Leu?Asn?Glu
370 375 380
Val?Ala?Lys?Asn?Leu?Asn?Glu?Ser?Leu?Ile?Asp?Leu?Gln?Glu?Leu?Gly
385 390 395 400
Lys?Tyr

Claims (14)

1. derive from reorganization SARS virus S, S1, S1-1, S1-2, S1-3, S2, S2-1, S2-2, S2-3, S2-4, S2-5 gene that multiple-shaped nuohan inferior yeast (Hansenula polymorpha) is expressed, their nucleotide sequence is respectively by forming corresponding to the nucleotide sequence shown in the SEQ ID NO:1-11.
2. gene according to claim 1, multiple-shaped nuohan inferior yeast (Hansenulapolymorpha) the reorganization SARS virus S gene of expressing wherein, its nucleotide sequence is made up of the nucleotide sequence shown in the SEQ ID NO:1, and the aminoacid sequence of its encoded protein is made up of the aminoacid sequence shown in SEQ ID NO:12.
3. gene according to claim 1, multiple-shaped nuohan inferior yeast (Hansenulapolymorpha) the reorganization SARS virus S1 gene of expressing wherein, its nucleotide sequence is made up of the nucleotide sequence shown in the SEQ ID NO:2, and the aminoacid sequence of its encoded protein is made up of the aminoacid sequence shown in SEQ ID NO:13.
4. gene according to claim 1, multiple-shaped nuohan inferior yeast (Hansenulapolymorpha) the reorganization SARS virus S1-1 gene of expressing wherein, its nucleotide sequence is made up of the nucleotide sequence shown in the SEQ IDNO:3, and the aminoacid sequence of its encoded protein is made up of the aminoacid sequence shown in SEQ ID NO:14.
5. gene according to claim 1, multiple-shaped nuohan inferior yeast (Hansenulapolymorpha) the reorganization SARS virus S1-2 gene of expressing wherein, its nucleotide sequence is made up of the nucleotide sequence shown in the SEQ IDNO:4, and the aminoacid sequence of its encoded protein is made up of the aminoacid sequence shown in SEQ ID NO:15.
6. gene according to claim 1, multiple-shaped nuohan inferior yeast (Hansenulapolymorpha) the reorganization SARS virus S1-3 gene of expressing wherein, its nucleotide sequence is made up of the nucleotide sequence shown in the SEQ IDNO:5, and the aminoacid sequence of its encoded protein is made up of the aminoacid sequence shown in SEQ ID NO:16.
7. gene according to claim 1, multiple-shaped nuohan inferior yeast (Hansenulapolymorpha) the reorganization SARS virus S2 gene of expressing wherein, its nucleotide sequence is made up of the nucleotide sequence shown in the SEQ ID NO:6, and the aminoacid sequence of its encoded protein is made up of the aminoacid sequence shown in SEQ ID NO:17.
8. gene according to claim 1, multiple-shaped nuohan inferior yeast (Hansenulapolymorpha) the reorganization SARS virus S2-1 gene of expressing wherein, its nucleotide sequence is made up of the nucleotide sequence shown in the SEQ IDNO:7, and the aminoacid sequence of its encoded protein is made up of the aminoacid sequence shown in SEQ ID NO:18.
9. gene according to claim 1, multiple-shaped nuohan inferior yeast (Hansenulapolymorpha) the reorganization SARS virus S2-2 gene of expressing wherein, its nucleotide sequence is made up of the nucleotide sequence shown in the SEQ IDNO:8, and the aminoacid sequence of its encoded protein is made up of the aminoacid sequence shown in SEQ ID NO:19.
10. gene according to claim 1, multiple-shaped nuohan inferior yeast (Hansenulapolymorpha) the reorganization SARS virus S2-3 gene of expressing wherein, its nucleotide sequence is made up of the nucleotide sequence shown in the SEQ IDNO:9, and the aminoacid sequence of its encoded protein is made up of the aminoacid sequence shown in SEQ ID NO:20.
11. gene according to claim 1, multiple-shaped nuohan inferior yeast (Hansenulapolymorpha) the reorganization SARS virus S2-4 gene of expressing wherein, its nucleotide sequence is made up of the nucleotide sequence shown in the SEQ IDNO:10, and the aminoacid sequence of its encoded protein is made up of the aminoacid sequence shown in SEQ ID NO:21.
12. gene according to claim 1, multiple-shaped nuohan inferior yeast (Hansenulapolymorpha) the reorganization SARS virus S2-5 gene of expressing wherein, its nucleotide sequence is made up of the nucleotide sequence shown in the SEQ IDNO:11, and the aminoacid sequence of its encoded protein is made up of the aminoacid sequence shown in SEQ ID NO:22.
13. a proteic method for preparing the described genes encoding of claim 1 may further comprise the steps:
A) according to multiple-shaped nuohan inferior yeast cance high-expression gene codon usage optimization design SARS virus S, S1, S1-1, S1-2, S1-3, S2, S2-1, S2-2, S2-3, S2-4, S2-5 gene;
B) pass through gene machine, the newly-designed S of synthetic, S1, S1-1, S1-2, S1-3, S2, S2-1, S2-2, S2-3, S2-4, S2-5 gene, their nucleotide sequence are respectively by forming corresponding to the nucleotide sequence shown in the SEQ ID NO:1-11;
C) utilize multiple-shaped nuohan inferior yeast expression vector or other any can be in debaryomyces hansenii the carrier for expression of eukaryon of integrative gene expression, make up the recombinant expression vector that contains the SARS virus S, the S1 that have optimized, S1-1, S1-2, S1-3, S2, S2-1, S2-2, S2-3, S2-4, S2-5 gene;
D) abduction delivering of multiple-shaped nuohan inferior yeast reorganization SARS virus S, S1, S1-1, S1-2, S1-3, S2, S2-1, S2-2, S2-3, S2-4, S2-5 gene coded protein;
E) expression product is identified and the biologic activity evaluation.
14. according to reorganization SARS virus S, S1, S1-1, S1-2, S1-3, S2, S2-1, S2-2, S2-3, S2-4, the S2-5 gene that the described multiple-shaped nuohan inferior yeast of claim 1-12 (Hansenula polymorpha) is expressed, the application of optional one of them gene in sick curative drug of preparation SARS virus and preventative vaccine.
CNB031411584A 2003-06-12 2003-06-12 Expression of recombination SARS virus gene in pleiomorphic Hansen yeast and its use Expired - Fee Related CN1244698C (en)

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