CN1981032A - A novel strain of sars-associated coronavirus and use thereof - Google Patents

Abstract

The invention relates to a novel strain of severe acute respiratory syndrome (SARS)-associated coronavirus, resulting from a sample collected in Hanoi (Vietnam), reference number 031589, nucleic acid molecules originating from the genome of same, proteins and peptides coded by said nucleic acid molecules and, more specifically, protein N and the applications thereof, for example, as diagnostic reagents and/or as a vaccine.

Description

The novel strain of SARS associated coronavirus and application thereof

Technical field

The present invention relates to a novel strain of severe acute respiratory syndrome (SARS) associated coronavirus, this virus strain is derived from one and is registered as No.031589's and is collected in the sample of Ha Noi (Vietnam), the invention still further relates to the nucleic acid molecule that gets by its genome, also relate to protein and the peptide and the application thereof of described nucleic acid molecule encoding, particularly as diagnostic reagent and/or vaccine.

Background technology

Coronavirus is a kind of virus that contains the straight polarity single stranded RNA of about 30,000 bases, and its RNA duplicates in the tenuigenin of host cell; Its genomic 5 ' end has cap structure, and 3 ' end comprises the polyadenylic acid afterbody.This virus is wrapped in the coating, and contains the raised structures that is called thorn (spicule) on the surface.

This genome from its 5 ' hold 3 ' end comprise following opening code-reading frame or ORF: corresponding to the proteic ORF1a and the ORF1b that transcribe-duplicate mixture, corresponding to ORF-S, ORF-E, ORF-M and the ORF-N of structural protein S, E, M and N.It also comprises corresponding to coding unknown function proteic ORF: between ORF-S and ORF-E and with latter's overlapping areas; Zone between ORF-M and ORF-N; Be included in the zone of ORF-N.

S albumen is a kind of membrane glycoprotein (200-220kDa), and it is present in from the form of the thorn of peplos surface formation or furcella.It is attached to the acceptor of host cell and induces in peplos and the process that cytolemma merges mutually in virus and works.

Little envelope protein (E) is also referred to as sM (smallmembrane), is a kind of non-glycosylated transmembrane protein of about 10kDa size, is the minimum protein of content in the virosome.It has played vital role coronavirus in the process of sprouting of the region intermediate (compartiment interm é diaire) of endoplasmic reticulum and golgi body.

M albumen or stromatin (25-30kDa) are the more membrane glycoproteins of a kind of content, and it interacts by M/E and is integrated in the virion, and S albumen is attached in the virion by the S/M interaction.M albumen be it seems for the maturation of coronavirus and very important for the decision of virion packaging site.

N albumen or nucleocapsid protein (45-50kDa) are the most conservative in the coronavirus structural protein, it for the encapsidate of geneome RNA and domination its to combine with virosome all be essential.This albumen may also participate in duplicating of RNA.

When host cell is infected, the reading frame (ORF) that is positioned at viral genome 5 ' end is translated into a kind of polyprotein, this polyprotein discharges several Nonstructural Proteins after being cut by the proteolytic enzyme of virus, for example the RNA polymerase (Rep) and the ATP enzyme helicase (Hel) of RNA dependence.These two kinds of albumen duplicate and to be used for the generation of transcript of synthetic virus protein relevant with virus genomic.The generation mechanism of these subgenomic mRNAs also imperfectly understands.Yet the nearest fact shows that the sequence of the transcriptional control of 5 ' end of each gene has been represented the discontinuous signal of transcribing of regulating subgenomic mRNA.

Virus membrane antigen (S, E and M albumen) is inserted in the region intermediate, and RNA that duplicates (normal chain) and the assembling of N (nucleocapsid) albumen.When the nucleocapsid mixture sprouted in endoplasmic reticulum, this protein-RNA mixture and the M protein combination that is contained in the endoplasmic reticulum formed virion.The virus migration is passed Golgi complex and is finally left cell by for example exocytosis then.Virus is attached to the site of host cell just on the proteic position of S.

15% is caused by coronavirus to 30% in the human flu, it causes that also respiratory organs of animal and digestion organs infect, especially cat (FIPV: feline infectious peritonitis virus (Felineinfectious peritonitis virus)), poultry (IBV: birds infectious bronchitis virus (Avian infectious bronchitis virus)), mouse (MHV: murine hepatitis virus (Mouse hepatitis virus)), pig (TGEV: infectivity gastroenteritis virus (Transmissible gastroenterititis virus), PEDV: Porcine epidemic diarrhea virus (Pocine Epidemic Diarrhea virus), blood coagulation type encephalomyelitis virus (Hemagglutinating encephalomyelitis virus)) and ox (BCoV: bovine coronavirus (Bovine coronavirus)) PRCoV: PRCV (Porcine Respiratory Coronavirus) (Pocine Respiratory Coronavirus), HEV:.

Usually, every kind of coronavirus only infects species; In immunocompetent individuality, infection can be chosen wantonly and induce neutralizing antibody and the cellular immunization that can eliminate cells infected.

A kind of severe acute respiratory syndrome that is called severe acute respiratory syndrome (SARS) popular in appearing at the initial main epidemic-stricken area of China 2002 seasons at the year end spread to many countries (Vietnam, Hong Kong, Singapore, Thailand and Canada) the first quarter in 2003.To such an extent as to the very serious mortality ratio of this disease is 3% to 6%.Numerous in the world laboratories is all in the generation reason of studying this disease.

In March, 2003, a kind of new coronavirus that is associated with the case of severe acute respiratory syndrome (SARS-CoV or SARS virus) (T.G.KSIAZEK et al., TheNew England Journal of Medicine, 2003 have been separated, 348,1319-1330; C.DROSTENet al., The New Englnd Journal of Medicine, 2003,348,1967-1976; Peiris et al., Lancet, 2003,361,1319).

Then obtained the genome sequence of this novel coronavirus, particularly be that (May 1 for Genbank numbering AY274119.3 and A.MARRA et al., Science for the Urbani strain isolated, 2003,300,1399-1404) with Toronto strain isolated (Tor2, Genbank numbering AY278741 and A.ROTA et al., Science, 2003,300, sequence 1394-1399).

This genomic structure and other known coronavirus are similar, belong to coronaviridae (Coronaviridae) so can determine SARS-CoV; Following opening code-reading frame: ORF1a and ORF1b have specifically been identified and corresponding to the proteic opening code-reading frame of S, E, M and N; Proteins encoded the zone between ORF-S and the ORF-E (ORF3), between ORF-S and the ORF-E and and ORF-E overlapping areas (ORF4), between ORF-M and ORF-N zone (ORF7 to ORF11) and corresponding to the zone (ORF13 and ORF14) of ORF-N.

Seven place's differences between the sequence of Tor2 and Urbani strain isolated have been identified; 3 places corresponding silent mutation (16622 the c/t of ORF1b and 19064 a/g, 24872 the t/c of ORF-S), its aminoacid sequence has been changed at 4 places respectively: ORF1a encoded protein (7919 c/t correspondence A/V sudden change), S albumen (23220 g/t correspondence A/S sudden change), ORF3 encoded protein (25298 a/g correspondence R/G sudden change) and M albumen (26857 t/c correspondence S/P sudden change).

In addition, it is far that Phylogenetic Analysis shows that SARS-CoV and other coronavirus concern, it neither similarly is the mutant of people's respiratory organs coronavirus, the recombinant chou that also similarly is not known coronavirus (sees Holmes about its summary, J.C.I., 2003,111,1605-1609).

Measure and pay attention to new mutation and significance is all arranged for the immunogenic composition that exploitation has enough susceptibility and specific SARS check and diagnostic reagent and exploitation can protect people that SARS is spread.

Summary of the invention

The contriver has now identified another kind and Tor2 and the related coronavirus strain of the distinguishing SARS of Urbani strain isolated.

Therefore theme of the present invention is human coronary virus's strain that a kind of severe acute respiratory syndrome isolating or purifying is associated, it is characterized in that containing in its genome and occur: the codon glycine of the Serine codon of S protein gene 23220-23222 position or ORF3 gene 25298-25300 position with the complementary DNA form, and the Serine codon of the L-Ala codon of the 7918-7920 position of ORF1a or M protein gene 26857-26859 position, described position is represented with reference to Genbank sequence A Y274119.3.

An embodiment preferred according to described virus strain, its genomic DNA Equivalent contains the sequence corresponding to sequence SEQ ID No:1, this coronavirus strain derives from the sample of collecting the bronchoalveolar lavage fluid (lavage bronchoalv é olaire) from a SARS patient, and this sample record is No.031589 and is collected in Ha Noi (Vietnam) French hospital.

According to the present invention, described sequence SEQ ID No:1 is the thymus nucleic acid corresponding to the ribonucleic acid molecule of above-mentioned isolating coronavirus pnca gene group.

The difference part of sequence A Y274119.3 among sequence SEQ ID No:1 and the Genbank (Tor2 strain isolated) is that it has comprised following sudden change:

The g/t of-23220 positions; The L-Ala codon (gct) of 577 positions replaces with Serine codon (tct) in the proteic aminoacid sequence of the S of Tor2,

The a/g of-25298 positions; The arginine codon (aga) of 11 positions replaces with codon glycine (gga) in the aminoacid sequence of the ORF3 encoded protein of Tor2.

In addition, the difference part of the sequence A Y278741 among sequence SEQ ID No:1 and the Genbank (Urbani strain isolated) is that it has comprised following sudden change:

The t/c of-7919 positions; The Xie Ansuan codon (gtt) of 2552 positions replaces with L-Ala codon (gct) in the aminoacid sequence of ORF1a encoded protein,

The t/c of-16622 positions; This sudden change does not change the aminoacid sequence (silent mutation) of ORF1b encoded protein,

The g/a of-19064 positions; This sudden change does not change the aminoacid sequence (silent mutation) of ORF1b encoded protein,

The c/t of-24872 positions; This sudden change does not change the proteic aminoacid sequence of S, and

The c/t of-26857 positions; The proline(Pro) codon (ccc) of 154 positions replaces with Serine codon (tcc) in the proteic aminoacid sequence of M.

Unless spell out, otherwise the position of Nucleotide and peptide sequence is represented with reference to the sequence A Y274119.3 among the Genbank.

Theme of the present invention also has one section isolating or purified polynucleotides, it is characterized in that its sequence is the genomic sequence of the defined isolating coronavirus strain in front.

According to an embodiment preferred of polynucleotide of the present invention, it comprises sequence SEQID No:1.

Theme of the present invention also has one section isolating or purified polynucleotides, it is characterized in that its sequence can hybridize under highly tight condition with the defined polynucleotide sequence in front.

Term " isolating or purifying " means " through artificial " and modifies from state of nature, if an object exists at occurring in nature in other words, and promptly from its physical environment, the modifying and/or extract of so-called isolating or purifying.For example, a kind of natural be present in polynucleotide in the organism alive or proteins/peptides be exactly do not separate, unpurified; On the other hand, be this polynucleotide or proteins/peptides equally, separately obtained from the molecule that it coexists nature by method with clone, amplification and/or chemosynthesis, it is exactly isolating in the intent of the present invention.Furthermore, a kind ofly being introduced into polynucleotide or the proteins/peptides of organism with conversion, genetic manipulation or any other method, also is " isolating ", even if it is present in the described organism.The term purifying is used in to mean among the present invention according to proteins/peptides of the present invention and does not combine with other protein or polypeptide basically, for example the product of purifying or from the product of non-recombinant sources purifying from the recombinant host cell culture.

According to the intent of the present invention, highly tight hybridization conditions should be understood that the temperature selected and ionic strength conditions make and keep specific between the complementary polynucleotide and optionally hybridize.

As example, the favourable height stringent condition that is used to define above-mentioned polynucleotide is as follows: DNA-DNA or DNA-RNA hybridization are carried out in two steps: (1) is at the phosphoric acid buffer (20mM that contains 5 * SSC (1 * SSC is the 0.15MNaCl+0.015M sodium citrate solution), 50% methane amide, 7% sodium lauryl sulphate (SDS), 10 * denhardt solution (Denhardt ' s), 5% T 500,1% salmon sperm dna, pH7.5) in, in 42 ℃ of prehybridizations 3 hours; (2) 42 ℃ of hybridization 20 hours uses 2 * SSC+2%SDS in 20 ℃ of washed twice, each 20 minutes then, uses 0.1 * SSC+0.1%SDS in 20 ℃ of washings 20 minutes again, uses 0.1 * SSC+0.1%SDS in 60 ℃ of washings 30 minutes at last.

Theme of the present invention also has the representative segment of above-mentioned polynucleotide, it is characterized in that described fragment can be obtained by following method: can discern and cut the restriction enzyme in the site that exists in the above-mentioned polynucleotide by using, or by using the specific oligonucleotide primer amplification of above-mentioned polynucleotide, or by in-vitro transcription, or by chemosynthesis.

According to a described segmental embodiment preferred, it is selected from: at least corresponding to one of them cDNA:ORF1a, ORF1b, ORF-S, ORF-E, ORF-M, ORF-N, ORF3, ORF4, ORF7 to ORF11, ORF13 and ORF14 of following opening code-reading frame, and corresponding to the non-coding 5 ' end of described polynucleotide or the cDNA of 3 ' end.

According to a preferred feature of above-mentioned embodiment, described fragment contains the sequence that is selected from following sequence:

-representative is corresponding to the sequence SEQ ID No:2 and 4 of the cDNA of coding S proteic ORF-S,

-representative is corresponding to the sequence SEQ ID No:13 and 15 of the cDNA of coding E proteic ORF-E,

-representative is corresponding to the sequence SEQ ID No:16 and 18 of the cDNA of coding M proteic ORF-M,

-representative is corresponding to the sequence SEQ ID No:36 and 38 of the cDNA of coding N proteic ORF-N,

-representative corresponds respectively to the sequence of the cDNA of following reading frame: ORF-1a and ORF-1b (ORF-1ab, SEQ ID No:31), ORF3 and ORF4 (SEQ ID No:7,8), ORF7 to 11 (SEQ ID No:19,20), ORF13 (SEQ ID No:32) and ORF14 (SEQID No:34), and

-representative corresponds respectively to the sequence of the noncoding 5 ' end (SEQ ID No:39 and 72) of described polynucleotide and 3 ' end (SEQ ID No:40,73) cDNA.

The theme of the present invention proteic cDNA fragment of above-mentioned S of encoding in addition is characterized in that it contains the sequence that is selected from sequence SEQ ID No:5 and 6 (Sa and Sb fragment).

Theme of the present invention also has the cDNA fragment corresponding to above-mentioned ORF-1a and ORF-1b, it is characterized in that it contains the sequence that is selected from sequence SEQ ID No:41 to 54 (L0 to L12 fragment).

Theme of the present invention also has above-mentioned polynucleotide passage, it is characterized in that it contains at least 15 the successive bases or the base pair of described virus strain genome sequence, this successive base or base pair comprise at least one following position: 7979,16622,19064,23220,24872,25298 and 26857.The fragment of 20 to 2500 base or base pair preferably, preferred 20 to 400.

According to a described segmental embodiment preferred, it comprises at least two base or base pairs in groups corresponding to following column position: 7919 and 23220,7919 and 25298,16622 and 23220,1,906 4 and 23220,16622 and 25298,19064 and 25298,23220 and 24872,23220 and 26857,24872 and 25298,25298 and 26857.

Theme of the present invention also has the primer of at least 18 bases, its can increase fragment of SARS associated coronavirus genome or its DNA Equivalent.

According to an embodiment of described primer, they are selected from:

-No.1 primer is right, corresponds respectively to 28507 to 28522 positions (sense primer, SEQ ID No:60) and 28774 to 28759 positions (antisense primer, the SEQ ID No:61) of above-mentioned polynucleotide sequence,

-No.2 primer is right, correspond respectively to 28375 to 28390 positions (sense primer, SEQ ID No:62) of above-mentioned polynucleotide sequence and 28702 to 28687 positions (antisense primer, SEQ ID No:63) and

-right by primer SEQ ID No:55 and 56 primers that constitute.

Theme of the present invention can detect the probe whether SARS associated coronavirus genome or its fragment exist in addition, it is characterized in that it is selected from: above-mentioned fragment and corresponding to the fragment of the following position of above-mentioned polynucleotide sequence: 28561 to 28586,28588 to 28608,28541 to 28563 and 28565 to 28589 (SEQ ID No:64 to 67).

Can be according to probe of the present invention and primer with method well-known to those skilled in the art with radioactivity or the direct or indirect ground mark of inactive compound, so that acquisition can detect and/or can quantitative signal.In the radio isotope that uses, can mention ³²P, ³³P, ³⁵S, ³H or ¹²⁵I.The on-radiation entity can be selected from aglucons such as biological example element, avidin, Streptavidin, digoxin (digoxyg é nine), haptens, dyestuff; Luminescence reagents such as for example radioluminescence, chemoluminescence, noclilucence, fluorescence and phosphorescence reagent.

The present invention includes deutero-label probe and primer from aforementioned sequence.

Described probe and primer can be used for SARS associated coronavirus diagnosis of infection.

Theme of the present invention also has the method that detects the SARS associated coronavirus from biological specimen, the method is characterized in which comprises at least:

(a) be present in the extraction of the nucleic acid in the described biological specimen,

(b) use above-mentioned primer to RT-PCR to the segmental amplification of ORF-N and

(c) use of the detection of suitable means to the amplified production of gained in (b).

(b) amplified production in (amplicon) is 268bp when using primer to No.1, is 328bp when using primer to No.2.

According to an embodiment preferred of described method, (b) step of detection method is used at least one probe corresponding to following column position: 28561 to 28586,28588 to 28608,28541 to 28563 and 28565 to 28589 positions of above-mentioned polynucleotide sequence.

Preferably, genomic detection of SARS associated coronavirus and optional quantitative Application PCR in real time, use the No.2 primer to corresponding to 28541 to 28563 positions with corresponding to the probe of 28565 to 28589 positions, probe is with different compound marks, particularly different fluorescent reagents.

Use the PCR in real time of above-mentioned primer and probe very sensitive, it can detect 10 ²The RNA of copy, even can reach 10 RNA that copy; And it is reliable and repeatably.

The present invention includes the polydeoxyribonucleotide and the polyribonucleotide of strand, two strands and three chains, they are corresponding to the genome and the fragments sequence thereof of above-mentioned coronavirus isolated strain, and justice or antisense complementary sequence, particularly corresponding to the RNA and the DNA of said gene group and fragments sequence thereof.

The present invention also comprises the amplified fragments that uses specificity to obtain at the genomic primer of virus strain above-mentioned purifying or isolating, particularly use above-mentioned primer or primer right, this restriction fragment comprises following fragment or is made of following fragments sequence: above-mentioned fragment sequence, the fragment that obtains from comprising SEQ IDNo:1 sequence or front to define segmental carrier in-vitro transcription, the fragment that obtains by chemosynthesis.Derive in the restriction map of the example of restriction fragment by sequence SEQ ID No:1 shown in Figure 13.According to the present invention, described fragment can be the form of isolated fragment, also can be the form of fragment mixture.The present invention also comprises modified fragment, fragment with respect to the front, it removes or has added the Nucleotide of about 15% ratio of above-mentioned relatively fragment length and/or modify in the Nucleotide properties, as long as modified nucleotide fragments keeps and the genome of above-mentioned strain isolated or the ability of inverted defined gene group RNA sequence hybridization.

Obtain by known ordinary method own according to nucleic acid molecule of the present invention, for example be described in Current Protocols in Molecular Biology (Frederick M. AUSUBEL according to following standard step, 2000, Wiley and son Inc., Library ofCongress, USA).For example, they can be by PCR or the RT-PCR amplification or all or part of chemosynthesis acquisition of nucleotide sequence.

Theme of the present invention also has DNA or RNA chip or filter, it is characterized in that it comprises one of at least one above-mentioned polynucleotide or its fragment.

, for example oligonucleotide is transplanted on the carrier of glass or nylon with chemistry or electrochemical method by known ordinary method preparation itself according to DNA of the present invention or RNA chip or filter.

Theme of the present invention also has the clone and/or the expression vector of reorganization, particularly comprises plasmid, virus, virus vector or the phage of above-mentioned nucleic acid fragment.Preferred described recombinant vectors is for placing described nucleic acid fragment suitable element control down to regulate and control the expression vector that it is transcribed and translates.In addition, described carrier can comprise and the 5 ' end of described inset and/or the sequence (label) of 3 ' end homophase (en phase) fusion that it is used for proteinic fixing and/or the detection and/or the purifying of described vector expression.

These carriers are by itself known conventional recombinant DNA and gene engineering method structure and introducing host cell.There are many known carriers itself to insert wherein, so that nucleic acid molecule is introduced host cell and in host cell, keep for the nucleic acid molecule that will be paid close attention to; The selection of suitable carrier depends on the intended purpose (for example expression of the duplicating of the sequence of being paid close attention to, sequence, sequence are with the reservation of extrachromosomal form or be integrated in host's the chromosomal material) of carrier and the character of host cell.

According to the present invention, described plasmid is selected from following plasmid especially:

-be called the plasmid of SARS-S, be contained in (CollectionNationale de Cultures de Microorganismes at French microbial preservation center, 25 rue du Docteur Roux, 75724 Paris Cedex 15) (CNCM) in the No.I-3059 bacterial isolates of preservation on June 20 in 2003; This plasmid comprises the S proteic cDNA sequence of SARS-CoV virus strain of coding source from the sample that is recorded as No.031589, and with reference to Genbank sequence A Y274119.3, described sequence is corresponding to the Nucleotide of 21406 to 25348 (SEQ ID No:4) position,

-be called the plasmid of SARS-S1, be contained at French microbial preservation center in the No.I-3020 bacterial isolates of preservation on May 12 in 2003; This plasmid comprises 5 ' fragment of the proteic cDNA sequence of S of the above-mentioned SARS-CoV virus strain that is derived from the sample that is recorded as No.031589 of coding, with reference to Genbank sequence A Y274119.3 Tor2, described fragment is corresponding to the Nucleotide of 21406 to 23454 (SEQ ID No:5) position

-be called the plasmid of SARS-S2, be contained at French microbial preservation center in the No.I-3019 bacterial isolates of preservation on May 12 in 2003; This plasmid comprises 3 ' fragment of the proteic cDNA sequence of S of the above-mentioned SARS-CoV virus strain that is derived from the sample that is recorded as No.031589 of coding, with reference to Genbank sequence numbering AY274119.3, described fragment is corresponding to the Nucleotide of 23322 to 25348 (SEQ ID No:6) position

-be called the plasmid of SARS-SE, be contained at French microbial preservation center in the No.I-3126 bacterial isolates of preservation on November 13 in 2003; This plasmid comprise corresponding to the above-mentioned SARS-CoV virus strain that is derived from the sample that is recorded as No.031589 between ORF-S and ORF-E and with the cDNA in the equitant zone of ORF-E, with reference to Genbank sequence numbering AY274119.3, described zone is corresponding to the Nucleotide of 25110 to 26244 (SEQ ID No:8) position

-be called the plasmid of SARS-E, be contained at French microbial preservation center in the No.I-3046 bacterial isolates of preservation on May 28 in 2003; This plasmid comprises the above-mentioned proteic cDNA sequence of E that is derived from the SARS-CoV virus strain of the sample that is recorded as No.031589 of coding, with reference to Genbank sequence numbering AY274119.3, described sequence is corresponding to the Nucleotide of 26082 to 26413 (SEQ IDNo:15) position

-be called the plasmid of SARS-M, be contained at French microbial preservation center in the No.I-3047 bacterial isolates of preservation on May 28 in 2003; This plasmid comprises the above-mentioned proteic cDNA sequence of M that is derived from the SARS-CoV virus strain of the sample that is recorded as No.031589 of coding, with reference to Genbank sequence numbering AY274119.3, described sequence is corresponding to the Nucleotide of 26330 to 27098 (SEQ IDNo:18) position

-be called the plasmid of SARS-MN, be contained at French microbial preservation center in the No.I-3125 bacterial isolates of preservation on November 13 in 2003; This plasmid comprises the cDNA sequence corresponding to the zone between ORF-M and ORF-N of the above-mentioned SARS-CoV virus strain that is derived from the sample of collecting in Ha Noi that is recorded as No.031589, with reference to Genbank sequence numbering AY274119.3, described sequence is corresponding to the Nucleotide of 26977 to 28218 (SEQ ID No:20) position

-be called the plasmid of SARS-N, be contained at French microbial preservation center in the No.I-3048 bacterial isolates of preservation on June 5 in 2003; This plasmid comprises the above-mentioned proteic cDNA of N that is derived from the SARS-CoV virus strain of the sample that is recorded as No.031589 of coding, and with reference to Genbank sequence numbering AY274119.3, described sequence is corresponding to the Nucleotide of 28054 to 29430 (SEQ ID No:38) position; Therefore this plasmid comprises the inset of sequence SEQ ID No:38, and is contained at French microbial preservation center in the No.I-3048 bacterial isolates of preservation on June 5 in 2003,

-be called the plasmid of SARS-5 ' NC, be contained at French microbial preservation center in the No.I-3124 bacterial isolates of preservation on November 7 in 2003; This plasmid comprises the cDNA that holds corresponding to the above-mentioned genomic non-coding 5 ' that is derived from the SARS-CoV virus strain of the sample that is recorded as No.031589, with reference to Genbank sequence numbering AY274119.3, described sequence is corresponding to the Nucleotide of 1 to 204 (SEQ ID No:39) position

-be called the plasmid of SARS-3 ' NC, be contained at French microbial preservation center in the No.I-3123 bacterial isolates of preservation on November 7 in 2003; This plasmid comprises the cDNA sequence of holding corresponding to the above-mentioned genomic non-coding 3 ' that is derived from the SARS-CoV virus strain of the sample that is recorded as No.031589, with reference to Genbank sequence numbering AY274119.3, described sequence is corresponding to the Nucleotide that is positioned between 28933 to 29727 positions (SEQ ID No:40), ending is a series of Nucleotide a.

-be called the expression plasmid of pIV2.3N, comprise the cDNA fragment of C end with the fusion rotein of polyhistidine label of coding N albumen (SEQ ID No:37),

-be called pIV2.3S _CExpression plasmid, comprise the cDNA fragment of coding corresponding to segmental C end with the fusion rotein of polyhistidine label of 475 to 1193 positions of S albumen (SEQ ID No:3) aminoacid sequence,

-expression plasmid pIV2.3S _L, comprise the cDNA fragment of coding corresponding to segmental C end with the fusion rotein of polyhistidine label of 14 to 1193 positions of S albumen (SEQ ID No:3) aminoacid sequence,

-be called the expression plasmid of pIV2.4N, comprise the cDNA fragment of N end with the fusion rotein of polyhistidine label of coding N albumen (SEQ ID No:3),

-be called pIV2.4S _COr pIV2.4S ₁Expression plasmid, comprise coding corresponding to the inset of the segmental N end of 475 to 1193 positions of S albumen (SEQID No:3) aminoacid sequence and the fusion rotein of polyhistidine label and

-be called pIV2.4S _LExpression plasmid, comprise the cDNA fragment of coding corresponding to segmental N end with the fusion rotein of polyhistidine label of 14 to 1193 positions of S albumen (SEQ ID No:3) aminoacid sequence.

According to a preferred feature of above-mentioned expression plasmid, it is contained at French microbial preservation center in the No.I-3117 bacterial isolates of preservation on October 23 in 2003.

According to another preferred feature of above-mentioned expression plasmid, it is contained at French microbial preservation center in the No.I-3118 bacterial isolates of preservation on October 23 in 2003.

According to another preferred feature of above-mentioned expression plasmid, it is contained in the bacterial isolates of CNCM preservation, and numbering is listed as follows:

A) strain number No.I-3118, preservation date on October 23rd, 2003,

B) strain number No.I-3019, preservation date on May 12nd, 2003,

C) strain number No.I-3020, preservation date on May 12nd, 2003,

D) strain number No.I-3059, preservation date on June 20th, 2003,

E) strain number No.I-3323, preservation date on November 22nd, 2004,

F) strain number No.I-3324, preservation date on November 22nd, 2004,

G) strain number No.I-3326, preservation date on December 1st, 2004,

H) strain number No.I-3327, preservation date on December 1st, 2004,

I) strain number No.I-3332, preservation date on December 1st, 2004,

J) strain number No.I-3333, preservation date on December 1st, 2004,

K) strain number No.I-3334, preservation date on December 1st, 2004,

L) strain number No.I-3335, preservation date on December 1st, 2004,

M) strain number No.I-3336, preservation date on December 1st, 2004,

N) strain number No.I-3337, preservation date on December 1st, 2004,

O) strain number No.I-3338, preservation date on December 2nd, 2004,

P) strain number No.I-3339, preservation date on December 2nd, 2004,

Q) strain number No.I-3340, preservation date on December 2nd, 2004,

R) strain number No.I-3341, preservation date on December 2nd, 2004.

Theme of the present invention also has the nucleic acid inset of viral source, it is characterized in that it is contained in any bacterial strain of top a)-r) definition.

Theme of the present invention has comprised synthetic gene in addition so that S albumen obtains the nucleic acid of optimum expression in eukaryotic cell, it is characterized in that it contains SEQ ID No:140 sequence.

Theme of the present invention comprises the expression of nucleic acids carrier in addition, and this nucleic acid comprises synthetic gene so that S albumen obtains optimum expression, and this carrier is contained at CNCM in the No.I-3333 bacterial isolates of preservation on December 1 in 2004.

According to an embodiment of described expression vector, it is the virus vector of virion form or recombination group form.

A preferred feature according to this embodiment; it is can be by above-mentioned g in suitable cell system), h) and k) to r) plasmid transfection of the section recombinant virus particle or the recombinant virus genomes that obtain; that is to say; for example by the cell of one or more other plasmid transfections, some lacks in carrier and is that virion forms necessary viral function to be intended to trans-complementation (trancompl é menter).

The statement of " S protein family " is interpreted as the fragment of complete S albumen, its ectodomain (ectodomaine) and ectodomain here, and they preferably produce in eukaryotic cell system.

Encode the in addition lentiviral vectors of polypeptide of above-mentioned S protein family of theme of the present invention.

Encode the in addition recombinant measles virus of polypeptide of above-mentioned S protein family of theme of the present invention.

Encode the in addition recombined vaccinia virus of polypeptide of above-mentioned S protein family of theme of the present invention.

The also with good grounds front e of theme of the present invention) to r) carrier of section or comprise that the carrier of the proteic synthetic gene of above-mentioned S produces SARS associated coronavirus S albumen or its segmental purposes in eukaryotic cell system.

Theme of the present invention also has produces the proteic method of S in eukaryotic cell system, be included in the eukaryotic cell culture step with the carrier transfectional cell, described carrier is for being selected from above-mentioned e) to r) carrier that bacterial strain comprised mentioned in the section or comprised synthetic gene so that S albumen obtains the carrier of optimum expression.

Theme of the present invention also has the cDNA library, it is characterized in that it comprises above-mentioned fragment, particularly is cloned into amplified fragments or restriction fragment (expression library) in the recombinant vectors (particularly expression vector).

Theme of the present invention also has cell, particularly the prokaryotic cell prokaryocyte of modifying through above-mentioned recombinant vectors.

Theme of the present invention also has the eukaryotic cell of expressing above-mentioned albumen or polypeptide through genetic modification.Clearly, the cell through the wild-type virus modification do not represented in term " through the eukaryotic cell of genetic modification ".

According to an embodiment preferred of described cell, it can be by above-mentioned i) to l) transfection of any carrier of section obtains.

According to a preferred feature of this embodiment, this cell is FRhK4-Ssol-30, is preserved in CNCM, preservation date on November 22nd, 2004, numbering No.I-3325.

Above-mentioned recombinant vectors and can be advantageously used in the production of respective egg white matter and peptide by described expression vector cell transformed.From described carrier deutero-expression library with can be advantageously used in identifying the proteic immunogenicity epi-position (B and T epi-position) of SARS associated coronavirus with described expression library cell transformed.

Theme of the present invention also has protein purifying or isolating and peptide, it is characterized in that they are coded by above-mentioned polynucleotide or an one fragment.

According to an embodiment preferred of the present invention, described protein is selected from:

-have S albumen or its ectodomain of sequence SEQ ID No:3

-have the E albumen of sequence SEQ ID No:14

-have the M albumen of sequence SEQ ID No:17

-have the N albumen of sequence SEQ ID No:37

-by the coded albumen of following opening code-reading frame: have sequence SEQ ID No:74,75,10,12,22,24,26,28,30,33 and 35 ORF1a, ORF1b, ORF3, ORF4, ORF7 to ORF11, ORF13 and ORF14 respectively.

Term " the proteic ectodomain of S " and " the proteic soluble form of S " use hereinafter interchangeably.

According to an embodiment preferred of the present invention, described polypeptide is made of the amino acid corresponding to S Argine Monohydrochloride sequence 1 to 1193 position.

According to another embodiment preferred of the present invention, described peptide is selected from:

A) corresponding to the peptide of S Argine Monohydrochloride sequence 14 to 1193 positions and 475 to 1193 positions,

B) corresponding to the peptide of M Argine Monohydrochloride sequence 2 to 14 positions (SEQ ID No:69) and 100 to 221 positions, these peptides respectively the proteic ectodomains of corresponding M and born of the same parents' intracellular domain (endodomaine) and

C) corresponding to the peptide of E Argine Monohydrochloride sequence 1 to 12 position (SEQ ID No:70) and 53 to 76 positions (SEQ ID No:71), these peptides respectively the proteic ectodomains of corresponding E and C end and

D) 5 to 50 continuous amino acids, preferred 10 to 30 amino acid whose peptides, comprise a), b) c) in the definition peptide sequence or with they part or all of eclipsed sequences.

Theme of the present invention also has peptide, it is characterized in that it contains 7 to 50 amino acid whose sequences, and this sequence contains and is selected from following amino-acid residue:

-be positioned at the L-Ala of aminoacid sequence 2552 positions of ORF1a encoded protein matter,

-be positioned at the Serine of the proteic aminoacid sequence of S 577 positions of above-mentioned SARS-CoV virus strain,

-be positioned at the glycine of aminoacid sequence 11 positions of the ORF3 encoded protein matter of above-mentioned SARS-CoV virus strain,

-be positioned at the Serine of the proteic aminoacid sequence of M 154 positions of above-mentioned SARS-CoV virus strain.

Theme of the present invention also has polyclone or monoclonal antibody or antibody fragment, can obtain animal immune by using above-mentioned recombinant vectors, above-mentioned cDNA library or above-mentioned albumen or peptide, it is characterized in that the above-mentioned SARS-CoV encoded protein of it and at least one matter combines.

The present invention includes the chimeric antibody and their fragment (Fab, Fv, scFv) of polyclonal antibody, monoclonal antibody, for example humanized antibody.

Theme of the present invention also has the hybridoma of producing at the proteic monoclonal antibody of N, it is characterized in that it is selected from following hybridoma:

The hybridoma of-manufacture order clonal antibody 87 is preserved in CNCM, preservation date on December 1st, 2004, and numbering I-3328,

The hybridoma of-manufacture order clonal antibody 86 is preserved in CNCM, preservation date on December 1st, 2004, and numbering I-3329,

The hybridoma of-manufacture order clonal antibody 57 is preserved in CNCM, preservation date on December 1st, 2004, numbering I-3330 and

The hybridoma of-manufacture order clonal antibody 156 is preserved in CNCM, preservation date on December 1st, 2004, numbering I-3331.

Theme of the present invention also has at the proteic polyclonal antibody of N or monoclonal antibody or antibody fragment, it is characterized in that it is by above-mentioned hybridoma production.

According to the intent of the present invention, the statement of chimeric antibody should be understood that, in contrast to the antibody of particular animal species or the antibody of certain kinds, chimeric antibody means antibody or the heavy chain of another kind of antibody and/or all or part of antibody of light chain that comprises another animal species.

According to the intent of the present invention, the statement of humanized antibody should be understood that human immunoglobulin (Ig), and the residue that wherein constitutes the CDR (complementary determining region) of antigen binding site is had the corresponding section replacement of needed specificity, avidity or active inhuman monoclonal antibody.Consider that nearly all residue of the FR district (framework region) of humanized antibody in fact and constant region (Fc) is the residue of the consensus sequence of human immunoglobulin, compare with inhuman antibody, their immunogenicities in human body are less and the long transformation period is arranged, because they have only the non-human sequence of small portion.

Theme of the present invention also has protein chip or filter, it is characterized in that it comprises above-mentioned protein, peptide or antibody.

Pass through itself known ordinary method preparation according to protein chip of the present invention.In the suitable upholder that protein can be fixed thereon, can mention the form of the upholder that plastics or glass are manufactured, particularly microtest plate here.

Theme of the present invention also has deutero-reagent from derive from the SARS associated coronavirus strain isolated of sample that recording mechanism is No.031589, can be used for research and diagnosis that the SARS associated coronavirus infects, and described reagent is selected from:

(a) above-mentioned primer is to, probe or DNA chip,

(b) above-mentioned recombinant vectors or modified cell,

(c) above-mentioned isolating coronavirus strain or polynucleotide,

(d) above-mentioned protein or peptide,

(e) above-mentioned antibody or antibody fragment and

(f) above-mentioned protein chip.

These different reagent can use and for example be described in Current Protocols in MolecularBiology (Frederick M. AUSUBEL, 2000, Wiley and Son Inc., Libraryof Congress, USA), Current Protocols in Immunology (John E.Cologan, 2000, Wiley and Son Inc., Library of Congress, USA) and Antibodies:A Laboratory Manual (E. Howell and D. Lane, ColdSpring Harbor Laboratory, 1998) conventional molecular biology in and the preparation of immunological technique secundum legem step and use.

Also prepare and use according to nucleic acid fragment of the present invention according to above-mentioned routine techniques.Prepare by recombinant DNA technology well known to those skilled in the art according to peptide of the present invention and protein, particularly utilize above-mentioned recombinant vectors preparation.Perhaps, can be according to peptide of the present invention by conventional solid phase well known by persons skilled in the art or the preparation of liquid phase synthetic technology.

Polyclonal antibody is by with the suitable animal preparation of above-mentioned protein or peptide immunity, randomly can for example (fully or not exclusively) freund adjuvant or aluminium hydroxide make up with KLH or albumin coupling and/or with suitable adjuvant; After reaching satisfied antibody titer, the serum of collecting immune animal according to routine techniques also precipitates with enrichment IgG with results antibody, randomly use affinity chromatography to come purifying special being attached with on described peptide and the described proteic suitable post, to obtain the IgG goods of monospecific at the proteic IgG of SARS-CoV.

Monoclonal antibody is produced from hybridoma, hybridoma be according to K_hler and Milstein (Nature, 1975,256, technology 495-497) is used to be merged by the bone-marrow-derived lymphocyte of the animal of above-mentioned albumen or peptide immunity and myeloma cell and is made; Hybridoma can be in vitro culture, particularly in fermentor tank, or in vivo with the form production of ascites.Perhaps, described monoclonal antibody can be with as U.S. Pat 4,816, the gene engineering method production of description in 567.

Humanized antibody can be used the ordinary method production of describing as in the International Application No. WO 98/45332.

The fragment of antibody can be used from hybridoma or be cloned V the mRNA of the splenic lymphocyte of immune mouse _HAnd V _LThe method production in district; For example according to Winter and Milstein (Nature, 1991,349, technology 293-299) is in filobactivirus surface expression Fv, scFv or Fab fragment; Through separable specificity after some screening steps at antigenic antibody fragment, and by clone and technology expressing antibodies fragment in suitable expression system of express recombinant DNA.

Above-mentioned antibody or its fragment are with routine techniques purifying well known by persons skilled in the art, for example affinity chromatography.

Theme of the present invention also has product to be used for the detection of SARS associated coronavirus and the purposes of the reagent of gene type/serotype randomly in preparation in addition, and described product is selected from: above-mentioned primer is to, probe, DNA chip, recombinant vectors, modified cell, isolating coronavirus strain, polynucleotide, protein or peptide, antibody or antibody fragment and protein chip.

Can be according to protein of the present invention and peptide by specificity at the antibody recognition of SARS associated coronavirus and/or can induce this production of antibodies, and can be used for the diagnosis of above-mentioned coronavirus infection; Use suitable technique--particularly EIA, ELISA, RIA, immunofluorescence--from detecting infection conditions the infected individual biological specimen of gathering.

According to a preferred feature of described purposes, described protein is selected from above-mentioned S, E, M and/or N albumen and peptide.

Above-mentioned S, E, M and/or N albumen and by these protein derived peptides, for example N albumen can be used for the indirect diagnosis (serodiagnosis that the SARS associated coronavirus infects; Specificity is at the detection of antibodies of SARS-CoV), particularly use immunoenzymology method (ELISA).

According to antibody of the present invention and antibody fragment,, can be used for the direct diagnosis that the SARS associated coronavirus infects particularly at the antibody and the antibody fragment of above-mentioned S, E, M and/or N albumen and institute's deutero-peptide; The proteinic detection of SARS-CoV carried out the infected individual biological specimen of gathering from possibility by suitable technique (particularly EIA, ELISA, RIA, immunofluorescence technique).

Theme of the present invention also has the detection method of SARS associated coronavirus in the biological specimen, the method is characterized in which comprises at least:

(a) make the chip of described biological specimen and at least a above-mentioned antibody or antibody fragment, protein, peptide or protein or peptide or filter contacts and

(b) make the antigen-antibody complex that forms in (a) visual with suitable means, for example use EIA, ELISA, RIA or immunofluorescence technique.

According to an embodiment preferred of described step, step (a) comprising:

(a ₁) make described biological specimen and contact attached at least a first antibody on the suitable upholder, particularly microtest plate or antibody fragment,

(a ₂) washing solid phase and

(a ₃) add at least a second antibody or antibody fragment, different with first antibody, depend on the circumstances, should be on described second antibody or the antibody fragment by mark suitably.

This method that can catch the virion that exists in the biological specimen is also referred to as the immunocapture method.

For example:

-(a ₁) used at least a first mono-clonal or polyclonal antibody or its fragment in the step, they are at S, M and/or E albumen and/or corresponding to these proteic one of them (M2-14 or E1-12 peptides) of peptide of ectodomain

-(a ₃) used at least a antibody or antibody fragment in the step, they at be another epi-position of same protein or preferably at another albumen, preferably at for example inside albumen of the proteic born of the same parents' intracellular domain of N nucleoprotein or E or M, more preferably they are at proteic antibody of the very abundant N of content in virion or antibody fragment; When using at the antibody of inner albumen (N) or E or the proteic born of the same parents' intracellular domain of M or antibody fragment, described antibody should be cultivated under the condition that washing composition exists, the example of washing composition such as Tween20, and concentration is about 0.1%.

-in the visual step of the antigen-antibody complex that makes formation (b), can directly use by vitamin H or the suitable enzyme such as the second antibody of peroxidase or alkali phosphatase enzyme mark, also can use the serum of the anti-immunoglobulin that has above-mentioned mark indirectly.The mixture of Xing Chenging is visual by suitable substrate like this.

This embodiment preferred on the one hand according to the present invention, biological specimen with catch before monoclonal antibody contact, mix earlier with visual monoclonal antibody.When needing, the visual mixtures of antibodies of serum was at room temperature cultivated 10 minutes before being applied to culture plate at least.

Theme of the present invention also has by detecting the immunocapture detection that natural nucleus albumen (N albumen) checks the SARS associated coronavirus to infect, and the antibody that particularly it is characterized in that being used to catching natural viral nucleoprotein is special monoclonal antibody at central section and/or conformational epitope.

According to an embodiment of described detection, being used to catch the proteic antibody of N is monoclonal antibody mAb87, and it is by the hybridoma production that was preserved in CNCM, numbering I-3328 on December 1st, 2004.

According to another embodiment that described immunocapture detects, being used to catch the proteic antibody of N is monoclonal antibody mAb86, and it is by the hybridoma production that was preserved in CNCM, numbering I-3329 on December 1st, 2004.

According to another embodiment that described immunocapture detects, monoclonal antibody mAb86 and mAb87 are used to catch N albumen.

Detect according to immunocapture of the present invention, in N albumen visualization step, can use on December 1st, 2004 and be preserved in the mAb57 monoclonal antibody that the hybridoma of the numbering I-3330 of CNCM is produced, described antibody is puted together mutually with visual molecule or particle.

Detect according to described immunocapture, it is proteic visual that the mAb57 that puts together mutually with visual molecule or particle and the combination of mAb87 antibody are used for N.

Visual molecule can be radioactive atom, dyestuff, fluorescence molecule, fluorophore, enzyme; Visual particle can be for for example: Radioactive colloidal gold, magnetic-particle or latex beads.

Theme of the present invention detects the reagent of SARS associated coronavirus in addition, it is characterized in that it is selected from:

(a) above-mentioned primer to or probe,

(b) above-mentioned recombinant vectors or modified cell,

(c) above-mentioned isolating coronavirus strain or polynucleotide,

(d) above-mentioned antibody or antibody fragment,

(e) the above-mentioned antibody that comprises monoclonal antibody mAb86 and/or mAb87 and monoclonal antibody mAb57 makes up,

(f) above-mentioned chip or filter.

Theme of the present invention also has by using the proteic indirect IgG ELISA of N to detect the method that the SARS associated coronavirus infects from biological specimen, the method is characterized in that with N protein solution sensitization culture plate, the N protein concentration is between 0.5 and 4 μ g/ml, be preferably 2 μ g/ml, be dissolved in contain 0.25ml/l phenol red, pH is in 7.2 the 10mM PBS damping fluid.

Theme of the present invention also has in addition by using two epi-position ELISA to detect the method that the SARS associated coronavirus infects from biological specimen, it is characterized in that serum to be measured mixes mutually with visual antigen, contacts described mixture then with the antigen that is attached to solid support.

According to a version of the detection of SARS associated coronavirus, these detections combine uses the proteic ELSA of N and another to use the proteic ELSA of S, as mentioned below.

Theme of the present invention also has by above-mentioned polyclone or the protein of monoclonal antibody or antibody fragment and SARS associated coronavirus or the immunocomplex that peptide forms.

Theme of the present invention also has SARS associated coronavirus detection kit in addition, it is characterized in that it comprises at least a following reagent that is selected from: above-mentioned primer is to, probe, DNA or RNA chip, recombinant vectors, modified cell, isolating coronavirus strain, polynucleotide, protein or peptide, antibody, protein chip.

Theme of the present invention also has immunogenic composition in addition, it is characterized in that comprising at least a following product that is selected from:

A) above-mentioned protein or peptide,

B) above-mentioned DNA or RNA type polynucleotide or its representative segment, its sequence is selected from:

(i) SEQ ID No:1 sequence or its RNA Equivalent

The (ii) sequence that can under the height stringent condition, hybridize with SEQ ID No:1 sequence,

(iii) with SEQ ID No:1 sequence or the sequence complementary sequence that can under the height stringent condition, hybridize with SEQ ID No:1 sequence,

(iv) above-mentioned (i), (ii) or the nucleotide sequence of the representative segment of the polynucleotide (iii),

(v) above-mentioned (i), (ii), (iii) or (iv) the sequence after modifying and

C) comprise b) described in polynucleotide recombinant expression vector and

D) above-mentioned cDNA library,

Described immunogenic composition can inducing specific at the immunity of the body fluid or the cell of the protectiveness of SARS associated coronavirus, particularly produce antibody at SARS associated coronavirus specific epitopes.

Above-mentioned protein and peptide, particularly S, M, E and/or N albumen and deutero-peptide, and the nucleic acid molecule of code for said proteins or described peptide (DNA or RNA) is good candidate vaccine and can uses at the immunogenic composition that is used for producing at the vaccine of SARS associated coronavirus.

According to an embodiment preferred of composition of the present invention, they also comprise at least a pharmaceutically useful vehicle and optional carrier substance and/or adjuvant in addition.

Pharmaceutically useful vehicle, carrier substance and adjuvant are conventional the uses.

Adjuvant preferably is selected from oil emulsion, saponin, mineral substance, bacterial extract, aluminium hydroxide and squalene.

Carrier substance preferably is selected from unilamellar liposome, multilamellar liposome, saponin particulate or sugar or contains golden microspheres with solid.

Composition according to the present invention is with conventional route administration, particularly intramuscular or subcutaneous route or local approach, particularly nose (aerosol) approach.

The albumen of the also separative or purifying of theme of the present invention or peptide are forming application in the immunocomplex with specificity at the antibody of the epi-position of SARS associated coronavirus, and described albumen or peptide have and be selected from following sequence: SEQ ID No:3,10,12,14,17,22,24,26,28,30,33,35,37,69,70,71,74 and 75.

Theme of the present invention also has immunocomplex, this immunocomplex is formed by the albumen of isolating or purifying or peptide and the specificity antibody at the epi-position of SARS associated coronavirus, and described albumen or peptide have and be selected from following sequence: SEQ ID No:3,10,12,14,17,22,24,26,28,30,33,35,37,69,70,71,74 and 75.

But theme of the present invention is also separative or the albumen of purifying or the application of peptide in the antibody of the epi-position of inducing generation specific recognition SARS associated coronavirus, and described albumen or peptide have and be selected from following sequence: SEQ ID No:3,10,12,14,17,22,24,26,28,30,33,35,37,69,70,71,74 and 75.

Theme of the present invention is also separative or but purified polynucleotides is inducing generation at the application in the antibody of the epi-position of the albumen of described polynucleotide encoding and specific recognition SARS associated coronavirus, and described polynucleotide have and are selected from following sequence: SEQ ID No:1,2,4,7,8,13,15,16,18,19,20,31,36 and 38.

The proteic monoclonal antibody of natural S of SARS associated coronavirus can be discerned in addition in theme of the present invention.

Theme of the present invention also has albumen or the polypeptide or the above-mentioned application of the proteic antibody of the natural S of identification in detect the infection of SARS associated coronavirus from biological specimen of above-mentioned S protein family.

Theme of the present invention also has the method that detects the infection of SARS associated coronavirus from biological specimen, it is characterized in that this detection is to be undertaken by the ELISA that uses the recombinant s protein of expressing in the eukaryotic cell system.

According to an embodiment preferred of described method, it is two epi-position ELISA methods, and serum to be measured mixes mutually with visual antigen, then described mixture is contacted with the antigen that is attached to solid support.

Theme of the present invention also has immunocomplex, and this immunocomplex is made of albumen or the peptide that can discern proteic monoclonal antibody of natural S or antibody fragment and SARS associated coronavirus.

Theme of the present invention also has immunocomplex, and this immunocomplex is made of the albumen of above-mentioned S protein family or polypeptide and the specificity antibody at the epi-position of SARS associated coronavirus.

Theme of the present invention also comprises the detection kit or the box of SARS associated coronavirus in addition, it is characterized in that comprising at least a following reagent that is selected from: albumen or the cell of polypeptide or the proteic antibody of natural S of identification SARS associated coronavirus of the albumen of the albumen of above-mentioned S protein family or polypeptide, above-mentioned coding S protein family or the nucleic acid of polypeptide, above-mentioned expression S protein family.

Theme of the present invention also has immunogenicity and/or vaccine composition, it is characterized in that being included in the polypeptide or the recombinant protein of the above-mentioned S protein family that obtains in the eukaryotic cell expression system.

Theme of the present invention also has immunogenicity and/or vaccine composition, it is characterized in that comprising the albumen of above-mentioned expressed S protein family or the carrier or the recombinant virus of polypeptide.

Except above-mentioned feature, the present invention also comprises other features that manifest in the following description, be derived from reference to representative the sample that is recorded as No. 031589 the genomic polynucleotide of SARS-CoV strain application embodiment and as the embodiment of the segmental application of cDNA of deriving of theme of the present invention, the tabulation of Table I display sequence:

Table I: sequence table

Identify numbering	Sequence	Position with reference to the cDNA of Genbank AY274119.3	Corresponding plasmid is at the deposit number of CNCM
				SEQ?ID?No：1	Be derived from the genome of the virus strain of sample 031589	-	-
SEQ?ID?No：2	ORF-S *	21406-25348	-
				SEQ?ID?No：3	S albumen	-	-
SEQ?ID?No：4	ORF-S **	21406-25348	I-3059
				SEQ?ID?No：5	The Sa fragment	21406-23454	I-3020
SEQ?ID?No：6	The Sb fragment	23322-25348	I-3019
				SEQ?ID?No：7	ORF-3+ORF-4 *	25110-26244	-
SEQ?ID?No：8	ORF-3+ORF-4 **	25110-26244	I-3126
				SEQ?ID?No：9	ORF3	-	-
SEQ?ID?No：10	ORF-3 albumen	-	-
				SEQ?ID?No：11	ORF4	-	-
SEQ?ID?No：12	ORF-4 albumen	-	-
				SEQ?ID?No：13	ORF-E *	26082-26413	-
SEQ?ID?No：14	E albumen	-	-
				SEQ?ID?No：15	ORF-E **	26082-26413	I-3046
SEQ?ID?No：16	ORF-M *	26330-27098	-
				SEQ?ID?No：17	M albumen	-	-
SEQ?ID?No：18	ORF-M **	26330-27098	I-3047
				SEQ?ID?No：19	ORF7 to 11 *	26977-28218	-
SEQ?ID?No：20	ORF7 to 11 **	26977-28218	I-3125
				SEQ?ID?No：21	ORF7	-	-
SEQ?ID?No：22	ORF7 albumen	-	-
				SEQ?ID?No：23	ORF8	-	-
SEQ?ID?No：24	ORF8 albumen	-	-
				SEQ?ID?No：25	ORF9	-	-
SEQ?ID?No：26	ORF9 albumen	-	-
				SEQ?ID?No：27	ORF10	-	-

SEQ?ID?No：28	ORF10 albumen	-	-
				SEQ?ID?No：29	ORF11	-	-
SEQ?ID?No：30	ORF11 albumen	-	-
				SEQ?ID?No：31	ORF1ab	265-21485	-
SEQ?ID?No：32	ORF13	28130-28426	-
				SEQ?ID?No：33	ORF13 albumen	-	-
SEQ?ID?No：34	ORF14	-	-
				SEQ?ID?No：35	ORF14 albumen	28583-28795	-
SEQ?ID?No：36	ORF-N *	28054-29430	-
				SEQ?ID?No：37	N albumen	-	-
SEQ?ID?No：38	ORF-N **	28054-29430	I-3048
				SEQ?ID?No：39	Non-coding 5 ' end **	1-204	I-3124
SEQ?ID?No：40	Non-coding 3 ' end **	28933-29727	I-3123
				SEQ?ID?No：41	ORF1ab fragment L0	30-500	-
SEQ?ID?No：42	Fragment L1	211-2260	-
				SEQ?ID?No：43	Fragment L2	2136-4187	-
SEQ?ID?No：44	Fragment L3	3892-5344	-
				SEQ?ID?No：45	Fragment L4b	4932-6043	-
SEQ?ID?No：46	Fragment L4	5305-7318	-
				SEQ?ID?No：47	Fragment L5	7275-9176	-
SEQ?ID?No：48	Fragment L6	9032-11086	-
				SEQ?ID?No：49	Fragment L7	10298-12982	-
SEQ?ID?No：50	Fragment L8	12815-14854	-
				SEQ?ID?No：51	Fragment L9	14745-16646	-
SEQ?ID?No：52	Fragment L10	16514-18590	-
				SEQ?ID?No：53	Fragment L11	18500-20602	-
SEQ?ID?No：54	Fragment L12	20319-22224	-
				SEQ?ID?No：55	Justice N primer	-	-
SEQ?ID?No：56	Antisense N primer	-	-
				SEQ?ID?No：57	Justice S CPrimer	-	-

SEQ?ID?No：58	Justice S LPrimer	-	-
				SEQ?ID?No：59	Antisense S CAnd S LPrimer	-	-
SEQ?ID?No：60	Sense primer series 1	28507-28522	-
				SEQ?ID?No：61	Antisense primer series 1	28774-28759	-
SEQ?ID?No：62	Sense primer series 2	28375-28390	-
				SEQ?ID?No：63	Antisense primer series 2	28702-28687	-
SEQ?ID?No：64	Probe 1/ series 1	28561-28586	-
				SEQ?ID?No：65	Probe 2/ series 1	28588-28608	-
SEQ?ID?No：66	Probe 1/ series 2	28541-28563	-
				SEQ?ID?No：67	Probe 2/ series 2	28565-28589	-
SEQ?ID?No：68	Anchor primer 14T		-
				SEQ?ID?No：69	Peptide M2-14	-	-
SEQ?ID?No：70	Peptide E1-12	-	-
				SEQ?ID?No：71	Peptide E53-76	-	-
SEQ?ID?No：72	Non-coding 5 ' end *	1-204	-
				SEQ?ID?No：73	Non-coding 3 ' end *	28933-29727	-
SEQ?ID?No：74	ORF1a albumen	-	-
				SEQ?ID?No：75	ORF1b albumen	-	-
SEQ?ID?No：76-139	Primer
				SEQ?ID?No：140	The pseudogene of S
SEQ?ID?No：141-148	Primer
				SEQ?ID?No：149	The Aa1-13 of S
SEQ?ID?No：150	Polypeptide
				SEQ?ID?No：151-158	Primer

* pcr amplification product (amplicon)

The * clone enters the inset in the plasmid that is preserved in CNCM

Description of drawings

The recombinant protein N that is to use expression vector pIVEX, S that-Fig. 1 shows _CAnd S _LProtein immunoblotting (Western-blot) analytical results of vivoexpression.Swimming lane 1:pIV2.3N.Swimming lane 2:pIV2.3S _cSwimming lane 3:pIV2.3S _LSwimming lane 4:pIV2.4N.Swimming lane 5:pIV2.4S ₁Or pIV2.4S _CSwimming lane 6:pIV2.4S _LUse same vehicle to express the proteic expression of results of GFP with comparing.

-Fig. 2 shows is to use polyacrylamide gel electrophoresis (SDS-PAGE) under the sex change condition and coomassie brilliant blue staining to the proteic analytical results of expression vector pIVEX expression in vivo N.E. coli bl21 (DE3) the pDIA17 bacterial strain that uses recombinant vectors pIVEX to transform uses on 30 ℃, LB substratum or does not use inductor (1mM IPTG) to cultivate.Swimming lane 1:pIV2.3N.Swimming lane 2:pIV2.4N.

-Fig. 3 shows is to use polyacrylamide gel electrophoresis (SDS-PAGE) under the sex change condition and coomassie brilliant blue staining to expression vector pIVEX expression in vivo S _CAnd S _LThe analytical results of polypeptide.E. coli bl21 (DE3) the pDIA17 bacterial strain that uses recombinant vectors pIVEX to transform uses on 30 ℃, LB substratum or does not use inductor (1mM IPTG) to cultivate.Swimming lane 1:pIV2.3S _CSwimming lane 2:pIV2.3S _LSwimming lane 3:pIV2.4S ₁Swimming lane 4:pIV2.4S _L

Reorganization N, S that e. coli bl21 (DE3) the pDIA17 bacterial strain that is to use recombinant vectors pIVEX to transform that-Fig. 4 shows is produced _CAnd S _LProteic antigenic activity.A: the electrophoresis of bacterium lysate (SDS-PAGE).B and C: the Western-blot from the same patient's who is infected by SARS-CoV serum analyzes, and (B: (C: serum M13) serum M12) and after 29 days samples respectively after 8 days of SARS symptom occurring.Swimming lane 1:pIV2.3N.Swimming lane 2:pIV2.4N.Swimming lane 3:pIV2.3S _CSwimming lane 4:pIV2.4S ₁Swimming lane 5:pIV2.3S _LSwimming lane 6:pIV2.4S _L

The result of recombinant N protein purifying on the Ni-NTA agarose column that e. coli bl21 (DE3) the pDIA17 bacterial strain that is to use carrier pIV2.3N to transform that-Fig. 5 shows is produced.Swimming lane 1: total bacterial extract.Swimming lane 2: soluble extract.Swimming lane 3: insoluble extract.Swimming lane 4: retain in the extract on the Ni-NTA post.Swimming lane 5: not conjugated protein.Swimming lane 6: peak 1 component.Swimming lane 7: peak 2 components.

-Fig. 6 shows is to use recombinant plasmid pIV2.4S ₁The reorganization S that transformed into escherichia coli BL21 (DE3) pDIA17 bacterial strain produces in inclusion body _CProteic purification result.A: use TritonX-100 (2%) to handle: swimming lane 1: total bacterial extract.Swimming lane 2: soluble extract.Swimming lane 3: insoluble extract.Swimming lane 4: use the supernatant liquor after Triton X-100 (2%) handles.Swimming lane 5 and 6: use the flaky precipitate after Triton X-100 (2%) handles.B: the urea processing soluble and the insoluble extract that use 4M, 5M, 6M and 7M.

-Fig. 7 has represented to use the split product of the cell that is infected by SARS-CoV and the immunoblotting of the patient's who suffers from atypical pneumonia serum generation.

The split product that-Fig. 8 has represented to use the cell that is infected by SARS-CoV and specificity are at nucleoprotein N (A) and sting the immunoblotting of the rabbit immune serum generation of Protein S (B).I.S.: immune serum.P.i.: preimmune serum.Anti-N protein immunization serum uses with 1/50000, and anti-S protein immunization serum uses with 1/10000.

-Fig. 9 shows is at the rabbit monospecific polyclonal antibody serum of N albumen or the proteic short-movie section of S (SC) the ELISA reactivity to the recombinant protein that is used for immunity accordingly.A: rabbit P13097, the P1308 1 and the P13031 that produce with the recombinant N protein immunity of purifying.B: use corresponding to the proteic short-movie section of S (S _C) inclusion body preparation immunity and rabbit P11135, the P13042 and the P14001 that produce.I.S.: immune serum.P.i.: preimmune serum.

-Figure 10 shows is the ELISA reactivity of the recombinant N protein of purifying to the atypical pneumonia patient's that caused by SARS-CoV serum.Figure 10 a:ELISA plate is the N albumen bag quilt of 4 μ g/ml and 2 μ g/ml with concentration.Figure 10 b:ELISA plate is the N albumen bag quilt of 1 μ g/ml with concentration.The serum that is denoted as A, B, D, E, F, G, H is corresponding with Table IV.

-Figure 11 shows is the synthetic RNA (10 of the SARS-CoV N gene that successively decreases of quantity ⁷To 1 the copy) the RT-PCR amplification, this amplification used the No.1 primer to (N/+/28507, N/-/28774) (A) and the No.2 primer to (N/+/28375, N/-/28702) (B).T: the amplification of no RNA.The MW:DNA molecular weight marker.

Swimming lane 16 to 29) and be diluted to 1/20 * 10-Figure 12 shows is real-time RT-PCR amplification for the synthetic RNA of SARS-CoV N gene: the synthetic RNA that quantity is successively decreased is as duplicate (duplicate: ^-4Viral RNA (swimming lane 32) is with the method amplification of real-time RT-PCR, the primer that " Light Cycler RNA Amplification Kit Hybridization Probes " test kit and No.2 series are used in this amplification to and probe, experiment condition is as described in the embodiment 8.

-Figure 13 (Figure 13 .1 to 13.7) has represented the restriction map of SEQ ID No:1 sequence, and described SEQ ID No:1 sequence is corresponding to the genomic DNA Equivalent of SARS-CoV virus strain that from recording mechanism is 031589 sample.

The SARS serology detected result that is to use indirect N protein ELISA (having tested the serum of first series) that-Figure 14 shows.

The SARS serology detected result (having tested the serum of second series) that is to use indirect N protein ELISA that-Figure 15 shows.

The SARS serology detected result (having tested the serum of first series) that is to use two epi-position N protein ELISAs of-Figure 16 representative.

The SARS serology detected result (having tested the serum of second series) that is to use two epi-position N protein ELISAs that-Figure 17 shows.

The anti-N protein monoclonal antibody that is to use ELISA mensuration that-Figure 18 shows is to the reactive result of the natural nucleus albumen N of SARS-CoV.The raying lysate of the Vero E6 cell that use is infected by SARS-CoV is as antigen, measured antibody (SARS clastogram) in the form of Hybridoma Cell Culture thing supernatant liquor with indirect ELISA.For every kind of antibody, with the lysate of the Vero E6 cell of uninfection as reactive negative control (negative clastogram).Some known specific monoclonal antibodies are as negative control antibody: at the antigenic para1-3 antibody of 1-3 type parainfluenza virus (Bio-Rad) with at the influenza B antibody (Bio-Rad) of Type B influenza antigen.

The N protein monoclonal antibody of the anti-SARS-CoV that is to use ELISA mensuration that-Figure 19 shows is to the reactive result of the natural antigen of human coronary virus 229E (HCoV-229E).The lysate of the MRC-5 cell that use is infected by human coronary virus 229E is as antigen, measured antibody (229E clastogram) in the form of Hybridoma Cell Culture thing supernatant liquor with indirect ELISA.For every kind of antibody, with the lysate of the MRC-5 cell of uninfection as immunoreactive negative control (negative clastogram).At the proteic monoclonal antibody 5-11H.6 of the S of human coronary virus 229E (Sizun et al. 1998, J. Virol. Met.72:145-152) as positive control antibody.In the test group of monoclonal antibody, added at the antigenic para1-3 antibody of 1-3 type parainfluenza virus (Bio-Rad) with at the influenza B antibody (Bio-Rad) of Type B influenza antigen.

The N protein monoclonal antibody of the anti-SARS-CoV that is to use the protein immunoblotting assay determination that-Figure 20 shows is for the reactivity of the SARS-CoV natural nucleus albumen N of sex change.The lysate of the Vero E6 cell that will be infected by SARS-CoV according to the method for Laemmli places sample loading buffer and at 12%SDS polyacrylamide gel electrophoresis, then with protein transduction to pvdf membrane.Tested anti-N protein monoclonal antibody is used for immunoassay with the concentration of 0.05 μ g/ml.Visualization process is used the peroxidase anti-mouse IgG of link coupled (H+L) antibody, and (NA93IV Amersham) carries out with the ECL+ system.Used two kinds of monoclonal antibodies as reactive negative control: at the influenza B antibody (Bio-Rad) of Type B influenza antigen with at the antigenic para1-3 antibody of 1-3 type parainfluenza virus (Bio-Rad).

-Figure 21 has represented to be used for to express the proteic plasmid of SARS-CoV S at mammalian cell.With the proteic cDNA of SARS-CoV S be inserted into expression plasmid pcDNA3.1 (+) (Clontech) BamH1 and the Xho1 site between obtain plasmid pcDNA-S, this cDNA is inserted between the Nhe1 of expression plasmid pCI (Promega) and the Xho1 site obtains plasmid pCI-S.WPRE and CTE sequence are inserted between the Xho1 and Xba1 site of plasmid pcDNA-S and pCI-S separately, obtain plasmid pcDNA-S-CTE, pcDNA-S-WPRE, pCI-S-CTE and pCI-S-WPRE respectively.

SP: use signalP v2.0 software (Nielsen et al., 1997.ProteinEngineering, 10:1-6) Yu Ce signal peptide (amino acid/11-13)

TM: use TMHMM v2.0 software (Sonnhammer et al., 1998, Proc.ofSixth Int. Conf. on Intelligent Systems for Molecular Biology, pp. 175-182, AAAI Press) prediction stride diaphragm area (amino acid/11 196-1218).Should notice that amino acid W1194 and P1195 may be parts of striding diaphragm area, its probability is respectively 0.13 and 0.42

P-CMV: cytomegalovirus immediately/early promoter.BGH pA: the polyadenylation signal of bovine growth hormone gene

SV40 pA:SV40 in late period virus polyadenylation signal in late period

SD/SA: splicing donor and acceptor site

WPRE: the sequence of " woodchuck hepatitis virus post-transcriptional control element (the Woodchuck Hepatitis Virus posttranscriptional regulatoryelement) " of woodchuck hepatitis virus

CTE: Ma Sen-gown is pricked the sequence of (Mason-Pfizer) monkey retroviral " composing type transhipment element (constitutive transport element) "

What-Figure 22 showed is the proteic expression of S behind transfection Vero E6 cell.Prepare cell extract with plasmid pcDNA, pcDNA-S, pCI and pCI-S transfection Vero E6 cell after 48 hours.Also infect and plasmid pcDNA or pcDNA-S transfection prepared cell extract after 18 hours with recombined vaccinia virus VV-TF7.3.Infect the VeroE6 cell in the infection multiplicity of using SARS-CoV with 3 and prepare cell extract in contrast after 8 hours.The said extracted thing separates on 8% SDS acrylamide gel, and (NA934V Amersham) has carried out the protein immunoblotting analysis to use anti-S albumen rabbit polyclonal antibody and the anti-rabbit igg of peroxidase link coupled (H+L) polyclonal antibody.Shown molecular weight gradient mark (kDa) among the figure.

SARS-CoV: the extract of the Vero E6 cell that is infected by SARS-CoV

Stand-in: the reference extract of uninfection cell

-Figure 23 shows is to the influence of S protein expression behind CTE and WPRE sequence transfection Vero E6 and the 293T cell.Prepare cell extract with plasmid pcDNA, pcDNA-S, pcDNA-S-CTE, pcDNA-S-WPRE, pCI-S, pCI-S-CTE and pCI-S-WPRE transfection Vero E6 cell (A) or 293T cell (B) after 48 hours, the said extracted thing separates on 8% SDS acrylamide gel, and (NA934V Amersham) has carried out the protein immunoblotting analysis to use anti-S albumen rabbit polyclonal antibody and the anti-rabbit igg of peroxidase link coupled (H+L) polyclonal antibody.Shown molecular weight gradient mark (kDa) among the figure.

SARS-CoV: usefulness SARS-CoV infects the extract of Vero E6 cell after 8 hours with 3 infection multiplicity

Stand-in: the reference extract of the Vero E6 cell of uninfection

-Figure 24 has represented to be used to express the proteic defective type lentiviral vectors with center DNA lobe of SARS-CoV S.The proteic cDNA of SARS-CoV S is cloned among the plasmid pTRIP Δ 3-CMV with the segmental form of BamH1-Xho1 and obtains plasmid pTRIP-S, described pTRIP Δ 3-CMV plasmid comprises defective type lentiviral vectors TRIP (the Sirven et al. with center DNA lobe, 2001, Mol. Ther., 3:438-448).By CMV virus immediately/early promoter, splicing signal, S albumen cDNA and transcribe the suitableeest expression cassette that back signal CTE or WPRE form and replaced and have center DNA lobe TRIP Δ U3-EF1 α (Sirven et al., 2001, Mol. Ther., expression cassette EF1 α-EGFP in defective type lentiviral vectors 3:438-448), thus plasmid pTRIP-SD/SA-S-CTE and pTRIP-SD/SA-S-WPRE obtained.

SP: signal peptide

TM: stride diaphragm area

P-CMV: cytomegalovirus immediately/early promoter

P-EF1 α: EF1 α gene promoter

SD/SA: splicing donor and acceptor site

WPRE: the sequence of " the woodchuck hepatitis virus post-transcriptional control element " of woodchuck hepatitis virus

CTE: Ma Sen-gown is pricked the sequence of monkey retroviral " composing type transhipment element "

LTR: " long terminal repeat "

Δ U3: the long terminal repeat of disappearance " promotor/enhanser " sequence

CPPT: " polypurine tract cis acting sequence "

CTS: " middle terminal sequence (central termination sequence) "

-Figure 25 shows is with lentiviral vectors TRIP-SD/SA-S-CTE and the TRIP-SD/SA-S-WPRE proteic expression of its SARS-CoVS after the clone of transduceing with the protein immunoblotting analysis.Set up clone FrhK4-S-CTE and FrhK4-S-WPRE after transduceing respectively with lentiviral vectors TRIP-SD/SA-S-CTE and TRIP-SD/SA-S-WPRE, and the preparation cell extract.The said extracted thing separates on 8% SDS acrylamide gel, and uses anti-S albumen rabbit polyclonal antibody and the anti-rabbit igg of peroxidase link coupled (H+L) polyclonal antibody to carry out the protein immunoblotting analysis.Shown molecular weight gradient mark (kDa) among the figure.

The reference extract of T-:FrhK-4 cell

T+: usefulness SARS-CoV infects the extract of FrhK-4 cell after 24 hours with 3 infection multiplicity

-Figure 26 relates to the transduce analysis of its Ssol expression of polypeptides after the clone with lentiviral vectors TRIP-SD/SA-Ssol-CTE and TRIP-SD/SA-Ssol-WPRE.Behind lentiviral vectors TRIP-SD/SA-Ssol-CTE and TRIP-SD/SA-Ssol-WPRE transduction FRhK-4 cell, separated a series of cell clone and measured the secretion of the Ssol polypeptide in its supernatant liquor.According to the method for Laemmli the supernatant liquor of 5 μ l is diluted 1/2 and carry out the protein immunoblotting analysis in sample loading buffer, (M2 Sigma) carries out visual with the anti-mouse IgG of peroxidase link coupled (H+L) antibody conjugates to use anti-FLAG monoclonal antibody.T-: the supernatant liquor of parental generation FRhK-4 clone.T+: the supernatant liquor of using the bhk cell of the recombinant vaccinia virus infection of expressing the Ssol polypeptide.Filled arrows is represented the Ssol polypeptide, and hollow arrow is represented the cell proteinic cross reaction of originating.

-Figure 27 shows the correlated results of analysis of the Ssol polypeptide of purifying

A. use 8,2,0.5 of anti-FLAG affinity chromatography and gel-filtration (G75) purifying on the 8%SDS polyacrylamide gel, to separate with the Ssol recombinant polypeptide of 0.125 μ g.The molecular weight marker (MM) of Ssol polypeptide and different amounts with cma staining (Gelcode SilverSNAP stain kitII, Pierce) and visual.

B. the standard marker of SELDI-TOF mass spectroscopy

IgG: ox IgG is the mark of molecular weight 147300

ConA: conalbumin is the mark of molecular weight 77490

HRP: horseradish peroxidase is the mark of molecular weight 43240 also in contrast

C. the mass spectrum (SELDI-TOF) of Ssol polypeptide of recombinating is analyzed.

Peak A and B represent respectively and are with single electric charge and doubly charged Ssol polypeptide.

D. the recombinate N end order-checking of Ssol polypeptide.Upward 5 Edman degraded circulations have been carried out in the liquid phase at ABI494 sequenator (AppliedBiosystems).

-Figure 28 shows is the influence that splicing signal and CTE and WPRE sequence are renderd a service for the genetic immunization that uses encoding SARS-proteic plasmid DNA of CoV S.

A. with pCI, pcDNA-S, pCI-S, pcDNA-N and the pCI-HA plasmid DNA immunity of several groups of BALB/c mouse (7/group), with twice of the timed interval immunity in 4 weeks with 50 μ g.

B. with pCI, pCI-S, pCI-S-CTE and the pCI-S-WPRE plasmid DNA immunity of several groups of BALB/c mouse (6/group), with twice of the timed interval immunity in 4 weeks with 2 μ g, 10 μ g or 50 μ g.

3 weeks of immunity back collecting immune serum for the second time, and using the VeroE6 cell lysate that is infected by SARS-CoV to carry out the indirect ELISA analysis as antigen.With the anti-mouse IgG of peroxidase link coupled polyclonal antibody (NA931V, Amersham) and TMB (KPL) carry out visual after, the inverse of the antibody dilution when producing specific OD value 0.5 calculates anti-SARS-CoV antibody titer.

After-Figure 29 was presented at the genetic immunization of S albumen plasmid of use encoding SARS-CoV, inductive antibody was to infective serum neutralization of SARS-CoV in the mouse body.For the second time 3 weeks of immunity back collecting each part immune serums, be used for described each the group experiment of Figure 28, and estimating it for the SARS-CoV of 100TCID50 infective neutralising capacity to the FRhK-4 cell.Since 1/20 doubling dilution serum one by one, each 4 hole of doubling dilution test.According to the method for Reed and Munsch, with can in and the inverse of infective serum dilution of two in 4 holes calculate the serum neutralization and tire.

A. with of pCI, pcDNA-S, pCI-S, pcDNA-N and the pCI-HA plasmid DNA immunity of several groups of BALB/c mouse, with twice of the timed interval immunity in 4 weeks with 50 μ g.: preimmune serum.■: immune serum.

B. with of pCI, pCI-S, pCI-S-CTE and the pCI-S-WPRE plasmid DNA immunity of several groups of BALB/c mouse, with twice of the timed interval immunity in 4 weeks with 2 μ g, 10 μ g or 50 μ g.

What-Figure 30 showed is the immunoreactivity of reorganization Ssol polypeptide for the SARS patients serum.Reorganization Ssol polypeptide with purifying prepares the reactivity that solid phase is also used indirect ELISA test patient serum.Antibody and solid state reaction with being diluted to 1/400 patient add H with the anti-IgG of peroxidase link coupled people (H+L) polyclonal antibody (Amersham NA933V) and TMB ₂O ₂(KPL) make the antibody of reaction visual.The serum of SARS suspected case suitably according to the series number of France national influenza virus literature centre (Centre National de R é f é rence des Virus Influenzae) and patient's name's abbreviation and after symptom occurring through fate define.The trier's serum T V that gathered in France before SARS in 2003 is popular is serum in contrast.

-Figure 31 shows is after with reorganization Ssol polypeptide immune, at inducing of the antibody of SARS-CoV.With reorganization Ssol polypeptide and the aluminum hydroxide adjuvant (Ssol group) of 10 μ g or use the immune two groups of mouse of adjuvant (simulation group) in contrast separately, every group of 6 mouse, the immunity timed interval was 3 weeks.Continuous immunity three times was also gathered immune serums (IS1, IS2, IS3) in three weeks after each immunity.Use the Vero E6 cell lysate that SARS-CoV infects as antigen, analyzed every part of immune serum in two groups with indirect ELISA.With the anti-mouse IgG of peroxidase link coupled polyclonal antibody (Amersham) and TMB (KPL) carry out visual after, the inverse of the antibody dilution when producing specific OD value 0.5 calculates the antibody titer of anti-SARS-CoV.

-Figure 32 has represented the Nucleotide comparison result of sequence of the wild type gene of synthetic gene 040530 sequence and SARS-CoV strain isolated 031589.I-3059 is numbered the Nucleotide 21406-25348 (SEQ ID No:4, plasmid pSARS-S) of the SARS-CoV strain isolated 031589 of I-3059 corresponding to being preserved in CNCM, and S-040530 is the sequence of synthetic gene 040530.

What-Figure 33 showed is the application of synthetic gene in expressing SARS-CoV S albumen.Prepare cell extract with plasmid pCI, pCI-S, pCI-S-CTE, pCI-S-WPRE and pCI-Ssynth transfection Vero E6 cell (A) or 293T cell (B) after 48 hours, the said extracted thing separates on 8% SDS acrylamide gel, and (NA934V Amersham) has carried out the protein immunoblotting analysis to use anti-S albumen rabbit polyclonal antibody and the anti-rabbit igg of peroxidase link coupled (H+L) polyclonal antibody.The protein immunoblotting analysis by luminescence method (ECL+, Amersham) and digital imaging apparatus (FluorS, BioRad) collection and visual.The proteic expression level of S is by quantitatively measuring two remarkable bands identifying on the image.

-Figure 34 is for making up the synoptic diagram of recombined vaccinia virus VV-TG-S, VV-TG-Ssol, VV-TN-S and VV-TN-Ssol.

A. the cDNA of the S albumen of SARS-CoV and Ssol polypeptide is inserted between the BamH1 of transferring plasmid pTG186 and the Sma1 restriction enzyme site and obtains plasmid pTG-S and pTG-Ssol.

B. then with the sequence of synthetic promoter 480 by exchanging 7.5 promoter sequences that the segmental mode of Nde1-Pst1 is replaced plasmid pTG186poly, pTG-S and pTG-Ssol, obtain transferring plasmid pTN480, pTN-S and pTN-Ssol.

The sequence of the synthetic promoter 480 in the transferring plasmid of C.pTN series between Nde1 and Pst1 restriction enzyme site.For the convenience of subsequent disposal has been inserted an Asc1 restriction enzyme site.This restriction enzyme site and promoter sequence mark with underscore.

D. between the TK gene of Copenhagen (Copenhague) strain of the TK expression cassette of the transferring plasmid of pTG and pTN series and vaccinia virus, carry out reorganization in two autoploids, and obtain recombined vaccinia virus.

SP: use signalP v2.0 software (Nielsen et al., 1997. ProteinEngineering, 10:1-6) Yu Ce signal peptide (amino acid/11-13)

TM: use TMHMM v2.0 software (Sonnhammer et al., 1998, Proc. ofSixth Int. Conf. on Intelligent Systems for Molecular Biology, pp.175-182, AAAIPress) prediction strides diaphragm area (amino acid/11 196-1218).Should notice that amino acid W1194 and P1195 may be parts of striding diaphragm area, its probability is respectively 0.13 and 0.42.

TK-L, TK-R: the left hand and the right hand portion of vaccinia virus thymidine kinase gene.

MCS: multiple clone site

PE: early promoter

PL: late promoter

PL synth: synthetic late promoter 480

What-Figure 35 showed is the proteic expression of analyzing through protein immunoblotting of recombined vaccinia virus S.Use recombined vaccinia virus VV-TG, VV-TG-S and VV-TN-S infect the CV1 cell with 2 infection multiplicity, prepare cell extract (A) after 18 hours.In contrast, usefulness SARS-CoV infects Vero E6 cell with 2 infection multiplicity, prepares cell extract after 8 hours.Also to use recombined vaccinia virus VV-TG-S, VV-TG-Ssol, VV-TN, VV-TN-S and VV-TN-Ssol to infect the CV1 cell, and after 18 hours, prepare cell extract (B).The said extracted thing separates on 8% SDS acrylamide gel, and (NA934V Amersham) has carried out the protein immunoblotting analysis to use anti-S albumen rabbit polyclonal antibody and the anti-rabbit igg of peroxidase link coupled (H+L) polyclonal antibody." 1 μ l " and " 10 μ l " is illustrated in the amount that goes up the cell extract of sample in the gel.Shown molecular weight gradient mark (kDa) among the figure.

SARS-CoV: the extract of the Vero E6 cell that is infected by SARS-CoV

Stand-in: the reference extract of non-infected cells

-Figure 36 has shown the protein immunoblotting analytical results of recombined vaccinia virus secretion Ssol polypeptide.

A. infect the CV1 cell in recombined vaccinia virus VV-TN, different clones' VV-TN-Ssol virus and VV-TG-Ssol or VV-TN-Sflag virus with 2 infection multiplicity, infects 18 hours collection CV1 cell conditioned medium liquid later on.

B. infect twice (1,2) 293T, FRhK-4, BHK-1 and CV1 cell with recombined vaccinia virus VV-TN-Ssol with 2 infection multiplicity, infects 18 hours collection supernatant liquors later on.The supernatant liquor of the viral VV-TN infection of same collection CV1 cell is (M) in contrast.

All supernatant liquors all separate on the 8%SDS acrylamide gel according to the method for Laemmli, and use anti-FLAG mouse monoclonal antibody and the anti-mouse IgG of peroxidase link coupled (H+L) polyclonal antibody (NA931V, Amersham) (A) or use anti-S albumen rabbit polyclonal antibody and the anti-rabbit igg of peroxidase link coupled (H+L) polyclonal antibody (NA934V, Amersham) (B) carries out the protein immunoblotting analysis.

Shown molecular weight gradient mark (kDa) among the figure.

-Figure 37 has shown the analysis to the Ssol polypeptide of purifying on sds page.

On 4 to 15% gradient sds page, separate with the reorganization Ssol polypeptide of 2 μ l with 10,5 of anti-FLAG affinitive layer purification.The molecular weight marker (MM) of Ssol polypeptide and different amounts with cma staining (Gelcode SilverSNAP stain kit II, Pierce) and visual.

-Figure 38 shows is the reorganization Ssol polypeptide produced by the recombined vaccinia virus VV-TN-Ssol immunoreactivity for the SARS patients serum.Reorganization Ssol polypeptide with purifying prepares the reactivity that solid phase is also used indirect ELISA test patient serum.Antibody and solid state reaction with being diluted to 1/100 and 1/400 patient add H with the anti-IgG of peroxidase link coupled people (H+L) polyclonal antibody (Amersham NA933V) and TMB ₂O ₂(KPL) make the antibody of reaction visual.The serum of SARS suspected case suitably according to the series number of France national influenza virus literature centre and patient's name's abbreviation and after symptom occurring through fate define.The trier's serum T V that gathered in France before SARS in 2003 is popular is serum in contrast.

What-Figure 39 showed is the intravital anti-SARS-CoV antibody response of mouse after using the recombined vaccinia virus immunity.Every group of 7 BALB/c mouse are carried out immunity with recombined vaccinia virus VV-TG, VV-TG-HA, VV-TG-S, VV-TG-Ssol, VV-TN, VV-TN-S, the VV-TN-Ssol of 106pfu through the intravenous injection approach respectively, immune secondary, and the timed interval was 4 weeks.

A. all immune serums of gathering in each group of preparation of three after the immunity each time, and the Vero6 cell lysate that uses the SARS-CoV infection are as antigen, and the immune serum of having analyzed in each group with indirect ELISA converges liquid.With the anti-mouse IgG of peroxidase link coupled polyclonal antibody (NA931V, Amersham) and TMB (KPL) carry out visual after, the inverse of the antibody dilution when producing specific OD value 0.5 calculates the antibody titer of anti-SARS-CoV.

B. estimated immune serum and converged liquid for the SARS-CoV of 100TCID50 infective serum neutralising capacity to the FRhK-4 cell.Since 1/20 doubling dilution serum one by one, each 4 hole of doubling dilution test.According to the method for Reed and Munsch, with can in and the inverse of infective serum dilution of two in 4 holes calculate the serum neutralization and tire.

-Figure 40 has described the construction process of recombinant virus MVSchw2-SARS-S and MVSchw2-SARS-Ssol.

A. in the complete genome group of the Schwarz vaccine strain of Measles virus (MV), introduce an additional transcription unit constitute the measles carrier (Combredet, 2003, Journal of Virology, 77:11546-11554).The expression of this additional opening code-reading frame is regulated and control by cis-acting elements, described cis-acting elements by transcribe, the formation and the genetically modified polyadenylic acidization of cap be essential, described transgenosis copy is from the element that is present in the N/P joint.Can insert in two different carriers, described insertion is on the one hand between P (phosphorprotein) and M (matrix) gene, on the other hand between H (hemagglutinin) and L (polysaccharase) gene.

B. the structure of Measles virus recombination group MVSchw2-SARS-S and MVSchw2-SARS-Ssol is that opening code-reading frame with S albumen and Ssol polypeptide is inserted among the additional transcription unit, and this additional transcriptional units is between the P and M gene of carrier.

The a plurality of genes that shown Measles virus (MV) among the figure: N (nucleoprotein), PVC (V/C phosphorprotein and albumen), M (matrix), F (syzygy), H (hemagglutinin), L (polysaccharase).T7=T7 rna polymerase promoter, hh=hammerhead ribozyme, T7t=T7 phage rna polymerase terminator sequence, the ribozyme of δ=hepatitis δ virus, (2), (3)=additional transcription unit (ATU).

MV genome size: 15894 Nucleotide.

SP: signal peptide

TM: stride diaphragm area

The FLAG:FLAG mark

The recombinant measles virus that is to use that-Figure 41 shows is expressed the proteic result of S, has used protein immunoblotting method analysis.

Respectively with different viral MVSchw2-SARS-S and MVSchw2-SARS-Ssol and the wild-type MWSchw virus infection Vero cells in contrast that go down to posterity and prepare the kytoplasm extract.Infect Vero E6 cells at SARS-CoV with infection multiplicity 3 and also prepare cell extract in the sample loading buffer after 8 hours according to Laemmli.The said extracted thing separates on 8% SDS acrylamide gel, and (NA934V Amersham) has carried out the protein immunoblotting analysis to use anti-S albumen rabbit polyclonal antibody and the anti-rabbit igg of peroxidase link coupled (H+L) polyclonal antibody.

Shown molecular weight gradient mark (kDa) among the figure.

Pn: with 293-3-46 and Vero co-culture of cells after the virus in n generation

SARS-CoV: the Vero E6 cell extract that is infected by SARS-CoV

Stand-in: the Vero E6 cell reference extract of Gan Raning not.

-Figure 42 has shown the proteic result of use recombinant measles virus expression S, has used immunofluorescence technique analysis.

Be laid on individual layer Vero cell on the glass slide with wild-type MWSchw virus (A) or MVSchw2-SARS-S (B) and MVSchw2-SARS-Ssol (C) virus infection.When synplasm reach 30-40% (A., B.) or 90-100% (C) converge rate after, with cell fixation, change thoroughly and carry out mark with anti-SARS-CoV rabbit polyclonal antibody and the anti-rabbit igg of FITC (Jackson) link coupled (H+L).

What-Figure 43 showed is the immunoreactive protein immunoblotting analytical results of rabbit anteserum at peptide E1-12, E53-76 and M2-14.The rabbit of numbering 20047 uses KLH link coupled peptide E1-12 to carry out immunity.The rabbit of numbering 22234 and 22240 uses KLH link coupled peptide E53-76 to carry out immunity.The rabbit of numbering 20013 and 20080 uses KLH link coupled peptide M2-14 to carry out immunity.The cell extract of the recombinant vaccinia virus infection of the cell extract that use is infected by SARS-CoV (B) or expression SARS-CoV031589 strain isolated E albumen (A) or M albumen (C) has carried out the protein immunoblotting analysis to immune serum.Immunoblotting use the anti-rabbit igg of peroxidase link coupled (H+L) conjugate (NA934V, Amersham) carry out visual.

The proteic position of E and M illustrates with arrow.

Shown molecular weight gradient mark (kDa) among the figure.

Embodiment

Yet should be appreciated that these embodiment that provide only are the intentions for explanation theme of the present invention, and be construed as limiting never in any form.

Embodiment 1: derive from recording mechanism and be the genomic clone of SARS-CoV virus strain and the order-checking of 031589 sample

The French hospital of (Vietnam) carries out bronchoalveolar lavage to a SARS patient in Ha Noi, and is registered as the RNA that extracts the SARS-CoV virus strain in 031589 the irrigating solution sample at this.

Isolating RNA is used as amplification template, with the cDNA of amplification corresponding to the different opening code-reading frames (ORF1a, ORF1b, ORF-S, ORF-E, ORF-M, ORF-N (comprising ORF-13 and ORF-14), ORF3, ORF4, ORF7 to ORF11) of genome and non-coding 5 ' and 3 ' end.Being used to the primer that increases/detect and the sequence of probe determines based on obtainable SARS-CoV nucleotide sequence.

Hereinafter, primer and probe are defined as follows: alphabetical S, and (L is the 5 ' end that comprises ORF1a and ORF1b with the letter that expression genome respective regions is arranged in the back; S, M and N are ORF-S, ORF-M, ORF-N, SE and MN are corresponding intergenic region), then randomly with Fn, Rn are arranged, n is between 1 to 6, (F1+R1 is that first amplimer is right, and F2+R2 is that second amplimer is right, by that analogy) to the primer that is applied to nest-type PRC, follow the back then has /+/ or/-/corresponding justice or antisense primer, backmost with the primer location that has with reference to Genbank sequence A Y27411.3; Only for the justice and S and N primer and other sense primers of antisense, when showing an independent position, its correspondence be the 5 ' position of holding of the probe or the primer of about 20 bases; For the antisense primer beyond S and the N primer, when showing an independent position, its correspondence be the position of 3 ' end of the probe of about 20 bases or primer.

The amplified production that produces like this uses the Auele Specific Primer order-checking to derive from the genomic complete sequence of SARS-CoV virus strain that is registered as 031589 sample with mensuration.These amplified productions are except corresponding to ORF1a and the ORF1b, and remaining is cloned into subsequently and is used to produce corresponding viral protein in the expression vector and at these proteic antibody, particularly is by the immunity based on DNA.

1.RNA extraction

According to the recommendation of manufacturer, use the QIamp viral RNA to extract miniature test kit (QIAGEN) and extract RNA.Process is as follows more specifically: 140 μ l samples and 560 μ lAVL damping fluids were acutely mixed 15 seconds, at room temperature cultivated 10 minutes, use top speed of short duration centrifugal then.100% ethanol that adds 560 μ l in supernatant liquor was with the mixture vigorous stirring that obtains like this 15 seconds.Mixture with 630 μ l is loaded on the post then.

Post is placed in the 2ml pipe, 8000 rev/mins centrifugal 1 minute, then remaining said mixture is loaded on the same post, again 8000 rev/mins centrifugal 1 minute, post is transferred in the clean 2ml pipe.Next the AW1 damping fluid with 500 μ l is added on the post, 8000 rev/mins centrifugal 1 minute and discard elutant.The AW2 damping fluid of 500 μ l is added on the post, 14000 rev/mins centrifugal 3 minutes, post is transferred in the 1.5ml pipe.AVE damping fluid with 60 μ l is added on the post at last, cultivated under the room temperature 1 to 2 minute, then 8000 rev/mins centrifugal 1 minute.Collection is corresponding to the elutant of purified RNA and frozen in-20 ℃.

2.cDNA amplification, order-checking and clone

2.1) the proteic cDNA of coding S

The RNA that will extract from sample carries out reverse transcription, and described reverse transcription uses six poly-oligonucleotide (pdN6) of stochastic sequence to produce the cDNA fragment.

It is two eclipsed dna fragmentation forms that the sequence of the S glycoprotein of encoding SARS-CoV is amplified: 5 ' fragment (SARS-Sa, SEQ ID No:5) and 3 ' fragment (SARS-Sb, SEQ IDNo:6), they are got by two successive amplifications using nested primer.The amplicon that obtains is like this checked order and is cloned into PCR plasmid vector 2.1-TOPO ^TM(INVITROGEN) in, measured the sequence of clone cDNA then.

A) segmental clone of Sa and Sb and order-checking

A.1) cDNA's is synthetic

Reaction mixture comprises: RNA (5 μ l), injection H ₂O (3.5 μ l), 5 * ThermoScript II damping fluid (4 μ l), 5mM dNTP (2 μ l), pdN6 100 μ g/ml (4 μ l), ribonuclease inhibitor (Rnasin) 40IU/ μ l (0.5 μ l) and ThermoScript II AMV-RT10IU/ μ l, PROMEGA (1 μ l), place thermo cycler to cultivate said mixture by following condition: 42 ℃ 45 minutes, 55 ℃ 15 minutes, 95 ℃ 5 minutes, then the cDNA that obtains is stored in+4 ℃.

A.2) pcr amplification for the first time

5 ' and 3 ' end of S gene uses primer to S/F1/+/21350-21372 and S/R1/-/23518-23498 respectively, and S/F3/+/23258-23277 and S/R3/-/25382-25363 increase.The reaction mixture of 50 μ l comprises: cDNA (2 μ l), 50 μ M primers (0.5 μ l), 10 * damping fluid (5 μ l), 5mM dNTP (2 μ l), Taq high-fidelity amplification enzyme Roche (0.75 μ l) and H ₂O (39.75 μ l), place thermo cycler to increase by following condition said mixture: the first step is 94 ℃ of sex change 2 minutes, be 40 circulations that may further comprise the steps then: 30 seconds, 72 ℃ of 30 seconds, 55 ℃ annealing of 94 ℃ of sex change are extended 2 minutes 30 seconds, every circulations and were additionally extended 10 seconds again, the extension of final step be 72 ℃ 5 minutes.

A.3) pcr amplification for the second time

The product of pcr amplification carries out the pcr amplification second time (nest-type PRC) with the first time, the condition of amplification is with for the first time identical for the second time, use primer to S/F2/+/21406-21426 and S/R2/-/23454-23435 respectively, S/F4/+/23322-23341 and S/R4/-/25348-25329 amplification 5 ' amplicon and 3 ' amplicon.

A.4) segmental clone of Sa and Sb and order-checking

With the Sa (5 ' end) that obtains like this and Sb (3 ' holds) amplicon specification sheets according to manufacturer, use QIAquick PCR purification kit to carry out purifying, then they are cloned among the carrier PCR 2.1-TOPO (INVITROGEN test kit), to obtain to be called the plasmid of SARS-S1 and SARS-S2.

Separate Sa and Sb clone's DNA, corresponding inset is checked order, according to manufacturers instruction, Big Dye test kit, Applied Biosystem have been used in order-checking ^_With M13 forward and the reverse universal primer of M13 and following primer: S/S/+/21867, S/S/+/22353, S/S/+/22811, S/S/+/23754, S/S/+/24207, S/S/+/24699, S/S/+/24348, S/S/-/24209, S/S/-/23630, S/S/-/23038, S/S/-/22454, S/S/-/21815, S/S/-/24784, S/S/+/21556, S/S/+/23130 and S/S/+/24465; Sa of Huo Deing and Sb fragments sequence are corresponding to the sequence as SEQ ID No:5 in the sequence table of annex and SEQ ID No:6 like this.

The plasmid that is called SARS-S1 is preserved in French microbial preservation center, and the date is on May 12nd, 2003, is numbered No.I-3020; This plasmid comprises 5 ' end fragment of the S gene order of the above-mentioned SARS-CoV virus strain that is derived from the sample that is recorded as No.031589, with reference to Genbank sequence A Y274119.3 Tor2, the described Nucleotide that is called the fragment of Sa corresponding to 21406 to 23454 (SEQID No:5) position.

The plasmid that is called TOP10F '-SARS-S2 is preserved in French microbial preservation center, and the date is on May 12nd, 2003, numbering No.I-3019; This plasmid comprises 3 ' end fragment of the S gene order of the above-mentioned SARS-CoV virus strain that is derived from the sample that is recorded as No.031589, with reference to Genbank sequence numbering AY274119.3, the described Nucleotide that is called the fragment of Sb corresponding to 23322 to 25348 (SEQ ID No:6) position.

B) clone and the order-checking of global cDNA (the SARS-S clone of 4kb)

From above-mentioned SARS-S1 and SARS-S2 clone, obtained complete ScDNA by following manner:

1) use above-mentioned primer S/R4/-/25348-25329 and primer S/S/+/24696-24715 that the SARS-S2 clone is carried out pcr amplification reaction: obtain the amplicon of a 633bp,

2) use above-mentioned primer S/F4/+/23322-23341 and primer S/S/-/24803-24784 that another SARS-S2 clone is carried out another time pcr amplification reaction: the amplicon that obtains a 1481bp.

This amplified reaction carries out under the condition of above-mentioned amplification Sa and the reaction of Sb fragment, and different with previous reaction is to comprise 94 ℃ of sex change 20 seconds, 72 ℃ of 30 amplification cycles of extending 2 minutes and 30 seconds.

3) above-mentioned two amplicons (633bp and 1481bp) carry out purifying by above-mentioned purifying Sa and the segmental condition of Sb.

4) to 3) in the purifying amplicon that obtains carry out another pcr amplification reaction, above-mentioned primer S/F4/+/23322-23341 and S/R4/-/25348-25329 are used in this reaction.This amplified reaction carries out under the condition of above-mentioned amplification Sa and the reaction of Sb fragment, and different with previous reaction is to carry out 30 amplification cycles.

To carrier PCR2.1-TOPO, use above-mentioned Sa and the used primer of Sb sequencing fragment to check order as stated above then the amplicon purifying rear clone of the 2026bp that obtains like this.So the clone who obtains is called clone 3 '.

5) SARS-S1 clone that will obtain above and clone 3 ' and digest with EcoRI, the band of about 2kb of gained carries out pcr amplification to it after gel-purified, above-mentioned primer S/F2/+/21406-21426 and the S/R4/-/25348-25329 of described amplification use.This amplified reaction carries out under the condition of above-mentioned amplification Sa and the reaction of Sb fragment, and different with previous reaction is to carry out 30 amplification cycles.Approximately the amplicon of 4kb is purified and checks order.Then with this amplicons cloned to the carrier PCR2.1-TOPO to obtain being called the plasmid of SARS-S, the inset that comprises in this plasmid uses above-mentioned Sa and the used primer of Sb sequencing fragment to check order as stated above.The cDNA sequence of the cDNA sequence of this inset and coding S proteic amplicon corresponds respectively to the sequence as SEQ ID No:4 in the sequence table of annex and SEQ ID No:2, their the S albumen (SEQ ID No:3) of encoding.

Compare with the corresponding sequence of Urbani strain isolated with Tor2 respectively from the sequence of the amplicon of the proteic cDNA of S of the SARS-CoV virus strain of the sample that is recorded as No.031589 corresponding to coding source, the sudden change of following two places is arranged, and the position of sudden change illustrates with reference to the complete genome group sequence (Genbank AY274119.3) of Tor2 strain isolated:

The g/t of-23220 positions; The L-Ala codon (gct) of 577 positions replaces with Serine codon (tct) in the proteic aminoacid sequence of the S of Tor2,

The c/t of-24872 positions; This sudden change does not change the proteic aminoacid sequence of S, and

The plasmid that is called SARS-S is preserved in French microbial preservation center, and the date is on June 20th, 2003, is numbered No.I-3059; This plasmid comprises the S proteic cDNA sequence of coding source from the SARS-CoV virus strain of the sample that is recorded as No.031589, and with reference to Genbank sequence A Y274119.3, described sequence is corresponding to the Nucleotide of 21406 to 25348 (SEQ ID No:4) position.

2.2) coding M and the proteic cDNA of E

The RNA that extracts with aforesaid method that is derived from sample 031589 is carried out reverse transcription, and this reverse transcription reaction is (Titan One Step RT-PCR in same steps as ^_Test kit Roche) combines pcr amplification reaction, and has used following primer right:

-S/E/F1/+/26051-26070 and S/E/R1/-/26455-26436 reach with amplification ORF-E

-S/M/F1/+/26225-26244 and S/M/R1/-/27148-27129 are with amplification ORF-M.

First reaction mixture comprises: the injection H of 8.6 μ l ₂The ribonuclease inhibitor (40IU/ μ l) of the DTT (100mM) of every kind of primer of the dNTP of O, 1 μ l (5mM), 0.2 μ l (50 μ M), 1.25 μ l and 0.25 μ l, second reaction mixture comprises: the injection H of the RNA of 1 μ l, 7 μ l ₂5 * RT-PCR the damping fluid of O, 5 μ l and the enzyme mixture of 0.5 μ l, with these two kinds of reaction mixture remix and place thermo cycler to cultivate by following condition: before this 42 ℃ 30 minutes, 55 ℃ 10 minutes, 94 ℃ 2 minutes, be 40 circulations that may further comprise the steps then: 30 seconds, 68 ℃ of 10 seconds, 55 ℃ annealing of 94 ℃ of sex change are extended 45 seconds, every circulations and are additionally increased by 3 seconds again, the end of final step extend 68 ℃ 7 minutes.

The product (M and E amplicon) of the amplification of acquisition is like this carried out the pcr amplification second time (nest-type PRC), and Expand High-Fi has been used in amplification for the second time ^_Test kit (Roche), and use following primer right:

-for the E amplicon, be S/E/F2/+/26082-26101 and S/E/R2/-/26413-26394, and

-for the M amplicon, be S/M/F2/+/26330-26350 and S/M/R2/-/27098-27078.

Reaction mixture comprises: the PCR product first time of 2 μ l, the injection H of 39.25 μ l ₂O, 5 μ l contain MgCl ₂Every kind of primer (50 μ M) of dNTP (5mM), 0.5 μ l of 10 * damping fluid, 2 μ l and the enzyme mixture of 0.75 μ l, place thermo cycler to cultivate said mixture: 94 ℃ of sex change of the first step 2 minutes by following condition, be 30 circulations that may further comprise the steps then: 30 seconds, 72 ℃ of 15 seconds, 60 ℃ annealing of 94 ℃ of sex change are extended 45 seconds, every circulations and are additionally increased by 3 seconds again, the end of final step extend be 72 ℃ 7 minutes.Correspondence coding E that obtains and the proteic cDNA amplified production of M also use following primer to check order according to the method described above: above-mentioned S/E/F2/+/26082, S/E/R2/-/26394, S/M/F2/+/26330 and S/M/R2/-/27078, and primer S/M/+/26636-26655 and S/M/-/26567-26548.Then they are cloned and obtain being called the plasmid of SARS-E and SARS-M according to the method described above.Separate these clones' DNA and it is checked order, M13 forward and reverse universal primer of M13 and above-mentioned S/M/+/26636 and S/M/-/26548 primers have been used in described order-checking.

The sequence of amplicon of cDNA that representative coding is above-mentioned to be derived from the E albumen (SEQ ID No:13) of the SARS-CoV virus strain of the sample that is recorded as No.031589 does not have difference with respect to the corresponding sequence of AY274119.3-Tor2 and AY278741-Urbani strain isolated.The E protein sequence of 031589 virus strain of SARS-CoV is corresponding to as the SEQ ID No:14 sequence in the sequence table of annex.

The plasmid that is called SARS-E, be preserved in French microbial preservation center, May 28 2003 date, numbering No.I-3046, this plasmid comprises the above-mentioned proteic cDNA sequence of E that is derived from the SARS-CoV virus strain of the sample that is recorded as No.031589 of coding, with reference to Genbank sequence numbering AY274119.3, described sequence is corresponding to the Nucleotide of 26082 to 26413 (SEQ ID No:15) position.

The sequence of amplicon of cDNA that representative coding is above-mentioned to be derived from the M albumen (SEQ ID No:16) of the SARS-CoV virus strain of the sample that is recorded as No.031589 does not have difference with respect to the corresponding sequence of AY274119.3-Tor2 strain isolated.But on 26857 positions, the AY278741-Urbani strain isolated comprises a c and the sequence that is derived from the SARS-CoV virus strain of the sample that is recorded as No.031589 has comprised a t.This sudden change has caused the change of the aminoacid sequence of respective egg white matter: on 154 positions of the SARS-CoV virus strain aminoacid sequence that is derived from the sample that is recorded as No.031589, proline(Pro) (AY278741-Urbani) has become Serine.The M protein sequence of SARS-CoV virus strain that is derived from the sample that is recorded as No.031589 is corresponding to as the SEQ ID No:17 sequence in the sequence table of annex.

The plasmid that is called SARS-M is preserved in French microbial preservation center, May 28 2003 date, numbering No.I-3047; This plasmid comprises the above-mentioned proteic cDNA sequence of M that is derived from the SARS-CoV virus strain of the sample that is recorded as No.031589 of coding, with reference to Genbank sequence numbering AY274119.3, described sequence is corresponding to the Nucleotide of 26330 to 27098 (SEQ ID No:18) position.

2.3) corresponding to the cDNA of ORF3, ORF4, ORF7 to ORF11

Use the same amplification, clone and order-checking strategy to obtain the cDNA fragment of corresponding following ORF respectively: ORF3, ORF4, ORF7, ORF8, ORF9, ORF10 and ORF11.The primer that amplification is for the first time used is to being:

-ORF3 and ORF4:S/SE/F1/+/25069-25088 and S/SE/R1/-/26300-26281

-ORF7 to ORF11:S/MN/F1/+/26898-26917 and S/MN/R1/-/28287-28266

The primer that amplification is for the second time used is to being:

-ORF3 and ORF4:S/SE/F2/+/25110-25129 and S/SE/R2/-/26244-26225

-ORF7 to ORF11:S/MN/F2/+/26977-26996 and S/MN/R2/-/28218-28199

For the first time the condition of amplification (RT-PCR) is as follows: before this 42 ℃ 45 minutes, 55 ℃ 10 minutes, 94 ℃ 2 minutes, be 40 circulations that may further comprise the steps then: 30 seconds, 68 ℃ of 15 seconds, 58 ℃ annealing of 94 ℃ of sex change are extended 1 minute, every circulations and are additionally increased by 5 seconds again, the end of final step extend 68 ℃ 7 minutes.

The condition of nest-type PRC is as follows: 94 ℃ of sex change of the first step 2 minutes, be 40 circulations that may further comprise the steps then: 30 seconds, 72 ℃ of 20 seconds, 58 ℃ annealing of 94 ℃ of sex change are extended 50 seconds, every circulations and are additionally increased by 4 seconds again, the end of final step extend be 72 ℃ 7 minutes.

The amplified production corresponding to the cDNA that comprises ORF3 and ORF4 and ORF7 to ORF11 respectively that is obtained uses following primer order-checking: S/SE/+/25363, S/SE/+/25835, S/SE/-/25494, S/SE/-/25875, S/MN/+/27839, S/MN/+/27409, S/MN/-/27836, S/MN/-/27799, amplified production is cloned with the method for above-mentioned other OFR of clone, to obtain being called the plasmid of SARS-SE and SARS-MN.Separate these clones' DNA and check order the above-mentioned primer that described order-checking use is same and M13 justice and M13 antisense universal primer.

Representative comprises that the sequence of amplicon of the cDNA in the ORF3 of the above-mentioned SARS-CoV virus strain that is derived from the sample that is recorded as No.031589 and ORF4 (SEQ ID No:7) zone has the difference of a Nucleotide with respect to the corresponding sequence of AY274119-Tor2 strain isolated.This sudden change in 25298 positions has caused the change of respective egg white matter (ORF3) aminoacid sequence: be derived from 11 positions of SARS-CoV virus strain aminoacid sequence of the sample that is recorded as No.031589, arginine (AY274119-Tor2) has become glycine.But, compare not discovery sudden change with the corresponding sequence of AY278741-Urbani strain isolated.Being derived from the ORF3 of SARS-CoV virus strain of sample of No.031589 and 4 sequence corresponds respectively to as SEQ ID No:10 in the sequence table of annex and 12 sequences.

The plasmid that is called SARS-SE is preserved in French microbial preservation center, November 13 2003 date, numbering No.I-3126; This plasmid comprise corresponding to the SARS-CoV virus strain of the sample of the above-mentioned No.031589 of being derived from record between ORF-S and ORF-E and with the cDNA in the equitant zone of ORF-E, with reference to Genbank sequence numbering AY274119.3, described zone is corresponding to the Nucleotide of 25110 to 26244 (SEQ ID No:8) position

Representative does not have difference corresponding to the sequence of the amplicon of the cDNA that comprises above-mentioned ORF7 to ORF11 (the SEQ ID No:19) zone that is derived from the SARS-CoV virus strain of the sample that is recorded as No.031589 with respect to the corresponding sequence of AY274119-Tor2 and AY278741-Urbani strain isolated.ORF7 to the ORF11 sequence of SARS-CoV virus strain that is derived from the sample of No.031589 corresponds respectively to as the SEQ ID No:22 in the sequence table of annex, 24,26,28 and 30 sequences.

The plasmid that is called SARS-MN is preserved in French microbial preservation center, November 13 2003 date, numbering No.I-3125; This plasmid comprises the cDNA sequence corresponding to the zone between ORF-M and ORF-N of the SARS-CoV virus strain of the above-mentioned sample that is recorded as No.031589 that picks up from Ha Noi, with reference to Genbank sequence numbering AY274119.3, described sequence is corresponding to the Nucleotide of 26977 to 28218 (SEQ ID No:20) position.

Representative does not have difference corresponding to the sequence of the amplicon of the cDNA that comprises above-mentioned ORF7 to ORF11 (the SEQ ID No:19) zone that is derived from the SARS-CoV virus strain of the sample that is recorded as No.031589 with respect to the corresponding sequence of AY274119-Tor2 and AY278741-Urbani strain isolated.ORF7 to the ORF11 sequence of SARS-CoV virus strain that is derived from the sample of No.031589 corresponds respectively to as the SEQ ID No:22 in the sequence table of annex, 24,26,28 and 30 sequences.

2.4) coding N albumen and comprise ORF13 and the cDNA of ORF14

Above-mentioned Sa and the segmental similar approach of Sb are used in the synthetic and amplification of cDNA.More specifically, reaction mixture comprises: the RNA of 5 μ l, 5 μ l injection H ₂The ribonuclease inhibitor (40IU/ μ l) of the dNTP (5mM) of 5 * ThermoScript II damping fluid of O, 4 μ l, 2 μ l, the oligonucleotide 20T of 2 μ l (5 μ M), 0.5 μ l and AMV-RT (the 10IU/ μ l of 1.5 μ l, Promega), place thermo cycler to cultivate said mixture according to following condition: 42 ℃ 45 minutes, 55 ℃ 15 minutes, 95 ℃ 5 minutes, place then+4 ℃ of preservations.

Pcr amplification uses primer that S/N/F3/+/28023 and S/N/R3/-/29480 are carried out for the first time.

Increase S1 and the segmental reaction mixture of S2 of above-mentioned being used for placed thermo cycler and cultivate according to following condition: 94 ℃ of sex change of the first step 2 minutes, be 40 circulations that may further comprise the steps then: 30 seconds, 72 ℃ of 20 seconds, 55 ℃ annealing of 94 ℃ of sex change are extended 1 minute 30 seconds, every circulations and were additionally extended 10 seconds again, the extension of final step be 72 ℃ 5 minutes.

The amplicon that the first time, pcr amplification obtained is carried out the pcr amplification step second time (nest-type PRC), and amplification for the second time uses primer to S/N/F4/+/28054 and S/N/R4/-/29430, and reaction conditions increases identical with the first time.

What obtain uses following primer to check order corresponding to coding source from the proteic cDNA amplified production of the N of the SARS-CoV of the sample of No.031589 virus strain: S/N/F4/+/28054, S/N/R4/-/29430, S/N/+/28468, S/N/+/28918 and S/N/-/28607, with they method clones, obtain being called the plasmid of SARS-N according to above-mentioned other ORF of clone.Separate these clones' DNA and it is checked order, M13 justice and M13 antisense universal primer and S/N/+/28468, S/N/+/28918 and S/N/-/28607 primers have been used in described order-checking.

Representative is corresponding to the ORF-N of the SARS-CoV virus strain of the sample that is derived from No.031589 and comprise that the sequence of amplicon of the cDNA of ORF13 and ORF14 (SEQ ID No:36) does not have difference with respect to the corresponding sequence of AY274119.3-Tor2 and AY278741-Urbani strain isolated.The N protein sequence of SARS-CoV virus strain of sample that is derived from No.031589 is corresponding to as the SEQ ID No:37 sequence in the sequence table of annex.

The ORF13 and the ORF14 sequence of SARS-CoV virus strain that is derived from the sample of No.031589 corresponds respectively to as SEQ ID No:32 in the sequence table of annex and 34 sequences.

The plasmid that is called SARS-N is preserved in French microbial preservation center, June 5 2003 date, numbering No.I-3048; This plasmid comprises the proteic cDNA of N of SARS-CoV virus strain of the sample of the above-mentioned No.031589 of being derived from that encodes, and with reference to Genbank sequence numbering AY274119.3, described sequence is corresponding to the Nucleotide of 28054 to 29430 (SEQ ID No:38) position.

2.5) non-coding 5 ' and 3 ' end

A) non-coding 5 ' end (5 ' NC)

A1) CDNA's is synthetic

The RNA that is derived from sample 031589 that extracts is as stated above carried out reverse transcription by following condition:

Place 75 ℃ to cultivate 10 minutes RNA (15 μ l) and primer S/L/-/443 (3 μ l, concentration is 5 μ M).

(6 μ l INVITROGEN), 10mM dNTP (1 μ l), 0.1M DTT (3 μ l), place 50 ℃ to cultivate 3 minutes in mixture then to add 5 * ThermoScript II damping fluid.

At last with the ThermoScript II (Superscript of 3 μ l ^_, INVITROGEN) add in the previous mixture 50 ℃ of cultivations 1 hour 30 minutes, then 90 ℃ of cultivations 2 minutes.

With the cDNA that obtains like this recommendation, use QIAquick PCR purification kit (QIAGEN) to carry out purifying according to manufacturer.

A2) The terminal enzyme (DNA) reaction(TdT)

CDNA (10 μ l) in 100 ℃ of cultivations 2 minutes, is stored on ice, add following material: H then ₂O (2.5 μ l), 5 * TdT damping fluid (4 μ l, AMERSHAM), 5Mm dATP (2 μ l) and TdT (1.5 μ l, AMERSHAM).The mixture that obtains is like this cultivated in 37 ℃ and was cultivated 2 minutes in 65 ℃ then in 45 minutes.

Products therefrom is by the amplification of PCR reaction for the first time, and the primer that described amplification is used is: S/L/-/225-206 and anchor primer 14T:5 '-AGATGAATTCGGTACCTTTTTTTTTTTTTT-3 ' (SEQ ID No:68).Amplification condition is as follows: 94 ℃ of sex change of the first step 2 minutes, be 10 circulations that may further comprise the steps then: 10 seconds, 45 ℃ annealing of 94 ℃ of sex change were extended 30 seconds for 30 seconds, 72 ℃, be 30 circulations that may further comprise the steps again then: 10 seconds, 50 ℃ annealing of 94 ℃ of sex change were extended 30 seconds for 30 seconds, 72 ℃, the extension of final step be 72 ℃ 5 minutes.

The product of pcr amplification carries out the amplification step second time with the first time, and primer S/L/-/204-185 and above-mentioned anchor primer 14T are used in amplification for the second time, and reaction conditions increases identical with the first time.The purified back of gained amplicon uses primer S/L/-/182-163 to check order, and they are cloned according to the method for the different ORF of above-mentioned clone, obtain being called the plasmid of SARS-5 ' NC.Separate this clone's DNA and it is checked order, M13 justice and M13 antisense universal primer and above-mentioned S/L/-/182-163 primer have been used in described order-checking.

Representative corresponding to the amplicon of the cDNA of 5 ' NC of the SARS-CoV virus strain that is derived from the sample that is recorded as No.031589 end corresponding to as the SEQ ID No:72 sequence in the sequence table of annex.This sequence does not have difference with respect to the corresponding sequence of AY274119.3-Tor2 and AY278741-Urbani strain isolated.

The plasmid that is called SARS-5 ' NC is preserved in French microbial preservation center, November 7 2003 date, numbering No.I-3124; This plasmid comprises the cDNA that holds corresponding to the above-mentioned genomic non-coding 5 ' that is derived from the SARS-CoV virus strain of the sample that is recorded as No.031589, with reference to Genbank sequence numbering AY274119.3, described sequence is corresponding to the Nucleotide of 1 to 204 (SEQ ID No:39) position

B) Non-coding 3 ' end(3 ' NC)

B1) CDNA's is synthetic

The RNA that is derived from sample 031589 that extracts is as stated above carried out reverse transcription by following operation: reaction mixture comprises: RNA (5 μ l), H ₂O (5 μ l), 5 * ThermoScript II damping fluid (4 μ l), 5mM dNTP (2 μ l), the ribonuclease inhibitor (0.5 μ l) of 5 μ M oligonucleotide 20T (2 μ l), 40IU/ μ l and RT-AMV (the 1.5 μ l of 10IU/ μ l, PROMEGA), place thermo cycler to cultivate said mixture according to following condition: 42 ℃ 45 minutes, 55 ℃ 15 minutes, 95 ℃ 5 minutes, place then+4 ℃ of preservations.

The cDNA of gained is by the amplification of PCR reaction for the first time, and the primer that described amplification is used is S/N/+/28468-28487 and above-mentioned anchor primer 14T.Amplification condition is as follows: 94 ℃ of initial denaturing steps of 2 minutes before this, be 10 circulations that may further comprise the steps then: 20 seconds, 45 ℃ annealing of 94 ℃ of sex change were extended 50 seconds for 30 seconds, 72 ℃, be 30 circulations that may further comprise the steps again then: 20 seconds, 50 ℃ annealing of 94 ℃ of sex change were extended 50 seconds for 30 seconds, 72 ℃, the extension of final step be 72 ℃ 5 minutes.

The product of pcr amplification carries out the amplification step second time with the first time, and primer S/N/+/28933-28952 and above-mentioned anchor primer 14T are used in amplification for the second time, and reaction conditions increases identical with the first time.The purified back of gained amplicon uses primer S/N/+/29257-29278 to check order, and they are cloned according to the method for the different ORF of above-mentioned clone, obtain being called the plasmid of SARS-3 ' NC.Separate these clones' DNA and it is checked order, M13 justice and M13 antisense universal primer and above-mentioned S/N/+/29257-29278 primer have been used in described order-checking.

Representative corresponding to the amplicon of the cDNA of 3 ' NC of the SARS-CoV virus strain that is derived from the sample that is recorded as No.031589 end corresponding to as the SEQ ID No:73 sequence in the sequence table of annex.This sequence does not have difference with respect to the corresponding sequence of AY274119.3-Tor2 and AY278741-Urbani strain isolated.

The plasmid that is called SARS-3 ' NC is preserved in French microbial preservation center, November 7 2003 date, numbering No.I-3123; This plasmid comprises the cDNA sequence of holding corresponding to the above-mentioned genomic non-coding 3 ' that is derived from the SARS-CoV virus strain of the sample that is recorded as No.031589, with reference to Genbank sequence numbering AY274119.3, described sequence is corresponding to the Nucleotide that is positioned at the position between 28933 to 29727 (the SEQ ID No:40), and ending is a series of Nucleotide a.

2.6) ORF1a and ORF1b

RT-PCR reaction and nest-type PRC subsequently with the same principle of above-mentioned other ORF of amplification have been used in the amplification in the 5 ' zone that comprises genomic ORF1a of the SARS-CoV that is derived from 031589 sample and ORF1b.Amplified fragments has the overlapping of a few tenths of base, can rebuild this part genomic complete sequence with computer like this.On average, amplified fragments has 2,000 bases.

14 overlapping fragmentses that are called as L0 to L12 are used following primer amplification:

Table II: the primer in the 5 ' zone (ORF1a and ORF1b) that is used to increase

The zone (not comprising primer) that is amplified and is checked order	The RT-PCR sense primer	The RT-PCR antisense primer	The nest-type PRC sense primer	The nest-type PRC antisense primer
					L0	S/L0/F1/+30	S/L0/R1/-481

50-480
					L1 231-2240	S/L1/F1/+147	S/L1/R1/-2336	S/L1/F2/+211	S/L1/R2/-2241
L2 2156-4167	S/L2/F1/+2033	S/L2/R1/-4192	S/L2/F2/+2136	S/L2/R2/-4168
					L3 3913-5324	S/L3bis/F1/+3850	S/L3bis/R1/-5365	S/L3bis/F2/+3892	S/L3bis/R2/-5325
L4b 4952-6023	S/L4b/F1/+4878	S/L4b/R1/-6061	S/L4b/F2/+4932	S/L4b/R2/-6024
					L4 5325-7318	S/L4/F1/+5272	S/L4/R1/-7392	S/L4/F2/+5305	S/L4/R2/-7323
L5 7296-9156	S/L5/F1/+7111	S/L5/R1/-9253	S/L5/F2/+7275	S/L5/R2/-9157
					L6 9053-11066	S/L6/F1/+8975	S/L6/R1/-11151	S/L6/F2/+9032	S/L6/R2/-11067
L7 10928-12962	S/L7/F1/+10883	S/L7/R1/-13050	S/L7/F2/+10928	S/L7/R2/-12963
					L8 12835-14834	S/L8/F1/+12690	S/L8/R1/-14857	S/L8/F2/+12815	S/L8/R2/-14835
L9 14765-16624	S/L9/F1/+14688	S/L9/R1/-16678	S/L9/F2/+14745	S/L9/R2/-16625
					L10 16534-18570	S/L10/F1/+16451	S/L10/R1/-18594	S/L10/F2/+16514	S/L10/R2/-18571
L11 18521-20582	S/L11/F1/+18441	S/L11/R1/-20612	S/L11/F2/+18500	S/L11/R2/-20583
					L12?20338-22205	S/L12/F1/+20279	S/L12/R1/-22229	S/L12/F2/+20319	S/L12/R2/-22206

Except the condition amplification of L0 fragment according to above-mentioned ORF-M, all the other all fragments increase according to following condition:

- RT-PCR: 42 ℃ 30 minutes, 55 ℃ 15 minutes, 94 ℃ 2 minutes, then gained cDNA is increased with following condition: 40 circulations that may further comprise the steps: 30 seconds, 68 ℃ of 15 seconds, 58 ℃ annealing of 94 ℃ of sex change are extended 1 minute 30 seconds, every circulations and are additionally increased by 5 seconds again, the extension of final step be 68 ℃ 7 minutes.

-nest-type PRC: 94 ℃ of sex change of the first step 2 minutes are 35 circulations that may further comprise the steps then: 30 seconds, 72 ℃ of 15 seconds, 60 ℃ annealing of 94 ℃ of sex change are extended 1 minute 30 seconds, every circulations and were additionally extended 5 seconds again, the extension of final step be 72 ℃ 7 minutes.

The primer that defines in the amplified production use Table III below checks order:

Table III: the primer that is used for the order-checking of 5 ' district (ORF1a and OR1b)

Title	Sequence (SEQ ID NO:76 to 139)
		S/L3/+/4932	5’-CCACACACAGCTTGTGGATA-3’
S/L4/+/6401	5’-CCGAAGTTGTAGGCAATGTC-3’
		S/L4/+/6964	5’-TTTGGTGCTCCTTCTTATTG-3’
S/L4/-/6817	5’-CCGGCATCCAAACATAATTT-3’
		S/L5/-/7633	5’-TGGTCAGTAGGGTTGATTGG-3’
S/L5/-/8127	5’-CATCCTTTGTGTCAACATCG-3’
		S/L5/-/8633	5’-GTCACGAGTGACACCATCCT-3’
S/L5/+/7839	5’-ATGCGACGAGTCTGCTTCTA-3’
		S/L5/+/8785	5’-TTCATAGTGCCTGGCTTACC-3’
S/L5/+/8255	5’-ATCTTGGCGCATGTATTGAC-3’
		S/L6/-/9422	5’-TGCATTAGCAGCAACAACAT-3’
S/L6/-/9966	5’-TCTGCAGAACAGCAGAAGTG-3’
		S/L6/-/10542	5’-CCTGTGCAGTTTGTCTGTCA-3’
S/L6/+/10677	5’-CCTTGTGGCAATGAAGTACA-3’
		S/L6/+/10106	5’-ATGTCATTTGCACAGCAGAA-3’
S/L6/+/9571	5’-CTTCAATGGTTTGCCATGTT-3’
		S/L7/-/11271	5’-TGCGAGCTGTCATGAGAATA-3’
S/L7/-/11801	5’-AACCGAGAGCAGTACCACAG-3’
		S/L7/-/12383	5’-TTTGGCTGCTGTAGTCAATG-3’
S/L7/+/12640	5’-CTACGACAGATGTCCTGTGC-3’
		S/L7/+/12088	5’-GAGCAGGCTGTAGCTAATGG-3’
S/L7/+/11551	5’-TTAGGCTATTGTTGCTGCTG-3’
		S/L8/-/13160	5’-CAGACAACATGAAGCACCAC-3’
S/L8/-/13704	5’-CGCTGACGTGATATATGTGG-3’

S/L8/-/14284	5’-TGCACAATGAAGGATACACC-3’
		S/L8/+/14453	5’-ACATAGCTCGCGTCTCAGTT-3’
S/L8/+/13968	5’-GGCATTGTAGGCGTACTGAC-3’
		S/L8/+/13401	5’-GTTTGCGGTGTAAGTGCAG-3’
S/L9/-/15098	5’-TAGTGGCGGCTATTGACTTC-3’
		S/L9/-/15677	5’-CTAAACCTTGAGCCGCATAG-3’
S/L9/-/16247	5’-CATGGTCATAGCAGCACTTG-3’
		S/L9/+/16323	5’-CCAGGTTGTGATGTCACTGAT-3’
S/L9/+/15858	5’-CCTTACCCAGATCCATCAAG-3’
		S/L9/+/15288	5’-CGCAAACATAACACTTGCTG-3’
S/L10/-/16914	5’-AGTGTTGGGTACAAGCCAGT-3’
		S/L10/-/17466	5’-GTTCCAAGGAACATGTCTGG-3’
S/L10/-/18022	5’-AGGTGCCTGTGTAGGATGAA-3’
		S/L10/+/18245	5’-GGGCTGTCATGCAACTAGAG-3’
S/L10/+/17663	5’-TCTTACACGCAATCCTGCTT-3’
		S/L10/+/17061	5’-TACCCATCTGCTCGCATAGT-3’
S/L11-/18877	5’-GCAAGCAGAATTAACCCTCA-3’
		S/L11/-/19396	5’-AGCACCACCTAAATTGCATC-3’
S/L11/-/20002	5’-TGGTCCCTTTGAAGGTGTTA-3’
		S/L11/+/20245	5’-TCGAACACATCGTTTATGGA-3’
S/L11/+/19611	5’-GAAGCACCTGTTTCCATCAT-3’
		S/L11/+/19021	5’-ACGATGCTCAGCCATGTAGT-3’
SARS/L1/F3/+800	5’-GAGGTGCAGTCACTCGCTAT-3’
		SARS/L1/F4/+1391	5’-CAGAGATTGGACCTGAGCAT-3’
SARS/L1/F5/+1925	5’-CAGCAAACCACTCAATTCCT-3’
		SARS/L1/R3/-1674	5’-AAATGATGGCAACCTCTTCA-3’
SARS/L1/R4/-1107	5’-CACGTGGTTGAATGACTTTG-3’
		SARS/L1/R5/-520	5’-ATTTCTGCAACCAGCTCAAC-3’
SARS/L2/F3/+2664	5’-CGCATTGTCTCCTGGTTTAC-3’
		SARS/L2/F4/+3232	5’-GAGATTGAGCCAGAACCAGA-3’
SARS/L2/F5/+3746	5’-ATGAGCAGGTTGTCATGGAT-3’

SARS/L2/R3/-3579	5’-CTGCCTTAAGAAGCTGGATG-3’
		SARS/L2/R4/-2991	5’-TTTCTTCACCAGCATCATCA-3’
SARS/L2/R5/-2529	5’-CACCGTTCTTGAGAACAACC-3’
		SARS/L3/F3/+4708	5’-TCTTTGGCTGGCTCTTACAG-3’
SARS/L3/F4/+5305	5’-GCTGGTGATGCTGCTAACTT-3’
		SARS/L3/F5/+5822	5’-CCATCAAGCCTGTGTCGTAT-3’
SARS/L3/R3/-5610	5’-CAGGTGGTGCAGACATCATA-3’
		SARS/L3/R4/-4988	5’-AACATCAGCACCATCCAAGT-3’
SARS/L3/R5/-4437	5’-ATCGGACACCATAGTCAACG-3’

L0 to the L12 fragments sequence that is derived from the SARS-CoV virus strain of the sample that is recorded as No.031589 corresponds respectively to as SEQ ID No:41 to the SEQ ID No:54 sequence in the sequence table of annex.In these sequences, only comprise the difference of a Nucleotide with respect to the corresponding sequence of AY278741-Urbani strain isolated corresponding to the L5 fragments sequence.This t/c sudden change that is positioned at 7919 positions has caused the change by the corresponding protein aminoacid sequence of ORF1a coding: on 2552 positions, (the gtt codon AY278741) has become L-Ala (gct codon) to Xie Ansuan in 031589 virus strain of SARS-CoV.Yet, do not have the sudden change of discovery with respect to the corresponding sequence of AY274119.3-Urbani strain isolated.Other fragment does not demonstrate difference with respect to the corresponding sequence of Tor2 and Urbani strain isolated.

Embodiment 2: the recombinant N protein and proteic production of S and the purifying that are derived from the SARS-CoV virus strain of the sample that is recorded as No.031589

Produced the complete N albumen and proteic two polypeptide fragments of S of the SARS-CoV virus strain that is derived from the sample that is recorded as No.031589 in intestinal bacteria, described albumen and fragment are the form generations with the fusion rotein of the polyhistidine label that comprises N end or C end.In these two S polypeptide, the hydrophobic sequence of S albumen n end and C end (signal peptide: 1 to 13 position and transbilayer helix: 1196 to 1218 positions) disappearance and two motifs (895 to 980 positions and 1155 to 1186 positions) of proteic β spiral of S (565 to 687 position) and coiled coil type all are retained.These two polypeptide are made of following part: corresponding to a long segment (S of 14 to 1193 positions of S Argine Monohydrochloride sequence _L) and corresponding to a short-movie section (S of 475 to 1193 positions of S Argine Monohydrochloride sequence _C).

1) with N, S _LAnd S _CCDNA be cloned in expression vector pIVEX2.3 and the pIVEX2.4

Corresponding to N albumen, S _LAnd S _CCDNA fragment increases by the PCR under standard conditions, and archaeal dna polymerase Platinum Pfx has been used in described amplification ^_(INVITROGEN).Use plasmid SARS-N and SARS-S as template and following oligonucleotide as primer:

5 '-CC CATATGTCTGATAATGGACCCCAATCAAAC-3 ' (N sense primer, SEQID NO:55)

5 '-CC CCCGGGTGCCTGAGTTGAATCAGCAGAAGC-3 ' (N antisense primer, SEQID NO:56)

5 '-CC CATATGAGTGACCTTGACCGGTGCACCAC-3 ' (S _CSense primer, SEQ IDNO:57)

5 '-CC CATATGAAACCTTGCACCCCACCTGCTC-3 ' (S _LSense primer, SEQ IDNO:58)

5 '-CC CCCGGGTTTAATATATTGCTCATATTTTCCC-3 ' (S _CAnd S _LAntisense primer, Seq ID NO:29)

Introduce a NdeI restriction enzyme site (underscore part) in the sense primer and introduced an XamI or SmaI restriction enzyme site (underscore part) in the antisense primer.(QIAquick PCR purification kit is cloned in the suitable carriers after QIAGEN) these 3 amplified productions through column purification.(QIAFilter Midi Plasmid test kit, plasmid DNA QIAGEN) is by digesting with enzyme NdeI and XamI after the sequence verification for purifying from these 3 constructs.Respectively will be corresponding to N, S _LAnd S _C3 fragments purifying on sepharose of cDNA, be inserted into then in advance by in good pIVEX2.3MCS of same enzymic digestion (C end polyhistidine label) and pIVEX2.4d (N the holds the polyhistidine label) plasmid.Through after the checking to construct, 6 expression vectors (pIV2.3N, pIV2.3S of Huo Deing like this _C, pIV2.3S _L, pIV2.4N, pIV2.4S _C(be also referred to as pIV2.4S ₁), pIV2.4S _L) be used for the outer protein expression of test body on the one hand, be used for transform bacteria bacterial strain BL21 (DE3) pDIA17 (NOVAGEN) on the other hand.The expection molecular weight of these construct encoded protein matter is as follows: pIV2.3N (47174Da), pIV2.3S _C(82897Da), pIV2.3S _L(132056Da), pIV2.4N (48996Da), pIV2.4S ₁(81076Da), pIV2.4S _L(133877Da).The bacterial strain that transforms with pIV2.3N is preserved in CNCM, and on October 23 2003 date, numbering I-3117 uses pIV2.4S ₁The bacterial strain that transforms is preserved in CNCM, October 23 2003 date, numbering I-3118.

2) analysis of recombinant protein expression in vitro and in vivo

At first in vitro system (RTS100, the Recombinant Protein Expression of having tested these 6 recombinant vectorss in Roche).Use protein immunoblotting method to analyze in 30 ℃ of protein of cultivating recombinant vectors external generation after pIVEX4 hour in the RTS100 system, that described protein immunoblotting method is used is peroxidase link coupled anti-(his) ₆Antibody.The result of vivoexpression (Fig. 1) shows no matter the polyhistidine label is at N end or C end position, all only has N albumen to obtain a large amount of expression.Second step, detected the proteic expression of N and S in vivo, described detection is at 30 ℃, LB substratum, existence or do not exist under the condition of inductor (1mM IPTG) and carry out.The expression of N albumen in this bacterial system fine (Fig. 2), and after the bacterium cracking, be mainly soluble part.On the contrary, the proteic long segment (S of S _L) expression very weak and soluble fully (Fig. 3).Short-movie section (S _C) solvability that shows is also very poor, but its expression level is than high many of long segment.This external C end position has merged the S of polyhistidine label _CThe size of construct is littler than expection.Use the immunodetection experiment of anti-polyhistidine antibody to show that this construct is incomplete.In a word, selecting to express the C end merges the proteic pIV2.3N construct of complete N of polyhistidine label and expresses the proteic pIV2.4S of short S that the N end merges the polyhistidine label ₁Construct produce these two kinds of albumen in a large number in case purifying they.Plasmid pIV2.3N and pIV2.4S ₁Be preserved in CNCM on October 23rd, 2003, numbering is respectively I-3117 and I-3118.

3) analysis of recombinant protein antigenic activity

Use protein immunoblotting method to test N, S _LAnd S _CProteic antigenic activity, described protein immunoassay have used from the patient of same infection SARS-CoV, two kinds of serum samples that 8 days (M12) and 29 days (M13) gathers after it the SARS symptom occurs.Experimental procedure is described in embodiment 3.The results are shown among Fig. 4, (i) is patient's seroconversion, shows that (ii) N albumen has than the high antigen reactivity of short S albumen.

4) from the proteic purifying of the N of pIV2.3N

Carried out the proteic experiment of N that some purifying are produced by carrier pIV2.3N according to following step.To place the 1 liter of substratum that contains the 0.1mg/ml penbritin 30 ℃ of cultivations, when cell density is equivalent to A with BL21 (DE3) the pDIA17 bacterium that expression vector pIV2.3N transforms ₆₀₀Induce with the IPTG of 1mM=0.8 o'clock (about 3 hours), cultivates after 2 hours centrifugal collecting cell (5000rpm, 10 minutes) and at lysis buffer (50mM NaH in the presence of inductor ₂PO ₄, 0.3M NaCl, 20mM imidazoles, pH8, contain protease inhibitor cocktail Complete ^_, resuspended in Roche), and with French press (12000psi) lysing cell.After bacterial lysate centrifugal (12000rpm, 15 minutes), with supernatant liquor (50ml) with the flow velocity of 1ml/min pack into the good metal chelating column of lysis buffer balance (15ml) (Ni-NTA superflow, Qiagen) on.After using the lysis buffer washing column of 200ml, with imidazoles gradient (20 → 250mM) the wash-out N albumen of 10 times of column volumes.Collection comprises the proteic component of N and analyzes with polyacrylamide gel electrophoresis under the sex change condition and Coomassie brilliant blue dyeing.The results are shown in Fig. 5, this result show with this step can purifying N protein to satisfied purity (95%), and average yield is every liter of nutrient solution 15mg albumen.

5) from pIV2.4S _C(pIV2.4S ₁) S _CProteic purifying

The short proteic step of S of following purifying has very big-difference with above-mentioned purification step, because this albumen is in bacterial system camber gathering (inclusion body).To use expression vector pIV2.4S ₁BL21 (DE3) the pDIA17 bacterium that transforms places the 1 liter of substratum that contains the 0.1mg/ml penbritin 30 ℃ of cultivations, when cell density is equivalent to A ₆₀₀Induce with the IPTG of 1mM=0.8 o'clock (about 3 hours), after cultivating 2 hours in the presence of the inductor, centrifugal collecting cell (5000rpm, 10 minutes) is also resuspended in lysis buffer (0.1M Tris-HCl, 1mM EDTA, pH7.5), and with French press (1200psi) lysing cell.After bacterial lysate centrifugal (12000rpm, 15 minutes), flaky precipitate is resuspended in the lysis buffer that contains 2%Triton X-100 and 10mM beta-mercaptoethanol, centrifugal 20 minutes then with 12000rpm.Again that flaky precipitate is resuspended in containing the 10mM Tris-HCl damping fluid of 7M urea, and mild stirring 30 minutes at room temperature.Final step is essential with the step of the urea washing inclusion body of 7M, can remove most like this and accumulative S _CThe intestinal bacteria membranin of albumen co-precipitation.Centrifugal 20 minutes at last, that last flaky precipitate is resuspended in 10mM Tris-HCl damping fluid with 12000rpm.The electrophoretic analysis result (Fig. 6) of prepared product shows that short S albumen is purified to satisfied purity (about 90%) from inclusion body (insoluble extract).

Embodiment 3: the proteic immundominance of N

By the protein immunoblotting method of carrying out under the following conditions, analyzed the antibody that exists among the atypical pneumonia patients serum who causes by SARS associated coronavirus (SARS-CoV) various proteic reactivity for this virus.

1) material

A) The lysate of the cell that is infected by SARS-CoV

With SARS-CoV (recording mechanism is the strain isolated of FFM/MA104) with 10 ^-1Or 10 ^-2Infection multiplicity (M.O.I.) infect Vero E6 cell (2 * 10 ⁶), cell is placed the DMEM substratum that contains 2%FCS, at 35 ℃, 5%CO ₂Condition under cultivate.Cultivate after 48 hours, with PBS washed cell layer and with the sample loading buffer lysing cell that contains beta-mercaptoethanol for preparing according to the Laemmli method of 500 μ l.Sample was boiled 10 minutes, and with supersound process 3 times, each 20 seconds.

B) Antibody

B1) Atypical pneumonia patient's serum

With the serum of the No.20033168 document name of France national influenza virus literature centre (north zone) the atypical pneumonia patient who is caused by SARS-CoV from a France, serum is collected in 38 days that occur after the symptom.The SARS-CoV diagnosis of infection is undertaken by nest-type PRC and quantitative PCR.

B2) At N albumen or the proteic monospecific rabbit polyclonal antibody of S serum

Serum is with recombinant N protein and S _CAlbumen (embodiment 2) is according to the immunization method production of describing among the embodiment 4; They are rabbit P13097 serum (anti-N albumen serum) and rabbit P11135 serum (anti-S albumen serum).

2) method

With 20 μ l by SARS-CoV with 10 ^-1With 10 ^-2The lysate of the cell that infects of infection multiplicity and the lysate of 20 μ l non-infected cells (stand-in) in contrast on the 10%SDS polyacrylamide gel, separate, go on the nitrocellulose filter then.After washing with the PBS solution sealing that contains 5% milk, 0.1%Tween and with the PBS that contains 0.1%Tween, spend the night with the hybridization of following serum at 4 ℃ with this film: (i) containing in the PBS damping fluid of 1%BSA and 0.1%Tween and be diluted to 1/300,1/1000 and 1/3000 No.20033168 immune serum, (ii) in same damping fluid, be diluted to 1/50000 rabbit P13097 serum (anti-N albumen serum) and (iii) in same damping fluid, be diluted to 1/10000 rabbit P11135 serum (anti-S albumen serum).After with the PBS/Tween washing, carry out the hybridization second time, sheep polyclonal antibody (the NA933V of peroxidase link coupled at people G heavy chain immunoglobulin and light chain used in hybridization for the second time, Amersham), or use the peroxidase link coupled at the donkey polyclonal antibody of rabbit G heavy chain immunoglobulin and light chain (NA934V, Amersham).The visual ECL+ test kit (Amersham) and the Hyperfilm MP radioautograph film (Amersham) of having used of binding antibody.Shown molecular weight gradient mark (kDa) among the figure.

3) result

Fig. 7 shows, in the extract of the cell that SARS-CoV infects, detected apparent molecular weight specifically and be 35,55 and three peptide species of 200kDa.

For identifying this several peptide species, use the rabbit polyclonal antibody of specificity at nucleoprotein N (rabbit P13097, Fig. 8 A) and thorn Protein S (rabbit P11135, Fig. 8 B), preparing two other immunoblotting (Fig. 8) on the same sample He under the similarity condition.This experiment shows the thorn glycoprotein S of the corresponding SARS-CoV of the polypeptide of this 200kDa, the corresponding nucleoprotein N of the polypeptide of this 55kDa and the polypeptide of this 35kDa may represent that N is proteic by the form of brachymemma or degraded.

Therefore, data presentation 20033168 serum among Fig. 7 and N albumen kickback and want much weak with the proteic reaction of the S of SARS-CoV, because use the immune serum of 1/300,1/1000 and 1/3000 dilution can make 35 and the polypeptide of 55kDa be shown as strong band, and as seen the polypeptide of 200kDa only faint when serum 1/300 dilution.It is further noted that the extent of dilution at 20033168 serum is detected greater than the SARS-CoV polypeptide that did not have other at 1/300 o'clock.

This experiment shows that specificity is better than the antibody response of specificity at other SARS-CoV polypeptide at the proteic antibody response of the N of SARS-CoV, particularly at the antibody response of S glycoprotein.This has illustrated the immundominance that infects nucleoprotein N in the human process at SARS-CoV.

Embodiment 4: at the N and the proteic monospecific Polyclonal Antibody Preparation of S of SARS associated coronavirus (SARS-CoV)

1) material and method

Use three rabbits of recombinant immunogenic polypeptide (P13097, P13081, P13031) of the corresponding complete nucleoprotein (N) of purifying, described recombinant polypeptide is according to the step preparation of describing among the embodiment 2.Injection is for the first time injected 0.35mg with complete freund adjuvant emulsive albumen (intradermal routes) for every rabbit, and 3 weeks and 4 weeks are carried out 3 booster immunization injections to animal more at interval then, and per injection 0.35mg is with incomplete freund adjuvant emulsive recombinant protein.

Use three rabbits of recombinant immunogenic polypeptide (P11135, P1304, P14001) of corresponding S albumen short-movie section (Sc), described recombinant polypeptide is according to the step preparation of describing among the embodiment 2.Since this peptide species in the kytoplasm of bacterium mainly the form with inclusion body exist, so animal is carried out 4 intradermal injections with the timed interval in 3-4 week, per injection with incomplete freund adjuvant emulsive, be equivalent to the inclusion body preparation of 0.5mg recombinant protein.The inclusion body preparation according to the step preparation of describing among the embodiment 2 is used in first three time injection, the 4th injection use according to the step preparation of describing among the embodiment 2 and through the inclusion body preparation of saccharose gradient purifying and 2%Triton X-100 washing.

To every rabbit, preparation its preimmune serum (p.i.) before the immunity first time, and at 5 week of the 4th immunity back preparation immune serum (I.S.).

At first, use the reactivity of having tested serum for the similar ELISA that is used for the recombinant protein preparation of immunity; Described ELISA test is according to the step of describing among the embodiment 6 and use the reagent among this embodiment to carry out.

Secondly, the reactivity of using the method (protein immunoblotting method) of the immunoblotting of the cell lysate that preparation infected by SARS-CoV to test serum, described test is carried out according to the step of describing among the embodiment 3.

2) result

The result (Fig. 9) of ELISA test proves recombinant N protein preparation and S albumen short-movie section (S _C) the inclusion body preparation have immunogenicity in animal body, and the tiring very high (being higher than 1/25000) of immune serum.

Immunoblotting result (Fig. 8) demonstration rabbit P13097 immune serum is discerned two peptide species in the lysate that is present in the cell that is infected by SARS-CoV: a kind of apparent molecular weight (being 50-55kDa based on experiment) and nucleoprotein N (422 residues, estimated molecular weight is 46kDa) close polypeptide and a kind of N that may represent be proteic by the polypeptide of the 35kDa of brachymemma or degraded form.

This experiment shows that also rabbit P11135 serum mainly discerns a peptide species, its apparent molecular weight (being 180-220kDa) and S albumen (1255 residues based on experiment, the non-glycosylated polypeptide chain of 139kDa) glycosylation form is close, also may represent the proteic polypeptide by brachymemma or nonglycosylated form of S close with lighter.

In a word, all these evidences are expressed in N and the proteic recombinant polypeptide of S of intestinal bacteria and corresponding SARS-CoV can induce polyclonal antibody in animal body, and described antibody can be discerned these proteic natural forms.

Embodiment 5: at the M and the proteic monospecific Polyclonal Antibody Preparation of E of SARS associated coronavirus (SARS-CoV)

1) the proteic structural analysis of M and E

A) E albumen

The structure of the E albumen of SARS-CoV (76 amino acid) is to use for example signalP v1.1, NetNGlyc 1.0, THMM 1.0 and 2.0 (Krogh et al., 2001, J. Mol. Biol., 305 (3): 567-580) or TOPPRED (von Heijne, 1992, J. Mol. Biol.225, various software bag 487-494) is analyzed by computer.Analytical results shows that this nonglycosylated polypeptide is a kind ofly to comprise that an independent transbilayer helix is (according to THMM, 12-34 amino acid is arranged) 1 type membranin, the major part of its hydrophilic-structure territory (42 residues) is positioned at C end and probably in virion (born of the same parents' intracellular domain).Can notice by the topological framework of 1.0 editions (N holds externally) and 2.0 editions (N holds in inside) of THMM software prediction and put upside down, but other algorithm, TOPPRED and THUMBUP (Zhou et Zhou particularly, 2003, Protein Science 12:1547-1555), determined the location, outside of E albumen n end.

B) M albumen

The similar analysis that the M albumen (221 amino acid) of SARS CoV is carried out shows do not have signal peptide in this polypeptide (according to signalP v1.1 software), but there are three membrane spaning domains (, 15-37,50-72,77-99 residue being arranged) and a big hydrophilic-structure territory (100-221 amino acid) to be positioned at virion inside (born of the same parents' intracellular domain) according to THMM2.0 software.Probably at 4 l-asparagine by glycosylation (according to NetNGlyc 1.0).

Thereby, consistent with the known experimental data of other coronavirus, it should be noted that these two albumen of M and E show born of the same parents' intracellular domain corresponding to most polypeptide, and ectodomain is very little.

The proteic ectodomain of-E may be corresponding to this proteic 1 to 11 or 1 to 12 residue: MYSFVSEETGT (L), SEQ ID NO:70.In fact, to be positioned at the probability of striding the film district be medium level (is 0.56 according to THMM 2.0) to residue 12.

The proteic ectodomain of-M may be corresponding to these proteic 2 to 14 residue: ADNGTITVEELKQ, SEQ ID NO:69.In fact, the methionine(Met) of M albumen n end has probably been excised from sophisticated polypeptide because 2 residue be a L-Ala (Varshavsky, 1996,93:12142-12149).

In addition, the proteic hydrophobicity analysis of E (Kyte ﹠amp; Doolittle, Hopp ﹠amp; Woods) the C end of the proteic born of the same parents' intracellular domain of confirmation E is hydrophilic, so probably be exposed to the surface of this structural domain.Therefore the synthetic peptide corresponding to this end is the good immunogenicity material standed for, and it can be used for inducing in animal body the antibody at E albumen born of the same parents intracellular domain.Therefore, synthesized a peptide corresponding to proteic 24 C end of E residue.

2) at the preparation of the antibody of M and E albumen ectodomain and E albumen born of the same parents intracellular domain

Peptide M2-14 (ADNGTITVEELKQ, SEQ ID NO:69), E1-12 (MYSFVSEETGTL, SEQ ID NO:70) and E53-76 (KPTVYVYSRV KNLNSSEGVPDLLV, SEQ ID NO:71) have been synthesized by Neosystem.Use MBS (m-maleimide-benzoyl-N-hydroxy-succinamide ester, m-maleimido-benzoyl-N-hydroxysuccinimide ester) by adding halfcystine in the building-up process, the N of above-mentioned synthetic peptide end (for E53-76) or (for M2-14 and the E1-12) coupling of C end KLH (keyhole limpet hemocyanin, KeyholeLimpetHemocyanin).

According to following immune step, with every kind of conjugate two rabbits are carried out immunity: injection is for the first time injected 0.5mg with complete freund adjuvant emulsive KLH link coupled peptide (intradermal routes) for every rabbit, 3 weeks or 4 weeks are carried out 2 to 4 booster immunization injections to animal more at interval then, and per injection 0.25mg is with incomplete freund adjuvant emulsive KLH link coupled peptide.

To every rabbit, preparation its preimmune serum (p.i.) before the immunity first time, and at 3 to 5 week of booster immunization injection back preparation immune serum (I.S.).

Analyzed the reactivity of serum by protein immunoblotting method, described immunoblotting assay uses the cell extract (Figure 43 B) that is infected by SARS-CoV or is expressed the E (VV-TG-E of SARS-CoV 031589 strain isolated, Figure 43 A) or the cell extract of the proteic recombinant vaccinia virus infection of M (VV-TN-M, Figure 43 C).

Discern about polypeptide of 9 to 10kD with conjugate KLH-E53-76 immunize rabbit 22234 and 22240 immune serums that produce, in the cell extract that is infected by SARS-CoV, contain described polypeptide, but then do not have (Figure 43 B) in the extract of the cell that does not infect.The apparent molecular weight of this polypeptide is close with the proteic molecular weight of E that is predicted as 8.4kD.Similarly, the immune serum that produces with conjugate KLH-E1-12 immunize rabbit 20047 is identified in by the polypeptide that contains in the cell extract of VV-TG-E virus infection its apparent molar mass and E proteic close (Figure 43 A).

Be identified in by the polypeptide that contains in the cell extract of VV-TN-M virus infection (Figure 43 C) with conjugate KLH-M2-14 immunize rabbit 20013 and 20080 immune serums that produce, its apparent molar mass (about 18kD) is close with the M glycoprotein of 25.1kD, and M glycoprotein has higher isoelectric point (iso-electric point of the polypeptide of unmodified is 9.1).

These results confirm, are peptide E1-12 and E53-76 on the one hand, are peptide M2-14 on the other hand, can induce polyclonal antibody in animal body, and described antibody can be discerned E and the proteic natural form of M of SARS-CoV respectively.

Embodiment 6: recombinant N protein is analyzed for SARS patients serum's ELISA is reactive

1) material

The antigen that is used to prepare solid phase is the purification of Recombinant nucleoprotein N of the step preparation described according to embodiment 2.

Test serum (Table IV) is selected based on their reactive immunofluorescence analysis results (IF-SARS tires) to the cell that infected by SARS-CoV.

Table IV: with the serum of ELISA mensuration

Reference number	The serum numbering	Serotype	Serum is gathered the date ***	IF-SARS tires
					3050	A	Contrast	na *	nt **
3048	B	Contrast	na	nt
					033168	D	SARS patient	1	04/27/03(D38)	320
033397	E	SARS patient	1	05/11/05(D52)							320
					032632	F	SARS patient	2	03/21/03(D17)	2500
032791	G	SARS patient	3	04/04/03(D3)							＜40
					033258	H	SARS patient	3	04/28/03(D27)	160

* na: inapplicable.* nt: not test.The date that * * points out is corresponding to the fate that occurs after the SARS symptom.

2) method

N albumen is diluted to different concns (1,2 or 4 μ g/ml) with the carbonate buffer solution of 0.1M, pH9.6, divides in each hole that is filled to the ELISA flat board (100 μ l), and place the laboratory temperature overnight incubation.Dull and stereotyped with the washing of PBS-Tween damping fluid also with the sealing of PBS-skimmed milk-sucrose (5%) damping fluid.Add the test sera (100 μ l) of dilution (1/50,1/100,1/200,1/400,1/800,1/1600 and 1/3200) in advance, then flat board is placed 37 ℃ to cultivate 1 hour.After washing 3 times, (reference number 209-035-098 JACKSON), places flat board 37 ℃ to cultivate 1 hour then to add the anti-human IgG conjugate be diluted to 1/18000 peroxidase labelling.After washing 4 times, add chromogen (TMB) and substrate (H ₂O ₂), place the room temperature lucifuge to cultivate 30 minutes flat board.Termination reaction and on the automatic reading instrument, measure the absorbancy at 450nm place subsequently.

3) result

The result (Figure 10) of ELISA test confirms, antibodies specific ground identification among the SARS patients serum that the recombinant N protein preparation can infected late period (〉=occur symptom after 17 days), and antibody among the SARS patients serum of can not be infected early stage (occur symptom after 3 days) or the control serum that do not infected the experimenter of SARS are discerned significantly.

Embodiment 7: for the SARS associated coronavirus in high specific and the susceptibility ground detection patient serum infects the ELISA test of preparing.

1) indirect ELISA IgG test

A) Reagent

Dull and stereotyped preparation

The PBS damping fluid sensitization of 10mM, pH7.2 that N albumen, the 0.25ml/l that dull and stereotyped use contains 2 μ g/ml is phenol red.Every hole adds 100 μ l solution and places incubated at room temperature to spend the night.With the 10mM PBS damping fluid pre-wash that contains 0.1%Tween, then wash and seal flat board with the PBS lock solution of 25% milk/sucrose.

Serum dilution

Damping fluid contains 0.48g/l TRIS, 10mM PBS, 3.7g/l EDTA, 15%v/v milk, pH6.7

The conjugate diluent

Citrate buffer (15g/l) contains 0.5%Tween, 25% bovine serum, 12%NaCl, 6%v/v skimmed milk, pH6.5

Conjugate

50 * anti-human IgG conjugate is available from Bio-Rad company: Platelia H. pylori test kit, reference number 72778

Other solution:

Washings R2, with TMB R8 diluent one be used from visual solution, R9 chromogen, R10 stop buffer: reagent is available from Bio Rad company (for example: Platelia H.pylori test kit, reference number 72778)

B) Step

Serum is diluted to 1/200 in the sample diluent

The every hole 100 μ l of packing

Cultivated 1 hour for 37 ℃

10 * washings R2 in advance with 10 times (i.e. 1 * washings) of demineralized water dilution, is washed 3 times

The conjugate of packing 100 μ l (50 * conjugate is diluted in the conjugate diluent that is provided with preceding shortly)

Cultivated 1 hour for 37 ℃

With 1 * washings washing 4 times

The visual solution of the every hole 200 μ l of packing (promptly use preceding dilution, for example: the R9 of 1ml is diluted in the R8 of 10ml)

In the dark incubated at room temperature is 30 minutes

R10 termination reaction with every hole 100 μ l

Read the absorbancy of 450/620nm

Can explain the result of test with the reaction of threshold value serum, the serum that is higher than threshold value can be judged as the positive.Threshold value serum is selected and dilutes, so that produce the signal more much better than than background noise.

2) two epi-position ELISA tests

A) Reagent

Dull and stereotyped preparation

The PBS damping fluid sensitization of 10mM, pH7.2 that N albumen, the 0.25m1/l that dull and stereotyped use contains 1 μ g/ml is phenol red.Every hole adds 100 μ l solution and places incubated at room temperature to spend the night.With the PBS damping fluid pre-wash of 10mM, 0.1%Tween, then wash and seal flat board with the PBS lock solution of 10mM, 25% (V/V) milk.

Serum and conjugate diluent

50mM TRIS salt buffer, pH8,2% milk

Conjugate

Conjugate is for being 1/2 peroxidase link coupled purification of Recombinant N albumen (Nakane P.K.and Kawaoi A. according to the step of Nakane with mol ratio separately; (1974): Peroxydase-labeled antibody, a new method of conjugation.TheJournal of Histochemistry and Cytochemistry Vol. 22, N) 23, pp.1084-1091).This ProtN POD conjugate concentration in serum/conjugate diluent in use is 2 μ g/ml.

Other solution:

Washings R2, with TMB R8 one be used from visual solution, diluent, R9 chromogen, R10 stop buffer: reagent is available from Bio-Rad company (for example: Platelia pylori test kit, reference number 72778).

B) Step

The first step is in " pre-dilution " flat board

● on pre-dilution plate, every part of serum is diluted to 1/5 (48 μ l diluents+12 μ l serum)

● diluted after all serum the conjugate of every hole packing 60 μ l

● when needing, second step of mixture of cultivating serum+conjugate is in " reaction " flat board

● mixture is transferred in the reaction plate every hole 100 μ l

● cultivated 1 hour for 37 ℃

● 10 * washings R2 in advance with 10 times (i.e. 1 * washings) of demineralized water dilution, is washed 5 times

● the visual solution of the every hole 200 μ l of packing (promptly use preceding dilution, for example: the R9 of 1ml is diluted in the R8 of 10ml)

● the room temperature lucifuge was cultivated 30 minutes

● with the R10 termination reaction of every hole 100 μ l

● read the absorbancy of 450/620nm

Similar to the indirect ELISA test, can use " threshold value " serum explain the result of test.The serum that any reaction is higher than threshold value serum can be judged as the positive.

2) result

Use indirect IgG-N test and two epi-position N test, analyzed the serum that is divided into SARS suspected case or the patient relevant of the French hospital that is derived from HANOI, Vietnam with the French hospital (JYK) in Ha Noi.

The result of IgG-N test (Figure 14 and Figure 15) and two epi-position N tests (Figure 16 and Figure 17) shows indirectly, relatively serum is for lysate (the ELISA-SARS-CoV lysate that infects or do not infect the Vero E6 cell of SARS-CoV, V sees the following form) reactivity, have good dependency between them and between the test of they and indirect ELISA.All serum of gathering more than 12 days or 12 days after symptom occurring all are male, comprise that those can not confirm the patient's of SARS-CoV virus infection serum with the respiratory organs sample of RT-PCR methods analyst, this is likely because sample collection gets too late in course of infection (〉=D12).With regard to patient TTH, its nose sample of gathering in the 7th day is negative in RT-PCR analyzes, and the quality of sample may be doubtful.

Some serum is negative and has detected the wherein existence of SARS-CoV with the RT-PCR method.They all be the early stage blood serum gathered when symptom occurring less than 10 days (for example: serum #032637).With regard to patient PTTH, in (serum #032673), when gathering sample, only be the suspection that has proposed SARS.

In a word, the two epi-position serology tests of IgG-N and N were infected back 12 days or serum for more time for being collected in indirectly, can confirm the infection of SARS-CoV in all patients.

The result of Table V: ELISA test

Catalogue number(Cat.No.)	The patient	Fate	PCR-SARS(1)	The ELISA of SARS-CoV lysate (2)	IgG-N (second series)	Two epi-position tests (second series)
							033168	JYK	38	Positive	+++	＞5000	NT
033597	JYK	74	Positive	NT	＝5000	NT
							032552	VTT	8	Negative D3 and D8 and D12	Negative	＜200	＜5
032544	CTP	16	Negative D16 and D20	++	＞5000	＞＞20
							032546	CJF	15	Negative D15 and D19	++	＞5000	＞＞20
032548	PTL	17	Negative D17 and D21	++	＞5000	＞＞20
							032550	NTH	17	Negative D17 and D21	++	＞5000	＞＞20

032553	VTT	8	Negative D3 and D8 and D12	Negative	＜200	＜5
							032554	NTBV	4	Positive	Negative	＜200	＜5
032555	NTBV	4	Positive	Negative	＜200
							032564	NTP	15	Positive	++	＞5000	＞＞20
032629	NVH	4	Positive	Negative	＜200	＜5
							032631	BTTX	9	Positive	Negative	＜200	＜5
032635	NHH	4	Positive	Negative	＜200	＜5
							032637	NHB	10	Positive	Negative	＜200	＜5
032642	BTTX	9	Positive	Negative	＜200	＜5
							032643	LTDH	1	Positive	Negative	＜200	＜5
032644	NTBV	4	Positive	Negative	＜200	＜5
							032646	TTH	12	Negative D7 and D12 and D16	++	＞5000	＞＞20
032647	DTH	17	Negative D17 and D21	++	＞5000	＞＞20
							032648	NNT	15	Negative D15 and D19	++	＞5000	＞＞20
032649	PTH	17	Negative D17 and D21	++	＞5000	＞＞20
							032672	LVV	16	Negative D16 and D20	+	＞5000	＞＞20
032673	PTTH	NA	Negative	Negative	＜200	＜5
							032674	PNB	17	Negative D17 and D21	++	＞5000	＞＞20
032682	VTH	12	Negative D12 and D16	++	＞5000	＞＞20
							032683	DTV	17	Negative D17 and D21	+	＞1000	＞＞20

Annotate:

(1): RT-PCR analyzes and uses nido RT-PCR BNI, LC Artus and LC-N, and nose or pharyngeal cleaning piece are analyzed.At least one sample of positive this patient of expression is positive.

(2): the reactivity of using the ELISA of the cell lysate that is infected by SARS-CoV to test serum, the OD value that obtains according to tested diluent are divided into high reactivity (+++), hyperergy (++), responding property (+) and feminine gender.

Embodiment 8: with the detection of RT-PCR to SARS associated coronavirus (SARS-CoV)

1) use is for the exploitation of the real-time RT-PCR condition of the Auele Specific Primer of nucleocapsid gene--" light circulation N albumen (Light Cycler N) " test

A) The design of primer and probe

Use " light circle probe design (Roche) " program, be recorded as 031589 SARS-CoV virus strain genome sequence design primer and probe according to being derived from.Therefore, selected the primer and the probe of following two series:

- Series 1(SEQ ID NO:60,61,64,65):

- Sense primer: N/+/28507:5 '-GGC ATC GTA TGG GTT G-3 ' is (28507-28522)

- Antisense primer: N/-/28774:5 '-CAG TTT CAC CAC CTC C-3 ' is (28774-28759)

- Probe 1: 5 '-GGC ACC CGC AAT CCT AAT AAC AAT GC-fluorescein, 3 ' (28561-28586)

- Probe 2: 5 ' Red705-GCC ACC GTG CTA CAA CTT CCT-phosphoric acid salt (28588-28608)

- Series 2(SEQ ID NO:62,63,66,67)

- Sense primer: N/+/28375:5 '-GGC TAC TAC CGA AGA G-3 ' is (28375-28390)

- Antisense primer: N/-/28702:5 '-AAT TAC CGC GAC TAC G-3 ' is (28702-28687)

- Probe 1: SARS/N/FL:5 '-ATA CAC CCA AAG ACC ACA TTG GC-fluorescein 3 ' (28541-28563)

- Probe 2: SARS/N/LC705:5 ' Red705-CCC GCA ATC CTA ATA ACA ATGCTG C-phosphoric acid salt 3 ' (28565-28589)

B) Two efficiency analysiss that primer is right

For testing the right efficient separately of these two primers, a synthetic RNA has been carried out the RT-PCR amplification, described synthetic RNA correspondence is derived from the genomic 28054-29430 Nucleotide of SARS-CoV virus strain that is recorded as 031589 sample, and comprises the sequence of N gene.

More specifically:

This synthetic RNA prepares by in-vitro transcription, and described in-vitro transcription has been used the T7 phage rna polymerase, and is template with plasmid SARS-N by the DNA of BamH1 enzyme linearizing gained.After having removed dna profiling,, then use twice of ammonium acetate and Virahol continuous precipitation with phenol-chloroform extracting and purifying synthetic RNA with 1 digestion of DNA enzyme.The synthetic RNA of institute comes quantitative analysis by the absorbancy of measuring the 260nm place, and the ratio and the agarose gel electrophoresis of the absorbancy by 260nm and 280nm place are checked its quality.Like this, the concentration that is used for the synthetic RNA goods of these researchs is 1.6mg/ml, should be 2.1 * 10 mutually ¹⁵The RNA of copy/ml.

According to the synthetic RNA that supplier's specification sheets use RT-PCR amplification quantity is successively decreased, that described amplification is used is " Superscript ^TMOne-Step RT-PCR with Platinum ^_Taq " test kit and No.1 (N/+/28507, N/-/28774) (Figure 1A) and No.2 (N/+/28375, N/-/28702) primer to (Figure 1B).The amplification condition that uses is as follows: for synthetic cDNA, cultivated 45 ℃ 30 minutes, 55 ℃ 15 minutes and 94 ℃ before this 2 minutes, for the cDNA that increases, be 5 circulations that comprise the steps then: 15 seconds, 45 ℃ annealing of 94 ℃ of sex change were extended 30 seconds for 30 seconds, 72 ℃, be 35 circulations that may further comprise the steps again then: 15 seconds, 55 ℃ annealing of 94 ℃ of sex change were extended 30 seconds for 30 seconds, 72 ℃, each circulation was additionally extended 2 seconds again, the extension of final step be 72 ℃ 5 minutes.The amplified production of gained places 10 ℃ of preservations.

Result among Figure 11 shows, uses the No.2 primer can detect even reach 10 copies (band of weak strength) or 10 to (N/+/28375, N/-/28702) ²The RNA of copy (band that intensity is good), and what use that the No.1 primer can detect (N/+/28507, N/-/28774) is 10 ⁴Copy.Amplicon is respectively 268bp (primer is to 1) and 328bp (primer is to 2).

C) The exploitation of real-time RT-PCR

Use the No.2 primer to the probe that constitutes by SRAS/N/FL and SRAS/N/LC705 to having developed real-time RT-PCR (Fig. 2).

" Light Cycler RNA Amplification Kit HybridizationProbes " test kit has been used in amplification, and (reference number 2015145 is Roche) and at LightCycler ^TM(Roche) carry out on the instrument, optimized reaction conditions as described later.Reaction mixture comprises: H ₂O (6.8 μ l), 25mM MgCl ₂(0.8 μ l, Mg ²⁺Final concentration be 4 μ M), 5 * reaction mixture (4 μ l), 3 μ M probe SRAS/N/FL (0.5 μ l, final concentration is 0.075 μ M), 3 μ M probe SRAS/N/LC705 (0.5 μ l, final concentration is 0.075 μ M), 10 μ M primer N/+/28375 (1 μ l, final concentration is 0.5 μ M), 10 μ M primer N/-/28702 (1 μ l, final concentration is 0.5 μ M), enzyme mixture (0.4 μ l) and sample (viral RNA, 0.5 μ l), the amplification of reaction mixture is carried out according to follow procedure:

-reverse transcription: 50 ℃ 10 minutes, analytical model: do not have

-sex change: 95 ℃ 30 seconds * 1, analytical model: do not have

-amplification: 95 ℃ 2 seconds

50 ℃ of 15 seconds analytical models: Quantitatively* } * 45

2.0 ℃/second of 72 ℃ of 13 seconds rate of temperature changes }

-annealing: 40 ℃ 30 seconds * 1, analytical model: do not have

* all measure fluorescence (pattern separately) when annealing finishes and in each circulation.

Result among Figure 12 shows that this real-time RT-PCR is very sensitive, because it can detect 10 in 100% ground in 5 analyzed samples ²The synthetic RNA (8 29/29 sample in experiment) of copy, and can be in 5 analyzed samples 100% ground detect even reach the RNA (8 40/45 sample in experiment) of 10 copies.The result shows that also this real-time RT-PCR can detect the SARS-CoV genome that exists in the sample, and can quantitatively have genomic quantity.For example, the viral RNA of the SARS-CoV virus stock of cultivating in Vero E6 cell extracts with " Qiamp viral RNAextraction " test kit (Qiagen), is diluted to 0.05 * 10 ^-14And analyze according to above-mentioned step with real-time RT-PCR; Analytical results among Figure 12 shows that virus stock comprises 6.5 * 10 ⁹Genome-Equivalent/ml (geq/ml), this and " RealArt that uses available from Artus company ^TMHPA-Coronavirus LC RT PCR Reagents " result 1.0 * 10 that measures of test kit ¹⁰The value of geq/ml is approximate fully.

2) be oriented to the exploitation of the nido RT-PCR condition of rna polymerase gene--" CDC (CDC)/IP nido RT-PCR " test

A) The extraction of viral RNA

Clinical sample: the specification sheets according to manufacturer uses QIAmp viral RNA Mini Kit test kit (QIAGEN) or similar technology.With the volume wash-out of RNA with 60 μ l.

B) " SNE/SAR " nest-type PRC

The first step: " SNE " link coupled RT-PCR

Used " the Superscript of Invitrogen company ^TMOne-Step RT-PCR withPlatinum ^_Taq " test kit, however use " Titan " test kit of Roche Boehringer company also can obtain similar result here.

Oligonucleotide:

-SNE-S1：5’GGT?TGG?GAT?TAT?CCA?AAA?TGT?GA?3’

-SNE-AS1：5’GCA?TCA?TCA?GAA?AGA?ATC?ATC?ATG?3’

→ expection size: 440bp

1. preparation mixture:

H ₂O 6.5μl

Reaction mixture 2 * 12.5 μ l

Oligonucleotide SNE-S1 50 μ M 0.2 μ l

Oligonucleotide SNE-AS1 50 μ M 0.2 μ l

Ribonuclease inhibitor 40U/ μ l 0.12 μ l

RT/Platinum Taq enzyme mixture 0.5 μ l

2. in the said mixture of 20 μ l, add the RNA of 5 μ l, on thermo cycler (ABI 9600 conditions), increase:

2.1.45 ℃ 30 minutes, 55 ℃ 15 minutes, 94 ℃ 2 minutes

(2.2. 94 ℃ 15 seconds, 45 ℃ 30 seconds, 72 ℃ 30 seconds) * 5 circulations

(2.3. 94 ℃ 15 seconds, 55 ℃ 30 seconds, 72 ℃ 30 seconds+2 seconds/circulations) * 35 circulations

2.4.72 ℃ 5 minutes

2.5.10 ℃ in unlimited time

Place+4 ℃ of storages

Use is available from the ribonuclease inhibitor (N2511/N2515) of the Promega company inhibitor as the RNA enzyme.

With synthetic RNA as positive control.In contrast, increased 10 in each experiment ³, 10 ²Synthetic RNA R with 10 copies _SNE

Second step: " SAR " nest-type PRC

Oligonucleotide:

-SAR1-S：5’CCT?CTC?TTG?TTC?TTG?CTC?GCA?3’

-SAR1-AS：5’TAT?AGT?GAG?CCG?CCA?CAC?ATG?3’

→ expection size: 121bp

1. preparation mixture:

H ₂O 35.8μl

Taq damping fluid 10 * 5 μ l

MgCl ₂?25mM 4μl

Mix dNTP 5mM 2 μ l

Oligonucleotide SAR1-S 50 μ M 0.5 μ l

Oligonucleotide SAR1-AS 50 μ M 0.5 μ l

Taq archaeal dna polymerase 5U/ μ l 0.25 μ l

Used Ampliaq archaeal dna polymerase (no MgCl available from Applied Biosystems company ₂10 * damping fluid, reference number 27216601).

2. the product and increase (the ABI9600 condition) that in the said mixture of 48 μ l, add 2 μ l the first step PCR:

2.1.94 ℃ 2 minutes

(2.2. 94 ℃ 30 seconds, 45 ℃ 45 seconds, 72 ℃ 30 seconds) * 5 circulations

(2.3. 94 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ 30 seconds+1 second/circulations) * 35 circulations

2.4.72 ℃ 5 minutes

2.5.10 ℃ in unlimited time

3. the reaction product with 10 μ l is containing (Seakem, GTG type) analysis on " low unwinding " gel of 3% agarose.

The susceptibility of nest-type PRC test is the RNA of 10 copies as a rule under these conditions.

4. the gained fragment can be used QIAquick PCR test kit (QIAGEN) purifying, and checks order with oligonucleotide SAR1-S and SAR1-AS.

3) use PCR method from the respiratory organs sample, to detect the RNA of SARS-CoV

A) The first step comparative studies

A series of respiratory organs samples that may comprise SARS-CoV that national influenza virus literature centre (zone, the north) is collected compare research.For carrying out this research, use " Qiampviral RNA extraction " test kit (Qiagen) extracts RNA and analyzes with real-time RT-PCR from sample, the primer that uses on the one hand No.2 series to probe to and above-mentioned experiment condition, use " LightCycler SARS-CoVQuantification kit " test kit (reference number 03 604 438) on the other hand available from Roche company.The result is summarized in down Table VI.Be presented in the table in 26 samples, have 18 for what these two test kits all were negative, that all is positive has 5, and has 1 sample only the Roche test kit to be positive, and has 2 samples only " series 2 " N reagent to be positive.In addition, when using the reagent (probe and primer) of No.2 series, there is the RNA detected level in 3 samples (20032701,20032712,20032714) obviously to improve.These results show that " series 2 " N primer and probe are higher than the sensitivity of present available test kit for the genomic detection of the SARS-CoV in the biological specimen.

Table VI: the primer that uses No.2 series (" series 2 " N) to probe to or use " LightCycler SARS-CoV Quantification kit " test kit (Roche), the real-time RT-PCR analytical results that the RNA that extracts is carried out from a series of samples from 5 patients.In two tests each all shows sample type and the quantity of the viral genome copy that records.Negative: negative RT-PCR.

Sample number	The patient	Sample type	The ROCHE test kit	" series 2 " N
					20033082 20033083 20033086 20033087 20032802 20032803 20032806 20031746ARN2 20032711 20032910 20032911 20033356 20033357 20031725 20032657 20032698 20032720 20033074 20032701 20032702 20031747ARN2 20032712 20032714 20032800 20033353 20033384	K K K K M M M C C B B V V K K K K K M M C C C B V V	The pharyngeal nose of nose pharyngeal nose expectoration nose or pharyngeal nose or the pharyngeal nose nose of the pharyngeal expectoration expectoration of pharyngeal nose Endotracheal aspirate Endotracheal aspirate Endotracheal aspirate pharyngeal the unknown of the pharyngeal expectoration of Endotracheal aspirate ight soil nose	Negative 39 negative negative negative 3 115 443 negative 634 17 is negative negative	Negative negative negative 150 negative 5 257 1,676 249 negative 6,914 223 is negative negative

B) The second step comparative studies

121 the respiratory organs samples SARS suspected case, that gather between the 4th to 17 day after symptom occurring from the French hospital of HANOI, Vietnam various nest-type PRCs and real-time RT-PCR and the effect of these methods have relatively been carried out.In these samples, use has 14 samples to be positive at the first time of the nido RT-PCR method of ORF1b (coding replicative enzyme) in the check, and described nido RT-PCR method is described in Bernhard Nocht Institute (BNI nido RT-PCR) at first.Information about this test can obtain on the internet, and network address is Http:// www15.bni-hamburg.de/bni2/neu2/getfile.acgi? area Engl=diagnostic﹠amp; Pid=4112

The various tests of comparing in this research are as follows:

-according to quantitative RT-PCR method of the present invention, this method has been used above-mentioned " series 2 " N primer and probe (LightCycler N column).

-above-mentioned nido RT-PCR the test at rna polymerase gene, this method is by CDC, BNI and Institut Pasteur exploitation (CDC/IP nido RT-PCR).

-reference number is the test kit of the ARTUS company of " HPA Corona LC RT-PCR Kit # 5601-02 ", and this test kit is the real-time RT-PCR test at the ORF1b gene.

-BNI nido RT-PCR test, this test also are at above-mentioned rna polymerase gene.

The contriver observes:

1) variation within batch in the same technology, this is relevant with the palliating degradation degree of RNA goods in the repetition thaw process, particularly for the extremely low sample of rna content,

2) specific sensitivity is lower mutually with BNI nido RT-PCR for CDC/IP nido RT-PCR, and

3) compare with LightCycler (LC) test of Artus company according to quantitative RT-PCR test of the present invention (LightCycler N), sensitivity is close.

These results of VII of being listed in the table below show that quantitative RT-PCR test according to the present invention provides excellent replenishing or alternatives for general test now.In fact the SARS associated coronavirus is a kind of burst virus that may make a variation fast.Particularly, present most of universal tests at the rna polymerase gene of SARS associated coronavirus, may recombinate with the rna polymerase gene of other non-SARS associated coronavirus.So only use test will cause false-negative generation at this gene.

Because what quantitative RT-PCR testing needle of the present invention was right is coding N proteic gene, it with ARTUS company test kit at genome area different.By carrying out two heterogeneic diagnostic tests at the SARS associated coronavirus, therefore hope can avoid due to illness virus gene evolution and the false negative type result of generation.

And, at the gene of nucleocapsid protein special advantage is arranged because this gene since high selective pressure and very stable, this high selective pressure limits relevant with this proteic attach structure.

Table VII: 121 samples from SARS (popular in the 2003) suspected case of the French hospital of HANOI, Vietnam are carried out the comparison of the whole bag of tricks of gene amplification analysis

The NRC numbering	Sample type (1)	The sample collection fate	The patient	CDC/IP nido RT-PCR	BNI nido RT-PCR	Artus Light Cyler test kit	Light Cycler N(IP)
								107 samples	N and P			Negative	Negative	Negative	Negative
032529	P	10	NHB	Negative	Positive	Negative	Negative
								032530	N	10	NHB	Positive	Positive	3.10E+01	4.20E+01
032531	P	7	LP	Positive	Positive	7.70E+00	3.10E+00
								032534	N	15	BND	Positive	Positive	1.60E+00	Negative
032600	P	4	NHH	Negative	Positive	Negative	1.30E+02
								032612	P	17	NTS	Negative	Positive	Negative	Negative
032688	P	9	BTX	Positive	Positive	Negative	Negative
								032689	N	4	NVH	Positive	Positive	1.20E+01	2.30E+02
032690	P	4	NVH	Negative	Positive	1.60E+00	Negative
								032727	P	8	NVH	Positive	Positive	2.30E+02	4.00E+02
032728	N	8	NVH	Positive	Positive	1.10E+03	1.60E+04
								032729	P	14	NHB	Positive	Positive	5.90E+00	3.40E+01
032730	N	14	NHB	Positive	Positive	1.30E+02	4.80E+02
								032741	P	8	NHH	Positive	Positive	2.10E+02	1.30E+02
Positive number				10	14	10	9
								Detect ratio in 14 positives				71.4％	100.0％	71.4％	64.3％

(1) the pharyngeal cleaning piece of P=

N=nose cleaning piece

Embodiment 9: at the production and the evaluation of the proteic monoclonal antibody of N

Use the recombinant N protein immunity BALB/c mouse of purifying, and their splenocyte and suitable muroid myeloma cell are merged according to the technology of K_hler and Milstein.

Prescreen goes out the hybridoma cell strains of 19 anti-N protein antibodies of secretion and has measured their immunoreactivity.These antibody have been discerned recombinant N protein (in the ELISA test) preferably with varying strength, and discern natural virus N albumen in ELISA test and/or protein immunoblotting analysis.Figure 18 to 20 has shown 15 above-mentioned test result in 19 monoclonal antibodies.

Following hyperergy clone has been carried out subclone: clone 12,17,28,57,72,76,86,87,98,103,146,156,166,170,199,212,218,219 and 222.Use proper tools to carry out The specificity, with the epi-position of measuring their identification and confirm that they do not have reactivity for other people coronavirus and some Respirovirus.

The existence that the epi-position collection of illustrative plates research of natural N albumen being carried out with the protein immunoblotting analysis (overlapping peptide by fifteen amino acid carries out on spot film (membrane spot)) and other researchs have disclosed 4 groups of following monoclonal antibodies:

1. specificity is at being positioned at N end position (75-81, sequence: the monoclonal antibody of main linear epitope INTNSVP).

The representative of this group antibody is an antibody 156.The hybridoma of producing this antibody be preserved in the French microbial preservation center (CNCM) of Pasteur's Institute (Paris, France), preservation date on December 1st, 2004, numbering I-3331.This same epi-position also can be discerned by the rabbit anteserum that obtains with identical N albumen routine immunization (how anti-anti-N albumen is).

2. specificity is at being positioned at central position (217-224 position, sequence: the monoclonal antibody of main linear epitope ETALALL); The representative of this group antibody is monoclonal antibody 87 and 166.The hybridoma of producing antibody 87 is preserved in CNCM, preservation date on December 1st, 2004, numbering I-3328.

3. (the 403-408 position, sequence: the monoclonal antibody of main linear epitope DFFRQL), the representative of this group antibody is an antibody 28,57 and 143 to specificity at being positioned at the C end position.The hybridoma of producing antibody 57 is preserved in CNCM, preservation date on December 1st, 2004, numbering I-3330.

4. specificity is at the monoclonal antibody of a discontinuous conformational epitope.This group antibody nonrecognition is distributed in any peptide on the N protein sequence, but reacts consumingly with unmodified native protein.This last the group antibody representative be antibody 86.The hybridoma of producing this antibody is preserved in CNCM, preservation date on December 1st, 2004, numbering I-3329.

Following Table VIII has been summarized the result that epi-position collection of illustrative plates institute gets:

Table VIII: the epi-position collection of illustrative plates of monoclonal antibody

Antibody	Epi-position	The position	The zone
				28	DFSRQL?Q	403-408	The C end

143	DFSRQL?Q
				76	DFSRQL?Q
57	DFSRQL?Q
					FFGMS?RI	315-319
146	LPQRQ	383-387
				166	ETALALL?LL	217-224	The center
87	ETALALL	217-224
			156	INTNSGP	75-81	The N end
86	Conformation
			212	Conformation
170	Conformation

In addition, specifying in Figure 18 and 19, these antibody N albumen for human corona virus 229E in ELISA and/or protein immunoblotting analysis does not show reactivity.

Embodiment 10: be used for the monoclonal antibody combination of the exploitation that sensitive immunocapture detects, it is that to be specifically designed to detection proteic by the virus N in the patients serum of SARS-CoV virus infection or the body fluid that this sensitivity immunocapture detects.

Selected following antibody according to the antibody characteristic, their characteristic is applicable to that proteic other of the virus N among experimenter or the patients serum catch and detect research.

These antibody produce and use affinitive layer purification in the ascites of mouse, they can be used alone or in combination as capture antibody and signal antibody.

The antibody of selecting is listed as follows:

-anti-C end regions antibody (No.28,57,143)

-anti-central zone antibody (No.87,166)

-anti-N end regions antibody (No.156)

-anti-discontinuous conformational epitope antibody (86)

1) preparation of reagent

A) immunocapture ELISA flat board

Dull and stereotyped carbonate buffer solution sensitization with the 0.1M, the pH9.6 that contain 5 μ g/ml antibody.Every hole adds 100 μ l (monovalent or polyvalent) antibody-solutions and spends the night in incubated at room temperature.Use PBS damping fluid (10mM, pH7.4, interpolation 0.1%Tween20) washing dull and stereotyped then also with the PBS solution sealing that contains 0.3%BSA and 5% sucrose.Behind the dry flat board flat board is incorporated with in the bag of siccative stand-by.

B) conjugate

Antibody purified is according to method (Nakane et al. 1974, J.of Histoand cytochemistry, the vol. of Nakane 22, pp.1084-1091) with the peroxidase coupling, the link coupled ratio is the peroxidase of the IgG of 1 molecule to 3 molecules.With these conjugates with the exclusion chromatography purifying and add 50% glycerine and concentrate (concentration be 1 to 2mg/ml) and be stored in-20 ℃.When being used to analyze they being diluted to final concentration with the PBS damping fluid (pH7.4) that contains 1%BSA is 1 or 2 μ g/ml.

C) other reagent

-human serum that all serum markers of HIV, HBV, HCV and THLV virus all are negative

The negative human serum mixture of-interpolation 0.5%Triton X-100

The virus of A g of-inactivation: the virus culture supernatant liquor is determined its inactivation with radiating method inactivation and with the method that is placed in the sensitive cells culture--tiring of suspension is about every milliliter 10 before the inactivation ⁷Infectious particles or every milliliter of antigen about 5 * 10 ⁹The physics virion

-Ag sample dilutes with negative human serum: specimen preparation is 1: 100 dilution and then by 5 times of gradient dilutions.

These non-infectious samples simulations are considered to contain people's sample of the virus nucleoprotein N of lower concentration or extremely low concentration.This sample is not suitable for conventional study.

-washings R2, visual TMB R8 solution, chromogen R9 and stop buffer R10, these reagent are available from the general reagent in the ELISA test kit of Bio-Rad company (for example: Platelia pylori test kit, reference number 72778).

2) step

With the sample of human serum that adds inactivation virus of A g with every hole 100 μ l direct packaging to stand-by flat board, place 37 ℃ to cultivate 1 hour (Bio-Rad IPS incubator) then.

Wash 3 times to remove not and solid phase bonded material (with the R2 washings of dilution, LP35 washer washing automatically).

With the every hole 100 μ l of the suitable conjugate packing that is diluted to 1 or 2 μ g/ml, and then place 37 ℃ to cultivate 1 hour (IPS incubator).

Continuous washing 4 times is to remove unnecessary conjugate (the R2 washings-LP35 washer of dilution).

Add the visual solution (R9 of 1ml and 10mlR8) for preparing before the usefulness of 100 μ l, place the room temperature lucifuge to cultivate 30 minutes, make that the conjugate that is attached on the flat board is visual.

R10 reagent (the 1N H that in all holes, adds 100 μ l ₂SO ₄) with final termination enzyme reaction.

Use suitable microtest plate reader to carry out reading with dual wavelength (450/620nm).

Test result can explain with interim threshold value, and interim threshold value multiply by the factor 2 for the mean value of at least two negative controls or is that the mean value of 100 negative serums adds the increment that is equivalent to 6SD (standard deviation that is calculated by 100 single meters).

3) result

Based on the property detection of selecting antibody different capture antibody and signal antibody combination, and avoid in solid phase and conjugate special at identical epi-position antibody and as the combination of the antibody of conjugate.

The result that following 4 combinations obtain is best, and these results also are listed in the table below among the IX again.

1. make up F/28

Solid phase (antibody 166+87 central zone): conjugate antibody 28 (C end)

2. make up G/28

Solid phase (antibody 86-conformational epitope): conjugate antibody 28 (C end)

3. make up H/28

Solid phase (antibody 86,166 and 87, central zone and conformational epitope): conjugate antibody 28 (C end)

4. make up H/28+87

Solid phase (antibody 86,166 and 87, central zone and conformational epitope): mix conjugate antibody 28 (C end) and 87 (centers)

5. make up G/87

Solid phase (antibody 86-conformational epitope): conjugate antibody 87 (center)

Preceding 4 combinations demonstrate of equal value and repeatably and than the high performance level of using of other combinations (for example making up G/87).Certainly in these combinations, monoclonal antibody can be discerned the antibody surrogate of identical epi-position by other.Therefore also to mention following version:

6. make up the version of F/28

Solid phase (only using antibody 87): conjugate antibody 57 (C end)

7. make up the version of G/28

Solid phase (antibody 86-conformational epitope): conjugate antibody 57 (C end)

8. make up the version of H/28

Solid phase (antibody 86 and 87, central zone and conformational epitope): conjugate antibody 57 (C end)

9. make up the version of H/28+87

Solid phase (antibody 86 and 87, central zone and conformational epitope): mix conjugate antibody 57 (C end) and 87 (centers)

Table I X: the immune response property testing of anti-SARS-CoV nucleoprotein antibody: the optical density(OD) that every kind of antibody measuring according to the extent of dilution of inactivation virus antigen makes up

Numbering	Extent of dilution	F/28	G/28	G/87	H/28	H/28+87
							0 1 2 3 4 5 6 7	1/,100 1/,500 1/2,500 1,/12,500 1,/62,500 1/312500 contrast contrast	5 3.795 2.815 0.987 0.404 0.285 0.210 0.269	5 3.814 2.950 1.038 0.348 0.211 0.200 0.153	3.495 1.379 0.275 0.135 0.125 0.123 0.098 0.104	3.900 3.702 3.268 1.374 0.480 0.240 0.186 0.193	5 3.804 2.680 0.865 0.328 0.215 0.156 0.202

The detection limit of these 4 experiments is corresponding to antigen extent of dilution of 1: 62500 in negative serum.Can infer apace can detect in every milliliter of serum thus and be less than 10 ³Infectious particles.

Can it is evident that from this research the only antibody that is used to catch natural viral nucleoprotein is specificity at the central zone and/or at the antibody of conformational epitope, this two antibody-like also will be selected with regard to its high-affinity to natural antigen.

Determined to be used for the optimum antibody of solid compositions, preferentially selected specificity to be used to detect the antigenic antibody that is attached to solid phase at the complementary antibody conduct of the dominance epi-position of C end regions.Use specificity all can cause general or bad result at other any complementary antibody of the epi-position that is positioned at this albumen n end zone.

Embodiment 11: the proteic eukaryotic expression system of thorn (S) that is used to express SARS associated coronavirus (SARS-CoV)

1) optimization of SARS-CoV S albumen expression condition in mammalian cell

Optimized the condition of SARS-CoV thorn (S) albumen at mammalian cell (293T, Vero E6) transient expression.

For this reason, with pcr amplification contain the dna fragmentation of SARS-CoV S albumen cDNA, oligonucleotide 5 '-ATAGGATCCA CCATGTTTAT TTTCTTATTA TTTCTTACTC TCACT-3 ' and the 5 '-ATACTCGAGTT ATGTGTAATG TAATTTGACA CCCTTG-3 ' that derives from plasmid pSARS-S (CNCM.No.I-3059) used in described amplification, then the fragment that amplifies is inserted into the plasmid pTRIP Δ U3-CMV (Sirven that contains lentiviral vectors TRIP, 2001, Mol.Ther., 3, between BamH1 438-448) and the Xho1 restriction enzyme site, thereby obtain plasmid pTRIP-S.To contain the BamH1 of S albumen cDNA and Xho1 fragment subclone then between eukaryon expression plasmid pcDNA3.1 (+) BamH1 and Xho1 restriction enzyme site (Clontech), thereby obtain plasmid pcDNA-S.To contain the Nhe1 of S albumen cDNA and Xho1 fragment subclone then between the corresponding restriction enzyme site of expression plasmid pCI (Promega), thereby obtain plasmid pCI-S.WPRE sequence (" woodchuck hepatitis virus post-transcriptional control element (Woodchuck Hepatitis Virus posttranscriptionalregula tory element) ") and Ma Sen-gown bundle monkey retrovirus CTE sequence (" composing type transhipment element (constitutive transport element) ") of woodchuck hepatitis virus are inserted between the Xho1 and Xba1 restriction enzyme site of plasmid pcDNA-S and pCI-S separately, obtain plasmid pcDNA-S-CTE, pcDNA-S-WPRE, pCI-S-CTE and pCI-S-WPRE (Figure 21) respectively.Plasmid pCI-S-WPRE is preserved in CNCM, preservation date on November 22nd, 2004, numbering I-3323.All insets all use BigDye Terminator v1.1 test kit (AppliedBiosystems) and ABI377 automatic sequencer to check order.

The ability (Figure 22) that they instruct the S albumen of SARS-CoV to express has been assessed in the transfection of plasmid construction body behind the Vero E6 cell in mammalian cell.In this experiment, according to the specification sheets of manufacturer, with 5 * 10 in following material and 6 μ l Fugene6 reagent (Roche) the transfection 35mm Petri culture dish ⁵The individual layer of individual Vero E6 cell: plasmid pcDNA (in contrast), pcDNA-S, pCI and the pCI-S of 2 μ g.At 37 ℃ and 5%CO ₂Condition under cultivate after 48 hours, cell extract placed sample loading buffer and on the 8%SDS polyacrylamide gel, separate according to the method for Laemmli, be transferred to then on the pvdf membrane (BioRad).Use anti-S albumen rabbit polyclonal serum (from the immune serum of rabbit P11135, referring to the foregoing description 4) and the donkey polyclonal antibody of the anti-rabbit igg of peroxidase link coupled (NA934V Amersham) has carried out immunoblotting (protein immunoblotting analysis) and has detected.Visual ECL+ test kit (Amersham) and the radioautograph film Hyperfilm MP (Amersham) of having used of the luminescence method of binding antibody.

This experiment (Figure 22) shows that plasmid pcDNA-S can not instruct the horizontal expression of S albumen detecting of SARS-CoV, and plasmid pCI-S faintly expresses, and near detection limit, this expression can detect when the film overexposure.When using immunofluorescence technique to observe the S protein expression, also can obtain similar result (data not shown goes out).Detection can not be attributed to employed detection technique less than the proteic effective expression of S because the cell extract that is infected by SARS-CoV or infected by recombined vaccinia virus VV-TF7.3 and by the extract of the Vero E6 cell of plasmid pcDNA-S transfection in can detect the S albumen of expection size (180kDa).In the experiment of back, viral VV-TF7.3 expresses the RNA polymerase of T7 phage and makes that the kytoplasm transcription product of non-cap RNA can be translated effectively.This experiment shows that above-mentioned expression defective should be summed up as: when when the nuclear level is transcribed into the step of messenger RNA(mRNA), owing to S albumen cDNA intrinsic reason makes it can not effective expression.

In second experiment, after with step transfection Vero E6 (Figure 23 A) similar to the above and 293T (Figure 23 B) cell, assessed CTE and WPRE signal effect for the S protein expression.Detecting less than the proteic expression of S with after being derived from the plasmid pcDNA-S-CTE of pcDNA-S and pcDNA-S-WPRE transfection, and the insertion of WPRE and CTE signal has improved the proteic expression of S greatly under the background of expression plasmid pCI-S.

For describing the experiment that this result has carried out another series in detail, (FluorS, BioRad) mode of Cai Jiing is visual quantitatively with immunoblotting with luminescence method and digital image-forming device.The result's that QuantityOne v4.2.3 software (BioRad) is obtained analysis shows that WPRE and CTE sequence make the expression of S albumen in Vero E6 cell improve 20 to 42 times and 10 to 26 times (Table X) respectively.In the 293T cell (Table X), the effect milder of CTE sequence a little (4 to 5 times).And the effect of WPRE sequence still very strong (13 to 28 times).

Table X: CTE and WPRE signal are for the quantitative analysis of the effect of the S protein expression of SARS-CoV

Preparing cell extract with plasmid pCI, pCI-S, pCI-S-CTE and pCI-S-WPRE transfection Vero E6 or 293T cell after 48 hours, and analyzing with the protein immunoblotting method in the explanation that is described in Figure 22.(ECL+, Amersham) (FluorS, BioRad) mode of Cai Jiing is visual with immunoblotting with the digital image-forming device with luminescence method.Represent expression level with level relatively, numerical value 1 representative is with the expression level that records after the plasmid pCI-S transfection.

Each of this two cellular types has been carried out independently experiment.In the experiment 1 that Vero E6 cell is carried out, transfection has carried out twice, and the result is shown as the mean value and the standard deviation of the expression level that records.

Plasmid	Cell	Experiment	1	Experiment 2
					pCI	Vero?E6	0.0	0.0
pCI-S	Vero?E6	1.0±0.1	1.0
				pCI-S-CTE	Vero?E6	9.8±0.9	26.4

pCI-S-WPRE	Vero?E6	20.1±2.0	42.3
				pCI	293T	0.0	0.0
pCI-S	293T	1.0	1.0
				pCI-S-CTE	293T	4.6	4.0
pCI-S-WPRE	293T	27.6	12.8

All these results show in a word, if will make the S albumen cDNA of SARS-CoV be subjected to the expression of rna plymerase ii promoter sequence regulation and control efficient in mammalian cell, then need among a splicing signal and sequence WPRE and the CTE one expression.

2) can obtain the generation of stable cell lines of the S protein expression of SARS-CoV

The S albumen cDNA of SARS-CoV is cloned into the segmental form of BamH1-Xho1 among the plasmid pTRIP Δ U3-CMV, obtain pTRIP-S plasmid (Figure 24), pTRIP Δ U3-CMV plasmid contains defective type lentiviral vectors TRIP (the Sirven et al. of band center DNA lobe, 2001, Mol.Ther., 3:438-448).Method (2000 according to people such as Zennou, Cell, 101:173-185), above-mentioned plasmid, encapsidate plasmid (p8.2) and plasmid (pHCMV-G) transient cotransfection of expressing VSV envelope glycoprotein G in the 293T cell, can be prepared the false particle of retrovirus of the false type that comprises carrier TRIP-S and envelope protein G like this.These false type TRIP-S carriers are used to translate 293T and FRhK-4 cell: use protein immunoblotting and immunofluorescence method to detect in transducer cell less than the proteic expression of S (data not shown goes out).

Use by CMV virus immediately/early promoter, splicing signal, S albumen cDNA and above-mentioned transcribe the suitableeest expression cassette replacement that back signal CTE or WPRE form and have center DNA lobe TRIP Δ U3-EF1 α (Sirven et al., 2001, Mol. Ther., the expression cassette EF1 α-EGFP (Figure 25) in defective type slow virus expression vector 3:438-448).These a series of continuous subclones of replacing by S protein expression box carry out, described S protein expression box is respectively from plasmid pCT-S-CTE (BglII-Apa1 site) or plasmid pCI-S-WPRE (BglII-Sal1 site) excision, be inserted into respectively then between the Mlu1 and Kpn1 site or Mlu1 or Xho1 site of plasmid TRIP Δ U3-EF1 α, thereby obtain plasmid pTRIP-SD/SA-S-CTE and pTRIP-SD/SA-S-WPRE, two plasmids of gained are preserved in CNCM, preservation date on December 1st, 2004, numbering is respectively I-3336 and I-3334.Pseudotyped vector is according to the method (2000 according to people such as Zennou, Cell, 101:173-185) produce, and in transduceed 293T cell (10000 cell) and the FRhK-4 cell (15000 cell), described transduction is a series of 5 transduction circulations continuously, and the p24 carrier amount that each recycles is equivalent to 25ng (pTRIP-SD/SA-S-CTE) or 22ng (pTRIP-SD/SA-S-WPRE).

The cell of transduction is cloned by limiting dilution, with the S proteic expression (data not shown go out) of a series of clone, carry out quantitative analysis (Figure 25) by the protein immunoblotting method of using anti-S albumen rabbit polyclonal serum then with its SARS-CoV of immunofluorescence technique qualitative analysis.Result among Figure 25 shows, compare with the S protein expression of observed SARS-CoV in the SARS-CoV course of infection, with the clone 2 of the FRhK4-S-CTE cell of TRIP-SD/SA-S-CTE transduction and 15 and with the clone 4,9 and 12 of the FRhK4-S-WPRE cell of TRIP-SD/SA-S-WPRE transduction respectively with the S albumen of lower or medium horizontal expression SARS-CoV.

In a word, can produce the proteic FRhK-4 cell of S of expression SARS-CoV and the stable clone of similar 293T cell with carrier TRIP-SD/SA-S-CTE and TRIP-SD/SA-S-WPRE, yet use the analysis of carrying out of " parental generation " carrier TRIP-S is that all right merit, this analysiss confirms to express generation that the proteic stabilized cell of S clones needs among splicing signal and sequence C TE and the WPRE one.

These of carrier TRIP are modified (insertion of transcribing the back signal of splicing signal and for example CTE and WPRE) and can be confirmed the advantage aspect the expression that improves other cDNA except that S albumen in addition.

3) can express the production of the proteic stable cell lines of S of the SARS-CoV of soluble form.The purifying of this recombinant antigen.

Obtained the cDNA of coding S albumen soluble form (Ssol) by following manner: the BspE1 joint by a coding SG dipeptides merges the sequence of proteins encoded ectodomain (1 to 1193 amino acid) mutually with flag sequence (FLAG:DYKDDDDK).In fact, in order to obtain plasmid pcDNA-Ssol, with oligonucleotide 5 '-ATAGGATCCA CCATGTTTAT TTTCTTATTATTTCTTACTC TCACT-3 ' and 5 '-ACCTCCGGAT TTAATATATT GCTCATATTTTCCCAA-3 ', by from plasmid pcDNA-S, the increased dna fragmentation of S albumen ectodomain of encoding SARS-CoV of PCR, be inserted into then between modified eukaryon expression plasmid pcDNA3.1 (+) the unique BamH1 and BspE1 restriction enzyme site (Clontech), this pcDNA3.1 (+) plasmid comprises sequence label FLAG between its BamH1 and Xho1 site:

//?GGATCC...nnn...TCC?GGA?GAT?TAT?AAA?GAT?GAC?GAC?GAT?AAA?TAA?CTCGAG//

The terminal Xho1 of BammH1 S G D Y K D D D D K

The Nhe1-Xho1 and the BamH1-Xho1 fragment that contain S albumen cDNA then from plasmid pcDNA-Ssol excision, and with their respectively subclone in the corresponding site of plasmid pTRIP-SD/SA-S-CTE and pTRIP-SD/SA-S-WPRE, thereby obtain plasmid pTRIP-SD/SA-Ssol-CTE and pTRIP-SD/SA-Ssol-WPRE, these two plasmids are preserved in CNCM, preservation date on December 1st, 2004, numbering is respectively I-3337 and I-3335.

Pseudotyped vector is according to people's such as Zennou method (2000, Cell, 101:173-185) produce, and in the FRhK-4 cell (15000 cell) of being transduceed, described transduction is a series of 5 transduction circulations (15000 cell) continuously, and the p24 carrier amount that each recycles is equivalent to 24ng (pTRIP-SD/SA-Ssol-CTE) or 40ng (pTRIP-SD/SA-Ssol-WPRE).The cell of transduction is cloned by limiting dilution, and the protein immunoblotting method of the monoclonal antibody by using anti-FLAG carried out Ssol expression of polypeptides analysis (Figure 26 and have data not shown to go out) to a series of 15 clones of a series of 16 clones of TRIP-SD/SA-Ssol-CTE transduction and TRIP-SD/SA-Ssol-WPRE transduction, and the special ELISA that catches at the Ssol polypeptide that has also used exploitation has for this purpose carried out analyzing (Table X I and have data not shown to go out).The best secretion clone's of screening procedure division is shown in Figure 26.Catch ELISA based on bag in solid phase by with the rabbit polyclonal antibody of the SARS-CoV immunity of purifying and inactivation.Such solid phase can catching secretion to the Ssol polypeptide of cell conditioned medium liquid, make that with a series of consecutive steps the Ssol polypeptide is visual then, these consecutive steps comprise: anti-FLAG monoclonal antibody (M2, the adhering to and chromogen and substrate (TMB+H of the adhering to of adhering to SIAGMA), anti-mouse IgG (H+L) biotinylation rabbit polyclonal antibody (Jackson), Streptavidin-peroxidase conjugated thing (Amersham) ₂O ₂, adding KPL).

Table X I: the analysis of the Ssol expression of polypeptides of the clone of lentiviral vectors TRIP-SD/SA-Ssol-WPRE and TRIP-SD/SA-Ssol-CTE transduction.

After with lentiviral vectors TRIP-SD/SA-Ssol-WPRE and TRIP-SD/SA-Ssol-CTE transduction FRhK-4 cell, separated a series of cell clone and measured the secretion of Ssol polypeptide in its supernatant liquor.Supernatant liquor is diluted to 1/50 back the test at the proteic ELISA of catching of the S of SARS-CoV by specificity and analyzes.

Carrier	The clone	OD(450nm)
			Contrast	-	0.031
TRIP-SD/SA-Ssol-CTE	CTE2	0.547
			CTE3	0.668
	CTE9	0.171
			CTE12	0.208
	CTE13	0.133
			TRIP-SD/SA-Ssol-WPRE	WPRE1	0.061
WPRE10	0.134

The clone of secretion maximum amount Ssol polypeptide is FRhK4-Ssol-CTE3 clone in culture supernatant.This clone is cloned then with the TRIP-SD/SA-Ssol-CTE carrier carries out second series under condition similar to the above 5 round-robin transductions.Use protein immunoblotting and catch the subclone that the elisa assay combined sorting goes out to secrete maximum amount Ssol: subclone FRhK4-Ssol-30 is preserved in CNCM, preservation date on November 22nd, 2004, numbering I-3325.

Can produce polypeptide quantitatively with FRhK4-Ssol-30 clone with purification of Recombinant Ssol.In a model experiment of optimizing growth, generation and purification condition, FRhK4-Ssol-30 clone is at standard medium (the DMEM substratum of no pyruvate salt, contain 4.5g/l glucose, add 5%FCS, 100U/ml penicillin, 100 μ g/ml Streptomycin sulphates) go up with minute junction individual layer (every 100cm ²10 ⁶Individual cell, 20ml substratum) form inoculation.After reaching junction, be kept to every 100cm of 0.5% with FCS content ²The amount of substratum is kept to the secretion substratum of 16ml and replaces standard medium.Remove culture supernatant after under 35 ℃, the condition of 5%CO2, cultivating 4 to 5 days.Reorganization Ssol polypeptide is by following consecutive steps purifying from supernatant liquor: 0.1 μ m polyethersulfone (PES) membrane filtration, the PES membrane ultrafiltration of cutting apart with 50kD concentrate, on anti-FLAG matrix affinity chromatography, with contain TBS solution (50mM tris, 150mM NaCl, the pH7.4) wash-out of 100 μ g/ml FLAG peptides (DYKDDDDK), in TBS solution on sephadex G-75 microballoon post (Pharmacia) gel permeation chromatography.Test the concentration that (Pierce) measured purification of Recombinant Ssol polypeptide with micro-BCA, analyzed its biochemical characteristic then.

8%SDS acrylamide gel electrophoresis analytical results with cma staining shows that dominant polypeptide molecular weight is about 180kD, and purity is estimated as 98% (Figure 27 A).Use SELDI-TOF mass spectrometer (Cyphergen) to detect two main peaks: they respectively correspondence be with single electric charge or doubly charged advantage polypeptide, recording its molecular weight like this is 182.6 ± 3.7kD (Figure 27 B and C).Be transferred to the Prosorb film and with after the 0.1%TFA rinsing, with the Edman edman degradation Edman 5 terminal residues of N end of Ssol polypeptide having been carried out checking order in liquid phase, (ABI494, AppliedBiosystems), and to measure them be SDLDR (Figure 27 D).The above results confirms, (Nielsen et al. according to software signalP v2.0 analysis, 1997, ProteinEngineering 10:1-6) is positioned at the S albumen n end end of SARS-CoV, the signal peptide of being made up of 1-13 amino acids (MFIFLLFLTLTSG) has been excised from ripe Ssol polypeptide.So this reorganization Ssol polypeptide has comprised proteic 14 to 1193 amino acid of the S of SARS-CoV, the C of this sequence end and the sequence SG that contains FLAG label (underscore part) DYKDDDDKMerge mutually.Difference between the theoretical molecular (132.0kD) of exposed Ssol polypeptide and the true molar mass (182.6kD) of mature polypeptide shows that the Ssol polypeptide is by glycosylated.

Prepared purifying Ssol polypeptide and used the micro-BCA measurements determination its protein concentration, so just can prepare calibration series, caught the characteristic that ELISA tests the Ssol peptide concentration of measuring in the culture supernatant and investigates secretory cell system thereby use.According to this test, after join cultivating 4 to 5 days, the polypeptide of FRhK4-Ssol-CT3 clone secretion 4 to 6 μ g/ml and the Ssol polypeptide of RhK4-Ssol-30 clone secretion 9 to 13 μ g/ml.In addition, use above-mentioned every liter of culture supernatant of purification step Ssol polypeptide of purifying 1 to 2mg routinely.

Embodiment 12: the proteic genetic immunization of thorn (S) that comprises SARS associated coronavirus (SARS-CoV)

Splicing signal and the effect of transcribing back signal WPRE and CTE after the genetic immunization of BALB/c mouse, have been analyzed.

For this reason, the timed interval with 4 weeks is carried out immunity to BALB/c mouse, salts solution to the plasmid DNA of the injection pcDNA-S of 50 μ g and pCI-S in the mouse tibialis anterior muscle, and the plasmid DNA of injecting the pcDNA-N (instructing the proteic expression of N of SARS-CoV) of 50 μ g or pCI-HA (instructing the expression of the HA of influenza virus A/PR/8/34) is in contrast, injecting for the second time 3 week the back gather immune serums.Use indirect ELISA to measure at the existence of the proteic antibody of S of SARS-CoV, described indirect ELISA uses the Vero E6 cell lysate that is infected by SARS-CoV as antigen, and uses the Vero E6 cell lysate that do not infect in contrast.(NA931V is Amersham) with interpolation H with the anti-mouse IgG of peroxidase link coupled polyclonal antibody ₂O ₂TMB (KPL) carry out visual after, the inverse of the antibody dilution when producing specific OD value 0.5 (difference between the observed value OD of infected cell lysate and the observed value OD of non-infected cells lysate) calculates tire (TI) (Figure 28 A) of anti-SARS-CoV antibody.

Under these conditions, to 3 in 6 mouse, expression plasmid pcDNA-S only can induce the proteic antibody (LOG of the S at SARS-CoV of low liter ₁₀(TI)=1.9 ± 0.6), and plasmid pcDNA-N can be in all animals the anti-N protein antibodies (LOG of inducement efficient valency ₁₀(TI)=3.9 ± 0.3), control plasmid (pCI, pCI-HA) can not be induced any antibody (LOG that detects ₁₀(TI)＜1.7).Assembled the antibody (LOG that the plasmid pCI-S of splicing signal can the inducement efficient valency ₁₀(TI)=3.7 ± 0.2), this is than having tired about 60 times of (p＜10 high with plasmid pcDNA-S injection back is observed ^-5).

Transcribe the effect of back signal and test by dose-response and study, studied as the quantity of immunogenic plasmid DNA (2 μ g, 10 μ g and 50 μ g) and the funtcional relationship between the anti-S protein antibodies of institute's inductive is tired in the BALB/c mouse body.This research (Figure 28 B) confirms to transcribe back signal WPRE can significantly improve genetic immunization when using low dose of DNA effect and (is p＜10 when the DNA of 2 μ g measures ^-5, when the DNA of 10 μ g amount, be p＜10 ^-2), and the effect of CTE signal still very weak (p=0.34 when the DNA of 2 μ g amount)

At last, inductive antibody is in the infectivity (Figure 29 A and 29B) of the external SARS-CoV that neutralized behind the genetic immunization, and it tires consistent with tiring of recording with ELISA.

In a word, use transcribing back signal WPRE and can obviously improving behind the plasmid DNA genetic immunization that the S of instruction SARS-CoV albumen cDNA expresses inducing of splicing signal and woodchuck hepatitis virus at the neutralizing antibody of SARS-CoV.

Embodiment 13: the proteic diagnostic use of S

Analyzed reorganization Ssol polypeptide for ELISA reactivity from SARS patient's serum.

Test serum from the SARS suspected case is selected based on its atopic analytical results (positive or negative) at the natural antigen of SARS-CoV, and described analysis is by being that antigenic indirect ELISA test is carried out for the test of the immunofluorescence of the Vero E6 cell that is infected by SARS-CoV and/or with the lysate of the Vero E6 cell that infected by SARS-CoV.These patients' serum with series number and patient's name's abbreviation of national influenza virus literature centre and after symptom occurring through fate define.Except 032552 serum of patient VTT, the serum of all suspected cases (seeing Table XII) is all discerned the natural antigen of SARS-CoV, and the SARS-CoV of this patient VTT infects and can not make a definite diagnosis by the RT-PCR to 3,8 and 12 days respiratory organs sample.Use one group of control serum (TV serum) in contrast: they are collected in the popular preceding France of SARS in 2003.

The serum of Table X II:SARS suspected case

Serum	The patient	The sample collection fate
			031724	JYK	7
033168	JYK	38
			033597	JYK	74
032632	NTM	17
			032634	THA	15
032541	PHV	10
			032542	NIH	17
032552	VTT	8
			032633	PTU	16
032791	JLB	3

033258	JLB	27
			032703	JCM	8
033153	JCM	29

The absorption preparation of PBS solution in the ELISA plate well of the purifying Ssol polypeptide of the solid phase of using reorganization Ssol polypeptide sensitization by containing 2 μ g/ml, dull and stereotyped then 4 ℃ of overnight incubation, use PBS-Tween damping fluid (PBS, 0.1%Tween 20) washing again.Dull and stereotyped with PBS-10% skimmed milk (weight/volume) solution sealing ELISA and with the PBS-Tween washing after, test serum (100 μ l) is diluted to 1/400 adds to then in the hole of ELISA flat board of sensitization in PBS-skimmed milk-Tween damping fluid (PBS, 3% skimmed milk, 0.1%Tween).Flat board places 37 ℃ to cultivate 1 hour.After with PBS-Tween damping fluid washing 3 times, be added in anti-human IgG antibody's conjugate (the reference number NA933V that is diluted to 1/4000 peroxidase labelling in PBS-skimmed milk-Tween damping fluid, Amersham), then flat board is placed 37 ℃ to cultivate 1 hour.After with PBS-Tween damping fluid washing 6 times, add chromogen (TMB) and substrate (H ₂O ₂) and with dull and stereotyped lucifuge cultivation 10 minutes.Add 1NH ₃PO ₄The solution termination reaction is the absorbancy at the 450nm place of reference measuring with the 620nm place then.

The result (Figure 30) of ELISA test confirms that the serum antibody that reorganization Ssol polypeptide can be collected in and infected mid-term or late period the SARS patient of (occur after the symptom 〉=10 days) discerns specifically, and the control serum that can not be collected in 2 patients' (JLB and JCM) that infect early stage (occur symptom after 3 to 8 days) serum antibody or not infect the experimenter of SARS is discerned significantly.Seroconversion for the first time appears in the serum antibody of patient JLB and JCM between 3 days and 27 days after symptom occurring, occurred seroconversion for the second time between 8 days and 29 days, and this has confirmed the reactive specificity of these serum for the Ssol polypeptide.

In a word, The above results confirms that reorganization Ssol polypeptide can use as antigen in the exploitation of the serodiagnostic ELISA test that SARS-CoV infects.

Embodiment 14: the proteic vaccine of recombinant soluble S is used

In the mouse body, studied the immunogenicity of reorganization Ssol polypeptide.

For this reason, with timed interval in 3 weeks be used in the 10 μ g reorganization Ssol polypeptide that dilutes among the PBS and 1mg aluminum hydroxide adjuvant (Alu-gel-S, Serva) immunity one group of 6 mouse.Carry out three immunity continuously and gather immune serum (IS1, IS2, IS3) in 3 weeks of each immunity back.Only according to one group of mouse (simulation group) of same steps as immunity in contrast with aluminium hydroxide.

The immune serum of each group in two groups is mixed the back analyze with indirect ELISA, described indirect ELISA uses the Vero E6 cell lysate that infected by SARS-CoV as antigen, the Vero E6 cell lysate that do not infect in contrast.(NA931V is Amersham) with interpolation H with the anti-mouse IgG of peroxidase link coupled (H+L) polyclonal antibody ₂O ₂TMB (KPL) carry out visual after, the inverse of the antibody dilution when producing specific OD value 0.5 calculates tiring of anti-SARS-CoV antibody.This analysis (Figure 31) shows, from immunity for the first time, uses the immunity of Ssol polypeptide just can induce the antibody that stings the albumen natural form at the SARS-CoV that exists in the infected Vero E6 cell lysate in the mouse body.After 2 times and 3 immunity, tiring of anti-S protein antibodies becomes very high.

With the serum mixing post analysis of each group in two groups in its serum and the infective ability of SARS-CoV.Since 1/20 doubling dilution serum one by one, 4 serum neutral holes of each doubling dilution test FRhK-4 cell (SARS-CoV of 100 TCID50).According to the method for Reed and Munsch, with can in and the inverse of infective serum dilution of two in 4 holes calculate the serum neutralization and tire.It is neutrality that this analysis shows with Ssol polypeptide inductive antibody in the mouse body: observed tiring after 2 times and 3 immunity very high (respectively greater than 2560 and 5120, Table X III).

Table X III: with inducing at the antibody of SARS-CoV behind the reorganization Ssol polypeptide immune

With the immune serum mixing post analysis of each group in two groups in its serum and the SARS-CoV of 100 TCID50 for the ability of FRhK-4 cell infection.Since 1/20 doubling dilution serum one by one, each 4 hole of doubling dilution test.According to the method for Reed and Munsch, with can in and the inverse of infective serum dilution of two in 4 holes calculate the serum neutralization and tire.

Group	Serum	Neutralizing antibody
			The simulation group	pi	＜20
IS1	＜20
		IS2		＜20
IS3	＜20
		The Ssol group		pi	＜20
IS1	57
			IS2	＞2560
IS3	＞5120

When using Ssol polypeptide immune mouse observed neutralization tire the level that reaches more than people such as Yang in the mouse body (2004; Nature; 428:561-564) and Buchholz in the hamster body (2004; PNAS, 101:9804-9809) observed, can protect mouse and hamster not to be subjected to tiring of antibody that SARS-CoV infects much higher respectively.So may in the mouse body, can protect these animals not to be subjected to the infection of SARS-CoV with Ssol polypeptide immune inductive neutralizing antibody.

Embodiment 15: can in mammalian cell, express SARS associated coronavirus (SARS-CoV) thorn (S) proteic optimization synthetic gene

1) design of synthetic gene

(plasmid pSARS-S has designed encoding SARS-CoV in CNCM.No.I-3059) and has stung proteic synthetic gene, so that at mammalian cell, particularly obtains high-caliber expression in the human archeocyte from the gene of 031589 strain isolated.

For this reason:

-the codon of having modified the wild type gene of strain isolated 031589 is selected, so that its translation effect that becomes and approach observed preference among the mankind and improve corresponding mRNA

-improved the whole GC content of gene, to prolong the transformation period of corresponding mRNA

-lacked can interference base because of the optional implicit motif (splicing donor and acceptor site, polyadenylation signal, the sequence that highly is rich in (＞80%) GC or GC content very low (＜30%), tumor-necrosis factor glycoproteins, participation form sequence, the TATA box of secondary RNA structure) of effective expression

-added second terminator codon so that translation can stop effectively.

In addition, in gene, introduced the CpG motif so that improve its immunogenicity as dna vaccination.For making things convenient for the operation of synthetic gene, placed two BamH1 and Xho1 restriction enzyme site on the both sides of S albumen opening code-reading frame, and in synthetic gene, avoided BamH1, Xho1, Nhe1, Kpn1, BspE1 and Sal1 restriction enzyme site.

The sequence of the synthetic gene (gene 040530) of design provides in SEQ ID No:140.

Synthetic gene 040530 is shown in Figure 32 with the sequence alignment of wild type gene, and described wild type gene is the gene that is preserved in SARS-CoV strain isolated 031589 (SEQID NO:4, plasmid pSRAS-S) CNCM., numbering I-3059.

2) plasmid construction body

Synthetic gene SEQ ID No:140 is assembled and is cloned between the Kpn1 and Sac1 restriction enzyme site of plasmid pUC-Kana by the synthetic oligonucleotide, thereby obtains plasmid 040530pUC-Kana.Confirmed the nucleotide sequence of the inset of plasmid 040530pUC-Kana with automatic sequencer (Applied).

The Kpn1-Xho1 fragment that will contain synthetic gene 040530 is excised from plasmid 040530pUC-Kana, and subclone is between the Nhe1 and Xho1 restriction enzyme site of expression plasmid pCI (Promega), thereby obtain plasmid pCI-Ssynth, this plasmid pCI-Ssynth is preserved in CNCM, preservation date on December 1st, 2004, numbering I-3333.

Obtained the synthetic gene of coding S albumen soluble form by following manner: merge mutually with sequence label (FLAG:DYKDDDDK) by will the encode sequence of S albumen ectodomain (1 to 1193 amino acid) of the BspE1 joint of a coding SG dipeptides.In fact, with oligonucleotide 5 '-ACTA GCTAGC GGATCCACC ATGTTCATCTT CCTG-3 ' and 5 '-AGTA TCCGGACTTGATGTACT GCTCGTACTTGC-3 ', by from plasmid 040530pUC-Kana, the increased dna fragmentation of S albumen ectodomain of encoding SARS-CoV of PCR, with being inserted between the unique N he1 and BspE1 restriction enzyme site of plasmid pCI-Ssol after Nhe1 and the BspE1 digestion, thereby obtain plasmid pCI-Scube, this plasmid pCI-Scube is preserved in CNCM, preservation date on December 1st, 2004, numbering I-3332.By distinguishing subclone between the Nhe1 and Xho1 restriction enzyme site of plasmid pCI, pCI-S-CTE and pCI-S-WPRE from the Kpn1-Xhol fragment (referring to technical manual DI 2004-106) of plasmid pcDNA-Ssol excision, obtain plasmid pCI-Ssol, pCI-Ssol-CTE and pCI-Ssol-WPRE in advance and (be preserved in CNCM, preservation date on November 22nd, 2004, numbering I-3324).

Plasmid pCI-Scube and the identical reorganization Ssol polypeptide of pCI-Ssol coding.

3) result

After primate cell (Vero E6) and human cell's (293T) the transient transfection, compared the ability that the S albumen that effectively instructs SARS-CoV of coding proteic synthetic gene of S and wild type gene is expressed in mammalian cell.

In the experiment of Figure 33 and Table X IV demonstration, according to manufacturers instruction (Roche), with 5 * 10 in the Fugene6 reagent transfection 35mm Petri culture dish of plasmid pCI (contrast), pCI-S, pCI-S-CTE, pCI-S-WPRE and the pCI-S-Ssynth of 2 μ g and 6 μ l ⁵Vero E6 or 7 * 10 ⁵The cell monolayer of 293T.At 37 ℃ and 5%CO ₂Condition under cultivate after 48 hours, cell extract placed sample loading buffer and on the 8%SDS polyacrylamide gel, separate according to the method for Laemmli, be transferred to then on the pvdf membrane (BioRad).Use anti-S albumen rabbit polyclonal serum (from the immune serum of rabbit P11135, referring to the foregoing description 4) and the donkey polyclonal antibody of the anti-rabbit igg of peroxidase link coupled (NA934V Amersham) has carried out immunoblotting (protein immunoblotting analysis) and has detected.(FluorS, BioRad) mode of Cai Jiing is visual quantitatively with immunoblotting with the luminescence method that uses ECL+ test kit (Amersham) and digital image-forming device.

The result's that QuantityOne v4.2.3 software (BioRad) is obtained analysis shows, in this experiment, plasmid pCI-Ssynth can make S albumen high level ground transient expression in Vero E6 and 293T cell, and plasmid pCI-S can not induce the expression of enough detected levels.Observed expression amount has reached about twice of observed expression amount when using plasmid pCI-S-WPRE.

Table X IV: synthetic gene is used for the proteic expression of S of SARS-CoV

Use plasmid pCI, pCI-S, pCI-S-CTE, pCI-S-WPRE and pCI-S-Ssynth transfection Vero E6 or 293T cell after 48 hours, the preparation cell extract also separates on the 8%SDS acrylamide gel, (NA934V Amersham) has carried out the protein immunoblotting analysis to use the polyclonal antibody of anti-S albumen rabbit polyclonal antibody and the anti-rabbit igg of peroxidase link coupled (H+L).(ECL+, Amersham) (FluorS, BioRad) mode of Cai Jiing is visual with the digital image-forming device with luminescence method for protein immunoblotting.Measure the proteic expression level of S in the mode that quantizes two definite remarkable bands of (seeing Figure 33) among the figure, and illustrate according to level relatively, numerical value 1 is represented the expression level that records after the plasmid pCI-S-WPRE transfection.

Plasmid	Vero?E6	293T
			pCI	0.0	0.0
pCI-S	≤0.1	≤0.1
			pCI-S-CTE	0.5	≤0.1
pCI-S-WPRE	1.0	1.0
			pCI-S-Ssynth	1.8	1.9

In second example, after hamster cell (BHK-21) and human cell's (293T) transient transfection, the Ssol polypeptide that effectively instructs that has compared synthetic gene Scube and wild type gene is synthesized and the excretory ability by mammalian cell.

In the experiment that Table X V shows, according to manufacturers instruction (Roche), with 6 * 10 in the Fugene6 reagent transfection 35mm Petri culture dish of plasmid pCI (contrast), pCI-Ssol, pCI-Ssol-CTE, pCI-Ssol-WPRE and the pCI-Scube of 2 μ g and 6 μ l ⁵BHK-21 and 7 * 10 ⁵The cell monolayer of 293T.At 37 ℃ and 5%CO ₂Condition under cultivate after 48 hours collecting cell supernatant liquor and by the secretion of catching ELISA test quantitative analysis Ssol polypeptide of specificity at the Ssol polypeptide.

Analysis to The above results shows that in this experiment, plasmid pCI-Scube makes Ssol polypeptide expression amount improve 8 times (BHK-21 cells) to 20 times (293T cells) than plasmid pCI-Ssol.Observed expression level has reached about twice (293T cell) of observed expression amount when using plasmid pCI-Ssol-WPRE to 5 times (BHK-21 cells).

Table X V: synthetic gene is used for the Ssol polypeptide expression

After using plasmid pCI, pCI-Ssol, pCI-Ssol-CTE, pCI-Ssol-WPRE and pCI-Scube transfection BHK or 293T cell 48 hours, collecting cell supernatant liquor and by the secretion of catching ELISA test quantitative analysis Ssol polypeptide of specificity at the Ssol polypeptide.Transfection has carried out twice, and the result is shown as the mean value of concentration (ng/ml) of the Ssol polypeptide that records and the form of standard deviation in supernatant liquor.

Plasmid	BHK		293T
				pCI	＜20	＜20
pCI-Ssol	＜20	56±10
			pCI-Ssol-CTE	＜20	63±8
pCI-Ssol-WPRE	28±1	531±15
			pCI-Scube	152±6	1140±20

In a word, The above results shows that the proteic synthetic gene 040530 of the S of encoding SARS-CoV is subjected to the expression of rna plymerase ii promoter sequence regulation and control more much effective than the expression of the wild type gene of 03159 strain isolated in mammalian cell.The expression of described synthetic gene very also will have than the expression that the wild type gene of the WPRE sequence that is contained woodchuck hepatitis virus instructs.

4) use

The use of its Scube varient of proteic synthetic gene 040530 of the S of encoding SARS-CoV or coding Ssol polypeptide can must advantageously substitute wild type gene in the application of high level expression at a lot of S albumen.Particularly for:

-even to improve plasmid pCI-Ssynth be the efficient of pCI-Ssynth-CTE or pCI-Ssynth-WPRE type genetic immunization

-by the recombined lentivirus vector that carries Ssynth gene or Scube gene respectively, set up and to express more the S albumen of volume or the new clone of Ssol polypeptide

-improvement can be expressed the immunogenicity of the recombined lentivirus vector of S albumen or Ssol polypeptide

-improving the immunogenicity of the live vector that can express S albumen or Ssol polypeptide, described live vector is recombined vaccinia virus or recombinant measles virus (stating embodiment 16 and 17 as follows) for example.

Embodiment 16: use SARS associated coronavirus (SARS-CoV) thorn (S) proteic expression of recombined vaccinia virus

Vaccine is used

The application of production solubility thorn (S) albumen and the test of design SARS serology

1) introduces

The purpose of present embodiment is that the ability that the antigenic recombined vaccinia virus of various SARS associated coronavirus (SARS-CoV) (VV) constitute new anti-SARS candidate vaccine is expressed in assessment, and becomes the ability of producing the means of recombinant antigen in mammalian cell.

For this reason, the contriver focuses on SARS-CoV thorn (S) albumen, during it can be induced after to the animal gene immunity and the infective antibody of SARS-CoV; Also focus on proteic soluble form of S and secreted form--Ssol polypeptide, this polypeptide is made up of by the BspE1 joint and FLAG label (DYKDDDDK) fusion of a coding SG dipeptides at its C end the proteic ectodomain of S (1-1193 amino acid).This Ssol polypeptide shows the antigenicity with the S protein similar, and behind albumen and aluminum hydroxide adjuvant injection mouse with purifying, anti-SARS-CoV neutralizing antibody that can the inducement efficient valency.

The various forms of S gene is placed under the control of promotor of 7.5K gene, and be introduced in thymidine kinase (TK) locus of vaccinia virus Copenhagen strain by the mode of two homologous recombination in the body.For improving the immunogenicity of recombined vaccinia virus, selected the synthetic late promoter to replace the 7.5K promotor, so that improve the output of S albumen and Ssol in the later stage of viral life cycle.

After separated recombined vaccinia virus and confirmed its antigenic ability of S of expressing SARS-CoV, tested it and in the mouse body, induced ability at the immunne response of SARS.At purifying from the supernatant liquor of infected cell behind the Ssol antigen, designed and be used for the serodiagnostic ELISA of SARS test, and used and assessed its effect from the serum of SARS suspected case.

2) structure of recombinant virus

The S glycoprotein and the recombined vaccinia virus solvable and that secreted form Ssol polypeptide is expressed thereof of 031589 strain isolated of SARS-CoV have been obtained to instruct under the control of 7.5K promotor.Be to improve S albumen and Ssol polypeptide expression level, also obtained the recombinant virus under the control that the cDNA of S albumen and Ssol wherein is placed in the synthetic promoter in late period.

Plasmid pTG186poly be the transferring plasmid that is used for the structure of recombined vaccinia virus (Kieny, 1986, Biotechnology, 4:790-795).Thereby it contains VV thymidine kinase gene, has inserted 7.5K gene promoter and a multiple clone site (Figure 34 A) that allows heterologous gene to insert in this gene.7.5K in fact gene promoter comprises the series connection of two promoter sequences, these two promoter sequences are respectively at the early stage (P of vaccinia viral replication round-robin _E) and later stage (P _L) activity arranged.Excise the BamH1-Xho1 fragment from plasmid pTRIP-S and pcDNA-Ssol respectively, and be inserted between the BamH1 and Sma1 restriction enzyme site of plasmid pTG186poly, thereby obtain plasmid pTG-S and pTG-Ssol (Figure 34 A).Plasmid pTG-S and pTG-Ssol are preserved in CNCM, and preservation date on December 2nd, 2004, numbering is respectively I-3338 and I-3339.

The Nde1-Pst1 fragment that contains the 7.5K promotor among plasmid pTG186poly, pTG-S and the pTG-Ssol is replaced with the dna fragmentation that contains synthetic late promoter 480, thereby obtain plasmid pTN480, pTG-S and pTG-Ssol respectively, described synthetic late promoter 480 is obtained (Figure 34 B) by the hybridization of oligonucleotide 5 '-TATGAGCTTT TTTTTTTTTT TTTTTTTGGC ATATAAATAG ACTCGGCGCGCCATCTGCA-3 ' and 5 '-GATGGCGCGC CGAGTCTATT TATATGCCAA AAAAAAAAAAAAAAAAAAGC TCA-3 '.This inset uses BigDyeTerminator vl.1 test kit (Applied Biosystems) and automatic sequencer ABI377 to check order.Synthetic promoter 480 the sequence in late period of being cloned in the transferring plasmid of pTN series is shown in Figure 34 C.Plasmid pTN-S and pTN-Ssol are preserved in CNCM, and preservation date on December 2nd, 2004, numbering is respectively I-3340 and I-3341.

Recombined vaccinia virus obtains by following mode: according to the method (1984 of people such as Kieny description, Nature, 312:163-166), between the TK gene of the TK box of pTG and pTN series transferring plasmid and vaccinia virus Copenhagen strain, carry out two homologous recombination in the body.In brief, with transferring plasmid transfection CV-1 cell under the help of DOTAP reagent (Roche) of the genomic dna of above-mentioned vaccinia virus Copenhagen strain and each pTG and pTN series, and then dyed 24 hours 33 ℃ of excess revolutions with auxiliary vaccinia virus VV-ts7.Helper virus cultivates by 40 ℃ that 2 days mode is counter selects then that recombinant virus (TK-phenotype) screens by two the clone's circulations to the 143Btk-cell on the nutrient agar that contains BuDr (25 μ g/ml).6 viral VV-TG, VV-TG-S, VV-TG-Ssol, VV-TN, VV-TN-S and VV-TN-Ssol use transferring plasmid pTG186poly, pTG-S, pTG-Ssol, pTN480, pTN-S and pTN-Ssol respectively and obtain.Thereby VV-TG and VV-TN virus are not expressed any heterologous gene and are contrasted as TK-in experiment.On the cell monolayer of CV-1 or BHK-21, carry out the preparation of recombinant virus, and according to the method (1998 of Earl and Moss, Current Protocols in MolecularBiology 16.16.1-16.16.13) measures tiring with plaque-forming unit (p.f.u.) expression on the CV-1 cell.

3) evaluation of recombinant virus

The genetically modified expression of coding S albumen and Ssol polypeptide is measured by protein immunoblotting method.

Use various recombined vaccinia virus VV-TG, VV-TG-S, VV-TG-Ssol, VV-TN, VV-TN-S and VV-TN-Ssol to infect the cell monolayer of CV-1 with infection multiplicity 2.At 37 ℃ and 5%CO ₂Condition under cultivate after 18 hours, cell extract placed sample loading buffer and on the 8%SDS polyacrylamide gel, separate according to the method for Laemmli, be transferred to then on the pvdf membrane (BioRad).(NA934V Amersham) has carried out immunoblotting (protein immunoblotting analysis) and has detected to use the donkey polyclonal antibody of anti-S albumen rabbit polyclonal serum (from the immune serum of rabbit P11135, referring to embodiment 4) and the anti-rabbit igg of peroxidase link coupled.Visual ECL+ test kit (Amersham) and the radioautograph film Hyperfilm MP (Amersham) of having used of the luminescence method of binding antibody.

Shown in Figure 35 A, the S protein expression level that recombinant virus VV-TN-S instructs is with infect after 8 hours observed expression level with SARS-CoV close, and this is more much higher than infect the observed infection level in back with VV-TG-S.In second experiment (Figure 35 B), the analysis of the cell extracts of different amounts shows, infecting the observed expression level in back with the virus (VV-TN-S and VV-TN-Ssol) of TN series is after virus (corresponding VV-TG-S and VV-TG-Ssol) with TG series infects observed about 10 times.In addition, by the CV-1 cell of VV-TN-Ssol virus infection than can more effectively being secreted the Ssol polypeptide to its supernatant liquor (Figure 36 A) by the cell of VV-TG-Ssol virus infection.In this experiment, use VV-TN-Sflag virus in contrast, because it can be expressed in the proteic form membrane of S that the C end merges the FLAG label.In the cell conditioned medium liquid that is infected by VV-TN-Sflag, do not detect Sflag albumen, confirm that the Ssol polypeptide is secreted really actively after infecting with VV-TN-Ssol.

These results confirm that recombined vaccinia virus is genetically modified suitable carrier, and can make SARS glycoprotein with its form membrane (S) or solvable or secreted form (Ssol) expression.Carry S protein expression that the vaccinia virus of synthetic promoter 480 allows and Ssol excretory level than carrying the viral much higher of 7.5K gene promoter.

4) application in the S albumen soluble form of producing SARS-CoV.The purifying of this recombinant antigen and diagnostic use

BHK-21 cells be in the clone of surveying (BHK-21, CV1,293T and FRhK-4, Figure 36 B) after infecting VV-TN-Ssol the highest clone of secretory volume of Ssol polypeptide; It can be used to recombinate mass production and purifying of Ssol polypeptide.In a model experiment of optimizing infection, production and purification condition, the BHK-21 cell is at standard medium (the DMEM substratum of no pyruvate salt, contain 4.5g/l glucose, add 5%TPB, 5%FCS, 100U/ml penicillin, 100 μ g/ml Streptomycin sulphates) go up with minute junction individual layer (every 100cm ²10 ⁷Individual cell, 25ml substratum) form inoculation.After under 37 ℃, the condition of 5%CO2, cultivating 24 hours, the infection multiplicity cells infected with 0.03, and be kept to 0.5%, do not contain the secretion substratum replacement standard medium of TPB with FCS content.Remove culture supernatant and add Triton X-100 (0.1%) after under 35 ℃, the condition of 5%CO2, cultivating 2.5 days and make the vaccinia virus inactivation.After with 0.1 μ m polyethersulfone (PES) membrane filtration, reorganization Ssol polypeptide is by following step purifying: affinity chromatography, usefulness contain TBS solution (50mM Tris, pH7.4, the 150mM NaCl) wash-out of 100 μ g/ml FLAG peptides (DYKDDDDK) on anti-FLAG matrix.

8%SDS acrylamide gel electrophoresis analysis with cma staining is determined: dominant polypeptide molecular weight is about 180kD, and purity is greater than 90% (Figure 37).The concentration of the Ssol recombinant polypeptide of purifying is by measuring with the comparison of molecular weight marker, and estimated concentration is 24ng/ μ l.

The Ssol polypeptide product of purifying makes and can prepare calibration series, catches ELISA and tests the Ssol concentration of measuring in the culture supernatant thereby use.According to this test, under above-mentioned working condition, BHK-21 cells is secreted the Ssol polypeptide of about 1 μ g/ml.In addition, use the Ssol polypeptide that above-mentioned every liter of culture supernatant of purification step can the about 160 μ g of purifying.

Analyzed reorganization Ssol polypeptide for ELISA reactivity from SARS patient's serum.

Test serum from the SARS suspected case is selected based on its atopic analytical results (positive or negative) at the natural antigen of SARS-CoV, and described analysis is by being that antigenic indirect ELISA test is carried out for the test of the immunofluorescence of the Vero E6 cell that is infected by SARS-CoV and/or with the lysate of the Vero E6 cell that infected by SARS-CoV.These patients' serum with series number and patient's name's abbreviation of national influenza virus literature centre and after symptom occurring through fate define.Except 032552 serum of patient VTT, the serum of all suspected cases (seeing Table XVI) is all discerned the natural antigen of SARS-CoV, and the SARS-CoV of this patient VTT infects and can not make a definite diagnosis by the RT-PCR to 3,8 and 12 days respiratory organs sample.Use one group of control serum (TV serum) in contrast: they are collected in the popular preceding France of SARS in 2003.

The serum of Table X VI:SARS suspected case

Serum	The patient	The sample collection fate
			033168	JYK	38
033597	JYK	74
			032632	NTM	17
032634	THA	15
			032541	PHV	10
032542	NIH	17
			032552	VTT	8
032633	PTU	16

The absorption preparation of PBS solution in the ELISA plate well of the purifying Ssol polypeptide of the solid phase of using reorganization Ssol polypeptide sensitization by containing 4 μ g/ml.Dull and stereotyped 4 ℃ of overnight incubation, use PBS-Tween damping fluid (PBS, 0.1%Tween20) washing then.With after the PBS-Tween washing, test serum (100 μ l) is diluted to 1/100 and 1/400 in PBS-skimmed milk-Tween damping fluid (PBS, 3% skimmed milk, 0.1%Tween), add to then in the hole of ELISA flat board of sensitization.Flat board places 37 ℃ to cultivate 1 hour.After with PBS-Tween damping fluid washing 3 times, be added in anti-human IgG conjugate (the reference number NA933V that in PBS-skimmed milk-Tween damping fluid, is diluted to 1/4000 peroxidase labelling, Amersham), then flat board is placed 37 ℃ to cultivate 1 hour.After with PBS-Tween damping fluid washing 6 times, add chromogen (TMB) and substrate (H ₂O ₂) and with dull and stereotyped lucifuge cultivation 10 minutes.Add 1MH ₃PO ₄The solution termination reaction is the absorbancy at the 450nm place of reference measuring with the 620nm place then.

The result (Figure 38) of ELISA test confirms that the serum antibody that reorganization Ssol polypeptide can be collected in and infected mid-term or late period the SARS patient of (occur after the symptom 〉=10 days) discerns specifically, and the control serum that can be infected the experimenter of SARS is discerned significantly.

In a word, The above results confirms that reorganization Ssol polypeptide can be purified from the mammalian cell supernatant liquor that is infected by recombined vaccinia virus VV-TN-Ssol, and can use as antigen in the serodiagnostic ELISA test that exploitation SARS-CoV infects.

5) vaccine is used

In the mouse body, studied the immunogenicity of recombined vaccinia virus.

For this reason, with the intravenous injection approach several groups of (7 every group) BALB/c mouse have been carried out twice immunity with the timed interval in 4 weeks, immunity has used 10 ⁶P.f.u. recombined vaccinia virus VV-TG, VV-TG-S, VV-TG-Ssol, VV-TN, VV-TN-S and VV-TN-Ssol, and used VV-TG-HA that the hemagglutinin that instructs influenza virus A/PR/8/34 strain expresses in contrast.Gather immune serum (IS1, IS2) in 3 weeks of each immunity back.

The immune serum of each group in each group is mixed the back analyze with indirect ELISA, described indirect ELISA uses the Vero E6 cell lysate that infected by SARS-CoV as antigen, the Vero E6 cell lysate that do not infect in contrast.(NA931V is Amersham) with interpolation H with the anti-mouse IgG of peroxidase link coupled (H+L) polyclonal antibody ₂O ₂TMB (KPL) carry out visual after, the inverse of the antibody dilution when producing specific OD value 0.5 calculates tire (TI) of anti-SARS-CoV antibody.This analysis (Figure 39 A) shows, from immunity for the first time, uses the immunity of VV-TG-S and VV-TN-S virus just can induce the antibody that stings the albumen natural form at the SARS-CoV that exists in the infected Vero E6 cell lysate in the mouse body.Stronger by replying of VV-TN-S virus induction than replying of VV-TG-S virus induction, after immunity (TI is respectively 740 and 270) for the first time and immunity (TI is respectively 3230 and 600) for the second time, all be like this.VV-TN-Ssol virus is at the anti-SARS-CoV antibody (TI=640) of twice immunity back inducement efficient valency, and the replying of VV-TG-Ssol virus induction only on detection limit (TI=40).

With each the group in each group immune serum mixings post analysis in its serum with the infective ability of SARS-CoV.Since 1/20 doubling dilution serum one by one, 4 serum neutral holes of each doubling dilution test FRhK-4 cell (SARS-CoV of 100 TCID50).According to the method for Reed and Munsch, with can in and the inverse of infective serum dilution of two in 4 holes calculate the serum neutralization and tire.This analysis shows that the vaccinia virus inductive antibody in the mouse body to express S albumen or Ssol polypeptide is neutrality, and the virus of carrying synthetic promoter and the virus of carrying the 7.5K promotor to compare immunogenicity stronger: after using the VV-TN-S virus immunity 2 times, observed the highest tiring (640) (Figure 39 B).

Use the SARS-CoV challenge infection to assess after using the recombined vaccinia virus immunity protection of inductive neutralizing antibody in the mouse body.

6) other application

As mentioned above, replace with the mode of in mammalian cell, expressing optimized synthetic gene, made up third generation recombined vaccinia virus by wild-type sequence with S and Ssol gene.These recombined vaccinia virus can be expressed more substantial S and Ssol antigen, therefore have higher immunogenicity.

Recombined vaccinia virus VV-TN-Ssol can be used for antigenic mass production of Ssol and purifying, and this Ssol antigen can be used for diagnosis (ELISA serologic test) and vaccine (subunit vaccine) purposes

Embodiment 17: express SARS associated coronavirus (SARS-CoV) thorn (S) proteic recombinant measles virus.Vaccine is used.

1) introduces

Measles Vaccine (MV) once separately can be in human body after the injection inducing sustained protective immunity (Hilleman, 2002, Vaccine, 20:651-655).The provide protection quite stable that is produced, and this provide protection is based on inducing that antibody response and CD4 and cd8 cell are replied.The MV genome is highly stable and never observe the pathogenic reverse of vaccine strain.Measles virus belongs to paramyxovirus (Paramyxoviridae) section Measles virus (Morbillivirus) and belongs to; It is a kind of enveloped virus, and its genome is 16kb strand negative polarity RNA (Figure 40 A), and the kytoplasm replication cycle of its uniqueness has also been got rid of it and is integrated into any possibility of host genome.Therefore Measles Vaccine is one of the most effective and safest living vaccine that is used for the crowd really.The research group of Fr é d é ricTangy has developed a kind of expression vector recently on the basis of Measles virus Schwarz strain, Measles virus Schwarz strain is safest attenuated strain and is used most widely for the mankind as the vaccine of anti-measles.This vaccine strain can separate from infective molecular cloning, and kept it to infecting responsive primates and the immunogenicity in the mouse.After inserting additional transcription unit, it constituted an expressing heterologous sequence carrier (Combredet, 2003, J.Virol.77:11546-11554).In addition; express west nile virus (virus West Nile; the reorganization MV Schwarz strain of envelope glycoprotein WNV) can inducing sustained effective antibody response; described antibody response can protect mouse not to be subjected to mortality challenge infection (the Despres et al. of WNV; 2004; J.Infect.Dis., wait to publish).All above-mentioned characteristics make the attenuated strain Schwarz of Measles virus become candidate's carrier that is hopeful very much to be used for new live recombined vaccines structure.

The purpose of present embodiment is that the ability that the antigenic recombinant measles virus of various SARS associated coronavirus (SARS-CoV) (MV) constitute new anti-SARS candidate vaccine is expressed in assessment.

For this reason, the contriver focuses on SARS-CoV thorn (S) albumen, during it can be induced after to the animal gene immunity and the infective antibody of SARS-CoV; Also focus on proteic soluble form of S and secreted form--Ssol polypeptide, this polypeptide is made up of by the BspE1 joint and FLAG label (DYKDDDDK) fusion of a coding SG dipeptides at its C end the proteic ectodomain of S (1-1193 amino acid).This Ssol polypeptide shows the antigenicity with the S protein similar, and behind albumen and aluminum hydroxide adjuvant injection mouse with purifying, anti-SARS-CoV neutralizing antibody that can the inducement efficient valency.

The various forms of S gene is introduced above-mentioned MV Schwarz strain (Combredet, 2003, J. Virol.77:11546-11554 with the form of the additional transcription unit between P (phosphorprotein) and M (matrix) gene; The european patent application No.02291550.8 in the european patent application No.02291551.6 on June 20th, 2002 and on June 20th, 2002) among the cDNA.

After having separated recombinant virus MVSchw2-SARS-S and MVSchw2-SARS-Ssol and having checked the antigenic ability of S of its expression SARS-CoV, tested it and in mouse and monkey body, induced the ability of the protective immune response of anti-SARS.

2) structure of recombinant virus

Plasmid pTM-MVSchw-ATU2 (Figure 40 B) contains the anti-genomic infectious CDNA corresponding to Measles virus (MV) Schwarz vaccine strain, introduced (the Combredet of an additional transcription unit (ATU) between P therein (phosphorprotein) and M (matrix) gene, 2003, Journalof Virology, 77:11546-11554).Recombination group MVSchw2-SARS-S of Measles virus and MVSchw2-SARS-Ssol make up be inserted into the additional transcription unit of MVSchw-ATU2 carrier by the ORF with S albumen and Ssol polypeptide among.

For this reason, with pcr amplification a dna fragmentation that contains the S albumen cDNA of SARS-CoV, described amplification is a template with plasmid pcDNA-S, and used following oligonucleotide: 5 '-ATACGTACGA CCATGTTTAT TTTCTTATTA TTTCTTACTC TCACT-3 ' and 5 '-ATAGCGCGCT CATTATGTGT AATGTAATTT GACACCCTTG-3 ' are inserted into plasmid pCR with the gained fragment then ^_2.1-TOPO (Invitrogen), thereby obtain plasmid pTOPO-S-MV.Two oligonucleotide that use above include restriction site BsiW1 and BssHII, this makes calling sequence can be inserted in the measles carrier subsequently, and the design of above-mentioned oligonucleotide makes can generate a sequence that comprises 3774 Nucleotide of initial sum terminator codon, so just can observe 6 rules, the genome length that this rule limits Measles virus must be divided exactly (Calain ﹠amp by 6; Roux, 1993, J.Virol., 67:4822-4830; Schneideret al., 1997, Virology, 227:314-322).Described inset uses BigDyeTerminator v1.1 test kit (Applied Biosystems) and ABI377 automatic sequencer to check order.

For expressing the proteic solvable and secreted form of S of SARS-CoV, obtained to comprise the plasmid of the cDNA of Ssol polypeptide, this cDNA corresponding at the C end by the BspE1 joint of a coding SG dipeptides and the proteic ectodomain of S (1-1193 amino acid) of the SARS-CoV of FLAG sequence label (DYKDDDDK) fusion.For this reason, use the following oligonucleotide dna fragmentation that from plasmid pcDNA-Ssol, increased: 5 '-CCATTTCAAC AATTTGGCCG-3 ' and 5 '-ATAGGATCC GCGCGCTCATT ATTTATCGTC GTCATCTTTA TAATC-3 ', the fragment with gained is inserted between the Sall and BamH1 restriction enzyme site of plasmid pTOPO-S-MV then, thereby obtains plasmid pTOPO-S-MV-SF.The length of sequence between BsiW1 and BssHII site that generates is 3618 Nucleotide, also observes 6 rules.Inset uses the aforesaid method order-checking.

The BsiW1-BssHII fragment that comprises S albumen and Ssol polypeptide cDNA can be excised from plasmid pTOPO-S-MV and pTOPO-S-MV-SF by digestion, and by subclone between the corresponding site of plasmid pTM-MVSchw-ATU2, thereby obtain plasmid pTM-MVSchw2-SARS-S and pTM-MVSchw2-SARS-Ssol (Figure 40 B).These two plasmids are preserved in CNCM., and preservation date on December 1st, 2004, numbering is respectively I-3326 and I-3327.

Obtained recombinant measles virus by following means: used reverse genetics method based on the system that uses auxiliary cell line corresponding to plasmid pTM-MVSchw2-SARS-S and pTM-MVSchw2-SARS-Ssol, this method describes (1995 by people such as Radecke, Embo.J., 14:5773-5784) and by people such as Parks improve (1999, J. Virol., 73:3560-3566).In brief, according to the calcium phosphate method, plasmid pEMC-La (being so kind as to give) the transfection helper 293-3-46 that instructs MV L polysaccharase to express with the plasmid pTM-MVSchw2-SARS-S of 5 μ g or pTM-MVSchw2-SARS-Ssol and 0.02 μ g by M.A.Billeter.After 37 ℃ of overnight incubation,, then cells transfected is transferred on the Vero cell monolayer in 43 ℃ of thermal shocks 2 hours.For each of this two plasmids, after synplasm comes across 2 to 3 days of common cultivation, synplasm is transferred to continuously reaches on the 70% Vero cell monolayer of joining, before this in the Petri of 35mm culture dish, be transferred to 25 and 75cm then ²Culturing bottle in.The meeting that reaches 80-90% when synplasm is fashionable, and frozen-thawed cell is once then with the scraper collecting cell.The supernatant liquor that will contain virus behind low-speed centrifugal is stored in-80 ℃ with aliquots containig.Recombinant virus MVSchw2-SARS-S and MVSchw2-SARS-Ssol tire according to limiting dilution on the Vero cell and the dosage (TCID that infects 50% hole ₅₀) determine, calculate according to the method for K_rber.

3) evaluation of recombinant virus

The genetically modified expression of coding S albumen and Ssol polypeptide is by protein immunoblotting method and determination of immunofluorescence method.

The Vero cell monolayer that uses two kinds of viral MVSchw2-SARS-S of different passage numbers and MVSchw2-SARS-Ssol and wild-type virus MWSchw in contrast to infect in the T-25 culturing bottle with infection multiplicity 0.05.The meeting that reaches 80-90% when synplasm is fashionable, (150mM NaCl, 50mM Tris-HCl, pH7.2,1%TritonX-100,0.1%SDS, 1%DOC) preparation kytoplasm extract in extracting damping fluid, in sample loading buffer, dilute the kytoplasm extract according to the method for Laemmli then and on the 8%SDS polyacrylamide gel, separate, be transferred to then on the pvdf membrane (BioRad).(NA934V Amersham) has carried out immunoblotting (protein immunoblotting analysis) and has detected to use the donkey polyclonal antibody of anti-S albumen rabbit polyclonal serum (from the immune serum of rabbit P11135, referring to embodiment 4) and the anti-rabbit igg of peroxidase link coupled.Visual ECL+ test kit (Amersham) and the radioautograph film Hyperfilm MP (Amersham) of having used of the luminescence method of binding antibody.

The Vero cell monolayer that uses two kinds of viral MVSchw2-SARS-S and MVSchw2-SARS-Ssol and wild-type virus MWSchw in contrast to infect on the glass slide with infection multiplicity 0.05.The meeting that reaches 90-100% (MVSchw2-SARS-Ssol virus) or 30-40% (MVSchw2-SARS-S, MVSchw) when synplasm is fashionable, with 4%PBS-PFA solution fixed cell, use the rabbit polyclonal antibody mark after changing processing thoroughly with the PBS solution that contains 0.2%Triton, this rabbit polyclonal antibody is with the SARS-CoV virosome hyperimmunization of purifying and inactivation, and then carries out mark with FITC link coupled goat anti-rabbit igg (H+L) antibody (Jackson) conjugate.

Shown in Figure 41 and 42, S albumen that recombinant virus MVSchw2-SARS-S and MVSchw2-SARS-Ssol instruct and Ssol expression of polypeptides level are respectively with infect after 8 hours observed expression level with SARS-CoV close.These polypeptide expression are still stable after recombinant virus went down to posterity for 3 generations in cell culture.The above results confirms that recombinant measles virus is suitable transgene carrier, and SARS glycoprotein is expressed with its form membrane (S) or soluble form (Ssol).When with behind the corresponding sequence transient transfection, the Ssol polypeptide is expressed (referring to the foregoing description 11) in mammalian cell, and according to this situation, the Ssol polypeptide is expected can be by by the emiocytosis of MVSchw2-SARS-Ssol virus infection.

4) use

After finding that viral MVSchw2-SARS-S and MVSchw2-SARS-Ssol can express the S albumen of SARS-CoV, to have assessed them and in the mouse body, induced ability at the protective immune response of SARS-CoV, described mouse be the CD46 that MV is infected sensitivity ^+/-IFN-α β R ^-/-Mouse.Use above-mentioned method by at the ELISA of SARS-CoV natural antigen test, assessed the antibody response of immune mouse and in external and the infective ability of SARS-CoV.After 2 days, coming the protection of assessment replies with SARS-CoV non-lethal challenge infection by the minimizing of measuring lung's virus load.

According to the method for describing in the foregoing description 15, replace with above-mentionedly in mammalian cell, expressing the mode of the synthetic gene of optimizing by wild-type sequence with S and Ssol gene, made up s-generation recombinant measles virus.These recombinant measles virus can be expressed more substantial S and Ssol antigen, therefore have higher immunogenicity.

Perhaps, the wild-type or the synthetic gene of coding S albumen or Ssol polypeptide can be inserted into the form of the additional transcription unit between H and the L gene among the measles carrier MVSchw-ATU3, produce and identify recombinant virus then in a similar fashion.This insertion can generate and have different qualities the recombinant virus of (propagation, the genetically modified expression level of virus), and this recombinant virus is compared with the recombinant virus that inserts the transgenosis gained between P and N gene, may have the immunogenicity of improvement.

Recombinant measles virus MVSchw2-SARS-Ssol can be used for antigenic mass production of Ssol and purifying, and this Ssol antigen can be used for diagnosis and vaccine use.

Embodiment 18: relevant proteic other application of S

A) lentiviral vectors that can express S albumen or Ssol (or even the proteic fragment of S) can constitute the recombiant vaccine of anti-SARS-CoV, thereby is used for the disease prevention of the mankind or beasts.For confirming the feasibility of this vaccine, in the mouse body, studied the immunogenicity of recombined lentivirus vector TRIP-SD/SA-S-WPRE and TRIP-SD/SA-Ssol-WPRE.

B) use reorganization Ssol polypeptide to produce monoclonal antibody.According to the result in the foregoing description 14, these antibody or at least the major part in them can discern the proteic natural form of S of SARS-CoV, and can be used in diagnosis and/or preventive use.

C) having developed with the Ssol polypeptide is the serology detection method of the SARS of antigen and two epi-position methods.

Sequence table

＜110〉Institute Pasteur

The State Scientific Research Centre

University Paris VII Denis Dideret

S. Fan Dewofu

N. Ai Sikeliao

B. crith Sen Zuo-Qian Geni

J-C. Ma Nugela

F. this spy of hole

B. Ka Lundete

J-M. shellfish Teng

V. Lorraine

S. Jie Erbaode

AM. Bai Erjiai

S. A Ze ratio

P. Cha Niao

F. smooth Ji

C. khoum mine-laying enlightening spy

J-F. moral glug Buddhist nun Austria

M. Martin

＜120〉the new strain and the application thereof of SARS associated coronavirus

<130>226-111ext

<150>FR0314152

<151>2003-12-02

<150>FR0314151

<151>2003-12-02

<160>158

<170>PatentIn?version?3.1

<210>1

<211>29746

<212>DNA

＜213〉coronavirus

<400>1

atattaggtt?tttacctacc?caggaaaagc?caaccaacct?cgatctcttg?tagatctgtt 60

ctctaaacga?actttaaaat?ctgtgtagct?gtcgctcggc?tgcatgccta?gtgcacctac 120

gcagtataaa?caataataaa?ttttactgtc?gttgacaaga?aacgagtaac?tcgtccctct 180

tctgcagact?gcttacggtt?tcgtccgtgt?tgcagtcgat?catcagcata?cctaggtttc 240

gtccgggtgt?gaccgaaagg?taagatggag?agccttgttc?ttggtgtcaa?cgagaaaaca 300

cacgtccaac?tcagtttgcc?tgtccttcag?gttagagacg?tgctagtgcg?tggcttcggg 360

gactctgtgg?aagaggccct?atcggaggca?cgtgaacacc?tcaaaaatgg?cacttgtggt 420

ctagtagagc?tggaaaaagg?cgtactgccc?cagcttgaac?agccctatgt?gttcattaaa 480

cgttctgatg?ccttaagcac?caatcacggc?cacaaggtcg?ttgagctggt?tgcagaaatg 540

gacggcattc?agtacggtcg?tagcggtata?acactgggag?tactcgtgcc?acatgtgggc 600

gaaaccccaa?ttgcataccg?caatgttctt?cttcgtaaga?acggtaataa?gggagccggt 660

ggtcatagct?atggcatcga?tctaaagtct?tatgacttag?gtgacgagct?tggcactgat 720

cccattgaag?attatgaaca?aaactggaac?actaagcatg?gcagtggtgc?actccgtgaa 780

ctcactcgtg?agctcaatgg?aggtgcagtc?actcgctatg?tcgacaacaa?tttctgtggc 840

ccagatgggt?accctcttga?ttgcatcaaa?gattttctcg?cacgcgcggg?caagtcaatg 900

tgcactcttt?ccgaacaact?tgattacatc?gagtcgaaga?gaggtgtcta?ctgctgccgt 960

gaccatgagc?atgaaattgc?ctggttcact?gagcgctctg?ataagagcta?cgagcaccag 1020

acacccttcg?aaattaagag?tgccaagaaa?tttgacactt?tcaaagggga?atgcccaaag 1080

tttgtgtttc?ctcttaactc?aaaagtcaaa?gtcattcaac?cacgtgttga?aaagaaaaag 1140

actgagggtt?tcatggggcg?tatacgctct?gtgtaccctg?ttgcatctcc?acaggagtgt 1200

aacaatatgc?acttgtctac?cttgatgaaa?tgtaatcatt?gcgatgaagt?ttcatggcag 1260

acgtgcgact?ttctgaaagc?cacttgtgaa?cattgtggca?ctgaaaattt?agttattgaa 1320

ggacctacta?catgtgggta?cctacctact?aatgctgtag?tgaaaatgcc?atgtcctgcc 1380

tgtcaagacc?cagagattgg?acctgagcat?agtgttgcag?attatcacaa?ccactcaaac 1440

attgaaactc?gactccgcaa?gggaggtagg?actagatgtt?ttggaggctg?tgtgtttgcc 1500

tatgttggct?gctataataa?gcgtgcctac?tgggttcctc?gtgctagtgc?tgatattggc 1560

tcaggccata?ctggcattac?tggtgacaat?gtggagacct?tgaatgagga?tctccttgag 1620

atactgagtc?gtgaacgtgt?taacattaac?attgttggcg?attttcattt?gaatgaagag 1680

gttgccatca?ttttggcatc?tttctctgct?tctacaagtg?cctttattga?cactataaag 1740

agtcttgatt?acaagtcttt?caaaaccatt?gttgagtcct?gcggtaacta?taaagttacc 1800

aagggaaagc?ccgtaaaagg?tgcttggaac?attggacaac?agagatcagt?tttaacacca 1860

ctgtgtggtt?ttccctcaca?ggctgctggt?gttatcagat?caatttttgc?gcgcacactt 1920

gatgcagcaa?accactcaat?tcctgatttg?caaagagcag?ctgtcaccat?acttgatggt 1980

atttctgaac?agtcattacg?tcttgtcgac?gccatggttt?atacttcaga?cctgctcacc 2040

aacagtgtca?ttattatggc?atatgtaact?ggtggtcttg?tacaacagac?ttctcagtgg 2100

ttgtctaatc?ttttgggcac?tactgttgaa?aaactcaggc?ctatctttga?atggattgag 2160

gcgaaactta?gtgcaggagt?tgaatttctc?aaggatgctt?gggagattct?caaatttctc 2220

attacaggtg?tttttgacat?cgtcaagggt?caaatacagg?ttgcttcaga?taacatcaag 2280

gattgtgtaa?aatgcttcat?tgatgttgtt?aacaaggcac?tcgaaatgtg?cattgatcaa 2340

gtcactatcg?ctggcgcaaa?gttgcgatca?ctcaacttag?gtgaagtctt?catcgctcaa 2400

agcaagggac?tttaccgtca?gtgtatacgt?ggcaaggagc?agctgcaact?actcatgcct 2460

cttaaggcac?caaaagaagt?aacctttctt?gaaggtgatt?cacatgacac?agtacttacc 2520

tctgaggagg?ttgttctcaa?gaacggtgaa?ctcgaagcac?tcgagacgcc?cgttgatagc 2580

ttcacaaatg?gagctatcgt?tggcacacca?gtctgtgtaa?atggcctcat?gctcttagag 2640

attaaggaca?aagaacaata?ctgcgcattg?tctcctggtt?tactggctac?aaacaatgtc 2700

tttcgcttaa?aagggggtgc?accaattaaa?ggtgtaacct?ttggagaaga?tactgtttgg 2760

gaagttcaag?gttacaagaa?tgtgagaatc?acatttgagc?ttgatgaacg?tgttgacaaa 2820

gtgcttaatg?aaaagtgctc?tgtctacact?gttgaatccg?gtaccgaagt?tactgagttt 2880

gcatgtgttg?tagcagaggc?tgttgtgaag?actttacaac?cagtttctga?tctccttacc 2940

aacatgggta?ttgatcttga?tgagtggagt?gtagctacat?tctacttatt?tgatgatgct 3000

ggtgaagaaa?acttttcatc?acgtatgtat?tgttcctttt?accctccaga?tgaggaagaa 3060

gaggacgatg?cagagtgtga?ggaagaagaa?attgatgaaa?cctgtgaaca?tgagtacggt 3120

acagaggatg?attatcaagg?tctccctctg?gaatttggtg?cctcagctga?aacagttcga 3180

gttgaggaag?aagaagagga?agactggctg?gatgatacta?ctgagcaatc?agagattgag 3240

ccagaaccag?aacctacacc?tgaagaacca?gttaatcagt?ttactggtta?tttaaaactt 3300

actgacaatg?ttgccattaa?atgtgttgac?atcgttaagg?aggcacaaag?tgctaatcct 3360

atggtgattg?taaatgctgc?taacatacac?ctgaaacatg?gtggtggtgt?agcaggtgca 3420

ctcaacaagg?caaccaatgg?tgccatgcaa?aaggagagtg?atgattacat?taagctaaat 3480

ggccctctta?cagtaggagg?gtcttgtttg?ctttctggac?ataatcttgc?taagaagtgt 3540

ctgcatgttg?ttggacctaa?cctaaatgca?ggtgaggaca?tccagcttct?taaggcagca 3600

tatgaaaatt?tcaattcaca?ggacatctta?cttgcaccat?tgttgtcagc?aggcatattt 3660

ggtgctaaac?cacttcagtc?tttacaagtg?tgcgtgcaga?cggttcgtac?acaggtttat 3720

attgcagtca?atgacaaagc?tctttatgag?caggttgtca?tggattatct?tgataacctg 3780

aagcctagag?tggaagcacc?taaacaagag?gagccaccaa?acacagaaga?ttccaaaact 3840

gaggagaaat?ctgtcgtaca?gaagcctgtc?gatgtgaagc?caaaaattaa?ggcctgcatt 3900

gatgaggtta?ccacaacact?ggaagaaact?aagtttctta?ccaataagtt?actcttgttt 3960

gctgatatca?atggtaagct?ttaccatgat?tctcagaaca?tgcttagagg?tgaagatatg 4020

tctttccttg?agaaggatgc?accttacatg?gtaggtgatg?ttatcactag?tggtgatatc 4080

acttgtgttg?taataccctc?caaaaaggct?ggtggcacta?ctgagatgct?ctcaagagct 4140

ttgaagaaag?tgccagttga?tgagtatata?accacgtacc?ctggacaagg?atgtgctggt 4200

tatacacttg?aggaagctaa?gactgctctt?aagaaatgca?aatctgcatt?ttatgtacta 4260

ccttcagaag?cacctaatgc?taaggaagag?attctaggaa?ctgtatcctg?gaatttgaga 4320

gaaatgcttg?ctcatgctga?agagacaaga?aaattaatgc?ctatatgcat?ggatgttaga 4380

gccataatgg?caaccatcca?acgtaagtat?aaaggaatta?aaattcaaga?gggcatcgtt 4440

gactatggtg?tccgattctt?cttttatact?agtaaagagc?ctgtagcttc?tattattacg 4500

aagctgaact?ctctaaatga?gccgcttgtc?acaatgccaa?ttggttatgt?gacacatggt 4560

tttaatcttg?aagaggctgc?gcgctgtatg?cgttctctta?aagctcctgc?cgtagtgtca 4620

gtatcatcac?cagatgctgt?tactacatat?aatggatacc?tcacttcgtc?atcaaagaca 4680

tctgaggagc?actttgtaga?aacagtttct?ttggctggct?cttacagaga?ttggtcctat 4740

tcaggacagc?gtacagagtt?aggtgttgaa?tttcttaagc?gtggtgacaa?aattgtgtac 4800

cacactctgg?agagccccgt?cgagtttcat?cttgacggtg?aggttctttc?acttgacaaa 4860

ctaaagagtc?tcttatccct?gcgggaggtt?aagactataa?aagtgttcac?aactgtggac 4920

aacactaatc?tccacacaca?gcttgtggat?atgtctatga?catatggaca?gcagtttggt 4980

ccaacatact?tggatggtgc?tgatgttaca?aaaattaaac?ctcatgtaaa?tcatgagggt 5040

aagactttct?ttgtactacc?tagtgatgac?acactacgta?gtgaagcttt?cgagtactac 5100

catactcttg?atgagagttt?tcttggtagg?tacatgtctg?ctttaaacca?cacaaagaaa 5160

tggaaatttc?ctcaagttgg?tggtttaact?tcaattaaat?gggctgataa?caattgttat 5220

ttgtctagtg?ttttattagc?acttcaacag?cttgaagtca?aattcaatgc?accagcactt 5280

caagaggctt?attatagagc?ccgtgctggt?gatgctgcta?acttttgtgc?actcatactc 5340

gcttacagta?ataaaactgt?tggcgagctt?ggtgatgtca?gagaaactat?gacccatctt 5400

ctacagcatg?ctaatttgga?atctgcaaag?cgagttctta?atgtggtgtg?taaacattgt 5460

ggtcagaaaa?ctactacctt?aacgggtgta?gaagctgtga?tgtatatggg?tactctatct 5520

tatgataatc?ttaagacagg?tgtttccatt?ccatgtgtgt?gtggtcgtga?tgctacacaa 5580

tatctagtac?aacaagagtc?ttcttttgtt?atgatgtctg?caccacctgc?tgagtataaa 5640

ttacagcaag?gtacattctt?atgtgcgaat?gagtacactg?gtaactatca?gtgtggtcat 5700

tacactcata?taactgctaa?ggagaccctc?tatcgtattg?acggagctca?ccttacaaag 5760

atgtcagagt?acaaaggacc?agtgactgat?gttttctaca?aggaaacatc?ttacactaca 5820

accatcaagc?ctgtgtcgta?taaactcgat?ggagttactt?acacagagat?tgaaccaaaa 5880

ttggatgggt?attataaaaa?ggataatgct?tactatacag?agcagcctat?agaccttgta 5940

ccaactcaac?cattaccaaa?tgcgagtttt?gataatttca?aactcacatg?ttctaacaca 6000

aaatttgctg?atgatttaaa?tcaaatgaca?ggcttcacaa?agccagcttc?acgagagcta 6060

tctgtcacat?tcttcccaga?cttgaatggc?gatgtagtgg?ctattgacta?tagacactat 6120

tcagcgagtt?tcaagaaagg?tgctaaatta?ctgcataagc?caattgtttg?gcacattaac 6180

caggctacaa?ccaagacaac?gttcaaacca?aacacttggt?gtttacgttg?tctttggagt 6240

acaaagccag?tagatacttc?aaattcattt?gaagttctgg?cagtagaaga?cacacaagga 6300

atggacaatc?ttgcttgtga?aagtcaacaa?cccacctctg?aagaagtagt?ggaaaatcct 6360

accatacaga?aggaagtcat?agagtgtgac?gtgaaaacta?ccgaagttgt?aggcaatgtc 6420

atacttaaac?catcagatga?aggtgttaaa?gtaacacaag?agttaggtca?tgaggatctt 6480

atggctgctt?atgtggaaaa?cacaagcatt?accattaaga?aacctaatga?gctttcacta 6540

gccttaggtt?taaaaacaat?tgccactcat?ggtattgctg?caattaatag?tgttccttgg 6600

agtaaaattt?tggcttatgt?caaaccattc?ttaggacaag?cagcaattac?aacatcaaat 6660

tgcgctaaga?gattagcaca?acgtgtgttt?aacaattata?tgccttatgt?gtttacatta 6720

ttgttccaat?tgtgtacttt?tactaaaagt?accaattcta?gaattagagc?ttcactacct 6780

acaactattg?ctaaaaatag?tgttaagagt?gttgctaaat?tatgtttgga?tgccggcatt 6840

aattatgtga?agtcacccaa?attttctaaa?ttgttcacaa?tcgctatgtg?gctattgttg 6900

ttaagtattt?gcttaggttc?tctaatctgt?gtaactgctg?cttttggtgt?actcttatct 6960

aattttggtg?ctccttctta?ttgtaatggc?gttagagaat?tgtatcttaa?ttcgtctaac 7020

gttactacta?tggatttctg?tgaaggttct?tttccttgca?gcatttgttt?aagtggatta 7080

gactcccttg?attcttatcc?agctcttgaa?accattcagg?tgacgatttc?atcgtacaag 7140

ctagacttga?caattttagg?tctggccgct?gagtgggttt?tggcatatat?gttgttcaca 7200

aaattctttt?atttattagg?tctttcagct?ataatgcagg?tgttctttgg?ctattttgct 7260

agtcatttca?tcagcaattc?ttggctcatg?tggtttatca?ttagtattgt?acaaatggca 7320

cccgtttctg?caatggttag?gatgtacatc?ttctttgctt?ctttctacta?catatggaag 7380

agctatgttc?atatcatgga?tggttgcacc?tcttcgactt?gcatgatgtg?ctataagcgc 7440

aatcgtgcca?cacgcgttga?gtgtacaact?attgttaatg?gcatgaagag?atctttctat 7500

gtctatgcaa?atggaggccg?tggcttctgc?aagactcaca?attggaattg?tctcaattgt 7560

gacacatttt?gcactggtag?tacattcatt?agtgatgaag?ttgctcgtga?tttgtcactc 7620

cagtttaaaa?gaccaatcaa?ccctactgac?cagtcatcgt?atattgttga?tagtgttgct 7680

gtgaaaaatg?gcgcgcttca?cctctacttt?gacaaggctg?gtcaaaagac?ctatgagaga 7740

catccgctct?cccattttgt?caatttagac?aatttgagag?ctaacaacac?taaaggttca 7800

ctgcctatta?atgtcatagt?ttttgatggc?aagtccaaat?gcgacgagtc?tgcttctaag 7860

tctgcttctg?tgtactacag?tcagctgatg?tgccaaccta?ttctgttgct?tgaccaagct 7920

cttgtatcag?acgttggaga?tagtactgaa?gtttccgtta?agatgtttga?tgcttatgtc 7980

gacacctttt?cagcaacttt?tagtgttcct?atggaaaaac?ttaaggcact?tgttgctaca 8040

gctcacagcg?agttagcaaa?gggtgtagct?ttagatggtg?tcctttctac?attcgtgtca 8100

gctgcccgac?aaggtgttgt?tgataccgat?gttgacacaa?aggatgttat?tgaatgtctc 8160

aaactttcac?atcactctga?cttagaagtg?acaggtgaca?gttgtaacaa?tttcatgctc 8220

acctataata?aggttgaaaa?catgacgccc?agagatcttg?gcgcatgtat?tgactgtaat 8280

gcaaggcata?tcaatgccca?agtagcaaaa?agtcacaatg?tttcactcat?ctggaatgta 8340

aaagactaca?tgtctttatc?tgaacagctg?cgtaaacaaa?ttcgtagtgc?tgccaagaag 8400

aacaacatac?cttttagact?aacttgtgct?acaactagac?aggttgtcaa?tgtcataact 8460

actaaaatct?cactcaaggg?tggtaagatt?gttagtactt?gttttaaact?tatgcttaag 8520

gccacattat?tgtgcgttct?tgctgcattg?gtttgttata?tcgttatgcc?agtacataca 8580

ttgtcaatcc?atgatggtta?cacaaatgaa?atcattggtt?acaaagccat?tcaggatggt 8640

gtcactcgtg?acatcatttc?tactgatgat?tgttttgcaa?ataaacatgc?tggttttgac 8700

gcatggttta?gccagcgtgg?tggttcatac?aaaaatgaca?aaagctgccc?tgtagtagct 8760

gctatcatta?caagagagat?tggtttcata?gtgcctggct?taccgggtac?tgtgctgaga 8820

gcaatcaatg?gtgacttctt?gcattttcta?cctcgtgttt?ttagtgctgt?tggcaacatt 8880

tgctacacac?cttccaaact?cattgagtat?agtgattttg?ctacctctgc?ttgcgttctt 8940

gctgctgagt?gtacaatttt?taaggatgct?atgggcaaac?ctgtgccata?ttgttatgac 9000

actaatttgc?tagagggttc?tatttcttat?agtgagcttc?gtccagacac?tcgttatgtg 9060

cttatggatg?gttccatcat?acagtttcct?aacacttacc?tggagggttc?tgttagagta 9120

gtaacaactt?ttgatgctga?gtactgtaga?catggtacat?gcgaaaggtc?agaagtaggt 9180

atttgcctat?ctaccagtgg?tagatgggtt?cttaataatg?agcattacag?agctctatca 9240

ggagttttct?gtggtgttga?tgcgatgaat?ctcatagcta?acatctttac?tcctcttgtg 9300

caacctgtgg?gtgctttaga?tgtgtctgct?tcagtagtgg?ctggtggtat?tattgccata 9360

ttggtgactt?gtgctgccta?ctactttatg?aaattcagac?gtgtttttgg?tgagtacaac 9420

catgttgttg?ctgctaatgc?acttttgttt?ttgatgtctt?tcactatact?ctgtctggta 9480

ccagcttaca?gctttctgcc?gggagtctac?tcagtctttt?acttgtactt?gacattctat 9540

ttcaccaatg?atgtttcatt?cttggctcac?cttcaatggt?ttgccatgtt?ttctcctatt 9600

gtgccttttt?ggataacagc?aatctatgta?ttctgtattt?ctctgaagca?ctgccattgg 9660

ttctttaaca?actatcttag?gaaaagagtc?atgtttaatg?gagttacatt?tagtaccttc 9720

gaggaggctg?ctttgtgtac?ctttttgctc?aacaaggaaa?tgtacctaaa?attgcgtagc 9780

gagacactgt?tgccacttac?acagtataac?aggtatcttg?ctctatataa?caagtacaag 9840

tatttcagtg?gagccttaga?tactaccagc?tatcgtgaag?cagcttgctg?ccacttagca 9900

aaggctctaa?atgactttag?caactcaggt?gctgatgttc?tctaccaacc?accacagaca 9960

tcaatcactt?ctgctgttct?gcagagtggt?tttaggaaaa?tggcattccc?gtcaggcaaa 10020

gttgaagggt?gcatggtaca?agtaacctgt?ggaactacaa?ctcttaatgg?attgtggttg 10080

gatgacacag?tatactgtcc?aagacatgtc?atttgcacag?cagaagacat?gcttaatcct 10140

aactatgaag?atctgctcat?tcgcaaatcc?aaccatagct?ttcttgttca?ggctggcaat 10200

gttcaacttc?gtgttattgg?ccattctatg?caaaattgtc?tgcttaggct?taaagttgat 10260

acttctaacc?ctaagacacc?caagtataaa?tttgtccgta?tccaacctgg?tcaaacattt 10320

tcagttctag?catgctacaa?tggttcacca?tctggtgttt?atcagtgtgc?catgagacct 10380

aatcatacca?ttaaaggttc?tttccttaat?ggatcatgtg?gtagtgttgg?ttttaacatt 10440

gattatgatt?gcgtgtcttt?ctgctatatg?catcatatgg?agcttccaac?aggagtacac 10500

gctggtactg?acttagaagg?taaattctat?ggtccatttg?ttgacagaca?aactgcacag 10560

gctgcaggta?cagacacaac?cataacatta?aatgttttgg?catggctgta?tgctgctgtt 10620

atcaatggtg?ataggtggtt?tcttaataga?ttcaccacta?ctttgaatga?ctttaacctt 10680

gtggcaatga?agtacaacta?tgaacctttg?acacaagatc?atgttgacat?attgggacct 10740

ctttctgctc?aaacaggaat?tgccgtctta?gatatgtgtg?ctgctttgaa?agagctgctg 10800

cagaatggta?tgaatggtcg?tactatcctt?ggtagcacta?ttttagaaga?tgagtttaca 10860

ccatttgatg?ttgttagaca?atgctctggt?gttaccttcc?aaggtaagtt?caagaaaatt 10920

gttaagggca?ctcatcattg?gatgctttta?actttcttga?catcactatt?gattcttgtt 10980

caaagtacac?agtggtcact?gtttttcttt?gtttacgaga?atgctttctt?gccatttact 11040

cttggtatta?tggcaattgc?tgcatgtgct?atgctgcttg?ttaagcataa?gcacgcattc 11100

ttgtgcttgt?ttctgttacc?ttctcttgca?acagttgctt?actttaatat?ggtctacatg 11160

cctgctagct?gggtgatgcg?tatcatgaca?tggcttgaat?tggctgacac?tagcttgtct 11220

ggttataggc?ttaaggattg?tgttatgtat?gcttcagctt?tagttttgct?tattctcatg 11280

acagctcgca?ctgtttatga?tgatgctgct?agacgtgttt?ggacactgat?gaatgtcatt 11340

acacttgttt?acaaagtcta?ctatggtaat?gctttagatc?aagctatttc?catgtgggcc 11400

ttagttattt?ctgtaacctc?taactattct?ggtgtcgtta?cgactatcat?gtttttagct 11460

agagctatag?tgtttgtgtg?tgttgagtat?tacccattgt?tatttattac?tggcaacacc 11520

ttacagtgta?tcatgcttgt?ttattgtttc?ttaggctatt?gttgctgctg?ctactttggc 11580

cttttctgtt?tactcaaccg?ttacttcagg?cttactcttg?gtgtttatga?ctacttggtc 11640

tctacacaag?aatttaggta?tatgaactcc?caggggcttt?tgcctcctaa?gagtagtatt 11700

gatgctttca?agcttaacat?taagttgttg?ggtattggag?gtaaaccatg?tatcaaggtt 11760

gctactgtac?agtctaaaat?gtctgacgta?aagtgcacat?ctgtggtact?gctctcggtt 11820

cttcaacaac?ttagagtaga?gtcatcttct?aaattgtggg?cacaatgtgt?acaactccac 11880

aatgatattc?ttcttgcaaa?agacacaact?gaagctttcg?agaagatggt?ttctcttttg 11940

tctgttttgc?tatccatgca?gggtgctgta?gacattaata?ggttgtgcga?ggaaatgctc 12000

gataaccgtg?ctactcttca?ggctattgct?tcagaattta?gttctttacc?atcatatgcc 12060

gcttatgcca?ctgcccagga?ggcctatgag?caggctgtag?ctaatggtga?ttctgaagtc 12120

gttctcaaaa?agttaaagaa?atctttgaat?gtggctaaat?ctgagtttga?ccgtgatgct 12180

gccatgcaac?gcaagttgga?aaagatggca?gatcaggcta?tgacccaaat?gtacaaacag 12240

gcaagatctg?aggacaagag?ggcaaaagta?actagtgcta?tgcaaacaat?gctcttcact 12300

atgcttagga?agcttgataa?tgatgcactt?aacaacatta?tcaacaatgc?gcgtgatggt 12360

tgtgttccac?tcaacatcat?accattgact?acagcagcca?aactcatggt?tgttgtccct 12420

gattatggta?cctacaagaa?cacttgtgat?ggtaacacct?ttacatatgc?atctgcactc 12480

tgggaaatcc?agcaagttgt?tgatgcggat?agcaagattg?ttcaacttag?tgaaattaac 12540

atggacaatt?caccaaattt?ggcttggcct?cttattgtta?cagctctaag?agccaactca 12600

gctgttaaac?tacagaataa?tgaactgagt?ccagtagcac?tacgacagat?gtcctgtgcg 12660

gctggtacca?cacaaacagc?ttgtactgat?gacaatgcac?ttgcctacta?taacaattcg 12720

aagggaggta?ggtttgtgct?ggcattacta?tcagaccacc?aagatctcaa?atgggctaga 12780

ttccctaaga?gtgatggtac?aggtacaatt?tacacagaac?tggaaccacc?ttgtaggttt 12840

gttacagaca?caccaaaagg?gcctaaagtg?aaatacttgt?acttcatcaa?aggcttaaac 12900

aacctaaata?gaggtatggt?gctgggcagt?ttagctgcta?cagtacgtct?tcaggctgga 12960

aatgctacag?aagtacctgc?caattcaact?gtgctttcct?tctgtgcttt?tgcagtagac 13020

cctgctaaag?catataagga?ttacctagca?agtggaggac?aaccaatcac?caactgtgtg 13080

aagatgttgt?gtacacacac?tggtacagga?caggcaatta?ctgtaacacc?agaagctaac 13140

atggaccaag?agtcctttgg?tggtgcttca?tgttgtctgt?attgtagatg?ccacattgac 13200

catccaaatc?ctaaaggatt?ctgtgacttg?aaaggtaagt?acgtccaaat?acctaccact 13260

tgtgctaatg?acccagtggg?ttttacactt?agaaacacag?tctgtaccgt?ctgcggaatg 13320

tggaaaggtt?atggctgtag?ttgtgaccaa?ctccgcgaac?ccttgatgca?gtctgcggat 13380

gcatcaacgt?ttttaaacgg?gtttgcggtg?taagtgcagc?ccgtcttaca?ccgtgcggca 13440

caggcactag?tactgatgtc?gtctacaggg?cttttgatat?ttacaacgaa?aaagttgctg 13500

gttttgcaaa?gttcctaaaa?actaattgct?gtcgcttcca?ggagaaggat?gaggaaggca 13560

atttattaga?ctcttacttt?gtagttaaga?ggcatactat?gtctaactac?caacatgaag 13620

agactattta?taacttggtt?aaagattgtc?cagcggttgc?tgtccatgac?tttttcaagt 13680

ttagagtaga?tggtgacatg?gtaccacata?tatcacgtca?gcgtctaact?aaatacacaa 13740

tggctgattt?agtctatgct?ctacgtcatt?ttgatgaggg?taattgtgat?acattaaaag 13800

aaatactcgt?cacatacaat?tgctgtgatg?atgattattt?caataagaag?gattggtatg 13860

acttcgtaga?gaatcctgac?atcttacgcg?tatatgctaa?cttaggtgag?cgtgtacgcc 13920

aatcattatt?aaagactgta?caattctgcg?atgctatgcg?tgatgcaggc?attgtaggcg 13980

tactgacatt?agataatcag?gatcttaatg?ggaactggta?cgatttcggt?gatttcgtac 14040

aagtagcacc?aggctgcgga?gttcctattg?tggattcata?ttactcattg?ctgatgccca 14100

tcctcacttt?gactagggca?ttggctgctg?agtcccatat?ggatgctgat?ctcgcaaaac 14160

cacttattaa?gtgggatttg?ctgaaatatg?attttacgga?agagagactt?tgtctcttcg 14220

accgttattt?taaatattgg?gaccagacat?accatcccaa?ttgtattaac?tgtttggatg 14280

ataggtgtat?ccttcattgt?gcaaacttta?atgtgttatt?ttctactgtg?tttccaccta 14340

caagttttgg?accactagta?agaaaaatat?ttgtagatgg?tgttcctttt?gttgtttcaa 14400

ctggatacca?ttttcgtgag?ttaggagtcg?tacataatca?ggatgtaaac?ttacatagct 14460

cgcgtctcag?tttcaaggaa?cttttagtgt?atgctgctga?tccagctatg?catgcagctt 14520

ctggcaattt?attgctagat?aaacgcacta?catgcttttc?agtagctgca?ctaacaaaca 14580

atgttgcttt?tcaaactgtc?aaacccggta?attttaataa?agacttttat?gactttgctg 14640

tgtctaaagg?tttctttaag?gaaggaagtt?ctgttgaact?aaaacacttc?ttctttgctc 14700

aggatggcaa?cgctgctatc?agtgattatg?actattatcg?ttataatctg?ccaacaatgt 14760

gtgatatcag?acaactccta?ttcgtagttg?aagttgttga?taaatacttt?gattgttacg 14820

atggtggctg?tattaatgcc?aaccaagtaa?tcgttaacaa?tctggataaa?tcagctggtt 14880

tcccatttaa?taaatggggt?aaggctagac?tttattatga?ctcaatgagt?tatgaggatc 14940

aagatgcact?tttcgcgtat?actaagcgta?atgtcatccc?tactataact?caaatgaatc 15000

ttaagtatgc?cattagtgca?aagaatagag?ctcgcaccgt?agctggtgtc?tctatctgta 15060

gtactatgac?aaatagacag?tttcatcaga?aattattgaa?gtcaatagcc?gccactagag 15120

gagctactgt?ggtaattgga?acaagcaagt?tttacggtgg?ctggcataat?atgttaaaaa 15180

ctgtttacag?tgatgtagaa?actccacacc?ttatgggttg?ggattatcca?aaatgtgaca 15240

gagccatgcc?taacatgctt?aggataatgg?cctctcttgt?tcttgctcgc?aaacataaca 15300

cttgctgtaa?cttatcacac?cgtttctaca?ggttagctaa?cgagtgtgcg?caagtattaa 15360

gtgagatggt?catgtgtggc?ggctcactat?atgttaaacc?aggtggaaca?tcatccggtg 15420

atgctacaac?tgcttatgct?aatagtgtct?ttaacatttg?tcaagctgtt?acagccaatg 15480

taaatgcact?tctttcaact?gatggtaata?agatagctga?caagtatgtc?cgcaatctac 15540

aacacaggct?ctatgagtgt?ctctatagaa?atagggatgt?tgatcatgaa?ttcgtggatg 15600

agttttacgc?ttacctgcgt?aaacatttct?ccatgatgat?tctttctgat?gatgccgttg 15660

tgtgctataa?cagtaactat?gcggctcaag?gtttagtagc?tagcattaag?aactttaagg 15720

cagttcttta?ttatcaaaat?aatgtgttca?tgtctgaggc?aaaatgttgg?actgagactg 15780

accttactaa?aggacctcac?gaattttgct?cacagcatac?aatgctagtt?aaacaaggag 15840

atgattacgt?gtacctgcct?tacccagatc?catcaagaat?attaggcgca?ggctgttttg 15900

tcgatgatat?tgtcaaaaca?gatggtacac?ttatgattga?aaggttcgtg?tcactggcta 15960

ttgatgctta?cccacttaca?aaacatccta?atcaggagta?tgctgatgtc?tttcacttgt 16020

atttacaata?cattagaaag?ttacatgatg?agcttactgg?ccacatgttg?gacatgtatt 16080

ccgtaatgct?aactaatgat?aacacctcac?ggtactggga?acctgagttt?tatgaggcta 16140

tgtacacacc?acatacagtc?ttgcaggctg?taggtgcttg?tgtattgtgc?aattcacaga 16200

cttcacttcg?ttgcggtgcc?tgtattagga?gaccattcct?atgttgcaag?tgctgctatg 16260

accatgtcat?ttcaacatca?cacaaattag?tgttgtctgt?taatccctat?gtttgcaatg 16320

ccccaggttg?tgatgtcact?gatgtgacac?aactgtatct?aggaggtatg?agctattatt 16380

gcaagtcaca?taagcctccc?attagttttc?cattatgtgc?taatggtcag?gtttttggtt 16440

tatacaaaaa?cacatgtgta?ggcagtgaca?atgtcactga?cttcaatgcg?atagcaacat 16500

gtgattggac?taatgctggc?gattacatac?ttgccaacac?ttgtactgag?agactcaagc 16560

ttttcgcagc?agaaacgctc?aaagccactg?aggaaacatt?taagctgtca?tatggtattg 16620

ccactgtacg?cgaagtactc?tctgacagag?aattgcatct?ttcatgggag?gttggaaaac 16680

ctagaccacc?attgaacaga?aactatgtct?ttactggtta?ccgtgtaact?aaaaatagta 16740

aagtacagat?tggagagtac?acctttgaaa?aaggtgacta?tggtgatgct?gttgtgtaca 16800

gaggtactac?gacatacaag?ttgaatgttg?gtgattactt?tgtgttgaca?tctcacactg 16860

taatgccact?tagtgcacct?actctagtgc?cacaagagca?ctatgtgaga?attactggct 16920

tgtacccaac?actcaacatc?tcagatgagt?tttctagcaa?tgttgcaaat?tatcaaaagg 16980

tcggcatgca?aaagtactct?acactccaag?gaccacctgg?tactggtaag?agtcattttg 17040

ccatcggact?tgctctctat?tacccatctg?ctcgcatagt?gtatacggca?tgctctcatg 17100

cagctgttga?tgccctatgt?gaaaaggcat?taaaatattt?gcccatagat?aaatgtagta 17160

gaatcatacc?tgcgcgtgcg?cgcgtagagt?gttttgataa?attcaaagtg?aattcaacac 17220

tagaacagta?tgttttctgc?actgtaaatg?cattgccaga?aacaactgct?gacattgtag 17280

tctttgatga?aatctctatg?gctactaatt?atgacttgag?tgttgtcaat?gctagacttc 17340

gtgcaaaaca?ctacgtctat?attggcgatc?ctgctcaatt?accagccccc?cgcacattgc 17400

tgactaaagg?cacactagaa?ccagaatatt?ttaattcagt?gtgcagactt?atgaaaacaa 17460

taggtccaga?catgttcctt?ggaacttgtc?gccgttgtcc?tgctgaaatt?gttgacactg 17520

tgagtgcttt?agtttatgac?aataagctaa?aagcacacaa?ggataagtca?gctcaatgct 17580

tcaaaatgtt?ctacaaaggt?gttattacac?atgatgtttc?atctgcaatc?aacagacctc 17640

aaataggcgt?tgtaagagaa?tttcttacac?gcaatcctgc?ttggagaaaa?gctgttttta 17700

tctcacctta?taattcacag?aacgctgtag?cttcaaaaat?cttaggattg?cctacgcaga 17760

ctgttgattc?atcacagggt?tctgaatatg?actatgtcat?attcacacaa?actactgaaa 17820

cagcacactc?ttgtaatgtc?aaccgcttca?atgtggctat?cacaagggca?aaaattggca 17880

ttttgtgcat?aatgtctgat?agagatcttt?atgacaaact?gcaatttaca?agtctagaaa 17940

taccacgtcg?caatgtggct?acattacaag?cagaaaatgt?aactggactt?tttaaggact 18000

gtagtaagat?cattactggt?cttcatccta?cacaggcacc?tacacacctc?agcgttgata 18060

taaagttcaa?gactgaagga?ttatgtgttg?acataccagg?cataccaaag?gacatgacct 18120

accgtagact?catctctatg?atgggtttca?aaatgaatta?ccaagtcaat?ggttacccta 18180

atatgtttat?cacccgcgaa?gaagctattc?gtcacgttcg?tgcgtggatt?ggctttgatg 18240

tagagggctg?tcatgcaact?agagatgctg?tgggtactaa?cctacctctc?cagctaggat 18300

tttctacagg?tgttaactta?gtagctgtac?cgactggtta?tgttgacact?gaaaataaca 18360

cagaattcac?cagagttaat?gcaaaacctc?caccaggtga?ccagtttaaa?catcttatac 18420

cactcatgta?taaaggcttg?ccctggaatg?tagtgcgtat?taagatagta?caaatgctca 18480

gtgatacact?gaaaggattg?tcagacagag?tcgtgttcgt?cctttgggcg?catggctttg 18540

agcttacatc?aatgaagtac?tttgtcaaga?ttggacctga?aagaacgtgt?tgtctgtgtg 18600

acaaacgtgc?aacttgcttt?tctacttcat?cagatactta?tgcctgctgg?aatcattctg 18660

tgggttttga?ctatgtctat?aacccattta?tgattgatgt?tcagcagtgg?ggctttacgg 18720

gtaaccttca?gagtaaccat?gaccaacatt?gccaggtaca?tggaaatgca?catgtggcta 18780

gttgtgatgc?tatcatgact?agatgtttag?cagtccatga?gtgctttgtt?aagcgcgttg 18840

attggtctgt?tgaataccct?attataggag?atgaactgag?ggttaattct?gcttgcagaa 18900

aagtacaaca?catggttgtg?aagtctgcat?tgcttgctga?taagtttcca?gttcttcatg 18960

acattggaaa?tccaaaggct?atcaagtgtg?tgcctcaggc?tgaagtagaa?tggaagttct 19020

acgatgctca?gccatgtagt?gacaaagctt?acaaaataga?ggaactcttc?tattcttatg 19080

ctacacatca?cgataaattc?actgatggtg?tttgtttgtt?ttggaattgt?aacgttgatc 19140

gttacccagc?caatgcaatt?gtgtgtaggt?ttgacacaag?agtcttgtca?aacttgaact 19200

taccaggctg?tgatggtggt?agtttgtatg?tgaataagca?tgcattccac?actccagctt 19260

tcgataaaag?tgcatttact?aatttaaagc?aattgccttt?cttttactat?tctgatagtc 19320

cttgtgagtc?tcatggcaaa?caagtagtgt?cggatattga?ttatgttcca?ctcaaatctg 19380

ctacgtgtat?tacacgatgc?aatttaggtg?gtgctgtttg?cagacaccat?gcaaatgagt 19440

accgacagta?cttggatgca?tataatatga?tgatttctgc?tggatttagc?ctatggattt 19500

acaaacaatt?tgatacttat?aacctgtgga?atacatttac?caggttacag?agtttagaaa 19560

atgtggctta?taatgttgtt?aataaaggac?actttgatgg?acacgccggc?gaagcacctg 19620

tttccatcat?taataatgct?gtttacacaa?aggtagatgg?tattgatgtg?gagatctttg 19680

aaaataagac?aacacttcct?gttaatgttg?catttgagct?ttgggctaag?cgtaacatta 19740

aaccagtgcc?agagattaag?atactcaata?atttgggtgt?tgatatcgct?gctaatactg 19800

taatctggga?ctacaaaaga?gaagccccag?cacatgtatc?tacaataggt?gtctgcacaa 19860

tgactgacat?tgccaagaaa?cctactgaga?gtgcttgttc?ttcacttact?gtcttgtttg 19920

atggtagagt?ggaaggacag?gtagaccttt?ttagaaacgc?ccgtaatggt?gttttaataa 19980

cagaaggttc?agtcaaaggt?ctaacacctt?caaagggacc?agcacaagct?agcgtcaatg 20040

gagtcacatt?aattggagaa?tcagtaaaaa?cacagtttaa?ctactttaag?aaagtagacg 20100

gcattattca?acagttgcct?gaaacctact?ttactcagag?cagagactta?gaggatttta 20160

agcccagatc?acaaatggaa?actgactttc?tcgagctcgc?tatggatgaa?ttcatacagc 20220

gatataagct?cgagggctat?gccttcgaac?acatcgttta?tggagatttc?agtcatggac 20280

aacttggcgg?tcttcattta?atgataggct?tagccaagcg?ctcacaagat?tcaccactta 20340

aattagagga?ttttatccct?atggacagca?cagtgaaaaa?ttacttcata?acagatgcgc 20400

aaacaggttc?atcaaaatgt?gtgtgttctg?tgattgatct?tttacttgat?gactttgtcg 20460

agataataaa?gtcacaagat?ttgtcagtga?tttcaaaagt?ggtcaaggtt?acaattgact 20520

atgctgaaat?ttcattcatg?ctttggtgta?aggatggaca?tgttgaaacc?ttctacccaa 20580

aactacaagc?aagtcaagcg?tggcaaccag?gtgttgcgat?gcctaacttg?tacaagatgc 20640

aaagaatgct?tcttgaaaag?tgtgaccttc?agaattatgg?tgaaaatgct?gttataccaa 20700

aaggaataat?gatgaatgtc?gcaaagtata?ctcaactgtg?tcaatactta?aatacactta 20760

ctttagctgt?accctacaac?atgagagtta?ttcactttgg?tgctggctct?gataaaggag 20820

ttgcaccagg?tacagctgtg?ctcagacaat?ggttgccaac?tggcacacta?cttgtcgatt 20880

cagatcttaa?tgacttcgtc?tccgacgcag?attctacttt?aattggagac?tgtgcaacag 20940

tacatacggc?taataaatgg?gaccttatta?ttagcgatat?gtatgaccct?aggaccaaac 21000

atgtgacaaa?agagaatgac?tctaaagaag?ggtttttcac?ttatctgtgt?ggatttataa 21060

agcaaaaact?agccctgggt?ggttctatag?ctgtaaagat?aacagagcat?tcttggaatg 21120

ctgaccttta?caagcttatg?ggccatttct?catggtggac?agcttttgtt?acaaatgtaa 21180

atgcatcatc?atcggaagca?tttttaattg?gggctaacta?tcttggcaag?ccgaaggaac 21240

aaattgatgg?ctataccatg?catgctaact?acattttctg?gaggaacaca?aatcctatcc 21300

agttgtcttc?ctattcactc?tttgacatga?gcaaatttcc?tcttaaatta?agaggaactg 21360

ctgtaatgtc?tcttaaggag?aatcaaatca?atgatatgat?ttattctctt?ctggaaaaag 21420

gtaggcttat?cattagagaa?aacaacagag?ttgtggtttc?aagtgatatt?cttgttaaca 21480

actaaacgaa?catgtttatt?ttcttattat?ttcttactct?cactagtggt?agtgaccttg 21540

accggtgcac?cacttttgat?gatgttcaag?ctcctaatta?cactcaacat?acttcatcta 21600

tgaggggggt?ttactatcct?gatgaaattt?ttagatcaga?cactctttat?ttaactcagg 21660

atttatttct?tccattttat?tctaatgtta?cagggtttca?tactattaat?catacgtttg 21720

gcaaccctgt?catacctttt?aaggatggta?tttattttgc?tgccacagag?aaatcaaatg 21780

ttgtccgtgg?ttgggttttt?ggttctacca?tgaacaacaa?gtcacagtcg?gtgattatta 21840

ttaacaattc?tactaatgtt?gttatacgag?catgtaactt?tgaattgtgt?gacaaccctt 21900

tctttgctgt?ttctaaaccc?atgggtacac?agacacatac?tatgatattc?gataatgcat 21960

ttaattgcac?tttcgagtac?atatctgatg?ccttttcgct?tgatgtttca?gaaaagtcag 22020

gtaattttaa?acacttacga?gagtttgtgt?ttaaaaataa?agatgggttt?ctctatgttt 22080

ataagggcta?tcaacctata?gatgtagttc?gtgatctacc?ttctggtttt?aacactttga 22140

aacctatttt?taagttgcct?cttggtatta?acattacaaa?ttttagagcc?attcttacag 22200

ccttttcacc?tgctcaagac?atttggggca?cgtcagctgc?agcctatttt?gttggctatt 22260

taaagccaac?tacatttatg?ctcaagtatg?atgaaaatgg?tacaatcaca?gatgctgttg 22320

attgttctca?aaatccactt?gctgaactca?aatgctctgt?taagagcttt?gagattgaca 22380

aaggaattta?ccagacctct?aatttcaggg?ttgttccctc?aggagatgtt?gtgagattcc 22440

ctaatattac?aaacttgtgt?ccttttggag?aggtttttaa?tgctactaaa?ttcccttctg 22500

tctatgcatg?ggagagaaaa?aaaatttcta?attgtgttgc?tgattactct?gtgctctaca 22560

actcaacatt?tttttcaacc?tttaagtgct?atggcgtttc?tgccactaag?ttgaatgatc 22620

tttgcttctc?caatgtctat?gcagattctt?ttgtagtcaa?gggagatgat?gtaagacaaa 22680

tagcgccagg?acaaactggt?gttattgctg?attataatta?taaattgcca?gatgatttca 22740

tgggttgtgt?ccttgcttgg?aatactagga?acattgatgc?tacttcaact?ggtaattata 22800

attataaata?taggtatctt?agacatggca?agcttaggcc?ctttgagaga?gacatatcta 22860

atgtgccttt?ctcccctgat?ggcaaacctt?gcaccccacc?tgctcttaat?tgttattggc 22920

cattaaatga?ttatggtttt?tacaccacta?ctggcattgg?ctaccaacct?tacagagttg 22980

tagtactttc?ttttgaactt?ttaaatgcac?cggccacggt?ttgtggacca?aaattatcca 23040

ctgaccttat?taagaaccag?tgtgtcaatt?ttaattttaa?tggactcact?ggtactggtg 23100

tgttaactcc?ttcttcaaag?agatttcaac?catttcaaca?atttggccgt?gatgtttctg 23160

atttcactga?ttccgttcga?gatcctaaaa?catctgaaat?attagacatt?tcaccttgct 23220

cttttggggg?tgtaagtgta?attacacctg?gaacaaatgc?ttcatctgaa?gttgctgttc 23280

tatatcaaga?tgttaactgc?actgatgttt?ctacagcaat?tcatgcagat?caactcacac 23340

cagcttggcg?catatattct?actggaaaca?atgtattcca?gactcaagca?ggctgtctta 23400

taggagctga?gcatgtcgac?acttcttatg?agtgcgacat?tcctattgga?gctggcattt 23460

gtgctagtta?ccatacagtt?tctttattac?gtagtactag?ccaaaaatct?attgtggctt 23520

atactatgtc?tttaggtgct?gatagttcaa?ttgcttactc?taataacacc?attgctatac 23580

ctactaactt?ttcaattagc?attactacag?aagtaatgcc?tgtttctatg?gctaaaacct 23640

ccgtagattg?taatatgtac?atctgcggag?attctactga?atgtgctaat?ttgcttctcc 23700

aatatggtag?cttttgcaca?caactaaatc?gtgcactctc?aggtattgct?gctgaacagg 23760

atcgcaacac?acgtgaagtg?ttcgctcaag?tcaaacaaat?gtacaaaacc?ccaactttga 23820

aatattttgg?tggttttaat?ttttcacaaa?tattacctga?ccctctaaag?ccaactaaga 23880

ggtcttttat?tgaggacttg?ctctttaata?aggtgacact?cgctgatgct?ggcttcatga 23940

agcaatatgg?cgaatgccta?ggtgatatta?atgctagaga?tctcatttgt?gcgcagaagt 24000

tcaatggact?tacagtgttg?ccacctctgc?tcactgatga?tatgattgct?gcctacactg 24060

ctgctctagt?tagtggtact?gccactgctg?gatggacatt?tggtgctggc?gctgctcttc 24120

aaataccttt?tgctatgcaa?atggcatata?ggttcaatgg?cattggagtt?acccaaaatg 24180

ttctctatga?gaaccaaaaa?caaatcgcca?accaatttaa?caaggcgatt?agtcaaattc 24240

aagaatcact?tacaacaaca?tcaactgcat?tgggcaagct?gcaagacgtt?gttaaccaga 24300

atgctcaagc?attaaacaca?cttgttaaac?aacttagctc?taattttggt?gcaatttcaa 24360

gtgtgctaaa?tgatatcctt?tcgcgacttg?ataaagtcga?ggcggaggta?caaattgaca 24420

ggttaattac?aggcagactt?caaagccttc?aaacctatgt?aacacaacaa?ctaatcaggg 24480

ctgctgaaat?cagggcttct?gctaatcttg?ctgctactaa?aatgtctgag?tgtgttcttg 24540

gacaatcaaa?aagagttgac?ttttgtggaa?agggctacca?ccttatgtcc?ttcccacaag 24600

cagccccgca?tggtgttgtc?ttcctacatg?tcacgtatgt?gccatcccag?gagaggaact 24660

tcaccacagc?gccagcaatt?tgtcatgaag?gcaaagcata?cttccctcgt?gaaggtgttt 24720

ttgtgtttaa?tggcacttct?tggtttatta?cacagaggaa?cttcttttct?ccacaaataa 24780

ttactacaga?caatacattt?gtctcaggaa?attgtgatgt?cgttattggc?atcattaaca 24840

acacagttta?tgatcctctg?caacctgagc?ttgactcatt?caaagaagag?ctggacaagt 24900

acttcaaaaa?tcatacatca?ccagatgttg?atcttggcga?catttcaggc?attaacgctt 24960

ctgtcgtcaa?cattcaaaaa?gaaattgacc?gcctcaatga?ggtcgctaaa?aatttaaatg 25020

aatcactcat?tgaccttcaa?gaattgggaa?aatatgagca?atatattaaa?tggccttggt 25080

atgtttggct?cggcttcatt?gctggactaa?ttgccatcgt?catggttaca?atcttgcttt 25140

gttgcatgac?tagttgttgc?agttgcctca?agggtgcatg?ctcttgtggt?tcttgctgca 25200

agtttgatga?ggatgactct?gagccagttc?tcaagggtgt?caaattacat?tacacataaa 25260

cgaacttatg?gatttgttta?tgagattttt?tactcttgga?tcaattactg?cacagccagt 25320

aaaaattgac?aatgcttctc?ctgcaagtac?tgttcatgct?acagcaacga?taccgctaca 25380

agcctcactc?cctttcggat?ggcttgttat?tggcgttgca?tttcttgctg?tttttcagag 25440

cgctaccaaa?ataattgcgc?tcaataaaag?atggcagcta?gccctttata?agggcttcca 25500

gttcatttgc?aatttactgc?tgctatttgt?taccatctat?tcacatcttt?tgcttgtcgc 25560

tgcaggtatg?gaggcgcaat?ttttgtacct?ctatgccttg?atatattttc?tacaatgcat 25620

caacgcatgt?agaattatta?tgagatgttg?gctttgttgg?aagtgcaaat?ccaagaaccc 25680

attactttat?gatgccaact?actttgtttg?ctggcacaca?cataactatg?actactgtat 25740

accatataac?agtgtcacag?atacaattgt?cgttactgaa?ggtgacggca?tttcaacacc 25800

aaaactcaaa?gaagactacc?aaattggtgg?ttattctgag?gataggcact?caggtgttaa 25860

agactatgtc?gttgtacatg?gctatttcac?cgaagtttac?taccagcttg?agtctacaca 25920

aattactaca?gacactggta?ttgaaaatgc?tacattcttc?atctttaaca?agcttgttaa 25980

agacccaccg?aatgtgcaaa?tacacacaat?cgacggctct?tcaggagttg?ctaatccagc 26040

aatggatcca?atttatgatg?agccgacgac?gactactagc?gtgcctttgt?aagcacaaga 26100

aagtgagtac?gaacttatgt?actcattcgt?ttcggaagaa?acaggtacgt?taatagttaa 26160

tagcgtactt?ctttttcttg?ctttcgtggt?attcttgcta?gtcacactag?ccatccttac 26220

tgcgcttcga?ttgtgtgcgt?actgctgcaa?tattgttaac?gtgagtttag?taaaaccaac 26280

ggtttacgtc?tactcgcgtg?ttaaaaatct?gaactcttct?gaaggagttc?ctgatcttct 26340

ggtctaaacg?aactaactat?tattattatt?ctgtttggaa?ctttaacatt?gcttatcatg 26400

gcagacaacg?gtactattac?cgttgaggag?cttaaacaac?tcctggaaca?atggaaccta 26460

gtaataggtt?tcctattcct?agcctggatt?atgttactac?aatttgccta?ttctaatcgg 26520

aacaggtttt?tgtacataat?aaagcttgtt?ttcctctggc?tcttgtggcc?agtaacactt 26580

gcttgttttg?tgcttgctgc?tgtctacaga?attaattggg?tgactggcgg?gattgcgatt 26640

gcaatggctt?gtattgtagg?cttgatgtgg?cttagctact?tcgttgcttc?cttcaggctg 26700

tttgctcgta?cccgctcaat?gtggtcattc?aacccagaaa?caaacattct?tctcaatgtg 26760

cctctccggg?ggacaattgt?gaccagaccg?ctcatggaaa?gtgaacttgt?cattggtgct 26820

gtgatcattc?gtggtcactt?gcgaatggcc?ggacactccc?tagggcgctg?tgacattaag 26880

gacctgccaa?aagagatcac?tgtggctaca?tcacgaacgc?tttcttatta?caaattagga 26940

gcgtcgcagc?gtgtaggcac?tgattcaggt?tttgctgcat?acaaccgcta?ccgtattgga 27000

aactataaat?taaatacaga?ccacgccggt?agcaacgaca?atattgcttt?gctagtacag 27060

taagtgacaa?cagatgtttc?atcttgttga?cttccaggtt?acaatagcag?agatattgat 27120

tatcattatg?aggactttca?ggattgctat?ttggaatctt?gacgttataa?taagttcaat 27180

agtgagacaa?ttatttaagc?ctctaactaa?gaagaattat?tcggagttag?atgatgaaga 27240

acctatggag?ttagattatc?cataaaacga?acatgaaaat?tattctcttc?ctgacattga 27300

ttgtatttac?atcttgcgag?ctatatcact?atcaggagtg?tgttagaggt?acgactgtac 27360

tactaaaaga?accttgccca?tcaggaacat?acgagggcaa?ttcaccattt?caccctcttg 27420

ctgacaataa?atttgcacta?acttgcacta?gcacacactt?tgcttttgct?tgtgctgacg 27480

gtactcgaca?tacctatcag?ctgcgtgcaa?gatcagtttc?accaaaactt?ttcatcagac 27540

aagaggaggt?tcaacaagag?ctctactcgc?cactttttct?cattgttgct?gctctagtat 27600

ttttaatact?ttgcttcacc?attaagagaa?agacagaatg?aatgagctca?ctttaattga 27660

cttctatttg?tgctttttag?cctttctgct?attccttgtt?ttaataatgc?ttattatatt 27720

ttggttttca?ctcgaaatcc?aggatctaga?agaaccttgt?accaaagtct?aaacgaacat 27780

gaaacttctc?attgttttga?cttgtatttc?tctatgcagt?tgcatatgca?ctgtagtaca 27840

gcgctgtgca?tctaataaac?ctcatgtgct?tgaagatcct?tgtaaggtac?aacactaggg 27900

gtaatactta?tagcactgct?tggctttgtg?ctctaggaaa?ggttttacct?tttcatagat 27960

ggcacactat?ggttcaaaca?tgcacaccta?atgttactat?caactgtcaa?gatccagctg 28020

gtggtgcgct?tatagctagg?tgttggtacc?ttcatgaagg?tcaccaaact?gctgcattta 28080

gagacgtact?tgttgtttta?aataaacgaa?caaattaaaa?tgtctgataa?tggaccccaa 28140

tcaaaccaac?gtagtgcccc?ccgcattaca?tttggtggac?ccacagattc?aactgacaat 28200

aaccagaatg?gaggacgcaa?tggggcaagg?ccaaaacagc?gccgacccca?aggtttaccc 28260

aataatactg?cgtcttggtt?cacagctctc?actcagcatg?gcaaggagga?acttagattc 28320

cctcgaggcc?agggcgttcc?aatcaacacc?aatagtggtc?cagatgacca?aattggctac 28380

taccgaagag?ctacccgacg?agttcgtggt?ggtgacggca?aaatgaaaga?gctcagcccc 28440

agatggtact?tctattacct?aggaactggc?ccagaagctt?cacttcccta?cggcgctaac 28500

aaagaaggca?tcgtatgggt?tgcaactgag?ggagccttga?atacacccaa?agaccacatt 28560

ggcacccgca?atcctaataa?caatgctgcc?accgtgctac?aacttcctca?aggaacaaca 28620

ttgccaaaag?gcttctacgc?agagggaagc?agaggcggca?gtcaagcctc?ttctcgctcc 28680

tcatcacgta?gtcgcggtaa?ttcaagaaat?tcaactcctg?gcagcagtag?gggaaattct 28740

cctgctcgaa?tggctagcgg?aggtggtgaa?actgccctcg?cgctattgct?gctagacaga 28800

ttgaaccagc?ttgagagcaa?agtttctggt?aaaggccaac?aacaacaagg?ccaaactgtc 28860

actaagaaat?ctgctgctga?ggcatctaaa?aagcctcgcc?aaaaacgtac?tgccacaaaa 28920

cagtacaacg?tcactcaagc?atttgggaga?cgtggtccag?aacaaaccca?aggaaatttc 28980

ggggaccaag?acctaatcag?acaaggaact?gattacaaac?attggccgca?aattgcacaa 29040

tttgctccaa?gtgcctctgc?attctttgga?atgtcacgca?ttggcatgga?agtcacacct 29100

tcgggaacat?ggctgactta?tcatggagcc?attaaattgg?atgacaaaga?tccacaattc 29160

aaagacaacg?tcatactgct?gaacaagcac?attgacgcat?acaaaacatt?cccaccaaca 29220

gagcctaaaa?aggacaaaaa?gaaaaagact?gatgaagctc?agcctttgcc?gcagagacaa 29280

aagaagcagc?ccactgtgac?tcttcttcct?gcggctgaca?tggatgattt?ctccagacaa 29340

cttcaaaatt?ccatgagtgg?agcttctgct?gattcaactc?aggcataaac?actcatgatg 29400

accacacaag?gcagatgggc?tatgtaaacg?ttttcgcaat?tccgtttacg?atacatagtc 29460

tactcttgtg?cagaatgaat?tctcgtaact?aaacagcaca?agtaggttta?gttaacttta 29520

atctcacata?gcaatcttta?atcaatgtgt?aacattaggg?aggacttgaa?agagccacca 29580

cattttcatc?gaggccacgc?ggagtacgat?cgagggtaca?gtgaataatg?ctagggagag 29640

ctgcctatat?ggaagagccc?taatgtgtaa?aattaatttt?agtagtgcta?tccccatgtg 29700

attttaatag?cttcttagga?gaatgacaaa?aaaaaaaaaa?aaaaaa 29746

<210>2

<211>3945

<212>DNA

＜213〉coronavirus

<220>

<221>CDS

<222>(89)..(3853)

<223>

<400>2

ttctcttctg?gaaaaaggta?ggcttatcat?tagagaaaac?aacagagttg?tggtttcaag 60

tgatattctt?gttaacaact?aaacgaac?atg?ttt?att?ttc?tta?tta?ttt?ctt 112

Met?Phe?Ile?Phe?Leu?Leu?Phe?Leu

1 5

act?ctc?act?agt?ggt?agt?gac?ctt?gac?cgg?tgc?acc?act?ttt?gat?gat 160

Thr?Leu?Thr?Ser?Gly?Ser?Asp?Leu?Asp?Arg?Cys?Thr?Thr?Phe?Asp?Asp

10 15 20

gtt?caa?gct?cct?aat?tac?act?caa?cat?cat?tca?tct?atg?agg?ggg?gtt 208

Val?Gln?Ala?Pro?Asn?Tyr?Thr?Gln?His?Thr?Ser?Ser?Met?Arg?Gly?Val

25 30 35 40

tac?tat?cct?gat?gaa?att?ttt?aga?tca?gac?act?ctt?tat?tta?act?cag 256

Tyr?Tyr?Pro?Asp?Glu?Ile?Phe?Arg?Ser?Asp?Thr?Leu?Tyr?Leu?Thr?Gln

45 50 55

gat?tta?ttt?ctt?cca?ttt?tat?tct?aat?gtt?aca?ggg?ttt?cat?act?att 304

Asp?Leu?Phe?Leu?Pro?Phe?Tyr?Ser?Asn?Val?Thr?Gly?Phe?His?Thr?Ile

60 65 70

aat?cat?acg?ttt?ggc?aac?cct?gtc?ata?cct?ttt?aag?gat?ggt?att?tat 352

Asn?His?Thr?Phe?Gly?Asn?Pro?Val?Ile?Pro?Phe?Lys?Asp?Gly?Ile?Tyr

75 80 85

ttt?gct?gcc?aca?gag?aaa?tca?aat?gtt?gtc?cgt?ggt?tgg?gtt?ttt?ggt 400

Phe?Ala?Ala?Thr?Glu?Lys?Ser?Asn?Val?Val?Arg?Gly?Trp?Val?Phe?Gly

90 95 100

tct?acc?atg?aac?aac?aag?tca?cag?tcg?gtg?att?att?att?aac?aat?tct 448

Ser?Thr?Met?Asn?Asn?Lys?Ser?Gln?Ser?Val?Ile?Ile?Ile?Asn?Asn?Ser

105 110 115 120

act?aat?gtt?gtt?ata?cga?gca?tgt?aac?ttt?gaa?ttg?tgt?gac?aac?cct 496

Thr?Asn?Val?Val?Ile?Arg?Ala?Cys?Asn?Phe?Glu?Leu?Cys?Asp?Asn?Pro

125 130 135

ttc?ttt?gct?gtt?tct?aaa?ccc?atg?ggt?aca?cag?aca?cat?act?atg?ata 544

Phe?Phe?Ala?Val?Ser?Lys?Pro?Met?Gly?Thr?Gln?Thr?His?Thr?Met?Ile

140 145 150

ttc?gat?aat?gca?ttt?aat?tgc?act?ttc?gag?tac?ata?tct?gat?gcc?ttt 592

Phe?Asp?Asn?Ala?Phe?Asn?Cys?Thr?Phe?Glu?Tyr?Ile?Ser?Asp?Ala?Phe

155 160 165

tcg?ctt?gat?gtt?tca?gaa?aag?tca?ggt?aat?ttt?aaa?cac?tta?cga?gag 640

Ser?Leu?Asp?Val?Ser?Glu?Lys?Ser?Gly?Asn?Phe?Lys?His?Leu?Arg?Glu

170 175 180

ttt?gtg?ttt?aaa?aat?aaa?gat?ggg?ttt?ctc?tat?gtt?tat?aag?ggc?tat 688

Phe?Val?Phe?Lys?Asn?Lys?Asp?Gly?Phe?Leu?Tyr?Val?Tyr?Lys?Gly?Tyr

185 190 195 200

caa?cct?ata?gat?gta?gtt?cgt?gat?cta?cct?tct?ggt?ttt?aac?act?ttg 736

Gln?Pro?Ile?Asp?Val?Val?Arg?Asp?Leu?Pro?Ser?Gly?Phe?Asn?Thr?Leu

205 210 215

aaa?cct?att?ttt?aag?ttg?cct?ctt?ggt?att?aac?att?aca?aat?ttt?aga 784

Lys?Pro?Ile?Phe?Lys?Leu?Pro?Leu?Gly?Ile?Asn?Ile?Thr?Asn?Phe?Arg

220 225 230

gcc?att?ctt?aca?gcc?ttt?tca?cct?gct?caa?gac?att?tgg?ggc?acg?tca 832

Ala?Ile?Leu?Thr?Ala?Phe?Ser?Pro?Ala?Gln?Asp?Ile?Trp?Gly?Thr?Ser

235 240 245

gct?gca?gcc?tat?ttt?gtt?ggc?tat?tta?aag?cca?act?aca?ttt?atg?ctc 880

Ala?Ala?Ala?Tyr?Phe?Val?Gly?Tyr?Leu?Lys?Pro?Thr?Thr?Phe?Met?Leu

250 255 260

aag?tat?gat?gaa?aat?ggt?aca?atc?aca?gat?gct?gtt?gat?tgt?tct?caa 928

Lys?Tyr?Asp?Glu?Asn?Gly?Thr?Ile?Thr?Asp?Ala?Val?Asp?Cys?Ser?Gln

265 270 275 280

aat?cca?ctt?gct?gaa?ctc?aaa?tgc?tct?gtt?aag?agc?ttt?gag?att?gac 976

Asn?Pro?Leu?Ala?Glu?Leu?Lys?Cys?Ser?Val?Lys?Ser?Phe?Glu?Ile?Asp

285 290 295

aaa?gga?att?tac?cag?acc?tct?aat?ttc?agg?gtt?gtt?ccc?tca?gga?gat 1024

Lys?Gly?Ile?Tyr?Gln?Thr?Ser?Asn?Phe?Arg?Val?Val?Pro?Ser?Gly?Asp

300 305 310

gtt?gtg?aga?ttc?cct?aat?att?aca?aac?ttg?tgt?cct?ttt?gga?gag?gtt 1072

Val?Val?Arg?Phe?Pro?Asn?Ile?Thr?Asn?Leu?Cys?Pro?Phe?Gly?Glu?Val

315 320 325

ttt?aat?gct?act?aaa?ttc?cct?tct?gtc?tat?gca?tgg?gag?aga?aaa?aaa 1120

Phe?Asn?Ala?Thr?Lys?Phe?Pro?Ser?Val?Tyr?Ala?Trp?Glu?Arg?Lys?Lys

330 335 340

att?tct?aat?tgt?gtt?gct?gat?tac?tct?gtg?ctc?tac?aac?tca?aca?ttt 1168

Ile?Ser?Asn?Cys?Val?Ala?Asp?Tyr?Ser?Val?Leu?Tyr?Asn?Ser?Thr?Phe

345 350 355 360

ttt?tca?acc?ttt?aag?tgc?tat?ggc?gtt?tct?gcc?act?aag?ttg?aat?gat 1216

Phe?Ser?Thr?Phe?Lys?Cys?Tyr?Gly?Val?Ser?Ala?Thr?Lys?Leu?Asn?Asp

365 370 375

ctt?tgc?ttc?tcc?aat?gtc?tat?gca?gat?tct?ttt?gta?gtc?aag?gga?gat 1264

Leu?Cys?Phe?Ser?Asn?Val?Tyr?Ala?Asp?Ser?Phe?Val?Val?Lys?Gly?Asp

380 385 390

gat?gta?aga?caa?ata?gcg?cca?gga?caa?act?ggt?gtt?att?gct?gat?tat 1312

Asp?Val?Arg?Gln?Ile?Ala?Pro?Gly?Gln?Thr?Gly?Val?Ile?Ala?Asp?Tyr

395 400 405

aat?tat?aaa?ttg?cca?gat?gat?ttc?atg?ggt?tgt?gtc?ctt?gct?tgg?aat 1360

Asn?Tyr?Lys?Leu?Pro?Asp?Asp?Phe?Met?Gly?Cys?Val?Leu?Ala?Trp?Asn

410 415 420

act?agg?aac?att?gat?gct?act?tca?act?ggt?aat?tat?aat?tat?aaa?tat 1408

Thr?Arg?Asn?Ile?Asp?Ala?Thr?Ser?Thr?Gly?Asn?Tyr?Asn?Tyr?Lys?Tyr

425 430 435 440

agg?tat?ctt?aga?cat?ggc?aag?ctt?agg?ccc?ttt?gag?aga?gac?ata?tct 1456

Arg?Tyr?Leu?Arg?His?Gly?Lys?Leu?Arg?Pro?Phe?Glu?Arg?Asp?Ile?Ser

445 450 455

aat?gtg?cct?ttc?tcc?cct?gat?ggc?aaa?cct?tgc?acc?cca?cct?gct?ctt 1504

Asn?Val?Pro?Phe?Ser?Pro?Asp?Gly?Lys?Pro?Cys?Thr?Pro?Pro?Ala?Leu

460 465 470

aat?tgt?tat?tgg?cca?tta?aat?gat?tat?ggt?ttt?tac?acc?act?act?ggc 1552

Asn?Cys?Tyr?Trp?Pro?Leu?Asn?Asp?Tyr?Gly?Phe?Tyr?Thr?Thr?Thr?Gly

475 480 485

att?ggc?tac?caa?cct?tac?aga?gtt?gta?gta?ctt?tct?ttt?gaa?ctt?tta 1600

Ile?Gly?Tyr?Gln?Pro?Tyr?Arg?Val?Val?Val?Leu?Ser?Phe?Glu?Leu?Leu

490 495 500

aat?gca?ccg?gcc?acg?gtt?tgt?gga?cca?aaa?tta?tcc?act?gac?ctt?att 1648

Asn?Ala?Pro?Ala?Thr?Val?Cys?Gly?Pro?Lys?Leu?Ser?Thr?Asp?Leu?Ile

505 510 515 520

aag?aac?cag?tgt?gtc?aat?ttt?aat?ttt?aat?gga?ctc?act?ggt?act?ggt 1696

Lys?Asn?Gln?Cys?Val?Asn?Phe?Asn?Phe?Asn?Gly?Leu?Thr?Gly?Thr?Gly

525 530 535

gtg?tta?act?cct?tct?tca?aag?aga?ttt?caa?cca?ttt?caa?caa?ttt?ggc 1744

Val?Leu?Thr?Pro?Ser?Ser?Lys?Arg?Phe?Gln?Pro?Phe?Gln?Gln?Phe?Gly

540 545 550

cgt?gat?gtt?tct?gat?ttc?act?gat?tcc?gtt?cga?gat?cct?aaa?aca?tct 1792

Arg?Asp?Val?Ser?Asp?Phe?Thr?Asp?Ser?Val?Arg?Asp?Pro?Lys?Thr?Ser

555 560 565

gaa?ata?tta?gac?att?tca?cct?tgc?tct?ttt?ggg?ggt?gta?agt?gta?att 1840

Glu?Ile?Leu?Asp?Ile?Ser?Pro?Cys?Ser?Phe?Gly?Gly?Val?Ser?Val?Ile

570 575 580

aca?cct?gga?aca?aat?gct?tca?tct?gaa?gtt?gct?gtt?cta?tat?caa?gat 1888

Thr?Pro?Gly?Thr?Asn?Ala?Ser?Ser?Glu?Val?Ala?Val?Leu?Tyr?Gln?Asp

585 590 595 600

gtt?aac?tgc?act?gat?gtt?tct?aca?gca?att?cat?gca?gat?caa?ctc?aca 1936

Val?Asn?Cys?Thr?Asp?Val?Ser?Thr?Ala?Ile?His?Ala?Asp?Gln?Leu?Thr

605 610 615

cca?gct?tgg?cgc?ata?tat?tct?act?gga?aac?aat?gta?ttc?cag?act?caa 1984

Pro?Ala?Trp?Arg?Ile?Tyr?Ser?Thr?Gly?Asn?Asn?Val?Phe?Gln?Thr?Gln

620 625 630

gca?ggc?tgt?ctt?ata?gga?gct?gag?cat?gtc?gac?act?tct?tat?gag?tgc 2032

Ala?Gly?Cys?Leu?Ile?Gly?Ala?Glu?His?Val?Asp?Thr?Ser?Tyr?Glu?Cys

635 640 645

gac?att?cct?att?gga?gct?ggc?att?tgt?gct?agt?tac?cat?aca?gtt?tct 2080

Asp?Ile?Pro?Ile?Gly?Ala?Gly?Ile?Cys?Ala?Ser?Tyr?His?Thr?Val?Ser

650 655 660

tta?tta?cgt?agt?act?agc?caa?aaa?tct?att?gtg?gct?tat?act?atg?tct 2128

Leu?Leu?Arg?Ser?Thr?Ser?Gln?Lys?Ser?Ile?Val?Ala?Tyr?Thr?Met?Ser

665 670 675 680

tta?ggt?gct?gat?agt?tca?att?gct?tac?tct?aat?aac?acc?att?gct?ata 2176

Leu?Gly?Ala?Asp?Ser?Ser?Ile?Ala?Tyr?Ser?Asn?Asn?Thr?Ile?Ala?Ile

685 690 695

cct?act?aac?ttt?tca?att?agc?att?act?aca?gaa?gta?atg?cct?gtt?tct 2224

Pro?Thr?Asn?Phe?Ser?Ile?Ser?Ile?Thr?Thr?Glu?Val?Met?Pro?Val?Ser

700 705 710

atg?gct?aaa?acc?tcc?gta?gat?tgt?aat?atg?tac?atc?tgc?gga?gat?tct 2272

Met?Ala?Lys?Thr?Ser?Val?Asp?Cys?Asn?Met?Tyr?Ile?Cys?Gly?Asp?Ser

715 720 725

act?gaa?tgt?gct?aat?ttg?ctt?ctc?caa?tat?ggt?agc?ttt?tgc?aca?caa 2320

Thr?Glu?Cys?Ala?Asn?Leu?Leu?Leu?Gln?Tyr?Gly?Ser?Phe?Cys?Thr?Gln

730 735 740

cta?aat?cgt?gca?ctc?tca?ggt?att?gct?gct?gaa?cag?gat?cgc?aac?aca 2368

Leu?Asn?Arg?Ala?Leu?Ser?Gly?Ile?Ala?Ala?Glu?Gln?Asp?Arg?Asn?Thr

745 750 755 760

cgt?gaa?gtg?ttc?gct?caa?gtc?aaa?caa?atg?tac?aaa?acc?cca?act?ttg 2416

Arg?Glu?Val?Phe?Ala?Gln?Val?Lys?Gln?Met?Tyr?Lys?Thr?Pro?Thr?Leu

765 770 775

aaa?tat?ttt?ggt?ggt?ttt?aat?ttt?tca?caa?ata?tta?cct?gac?cct?cta 2464

Lys?Tyr?Phe?Gly?Gly?Phe?Asn?Phe?Ser?Gln?Ile?Leu?Pro?Asp?Pro?Leu

780 785 790

aag?cca?act?aag?agg?tct?ttt?att?gag?gac?ttg?ctc?ttt?aat?aag?gtg 2512

Lys?Pro?Thr?Lys?Arg?Ser?Phe?Ile?Glu?Asp?Leu?Leu?Phe?Asn?Lys?Val

795 800 805

aca?ctc?gct?gat?gct?ggc?ttc?atg?aag?caa?tat?ggc?gaa?tgc?cta?ggt 2560

Thr?Leu?Ala?Asp?Ala?Gly?Phe?Met?Lys?Gln?Tyr?Gly?Glu?Cys?Leu?Gly

810 815 820

gat?att?aat?gct?aga?gat?ctc?att?tgt?gcg?cag?aag?ttc?aat?gga?ctt 2608

Asp?Ile?Asn?Ala?Arg?Asp?Leu?Ile?Cys?Ala?Gln?Lys?Phe?Asn?Gly?Leu

825 830 835 840

aca?gtg?ttg?cca?cct?ctg?ctc?act?gat?gat?atg?att?gct?gcc?tac?act 2656

Thr?Val?Leu?Pro?Pro?Leu?Leu?Thr?Asp?Asp?Met?Ile?Ala?Ala?Tyr?Thr

845 850 855

gct?gct?cta?gtt?agt?ggt?act?gcc?act?gct?gga?tgg?aca?ttt?ggt?gct 2704

Ala?Ala?Leu?Val?Ser?Gly?Thr?Ala?Thr?Ala?Gly?Trp?Thr?Phe?Gly?Ala

860 865 870

ggc?gct?gct?ctt?caa?ata?cct?ttt?gct?atg?caa?atg?gca?tat?agg?ttc 2752

Gly?Ala?Ala?Leu?Gln?Ile?Pro?Phe?Ala?Met?Gln?Met?Ala?Tyr?Arg?Phe

875 880 885

aat?ggc?att?gga?gtt?acc?caa?aat?gtt?ctc?tat?gag?aac?caa?aaa?caa 2800

Asn?Gly?Ile?Gly?Val?Thr?Gln?Asn?Val?Leu?Tyr?Glu?Asn?Gln?Lys?Gln

890 895 900

atc?gcc?aac?caa?ttt?aac?aag?gcg?att?agt?caa?att?caa?gaa?tca?ctt 2848

Ile?Ala?Asn?Gln?Phe?Asn?Lys?Ala?Ile?Ser?Gln?Ile?Gln?Glu?Ser?Leu

905 910 915 920

aca?aca?aca?tca?act?gca?ttg?ggc?aag?ctg?caa?gac?gtt?gtt?aac?cag 2896

Thr?Thr?Thr?Ser?Thr?Ala?Leu?Gly?Lys?Leu?Gln?Asp?Val?Val?Asn?Gln

925 930 935

aat?gct?caa?gca?tta?aac?aca?ctt?gtt?aaa?caa?ctt?agc?tct?aat?ttt 2944

Asn?Ala?Gln?Ala?Leu?Asn?Thr?Leu?Val?Lys?Gln?Leu?Ser?Ser?Asn?Phe

940 945 950

ggt?gca?att?tca?agt?gtg?cta?aat?gat?atc?ctt?tcg?cga?ctt?gat?aaa 2992

Gly?Ala?Ile?Ser?Ser?Val?Leu?Asn?Asp?Ile?Leu?Ser?Arg?Leu?Asp?Lys

955 960 965

gtc?gag?gcg?gag?gta?caa?att?gac?agg?tta?att?aca?ggc?aga?ctt?caa 3040

Val?Glu?Ala?Glu?Val?Gln?Ile?Asp?Arg?Leu?Ile?Thr?Gly?Arg?Leu?Gln

970 775 980

agc?ctt?caa?acc?tat?gta?aca?caa?caa?cta?atc?agg?gct?gct?gaa?atc 3088

Ser?Leu?Gln?Thr?Tyr?Val?Thr?Gln?Gln?Leu?Ile?Arg?Ala?Ala?Glu?Ile

985 990 995 1000

agg?gct?tct?gct?aat ctt?gct?gct?act?aaa atg?tct?gag?tgt?gtt 3133

Arg?Ala?Ser?Ala?Asn Leu?Ala?Ala?Thr?Lys Met?Ser?Glu?Cys?Val

1005 1010 1015

ctt?gga?caa?tca?aaa aga?gtt?gac?ttt?tgt gga?aag?ggc?tac?cac 3178

Leu?Gly?Gln?Ser?Lys Arg?Val?Asp?Phe?Cys Gly?Lys?Gly?Tyr?His

1020 1025 1030

ctt?atg?tcc?ttc?cca caa?gca?gcc?ccg?cat ggt?gtt?gtc?ttc?cta 3223

Leu?Met?Ser?Phe?Pro Gln?Ala?Ala?Pro?His Gly?Val?Val?Phe?Leu

1035 1040 1045

cat?gtc?acg?tat?gtg cca?tcc?cag?gag?agg aac?ttc?acc?aca?gcg 3268

His?Val?Thr?Tyr?Val Pro?Ser?Gln?Glu?Arg Asn?Phe?Thr?Thr?Ala

1050 1055 1060

cca?gca?att?tgt?cat gaa?ggc?aaa?gca?tac ttc?cct?cgt?gaa?ggt 3313

Pro?Ala?Ile?Cys?His Glu?Gly?Lys?Ala?Tyr Phe?Pro?Arg?Glu?Gly

1065 1070 1075

gtt?ttt?gtg?ttt?aat ggc?act?tct?tgg?ttt att?aca?cag?agg?aac 3358

Val?Phe?Val?Phe?Asn Gly?Thr?Ser?Trp?Phe Ile?Thr?Gln?Arg?Asn

1080 1085 1090

ttc?ttt?tct?cca?caa ata?att?act?aca?gac aat?aca?ttt?gtc?tca 3403

Phe?Phe?Ser?Pro?Gln Ile?Ile?Thr?Thr?Asp Asn?Thr?Phe?Val?Ser

1095 1100 1105

gga?aat?tgt?gat?gtc gtt?att?ggc?atc?att aac?aac?aca?gtt?tat 3448

Gly?Asn?Cys?Asp?Val Val?Ile?Gly?Ile?Ile Asn?Asn?Thr?Val?Tyr

1110 1115 1120

gat?cct?ctg?caa?cct gag?ctt?gac?tca?ttc aaa?gaa?gag?ctg?gac 3493

Asp?Pro?Leu?Gln?Pro Glu?Leu?Asp?Ser?Phe Lys?Glu?Glu?Leu?Asp

1125 1130 1135

aag?tac?ttc?aaa?aat cat?aca?tca?cca?gat gtt?gat?ctt?ggc?gac 3538

Lys?Tyr?Phe?Lys?Asn His?Thr?Ser?Pro?Asp Val?Asp?Leu?Gly?Asp

1140 1145 1150

att?tca?ggc?att?aac gct?tct?gtc?gtc?aac att?caa?aaa?gaa?att 3583

Ile?Ser?Gly?Ile?Asn Ala?Ser?Val?Val?Asn Ile?Gln?Lys?Glu?Ile

1155 1160 1165

gac?cgc?ctc?aat?gag gtc?gct?aaa?aat?tta aat?gaa?tca?ctc?att 3628

Asp?Arg?Leu?Asn?Glu Val?Ala?Lys?Asn?Leu Asn?Glu?Ser?Leu?Ile

1170 1175 1180

gac?ctt?caa?gaa?ttg gga?aaa?tat?gag?caa tat?att?aaa?tgg?cct 3673

Asp?Leu?Gln?Glu?Leu Gly?Lys?Tyr?Glu?Gln Tyr?Ile?Lys?Trp?Pro

1185 1190 1195

tgg?tat?gtt?tgg?ctc ggc?ttc?att?gct?gga cta?att?gcc?atc?gtc 3718

Trp?Tyr?Val?Trp?Leu Gly?Phe?Ile?Ala?Gly Leu?Ile?Ala?Ile?Val

1200 1205 1210

atg?gtt?aca?atc?ttg ctt?tgt?tgc?atg?act agt?tgt?tgc?agt?tgc 3763

Met?Val?Thr?Ile?Leu Leu?Cys?Cys?Met?Thr Ser?Cys?Cys?Ser?Cys

1215 1220 1225

ctc?aag?ggt?gca?tgc tct?tgt?ggt?tct?tgc tgc?aag?ttt?gat?gag 3808

Leu?Lys?Gly?Ala?Cys Ser?Cys?Gly?Ser?Cys Cys?Lys?Phe?Asp?Glu

1230 1235 1240

gat?gac?tct?gag?cca gtt?ctc?aag?ggt?gtc aaa?tta?cat?tac?aca 3853

Asp?Asp?Ser?Glu?Pro Val?Leu?Lys?Gly?Val Lys?Leu?His?Tyr?Thr

1245 1250 1255

taaacgaact?tatggatttg?tttatgagat?tttttactct?tggatcaatt?actgcacagc 3913

cagtaaaaat?tgacaatgct?tctcctgcaa?gt 3945

<210>3

<211>1255

<212>PRT

＜213〉coronavirus

<400>3

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?Gly?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?Ile?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>4

<211>3943

<212>DNA

＜213〉coronavirus

<400>4

ctcttctgga?aaaaggtagg?cttatcatta?gagaaaacaa?cagagttgtg?gtttcaagtg 60

atattcttgt?taacaactaa?acgaacatgt?ttattttctt?attatttctt?actctcacta 120

gtggtagtga?ccttgaccgg?tgcaccactt?ttgatgatgt?tcaagctcct?aattacactc 180

aacatacttc?atctatgagg?ggggtttact?atcctgatga?aatttttaga?tcagacactc 240

tttatttaac?tcaggattta?tttcttccat?tttattctaa?tgttacaggg?tttcatacta 300

ttaatcatac?gtttggcaac?cctgtcatac?cttttaagga?tggtatttat?tttgctgcca 360

cagagaaatc?aaatgttgtc?cgtggttggg?tttttggttc?taccatgaac?aacaagtcac 420

agtcggtgat?tattattaac?aattctacta?atgttgttat?acgagcatgt?aactttgaat 480

tgtgtgacaa?ccctttcttt?gctgtttcta?aacccatggg?tacacagaca?catactatga 540

tattcgataa?tgcatttaat?tgcactttcg?agtacatatc?tgatgccttt?tcgcttgatg 600

tttcagaaaa?gtcaggtaat?tttaaacact?tacgagagtt?tgtgtttaaa?aataaagatg 660

ggtttctcta?tgtttataag?ggctatcaac?ctatagatgt?agttcgtgat?ctaccttctg 720

gttttaacac?tttgaaacct?atttttaagt?tgcctcttgg?tattaacatt?acaaatttta 780

gagccattct?tacagccttt?tcacctgctc?aagacatttg?gggcacgtca?gctgcagcct 840

attttgttgg?ctatttaaag?ccaactacat?ttatgctcaa?gtatgatgaa?aatggtacaa 900

tcacagatgc?tgttgattgt?tctcaaaatc?cacttgctga?actcaaatgc?tctgttaaga 960

gctttgagat?tgacaaagga?atttaccaga?cctctaattt?cagggttgtt?ccctcaggag 1020

atgttgtgag?attccctaat?attacaaact?tgtgtccttt?tggagaggtt?tttaatgcta 1080

ctaaattccc?ttctgtctat?gcatgggaga?gaaaaaaaat?ttctaattgt?gttgctgatt 1140

actctgtgct?ctacaactca?acattttttt?caacctttaa?gtgctatggc?gtttctgcca 1200

ctaagttgaa?tgatctttgc?ttctccaatg?tctatgcaga?ttcttttgta?gtcaagggag 1260

atgatgtaag?acaaatagcg?ccaggacaaa?ctggtgttat?tgctgattat?aattataaat 1320

tgccagatga?tttcatgggt?tgtgtccttg?cttggaatac?taggaacatt?gatgctactt 1380

caactggtaa?ttataattat?aaatataggt?atcttagaca?tggcaagctt?aggccctttg 1440

agagagacat?atctaatgtg?cctttctccc?ctgatggcaa?accttgcacc?ccacctgctc 1500

ttaattgtta?ttggccatta?aatgattatg?gtttttacac?cactactggc?attggctacc 1560

aaccttacag?agttgtagta?ctttcttttg?aacttttaaa?tgcaccggcc?acggtttgtg 1620

gaccaaaatt?atccactgac?cttattaaga?accagtgtgt?caattttaat?tttaatggac 1680

tcactggtac?tggtgtgtta?actccttctt?caaagagatt?tcaaccattt?caacaatttg 1740

gccgtgatgt?ctctgatttc?actgattccg?ttcgagatcc?taaaacatct?gaaatattag 1800

acatttcacc?ttgctctttt?gggggtgtaa?gtgtaattac?acctggaaca?aatgcttcat 1860

ctgaagttgc?tgttctatat?caagatgtta?actgcactga?tgtttctaca?gcaatccatg 1920

cagatcaact?cacaccagct?tggcgcatat?attctactgg?aaacaatgta?ttccagactc 1980

aagcaggctg?tcttatagga?gctgagcatg?tcgacacttc?ttatgagtgc?gacattccta 2040

ttggagctgg?catttgtgct?agttaccata?cagtttcttt?attacgtagt?actagccaaa 2100

aatctattgt?ggcttatact?atgtctttag?gtgctgatag?ttcaattgct?tactctaata 2160

acaccattgc?tatacctact?aacttttcaa?ttagcattac?tacagaagta?atgcctgttt 2220

ctatggctaa?aacctccgta?gattgtaata?tgtacatctg?cggagattct?actgaatgtg 2280

ctaatttgct?tctccaatat?ggtagctttt?gcacacaact?aaatcgtgca?ctctcaggta 2340

ttgctgctga?acaggatcgc?aacacacgtg?aagtgttcgc?tcaagtcaaa?caaatgtaca 2400

aaaccccaac?tttgaaatat?tttggtggtt?ttaatttttc?acaaatatta?cctgaccctc 2460

taaagccaac?taagaggtct?tttattgagg?acttgctctt?taataaggtg?acactcgctg 2520

atgctggctt?catgaagcaa?tatggcgaat?gcctaggtga?tattaatgct?agagatctca 2580

tttgtgcgca?gaagttcaat?gggcttacag?tgttgccacc?tctgctcact?gatgatatga 2640

ttgctgccta?cactgctgct?ctagttagtg?gtactgccac?tgctggatgg?acatttggtg 2700

ctggcgctgc?tcttcaaata?ccttttgcta?tgcaaatggc?atataggttc?aatggcattg 2760

gagttaccca?aaatgttctc?tatgagaacc?aaaaacaaat?cgccaaccaa?tttaacaagg 2820

cgattagtca?aattcaagaa?tcacttacaa?caacatcaac?tgcattgggc?aagctgcaag 2880

acgttgttaa?ccagaatgct?caagcattaa?acacacttgt?taaacaactt?agctctaatt 2940

ttggtgcaat?ttcaagtgtg?ctaaatgata?tcctttcgcg?acttgataaa?gtcgaggcgg 3000

aggtacaaat?tgacaggcta?attacaggca?gacttcaaag?ccttcaaacc?tatgtaacac 3060

aacaactaat?cagggctgct?gaaatcaggg?cttctgctaa?tcttgctgct?actaaaatgt 3120

ctgagtgtgt?tcttggacaa?tcaaaaagag?ttgacttttg?tggaaagggc?taccacctta 3180

tgtccttccc?acaagcagcc?ccgcatggtg?ttgtcttcct?acatgtcacg?tatgtgccat 3240

cccaggagag?gaacttcacc?acagcgccag?caatttgtca?tgaaggcaaa?gcatacttcc 3300

ctcgtgaagg?tgtttttgtg?tttaatggca?cttcttggtt?tattacacag?aggaacttct 3360

tttctccaca?aataattact?acagacaata?catttgtctc?aggaaattgt?gatgtcgtta 3420

ttggcatcat?taacaacaca?gtttatgatc?ctctgcaacc?tgagcttgac?tcattcaaag 3480

aagagctgga?caagtacttc?aaaaatcata?catcaccaga?tgttgatctt?ggcgacattt 3540

caggcattaa?cgcttctgtc?gtcaacattc?aaaaagaaat?tgaccgcctc?aatgaggtcg 3600

ctaaaaattt?aaatgaatca?ctcattgacc?ttcaagaatt?gggaaaatat?gagcaatata 3660

ttaaatggcc?ttggtatgtt?tggctcggct?tcattgctgg?actaattgcc?atcgtcatgg 3720

ttacaatctt?gctttgttgc?atgactagtt?gttgcagttg?cctcaagggt?gcatgctctt 3780

gtggttcttg?ctgcaagttt?gatgaggatg?actctgagcc?agttctcaag?ggtgtcaaat 3840

tacattacac?ataaacgaac?ttatggattt?gtttatgaga?ttttttactc?ttggatcaat 3900

tactgcacag?ccagtaaaaa?ttgacaatgc?ttctcctgca?agt 3943

<210>5

<211>2049

<212>DNA

＜213〉coronavirus

<400>5

ctcttctgga?aaaaggtagg?cttatcatta?gagaaaacaa?cagagttgtg?gtttcaagtg 60

atattcttgt?taacaactaa?acgaacatgt?ttattttctt?attatttctt?actctcacta 120

gtggtagtga?ccttgaccgg?tgcaccactt?ttgatgatgt?tcaagctcct?aattacactc 180

aacatacttc?atctatgagg?ggggtttact?atcctgatga?aatttttaga?tcagacactc 240

tttatttaac?tcaggattta?tttcttccat?tttattctaa?tgttacaggg?tttcatacta 300

ttaatcatac?gtttggcaac?cctgtcatac?cttttaagga?tggtatttat?tttgctgcca 360

cagagaaatc?aaatgttgtc?cgtggttggg?tttttggttc?taccatgaac?aacaagtcac 420

agtcggtgat?tattattaac?aattctacta?atgttgttat?acgagcatgt?aactttgaat 480

tgtgtgacaa?ccctttcttt?gctgtttcta?aacccatggg?tacacagaca?catactatga 540

tattcgataa?tgcatttaat?tgcactttcg?agtacatatc?tgatgccttt?tcgcttgatg 600

tttcagaaaa?gtcaggtaat?tttaaacact?tacgagagtt?tgtgtttaaa?aataaagatg 660

ggtttctcta?tgtttataag?ggctatcaac?ctatagatgt?agttcgtgat?ctaccttctg 720

gttttaacac?tttgaaacct?atttttaagt?tgcctcttgg?tattaacatt?acaaatttta 780

gagccattct?tacagccttt?tcacctgctc?aagacatttg?gggcacgtca?gctgcagcct 840

attttgttgg?ctatttaaag?ccaactacat?ttatgctcaa?gtatgatgaa?aatggtacaa 900

tcacagatgc?tgttgattgt?tctcaaaatc?cacttgctga?actcaaatgc?tctgttaaga 960

gctttgagat?tgacaaagga?atttaccaga?cctctaattt?cagggttgtt?ccctcaggag 1020

atgttgtgag?attccctaat?attacaaact?tgtgtccttt?tggagaggtt?tttaatgcta 1080

ctaaattccc?ttctgtctat?gcatgggaga?gaaaaaaaat?ttctaattgt?gttgctgatt 1140

actctgtgct?ctacaactca?acattttttt?caacctttaa?gtgctatggc?gtttctgcca 1200

ctaagttgaa?tgatctttgc?ttctccaatg?tctatgcaga?ttcttttgta?gtcaagggag 1260

atgatgtaag?acaaatagcg?ccaggacaaa?ctggtgttat?tgctgattat?aattataaat 1320

tgccagatga?tttcatgggt?tgtgtccttg?cttggaatac?taggaacatt?gatgctactt 1380

caactggtaa?ttataattat?aaatataggt?atcttagaca?tggcaagctt?aggccctttg 1440

agagagacat?atctaatgtg?cctttctccc?ctgatggcaa?accttgcacc?ccacctgctc 1500

ttaattgtta?ttggccatta?aatgattatg?gtttttacac?cactactggc?attggctacc 1560

aaccttacag?agttgtagta?ctttcttttg?aacttttaaa?tgcaccggcc?acggtttgtg 1620

gaccaaaatt?atccactgac?cttattaaga?accagtgtgt?caattttaat?tttaatggac 1680

tcactggtac?tggtgtgtta?actccttctt?caaagagatt?tcaaccattt?caacaatttg 1740

gccgtgatgt?ctctgatttc?actgattccg?ttcgagatcc?taaaacatct?gaaatattag 1800

acatttcacc?ttgctctttt?gggggtgtaa?gtgtaattac?acctggaaca?aatgcttcat 1860

ctgaagttgc?tgttctatat?caagatgtta?actgcactga?tgtttctaca?gcaatccatg 1920

cagatcaact?cacaccagct?tggcgcatat?attctactgg?aaacaatgta?ttccagactc 1980

aagcaggctg?tcttatagga?gctgagcatg?tcgacacttc?ttatgagtgc?gacattccta 2040

ttggagctg 2049

<210>6

<211>2027

<212>DNA

＜213〉coronavirus

<400>6

catgcagatc?aactcacacc?agcttggcgc?atatattcta?ctggaaacaa?tgtattccag 60

actcaagcag?gctgtcttat?aggagctgag?catgtcgaca?cttcttatga?gtgcgacatt 120

cctattggag?ctggcatttg?tgctagttac?catacagttt?ctttattacg?tagtactagc 180

caaaaatcta?ttgtggctta?tactatgtct?ttaggtgctg?atagttcaat?tgcttactct 240

aataacacca?ttgctatacc?tactaacttt?tcaattagca?ttactacaga?agtaatgcct 300

gtttctatgg?ctaaaacctc?cgtagattgt?aatatgtaca?tctgcggaga?ttctactgaa 360

tgtgctaatt?tgcttctcca?atatggtagc?ttttgcacac?aactaaatcg?tgcactctca 420

ggtattgctg?ctgaacagga?tcgcaacaca?cgtgaagtgt?tcgctcaagt?caaacaaatg 480

tacaaaaccc?caactttgaa?atattttggt?ggttttaatt?tttcacaaat?attacctgac 540

cctctaaagc?caactaagag?gtcttttatt?gaggacttgc?tctttaataa?ggtgacactc 600

gctgatgctg?gcttcatgaa?gcaatatggc?gaatgcctag?gtgatattaa?tgctagagat 660

ctcatttgtg?cgcagaagtt?caatgggctt?acagtgttgc?cacctctgct?cactgatgat 720

atgattgctg?cctacactgc?tgctctagtt?agtggtactg?ccactgctgg?atggacattt 780

ggtgctggcg?ctgctcttca?aatacctttt?gctatgcaaa?tggcatatag?gttcaatggc 840

attggagtta?cccaaaatgt?tctctatgag?aaccaaaaac?aaatcgccaa?ccaatttaac 900

aaggcgatta?gtcaaattca?agaatcactt?acaacaacat?caactgcatt?gggcaagctg 960

caagacgttg?ttaaccagaa?tgctcaagca?ttaaacacac?ttgttaaaca?acttagctct 1020

aattttggtg?caatttcaag?tgtgctaaat?gatatccttt?cgcgacttga?taaagtcgag 1080

gcggaggtac?aaattgacag?gttaattaca?ggcagacttc?aaagccttca?aacctatgta 1140

acacaacaac?taatcagggc?tgctgaaatc?agggcttctg?ctaatcttgc?tgctactaaa 1200

atgtctgagt?gtgttcttgg?acaatcaaaa?agagttgact?tttgtggaaa?gggctaccac 1260

cttatgtcct?tcccacaagc?agccccgcat?ggtgttgtct?tcctacatgt?cacgtatgtg 1320

ccatcccagg?agaggaactt?caccacagcg?ccagcaattt?gtcatgaagg?caaagcatac 1380

ttccctcgtg?aaggtgtttt?tgtgtttaat?ggcacttctt?ggtttattac?acagaggaac 1440

ttcttttctc?cacaaataat?tactacagac?aatacatttg?tctcaggaaa?ttgtgatgtc 1500

gttattggcg?tcattaacaa?cacagtttat?gatcctctgc?aacctgagct?tgactcattc 1560

aaagaagagc?tggacaagta?cttcaaaaat?catacatcac?cagatgttga?tcttggcgac 1620

atttcaggca?ttaacgcttc?tgtcgtcaac?attcaaaaag?aaattgaccg?cctcaatgag 1680

gtcgctaaaa?atttaaatga?atcactcatt?gaccttcaag?aattgggaaa?atatgagcaa 1740

tatattaaat?ggccttggta?tgtttggctc?ggcttcattg?ctggactaat?tgccatcgtc 1800

atggttacaa?tcttgctttg?ttgcatgact?agttgttgca?gttgcctcaa?gggtgcatgc 1860

tcttgtggtt?cttgctgcaa?gtttgatgag?gatgactctg?agccagttct?caagggtgtc 1920

aaattacatt?acacataaac?gaacttatgg?atttgtttat?gagatttttt?actcttggat 1980

caattactgc?acagccagta?aaaattgaca?atgcttctcc?tgcaagt 2027

<210>7

<211>1096

<212>DNA

＜213〉coronavirus

<400>7

tcttgctttg?ttgcatgact?agttgttgca?gttgcctcaa?gggtgcatgc?tcttgtggtt 60

cttgctgcaa?gtttgatgag?gatgactctg?agccagttct?caagggtgtc?aaattacatt 120

acacataaac?gaacttatgg?atttgtttat?gagatttttt?actcttggat?caattactgc 180

acagccagta?aaaattgaca?atgcttctcc?tgcaagtact?gttcatgcta?cagcaacgat 240

accgctacaa?gcctcactcc?ctttcggatg?gcttgttatt?ggcgttgcat?ttcttgctgt 300

ttttcagagc?gctaccaaaa?taattgcgct?caataaaaga?tggcagctag?ccctttataa 360

gggcttccag?ttcatttgca?atttactgct?gctatttgtt?accatctatt?cacatctttt 420

gcttgtcgct?gcaggtatgg?aggcgcaatt?tttgtacctc?tatgccttga?tatattttct 480

acaatgcatc?aacgcatgta?gaattattat?gagatgttgg?ctttgttgga?agtgcaaatc 540

caagaaccca?ttactttatg?atgccaacta?ctttgtttgc?tggcacacac?ataactatga 600

ctactgtata?ccatataaca?gtgtcacaga?tacaattgtc?gttactgaag?gtgacggcat 660

ttcaacacca?aaactcaaag?aagactacca?aattggtggt?tattctgagg?ataggcactc 720

aggtgttaaa?gactatgtcg?ttgtacatgg?ctatttcacc?gaagtttact?accagcttga 780

gtctacacaa?attactacag?acactggtat?tgaaaatgct?acattcttca?tctttaacaa 840

gcttgttaaa?gacccaccga?atgtgcaaat?acacacaatc?gacggctctt?caggagttgc 900

taatccagca?atggatccaa?tttatgatga?gccgacgacg?actactagcg?tgcctttgta 960

agcacaagaa?agtgagtacg?aacttatgta?ctcattcgtt?tcggaagaaa?caggtacgtt 1020

aatagttaat?agcgtacttc?tttttcttgc?tttcgtggta?ttcttgctag?tcacactagc 1080

catccttact?gcgctt 1096

<210>8

<211>1135

<212>DNA

＜213〉coronavirus

<400>8

attgccatcg?tcatggttac?aatcttgctt?tgttgcatga?ctagttgttg?cagttgcctc 60

aagggtgcat?gctcttgtgg?ttcttgctgc?aagtttgatg?aggatgactc?tgagccagtt 120

ctcaagggtg?tcaaattaca?ttacacataa?acgaacttat?ggatttgttt?atgagatttt 180

ttactcttgg?atcaattact?gcacagccag?taaaaattga?caatgcttct?cctgcaagta 240

ctgttcatgc?tacagcaacg?ataccgctac?aagcctcact?ccctttcgga?tggcttgtta 300

ttggcgttgc?atttcttgct?gtttttcaga?gcgctaccaa?aataattgcg?ctcaataaaa 360

gatggcagct?agccctttat?aagggcttcc?agttcatttg?caatttactg?ctgctatttg 420

ttaccatcta?ttcacatctt?ttgcttgtcg?ctgcaggtat?ggaggcgcaa?tttttgtacc 480

tctatgcctt?gatatatttt?ctacaatgca?tcaacgcatg?tagaattatt?atgagatgtt 540

ggctttgttg?gaagtgcaaa?tccaagaacc?cattacttta?tgatgccaac?tactttgttt 600

gctggcacac?acataactat?gactactgta?taccatataa?cagtgtcaca?gatacaattg 660

tcgttactga?aggtgacggc?atttcaacac?caaaactcaa?agaagactac?caaattggtg 720

gttattctga?ggataggcac?tcaggtgtta?aagactatgt?cgttgtacat?ggctatttca 780

ccgaagttta?ctaccagctt?gagtctacac?aaattactac?agacactggt?attgaaaatg 840

ctacattctt?catctttaac?aagcttgtta?aagacccacc?gaatgtgcaa?atacacacaa 900

tcgacggctc?ttcaggagtt?gctaatccag?caatggatcc?aatttatgat?gagccgacga 960

cgactactag?cgtgcctttg?taagcacaag?aaagtgagta?cgaacttatg?tactcattcg 1020

tttcggaaga?aacaggtacg?ttaatagtta?atagcgtact?tctttttctt?gctttcgtgg 1080

tattcttgct?agtcacacta?gccatcctta?ctgcgcttcg?attgtgtgcg?tactg 1135

<210>9

<211>1096

<212>DNA

＜213〉coronavirus

<220>

<221>CDS

<222>(137)..(958)

<223>

<400>9

tcttgctttg?ttgcatgact?agttgttgca?gttgcctcaa?gggtgcatgc?tcttgtggtt 60

cttgctgcaa?gtttgatgag?gatgactctg?agccagttct?caagggtgtc?aaattacatt 120

acacataaac?gaactt?atg?gat?ttg?ttt?atg?aga?ttt?ttt?act?ctt?gga?tca 172

Met?Asp?Leu?Phe?Met?Arg?Phe?Phe?Thr?Leu?Gly?Ser

1 5 10

att?act?gca?cag?cca?gta?aaa?att?gac?aat?gct?tct?cct?gca?agt?act 220

Ile?Thr?Ala?Gln?Pro?Val?Lys?Ile?Asp?Asn?Ala?Ser?Pro?Ala?Ser?Thr

15 20 25

gtt?cat?gct?aca?gca?acg?ata?ccg?cta?caa?gcc?tca?ctc?cct?ttc?gga 268

Val?His?Ala?Thr?Ala?Thr?Ile?Pro?Leu?Gln?Ala?Ser?Leu?Pro?Phe?Gly

30 35 40

tgg?ctt?gtt?att?ggc?gtt?gca?ttt?ctt?gct?gtt?ttt?cag?agc?gct?acc 316

Trp?Leu?Val?Ile?Gly?Val?Ala?Phe?Leu?Ala?Val?Phe?Gln?Ser?Ala?Thr

45 50 55 60

aaa?ata?att?gcg?ctc?aat?aaa?aga?tgg?cag?cta?gcc?ctt?tat?aag?ggc 364

Lys?Ile?Ile?Ala?Leu?Asn?Lys?Arg?Trp?Gln?Leu?Ala?Leu?Tyr?Lys?Gly

65 70 75

ttc?cag?ttc?att?tgc?aat?tta?ctg?ctg?cta?ttt?gtt?acc?atc?tat?tca 412

Phe?Gln?Phe?Ile?Cys?Asn?Leu?Leu?Leu?Leu?Phe?Val?Thr?Ile?Tyr?Ser

80 85 90

cat?ctt?ttg?ctt?gtc?gct?gca?ggt?atg?gag?gcg?caa?ttt?ttg?tac?ctc 460

His?Leu?Leu?Leu?Val?Ala?Ala?Gly?Met?Glu?Ala?Gln?Phe?Leu?Tyr?Leu

95 100 105

tat?gcc?ttg?ata?tat?ttt?cta?caa?tgc?atc?aac?gca?tgt?aga?att?att 508

Tyr?Ala?Leu?Ile?Tyr?Phe?Leu?Gln?Cys?Ile?Asn?Ala?Cys?Arg?Ile?Ile

110 115 120

atg?aga?tgt?tgg?ctt?tgt?tgg?aag?tgc?aaa?tcc?aag?aac?cca?tta?ctt 556

Met?Arg?Cys?Trp?Leu?Cys?Trp?Lys?Cys?Lys?Ser?Lys?Asn?Pro?Leu?Leu

125 130 135 140

tat?gat?gcc?aac?tac?ttt?gtt?tgc?tgg?cac?aca?cat?aac?tat?gac?tac 604

Tyr?Asp?Ala?Asn?Tyr?Phe?Val?Cys?Trp?His?Thr?His?Asn?Tyr?Asp?Tyr

145 150 155

tgt?ata?cca?tat?aac?agt?gtc?aca?gat?aca?att?gtc?gtt?act?gaa?ggt 652

Cys?Ile?Pro?Tyr?Asn?Ser?Val?Thr?Asp?Thr?Ile?Val?Val?Thr?Glu?Gly

160 165 170

gac?ggc?att?tca?aca?cca?aaa?ctc?aaa?gaa?gac?tac?caa?att?ggt?ggt 700

Asp?Gly?Ile?Ser?Thr?Pro?Lys?Leu?Lys?Glu?Asp?Tyr?Gln?Ile?Gly?Gly

175 180 185

tat?tct?gag?gat?agg?cac?tca?ggt?gtt?aaa?gac?tat?gtc?gtt?gta?cat 748

Tyr?Ser?Glu?Asp?Arg?His?Ser?Gly?Val?Lys?Asp?Tyr?Val?Val?Val?His

190 195 200

ggc?tat?ttc?acc?gaa?gtt?tac?tac?cag?ctt?gag?tct?aca?caa?att?act 796

Gly?Tyr?Phe?Thr?Glu?Val?Tyr?Tyr?Gln?Leu?Glu?Ser?Thr?Gln?Ile?Thr

205 210 215 220

aca?gac?act?ggt?att?gaa?aat?gct?aca?ttc?ttc?atc?ttt?aac?aag?ctt 844

Thr?Asp?Thr?Gly?Ile?Glu?Asn?Ala?Thr?Phe?Phe?Ile?Phe?Asn?Lys?Leu

225 230 235

gvt?aaa?gac?cca?ccg?aat?gtg?caa?ata?cac?aca?atc?gac?ggc?tct?tca 892

Val?Lys?Asp?Pro?Pro?Asn?Val?Gln?Ile?His?Thr?Ile?Asp?Gly?Ser?Ser

240 245 250

gga?gtt?gct?aat?cca?gca?atg?gat?cca?att?tat?gat?gag?ccg?acg?acg 940

Gly?Val?Ala?Asn?Pro?Ala?Met?Asp?Pro?Ile?Tyr?Asp?Glu?Pro?Thr?Thr

255 260 265

act?act?agc?gtg?cct?ttg?taagcacaag?aaagtgagta?cgaacttatg 988

Thr?Thr?Ser?Val?Pro?Leu

270

tactcattcg?tttcggaaga?aacaggtacg?ttaatagtta?atagcgtact?tctttttctt?1048

gctttcgtgg?tattcttgct?agtcacacta?gccatcctta?ctgcgctt 1096

<210>10

<211>274

<212>PRT

＜213〉coronavirus

<400>10

Met?Asp?Leu?Phe?Met?Arg?Phe?Phe?Thr?Leu?Gly?Ser?Ile?Thr?Ala?Gln

1 5 10 15

Pro?Val?Lys?Ile?Asp?Asn?Ala?Ser?Pro?Ala?Ser?Thr?Val?His?Ala?Thr

20 25 30

Ala?Thr?Ile?Pro?Leu?Gln?Ala?Ser?Leu?Pro?Phe?Gly?Trp?Leu?Val?Ile

35 40 45

Gly?Val?Ala?Phe?Leu?Ala?Val?Phe?Gln?Ser?Ala?Thr?Lys?Ile?Ile?Ala

50 55 60

Leu?Asn?Lys?Arg?Trp?Gln?Leu?Ala?Leu?Tyr?Lys?Gly?Phe?Gln?Phe?Ile

65 70 75 80

Cys?Asn?Leu?Leu?Leu?Leu?Phe?Val?Thr?Ile?Tyr?Ser?His?Leu?Leu?Leu

85 90 95

Val?Ala?Ala?Gly?Met?Glu?Ala?Gln?Phe?Leu?Tyr?Leu?Tyr?Ala?Leu?Ile

100 105 110

Tyr?Phe?Leu?Gln?Cys?Ile?Asn?Ala?Cys?Arg?Ile?Ile?Met?Arg?Cys?Trp

115 120 125

Leu?Cys?Trp?Lys?Cys?Lys?Ser?Lys?Asn?Pro?Leu?Leu?Tyr?Asp?Ala?Asn

130 135 140

Tyr?Phe?Val?Cys?Trp?His?Thr?His?Asn?Tyr?Asp?Tyr?Cys?Ile?Pro?Tyr

145 150 155 160

Asn?Ser?Val?Thr?Asp?Thr?Ile?Val?Val?Thr?Glu?Gly?Asp?Gly?Ile?Ser

165 170 175

Thr?Pro?Lys?Leu?Lys?Glu?Asp?Tyr?Gln?Ile?Gly?Gly?Tyr?Ser?Glu?Asp

180 185 190

Arg?His?Ser?Gly?Val?Lys?Asp?Tyr?Val?Val?Val?His?Gly?Tyr?Phe?Thr

195 200 205

Glu?Val?Tyr?Tyr?Gln?Leu?Glu?Ser?Thr?Gln?Ile?Thr?Thr?Asp?Thr?Gly

210 215 220

Ile?Glu?Asn?Ala?Thr?Phe?Phe?Ile?Phe?Asn?Lys?Leu?Val?Lys?Asp?Pro

225 230 235 240

Pro?Asn?Val?Gln?Ile?His?Thr?Ile?Asp?Gly?Ser?Ser?Gly?Val?Ala?Asn

245 250 255

Pro?Ala?Met?Asp?Pro?Ile?Tyr?Asp?Glu?Pro?Thr?Thr?Thr?Thr?Ser?Val

260 265 270

Pro?Leu

<210>11

<211>1096

<212>DNA

＜213〉coronavirus

<220>

<221>CDS

<222>(558)..(1019)

<223>

<400>11

tcttgctttg?ttgcatgact?agttgttgca?gttgcctcaa?gggtgcatgc?tcttgtggtt 60

cttgctgcaa?gtttgatgag?gatgactctg?agccagttct?caagggtgtc?aaattacatt 120

acacataaac?gaacttatgg?atttgtttat?gagatttttt?actcttggat?caattactgc 180

acagccagta?aaaattgaca?atgcttctcc?tgcaagtact?gttcatgcta?cagcaacgat 240

accgctacaa?gcctcactcc?ctttcggatg?gcttgttatt?ggcgttgcat?ttcttgctgt 300

ttttcagagc?gctaccaaaa?taattgcgct?caataaaaga?tggcagctag?ccctttataa 360

gggcttccag?ttcatttgca?atttactgct?gctatttgtt?accatctatt?cacatctttt 420

gcttgtcgct?gcaggtatgg?aggcgcaatt?tttgtacctc?tatgccttga?tatattttct 480

acaatgcatc?aacgcatgta?gaattattat?gagatgttgg?ctttgttgga?agtgcaaatc 540

caagaaccca?ttacttt?atg?atg?cca?act?act?ttg?ttt?gct?ggc?aca?cac 590

Met?Met?Pro?Thr?Thr?Leu?Phe?Ala?Gly?Thr?His

1 5 10

ata?act?atg?act?act?gta?tac?cat?ata?aca?gtg?tca?cag?ata?caa?ttg 638

Ile?Thr?Met?Thr?Thr?Val?Tyr?His?Ile?Thr?Val?Ser?Gln?Ile?Gln?Leu

15 20 25

tcg?tta?ctg?aag?gtg?acg?gca?ttt?caa?cac?caa?aac?tca?aag?aag?act 686

Ser?Leu?Leu?Lys?Val?Thr?Ala?Phe?Gln?His?Gln?Asn?Ser?Lys?Lys?Thr

30 35 40

acc?aaa?ttg?gtg?gtt?att?ctg?agg?ata?ggc?act?cag?gtg?tta?aag?act 734

Thr?Lys?Leu?Val?Val?Ile?Leu?Arg?Ile?Gly?Thr?Gln?Val?Leu?Lys?Thr

45 50 55

atg?tcg?ttg?tac?atg?gct?att?tca?ccg?aag?ttt?act?acc?agc?ttg?agt 782

Met?Ser?Leu?Tyr?Met?Ala?Ile?Ser?Pro?Lys?Phe?Thr?Thr?Ser?Leu?Ser

60 65 70 75

cta?cac?aaa?tta?cta?cag?aca?ctg?gta?ttg?aaa?atg?cta?cat?tct?tca 830

Leu?His?Lys?Leu?Leu?Gln?Thr?Leu?Val?Leu?Lys?Met?Leu?His?Ser?Ser

80 85 90

tct?tta?aca?agc?ttg?tta?aag?acc?cac?cga?atg?tgc?aaa?tac?aca?caa 878

Ser?Leu?Thr?Ser?Leu?Leu?Lys?Thr?His?Arg?Met?Cys?Lys?Tyr?Thr?Gln

95 100 105

tcg?acg?gct?ctt?cag?gag?ttg?cta?atc?cag?caa?tgg?atc?caa?ttt?atg 926

Ser?Thr?Ala?Leu?Gln?Glu?Leu?Leu?Ile?Gln?Gln?Trp?Ile?Gln?Phe?Met

110 115 120

atg?agc?cga?cga?cga?cta?cta?gcg?tgc?ctt?tgt?aag?cac?aag?aaa?gtg 974

Met?Ser?Arg?Arg?Arg?Leu?Leu?Ala?Cys?Leu?Cys?Lys?His?Lys?Lys?Val

125 130 135

agt?acg?aac?tta?tgt?act?cat?tcg?ttt?cgg?aag?aaa?cag?gta?cgt 1019

Ser?Thr?Asn?Leu?Cys?Thr?His?Ser?Phe?Arg?Lys?Lys?Gln?Val?Arg

140 145 150

taatagttaa?tagcgtactt?ctttttcttg?ctttcgtggt?attcttgcta?gtcacactag 1079

ccatccttac?tgcgctt 1096

<210>12

<211>154

<212>PRT

＜213〉coronavirus

<400>12

Met?Met?Pro?Thr?Thr?Leu?Phe?Ala?Gly?Thr?His?Ile?Thr?Met?Thr?Thr

1 5 10 15

Val?Tyr?His?Ile?Thr?Val?Ser?Gln?Ile?Gln?Leu?Ser?Leu?Leu?Lys?Val

20 25 30

Thr?Ala?Phe?Gln?His?Gln?Asn?Ser?Lys?Lys?Thr?Thr?Lys?Leu?Val?Val

35 40 45

Ile?Leu?Arg?Ile?Gly?Thr?Gln?Val?Leu?Lys?Thr?Met?Ser?Leu?Tyr?Met

50 55 60

Ala?Ile?Ser?Pro?Lys?Phe?Thr?Thr?Ser?Leu?Ser?Leu?His?Lys?Leu?Leu

65 70 75 80

Gln?Thr?Leu?Val?Leu?Lys?Met?Leu?His?Ser?Ser?Ser?Leu?Thr?Ser?Leu

85 90 95

Leu?Lys?Thr?His?Arg?Met?Cys?Lys?Tyr?Thr?Gln?Ser?Thr?Ala?Leu?Gln

100 105 110

Glu?Leu?Leu?Ile?Gln?Gln?Trp?Ile?Gln?Phe?Met?Met?Ser?Arg?Arg?Arg

115 120 125

Leu?Leu?Ala?Cys?Leu?Cys?Lys?His?Lys?Lys?Val?Ser?Thr?Asn?Leu?Cys

130 135 140

Thr?His?Ser?Phe?Arg?Lys?Lys?Gln?Val?Arg

145 150

<210>13

<211>332

<212>DNA

＜213〉coronavirus

<220>

<221>CDS

<222>(36)..(263)

<223>

<400>13

tgcctttgta?agcacaagaa?agtgagtacg?aactt?atg?tac?tca?ttc?gtt?tcg 53

Met?Tyr?Ser?Phe?Val?Ser

1 5

gaa?gaa?aca?ggt?acg?tta?ata?gtt?aat?agc?gta?ctt?ctt?ttt?ctt?gct 101

Glu?Glu?Thr?Gly?Thr?Leu?Ile?Val?Asn?Ser?Val?Leu?Leu?Phe?Leu?Ala

10 15 20

ttc?gtg?gta?ttc?ttg?cta?gtc?aca?cta?gcc?atc?ctt?act?gcg?ctt?cga 149

Phe?Val?Val?Phe?Leu?Leu?Val?Thr?Leu?Ala?Ile?Leu?Thr?Ala?Leu?Arg

25 30 35

ttg?tgt?gcg?tac?tgc?tgc?aat?att?gtt?aac?gtg?agt?tta?gta?aaa?cca 197

Leu?Cys?Ala?Tyr?Cys?Cys?Asn?Ile?Val?Asn?Val?Ser?Leu?Val?Lys?Pro

40 45 50

acg?gtt?tac?gtc?tac?tcg?cgt?gtt?aaa?aat?ctg?aac?tct?tct?gaa?gga 245

Thr?Val?Tyr?Val?Tyr?Ser?Arg?Val?Lys?Asn?Leu?Asn?Ser?Ser?Glu?Gly

55 60 65 70

gtt?cct?gat?ctt?ctg?gtc?taaacgaact?aactattatt?attattctgt 293

Val?Pro?Asp?Leu?Leu?Val

75

ttggaacttt?aacattgctt?atcatggcag?acaacggta 332

<210>14

<211>76

<212>PRT

＜213〉coronavirus

<400>14

Met?Tyr?Ser?Phe?Val?Ser?Glu?Glu?Thr?Gly?Thr?Leu?Ile?Val?Asn?Ser

1 5 10 15

Val?Leu?Leu?Phe?Leu?Ala?Phe?Val?Val?Phe?Leu?Leu?Val?Thr?Leu?Ala

20 25 30

Ile?Leu?Thr?Ala?Leu?Arg?Leu?Cys?Ala?Tyr?Cys?Cys?Asn?Ile?Val?Asn

35 40 45

Val?Ser?Leu?Val?Lys?Pro?Thr?Val?Tyr?Val?Tyr?Ser?Arg?Val?Lys?Asn

50 55 60

Leu?Asn?Ser?Ser?Glu?Gly?Val?Pro?Asp?Leu?Leu?Val

65 70 75

<210>15

<211>332

<212>DNA

＜213〉coronavirus

<400>15

tgcctttgta?agcacaagaa?agtgagtacg?aacttatgta?ctcattcgtt?tcggaagaaa 60

caggtacgtt?aatagttaat?agcgtacttc?tttttcttgc?tttcgtggta?ttcttgctag 120

tcacactagc?catccttact?gcgcttcgat?tgtgtgcgta?ctgctgcaat?attgttaacg 180

tgagtttagt?aaaaccaacg?gtttacgtct?actcgcgtgt?taaaaatctg?aactcttctg 240

aaggagttcc?tgatcttctg?gtctaaacga?actaactatt?attattattc?tgtttggaac 300

tttaacattg?cttatcatgg?cagacaacgg?ta 332

<210>16

<211>708

<212>DNA

＜213〉coronavirus

<220>

<221>CDS

<222>(41)..(703)

<223>

<400>16

tattattatt?attctgtttg?gaactttaac?attgcttatc?atg?gca?gac?aac?ggt 55

Met?Ala?Asp?Asn?Gly

1 5

act?att?acc?gtt?gag?gag?ctt?aaa?caa?ctc?ctg?gaa?caa?tgg?aac?cta 103

Thr?Ile?Thr?Val?Glu?Glu?Leu?Lys?Gln?Leu?Leu?Glu?Gln?Trp?Asn?Leu

10 15 20

gta?ata?ggt?ttc?cta?ttc?cta?gcc?tgg?att?atg?tta?cta?caa?ttt?gcc 151

Val?Ile?Gly?Phe?Leu?Phe?Leu?Ala?Trp?Ile?Met?Leu?Leu?Gln?Phe?Ala

25 30 35

tat?tct?aat?cgg?aac?agg?ttt?ttg?tac?ata?ata?aag?ctt?gtt?ttc?ctc 199

Tyr?Ser?Asn?Arg?Asn?Arg?Phe?Leu?Tyr?Ile?Ile?Lys?Leu?Val?Phe?Leu

40 45 50

tgg?ctc?ttg?tgg?cca?gta?aca?ctt?gct?tgt?ttt?gtg?ctt?gct?gct?gtc 247

Trp?Leu?Leu?Trp?Pro?Val?Thr?Leu?Ala?Cys?Phe?Val?Leu?Ala?Ala?Val

55 60 65

tac?aga?att?aat?tgg?gtg?act?ggc?ggg?att?gcg?att?gca?atg?gct?tgt 295

Tyr?Arg?Ile?Asn?Trp?Val?Thr?Gly?Gly?Ile?Ala?Ile?Ala?Met?Ala?Cys

70 75 80 85

att?gta?ggc?ttg?atg?tgg?ctt?agc?tac?ttc?gtt?gct?tcc?ttc?agg?ctg 343

Ile?Val?Gly?Leu?Met?Trp?Leu?Ser?Tyr?Phe?Val?Ala?Ser?Phe?Arg?Leu

90 95 100

ttt?gct?cgt?acc?cgc?tca?atg?tgg?tca?ttc?aac?cca?gaa?aca?aac?att 391

Phe?Ala?Arg?Thr?Arg?Ser?Met?Trp?Ser?Phe?Asn?Pro?Glu?Thr?Asn?Ile

105 110 115

ctt?ctc?aat?gtg?cct?ctc?cgg?ggg?aca?att?gtg?acc?aga?ccg?ctc?atg 439

Leu?Leu?Asn?Val?Pro?Leu?Arg?Gly?Thr?Ile?Val?Thr?Arg?Pro?Leu?Met

120 125 130

gaa?agt?gaa?ctt?gtc?att?ggt?gct?gtg?atc?att?cgt?ggt?cac?ttg?cga 487

Glu?Ser?Glu?Leu?Val?Ile?Gly?Ala?Val?Ile?Ile?Arg?Gly?His?Leu?Arg

135 140 145

atg?gcc?gga?cac?tcc?cta?ggg?cgc?tgt?gac?att?aag?gac?ctg?cca?aaa 535

Met?Ala?Gly?His?Ser?Leu?Gly?Arg?Cys?Asp?Ile?Lys?Asp?Leu?Pro?Lys

150 155 160 165

gag?atc?act?gtg?gct?aca?tca?cga?acg?ctt?tct?tat?tac?aaa?tta?gga 583

Glu?Ile?Thr?Val?Ala?Thr?Ser?Arg?Thr?Leu?Ser?Tyr?Tyr?Lys?Leu?Gly

170 175 180

gcg?tcg?cag?cgt?gta?ggc?act?gat?tca?ggt?ttt?gct?gca?tac?aac?cgc 631

Ala?Ser?Gln?Arg?Val?Gly?Thr?Asp?Ser?Gly?Phe?Ala?Ala?Tyr?Asn?Arg

185 190 195

tac?cgt?att?gga?aac?tat?aaa?tta?aat?aca?gac?cac?gcc?ggt?agc?aac 679

Tyr?Arg?Ile?Gly?Asn?Tyr?Lys?Leu?Asn?thr?Asp?His?Ala?Gly?Ser?Asn

200 205 210

gac?aat?att?gct?ttg?cta?gta?cag?taagt 708

Asp?Asn?Ile?Ala?Leu?Leu?Val?Gln

215 220

<210>17

<211>221

<212>PRT

＜213〉coronavirus

<400>17

Met?Ala?Asp?Asn?Gly?Thr?Ile?Thr?Val?Glu?Glu?Leu?Lys?Gln?Leu?Leu

1 5 10 15

Glu?Gln?Trp?Asn?Leu?Val?Ile?Gly?Phe?Leu?Phe?Leu?Ala?Trp?Ile?Met

20 25 30

Leu?Leu?Gln?Phe?Ala?Tyr?Ser?Asn?Arg?Asn?Arg?Phe?Leu?Tyr?Ile?Ile

35 40 45

Lys?Leu?Val?Phe?Leu?Trp?Leu?Leu?Trp?Pro?Val?Thr?Leu?Ala?Cys?Phe

50 55 60

Val?Leu?Ala?Ala?Val?Tyr?Arg?Ile?Asn?Trp?Val?Thr?Gly?Gly?Ile?Ala

65 70 75 80

Ile?Ala?Met?Ala?Cys?Ile?Val?Gly?Leu?Met?Trp?Leu?Ser?Tyr?Phe?Val

85 90 95

Ala?Ser?Phe?Arg?Leu?Phe?Ala?Arg?Thr?Arg?Ser?Met?Trp?Ser?Phe?Asn

100 105 110

Pro?Glu?Thr?Asn?Ile?Leu?Leu?Asn?Val?Pro?Leu?Arg?Gly?Thr?Ile?Val

115 120 125

Thr?Arg?Pro?Leu?Met?Glu?Ser?Glu?Leu?Val?Ile?Gly?Ala?Val?Ile?Ile

130 135 140

Arg?Gly?His?Leu?Arg?Met?Ala?Gly?His?Ser?Leu?Gly?Arg?Cys?Asp?Ile

145 150 155 160

Lys?Asp?Leu?Pro?Lys?Glu?Ile?Thr?Val?Ala?Thr?Ser?Arg?Thr?Leu?Ser

165 170 175

Tyr?Tyr?Lys?Leu?Gly?Ala?Ser?Gln?Arg?Val?Gly?Thr?Asp?Ser?Gly?Phe

180 185 190

Ala?Ala?Tyr?Asn?Arg?Tyr?Arg?Ile?Gly?Asn?Tyr?Lys?Leu?Asn?Thr?Asp

195 200 205

His?Ala?Gly?Ser?Asn?Asp?Asn?Ile?Ala?Leu?Leu?Val?Gln

210 215 220

<210>18

<211>769

<212>DNA

＜213〉coronavirus

<400>18

cctgatcttc?tggtctaaac?gaactaacta?ttattattat?tctgtttgga?actttaacat 60

tgcttatcat?ggcagacaac?ggtactatta?ccgttgagga?gcttaaacaa?ctcctggaac 120

aatggaacct?agtaataggt?ttcctattcc?tagcctggat?tatgttacta?caatttgcct 180

attctaatcg?gaacaggttt?ttgtacataa?taaagcttgt?tttcctctgg?ctcttgtggc 240

cagtaacact?tgcttgtttt?gtgcttgctg?ctgtctacag?aattaattgg?gtgactggcg 300

ggattgcgat?tgcaatggct?tgtattgtag?gcttgatgtg?gcttagctac?ttcgttgctt 360

ccttcaggct?gtttgctcgt?acccgctcaa?tgtggtcatt?caacccagaa?acaaacattc 420

ttctcaatgt?gcctctccgg?gggacaattg?tgaccagacc?gctcatggaa?agtgaacttg 480

tcattggtgc?tgtgatcatt?cgtggtcact?tgcgaatggc?cggacactcc?ctagggcgct 540

gtgacattaa?ggacctgcca?aaagagatca?ctgtggctac?atcacgaacg?ctttcttatt 600

acaaattagg?agcgtcgcag?cgtgtaggca?ctgattcagg?ttttgctgca?tacaaccgct 660

accgtattgg?aaactataaa?ttaaatacag?accacgccgg?tagcaacgac?aatattgctt 720

tgctagtaca?gtaagtgaca?acagatgttt?catcttgttg?acttccagg 769

<210>19

<211>1231

<212>DNA

＜213〉coronavirus

<400>19

taccgtattg?gaaactataa?attaaataca?gaccacgccg?gtagcaacga?caatattgct 60

ttgctagtac?agtaagtgac?aacagatgtt?tcatcttgtt?gacttccagg?ttacaatagc 120

agagatattg?attatcatta?tgaggacttt?caggattgct?atttggaatc?ttgacgttat 180

aataagttca?atagtgagac?aattatttaa?gcctctaact?aagaagaatt?attcggagtt 240

agatgatgaa?gaacctatgg?agttagatta?tccataaaac?gaacatgaaa?attattctct 300

tcctgacatt?gattgtattt?acatcttgcg?agctatatca?ctatcaggag?tgtgttagag 360

gtacgactgt?actactaaaa?gaaccttgcc?catcaggaac?atacgagggc?aattcaccat 420

ttcaccctct?tgctgacaat?aaatttgcac?taacttgcac?tagcacacac?tttgcttttg 480

cttgtgctga?cggtactcga?catacctatc?agctgcgtgc?aagatcagtt?tcaccaaaac 540

ttttcatcag?acaagaggag?gttcaacaag?agctctactc?gccacttttt?ctcattgttg 600

ctgctctagt?atttttaata?ctttgcttca?ccattaagag?aaagacagaa?tgaatgagct 660

cactttaatt?gacttctatt?tgtgcttttt?agcctttctg?ctattccttg?ttttaataat 720

gcttattata?ttttggtttt?cactcgaaat?ccaggatcta?gaagaacctt?gtaccaaagt 780

ctaaacgaac?atgaaacttc?tcattgtttt?gacttgtatt?tctctatgca?gttgcatatg 840

cactgtagta?cagcgctgtg?catctaataa?acctcatgtg?cttgaagatc?cttgtaaggt 900

acaacactag?gggtaatact?tatagcactg?cttggctttg?tgctctagga?aaggttttac 960

cttttcatag?atggcacact?atggttcaaa?catgcacacc?taatgttact?atcaactgtc 1020

aagatccagc?tggtggtgcg?cttatagcta?ggtgttggta?ccttcatgaa?ggtcaccaaa 1080

ctgctgcatt?tagagacgta?cttgttgttt?taaataaacg?aacaaattaa?aatgtctgat 1140

aatggacccc?aatcaaacca?acgtagtgcc?ccccgcatta?catttggtgg?acccacagat 1200

tcaactgaca?ataaccagaa?tggaggacgc?a 1231

<210>20

<211>1242

<212>DNA

＜213〉coronavirus

<400>20

gcatacaacc?gctaccgtat?tggaaactat?aaattaaata?cagaccacgc?cggtagcaac 60

gacaatattg?ctttgctagt?acagtaagtg?acaacagatg?tttcatcttg?ttgacttcca 120

ggttacaata?gcagagatat?tgattatcat?tatgaggact?ttcaggattg?ctatttggaa 180

tcttgacgtt?ataataagtt?caatagtgag?acagttattt?aagcctctaa?ctaagaagaa 240

ttattcggag?ttagatgatg?aagaacctat?ggagttagat?tatccataaa?acgaacatga 300

aaattattct?cttcctgaca?ttgattgtat?ttacatcttg?cgagctatat?cactatcagg 360

agtgtgttag?aggtacgact?gtactactaa?aagaaccttg?cccatcagga?acatacgagg 420

gcaattcacc?atttcaccct?cttgctgaca?ataaatttgc?actaacttgc?actagcacac 480

actttgcttt?tgcttgtgct?gacggtactc?gacataccta?tcagctgcgt?gcaagatcag 540

tttcaccaaa?acttttcatc?agacaagagg?aggttcaaca?agagctctac?tcgccacttt 600

ttctcattgt?tgctgctcta?gtatttttaa?tactttgctt?caccattaag?agaaagacag 660

aatgaatgag?ctcactttaa?ttgacttcta?tttgtgcttt?ttagcctttc?tgctattcct 720

tgttttaata?atgcttatta?tattttggtt?ttcactcgaa?atccaggatc?tagaagaacc 780

ttgtaccaaa?gtctaaacga?acatgaaact?tctcattgtt?ttgacttgta?tttctctatg 840

cagttgcata?tgcactgtag?tacagcgctg?tgcatctaat?aaacctcatg?tgcttgaaga 900

tccttgtaag?gtacaacact?aggggtaata?cttatagcac?tgcttggctt?tgtgctctag 960

gaaaggtttt?accttttcat?agatggcaca?ctatggttca?aacatgcaca?cctaatgtta 1020

ctatcaactg?tcaagatcca?gctggtggtg?cgcttatagc?taggtgttgg?taccttcatg 1080

aaggtcacca?aactgctgca?tttagagacg?tacttgttgt?tttaaataaa?cgaacgaatt 1140

aaaatgtctg?ataatggacc?ccaatcaaac?caacgtagtg?ccccccgcat?tacatttggt 1200

ggacccacag?attcaactga?caataaccag?aatggaggac?gc 1242

<210>21

<211>1231

<212>DNA

＜213〉coronavirus

<220>

<221>CDS

<222>(86)..(274)

<223>

<400>21

taccgtattg?gaaactataa?attaaataca?gaccacgccg?gtagcaacga?caatattgct 60

ttgctagtac?agtaagtgac?aacag?atg?ttt?cat?ctt?gtt?gac?ttc?cag?gtt 112

Met?Phe?His?Leu?Val?Asp?Phe?Gln?Val

1 5

aca?ata?gca?gag?ata?ttg?att?atc?att?atg?agg?act?ttc?agg?att?gct 160

Thr?Ile?Ala?Glu?Ile?Leu?Ile?Ile?Ile?Met?Arg?Thr?Phe?Arg?Ile?Ala

10 15 20 25

att?tgg?aat?ctt?gac?gtt?ata?ata?agt?tca?ata?gtg?aga?caa?tta?ttt 208

Ile?Trp?Asn?Leu?Asp?Val?Ile?Ile?Ser?Ser?Ile?Val?Arg?Gln?Leu?Phe

30 35 40

aag?cct?cta?act?aag?aag?aat?tat?tcg?gag?tta?gat?gat?gaa?gaa?cct 256

Lys?Pro?Leu?Thr?Lys?Lys?Asn?Tyr?Ser?Glu?Leu?Asp?Asp?Glu?Glu?Pro

45 50 55

atg?gag?tta?gat?tat?cca?taaaacgaac?atgaaaatta?ttctcttcct 304

Met?Glu?Leu?Asp?Tyr?Pro

60

gacattgatt?gtatttacat?cttgcgagct?atatcactat?caggagtgtg?ttagaggtac 364

gactgtacta?ctaaaagaac?cttgcccatc?aggaacatac?gagggcaatt?caccatttca 424

ccctcttgct?gacaataaat?ttgcactaac?ttgcactagc?acacactttg?cttttgcttg 484

tgctgacggt?actcgacata?cctatcagct?gcgtgcaaga?tcagtttcac?caaaactttt 544

catcagacaa?gaggaggttc?aacaagagct?ctactcgcca?ctttttctca?ttgttgctgc 604

tctagtattt?ttaatacttt?gcttcaccat?taagagaaag?acagaatgaa?tgagctcact 664

ttaattgact?tctatttgtg?ctttttagcc?tttctgctat?tccttgtttt?aataatgctt 724

attatatttt?ggttttcact?cgaaatccag?gatctagaag?aaccttgtac?caaagtctaa 784

acgaacatga?aacttctcat?tgttttgact?tgtatttctc?tatgcagttg?catatgcact 844

gtagtacagc?gctgtgcatc?taataaacct?catgtgcttg?aagatccttg?taaggtacaa 904

cactaggggt?aatacttata?gcactgcttg?gctttgtgct?ctaggaaagg?ttttaccttt 964

tcatagatgg?cacactatgg?ttcaaacatg?cacacctaat?gttactatca?actgtcaaga 1024

tccagctggt?ggtgcgctta?tagctaggtg?ttggtacctt?catgaaggtc?accaaactgc 1084

tgcatttaga?gacgtacttg?ttgttttaaa?taaacgaaca?aattaaaatg?tctgataatg 1144

gaccccaatc?aaaccaacgt?agtgcccccc?gcattacatt?tggtggaccc?acagattcaa 1204

ctgacaataa?ccagaatgga?ggacgca 1231

<210>22

<211>63

<212>PRT

＜213〉coronavirus

<400>22

Met?Phe?His?Leu?Val?Asp?Phe?Gln?Val?Thr?Ile?Ala?Glu?Ile?Leu?Ile

1 5 10 15

Ile?Ile?Met?Arg?Thr?Phe?Arg?Ile?Ala?Ile?Trp?Asn?Leu?Asp?Val?Ile

20 25 30

Ile?Ser?Ser?Ile?Val?Arg?Gln?Leu?Phe?Lys?Pro?Leu?Thr?Lys?Lys?Asn

35 40 45

Tyr?Ser?Glu?Leu?Asp?Asp?Glu?Glu?Pro?Met?Glu?Leu?Asp?Tyr?Pro

50 55 60

<210>23

<211>1231

<212>DNA

＜213〉coronavirus

<220>

<221>CDS

<222>(285)..(650)

<223>

<400>23

taccgtattg?gaaactataa?attaaataca?gaccacgccg?gtagcaacga?caatattgct 60

ttgctagtac?agtaagtgac?aacagatgtt?tcatcttgtt?gacttccagg?ttacaatagc 120

agagatattg?attatcatta?tgaggacttt?caggattgct?atttggaatc?ttgacgttat 180

aataagttca?atagtgagac?aattatttaa?gcctctaact?aagaagaatt?attcggagtt 240

agatgatgaa?gaacctatgg?agttagatta?tccataaaac?gaac?atg?aaa?att?att 296

Met?Lys?Ile?Ile

1

ctc?ttc?ctg?aca?ttg?att?gta?ttt?aca?tct?tgc?gag?cta?tat?cac?tat 344

Leu?Phe?Leu?Thr?Leu?Ile?Val?Phe?Thr?Ser?Cys?Glu?Leu?Tyr?His?Tyr

5 10 15 20

cag?gag?tgt?gtt?aga?ggt?acg?act?gta?cta?cta?aaa?gaa?cct?tgc?cca 392

Gln?Glu?Cys?Val?Arg?Gly?Thr?Thr?Val?Leu?Leu?Lys?Glu?Pro?Cys?Pro

25 30 35

tca?gga?aca?tac?gag?ggc?aat?tca?cca?ttt?cac?cct?ctt?gct?gac?aat 440

Ser?Gly?Thr?Tyr?Glu?Gly?Asn?Ser?Pro?Phe?His?Pro?Leu?Ala?Asp?Asn

40 45 50

aaa?ttt?gca?cta?act?tgc?act?agc?aca?cac?ttt?gct?ttt?gct?tgt?gct 488

Lys?Phe?Ala?Leu?Thr?Cys?Thr?Ser?Thr?His?Phe?Ala?Phe?Ala?Cys?Ala

55 60 65

gac?ggt?act?cga?cat?acc?tat?cag?ctg?cgt?gca?aga?tca?gtt?tca?cca 536

Asp?Gly?Thr?Arg?His?Thr?Tyr?Gln?Leu?Arg?Ala?Arg?Ser?Val?Ser?Pro

70 75 80

aaa?ctt?ttc?atc?aga?caa?gag?gag?gtt?caa?caa?gag?ctc?tac?tcg?cca 584

Lys?Leu?Phe?Ile?Arg?Gln?Glu?Glu?Val?Gln?Gln?Glu?Leu?Tyr?Ser?Pro

85 90 95 100

ctt?ttt?ctc?att?gtt?gct?gct?cta?gta?ttt?tta?ata?ctt?tgc?ttc?acc 632

Leu?Phe?Leu?Ile?Val?Ala?Ala?Leu?Val?Phe?Leu?Ile?Leu?Cys?Phe?Thr

105 110 115

att?aag?aga?aag?aca?gaa?tgaatgagct?cactttaatt?gacttctatt 680

Ile?Lys?Arg?Lys?Thr?Glu

120

tgtgcttttt?agcctttctg?ctattccttg?ttttaataat?gcttattata?ttttggtttt 740

cactcgaaat?ccaggatcta?gaagaacctt?gtaccaaagt?ctaaacgaac?atgaaacttc 800

tcattgtttt?gacttgtatt?tctctatgca?gttgcatatg?cactgtagta?cagcgctgtg 860

catctaataa?acctcatgtg?cttgaagatc?cttgtaaggt?acaacactag?gggtaatact 920

tatagcactg?cttggctttg?tgctctagga?aaggttttac?cttttcatag?atggcacact 980

atggttcaaa?catgcacacc?taatgttact?atcaactgtc?aagatccagc?tggtggtgcg 1040

cttatagcta?ggtgttggta?ccttcatgaa?ggtcaccaaa?ctgctgcatt?tagagacgta 1100

cttgttgttt?taaataaacg?aacaaattaa?aatgtctgat?aatggacccc?aatcaaacca 1160

acgtagtgcc?ccccgcatta?catttggtgg?acccacagat?tcaactgaca?ataaccagaa 1220

tggaggacgc?a 1231

<210>24

<211>122

<212>PRT

＜213〉coronavirus

<400>24

Met?Lys?Ile?Ile?Leu?Phe?Leu?Thr?Leu?Ile?Val?Phe?Thr?Ser?Cys?Glu

1 5 10 15

Leu?Tyr?His?Tyr?Gln?Glu?Cys?Val?Arg?Gly?Thr?Thr?Val?Leu?Leu?Lys

20 25 30

Glu?Pro?Cys?Pro?Ser?Gly?Thr?Tyr?Glu?Gly?Asn?Ser?Pro?Phe?His?Pro

35 40 45

Leu?Ala?Asp?Asn?Lys?Phe?Ala?Leu?Thr?Cys?Thr?Ser?Thr?His?Phe?Ala

50 55 60

Phe?Ala?Cys?Ala?Asp?Gly?Thr?Arg?His?Thr?Tyr?Gln?Leu?Arg?Ala?Arg

65 70 75 80

Ser?Val?Ser?Pro?Lys?Leu?Phe?Ile?Arg?Gln?Glu?Glu?Val?Gln?Gln?Glu

85 90 95

Leu?Tyr?Ser?Pro?Leu?Phe?Leu?Ile?Val?Ala?Ala?Leu?Val?Phe?Leu?Ile

100 105 110

Leu?Cys?Phe?Thr?Ile?Lys?Arg?Lys?Thr?Glu

115 120

<210>25

<211>1231

<212>DNA

＜213〉coronavirus

<220>

<221>CDS

<222>(650)..(781)

<223>

<400>25

taccgtattg?gaaactataa?attaaataca?gaccacgccg?gtagcaacga?caatattgct 60

ttgctagtac?agtaagtgac?aacagatgtt?tcatcttgtt?gacttccagg?ttacaatagc 120

agagatattg?attatcatta?tgaggacttt?caggattgct?atttggaatc?ttgacgttat 180

aataagttca?atagtgagac?aattatttaa?gcctctaact?aagaagaatt?attcggagtt 240

agatgatgaa?gaacctatgg?agttagatta?tccataaaac?gaacatgaaa?attattctct 300

tcctgacatt?gattgtattt?acatcttgcg?agctatatca?ctatcaggag?tgtgttagag 360

gtacgactgt?actactaaaa?gaaccttgcc?catcaggaac?atacgagggc?aattcaccat 420

ttcaccctct?tgctgacaat?aaatttgcac?taacttgcac?tagcacacac?tttgcttttg 480

cttgtgctga?cggtactcga?catacctatc?agctgcgtgc?aagatcagtt?tcaccaaaac 540

ttttcatcag?acaagaggag?gttcaacaag?agctctactc?gccacttttt?ctcattgttg 600

ctgctctagt?atttttaata?ctttgcttca?ccattaagag?aaagacaga?atg?aat?gag 658

Met?Asn?Glu

1

ctc?act?tta?att?gac?ttc?tat?ttg?tgc?ttt?tta?gcc?ttt?ctg?cta?ttc 706

Leu?Thr?Leu?Ile?Asp?Phe?Tyr?Leu?Cys?Phe?Leu?Ala?Phe?Leu?Leu?Phe

5 10 15

ctt?gtt?tta?ata?atg?ctt?att?ata?ttt?tgg?ttt?tca?ctc?gaa?atc?cag 754

Leu?Val?Leu?Ile?Met?Leu?Ile?Ile?Phe?Trp?Phe?Ser?Leu?Glu?Ile?Gln

20 25 30 35

gat?cta?gaa?gaa?cct?tgt?acc?aaa?gtc?taaacgaaca?tgaaacttct 801

Asp?Leu?Glu?Glu?Pro?Cys?Thr?Lys?Val

40

cattgttttg?acttgtattt?ctctatgcag?ttgcatatgc?actgtagtac?agcgctgtgc 861

atctaataaa?cctcatgtgc?ttgaagatcc?ttgtaaggta?caacactagg?ggtaatactt 921

atagcactgc?ttggctttgt?gctctaggaa?aggttttacc?ttttcataga?tggcacacta 981

tggttcaaac?atgcacacct?aatgttacta?tcaactgtca?agatccagct?ggtggtgcgc 1041

ttatagctag?gtgttggtac?cttcatgaag?gtcaccaaac?tgctgcattt?agagacgtac 1101

ttgttgtttt?aaataaacga?acaaattaaa?atgtctgata?atggacccca?atcaaaccaa 1161

cgtagtgccc?cccgcattac?atttggtgga?cccacagatt?caactgacaa?taaccagaat 1221

ggaggacgca 1231

<210>26

<211>44

<212>PRT

＜213〉coronavirus

<400>26

Met?Asn?Glu?Leu?Thr?Leu?Ile?Asp?Phe?Tyr?Leu?Cys?Phe?Leu?Ala?Phe

1 5 10 15

Leu?Leu?Phe?Leu?Val?Leu?Ile?Met?Leu?Ile?Ile?Phe?Trp?Phe?Ser?Leu

20 25 30

Glu?Ile?Gln?Asp?Leu?Glu?Glu?Pro?Cys?Thr?Lys?Val

35 40

<210>27

<211>1231

<212>DNA

＜213〉coronavirus

<220>

<221>CDS

<222>(791)..(907)

<223>

<400>27

taccgtattg?gaaactataa?attaaataca?gaccacgccg?gtagcaacga?caatattgct 60

ttgctagtac?agtaagtgac?aacagatgtt?tcatcttgtt?gacttccagg?ttacaatagc 120

agagatattg?attatcatta?tgaggacttt?caggattgct?atttggaatc?ttgacgttat 180

aataagttca?atagtgagac?aattatttaa?gcctctaact?aagaagaatt?attcggagtt 240

agatgatgaa?gaacctatgg?agttagatta?tccataaaac?gaacatgaaa?attattctct 300

tcctgacatt?gattgtattt?acatcttgcg?agctatatca?ctatcaggag?tgtgttagag 360

gtacgactgt?actactaaaa?gaaccttgcc?catcaggaac?atacgagggc?aattcaccat 420

ttcaccctct?tgctgacaat?aaatttgcac?taacttgcac?tagcacacac?tttgcttttg 480

cttgtgctga?cggtactcga?catacctatc?agctgcgtgc?aagatcagtt?tcaccaaaac 540

ttttcatcag?acaagaggag?gttcaacaag?agctctactc?gccacttttt?ctcattgttg 600

ctgctctagt?atttttaata?ctttgcttca?ccattaagag?aaagacagaa?tgaatgagct 660

cactttaatt?gacttctatt?tgtgcttttt?agcctttctg?ctattccttg?ttttaataat 720

gcttattata?ttttggtttt?cactcgaaat?ccaggatcta?gaagaacctt?gtaccaaagt 780

ctaaacgaac?atg?aaa?ctt?ctc?att?gtt?ttg?act?tgt?att?tct?cta?tgc 829

Met?Lys?Leu?Leu?Ile?Val?Leu?Thr?Cys?Ile?Ser?Leu?Cys

1 5 10

agt?tgc?ata?tgc?act?gta?gta?cag?cgc?tgt?gca?tct?aat?aaa?cct?cat 877

Ser?Cys?Ile?Cys?Thr?Val?Val?Gln?Arg?Cys?Ala?Ser?Asn?Lys?Pro?His

15 20 25

gtg?ctt?gaa?gat?cct?tgt?aag?gta?caa?cac?taggggtaat?acttatagca 927

Val?Leu?Glu?Asp?Pro?Cys?Lys?Val?Gln?His

30 35

ctgcttggct?ttgtgctcta?ggaaaggttt?taccttttca?tagatggcac?actatggttc 987

aaacatgcac?acctaatgtt?actatcaact?gtcaagatcc?agctggtggt?gcgcttatag 1047

ctaggtgttg?gtaccttcat?gaaggtcacc?aaactgctgc?atttagagac?gtacttgttg 1107

ttttaaataa?acgaacaaat?taaaatgtct?gataatggac?cccaatcaaa?ccaacgtagt 1167

gccccccgca?ttacatttgg?tggacccaca?gattcaactg?acaataacca?gaatggagga 1227

cgca 1231

<210>28

<211>39

<212>PRT

＜213〉coronavirus

<400>28

Met?Lys?Leu?Leu?Ile?Val?Leu?Thr?Cys?Ile?Ser?Leu?Cys?Ser?Cys?Ile

1 5 10 15

Cys?Thr?Val?Val?Gln?Arg?Cys?Ala?Ser?Asn?Lys?Pro?His?Val?Leu?Glu

20 25 30

Asp?Pro?Cys?Lys?Val?Gln?His

35

<210>29

<211>1231

<212>DNA

＜213〉coronavirus

<220>

<221>CDS

<222>(876)..(1127)

<223>

<400>29

taccgtattg?gaaactataa?attaaataca?gaccacgccg?gtagcaacga?caatattgct 60

ttgctagtac?agtaagtgac?aacagatgtt?tcatcttgtt?gacttccagg?ttacaatagc 120

agagatattg?attatcatta?tgaggacttt?caggattgct?atttggaatc?ttgacgttat 180

aataagttca?atagtgagac?aattatttaa?gcctctaact?aagaagaatt?attcggagtt 240

agatgatgaa?gaacctatgg?agttagatta?tccataaaac?gaacatgaaa?attattctct 300

tcctgacatt?gattgtattt?acatcttgcg?agctatatca?ctatcaggag?tgtgttagag 360

gtacgactgt?actactaaaa?gaaccttgcc?catcaggaac?atacgagggc?aattcaccat 420

ttcaccctct?tgctgacaat?aaatttgcac?taacttgcac?tagcacacac?tttgcttttg 480

cttgtgctga?cggtactcga?catacctatc?agctgcgtgc?aagatcagtt?tcaccaaaac 540

ttttcatcag?acaagaggag?gttcaacaag?agctctactc?gccacttttt?ctcattgttg 600

ctgctctagt?atttttaata?ctttgcttca?ccattaagag?aaagacagaa?tgaatgagct 660

cactttaatt?gacttctatt?tgtgcttttt?agcctttctg?ctattccttg?ttttaataat 720

gcttattata?ttttggtttt?cactcgaaat?ccaggatcta?gaagaacctt?gtaccaaagt 780

ctaaacgaac?atgaaacttc?tcattgtttt?gacttgtatt?tctctatgca?gttgcatatg 840

cactgtagta?cagcgctgtg?catctaataa?acctc?atg?tgc?ttg?aag?atc?ctt 893

Met?Cys?Leu?Lys?Ile?Leu

1 5

gta?agg?tac?aac?act?agg?ggt?aat?act?tat?agc?act?gct?tgg?ctt?tgt 941

Val?Arg?Tyr?Asn?Thr?Arg?Gly?Asn?Thr?Tyr?Ser?Thr?Ala?Trp?Leu?Cys

10 15 20

gct?cta?gga?aag?gtt?tta?cct?ttt?cat?aga?tgg?cac?act?atg?gtt?caa 989

Ala?Leu?Gly?Lys?Val?Leu?Pro?Phe?His?Arg?Trp?His?Thr?Met?Val?Gln

25 30 35

aca?tgc?aca?cct?aat?gtt?act?atc?aac?tgt?caa?gat?cca?gct?ggt?ggt 1037

Thr?Cys?Thr?Pro?Asn?Val?Thr?Ile?Asn?Cys?Gln?Asp?Pro?Ala?Gly?Gly

40 45 50

gcg?ctt?ata?gct?agg?tgt?tgg?tac?ctt?cat?gaa?ggt?cac?caa?act?gct 1085

Ala?Leu?Ile?Ala?Arg?Cys?Trp?Tyr?Leu?His?Glu?Gly?His?Gln?Thr?Ala

55 60 65 70

gca?ttt?aga?gac?gta?ctt?gtt?gtt?tta?aat?aaa?cga?aca?aat 1127

Ala?Phe?Arg?Asp?Val?Leu?Val?Val?Leu?Asn?Lys?Arg?Thr?Asn

75 80

taaaatgtct?gataatggac?cccaatcaaa?ccaacgtagt?gccccccgca?ttacatttgg 1187

tggacccaca?gattcaactg?acaataacca?gaatggagga?cgca 1231

<210>30

<211>84

<212>PRT

＜213〉coronavirus

<400>30

Met?Cys?Leu?Lys?Ile?Leu?Val?Arg?Tyr?Asn?Thr?Arg?Gly?Asn?Thr?Tyr

1 5 10 15

Ser?Thr?Ala?Trp?Leu?Cys?Ala?Leu?Gly?Lys?Val?Leu?Pro?Phe?His?Arg

20 25 30

Trp?His?Thr?Met?Val?Gln?Thr?Cys?Thr?Pro?Asn?Val?Thr?Ile?Asn?Cys

35 40 45

Gln?Asp?Pro?Ala?Gly?Gly?Ala?Leu?Ile?Ala?Arg?Cys?Trp?Tyr?Leu?His

50 55 60

Glu?Gly?His?Gln?Thr?Ala?Ala?Phe?Arg?Asp?Val?Leu?Val?Val?Leu?Asn

65 70 75 80

Lys?Arg?Thr?Asn

<210>31

<211>21221

<212>DNA

＜213〉coronavirus

<400>31

atggagagcc?ttgttcttgg?tgtcaacgag?aaaacacacg?tccaactcag?tttgcctgtc 60

cttcaggtta?gagacgtgct?agtgcgtggc?ttcggggact?ctgtggaaga?ggccctatcg 120

gaggcacgtg?aacacctcaa?aaatggcact?tgtggtctag?tagagctgga?aaaaggcgta 180

ctgccccagc?ttgaacagcc?ctatgtgttc?attaaacgtt?ctgatgcctt?aagcaccaat 240

cacggccaca?aggtcgttga?gctggttgca?gaaatggacg?gcattcagta?cggtcgtagc 300

ggtataacac?tgggagtact?cgtgccacat?gtgggcgaaa?ccccaattgc?ataccgcaat 360

gttcttcttc?gtaagaacgg?taataaggga?gccggtggtc?atagctatgg?catcgatcta 420

aagtcttatg?acttaggtga?cgagcttggc?actgatccca?ttgaagatta?tgaacaaaac 480

tggaacacta?agcatggcag?tggtgcactc?cgtgaactca?ctcgtgagct?caatggaggt 540

gcagtcactc?gctatgtcga?caacaatttc?tgtggcccag?atgggtaccc?tcttgattgc 600

atcaaagatt?ttctcgcacg?cgcgggcaag?tcaatgtgca?ctctttccga?acaacttgat 660

tacatcgagt?cgaagagagg?tgtctactgc?tgccgtgacc?atgagcatga?aattgcctgg 720

ttcactgagc?gctctgataa?gagctacgag?caccagacac?ccttcgaaat?taagagtgcc 780

aagaaatttg?acactttcaa?aggggaatgc?ccaaagtttg?tgtttcctct?taactcaaaa 840

gtcaaagtca?ttcaaccacg?tgttgaaaag?aaaaagactg?agggtttcat?ggggcgtata 900

cgctctgtgt?accctgttgc?atctccacag?gagtgtaaca?atatgcactt?gtctaccttg 960

atgaaatgta?atcattgcga?tgaagtttca?tggcagacgt?gcgactttct?gaaagccact 1020

tgtgaacatt?gtggcactga?aaatttagtt?attgaaggac?ctactacatg?tgggtaccta 1080

cctactaatg?ctgtagtgaa?aatgccatgt?cctgcctgtc?aagacccaga?gattggacct 1140

gagcatagtg?ttgcagatta?tcacaaccac?tcaaacattg?aaactcgact?ccgcaaggga 1200

ggtaggacta?gatgttttgg?aggctgtgtg?tttgcctatg?ttggctgcta?taataagcgt 1260

gcctactggg?ttcctcgtgc?tagtgctgat?attggctcag?gccatactgg?cattactggt 1320

gacaatgtgg?agaccttgaa?tgaggatctc?cttgagatac?tgagtcgtga?acgtgttaac 1380

attaacattg?ttggcgattt?tcatttgaat?gaagaggttg?ccatcatttt?ggcatctttc 1440

tctgcttcta?caagtgcctt?tattgacact?ataaagagtc?ttgattacaa?gtctttcaaa 1500

accattgttg?agtcctgcgg?taactataaa?gttaccaagg?gaaagcccgt?aaaaggtgct 1560

tggaacattg?gacaacagag?atcagtttta?acaccactgt?gtggttttcc?ctcacaggct 1620

gctggtgtta?tcagatcaat?ttttgcgcgc?acacttgatg?cagcaaacca?ctcaattcct 1680

gatttgcaaa?gagcagctgt?caccatactt?gatggtattt?ctgaacagtc?attacgtctt 1740

gtcgacgcca?tggtttatac?ttcagacctg?ctcaccaaca?gtgtcattat?tatggcatat 1800

gtaactggtg?gtcttgtaca?acagacttct?cagtggttgt?ctaatctttt?gggcactact 1860

gttgaaaaac?tcaggcctat?ctttgaatgg?attgaggcga?aacttagtgc?aggagttgaa 1920

tttctcaagg?atgcttggga?gattctcaaa?tttctcatta?caggtgtttt?tgacatcgtc 1980

aagggtcaaa?tacaggttgc?ttcagataac?atcaaggatt?gtgtaaaatg?cttcattgat 2040

gttgttaaca?aggcactcga?aatgtgcatt?gatcaagtaa?ctatcgctgg?cgcaaagttg 2100

cgatcactca?acttaggtga?agtcttcatc?gctcaaagca?agggacttta?ccgtcagtgt 2160

atacgtggca?aggagcagct?gcaactactc?atgcctctta?aggcaccaaa?agaagtaacc 2220

tttcttgaag?gtgattcaca?tgacacagta?cttacctctg?aggaggttgt?tctcaagaac 2280

ggtgaactcg?aagcactcga?gacgcccgtt?gatagcttca?caaatggagc?tatcgttggc 2340

acaccagtct?gtgtaaatgg?cctcatgctc?ttagagatta?aggacaaaga?acaatactgc 2400

gcattgtctc?ctggtttact?ggctacaaac?aatgtctttc?gcttaaaagg?gggtgcacca 2460

attaaaggtg?taacctttgg?agaagatact?gtttgggaag?ttcaaggtta?caagaatgtg 2520

agaatcacat?ttgagcttga?tgaacgtgtt?gacaaagtgc?ttaatgaaaa?gtgctctgtc 2580

tacactgttg?aatccggtac?cgaagttact?gagtttgcat?gtgttgtagc?agaggctgtt 2640

gtgaagactt?tacaaccagt?ttctgatctc?cttaccaaca?tgggtattga?tcttgatgag 2700

tggagtgtag?ctacattcta?cttatttgat?gatgctggtg?aagaaaactt?ttcatcacgt 2760

atgtattgtt?ccttttaccc?tccagatgag?gaagaagagg?acgatgcaga?gtgtgaggaa 2820

gaagaaattg?atgaaacctg?tgaacatgag?tacggtacag?aggatgatta?tcaaggtctc 2880

cctctggaat?ttggtgcctc?agctgaaaca?gttcgagttg?aggaagaaga?agaggaagac 2940

tggctggatg?atactactga?gcaatcagag?attgagccag?aaccagaacc?tacacctgaa 3000

gaaccagtta?atcagtttac?tggttattta?aaacttactg?acaatgttgc?cattaaatgt 3060

gttgacatcg?ttaaggaggc?acaaagtgct?aatcctatgg?tgattgtaaa?tgctgctaac 3120

atacacctga?aacatggtgg?tggtgtagca?ggtgcactca?acaaggcaac?caatggtgcc 3180

atgcaaaagg?agagtgatga?ttacattaag?ctaaatggcc?ctcttacagt?aggagggtct 3240

tgtttgcttt?ctggacataa?tcttgctaag?aagtgtctgc?atgttgttgg?acctaaccta 3300

aatgcaggtg?aggacatcca?gcttcttaag?gcagcatatg?aaaatttcaa?ttcacaggac 3360

atcttacttg?caccattgtt?gtcagcaggc?atatttggtg?ctaaaccact?tcagtcttta 3420

caagtgtgcg?tgcagacggt?tcgtacacag?gtttatattg?cagtcaatga?caaagctctt 3480

tatgagcagg?ttgtcatgga?ttatcttgat?aacctgaagc?ctagagtgga?agcacctaac 3540

caagaggagc?caccaaacac?agaagattcc?aaaactgagg?agaaatctgt?cgtacagaag 3600

cctgtcgatg?tgaagccaaa?aattaaggcc?tgcattgatg?aggttaccac?aacactggaa 3660

gaaactaagt?ttcttaccaa?taagttactc?ttgtttgctg?atatcaatgg?taagctttac 3720

catgattctc?agaacatgct?tagaggtgaa?gatatgtctt?tccttgagaa?ggatgcacct 3780

tacatggtag?gtgatgttat?cactagtggt?gatatcactt?gtgttgtaat?accctccaaa 3840

aaggctggtg?gcactactga?gatgctctca?agagctttga?agaaagtgcc?agttgatgag 3900

tatataacca?cgtaccctgg?acaaggatgt?gctggttata?cacttgagga?agctaagact 3960

gctcttaaga?aatgcaaatc?tgcattttat?gtactacctt?cagaagcacc?taatgctaag 4020

gaagagattc?taggaactgt?atcctggaat?ttgagagaaa?tgcttgctca?tgctgaagag 4080

acaagaaaat?taatgcctat?atgcatggat?gttagagcca?taatggcaac?catccaacgt 4140

aagtataaag?gaattaaaat?tcaagagggc?atcgttgact?atggtgtccg?attcttcttt 4200

tatactagta?aagagcctgt?agcttctatt?attacgaagc?tgaactctct?aaatgagccg 4260

cttgtcacaa?tgccaattgg?ttatgtgaca?catggtttta?atcttgaaga?ggctgcgcgc 4320

tgtatgcgtt?ctcttaaagc?tcctgccgta?gtgtcagtat?catcaccaga?tgctgttact 4380

acatataatg?gatacctcac?ttcgtcatca?aagacatctg?aggagcactt?tgtagaaaca 4440

gtttctttgg?ctggctctta?cagagattgg?tcctattcag?gacagcgtac?agagttaggt 4500

gttgaatttc?ttaagcgtgg?tgacaaaatt?gtgtaccaca?ctctggagag?ccccgtcgag 4560

tttcatcttg?acggtgaggt?tctttcactt?gacaaactaa?agagtctctt?atccctgcgg 4620

gaggttaaga?ctataaaagt?gttcacaact?gtggacaaca?ctaatctcca?cacacagctt 4680

gtggatatgt?ctatgacata?tggacagcag?tttggtccaa?catacttgga?tggtgctgat 4740

gttacaaaaa?ttaaacctca?tgtaaatcat?gagggtaaga?ctttctttgt?actacctagt 4800

gatgacacac?tacgtagtga?agctttcgag?tactaccata?ctcttgatga?gagttttctt 4860

ggtaggtaca?tgtctgcttt?aaaccacaca?aagaaatgga?aatttcctca?agttggtggt 4920

ttaacttcaa?ttaaatgggc?tgataacaat?tgttatttgt?ctagtgtttt?attagcactt 4980

caacagcttg?aagtcaaatt?caatgcacca?gcacttcaag?aggcttatta?tagagcccgt 5040

gctggtgatg?ctgctaactt?ttgtgcactc?atactcgctt?acagtaataa?aactgttggc 5100

gagcttggtg?atgtcagaga?aactatgacc?catcttctac?agcatgctaa?tttggaatct 5160

gcaaagcgag?ttcttaatgt?ggtgtgtaaa?cattgtggtc?agaaaactac?taccttaacg 5220

ggtgtagaag?ctgtgatgta?tatgggtact?ctatcttatg?ataatcttaa?gacaggtgtt 5280

tccattccat?gtgtgtgtgg?tcgtgatgct?acacaatatc?tagtacaaca?agagtcttct 5340

tttgttatga?tgtctgcacc?acctgctgag?tataaattac?agcaaggtac?attcttatgt 5400

gcgaatgagt?acactggtaa?ctatcagtgt?ggtcattaca?ctcatataac?tgctaaggag 5460

accctctatc?gtattgacgg?agctcacctt?acaaagatgt?cagagtacaa?aggaccagtg 5520

actgatgttt?tctacaagga?aacatcttac?actacaacca?tcaagcctgt?gtcgtataaa 5580

ctcgatggag?ttacttacac?agagattgaa?ccaaaattgg?atgggtatta?taaaaaggat 5640

aatgcttact?atacagagca?gcctatagac?cttgtaccaa?ctcaaccatt?accaaatgcg 5700

agttttgata?atttcaaact?cacatgttct?aacacaaaat?ttgctgatga?tttaaatcaa 5760

atgacaggct?tcacaaagcc?agcttcacga?gagctatctg?tcacattctt?cccagacttg 5820

aatggcgatg?tagtggctat?tgactataga?cactattcag?cgagtttcaa?gaaaggtgct 5880

aaattactgc?ataagccaat?tgtttggcac?attaaccagg?ctacaaccaa?gacaacgttc 5940

aaaccaaaca?cttggtgttt?acgttgtctt?tggagtacaa?agccagtaga?tacttcaaat 6000

tcatttgaag?ttctggcagt?agaagacaca?caaggaatgg?acaatcttgc?ttgtgaaagt 6060

caacaaccca?cctctgaaga?agtagtggaa?aatcctacca?tacagaagga?agtcatagag 6120

tgtgacgtga?aaactaccga?agttgtaggc?aatgtcatac?ttaaaccatc?agatgaaggt 6180

gttaaagtaa?cacaagagtt?aggtcatgag?gatcttatgg?ctgcttatgt?ggaaaacaca 6240

agcattacca?ttaagaaacc?taatgagctt?tcactagcct?taggtttaaa?aacaattgcc 6300

actcatggta?ttgctgcaat?taatagtgtt?ccttggagta?aaattttggc?ttatgtcaaa 6360

ccattcttag?gacaagcagc?aattacaaca?tcaaattgcg?ctaagagatt?agcacaacgt 6420

gtgtttaaca?attatatgcc?ttatgtgttt?acattattgt?tccaattgtg?tacttttact 6480

aaaagtacca?attctagaat?tagagcttca?ctacctacaa?ctattgctaa?aaatagtgtt 6540

aagagtgttg?ctaaattatg?tttggatgcc?ggcattaatt?atgtgaagtc?acccaaattt 6600

tctaaattgt?tcacaatcgc?tatgtggcta?ttgttgttaa?gtatttgctt?aggttctcta 6660

atctgtgtaa?ctgctgcttt?tggtgtactc?ttatctaatt?ttggtgctcc?ttcttattgt 6720

aatggcgtta?gagaattgta?tcttaattcg?tctaacgtta?ctactatgga?tttctgtgaa 6780

ggttcttttc?cttgcagcat?ttgtttaagt?ggattagact?cccttgattc?ttatccagct 6840

cttgaaacca?ttcaggtgac?gatttcatcg?tacaagctag?acttgacaat?tttaggtctg 6900

gccgctgagt?gggttttggc?atatatgttg?ttcacaaaat?tcttttattt?attaggtctt 6960

tcagctataa?tgcaggtgtt?ctttggctat?tttgctagtc?atttcatcag?caattcttgg 7020

ctcatgtggt?ttatcattag?tattgtacaa?atggcacccg?tttctgcaat?ggttaggatg 7080

tacatcttct?ttgcttcttt?ctactacata?tggaagagct?atgttcatat?catggatggt 7140

tgcacctctt?cgacttgcat?gatgtgctat?aagcgcaatc?gtgccacacg?cgttgagtgt 7200

acaactattg?ttaatggcat?gaagagatct?ttctatgtct?atgcaaatgg?aggccgtggc 7260

ttctgcaaga?ctcacaattg?gaattgtctc?aattgtgaca?cattttgcac?tggtagtaca 7320

ttcattagtg?atgaagttgc?tcgtgatttg?tcactccagt?ttaaaagacc?aatcaaccct 7380

actgaccagt?catcgtatat?tgttgatagt?gttgctgtga?aaaatggcgc?gcttcacctc 7440

tactttgaca?aggctggtca?aaagacctat?gagagacatc?cgctctccca?ttttgtcaat 7500

ttagacaatt?tgagagctaa?caacactaaa?ggttcactgc?ctattaatgt?catagttttt 7560

gatggcaagt?ccaaatgcga?cgagtctgct?tctaagtctg?cttctgtgta?ctacagtcag 7620

ctgatgtgcc?aacctattct?gttgcttgac?caagctcttg?tatcagacgt?tggagatagt 7680

actgaagttt?ccgttaagat?gtttgatgct?tatgtcgaca?ccttttcagc?aacttttagt 7740

gttcctatgg?aaaaacttaa?ggcacttgtt?gctacagctc?acagcgagtt?agcaaagggt 7800

gtagctttag?atggtgtcct?ttctacattc?gtgtcagctg?cccgacaagg?tgttgttgat 7860

accgatgttg?acacaaagga?tgttattgaa?tgtctcaaac?tttcacatca?ctctgactta 7920

gaagtgacag?gtgacagttg?taacaatttc?atgctcacct?ataataaggt?tgaaaacatg 7980

acgcccagag?atcttggcgc?atgtattgac?tgtaatgcaa?ggcatatcaa?tgcccaagta 8040

gcaaaaagtc?acaatgtttc?actcatctgg?aatgtaaaag?actacatgtc?tttatctgaa 8100

cagctgcgta?aacaaattcg?tagtgctgcc?aagaagaaca?acataccttt?tagactaact 8160

tgtgctacaa?ctagacaggt?tgtcaatgtc?ataactacta?aaatctcact?caagggtggt 8220

aagattgtta?gtacttgttt?taaacttatg?cttaaggcca?cattattgtg?cgttcttgct 8280

gcattggttt?gttatatcgt?tatgccagta?catacattgt?caatccatga?tggttacaca 8340

aatgaaatca?ttggttacaa?agccattcag?gatggtgtca?ctcgtgacat?catttctact 8400

gatgattgtt?ttgcaaataa?acatgctggt?tttgacgcat?ggtttagcca?gcgtggtggt 8460

tcatacaaaa?atgacaaaag?ctgccctgta?gtagctgcta?tcattacaag?agagattggt 8520

ttcatagtgc?ctggcttacc?gggtactgtg?ctgagagcaa?tcaatggtga?cttcttgcat 8580

tttctacctc?gtgtttttag?tgctgttggc?aacatttgct?acacaccttc?caaactcatt 8640

gagtatagtg?attttgctac?ctctgcttgc?gttcttgctg?ctgagtgtac?aatttttaag 8700

gatgctatgg?gcaaacctgt?gccatattgt?tatgacacta?atttgctaga?gggttctatt 8760

tcttatagtg?agcttcgtcc?agacactcgt?tatgtgctta?tggatggttc?catcatacag 8820

tttcctaaca?cttacctgga?gggttctgtt?agagtagtaa?caacttttga?tgctgagtac 8880

tgtagacatg?gtacatgcga?aaggtcagaa?gtaggtattt?gcctatctac?cagtggtaga 8940

tgggttctta?ataatgagca?ttacagagct?ctatcaggag?ttttctgtgg?tgttgatgcg 9000

atgaatctca?tagctaacat?ctttactcct?cttgtgcaac?ctgtgggtgc?tttagatgtg 9060

tctgcttcag?tagtggctgg?tggtattatt?gccatattgg?tgacttgtgc?tgcctactac 9120

tttatgaaat?tcagacgtgt?ttttggtgag?tacaaccatg?ttgttgctgc?taatgcactt 9180

ttgtttttga?tgtctttcac?tatactctgt?ctggtaccag?cttacagctt?tctgccggga 9240

gtctactcag?tcttttactt?gtacttgaca?ttctatttca?ccaatgatgt?ttcattcttg 9300

gctcaccttc?aatggtttgc?catgttttct?cctattgtgc?ctttttggat?aacagcaatc 9360

tatgtattct?gtatttctct?gaagcactgc?cattggttct?ttaacaacta?tcttaggaaa 9420

agagtcatgt?ttaatggagt?tacatttagt?accttcgagg?aggctgcttt?gtgtaccttt 9480

ttgctcaaca?aggaaatgta?cctaaaattg?cgtagcgaga?cactgttgcc?acttacacag 9540

tataacaggt?atcttgctct?atataacaag?tacaagtatt?tcagtggagc?cttagatact 9600

accagctatc?gtgaagcagc?ttgctgccac?ttagcaaagg?ctctaaatga?ctttagcaac 9660

tcaggtgctg?atgttctcta?ccaaccacca?cagacatcaa?tcacttctgc?tgttctgcag 9720

agtggtttta?ggaaaatggc?attcccgtca?ggcaaagttg?aagggtgcat?ggtacaagta 9780

acctgtggaa?ctacaactct?taatggattg?tggttggatg?acacagtata?ctgtccaaga 9840

catgtcattt?gcacagcaga?agacatgctt?aatcctaact?atgaagatct?gctcattcgc 9900

aaatccaacc?atagctttct?tgttcaggct?ggcaatgttc?aacttcgtgt?tattggccat 9960

tctatgcaaa?attgtctgct?taggcttaaa?gttgatactt?ctaaccctaa?gacacccaag 10020

tataaatttg?tccgtatcca?acctggtcaa?acattttcag?ttctagcatg?ctacaatggt 10080

tcaccatctg?gtgtttatca?gtgtgccatg?agacctaatc?ataccattaa?aggttctttc 10140

cttaatggat?catgtggtag?tgttggtttt?aacattgatt?atgattgcgt?gtctttctgc 10200

tatatgcatc?atatggagct?tccaacagga?gtacacgctg?gtactgactt?agaaggtaaa 10260

ttctatggtc?catttgttga?cagacaaact?gcacaggctg?caggtacaga?cacaaccata 10320

acattaaatg?ttttggcatg?gctgtatgct?gctgttatca?atggtgatag?gtggtttctt 10380

aatagattca?ccactacttt?gaatgacttt?aaccttgtgg?caatgaagta?caactatgaa 10440

cctttgacac?aagatcatgt?tgacatattg?ggacctcttt?ctgctcaaac?aggaattgcc 10500

gtcttagata?tgtgtgctgc?tttgaaagag?ctgctgcaga?atggtatgaa?tggtcgtact 10560

atccttggta?gcactatttt?agaagatgag?tttacaccat?ttgatgttgt?tagacaatgc 10620

tctggtgtta?ccttccaagg?taagttcaag?aaaattgtta?agggcactca?tcattggatg 10680

cttttaactt?tcttgacatc?actattgatt?cttgttcaaa?gtacacagtg?gtcactgttt 10740

ttctttgttt?acgagaatgc?tttcttgcca?tttactcttg?gtattatggc?aattgctgca 10800

tgtgctatgc?tgcttgttaa?gcataagcac?gcattcttgt?gcttgtttct?gttaccttct 10860

cttgcaacag?ttgcttactt?taatatggtc?tacatgcctg?ctagctgggt?gatgcgtatc 10920

atgacatggc?ttgaattggc?tgacactagc?ttgtctggtt?ataggcttaa?ggattgtgtt 10980

atgtatgctt?cagctttagt?tttgcttatt?ctcatgacag?ctcgcactgt?ttatgatgat 11040

gctgctagac?gtgtttggac?actgatgaat?gtcattacac?ttgtttacaa?agtctactat 11100

ggtaatgctt?tagatcaagc?tatttccatg?tgggccttag?ttatttctgt?aacctctaac 11160

tattctggtg?tcgttacgac?tatcatgttt?ttagctagag?ctatagtgtt?tgtgtgtgtt 11220

gagtattacc?cattgttatt?tattactggc?aacaccttac?agtgtatcat?gcttgtttat 11280

tgtttcttag?gctattgttg?ctgctgctac?tttggccttt?tctgtttact?caaccgttac 11340

ttcaggctta?ctcttggtgt?ttatgactac?ttggtctcta?cacaagaatt?taggtatatg 11400

aactcccagg?ggcttttgcc?tcctaagagt?agtattgatg?ctttcaagct?taacattaag 11460

ttgttgggta?ttggaggtaa?accatgtatc?aaggttgcta?ctgtacagtc?taaaatgtct 11520

gacgtaaagt?gcacatctgt?ggtactgctc?tcggttcttc?aacaacttag?agtagagtca 11580

tcttctaaat?tgtgggcaca?atgtgtacaa?ctccacaatg?atattcttct?tgcaaaagac 11640

acaactgaag?ctttcgagaa?gatggtttct?cttttgtctg?ttttgctatc?catgcagggt 11700

gctgtagaca?ttaataggtt?gtgcgaggaa?atgctcgata?accgtgctac?tcttcaggct 11760

attgcttcag?aatttagttc?tttaccatca?tatgccgctt?atgccactgc?ccaggaggcc 11820

tatgagcagg?ctgtagctaa?tggtgattct?gaagtcgttc?tcaaaaagtt?aaagaaatct 11880

ttgaatgtgg?ctaaatctga?gtttgaccgt?gatgctgcca?tgcaacgcaa?gttggaaaag 11940

atggcagatc?aggctatgac?ccaaatgtac?aaacaggcaa?gatctgagga?caagagggca 12000

aaagtaacta?gtgctatgca?aacaatgctc?ttcactatgc?ttaggaagct?tgataatgat 12060

gcacttaaca?acattatcaa?caatgcgcgt?gatggttgtg?ttccactcaa?catcatacca 12120

ttgactacag?cagccaaact?catggttgtt?gtccctgatt?atggtaccta?caagaacact 12180

tgtgatggta?acacctttac?atatgcatct?gcactctggg?aaatccagca?agttgttgat 12240

gcggatagca?agattgttca?acttagtgaa?attaacatgg?acaattcacc?aaatttggct 12300

tggcctctta?ttgttacagc?tctaagagcc?aactcagctg?ttaaactaca?gaataatgaa 12360

ctgagtccag?tagcactacg?acagatgtcc?tgtgcggctg?gtaccacaca?aacagcttgt 12420

actgatgaca?atgcacttgc?ctactataac?aattcgaagg?gaggtaggtt?tgtgctggca 12480

ttactatcag?accaccaaga?tctcaaatgg?gctagattcc?ctaagagtga?tggtacaggt 12540

acaatttaca?cagaactgga?accaccttgt?aggtttgtta?cagacacacc?aaaagggcct 12600

aaagtgaaat?acttgtactt?catcaaaggc?ttaaacaacc?taaatagagg?tatggtgctg 12660

ggcagtttag?ctgctacagt?acgtcttcag?gctggaaatg?ctacagaagt?acctgccaat 12720

tcaactgtgc?tttccttctg?tgcttttgca?gtagaccctg?ctaaagcata?taaggattac 12780

ctagcaagtg?gaggacaacc?aatcaccaac?tgtgtgaaga?tgttgtgtac?acacactggt 12840

acaggacagg?caattactgt?aacaccagaa?gctaacatgg?accaagagtc?ctttggtggt 12900

gcttcatgtt?gtctgtattg?tagatgccac?attgaccatc?caaatcctaa?aggattctgt 12960

gacttgaaag?gtaagtacgt?ccaaatacct?accacttgtg?ctaatgaccc?agtgggtttt 13020

acacttagaa?acacagtctg?taccgtctgc?ggaatgtgga?aaggttatgg?ctgtagttgt 13080

gaccaactcc?gcgaaccctt?gatgcagtct?gcggatgcat?caacgttttt?aaacgggttt 13140

gcggtgtaag?tgcagcccgt?cttacaccgt?gcggcacagg?cactagtact?gatgtcgtct 13200

acagggcttt?tgatatttac?aacgaaaaag?ttgctggttt?tgcaaagttc?ctaaaaacta 13260

attgctgtcg?cttccaggag?aaggatgagg?aaggcaattt?attagactct?tactttgtag 13320

ttaagaggca?tactatgtct?aactaccaac?atgaagagac?tatttataac?ttggttaaag 13380

attgtccagc?ggttgctgtc?catgactttt?tcaagtttag?agtagatggt?gacatggtac 13440

cacatatatc?acgtcagcgt?ctaactaaat?acacaatggc?tgatttagtc?tatgctctac 13500

gtcattttga?tgagggtaat?tgtgatacat?taaaagaaat?actcgtcaca?tacaattgct 13560

gtgatgatga?ttatttcaat?aagaaggatt?ggtatgactt?cgtagagaat?cctgacatct 13620

tacgcgtata?tgctaactta?ggtgagcgtg?tacgccaatc?attattaaag?actgtacaat 13680

tctgcgatgc?tatgcgtgat?gcaggcattg?taggcgtact?gacattagat?aatcaggatc 13740

ttaatgggaa?ctggtacgat?ttcggtgatt?tcgtacaagt?agcaccaggc?tgcggagttc 13800

ctattgtgga?ttcatattac?tcattgctga?tgcccatcct?cactttgact?agggcattgg 13860

ctgctgagtc?ccatatggat?gctgatctcg?caaaaccact?tattaagtgg?gatttgctga 13920

aatatgattt?tacggaagag?agactttgtc?tcttcgaccg?ttattttaaa?tattgggacc 13980

agacatacca?tcccaattgt?attaactgtt?tggatgatag?gtgtatcctt?cattgtgcaa 14040

actttaatgt?gttattttct?actgtgtttc?cacctacaag?ttttggacca?ctagtaagaa 14100

aaatatttgt?agatggtgtt?ccttttgttg?tttcaactgg?ataccatttt?cgtgagttag 14160

gagtcgtaca?taatcaggat?gtaaacttac?atagctcgcg?tctcagtttc?aaggaacttt 14220

tagtgtatgc?tgctgatcca?gctatgcatg?cagcttctgg?caatttattg?ctagataaac 14280

gcactacatg?cttttcagta?gctgcactaa?caaacaatgt?tgcttttcaa?actgtcaaac 14340

ccggtaattt?taataaagac?ttttatgact?ttgctgtgtc?taaaggtttc?tttaaggaag 14400

gaagttctgt?tgaactaaaa?cacttcttct?ttgctcagga?tggcaacgct?gctatcagtg 14460

attatgacta?ttatcgttat?aatctgccaa?caatgtgtga?tatcagacaa?ctcctattcg 14520

tagttgaagt?tgttgataaa?tactttgatt?gttacgatgg?tggctgtatt?aatgccaacc 14580

aagtaatcgt?taacaatctg?gataaatcag?ctggtttccc?atttaataaa?tggggtaagg 14640

ctagacttta?ttatgactca?atgagttatg?aggatcaaga?tgcacttttc?gcgtatacta 14700

agcgtaatgt?catccctact?ataactcaaa?tgaatcttaa?gtatgccatt?agtgcaaaga 14760

atagagctcg?caccgtagct?ggtgtctcta?tctgtagtac?tatgacaaat?agacagtttc 14820

atcagaaatt?attgaagtca?atagccgcca?ctagaggagc?tactgtggta?attggaacaa 14880

gcaagtttta?cggtggctgg?cataatatgt?taaaaactgt?ttacagtgat?gtagaaactc 14940

cacaccttat?gggttgggat?tatccaaaat?gtgacagagc?catgcctaac?atgcttagga 15000

taatggcctc?tcttgttctt?gctcgcaaac?ataacacttg?ctgtaactta?tcacaccgtt 15060

tctacaggtt?agctaacgag?tgtgcgcaag?tattaagtga?gatggtcatg?tgtggcggct 15120

cactatatgt?taaaccaggt?ggaacatcat?ccggtgatgc?tacaactgct?tatgctaata 15180

gtgtctttaa?catttgtcaa?gctgttacag?ccaatgtaaa?tgcacttctt?tcaactgatg 15240

gtaataagat?agctgacaag?tatgtccgca?atctacaaca?caggctctat?gagtgtctct 15300

atagaaatag?ggatgttgat?catgaattcg?tggatgagtt?ttacgcttac?ctgcgtaaac 15360

atttctccat?gatgattctt?tctgatgatg?ccgttgtgtg?ctataacagt?aactatgcgg 15420

ctcaaggttt?agtagctagc?attaagaact?ttaaggcagt?tctttattat?caaaataatg 15480

tgttcatgtc?tgaggcaaaa?tgttggactg?agactgacct?tactaaagga?cctcacgaat 15540

tttgctcaca?gcatacaatg?ctagttaaac?aaggagatga?ttacgtgtac?ctgccttacc 15600

cagatccatc?aagaatatta?ggcgcaggct?gttttgtcga?tgatattgtc?aaaacagatg 15660

gtacacttat?gattgaaagg?ttcgtgtcac?tggctattga?tgcttaccca?cttacaaaac 15720

atcctaatca?ggagtatgct?gatgtctttc?acttgtattt?acaatacatt?agaaagttac 15780

atgatgagct?tactggccac?atgttggaca?tgtattccgt?aatgctaact?aatgataaca 15840

cctcacggta?ctgggaacct?gagttttatg?aggctatgta?cacaccacat?acagtcttgc 15900

aggctgtagg?tgcttgtgta?ttgtgcaatt?cacagacttc?acttcgttgc?ggtgcctgta 15960

ttaggagacc?attcctatgt?tgcaagtgct?gctatgacca?tgtcatttca?acatcacaca 16020

aattagtgtt?gtctgttaat?ccctatgttt?gcaatgcccc?aggttgtgat?gtcactgatg 16080

tgacacaact?gtatctagga?ggtatgagct?attattgcaa?gtcacataag?cctcccatta 16140

gttttccatt?atgtgctaat?ggtcaggttt?ttggtttata?caaaaacaca?tgtgtaggca 16200

gtgacaatgt?cactgacttc?aatgcgatag?caacatgtga?ttggactaat?gctggcgatt 16260

acatacttgc?caacacttgt?actgagagac?tcaagctttt?cgcagcagaa?acgctcaaag 16320

ccactgagga?aacatttaag?ctgtcatatg?gtattgccac?tgtacgcgaa?gtactctctg 16380

acagagaatt?gcatctttca?tgggaggttg?gaaaacctag?accaccattg?aacagaaact 16440

atgtctttac?tggttaccgt?gtaactaaaa?atagtaaagt?acagattgga?gagtacacct 16500

ttgaaaaagg?tgactatggt?gatgctgttg?tgtacagagg?tactacgaca?tacaagttga 16560

atgttggtga?ttactttgtg?ttgacatctc?acactgtaat?gccacttagt?gcacctactc 16620

tagtgccaca?agagcactat?gtgagaatta?ctggcttgta?cccaacactc?aacatctcag 16680

atgagttttc?tagcaatgtt?gcaaattatc?aaaaggtcgg?catgcaaaag?tactctacac 16740

tccaaggacc?acctggtact?ggtaagagtc?attttgccat?cggacttgct?ctctattacc 16800

catctgctcg?catagtgtat?acggcatgct?ctcatgcagc?tgttgatgcc?ctatgtgaaa 16860

aggcattaaa?atatttgccc?atagataaat?gtagtagaat?catacctgcg?cgtgcgcgcg 16920

tagagtgttt?tgataaattc?aaagtgaatt?caacactaga?acagtatgtt?ttctgcactg 16980

taaatgcatt?gccagaaaca?actgctgaca?ttgtagtctt?tgatgaaatc?tctatggcta 17040

ctaattatga?cttgagtgtt?gtcaatgcta?gacttcgtgc?aaaacactac?gtctatattg 17100

gcgatcctgc?tcaattacca?gccccccgca?cattgctgac?taaaggcaca?ctagaaccag 17160

aatattttaa?ttcagtgtgc?agacttatga?aaacaatagg?tccagacatg?ttccttggaa 17220

cttgtcgccg?ttgtcctgct?gaaattgttg?acactgtgag?tgctttagtt?tatgacaata 17280

agctaaaagc?acacaaggat?aagtcagctc?aatgcttcaa?aatgttctac?aaaggtgtta 17340

ttacacatga?tgtttcatct?gcaatcaaca?gacctcaaat?aggcgttgta?agagaatttc 17400

ttacacgcaa?tcctgcttgg?agaaaagctg?tttttatctc?accttataat?tcacagaacg 17460

ctgtagcttc?aaaaatctta?ggattgccta?cgcagactgt?tgattcatca?cagggttctg 17520

aatatgacta?tgtcatattc?acacaaacta?ctgaaacagc?acactcttgt?aatgtcaacc 17580

gcttcaatgt?ggctatcaca?agggcaaaaa?ttggcatttt?gtgcataatg?tctgatagag 17640

atctttatga?caaactgcaa?tttacaagtc?tagaaatacc?acgtcgcaat?gtggctacat 17700

tacaagcaga?aaatgtaact?ggacttttta?aggactgtag?taagatcatt?actggtcttc 17760

atcctacaca?ggcacctaca?cacctcagcg?ttgatataaa?gttcaagact?gaaggattat 17820

gtgttgacat?accaggcata?ccaaaggaca?tgacctaccg?tagactcatc?tctatgatgg 17880

gtttcaaaat?gaattaccaa?gtcaatggtt?accctaatat?gtttatcacc?cgcgaagaag 17940

ctattcgtca?cgttcgtgcg?tggattggct?ttgatgtaga?gggctgtcat?gcaactagag 18000

atgctgtggg?tactaaccta?cctctccagc?taggattttc?tacaggtgtt?aacttagtag 18060

ctgtaccgac?tggttatgtt?gacactgaaa?ataacacaga?attcaccaga?gttaatgcaa 18120

aacctccacc?aggtgaccag?tttaaacatc?ttataccact?catgtataaa?ggcttgccct 18180

ggaatgtagt?gcgtattaag?atagtacaaa?tgctcagtga?tacactgaaa?ggattgtcag 18240

acagagtcgt?gttcgtcctt?tgggcgcatg?gctttgagct?tacatcaatg?aagtactttg 18300

tcaagattgg?acctgaaaga?acgtgttgtc?tgtgtgacaa?acgtgcaact?tgcttttcta 18360

cttcatcaga?tacttatgcc?tgctggaatc?attctgtggg?ttttgactat?gtctataacc 18420

catttatgat?tgatgttcag?cagtggggct?ttacgggtaa?ccttcagagt?aaccatgacc 18480

aacattgcca?ggtacatgga?aatgcacatg?tggctagttg?tgatgctatc?atgactagat 18540

gtttagcagt?ccatgagtgc?tttgttaagc?gcgttgattg?gtctgttgaa?taccctatta 18600

taggagatga?actgagggtt?aattctgctt?gcagaaaagt?acaacacatg?gttgtgaagt 18660

ctgcattgct?tgctgataag?tttccagttc?ttcatgacat?tggaaatcca?aaggctatca 18720

agtgtgtgcc?tcaggctgaa?gtagaatgga?agttctacga?tgctcagcca?tgtagtgaca 18780

aagcttacaa?aatagaggaa?ctcttctatt?cttatgctac?acatcacgat?aaattcactg 18840

atggtgtttg?tttgttttgg?aattgtaacg?ttgatcgtta?cccagccaat?gcaattgtgt 18900

gtaggtttga?cacaagagtc?ttgtcaaact?tgaacttacc?aggctgtgat?ggtggtagtt 18960

tgtatgtgaa?taagcatgca?ttccacactc?cagctttcga?taaaagtgca?tttactaatt 19020

taaagcaatt?gcctttcttt?tactattctg?atagtccttg?tgagtctcat?ggcaaacaag 19080

tagtgtcgga?tattgattat?gttccactca?aatctgctac?gtgtattaca?cgatgcaatt 19140

taggtggtgc?tgtttgcaga?caccatgcaa?atgagtaccg?acagtacttg?gatgcatata 19200

atatgatgat?ttctgctgga?tttagcctat?ggatttacaa?acaatttgat?acttataacc 19260

tgtggaatac?atttaccagg?ttacagagtt?tagaaaatgt?ggcttataat?gttgttaata 19320

aaggacactt?tgatggacac?gccggcgaag?cacctgtttc?catcattaat?aatgctgttt 19380

acacaaaggt?agatggtatt?gatgtggaga?tctttgaaaa?taagacaaca?cttcctgtta 19440

atgttgcatt?tgagctttgg?gctaagcgta?acattaaacc?agtgccagag?attaagatac 19500

tcaataattt?gggtgttgat?atcgctgcta?atactgtaat?ctgggactac?aaaagagaag 19560

ccccagcaca?tgtatctaca?ataggtgtct?gcacaatgac?tgacattgcc?aagaaaccta 19620

ctgagagtgc?ttgttcttca?cttactgtct?tgtttgatgg?tagagtggaa?ggacaggtag 19680

acctttttag?aaacgcccgt?aatggtgttt?taataacaga?aggttcagtc?aaaggtctaa 19740

caccttcaaa?gggaccagca?caagctagcg?tcaatggagt?cacattaatt?ggagaatcag 19800

taaaaacaca?gtttaactac?tttaagaaag?tagacggcat?tattcaacag?ttgcctgaaa 19860

cctactttac?tcagagcaga?gacttagagg?attttaagcc?cagatcacaa?atggaaactg 19920

actttctcga?gctcgctatg?gatgaattca?tacagcgata?taagctcgag?ggctatgcct 19980

tcgaacacat?cgtttatgga?gatttcagtc?atggacaact?tggcggtctt?catttaatga 20040

taggcttagc?caagcgctca?caagattcac?cacttaaatt?agaggatttt?atccctatgg 20100

acagcacagt?gaaaaattac?ttcataacag?atgcgcaaac?aggttcatca?aaatgtgtgt 20160

gttctgtgat?tgatctttta?cttgatgact?ttgtcgagat?aataaagtca?caagatttgt 20220

cagtgatttc?aaaagtggtc?aaggttacaa?ttgactatgc?tgaaatttca?ttcatgcttt 20280

ggtgtaagga?tggacatgtt?gaaaccttct?acccaaaact?acaagcaagt?caagcgtggc 20340

aaccaggtgt?tgcgatgcct?aacttgtaca?agatgcaaag?aatgcttctt?gaaaagtgtg 20400

accttcagaa?ttatggtgaa?aatgctgtta?taccaaaagg?aataatgatg?aatgtcgcaa 20460

agtatactca?actgtgtcaa?tacttaaata?cacttacttt?agctgtaccc?tacaacatga 20520

gagttattca?ctttggtgct?ggctctgata?aaggagttgc?accaggtaca?gctgtgctca 20580

gacaatggtt?gccaactggc?acactacttg?tcgattcaga?tcttaatgac?ttcgtctccg 20640

acgcagattc?tactttaatt?ggagactgtg?caacagtaca?tacggctaat?aaatgggacc 20700

ttattattag?cgatatgtat?gaccctagga?ccaaacatgt?gacaaaagag?aatgactcta 20760

aagaagggtt?tttcacttat?ctgtgtggat?ttataaagca?aaaactagcc?ctgggtggtt 20820

ctatagctgt?aaagataaca?gagcattctt?ggaatgctga?cctttacaag?cttatgggcc 20880

atttctcatg?gtggacagct?tttgttacaa?atgtaaatgc?atcatcatcg?gaagcatttt 20940

taattggggc?taactatctt?ggcaagccga?aggaacaaat?tgatggctat?accatgcatg 21000

ctaactacat?tttctggagg?aacacaaatc?ctatccagtt?gtcttcctat?tcactctttg 21060

acatgagcaa?atttcctctt?aaattaagag?gaactgctgt?aatgtctctt?aaggagaatc 21120

aaatcaatga?tatgatttat?tctcttctgg?aaaaaggtag?gcttatcatt?agagaaaaca 21180

acagagttgt?ggtttcaagt?gatattcttg?ttaacaacta?a 21221

<210>32

<211>297

<212>DNA

＜213〉coronavirus

<400>32

atggacccca?atcaaaccaa?cgtagtgccc?cccgcattac?atttggtgga?cccacagatt 60

caactgacaa?taaccagaat?ggaggacgca?atggggcaag?gccaaaacag?cgccgacccc 120

aaggtttacc?caataatact?gcgtcttggt?tcacagctct?cactcagcat?ggcaaggagg 180

aacttagatt?ccctcgaggc?cagggcgttc?caatcaacac?caatagtggt?ccagatgacc 240

aaattggcta?ctaccgaaga?gctacccgac?gagttcgtgg?tggtgacggc?aaaatga 297

<210>33

<211>98

<212>PRT

＜213〉coronavirus

<400>33

Met?Asp?Pro?Asn?Gln?Thr?Asn?Val?Val?Pro?Pro?Ala?Leu?His?Leu?Val

1 5 10 15

Asp?Pro?Gln?Ile?Gln?Leu?Thr?Ile?Thr?Arg?Met?Glu?Asp?Ala?Met?Gly

20 25 30

Gln?Gly?Gln?Asn?Ser?Ala?Asp?Pro?Lys?Val?Tyr?Pro?Ile?Ile?Leu?Arg

35 40 45

Leu?Gly?Ser?Gln?Leu?Ser?Leu?Ser?Met?Ala?Arg?Arg?Asn?Leu?Asp?Ser

50 55 60

Leu?Glu?Ala?Arg?Ala?Phe?Gln?Ser?Thr?Pro?Ile?Val?Val?Gln?Met?Thr

65 70 75 80

Lys?Leu?Ala?Thr?Thr?Glu?Glu?Leu?Pro?Asp?Glu?Phe?Val?Val?Val?Thr

85 90 95

Ala?Lys

<210>34

<211>213

<212>DNA

＜213〉coronavirus

<400>34

atgctgccac?cgtgctacaa?cttcctcaag?gaacaacatt?gccaaaaggc?ttctacgcag 60

agggaagcag?aggcggcagt?caagcctctt?ctcgctcctc?atcacgtagt?cgcggtaatt 120

caagaaattc?aactcctggc?agcagtaggg?gaaattctcc?tgctcgaatg?gctagcggag 180

gtggtgaaac?tgccctcgcg?ctattgctgc?tag 213

<210>35

<211>70

<212>PRT

＜213〉coronavirus

<400>35

Met?Leu?Pro?Pro?Cys?Tyr?Asn?Phe?Leu?Lys?Glu?Gln?His?Cys?Gln?Lys

1 5 10 15

Ala?Ser?Thr?Gln?Arg?Glu?Ala?Glu?Ala?Ala?Val?Lys?Pro?Leu?Leu?Ala

20 25 30

Pro?His?His?Val?Val?Ala?Val?Ile?Gln?Glu?Ile?Gln?Leu?Leu?Ala?Ala

35 40 45

Val?Gly?Glu?Ile?Leu?Leu?Leu?Glu?Trp?Leu?Ala?Glu?Val?Val?Lys?Leu

50 55 60

Pro?Ser?Arg?Tyr?Cys?Cys

65 70

<210>36

<211>1377

<212>DNA

＜213〉coronavirus

<220>

<221>CDS

<222>(67)..(1335)

<223>

<400>36

atgaaggtca?ccaaactgct?gcatttagag?acgtacttgt?tgttttaaat?aaacgaacaa 60

attaaa atg?tct?gat?aat?gga?ccc?caa?tca?aac?caa?cgt?agt?gcc?ccc 108

Met?Ser?Asp?Asn?Gly?Pro?Gln?Ser?Asn?Gln?Arg?Ser?Ala?Pro

1 5 10

cgc?att?aca?ttt?ggt?gga?ccc?aca?gat?tca?act?gac?aat?aac?cag?aat 156

Arg?Ile?Thr?Phe?Gly?Gly?Pro?Thr?Asp?Ser?Thr?Asp?Asn?Asn?Gln?Asn

15 20 25 30

gga?gga?cgc?aat?ggg?gca?agg?cca?aaa?cag?cgc?cga?ccc?caa?ggt?tta 204

Gly?Gly?Arg?Asn?Gly?Ala?Arg?Pro?Lys?Gln?Arg?Arg?Pro?Gln?Gly?Leu

35 40 45

ccc?aat?aat?act?gcg?tct?tgg?ttc?aca?gct?ctc?act?cag?cat?ggc?aag 252

Pro?Asn?Asn?Thr?Ala?Ser?Trp?Phe?Thr?Ala?Leu?Thr?Gln?His?Gly?Lys

50 55 60

gag?gaa?ctt?aga?ttc?cct?cga?ggc?cag?ggc?gtt?cca?atc?aac?acc?aat 300

Glu?Glu?Leu?Arg?Phe?Pro?Arg?Gly?Gln?Gly?Val?Pro?Ile?Asn?Thr?Asn

65 70 75

agt?ggt?cca?gat?gac?caa?att?ggc?tac?tac?cga?aga?gct?acc?cga?cga 348

Ser?Gly?Pro?Asp?Asp?Gln?Ile?Gly?Tyr?Tyr?Arg?Arg?Ala?Thr?Arg?Arg

80 85 90

gtt?cgt?ggt?ggt?gac?ggc?aaa?atg?aaa?gag?ctc?agc?ccc?aga?tgg?tac 396

Val?Arg?Gly?Gly?Asp?Gly?Lys?Met?Lys?Glu?Leu?Ser?Pro?Arg?Trp?Tyr

95 100 105 110

ttc?tat?tac?cta?gga?act?ggc?cca?gaa?gct?tca?ctt?ccc?tac?ggc?gct 444

Phe?Tyr?Tyr?Leu?Gly?Thr?Gly?Pro?Glu?Ala?Ser?Leu?Pro?Tyr?Gly?Ala

115 120 125

aac?aaa?gaa?ggc?atc?gta?tgg?gtt?gca?act?gag?gga?gcc?ttg?aat?aca 492

Asn?Lys?Glu?Gly?Ile?Val?Trp?Val?Ala?Thr?Glu?Gly?Ala?Leu?Asn?Thr

130 135 140

ccc?aaa?gac?cac?att?ggc?acc?cgc?aat?cct?aat?aac?aat?gct?gcc?acc 540

Pro?Lys?Asp?His?Ile?Gly?Thr?Arg?Asn?Pro?Asn?Asn?Asn?Ala?Ala?Thr

145 150 155

gtg?cta?caa?ctt?cct?caa?gga?aca?aca?ttg?cca?aaa?ggc?ttc?tac?gca 588

Val?Leu?Gln?Leu?Pro?Gln?Gly?Thr?Thr?Leu?Pro?Lys?Gly?Phe?Tyr?Ala

160 165 170

gag?gga?agc?aga?ggc?ggc?agt?caa?gcc?tct?tct?cgc?tcc?tca?tca?cgt 636

Glu?Gly?Ser?Arg?Gly?Gly?Ser?Gln?Ala?Ser?Ser?Arg?Ser?Ser?Ser?Arg

175 180 185 190

agt?cgc?ggt?aat?tca?aga?aat?tca?act?cct?ggc?agc?agt?agg?gga?aat 684

Ser?Arg?Gly?Asn?Ser?Arg?Asn?Ser?Thr?Pro?Gly?Ser?Ser?Arg?Gly?Asn

195 200 205

tct?cct?gct?cga?atg?gct?agc?gga?ggt?ggt?gaa?act?gcc?ctc?gcg?cta 732

Ser?Pro?Ala?Arg?Met?Ala?Ser?Gly?Gly?Gly?Glu?Thr?Ala?Leu?Ala?Leu

210 215 220

ttg?ctg?cta?gac?aga?ttg?aac?cag?ctt?gag?agc?aaa?gtt?tct?ggt?aaa 780

Leu?Leu?Leu?Asp?Arg?Leu?Asn?Gln?Leu?Glu?Ser?Lys?Val?Ser?Gly?Lys

225 230 235

ggc?caa?caa?caa?caa?ggc?caa?act?gtc?act?aag?aaa?tct?gct?gct?gag 828

Gly?Gln?Gln?Gln?Gln?Gly?Gln?Thr?Val?Thr?Lys?Lys?Ser?Ala?Ala?Glu

240 245 250

gca?tct?aaa?aag?cct?cgc?caa?aaa?cgt?act?gcc?aca?aaa?cag?tac?aac 876

Ala?Ser?Lys?Lys?Pro?Arg?Gln?Lys?Arg?Thr?Ala?Thr?Lys?Gln?Tyr?Asn

255 260 265 270

gtc?act?caa?gca?ttt?ggg?aga?cgt?ggt?cca?gaa?caa?acc?caa?gga?aat 924

Val?Thr?Gln?Ala?Phe?Gly?Arg?Arg?Gly?Pro?Glu?Gln?Thr?Gln?Gly?Asn

275 280 285

ttc?ggg?gac?caa?gac?cta?atc?aga?caa?gga?act?gat?tac?aaa?cat?tgg 972

Phe?Gly?Asp?Gln?Asp?Leu?Ile?Arg?Gln?Gly?Thr?Asp?Tyr?Lys?His?Trp

290 295 300

ccg?caa?att?gca?caa?ttt?gct?cca?agt?gcc?tct?gca?ttc?ttt?gga?atg 1020

Pro?Gln?Ile?Ala?Gln?Phe?Ala?Pro?Ser?Ala?Ser?Ala?Phe?Phe?Gly?Met

305 310 315

tca?cgc?att?ggc?atg?gaa?gtc?aca?cct?tcg?gga?aca?tgg?ctg?act?tat 1068

Ser?Arg?Ile?Gly?Met?Glu?Val?Thr?Pro?Ser?Gly?Thr?Trp?Leu?Thr?Tyr

320 325 330

cat?gga?gcc?att?aaa?ttg?gat?gac?aaa?gat?cca?caa?ttc?aaa?gac?aac 1116

His?Gly?Ala?Ile?Lys?Leu?Asp?Asp?Lys?Asp?Pro?Gln?Phe?Lys?Asp?Asn

335 340 345 350

gtc?ata?ctg?ctg?aac?aag?cac?att?gac?gca?tac?aaa?aca?ttc?cca?cca 1164

Val?Ile?Leu?Leu?Asn?Lys?His?Ile?Asp?Ala?Tyr?Lys?Thr?Phe?Pro?Pro

355 360 365

aca?gag?cct?aaa?aag?gac?aaa?aag?aaa?aag?act?gat?gaa?gct?cag?cct 1212

Thr?Glu?Pro?Lys?Lys?Asp?Lys?Lys?Lys?Lys?Thr?Asp?Glu?Ala?Gln?Pro

370 375 380

ttg?ccg?cag?aga?caa?aag?aag?cag?ccc?act?gtg?act?ctt?ctt?cct?gcg 1260

Leu?Pro?Gln?Arg?Gln?Lys?Lys?Gln?Pro?Thr?Val?Thr?Leu?Leu?Pro?Ala

385 390 395

gct?gac?atg?gat?gat?ttc?tcc?aga?caa?ctt?caa?aat?tcc?atg?agt?gga 1308

Ala?Asp?Met?Asp?Asp?Phe?Ser?Arg?Gln?Leu?Gln?Asn?Ser?Met?Ser?Gly

400 405 410

gct?tct?gct?gat?tca?act?cag?gca?taa?acactcatga?tgaccacaca 1355

Ala?Ser?Ala?Asp?Ser?Thr?Gln?Ala

415 420

aggcagatgg?gctatgtaaa?cg 1377

<210>37

<211>422

<212>PRT

＜213〉coronavirus

<400>37

Met?Ser?Asp?Asn?Gly?Pro?Gln?Ser?Asn?Gln?Arg?Ser?Ala?Pro?Arg?Ile

1 5 10 15

Thr?Phe?Gly?Gly?Pro?Thr?Asp?Ser?Thr?Asp?Asn?Asn?Gln?Asn?Gly?Gly

20 25 30

Arg?Asn?Gly?Ala?Arg?Pro?Lys?Gln?Arg?Arg?Pro?Gln?Gly?Leu?Pro?Asn

35 40 45

Asn?Thr?Ala?Ser?Trp?Phe?Thr?Ala?Leu?Thr?Gln?His?Gly?Lys?Glu?Glu

50 55 60

Leu?Arg?Phe?Pro?Arg?Gly?Gln?Gly?Val?Pro?Ile?Asn?Thr?Asn?Ser?Gly

65 70 75 80

Pro?Asp?Asp?Gln?Ile?Gly?Tyr?Tyr?Arg?Arg?Ala?Thr?Arg?Arg?Val?Arg

85 90 95

Gly?Gly?Asp?Gly?Lys?Met?Lys?Glu?Leu?Ser?Pro?Arg?Trp?Tyr?Phe?Tyr

100 105 110

Tyr?Leu?Gly?Thr?Gly?Pro?Glu?Ala?Ser?Leu?Pro?Tyr?Gly?Ala?Asn?Lys

115 120 125

Glu?Gly?Ile?Val?Trp?Val?Ala?Thr?Glu?Gly?Ala?Leu?Asn?Thr?Pro?Lys

130 135 140

Asp?His?Ile?Gly?Thr?Arg?Asn?Pro?Asn?Asn?Asn?Ala?Ala?Thr?Val?Leu

145 150 155 160

Gln?Leu?Pro?Gln?Gly?Thr?Thr?Leu?Pro?Lys?Gly?Phe?Tyr?Ala?Glu?Gly

165 170 175

Ser?Arg?Gly?Gly?Ser?Gln?Ala?Ser?Ser?Arg?Ser?Ser?Ser?Arg?Ser?Arg

180 185 190

Gly?Asn?Ser?Arg?Asn?Ser?Thr?Pro?Gly?Ser?Ser?Arg?Gly?Asn?Ser?Pro

195 200 205

Ala?Arg?Met?Ala?Ser?Gly?Gly?Gly?Glu?Thr?Ala?Leu?Ala?Leu?Leu?Leu

210 215 220

Leu?Asp?Arg?Leu?Asn?Gln?Leu?Glu?Ser?Lys?Val?Ser?Gly?Lys?Gly?Gln

225 230 235 240

Gln?Gln?Gln?Gly?Gln?Thr?Val?Thr?Lys?Lys?Ser?Ala?Ala?Glu?Ala?Ser

245 250 255

Lys?Lys?Pro?Arg?Gln?Lys?Arg?Thr?Ala?Thr?Lys?Gln?Tyr?Asn?Val?Thr

260 265 270

Gln?Ala?Phe?Gly?Arg?Arg?Gly?Pro?Glu?Gln?Thr?Gln?Gly?Asn?Phe?Gly

275 280 285

Asp?Gln?Asp?Leu?Ile?Arg?Gln?Gly?Thr?Asp?Tyr?Lys?His?Trp?Pro?Gln

290 295 300

Ile?Ala?Gln?Phe?Ala?Pro?Ser?Ala?Ser?Ala?Phe?Phe?Gly?Met?Ser?Arg

305 310 315 320

Ile?Gly?Met?Glu?Val?Thr?Pro?Ser?Gly?Thr?Trp?Leu?Thr?Tyr?His?Gly

325 330 335

Ala?Ile?Lys?Leu?Asp?Asp?Lys?Asp?Pro?Gln?Phe?Lys?Asp?Asn?Val?Ile

340 345 350

Leu?Leu?Asn?Lys?His?Ile?Asp?Ala?Tyr?Lys?Thr?Phe?Pro?Pro?Thr?Glu

355 360 365

Pro?Lys?Lys?Asp?Lys?Lys?Lys?Lys?Thr?Asp?Glu?Ala?Gln?Pro?Leu?Pro

370 375 380

Gln?Arg?Gln?Lys?Lys?Gln?Pro?Thr?Val?Thr?Leu?Leu?Pro?Ala?Ala?Asp

385 390 395 400

Met?Asp?Asp?Phe?Ser?Arg?Gln?Leu?Gln?Asn?Ser?Met?Ser?Gly?Ala?Ser

405 410 415

Ala?Asp?Ser?Thr?Gln?Ala

420

<210>38

<211>1377

<212>DNA

＜213〉coronavirus

<400>38

atgaaggtca?ccaaactgct?gcatttagag?acgtacttgt?tgttttaaat?aaacgaacaa 60

attaaaatgt?ctgataatgg?accccaatca?aaccaacgta?gtgccccccg?cattacattt 120

ggtggaccca?cagattcaac?tgacaataac?cagaatggag?gacgcaatgg?ggcaaggcca 180

aaacagcgcc?gaccccaagg?tttacccaat?aatactgcgt?cttggttcac?agctctcact 240

cagcatggca?aggaggaact?tagattccct?cgaggccagg?gcgttccaat?caacaccaat 300

agtggtccag?atgaccaaat?tggctactac?cgaagagcta?cccgacgagt?tcgtggtggt 360

gacggcaaaa?tgaaagagct?cagccccaga?tggtacttct?attacctagg?aactggccca 420

gaagcttcac?ttccctacgg?cgctaacaaa?gaaggcatcg?tatgggttgc?aactgaggga 480

gccttgaata?cacccaaaga?ccacattggc?acccgcaatc?ctaataacaa?tgctgccacc 540

gtgctacaac?ttcctcaagg?aacaacattg?ccaaaaggct?tctacgcaga?gggaagcaga 600

ggcggcagtc?aagcctcttc?tcgctcctca?tcacgtagtc?gcggtaattc?aagaaattca 660

actcctggca?gcagtagggg?aaattctcct?gctcgaatgg?ctagcggagg?tggtgaaact 720

gccctcgcgc?tattgctgct?agacagattg?aaccagcttg?agagcaaagt?ttctggtaaa 780

ggccaacaac?aacaaggcca?aactgtcact?aagaaatctg?ctgctgaggc?atctaaaaag 840

cctcgccaaa?aacgtactgc?cacaaaacag?tacaacgtca?ctcaagcatt?tgggagacgt 900

ggtccagaac?aaacccaagg?aaatttcggg?gaccaagacc?taatcagaca?aggaactgat 960

tacaaacatt?ggccgcaaat?tgcacaattt?gctccaagtg?cctctgcatt?ctttggaatg 1020

tcacgcattg?gcatggaagt?cacaccttcg?ggaacatggc?tgacttatca?tggagccatt 1080

aaattggatg?acaaagatcc?acaattcaaa?gacaacgtca?tactgctgaa?caagcacatt 1140

gacgcataca?aaacattccc?accaacagag?cctaaaaagg?acaaaaagaa?aaagactgat 1200

gaagctcagc?ctttgccgca?gagacaaaag?aagcagccca?ctgtgactct?tcttcctgcg 1260

gctgacatgg?atgatttctc?cagacaactt?caaaattcca?tgagtggagc?ttctgctgat 1320

tcaactcagg?cataaacact?catgatgacc?acacaaggca?gatgggctat?gtaaacg 1377

<210>39

<211>204

<212>DNA

＜213〉coronavirus

<400>39

atattaggtt?tttacctacc?caggaaaagc?caaccaacct?cgatctcttg?tagatctgtt 60

ctctaaacga?actttaaaat?ctgtgtagct?gtcgctcggc?tgcatgccta?gtgcacctac 120

gcagtataaa?caataataaa?ttttactgtc?gttgacaaga?aacgagtaac?tcgtccctct 180

tctgcagact?gcttacggtt?tcgt 204

<210>40

<211>809

<212>DNA

＜213〉coronavirus

<400>40

actcaagcat?ttgggagacg?tggtccagaa?caaacccaag?gaaatttcgg?ggaccaagac 60

ctaatcagac?aaggaactga?ttacaaacat?tggccgcaaa?ttgcacaatt?tgctccaagt 120

gcctctgcat?tctttggaat?gtcacgcatt?ggcatggaag?tcacaccttc?gggaacatgg 180

ctgacttatc?atggagccat?taaattggat?gacaaagatc?cacaattcaa?agacaacgtc 240

atactgctga?acaagcacat?tgacgcatac?aaaacattcc?caccaacaga?gcctaaaaag 300

gacaaaaaga?aaaagactga?tgaagctcag?cctttgccgc?agagacaaaa?gaagcagccc 360

actgtgactc?ttcttcctgc?ggctgacatg?gatgatttct?ccagacaact?tcaaaattcc 420

atgagtggag?cttctgctga?ttcaactcag?gcataaacac?tcatgatgac?cacacaaggc 480

agatgggcta?tgtaaacgtt?ttcgcaattc?cgtttacgat?acatagtcta?ctcttgtgca 540

gaatgaattc?tcgtaactaa?acagcacaag?taggtttagt?taactttaat?ctcacatagc 600

aatctttaat?caatgtgtaa?cattagggag?gacttgaaag?agccaccaca?ttttcatcga 660

ggccacgcgg?agtacgatcg?agggtacagt?gaataatgct?agggagagct?gcctatatgg 720

aagagcccta?atgtgtaaaa?ttaattttag?tagtgctatc?cccatgtgat?tttaatagct 780

tcttaggaga?atgacaaaaa?aaaaaaaaa 809

<210>41

<211>448

<212>DNA

＜213〉coronavirus

<400>41

aatgaacaca?tagggctgtt?caagctgggg?cagtacgcct?ttttccagct?ctactagacc 60

acaagtgcca?tttttgaggt?gttcacgtgc?ctccgatagg?gcctcttcca?cagagtcccc 120

gaagccacgc?actagcacgt?ctctaacctg?aaggacaggc?aaactgagtt?ggacgtgtgt 180

tttctcgttg?acaccaagaa?caaggctctc?catcttacct?ttcggtcaca?cccggacgaa 240

acctaggtat?gctgatgatc?gactgcaaca?cggacgaaac?cgtaagcagt?ctgcagaaga 300

gggacgagtt?actcgtttct?tgtcaacgac?agtaaaattt?attattgttt?atactgcgta 360

ggtgcactag?gcatgcagcc?gagcgacagc?tacacagatt?ttaaagttcg?tttagagaac 420

agatctacaa?gagatcgagg?ttggttgg 448

<210>42

<211>2033

<212>DNA

＜213〉coronavirus

<400>42

atacctaggt?ttcgtccggg?tgtgaccgaa?aggtaagatg?gagagccttg?ttcttggtgt 60

caacgagaaa?acacacgtcc?aactcagttt?gcctgtcctt?caggttagag?acgtgctagt 120

gcgtggcttc?ggggactctg?tggaagaggc?cctatcggag?gcacgtgaac?acctcaaaaa 180

tggcacttgt?ggtctagtag?agctggaaaa?aggcgtactg?ccccagcttg?aacagcccta 240

tgtgttcatt?aaacgttctg?atgccttaag?caccaatcac?ggccacaagg?tcgttgagct 300

ggttgcagaa?atggacggca?ttcagtacgg?tcgtagcggt?ataacactgg?gagtactcgt 360

gccacatgtg?ggcgaaaccc?caattgcata?ccgcaatgtt?cttcttcgta?agaacggtaa 420

taagggagcc?ggtggtcata?gctatggcat?cgatctaaag?tcttatgact?taggtgacga 480

gcttggcact?gatcccattg?aagattatga?acaaaactgg?aacactaagc?atggcagtgg 540

tgcactccgt?gaactcactc?gtgagctcaa?tggaggtgca?gtcactcgct?atgtcgacaa 600

caatttctgt?ggcccagatg?ggtaccctct?tgattgcatc?aaagattttc?tcgcacgcgc 660

gggcaagtca?atgtgcactc?tttccgaaca?acttgattac?atcgagtcga?agagaggtgt 720

ctactgctgc?cgtgaccatg?agcatgaaat?tgcctggttc?actgagcgct?ctgataagag 780

ctacgagcac?cagacaccct?tcgaaattaa?gagtgccaag?aaatttgaca?ctttcaaagg 840

ggaatgccca?aagtttgtgt?ttcctcttaa?ctcaaaagtc?aaagtcattc?aaccacgtgt 900

tgaaaagaaa?aagactgagg?gtttcatggg?gcgtatacgc?ctctgtgtac?ctgttgcatc 960

tccacaggag?tgtaacaata?tgcacttgtc?taccttgatg?aaatgtaatc?attgcgatga 1020

agtttcatgg?cagacgtgcg?actttctgaa?agccacttgt?gaacattgtg?gcactgaaaa 1080

tttagttatt?gaaggaccta?ctacatgtgg?gtacctacct?actaatgctg?tagtgaaaat 1140

gccatgtcct?gcctgtcaag?acccagagat?tggacctgag?catagtgttg?cagattatca 1200

caaccactca?aacattgaaa?ctcgactccg?caagggaggt?aggactagat?gttttggagg 1260

ctgtgtgttt?gcctatgttg?gctgctataa?taagcgtgcc?tactgggttc?ctcgtgctag 1320

tgctgatatt?ggctcaggcc?atactggcat?tactggtgac?aatgtggaga?ccttgaatga 1380

ggatctcctt?gagatactga?gtcgtgaacg?tgttaacatt?aacattgttg?gcgattttca 1440

tttgaatgaa?gaggttgcca?tcattttggc?atctttctct?gcttctacaa?gtgcctttat 1500

tgacactata?aagagtcttg?attacaagtc?tttcaaaacc?attgttgagt?cctgcggtaa 1560

ctataaagtt?accaagggaa?agcccgtaaa?aggtgcttgg?aacattggac?aacagagatc 1620

agttttaaca?ccactgtgtg?gttttccctc?acaggctgct?ggtgttatca?gatcaatttt 1680

tgcgcgcaca?cttgatgcag?caaaccactc?aattcctgat?ttgcaaagag?cagctgtcac 1740

catacttgat?ggtatttctg?aacagtcatt?acgtcttgtc?gacgccatgg?tttatacttc 1800

agacctgctc?accaacagtg?tcattattat?ggcatatgta?actggtggtc?ttgtacaaca 1860

gacttctcag?tggttgtcta?atcttttggg?cactactgtt?gaaaaactca?ggcctatctt 1920

tgaatggatt?gaggcgaaac?ttagtgcagg?agttgaattt?ctcaaggatg?cttgggagat 1980

tctcaaattt?ctcattacag?gtgtttttga?catcgtcaag?ggtcaaatac?agg 2033

<210>43

<211>2018

<212>DNA

＜213〉coronavirus

<400>43

ggattgaggc?gaaacttagt?gcaggagttg?aatttctcaa?ggatgcttgg?gagattctca 60

aatttctcat?tacaggtgtt?tttgacatcg?tcaagggtca?aatacaggtt?gcttcagata 120

acatcaagga?ttgtgtaaaa?tgcttcattg?atgttgttaa?caaggcactc?gaaatgtgca 180

ttgatcaagt?cactatcgct?ggcgcaaagt?tgcgatcact?caacttaggt?gaagtcttca 240

tcgctcaaag?caagggactt?taccgtcagt?gtatacgtgg?caaggagcag?ctgcaactac 300

tcatgcctct?taaggcacca?aaagaagtaa?cctttcttga?aggtgattca?catgacacag 360

tacttacctc?tgaggaggtt?gttctcaaga?acggtgaact?cgaagcactc?gagacgcccg 420

ttgatagctt?cacaaatgga?gctatcgttg?gcacaccagt?ctgtgtaaat?ggcctcatgc 480

tcttagagat?taaggacaaa?gaacaatact?gcgcattgtc?tcctggttta?ctggctacaa 540

acaatgtctt?tcgcttaaaa?gggggtgcac?caattaaagg?tgtaaccttt?ggagaagata 600

ctgtttggga?agttcaaggt?tacaagaatg?tgagaatcac?atttgagctt?gatgaacgtg 660

ttgacaaagt?gcttaatgaa?aagtgctctg?tctacactgt?tgaatccggt?accgaagtta 720

ctgagtttgc?atgtgttgta?gcagaggctg?ttgtgaagac?tttacaacca?gtttctgatc 780

tccttaccaa?catgggtatt?gatcttgatg?agtggagtgt?agctacattc?tacttatttg 840

atgatgctgg?tgaagaaaac?ttttcatcac?gtatgtattg?ttccttttac?cctccagatg 900

aggaagaaga?ggacgatgca?gagtgtgagg?aagaagaaat?tgatgaaacc?tgtgaacatg 960

agtacggtac?agaggatgat?tatcaaggtc?tccctctgga?atttggtgcc?tcagctgaaa 1020

cagttcgagt?tgaggaagaa?gaagaggaag?actggctgga?tgatactact?gagcaatcag 1080

agattgagcc?agaaccagaa?cctacacctg?aagaaccagt?taatcagttt?actggttatt 1140

taaaacttac?tgacaatgtt?gccattaaat?gtgttgacat?cgttaaggag?gcacaaagtg 1200

ctaatcctat?ggtgattgta?aatgctgcta?acatacacct?gaaacatggt?ggtggtgtag 1260

caggtgcact?caacaaggca?accaatggtg?ccatgcaaaa?ggagagtgat?gattacatta 1320

agctaaatgg?ccctcttaca?gtaggagggt?cttgtttgct?ttctggacat?aatcttgcta 1380

agaagtgtct?gcatgttgtt?ggacctaacc?taaatgcagg?tgaggacatc?cagcttctta 1440

aggcagcata?tgaaaatttc?aattcacagg?acatcttact?tgcaccattg?ttgtcagcag 1500

gcatatttgg?tgctaaacca?cttcagtctt?tacaagtgtg?cgtgcagacg?gttcgtacac 1560

aggtttatat?tgcagtcaat?gacaaagctc?tttatgagca?ggttgtcatg?gattatcttg 1620

ataacctgaa?gcctagagtg?gaagcaccta?aacaagagga?gccaccaaac?acagaagatt 1680

ccaaaactga?ggagaaatct?gtcgtacaga?agcctgtcga?tgtgaagcca?aaaattaagg 1740

cctgcattga?tgaggttacc?acaacactgg?aagaaactaa?gtttcttacc?aataagttac 1800

tcttgtttgc?tgatatcaat?ggtaagcttt?accatgattc?tcagaacatg?cttagaggtg 1860

aagatatgtc?tttccttgag?aaggatgcac?cttacatggt?aggtgatgtt?atcactagtg 1920

gtgatatcac?ttgtgttgta?ataccctcca?aaaaggctgg?tggcactact?gagatgctct 1980

caagagcttt?gaagaaagtg?ccagttgatg?agtatata 2018

<210>44

<211>1442

<212>DNA

＜213〉coronavirus

<400>44

ttgatgaggt?taccacaaca?ctggaagaaa?ctaagtttct?taccaataag?ttactcttgt 60

ttgctgatat?caatggtaag?ctttaccatg?attctcagaa?catgcttaga?ggtgaagata 120

tgtctttcct?tgagaaggat?gcaccttaca?tggtaggtga?tgttatcact?agtggtgata 180

tcacttgtgt?tgtaataccc?tccaaaaagg?ctggtggcac?tactgagatg?ctctcaagag 240

ctttgaagaa?agtgccagtt?gatgagtata?taaccacgta?ccctggacaa?ggatgtgctg 300

gttatacact?tgaggaagct?aagactgctc?ttaagaaatg?caaatctgca?ttttatgtac 360

taccttcaga?agcacctaat?gctaaggaag?agattctagg?aactgtatcc?tggaatttga 420

gagaaatgct?tgctcatgct?gaagagacaa?gaaaattaat?gcctatatgc?atggatgtta 480

gagccataat?ggcaaccatc?caacgtaagt?ataaaggaat?taaaattcaa?gagggcatcg 540

ttgactatgg?tgtccgattc?ttcttttata?ctagtaaaga?gcctgtagct?tctattatta 600

cgaagctgaa?ctctctaaat?gagccgcttg?tcacaatgcc?aattggttat?gtgacacatg 660

gttttaatct?tgaagaggct?gcgcgctgta?tgcgttctct?taaagctcct?gccgtagtgt 720

cagtatcatc?accagatgct?gttactacat?ataatggata?cctcacttcg?tcatcaaaga 780

catctgagga?gcactttgta?gaaacagttt?ctttggctgg?ctcttacaga?gattggtcct 840

attcaggaca?gcgtacagag?ttaggtgttg?aatttcttaa?gcgtggtgac?aaaattgtgt 900

accacactct?ggagagcccc?gtcgagtttc?atcttgacgg?tgaggttctt?tcacttgaca 960

aactaaagag?tctcttatcc?ctgcgggagg?ttaagactat?aaaagtgttc?acaactgtgg 1020

acaacactaa?tctccacaca?cagcttgtgg?atatgtctat?gacatatgga?cagcagtttg 1080

gtccaacata?cttggatggt?gctgatgtta?caaaaattaa?acctcatgta?aatcatgagg 1140

gtaagacttt?ctttgtacta?cctagtgatg?acacactacg?tagtgaagct?ttcgagtact 1200

accatactct?tgatgagagt?tttcttggta?ggtacatgtc?tgctttaaac?cacacaaaga 1260

aatggaaatt?tcctcaagtt?ggtggtttaa?cttcaattaa?atgggctgat?aacaattgtt 1320

atttgtctag?tgttttatta?gcacttcaac?agcttgaagt?caaattcaat?gcaccagcac 1380

ttcaagaggc?ttattataga?gcccgtgctg?gtgatgctgc?taacttttgt?gcactcatac 1440

tc 1442

<210>45

<211>1050

<212>DNA

＜213〉coronavirus

<400>45

atatgtctat?gacatatgga?cagcagtttg?gtccaacata?cttggatggt?gctgatgtta 60

caaaaattaa?acctcatgta?aatcatgagg?gtaagacttt?ctttgtacta?cctagtgatg 120

acacactacg?tagtgaagct?ttcgagtact?accatactct?tgatgagagt?tttcttggta 180

ggtacatgtc?tgctttaaac?cacacaaaga?aatggaaatt?tcctcaagtt?ggtggtttaa 240

cttcaattaa?atgggctgat?aacaattgtt?atttgtctag?tgttttatta?gcacttcaac 300

agcttgaagt?caaattcaat?gcaccagcac?ttcaagaggc?ttattataga?gcccgtgctg 360

gtgatgctgc?taacttttgt?gcactcatac?tcgcttacag?taataaaact?gttggcgagc 420

ttggtgatgt?cagagaaact?atgacccatc?ttctacagca?tgctaatttg?gaatctgcaa 480

agcgagttct?taatgtggtg?tgtaaacatt?gtggtcagaa?aactactacc?ttaacgggtg 540

tagaagctgt?gatgtatatg?ggtactctat?cttatgataa?tcttaagaca?ggtgtttcca 600

ttccatgtgt?gtgtggtcgt?gatgctacac?aatatctagt?acaacaagag?tcttcttttg 660

ttatgatgtc?tgcaccacct?gctgagtata?aattacagca?aggtacattc?ttatgtgcga 720

atgagtacac?tggtaactat?cagtgtggtc?attacactca?tataactgct?aaggagaccc 780

tctatcgtat?tgacggagct?caccttacaa?agatgtcaga?gtacaaagga?ccagtgactg 840

atgttttcta?caaggaaaca?tcttacacta?caaccatcaa?gcctgtgtcg?tataaactcg 900

atggagttac?ttacacagag?attgaaccaa?aattggatgg?gtattataaa?aaggataatg 960

cttactatac?agagcagcct?atagaccttg?taccaactca?accattacca?aatgcgagtt 1020

ttgataattt?caaactcaca?tgttctaaca 1050

<210>46

<211>1995

<212>DNA

＜213〉coronavirus

<400>46

tttgtgcact?catactcgct?tacagtaata?aaactgttgg?cgagcttggt?gatgtcagag 60

aaactatgac?ccatcttcta?cagcatgcta?atttggaatc?tgcaaagcga?gttcttaatg 120

tggtgtgtaa?acattgtggt?cagaaaacta?ctaccttaac?gggtgtagaa?gctgtgatgt 180

atatgggtac?tctatcttat?gataatctta?agacaggtgt?ttccattcca?tgtgtgtgtg 240

gtcgtgatgc?tacacaatat?ctagtacaac?aagagtcttc?ttttgttatg?atgtctgcac 300

cacctgctga?gtataaatta?cagcaaggta?cattcttatg?tgcgaatgag?tacactggta 360

actatcagtg?tggtcattac?actcatataa?ctgctaagga?gaccctctat?cgtattgacg 420

gagctcacct?tacaaagatg?tcagagtaca?aaggaccagt?gactgatgtt?ttctacaagg 480

aaacatctta?cactacaacc?atcaagcctg?tgtcgtataa?actcgatgga?gttacttaca 540

cagagattga?accaaaattg?gatgggtatt?ataaaaagga?taatgcttac?tatacagagc 600

agcctataga?ccttgtacca?actcaaccat?taccaaatgc?gagttttgat?aatttcaaac 660

tcacatgttc?taacacaaaa?tttgctgatg?atttaaatca?aatgacaggc?ttcacaaagc 720

cagcttcacg?agagctatct?gtcacattct?tcccagactt?gaatggcgat?gtagtggcta 780

ttgactatag?acactattca?gcgagtttca?agaaaggtgc?taaattactg?cataagccaa 840

ttgtttggca?cattaaccag?gctacaacca?agacaacgtt?caaaccaaac?acttggtgtt 900

tacgttgtct?ttggagtaca?aagccagtag?atacttcaaa?ttcatttgaa?gttctggcag 960

tagaagacac?acaaggaatg?gacaatcttg?cttgtgaaag?tcaacaaccc?acctctgaag 1020

aagtagtgga?aaatcctacc?atacagaagg?aagtcataga?gtgtgacgtg?aaaactaccg 1080

aagttgtagg?caatgtcata?cttaaaccat?cagatgaagg?tgttaaagta?acacaagagt 1140

taggtcatga?ggatcttatg?gctgcttatg?tggaaaacac?aagcattacc?attaagaaac 1200

ctaatgagct?ttcactagcc?ttaggtttaa?aaacaattgc?cactcatggt?attgctgcaa 1260

ttaatagtgt?tccttggagt?aaaattttgg?cttatgtcaa?accattctta?ggacaagcag 1320

caattacaac?atcaaattgc?gctaagagat?tagcacaacg?tgtgtttaac?aattatatgc 1380

cttatgtgtt?tacattattg?ttccaattgt?gtacttttac?taaaagtacc?aattctagaa 1440

ttagagcttc?actacctaca?actattgcta?aaaatagtgt?taagagtgtt?gctaaattat 1500

gtttggatgc?cggcattaat?tatgtgaagt?cacccaaatt?ttctaaattg?ttcacaatcg 1560

ctatgtggct?attgttgtta?agtatttgct?taggttctct?aatctgtgta?actgctgctt 1620

ttggtgtact?cttatctaat?tttggtgctc?cttcttattg?taatggcgtt?agagaattgt 1680

atcttaattc?gtctaacgtt?actactatgg?atttctgtga?aggttctttt?ccttgcagca 1740

tttgtttaag?tggattagac?tcccttgatt?cttatccagc?tcttgaaacc?attcaggtga 1800

cgatttcatc?gtacaagcta?gacttgacaa?ttttaggtct?ggccgctgag?tgggttttgg 1860

catatatgtt?gttcacaaaa?ttcttttatt?tattaggtct?ttcagctata?atgcaggtgt 1920

tctttggcta?ttttgctagt?catttcatca?gcaattcttg?gctcatgtgg?tttatcatta 1980

gtattgtaca?aatgg 1995

<210>47

<211>1884

<212>DNA

＜213〉coronavirus

<400>47

aattcttggc?tcatgtggtt?tatcattagt?attgtacaaa?tggcacccgt?ttctgcaatg 60

gttaggatgt?acatcttctt?tgcttctttc?tactacatat?ggaagagcta?tgttcatatc 120

atggatggtt?gcacctcttc?gacttgcatg?atgtgctata?agcgcaatcg?tgccacacgc 180

gttgagtgta?caactattgt?taatggcatg?aagagatctt?tctatgtcta?tgcaaatgga 240

ggccgtggct?tctgcaagac?tcacaattgg?aattgtctca?attgtgacac?attttgcact 300

ggtagtacat?tcattagtga?tgaagttgct?cgtgatttgt?cactccagtt?taaaagacca 360

atcaacccta?ctgaccagtc?atcgtatatt?gttgatagtg?ttgctgtgaa?aaatggcgcg 420

cttcacctct?actttgacaa?ggctggtcaa?aagacctatg?agagacatcc?gctctcccat 480

tttgtcaatt?tagacaattt?gagagctaac?aacactaaag?gttcactgcc?tattaatgtc 540

atagtttttg?atggcaagtc?caaatgcgac?gagtctgctt?ctaagtctgc?ttctgtgtac 600

tacagtcagc?tgatgtgcca?acctattctg?ttgcttgacc?aagctcttgt?atcagacgtt 660

ggagatagta?ctgaagtttc?cgttaagatg?tttgatgctt?atgtcgacac?cttttcagca 720

acttttagtg?ttcctatgga?aaaacttaag?gcacttgttg?ctacagctca?cagcgagtta 780

gcaaagggtg?tagctttaga?tggtgtcctt?tctacattcg?tgtcagctgc?ccgacaaggt 840

gttgttgata?ccgatgttga?cacaaaggat?gttattgaat?gtctcaaact?ttcacatcac 900

tctgacttag?aagtgacagg?tgacagttgt?aacaatttca?tgctcaccta?taataaggtt 960

gaaaacatga?cgcccagaga?tcttggcgca?tgtattgact?gtaatgcaag?gcatatcaat 1020

gcccaagtag?caaaaagtca?caatgtttca?ctcatctgga?atgtaaaaga?ctacatgtct 1080

ttatctgaac?agctgcgtaa?acaaattcgt?agtgctgcca?agaagaacaa?catacctttt 1140

agactaactt?gtgctacaac?tagacaggtt?gtcaatgtca?taactactaa?aatctcactc 1200

aagggtggta?agattgttag?tacttgtttt?aaacttatgc?ttaaggccac?attattgtgc 1260

gttcttgctg?cattggtttg?ttatatcgtt?atgccagtac?atacattgtc?aatccatgat 1320

ggttacacaa?atgaaatcat?tggttacaaa?gccattcagg?atggtgtcac?tcgtgacatc 1380

atttctactg?atgattgttt?tgcaaataaa?catgctggtt?ttgacgcatg?gtttagccag 1440

cgtggtggtt?catacaaaaa?tgacaaaagc?tgccctgtag?tagctgctat?cattacaaga 1500

gagattggtt?tcatagtgcc?tggcttaccg?ggtactgtgc?tgagagcaat?caatggtgac 1560

ttcttgcatt?ttctacctcg?tgtttttagt?gctgttggca?acatttgcta?cacaccttcc 1620

aaactcattg?agtatagtga?ttttgctacc?tctgcttgcg?ttcttgctgc?tgagtgtaca 1680

atttttaagg?atgctatggg?caaacctgtg?ccatattgtt?atgacactaa?tttgctagag 1740

ggttctattt?cttatagtga?gcttcgtcca?gacactcgtt?atgtgcttat?ggatggttcc 1800

atcatacagt?ttcctaacac?ttacctggag?ggttctgtta?gagtagtaac?aacttttgat 1860

gctgagtact?gtagacatgg?taca 1884

<210>48

<211>2020

<212>DNA

＜213〉coronavirus

<400>48

cactcgttat?gtgcttatgg?atggttccat?catacagttt?cctaacactt?acctggaggg 60

ttctgttaga?gtagtaacaa?cttttgatgc?tgagtactgt?agacatggta?catgcgaaag 120

gtcagaagta?ggtatttgcc?tatctaccag?tggtagatgg?gttcttaata?atgagcatta 180

cagagctcta?tcaggagttt?tctgtggtgt?tgatgcgatg?aatctcatag?ctaacatctt 240

tactcctctt?gtgcaacctg?tgggtgcttt?agatgtgtct?gcttcagtag?tggctggtgg 300

tattattgcc?atattggtga?cttgtgctgc?ctactacttt?atgaaattca?gacgtgtttt 360

tggtgagtac?aaccatgttg?ttgctgctaa?tgcacttttg?tttttgatgt?ctttcactat 420

actctgtctg?gtaccagctt?acagctttct?gccgggagtc?tactcagtct?tttacttgta 480

cttgacattc?tatttcacca?atgatgtttc?attcttggct?caccttcaat?ggtttgccat 540

gttttctcct?attgtgcctt?tttggataac?agcaatctat?gtattctgta?tttctctgaa 600

gcactgccat?tggttcttta?acaactatct?taggaaaaga?gtcatgttta?atggagttac 660

atttagtacc?ttcgaggagg?ctgctttgtg?tacctttttg?ctcaacaagg?aaatgtacct 720

aaaattgcgt?agcgagacac?tgttgccact?tacacagtat?aacaggtatc?ttgctctata 780

taacaagtac?aagtatttca?gtggagcctt?agatactacc?agctatcgtg?aagcagcttg 840

ctgccactta?gcaaaggctc?taaatgactt?tagcaactca?ggtgctgatg?ttctctacca 900

accaccacag?acatcaatca?cttctgctgt?tctgcagagt?ggttttagga?aaatggcatt 960

cccgtcaggc?aaagttgaag?ggtgcatggt?acaagtaacc?tgtggaacta?caactcttaa 1020

tggattgtgg?ttggatgaca?cagtatactg?tccaagacat?gtcatttgca?cagcagaaga 1080

catgcttaat?cctaactatg?aagatctgct?cattcgcaaa?tccaaccata?gctttcttgt 1140

tcaggctggc?aatgttcaac?ttcgtgttat?tggccattct?atgcaaaatt?gtctgcttag 1200

gcttaaagtt?gatacttcta?accctaagac?acccaagtat?aaatttgtcc?gtatccaacc 1260

tggtcaaaca?ttttcagttc?tagcatgcta?caatggttca?ccatctggtg?tttatcagtg 1320

tgccatgaga?cctaatcata?ccattaaagg?ttctttcctt?aatggatcat?gtggtagtgt 1380

tggttttaac?attgattatg?attgcgtgtc?tttctgctat?atgcatcata?tggagcttcc 1440

aacaggagta?cacgctggta?ctgacttaga?aggtaaattc?tatggtccat?ttgttgacag 1500

acaaactgca?caggctgcag?gtacagacac?aaccataaca?ttaaatgttt?tggcatggct 1560

gtatgctgct?gttatcaatg?gtgataggtg?gtttcttaat?agattcacca?ctactttgaa 1620

tgactttaac?cttgtggcaa?tgaagtacaa?ctatgaacct?ttgacacaag?atcatgttga 1680

catattggga?cctctttctg?ctcaaacagg?aattgccgtc?ttagatatgt?gtgctgcttt 1740

gaaagagctg?ctgcagaatg?gtatgaatgg?tcgtactatc?cttggtagca?ctattttaga 1800

agatgagttt?acaccatttg?atgttgttag?acaatgctct?ggtgttacct?tccaaggtaa 1860

gttcaagaaa?attgttaagg?gcactcatca?ttggatgctt?ttaactttct?tgacatcact 1920

attgattctt?gttcaaagta?cacagtggtc?actgtttttc?tttgtttacg?agaatgcttt 1980

cttgccattt?actcttggta?ttatggcaat?tgctgcatgt 2020

<210>49

<211>2040

<212>DNA

＜213〉coronavirus

<400>49

agcatttcca?gcctgaagac?gtactgtagc?agctaaactg?cccagcacca?tacctctatt 60

taggttgttt?aagcctttga?tgaagtacaa?gtatttcact?ttaggccctt?ttggtgtgtc 120

tgtaacaaac?ctacaaggtg?gttccagttc?tgtgtaaatt?gtacctgtac?catcactctt 180

agggaatcta?gcccatttga?gatcttggtg?gtctgatagt?aatgccagca?caaacctacc 240

tcccttcgaa?ttgttatagt?aggcaagtgc?attgtcatca?gtacaagctg?tttgtgtggt 300

accagccgca?caggacatct?gtcgtagtgc?tactggactc?agttcattat?tctgtagttt 360

aacagctgag?ttggctctta?gagctgtaac?aataagaggc?caagccaaat?ttggtgaatt 420

gtccatgtta?atttcactaa?gttgaacaat?cttgctatcc?gcatcaacaa?cttgctggat 480

ttcccagagt?gcagatgcat?atgtaaaggt?gttaccatca?caagtgttct?tgtaggtacc 540

ataatcaggg?acaacaacca?tgagtttggc?tgctgtagtc?aatggtatga?tgttgagtgg 600

aacacaacca?tcacgcgcat?tgttgataat?gttgttaagt?gcatcattat?caagcttcct 660

aagcatagtg?aagagcattg?tttgcatagc?actagttact?tttgccctct?tgtcctcaga 720

tcttgcctgt?ttgtacattt?gggtcatagc?ctgatctgcc?atcttttcca?acttgcgttg 780

catggcagca?tcacggtcaa?actcagattt?agccacattc?aaagatttct?ttaacttttt 840

gagaacgact?tcagaatcac?cattagctac?agcctgctca?taggcctcct?gggcagtggc 900

ataagcggca?tatgatggta?aagaactaaa?ttctgaagca?atagcctgaa?gagtagcacg 960

gttatcgagc?atttcctcgc?acaacctatt?aatgtctaca?gcaccctgca?tggatagcaa 1020

aacagacaaa?agagaaacca?tcttctcgaa?agcttcagtt?gtgtcttttg?caagaagaat 1080

atcattgtgg?agttgtacac?attgtgccca?caatttagaa?gatgactcta?ctctaagttg 1140

ttgaagaacc?gagagcagta?ccacagatgt?gcactttacg?tcagacattt?tagactgtac 1200

agtagcaacc?ttgatacatg?gtttacctcc?aatacccaac?aacttaatgt?taagcttgaa 1260

agcatcaata?ctactcttag?gaggcaaaag?cccctgggag?ttcatatacc?taaattcttg 1320

tgtagagacc?aagtagtcat?aaacaccaag?agtaagcctg?aagtaacggt?tgagtaaaca 1380

gaaaaggcca?aagtagcagc?agcaacaata?gcctaagaaa?caataaacaa?gcatgataca 1440

ctgtaaggtg?ttgccagtaa?taaataacaa?tgggtaatac?tcaacacaca?caaacactat 1500

agctctagct?aaaaacatga?tagtcgtaac?gacaccagaa?tagttagagg?ttacagaaat 1560

aactaaggcc?cacatggaaa?tagcttgatc?taaagcatta?ccatagtaga?ctttgtaaac 1620

aagtgtaatg?acattcatca?gtgtccaaac?acgtctagca?gcatcatcat?aaacagtgcg 1680

agctgtcatg?agaataagca?aaactaaagc?tgaagcatac?ataacacaat?ccttaagcct 1740

ataaccagac?aagctagtgt?cagccaattc?aagccatgtc?atgatacgca?tcacccagct 1800

agcaggcatg?tagaccatat?taaagtaagc?aactgttgca?agagaaggta?acagaaacaa 1860

gcacaagaat?gcgtgcttat?gcttaacaag?cagcatagca?catgcagcaa?ttgccataat 1920

accaagagta?aatggcaaga?aagcattctc?gtaaacaaag?aaaaacagtg?accactgtgt 1980

actttgaaca?agaatcaata?gtgatgtcaa?gaaagttaaa?agcatccaat?gatgagtgca 2040

<210>50

<211>2012

<212>DNA

＜213〉coronavirus

<400>50

cttgtaggtt?tgttacagac?acaccaaaag?ggcctaaagt?gaaatacttg?tacttcatca 60

aaggcttaaa?caacctaaat?agaggtatgg?tgctgggcag?tttagctgct?acagtacgtc 120

ttcaggctgg?aaatgctaca?gaagtacctg?ccaattcaac?tgtgctttcc?ttctgtgctt 180

ttgcagtaga?ccctgctaaa?gcatataagg?attacctagc?aagtggagga?caaccaatca 240

ccaactgtgt?gaagatgttg?tgtacacaca?ctggtacagg?acaggcaatt?actgtaacac 300

cagaagctaa?catggaccaa?gagtcctttg?gtggtgcttc?atgttgtctg?tattgtagat 360

gccacattga?ccatccaaat?cctaaaggat?tctgtgactt?gaaaggtaag?tacgtccaaa 420

tacctaccac?ttgtgctaat?gacccagtgg?gttttacact?tagaaacaca?gtctgtaccg 480

tctgcggaat?gtggaaaggt?tatggctgta?gttgtgacca?actccgcgaa?cccttgatgc 540

agtctgcgga?tgcatcaacg?tttttaaacg?ggtttgcggt?gtaagtgcag?cccgtcttac 600

accgtgcggc?acaggcacta?gtactgatgt?cgtctacagg?gcttttgata?tttacaacga 660

aaaagttgct?ggttttgcaa?agttcctaaa?aactaattgc?tgtcgcttcc?aggagaagga 720

tgaggaaggc?aatttattag?actcttactt?tgtagttaag?aggcatacta?tgtctaacta 780

ccaacatgaa?gagactattt?ataacttggt?taaagattgt?ccagcggttg?ctgtccatga 840

ctttttcaag?tttagagtag?atggtgacat?ggtaccacat?atatcacgtc?agcgtctaac 900

taaatacaca?atggctgatt?tagtctatgc?tctacgtcat?tttgatgagg?gtaattgtga 960

tacattaaaa?gaaatactcg?tcacatacaa?ttgctgtgat?gatgattatt?tcaataagaa 1020

ggattggtat?gacttcgtag?agaatcctga?catcttacgc?gtatatgcta?acttaggtga 1080

gcgtgtacgc?caatcattat?taaagactgt?acaattctgc?gatgctatgc?gtgatgcagg 1140

cattgtaggc?gtactgacat?tagataatca?ggatcttaat?gggaactggt?acgatttcgg 1200

tgatttcgta?caagtagcac?caggctgcgg?agttcctatt?gtggattcat?attactcatt 1260

gctgatgccc?atcctcactt?tgactagggc?attggctgct?gagtcccata?tggatgctga 1320

tctcgcaaaa?ccacttatta?agtgggattt?gctgaaatat?gattttacgg?aagagagact 1380

ttgtctcttc?gaccgttatt?ttaaatattg?ggaccagaca?taccatccca?attgtattaa 1440

ctgtttggat?gataggtgta?tccttcattg?tgcaaacttt?aatgtgttat?tttctactgt 1500

gtttccacct?acaagttttg?gaccactagt?aagaaaaata?tttgtagatg?gtgttccttt 1560

tgttgtttca?actggatacc?attttcgtga?gttaggagtc?gtacataatc?aggatgtaaa 1620

cttacatagc?tcgcgtctca?gtttcaagga?acttttagtg?tatgctgctg?atccagctat 1680

gcatgcagct?tctggcaatt?tattgctaga?taaacgcact?acatgctttt?cagtagctgc 1740

actaacaaac?aatgttgctt?ttcaaactgt?caaacccggt?aattttaata?aagactttta 1800

tgactttgct?gtgtctaaag?gtttctttaa?ggaaggaagt?tctgttgaac?taaaacactt 1860

cttctttgct?caggatggca?acgctgctat?cagtgattat?gactattatc?gttataatct 1920

gccaacaatg?tgtgatatca?gacaactcct?attcgtagtt?gaagttgttg?ataaatactt 1980

tgattgttac?gatggtggct?gtattaatgc?ca 2012

<210>51

<211>1877

<212>DNA

＜213〉coronavirus

<400>51

gtacttcgcg?tacagtggca?ataccatatg?acagcttaaa?tgtttcctca?gtggctttga 60

gcgtttctgc?tgcgaaaagc?ttgagtctct?cagtacaagt?gttggcaagt?atgtaatcgc 120

cagcattagt?ccaatcacat?gttgctatcg?cattgaagtc?agtgacattg?tcactgccta 180

cacatgtgtt?tttgtataaa?ccaaaaacct?gaccattagc?acataatgga?aaactaatgg 240

gaggcttatg?tgacttgcaa?taatagctca?tacctcctag?atacagttgt?gtcacatcag 300

tgacatcaca?acctggggca?ttgcaaacat?agggattaac?agacaacact?aatttgtgtg 360

atgttgaaat?gacatggtca?tagcagcact?tgcaacatag?gaatggtctc?ctaatacagg 420

caccgcaacg?aagtgaagtc?tgtgaattgc?acaatacaca?agcacctaca?gcctgcaaga 480

ctgtatgtgg?tgtgtacata?gcctcataaa?actcaggttc?ccagtaccgt?gaggtgttat 540

cattagttag?cattacggaa?tacatgtcca?acatgtggcc?agtaagctca?tcatgtaact 600

ttctaatgta?ttgtaaatac?aagtgaaaga?catcagcata?ctcctgatta?ggatgttttg 660

taagtgggta?agcatcaata?gccagtgaca?cgaacctttc?aatcataagt?gtaccatctg 720

ttttgacaat?atcatcgaca?aaacagcctg?cgcctaatat?tcttgatgga?tctgggtaag 780

gcaggtacac?gtaatcatct?ccttgtttaa?ctagcattgt?atgctgtgag?caaaattcgt 840

gaggtccttt?agtaaggtca?gtctcagtcc?aacattttgc?ctcagacatg?aacacattat 900

tttgataata?aagaactgcc?ttaaagttct?taatgctagc?tactaaacct?tgagccgcat 960

agttactgtt?atagcacaca?acggcatcat?cagaaagaat?catcatggag?aaatgtttac 1020

gcaggtaagc?gtaaaactca?tccacgaatt?catgatcaac?atccctattt?ctatagagac 1080

actcatagag?cctgtgttgt?agattgcgga?catacttgtc?agctatctta?ttaccatcag 1140

ttgaaagaag?tgcatttaca?ttggctgtaa?cagcttgaca?aatgttaaag?acactattag 1200

cataagcagt?tgtagcatca?ccggatgatg?ttccacctgg?tttaacatat?agtgagccgc 1260

cacacatgac?catctcactt?aatacttgcg?cacactcgtt?agctaacctg?tagaaacggt 1320

gtgataagtt?acagcaagtg?ttatgtttgc?gagcaagaac?aagagaggcc?attatcctaa 1380

gcatgttagg?catggctctg?tcacattttg?gataatccca?acccataagg?tgtggagttt 1440

ctacatcact?gtaaacagtt?tttaacatat?tatgccagcc?accgtaaaac?ttgcttgttc 1500

caattaccac?agtagctcct?ctagtggcgg?ctattgactt?caataatttc?tgatgaaact 1560

gtctatttgt?catagtacta?cagatagaga?caccagctac?ggtgcgagct?ctattctttg 1620

cactaatggc?atacttaaga?ttcatttgag?ttatagtagg?gatgacatta?cgcttagtat 1680

acgcgaaaag?tgcatcttga?tcctcataac?tcattgagtc?ataataaagt?ctagccttac 1740

cccatttatt?aaatgggaaa?ccagctgatt?tatccagatt?gttaacgatt?acttggttgg 1800

cattaataca?gccaccatcg?taacaatcaa?agtatttatc?aacaacttca?actacgaata 1860

ggagttgtct?gatatca 1877

<210>52

<211>2051

<212>DNA

＜213〉coronavirus

<400>52

tcaggtccaa?tcttgacaaa?gtacttcatt?gatgtaagct?caaagccatg?cgcccaaagg 60

acgaacacga?ctctgtctga?caatcctttc?agtgtatcac?tgagcatttg?tactatctta 120

atacgcacta?cattccaggg?caagccttta?tacatgagtg?gtataagatg?tttaaactgg 180

tcacctggtg?gaggttttgc?attaactctg?gtgaattctg?tgttattttc?agtgtcaaca 240

taaccagtcg?gtacagctac?taagttaaca?cctgtagaaa?atcctagctg?gagaggtagg 300

ttagtaccca?cagcatctct?agttgcatga?cagccctcta?catcaaagcc?aatccacgca 360

cgaacgtgac?gaatagcttc?ttcgcgggtg?ataaacatat?tagggtaacc?attgacttgg 420

taattcattt?tgaaacccat?catagagatg?agtctacggt?aggtcatgtc?ctttggtatg 480

cctggtatgt?caacacataa?tccttcagtc?ttgaacttta?tatcaacgct?gaggtgtgta 540

ggtgcctgtg?taggatgaag?accagtaatg?atcttactac?agtccttaaa?aagtccagtt 600

acattttctg?cttgtaatgt?agccacattg?cgacgtggta?tttctagact?tgtaaattgc 660

agtttgtcat?aaagatctct?atcagacatt?atgcacaaaa?tgccaatttt?tgcccttgtg 720

atagccacat?tgaagcggtt?gacattacaa?gagtgtgctg?tttcagtagt?ttgtgtgaat 780

atgacatagt?catattcaga?accctgtgat?gaatcaacag?tctgcgtagg?caatcctaag 840

atttttgaag?ctacagcgtt?ctgtgaatta?taaggtgaga?taaaaacagc?ttttctccaa 900

gcaggattgc?gtgtaagaaa?ttctcttaca?acgcctattt?gaggtctgtt?gattgcagat 960

gaaacatcat?gtgtaataac?acctttgtag?aacattttga?agcattgagc?tgacttatcc 1020

ttgtgtgctt?ttagcttatt?gtcataaact?aaagcactca?cagtgtcaac?aatttcagca 1080

ggacaacggc?gacaagttcc?aaggaacatg?tctggaccta?ttgttttcat?aagtctgcac 1140

actgaattaa?aatattctgg?ttctagtgtg?cctttagtca?gcaatgtgcg?gggggctggt 1200

aattgagcag?gatcgccaat?atagacgtag?tgttttgcac?gaagtctagc?attgacaaca 1260

ctcaagtcat?aattagtagc?catagagatt?tcatcaaaga?ctacaatgtc?agcagttgtt 1320

tctggcaatg?catttacagt?gcagaaaaca?tactgttcta?gtgttgaatt?cactttgaat 1380

ttatcaaaac?actctacgcg?cgcacgcgca?ggtatgattc?tactacattt?atctatgggc 1440

aaatatttta?atgccttttc?acatagggca?tcaacagctg?catgagagca?tgccgtatac 1500

actatgcgag?cagatgggta?atagagagca?agtccgatgg?caaaatgact?cttaccagta 1560

ccaggtggtc?cttggagtgt?agagtacttt?tgcatgccga?ccttttgata?atttgcaaca 1620

ttgctagaaa?actcatctga?gatgttgagt?gttgggtaca?agccagtaat?tctcacatag 1680

tgctcttgtg?gcactagagt?aggtgcacta?agtggcatta?cagtgtgaga?tgtcaacaca 1740

aagtaatcac?caacattcaa?cttgtatgtc?gtagtacctc?tgtacacaac?agcatcacca 1800

tagtcacctt?tttcaaaggt?gtactctcca?atctgtactt?tactattttt?agttacacgg 1860

taaccagtaa?agacatagtt?tctgttcaat?ggtggtctag?gttttccaac?ctcccatgaa 1920

agatgcaatt?ctctgtcaga?gagtacttcg?cgtacagtgg?caataccata?tgacagctta 1980

aatgtttcct?cagtggcttt?gagcgtttct?gctgcgaaaa?gcttgagtct?ctcagtacaa 2040

gtgttggcaa?g 2051

<210>53

<211>2075

<212>DNA

＜213〉coronavirus

<400>53

tgcttgtagt?tttgggtaga?aggtttcaac?atgtccatcc?ttacaccaaa?gcatgaatga 60

aatttcagca?tagtcaattg?taaccttgac?cacttttgaa?atcactgaca?aatcttgtga 120

ctttattatc?tcgacaaagt?catcaagtaa?aagatcaatc?acagaacaca?cacattttga 180

tgaacctgtt?tgcgcatctg?ttatgaagta?atttttcact?gtgctgtcca?tagggataaa 240

atcctctaat?ttaagtggtg?aatcttgtga?gcgcttggct?aagcctatca?ttaaatgaag 300

accgccaagt?tgtccatgac?tgaaatctcc?ataaacgatg?tgttcgaagg?catagccctc 360

gagcttatat?cgctgtatga?attcatccat?agcgagctcg?agaaagtcag?tttccatttg 420

tgatctgggc?ttaaaatcct?ctaagtctct?gctctgagta?aagtaggttt?caggcaactg 480

ttgaataatg?ccgtctactt?tcttaaagta?gttaaactgt?gtttttactg?attctccaat 540

taatgtgact?ccattgacgc?tagcttgtgc?tggtcccttt?gaaggtgtta?gacctttgac 600

tgaaccttct?gttattaaaa?caccattacg?ggcgtttcta?aaaaggtcta?cctgtccttc 660

cactctacca?tcaaacaaga?cagtaagtga?agaacaagca?ctctcagtag?gtttcttggc 720

aatgtcagtc?attgtgcaga?cacctattgt?agatacatgt?gctggggctt?ctcttttgta 780

gtcccagatt?acagtattag?cagcgatatc?aacacccaaa?ttattgagta?tcttaatctc 840

tggcactggt?ttaatgttac?gcttagccca?aagctcaaat?gcaacattaa?caggaagtgt 900

tgtcttattt?tcaaagatct?ccacatcaat?accatctacc?tttgtgtaaa?cagcattatt 960

aatgatggaa?acaggtgctt?cgccggcgtg?tccatcaaag?tgtcctttat?taacaacatt 1020

ataagccaca?ttttctaaac?tctgtaacct?ggtaaatgta?ttccacaggt?tataagtatc 1080

aaattgtttg?taaatccata?ggctaaatcc?agcagaaatc?atcatattat?atgcatccaa 1140

gtactgtcgg?tactcatttg?catggtgtct?gcaaacagca?ccacctaaat?tgcatcgtgt 1200

aatacacgta?gcagatttga?gtggaacata?atcaatatcc?gacactactt?gtttgccatg 1260

agactcacaa?ggactatcag?aatagtaaaa?gaaaggcaat?tgctttaaat?tagtaaatgc 1320

acttttatcg?aaagctggag?tgtggaatgc?atgcttattc?acatacaaac?taccaccatc 1380

acagcctggt?aagttcaagt?ttgacaagac?tcttgtgtca?aacctacaca?caattgcatt 1440

ggctgggtaa?cgatcaacgt?tacaattcca?aaacaaacaa?acaccatcag?tgaatttatc 1500

gtgatgtgta?gcataagaat?agaagagttc?ctctattttg?taagctttgt?cactacatgg 1560

ctgagcatcg?tagaacttcc?attctacttc?agcctgaggc?acacacttga?tagcctttgg 1620

atttccaatg?tcatgaagaa?ctggaaactt?atcagcaagc?aatgcagact?tcacaaccat 1680

gtgttgtact?tttctgcaag?cagaattaac?cctcagttca?tctcctataa?tagggtattc 1740

aacagaccaa?tcaacgcgct?taacaaagca?ctcatggact?gctaaacatc?tagtcatgat 1800

agcatcacaa?ctagccacat?gtgcatttcc?atgtacctgg?caatgttggt?catggttact 1860

ctgaaggtta?cccgtaaagc?cccactgctg?aacatcaatc?ataaatgggt?tatagacata 1920

gtcaaaaccc?acagaatgat?tccagcaggc?ataagtatct?gatgaagtag?aaaagcaagt 1980

tgcacgtttg?tcacacagac?aacacgttct?ttcaggtcca?atcttgacaa?agtacttcat 2040

tgatgtaagc?tcaaagccat?gcgcccaaag?gacga 2075

<210>54

<211>1891

<212>DNA

＜213〉coronavirus

<400>54

aagattcacc?acttaaatta?gaggatttta?tccctatgga?cagcacagtg?aaaaattact 60

tcataacaga?tgcgcaaaca?ggttcatcaa?aatgtgtgtg?ttctgtgatt?gatcttttac 120

ttgatgactt?tgtcgagata?ataaagtcac?aagatttgtc?agtgatttca?aaagtggtca 180

aggttacaat?tgactatgct?gaaatttcat?tcatgctttg?gtgtaaggat?ggacatgttg 240

aaaccttcta?cccaaaacta?caagcaagtc?aagcgtggca?accaggtgtt?gcgatgccta 300

acttgtacaa?gatgcaaaga?atgcttcttg?aaaagtgtga?ccttcagaat?tatggtgaaa 360

atgctgttat?accaaaagga?ataatgatga?atgtcgcaaa?gtatactcaa?ctgtgtcaat 420

acttaaatac?acttacttta?gctgtaccct?acaacatgag?agttattcac?tttggtgctg 480

gctctgataa?aggagttgca?ccaggtacag?ctgtgctcag?acaatggttg?ccaactggca 540

cactacttgt?cgattcagat?cttaatgact?tcgtctccga?cgcagattct?actttaattg 600

gagactgtgc?aacagtacat?acggctaata?aatgggacct?tattattagc?gatatgtatg 660

accctaggac?caaacatgtg?acaaaagaga?atgactctaa?agaagggttt?ttcacttatc 720

tgtgtggatt?tataaagcaa?aaactagccc?tgggtggttc?tatagctgta?aagataacag 780

agcattcttg?gaatgctgac?ctttacaagc?ttatgggcca?tttctcatgg?tggacagctt 840

ttgttacaaa?tgtaaatgca?tcatcatcgg?aagcattttt?aattggggct?aactatcttg 900

gcaagccgaa?ggaacaaatt?gatggctata?ccatgcatgc?taactacatt?ttctggagga 960

acacaaatcc?tatccagttg?tcttcctatt?cactctttga?catgagcaaa?tttcctctta 1020

aattaagagg?aactgctgta?atgtctctta?aggagaatca?aatcaatgat?atgatttatt 1080

ctcttctgga?aaaaggtagg?cttatcatta?gagaaaacaa?cagagttgtg?gtttcaagtg 1140

atattcttgt?taacaactaa?acgaacatgt?ttattttctt?attatttctt?actctcacta 1200

gtggtagtga?ccttgaccgg?tgcaccactt?ttgatgatgt?tcaagctcct?aattacactc 1260

aacatacttc?atctatgagg?ggggtttact?atcctgatga?aatttttaga?tcagacactc 1320

tttatttaac?tcaggattta?tttcttccat?tttattctaa?tgttacaggg?tttcatacta 1380

ttaatcatac?gtttggcaac?cctgtcatac?cttttaagga?tggtatttat?tttgctgcca 1440

cagagaaatc?aaatgttgtc?cgtggttggg?tttttggttc?taccatgaac?aacaagtcac 1500

agtcggtgat?tattattaac?aattctacta?atgttgttat?acgagcatgt?aactttgaat 1560

tgtgtgacaa?ccctttcttt?gctgtttcta?aacccatggg?tacacagaca?catactatga 1620

tattcgataa?tgcatttaat?tgcactttcg?agtacatatc?tgatgccttt?tcgcttgatg 1680

tttcagaaaa?gtcaggtaat?tttaaacact?tacgagagtt?tgtgtttaaa?aataaagatg 1740

ggtttctcta?tgtttataag?ggctatcaac?ctatagatgt?agttcgtgat?ctaccttctg 1800

gttttaacac?tttgaaacct?atttttaagt?tgcctcttgg?tattaacatt?acaaatttta 1860

gagccattct?tacagccttt?tcacctgctc?a 1891

<210>55

<211>32

<212>DNA

＜213〉artificial sequence

<220>

＜223〉N sense primer

<400>55

cccatatgtc?tgataatgga?ccccaatcaa?ac 32

<210>56

<211>32

<212>DNA

＜213〉artificial sequence

<220>

＜223〉N antisense primer r

<400>56

cccccgggtg?cctgagttga?atcagcagaa?gc 32

<210>57

<211>31

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Sc sense primer

<400>57

cccatatgag?tgaccttgac?cggtgcacca?c 31

<210>58

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉SL sense primer

<400>58

cccatatgaa?accttgcacc?ccacctgctc 30

<210>59

<211>33

<212>DNA

＜213〉Sc and SL antisense primer r

<400>59

cccccgggtt?taatatattg?ctcatatttt?ccc 33

<210>60

<211>16

<212>DNA

＜213〉serial 1 primer of justice

<400>60

ggcatcgtat?gggttg 16

<210>61

<211>16

<212>DNA

＜213〉antisense series 2 (28774-28759) primer

<400>61

cagtttcacc?acctcc 16

<210>62

<211>16

<212>DNA

＜213〉justice series 2 (28375-28390) primer

<400>62

ggctactacc?gaagag 16

<210>63

<211>16

<212>DNA

＜213〉antisense series 2 (28702-28687) primer

<400>63

aattaccgcg?actacg 16

<210>64

<211>26

<212>DNA

＜213〉probe 1/ series 1 (28561-28586)

<400>64

ggcacccgca?atcctaataa?caatgc 26

<210>65

<211>21

<212>DNA

＜213〉probe 2/ series 1 (28588-28608)

<400>65

gccaccgtgc?tacaacttcc?t 21

<210>66

<211>23

<212>DNA

＜213〉probe 1/ serial 2/ probe N/FL (28541-28563)

<400>66

atacacccaa?agaccacatt?ggc 23

<210>67

<211>25

<212>DNA

＜213〉probe 2/ serial 2/ probe SARS/N/LC705 (28565-28589)

<400>67

cccgcaatcc taataacaat?gctgc 25

<210>68

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉anchor primer 14T

<400>68

agatgaattc?ggtacctttt?tttttttttt 30

<210>69

<211>13

<212>PRT

＜213〉artificial sequence

<220>

＜223〉M2-14 peptide

<400>69

Ala?Asp?Asn?Gly?Thr?Ile?Thr?Val?Glu?Glu?Leu?Lys?Gln

1 5 10

<210>70

<211>12

<212>PRT

＜213〉artificial sequence

<220>

＜223〉E1-12 peptide

<400>70

Met?Tyr?Ser?Phe?Val?Ser?Glu?Glu?Thr?Gly?Thr?Leu

1 5 10

<210>71

<211>24

<212>PRT

＜213〉artificial sequence

<220>

＜223〉E53-72 peptide

<400>71

Lys?Pro?Thr?Val?Tyr?Val?Tyr?Ser?Arg?Val?Lys?Asn?Leu?Asn?Ser?Ser

1 5 10 15

Glu?Gly?Val?Pro?Asp?Leu?Leu?Val

20

<210>72

<211>153

<212>DNA

＜213〉coronavirus

<400>72

gatattaggt?ttttacctac?ccaggaaaag?ccaaccaacc?tcgatctctt?gtagatctgt 60

tctctaaacg?aactttaaaa?tctgtgtagc?tgtcgctcgg?ctgcatgcct?agtgcaccta 120

cgcagtataa?acaataataa?attttactgt?cgt 153

<210>73

<211>410

<212>DNA

＜213〉coronavirus

<400>73

ttctccagac?aacttcaaaa?ttccatgagt?ggagcttctg?ctgattcaac?tcaggcataa 60

acactcatga?tgaccacaca?aggcagatgg?gctatgtaaa?cgttttcgca?attccgttta 120

cgatacatag?tctactcttg?tgcagaatga?attctcgtaa?ctaaacagca?caagtaggtt 180

tagttaactt?taatctcaca?tagcaatctt?taatcaatgt?gtaacattag?ggaggacttg 240

aaagagccac?cacattttca?tcgaggccac?gcggagtacg?atcgagggta?cagtgaataa 300

tgctagggag?agctgcctat?atggaagagc?cctaatgtgt?aaaattaatt?ttagtagtgc 360

tatccccatg?tgattttaat?agcttcttag?gagaatgaca?aaaaaaaaaa 410

<210>74

<211>4382

<212>PRT

＜213〉coronavirus

<400>74

Met?Glu?Ser?Leu?Val?Leu?Gly?Val?Asn?Glu?Lys?Thr?His?Val?Gln?Leu

1 5 10 15

Ser?Leu?Pro?Val?Leu?Gln?Val?Arg?Asp?Val?Leu?Val?Arg?Gly?Phe?Gly

20 25 30

Asp?Ser?Val?Glu?Glu?Ala?Leu?Ser?Glu?Ala?Arg?Glu?His?Leu?Lys?Asn

35 40 45

Gly?Thr?Cys?Gly?Leu?Val?Glu?Leu?Glu?Lys?Gly?Val?Leu?Pro?Gln?Leu

50 55 60

Glu?Gln?Pro?Tyr?Val?Phe?Ile?Lys?Arg?Ser?Asp?Ala?Leu?Ser?Thr?Asn

65 70 75 80

His?Gly?His?Lys?Val?Val?Glu?Leu?Val?Ala?Glu?Met?Asp?Gly?Ile?Gln

85 90 95

Tyr?Gly?Arg?Ser?Gly?Ile?Thr?Leu?Gly?Val?Leu?Val?Pro?His?Val?Gly

100 105 110

Glu?Thr?Pro?Ile?Ala?Tyr?Arg?Asn?Val?Leu?Leu?Arg?Lys?Asn?Gly?Asn

115 120 125

Lys?Gly?Ala?Gly?Gly?His?Ser?Tyr?Gly?Ile?Asp?Leu?Lys?Ser?Tyr?Asp

130 135 140

Leu?Gly?Asp?Glu?Leu?Gly?Thr?Asp?ProIle?Glu?Asp?Tyr?Glu?Gln?Asn

145 150 155 160

Trp?Asn?Thr?Lys?His?Gly?Ser?Gly?Ala?Leu?Arg?Glu?Leu?Thr?Arg?Glu

165 170 175

Leu?Asn?Gly?Gly?Ala?Val?Thr?Arg?Tyr?Val?Asp?Asn?Asn?Phe?Cys?Gly

180 185 190

Pro?Asp?Gly?Tyr?Pro?Leu?Asp?Cys?Ile?Lys?Asp?Phe?Leu?Ala?Arg?Ala

195 200 205

Gly?Lys?Ser?Met?Cys?Thr?Leu?Ser?Glu?Gln?Leu?Asp?Tyr?Ile?Glu?Ser

210 215 220

Lys?Arg?Gly?Val?Tyr?Cys?Cys?Arg?Asp?His?Glu?His?Glu?Ile?Ala?Trp

225 230 235 240

Phe?Thr?Glu?Arg?Ser?Asp?Lys?Ser?Tyr?Glu?His?Gln?Thr?Pro?Phe?Glu

245 250 255

Ile?Lys?Ser?Ala?Lys?Lys?Phe?Asp?Thr?Phe?Lys?Gly?Glu?Cys?Pro?Lys

260 265 270

Phe?Val?Phe?Pro?Leu?Asn?Ser?Lys?Val?Lys?Val?Ile?Gln?Pro?Arg?Val

275 280 285

Glu?Lys?Lys?Lys?Thr?Glu?Gly?Phe?Met?Gly?Arg?Ile?Arg?Ser?Val?Tyr

290 295 300

Pro?Val?Ala?Ser?Pro?Gln?Glu?Cys?Asn?Asn?Met?His?Leu?Ser?Thr?Leu

305 310 315 320

Met?Lys?Cys?Asn?His?Cys?Asp?Glu?Val?Ser?Trp?Gln?Thr?Cys?Asp?Phe

325 330 335

Leu?Lys?Ala?Thr?Cys?Glu?His?Cys?Gly?Thr?Glu?Asn?Leu?Val?Ile?Glu

340 345 350

Gly?Pro?Thr?Thr?Cys?Gly?Tyr?Leu?Pro?Thr?Asn?Ala?Val?Val?Lys?Met

355 360 365

Pro?Cys?Pro?Ala?Cys?Gln?Asp?Pro?Glu?Ile?Gly?Pro?Glu?His?Ser?Val

370 375 380

Ala?Asp?Tyr?His?Asn?His?Ser?Asn?Ile?Glu?Thr?Arg?Leu?Arg?Lys?Gly

385 390 395 400

Gly?Arg?Thr?Arg?Cys?Phe?Gly?Gly?Cys?Val?Phe?Ala?Tyr?Val?Gly?Cys

405 410 415

Tyr?Asn?Lys?Arg?Ala?Tyr?Trp?Val?Pro?Arg?Ala?Ser?Ala?Asp?Ile?Gly

420 425 430

Ser?Gly?His?Thr?Gly?Ile?Thr?Gly?Asp?Asn?Val?Glu?Thr?Leu?Asn?Glu

435 440 445

Asp?Leu?Leu?Glu?Ile?Leu?Ser?Arg?Glu?Arg?Val?Asn?Ile?Asn?Ile?Val

450 455 460

Gly?Asp?Phe?His?Leu?Asn?Glu?Glu?Val?Ala?Ile?Ile?Leu?Ala?Ser?Phe

465 470 475 480

Ser?Ala?Ser?Thr?Ser?Ala?Phe?Ile?Asp?Thr?Ile?Lys?Ser?Leu?Asp?Tyr

485 490 495

Lys?Ser?Phe?Lys?Thr?Ile?Val?Glu?Ser?Cys?Gly?Asn?Tyr?Lys?Val?Thr

500 505 510

Lys?Gly?Lys?Pro?Val?Lys?Gly?Ala?Trp?Asn?Ile?Gly?Gln?Gln?Arg?Ser

515 520 525

Val?Leu?Thr?Pro?Leu?Cys?Gly?Phe?Pro?Ser?Gln?Ala?Ala?Gly?Val?Ile

530 535 540

Arg?Ser?Ile?Phe?Ala?Arg?Thr?Leu?Asp?Ala?Ala?Asn?His?Ser?Ile?Pro

545 550 555 560

Asp?Leu?Gln?Arg?Ala?Ala?Val?Thr?Ile?Leu?Asp?Gly?Ile?Ser?Glu?Gln

565 570 575

Ser?Leu?Arg?Leu?Val?Asp?Ala?Met?Val?Tyr?Thr?Ser?Asp?Leu?Leu?Thr

580 585 590

Asn?Ser?Val?Ile?Ile?Met?Ala?Tyr?Val?Thr?Gly?Gly?Leu?Val?Gln?Gln

595 600 605

Thr?Ser?Gln?Trp?Leu?Ser?Asn?Leu?Leu?Gly?Thr?Thr?Val?Glu?Lys?Leu

610 615 620

Arg?Pro?Ile?Phe?Glu?Trp?Ile?Glu?Ala?Lys?Leu?Ser?Ala?Gly?Val?Glu

625 630 635 640

Phe?Leu?Lys?Asp?Ala?Trp?Glu?Ile?Leu?Lys?Phe?Leu?Ile?Thr?Gly?Val

645 650 655

Phe?Asp?Ile?Val?Lys?Gly?Gln?Ile?Gln?Val?Ala?Ser?Asp?Asn?Ile?Lys

660 665 670

Asp?Cys?Val?Lys?Cys?Phe?Ile?Asp?Val?Val?Asn?Lys?Ala?Leu?Glu?Met

675 680 685

Cys?Ile?Asp?Gln?Val?Thr?Ile?Ala?Gly?Ala?Lys?Leu?Arg?Ser?Leu?Asn

690 695 700

Leu?Gly?Glu?Val?Phe?Ile?Ala?Gln?Ser?Lys?Gly?Leu?Tyr?Arg?Gln?Cys

705 710 715 720

Ile?Arg?Gly?Lys?Glu?Gln?Leu?Gln?Leu?Leu?Met?Pro?Leu?Lys?Ala?Pro

725 730 735

Lys?Glu?Val?Thr?Phe?Leu?Glu?Gly?Asp?Ser?His?Asp?Thr?Val?Leu?Thr

740 745 750

Ser?Glu?Glu?Val?Val?Leu?Lys?Asn?Gly?Glu?Leu?Glu?Ala?Leu?Glu?Thr

755 760 765

Pro?Val?Asp?Ser?Phe?Thr?Asn?Gly?Ala?Ile?Val?Gly?Thr?Pro?Val?Cys

770 775 780

Val?Asn?Gly?Leu?Met?Leu?Leu?Glu?Ile?Lys?Asp?Lys?Glu?Gln?Tyr?Cys

785 790 795 800

Ala?Leu?Ser?Pro?Gly?Leu?Leu?Ala?Thr?Asn?Asn?Val?Phe?Arg?Leu?Lys

805 810 815

Gly?Gly?Ala?Pro?Ile?Lys?Gly?Val?Thr?Phe?Gly?Glu?Asp?Thr?Val?Trp

820 825 830

Glu?Val?Gln?Gly?Tyr?Lys?Asn?Val?Arg?Ile?Thr?Phe?Glu?Leu?Asp?Glu

835 840 845

Arg?Val?Asp?Lys?Val?Leu?Asn?Glu?Lys?Cys?Ser?Val?Tyr?Thr?Val?Glu

850 855 860

Ser?Gly?Thr?Glu?Val?Thr?Glu?Phe?Ala?Cys?Val?Val?Ala?Glu?Ala?Val

865 870 875 880

Val?Lys?Thr?Leu?Gln?Pro?Val?Ser?Asp?Leu?Leu?Thr?Asn?Met?Gly?Ile

885 890 895

Asp?Leu?Asp?Glu?Trp?Ser?Val?Ala?Thr?Phe?Tyr?Leu?Phe?Asp?Asp?Ala

900 905 910

Gly?Glu?Glu?Asn?Phe?Ser?Ser?Arg?Met?Tyr?Cys?Ser?Phe?Tyr?Pro?Pro

915 920 925

Asp?Glu?Glu?Glu?Glu?Asp?Asp?Ala?Glu?Cys?Glu?Glu?Glu?Glu?Ile?Asp

930 935 940

Glu?Thr?Cys?Glu?His?Glu?Tyr?Gly?Thr?Glu?Asp?Asp?Tyr?Gln?Gly?Leu

945 950 955 960

Pro?Leu?Glu?Phe?Gly?Ala?Ser?Ala?Glu?Thr?Val?Arg?Val?Glu?Glu?Glu

965 970 975

Glu?Glu?Glu?Asp?Trp?Leu?Asp?Asp?Thr?Thr?Glu?Gln?Ser?Glu?Ile?Glu

980 985 990

Pro?Glu?Pro?Glu?Pro?Thr?Pro?Glu?Glu?Pro?Val?Asn?Gln?Phe?Thr?Gly

995 1000 1005

Tyr?Leu?Lys?Leu?Thr?Asp?Asn?Val?Ala?Ile?Lys?Cys?Val?Asp?Ile

1010 1015 1020

Val?Lys?Glu?Ala?Gln?Ser?Ala?Asn?Pro?Met?Val?Ile?Val?Asn?Ala

1025 1030 1035

Ala?Asn?Ile?His?Leu?Lys?His?Gly?Gly?Gly?Val?Ala?Gly?Ala?Leu

1040 1045 1050

Asn?Lys?Ala?Thr?Asn?Gly?Ala?Met?Gln?Lys?Glu?Ser?Asp?Asp?Tyr

1055 1060 1065

Ile?Lys?Leu?Asn?Gly?Pro?Leu?Thr?Val?Gly?Gly?Ser?Cys?Leu?Leu

1070 1075 1080

Ser?Gly?His?Asn?Leu?Ala?Lys?Lys?Cys?Leu?His?Val?Val?Gly?Pro

1085 1090 1095

Asn?Leu?Asn?Ala?Gly?Glu?Asp?Ile?Gln?Leu?Leu?Lys?Ala?Ala?Tyr

1100 1105 1110

Glu?Asn?Phe?Asn?Ser?Gln?Asp?Ile?Leu?Leu?Ala?Pro?Leu?Leu?Ser

1115 1120 1125

Ala?Gly?Ile?Phe?Gly?Ala?Lys?Pro?Leu?Gln?Ser?Leu?Gln?Val?Cys

1130 1135 1140

Val?Gln?Thr?Val?Arg?Thr?Gln?Val?Tyr?Ile?Ala?Val?Asn?Asp?Lys

1145 1150 1155

Ala?Leu?Tyr?Glu?Gln?Val?Val?Met?Asp?Tyr?Leu?Asp?Asn?Leu?Lys

1160 1165 1170

Pro?Arg?Val?Glu?Ala?Pro?Lys?Gln?Glu?Glu?Pro?Pro?Asn?Thr?Glu

1175 1180 1185

Asp?Ser?Lys?Thr?Glu?Glu?Lys?Ser?Val?Val?Gln?Lys?Pro?Val?Asp

1190 1195 1200

Val?Lys?Pro?Lys?Ile?Lys?Ala?Cys?Ile?Asp?Glu?Val?Thr?Thr?Thr

1205 1210 1215

Leu?Glu?Glu?Thr?Lys?Phe?Leu?Thr?Asn?Lys?Leu?Leu?Leu?Phe?Ala

1220 1225 1230

Asp?Ile?Asn?Gly?Lys?Leu?Tyr?His?Asp?Ser?Gln?Asn?Met?Leu?Arg

1235 1240 1245

Gly?Glu?Asp?Met?Ser?Phe?Leu?Glu?Lys?Asp?Ala?Pro?Tyr?Met?Val

1250 1255 1260

Gly?Asp?Val?Ile?Thr?Ser?Gly?Asp?Ile?Thr?Cys?Val?Val?Ile?Pro

1265 1270 1275

Ser?Lys?Lys?Ala?Gly?Gly?Thr?Thr?Glu?Met?Leu?Ser?Arg?Ala?Leu

1280 1285 1290

Lys?Lys?Val?Pro?Val?Asp?Glu?Tyr?Ile?Thr?Thr?Tyr?Pro?Gly?Gln

1295 1300 1305

Gly?Cys?Ala?Gly?Tyr?Thr?Leu?Glu?Glu?Ala?Lys?Thr?Ala?Leu?Lys

1310 1315 1320

Lys?Cys?Lys?Ser?Ala?Phe?Tyr?Val?Leu?Pro?Ser?Glu?Ala?Pro?Asn

1325 1330 1335

Ala?Lys?Glu?Glu?Ile?Leu?Gly?Thr?Val?Ser?Trp?Asn?Leu?Arg?Glu

1340 1345 1350

Met?Leu?Ala?His?Ala?Glu?Glu?Thr?Arg?Lys?Leu?Met?Pro?Ile?Cys

1355 1360 1365

Met?Asp?Val?Arg?Ala?Ile?Met?Ala?Thr?Ile?Gln?Arg?Lys?Tyr?Lys

1370 1375 1380

Gly?Ile?Lys?Ile?Gln?Glu?Gly?Ile?Val?Asp?Tyr?Gly?Val?Arg?Phe

1385 1390 1395

Phe?Phe?Tyr?Thr?Ser?Lys?Glu?Pro?Val?Ala?Ser?Ile?Ile?Thr?Lys

1400 1405 1410

Leu?Asn?Ser?Leu?Asn?Glu?Pro?Leu?Val?Thr?Met?Pro?Ile?Gly?Tyr

1415 1420 1425

Val?Thr?His?Gly?Phe?Asn?Leu?Glu?Glu?Ala?Ala?Arg?Cys?Met?Arg

1430 1435 1440

Ser?Leu?Lys?Ala?Pro?Ala?Val?Val?Ser?Val?Ser?Ser?Pro?Asp?Ala

1445 1450 1455

Val?Thr?Thr?Tyr?Asn?Gly?Tyr?Leu?Thr?Ser?Ser?Ser?Lys?Thr?Ser

1460 1465 1470

Glu?Glu?His?Phe?Val?Glu?Thr?Val?Ser?Leu?Ala?Gly?Ser?Tyr?Arg

1475 1480 1485

Asp?Trp?Ser?Tyr?Ser?Gly?Gln?Arg?Thr?Glu?Leu?Gly?Val?Glu?Phe

1490 1495 1500

Leu?Lys?Arg?Gly?Asp?Lys?Ile?Val?Tyr?His?Thr?Leu?Glu?Ser?Pro

1505 1510 1515

Val?Glu?Phe?His?Leu?Asp?Gly?Glu?Val?Leu?Ser?Leu?Asp?Lys?Leu

1520 1525 1530

Lys?Ser?Leu?Leu?Ser?Leu?Arg?Glu?Val?Lys?Thr?Ile?Lys?Val?Phe

1535 1540 1545

Thr?Thr?Val?Asp?Asn?Thr?Asn?Leu?His?Thr?Gln?Leu?Val?Asp?Met

1550 1555 1560

Ser?Met?Thr?Tyr?Gly?Gln?Gln?Phe?Gly?Pro?Thr?Tyr?Leu?Asp?Gly

1565 1570 1575

Ala?Asp?Val?Thr?Lys?Ile?Lys?Pro?His?Val?Asn?His?Glu?Gly?Lys

1580 1585 1590

Thr?Phe?Phe?Val?Leu?Pro?Ser?Asp?Asp?Thr?Leu?Arg?Ser?Glu?Ala

1595 1600 1605

Phe?Glu?Tyr?Tyr?His?Thr?Leu?Asp?Glu?Ser?Phe?Leu?Gly?Arg?Tyr

1610 1615 1620

Met?Ser?Ala?Leu?Asn?His?Thr?Lys?Lys?Trp?Lys?Phe?Pro?Gln?Val

1625 1630 1635

Gly?Gly?Leu?Thr?Ser?Ile?Lys?Trp?Ala?Asp?Asn?Asn?Cys?Tyr?Leu

1640 1645 1650

Ser?Ser?Val?Leu?Leu?Ala?Leu?Gln?Gln?Leu?Glu?Val?Lys?Phe?Asn

1655 1660 1665

Ala?Pro?Ala?Leu?Gln?Glu?Ala?Tyr?Tyr?Arg?Ala?Arg?Ala?Gly?Asp

1670 1675 1680

Ala?Ala?Asn?Phe?Cys?Ala?Leu?Ile?Leu?Ala?Tyr?Ser?Asn?Lys?Thr

1685 1690 1695

Val?Gly?Glu?Leu?Gly?Asp?Val?Arg?Glu?Thr?Met?Thr?His?Leu?Leu

1700 1705 1710

Gln?His?Ala?Asn?Leu?Glu?Ser?Ala?Lys?Arg?Val?Leu?Asn?Val?Val

1715 1720 1725

Cys?Lys?His?Cys?Gly?Gln?Lys?Thr?Thr?Thr?Leu?Thr?Gly?Val?Glu

1730 1735 1740

Ala?Val?Met?Tyr?Met?Gly?Thr?Leu?Ser?Tyr?Asp?Asn?Leu?Lys?Thr

1745 1750 1755

Gly?Val?Ser?Ile?Pro?Cys?Val?Cys?Gly?Arg?Asp?Ala?Thr?Gln?Tyr

1760 1765 1770

Leu?Val?Gln?Gln?Glu?Ser?Ser?Phe?Val?Met?Met?Ser?Ala?Pro?Pro

1775 1780 1785

Ala?Glu?Tyr?Lys?Leu?Gln?Gln?Gly?Thr?Phe?Leu?Cys?Ala?Asn?Glu

1790 1795 1800

Tyr?Thr?Gly?Asn?Tyr?Gln?Cys?Gly?His?Tyr?Thr?His?Ile?Thr?Ala

1805 1810 1815

Lys?Glu?Thr?Leu?Tyr?Arg?Ile?Asp?Gly?Ala?His?Leu?Thr?Lys?Met

1820 1825 1830

Ser?Glu?Tyr?Lys?Gly?Pro?Val?Thr?Asp?Val?Phe?Tyr?Lys?Glu?Thr

1835 1840 1845

Ser?Tyr?Thr?Thr?Thr?Ile?Lys?Pro?Val?Ser?Tyr?Lys?Leu?Asp?Gly

1850 1855 1860

Val?Thr?Tyr?Thr?Glu?Ile?Glu?Pro?Lys?Leu?Asp?Gly?Tyr?Tyr?Lys

1865 1870 1875

Lys?Asp?Asn?Ala?Tyr?Tyr?Thr?Glu?Gln?Pro?Ile?Asp?Leu?Val?Pro

1880 1885 1890

Thr?Gln?Pro?Leu?Pro?Asn?Ala?Ser?Phe?Asp?Asn?Phe?Lys?Leu?Thr

1895 1900 1905

Cys?Ser?Asn?Thr?Lys?Phe?Ala?Asp?Asp?Leu?Asn?Gln?Met?Thr?Gly

1910 1915 1920

Phe?Thr?Lys?Pro?Ala?Ser?Arg?Glu?Leu?Ser?Val?Thr?Phe?Phe?Pro

1925 1930 1935

Asp?Leu?Asn?Gly?Asp?Val?Val?Ala?Ile?Asp?Tyr?Arg?His?Tyr?Ser

1940 1945 1950

Ala?Ser?Phe?Lys?Lys?Gly?Ala?Lys?Leu?Leu?His?Lys?Pro?Ile?Val

1955 1960 1965

Trp?His?Ile?Asn?Gln?Ala?Thr?Thr?Lys?Thr?Thr?Phe?Lys?Pro?Asn

1970 1975 1980

Thr?Trp?Cys?Leu?Arg?Cys?Leu?Trp?Ser?Thr?Lys?Pro?Val?Asp?Thr

1985 1990 1995

Ser?Asn?Ser?Phe?Glu?Val?Leu?Ala?Val?Glu?Asp?Thr?Gln?Gly?Met

2000 2005 2010

Asp?Asn?Leu?Ala?Cys?Glu?Ser?Gln?Gln?Pro?Thr?Ser?Glu?Glu?Val

2015 2020 2025

Val?Glu?Asn?Pro?Thr?Ile?Gln?Lys?Glu?Val?Ile?Glu?Cys?Asp?Val

2030 2035 2040

Lys?Thr?Thr?Glu?Val?Val?Gly?Asn?Val?Ile?Leu?Lys?Pro?Ser?Asp

2045 2050 2055

Glu?Gly?Val?Lys?Val?Thr?Gln?Glu?Leu?Gly?His?Glu?Asp?Leu?Met

2060 2065 2070

Ala?Ala?Tyr?Val?Glu?Asn?Thr?Ser?Ile?Thr?Ile?Lys?Lys?Pro?Asn

2075 2080 2085

Glu?Leu?Ser?Leu?Ala?Leu?Gly?Leu?Lys?Thr?Ile?Ala?Thr?His?Gly

2090 2095 2100

Ile?Ala?Ala?Ile?Asn?Ser?Val?Pro?Trp?Ser?Lys?Ile?Leu?Ala?Tyr

2105 2110 2115

Val?Lys?Pro?Phe?Leu?Gly?Gln?Ala?Ala?Ile?Thr?Thr?Ser?Asn?Cys

2120 2125 2130

Ala?Lys?Arg?Leu?Ala?Gln?Arg?Val?Phe?Asn?Asn?Tyr?Met?Pro?Tyr

2135 2140 2145

Val?Phe?Thr?Leu?Leu?Phe?Gln?Leu?Cys?Thr?Phe?Thr?Lys?Ser?Thr

2150 2155 2160

Ash?Ser?Arg?Ile?Arg?Ala?Ser?Leu?Pro?Thr?Thr?Ile?Ala?Lys?Asn

2165 2170 2175

Ser?Val?Lys?Ser?Val?Ala?Lys?Leu?Cys?Leu?Asp?Ala?Gly?Ile?Asn

2180 2185 2190

Tyr?Val?Lys?Ser?Pro?Lys?Phe?Ser?Lys?Leu?Phe?Thr?Ile?Ala?Met

2195 2200 2205

Trp?Leu?Leu?Leu?Leu?Ser?Ile?Cys?Leu?Gly?Ser?Leu?Ile?Cys?Val

2210 2215 2220

Thr?Ala?Ala?Phe?Gly?Val?Leu?Leu?Ser?Asn?Phe?Gly?Ala?Pro?Ser

2225 2230 2235

Tyr?Cys?Asn?Gly?Val?Arg?Glu?Leu?Tyr?Leu?Asn?Ser?Ser?Asn?Val

2240 2245 2250

Thr?Thr?Met?Asp?Phe?Cys?Glu?Gly?Ser?Phe?Pro?Cys?Ser?Ile?Cys

2255 2260 2265

Leu?Ser?Gly?Leu?Asp?Ser?Leu?Asp?Ser?Tyr?Pro?Ala?Leu?Glu?Thr

2270 2275 2280

Ile?Gln?Val?Thr?Ile?Ser?Ser?Tyr?Lys?Leu?Asp?Leu?Thr?Ile?Leu

2285 2290 2295

Gly?Leu?Ala?Ala?Glu?Trp?Val?Leu?Ala?Tyr?Met?Leu?Phe?Thr?Lys

2300 2305 2310

Phe?Phe?Tyr?Leu?Leu?Gly?Leu?Ser?Ala?Ile?Met?Gln?Val?Phe?Phe

2315 2320 2325

Gly?Tyr?Phe?Ala?Ser?His?Phe?Ile?Ser?Asn?Ser?Trp?Leu?Met?Trp

2330 2335 2340

Phe?Ile?Ile?Ser?Ile?Val?Gln?Met?Ala?Pro?Val?Ser?Ala?Met?Val

2345 2350 2355

Arg?Met?Tyr?Ile?Phe?Phe?Ala?Ser?Phe?Tyr?Tyr?Ile?Trp?Lys?Ser

2360 2365 2370

Tyr?Val?His?Ile?Met?Asp?Gly?Cys?Thr?Ser?Ser?Thr?Cys?Met?Met

2375 2380 2385

Cys?Tyr?Lys?Arg?Asn?Arg?Ala?Thr?Arg?Val?Glu?Cys?Thr?Thr?Ile

2390 2395 2400

Val?Asn?Gly?Met?Lys?Arg?Ser?Phe?Tyr?Val?Tyr?Ala?Asn?Gly?Gly

2405 2410 2415

Arg?Gly?Phe?Cys?Lys?Thr?His?Asn?Trp?Asn?Cys?Leu?Asn?Cys?Asp

2420 2425 2430

Thr?Phe?Cys?Thr?Gly?Ser?Thr?Phe?Ile?Ser?Asp?Glu?Val?Ala?Arg

2435 2440 2445

Asp?Leu?Ser?Leu?Gln?Phe?Lys?Arg?Pro?Ile?Asn?Pro?Thr?Asp?Gln

2450 2455 2460

Ser?Ser?Tyr?Ile?Val?Asp?Ser?Val?Ala?Val?Lys?Asn?Gly?Ala?Leu

2465 2470 2475

His?Leu?Tyr?Phe?Asp?Lys?Ala?Gly?Gln?Lys?Thr?Tyr?Glu?Arg?His

2480 2485 2490

Pro?Leu?Ser?His?Phe?Val?Asn?Leu?Asp?Asn?Leu?Arg?Ala?Asn?Asn

2495 2500 2505

Thr?Lys?Gly?Ser?Leu?Pro?Ile?Asn?Val?Ile?Val?Phe?Asp?Gly?Lys

2510 2515 2520

Ser?Lys?Cys?Asp?Glu?Ser?Ala?Ser?Lys?Ser?Ala?Ser?Val?Tyr?Tyr

2525 2530 2535

Ser?Gln?Leu?Met?Cys?Gln?Pro?Ile?Leu?Leu?Leu?Asp?Gln?Ala?Leu

2540 2545 2550

Val?Ser?Asp?Val?Gly?Asp?Ser?Thr?Glu?Val?Ser?Val?Lys?Met?Phe

2555 2560 2565

Asp?Ala?Tyr?Val?Asp?Thr?Phe?Ser?Ala?Thr?Phe?Ser?Val?Pro?Met

2570 2575 2580

Glu?Lys?Leu?Lys?Ala?Leu?Val?Ala?Thr?Ala?His?Ser?Glu?Leu?Ala

2585 2590 2595

Lys?Gly?Val?Ala?Leu?Asp?Gly?Val?Leu?Ser?Thr?Phe?Val?Ser?Ala

2600 2605 2610

Ala?Arg?Gln?Gly?Val?Val?Asp?Thr?Asp?Val?Asp?Thr?Lys?Asp?Val

2615 2620 2625

Ile?Glu?Cys?Leu?Lys?Leu?Ser?His?His?Ser?Asp?Leu?Glu?Val?Thr

2630 2635 2640

Gly?Asp?Ser?Cys?Asn?Asn?Phe?Met?Leu?Thr?Tyr?Asn?Lys?Val?Glu

2645 2650 2655

Asn?Met?Thr?Pro?Arg?Asp?Leu?Gly?Ala?Cys?Ile?Asp?Cys?Asn?Ala

2660 2665 2670

Arg?His?Ile?Asn?Ala?Gln?Val?Ala?Lys?Ser?His?Asn?Val?Ser?Leu

2675 2680 2685

Ile?Trp?Asn?Val?Lys?Asp?Tyr?Met?Ser?Leu?Ser?Glu?Gln?Leu?Arg

2690 2695 2700

Lys?Gln?Ile?Arg?Ser?Ala?Ala?Lys?Lys?Asn?Asn?Ile?Pro?Phe?Arg

2705 2710 2715

Leu?Thr?Cys?Ala?Thr?Thr?Arg?Gln?Val?Val?Asn?Val?Ile?Thr?Thr

2720 2725 2730

Lys?Ile?Ser?Leu?Lys?Gly?Gly?Lys?Ile?Val?Ser?Thr?Cys?Phe?Lys

2735 2740 2745

Leu?Met?Leu?Lys?Ala?Thr?Leu?Leu?Cys?Val?Leu?Ala?Ala?Leu?Val

2750 2755 2760

Cys?Tyr?Ile?Val?Met?Pro?Val?His?Thr?Leu?Ser?Ile?His?Asp?Gly

2765 2770 2775

Tyr?Thr?Asn?Glu?Ile?Ile?Gly?Tyr?Lys?Ala?Ile?Gln?Asp?Gly?Val

2780 2785 2790

Thr?Arg?Asp?Ile?Ile?Ser?Thr?Asp?Asp?Cys?Phe?Ala?Asn?Lys?His

2795 2800 2805

Ala?Gly?Phe?Asp?Ala?Trp?Phe?Ser?Gln?Arg?Gly?Gly?Ser?Tyr?Lys

2810 2815 2820

Asn?Asp?Lys?Ser?Cys?Pro?Val?Val?Ala?Ala?Ile?Ile?Thr?Arg?Glu

2825 2830 2835

Ile?Gly?Phe?Ile?Val?Pro?Gly?Leu?Pro?Gly?Thr?Val?Leu?Arg?Ala

2840 2845 2850

Ile?Asn?Gly?Asp?Phe?Leu?His?Phe?Leu?Pro?Arg?Val?Phe?Ser?Ala

2855 2860 2865

Val?Gly?Asn?Ile?Cys?Tyr?Thr?Pro?Ser?Lys?Leu?Ile?Glu?Tyr?Ser

2870 2875 2880

Asp?Phe?Ala?Thr?Ser?Ala?Cys?Val?Leu?Ala?Ala?Glu?Cys?Thr?Ile

2885 2890 2895

Phe?Lys?Asp?Ala?Met?Gly?Lys?Pro?Val?Pro?Tyr?Cys?Tyr?Asp?Thr

2900 2905 2910

Asn?Leu?Leu?Glu?Gly?Ser?Ile?Ser?Tyr?Ser?Glu?Leu?Arg?Pro?Asp

2915 2920 2925

Thr?Arg?Tyr?Val?Leu?Met?Asp?Gly?Ser?Ile?Ile?Gln?Phe?Pro?Asn

2930 2935 2940

Thr?Tyr?Leu?Glu?Gly?Ser?Val?Arg?Val?Val?Thr?Thr?Phe?Asp?Ala

2945 2950 2955

Glu?Tyr?Cys?Arg?His?Gly?Thr?Cys?Glu?Arg?Ser?Glu?Val?Gly?Ile

2960 2965 2970

Cys?Leu?Ser?Thr?Ser?Gly?Arg?Trp?Val?Leu?Asn?Asn?Glu?His?Tyr

2975 2980 2985

Arg?Ala?Leu?Ser?Gly?Val?Phe?Cys?Gly?Val?Asp?Ala?Met?Asn?Leu

2990 2995 3000

Ile?Ala?Asn?Ile?Phe?Thr?Pro?Leu?Val?Gln?Pro?Val?Gly?Ala?Leu

3005 3010 3015

Asp?Val?Ser?Ala?Ser?Val?Val?Ala?Gly?Gly?Ile?Ile?Ala?Ile?Leu

3020 3025 3030

Val?Thr?Cys?Ala?Ala?Tyr?Tyr?Phe?Met?Lys?Phe?Arg?Arg?Val?Phe

3035 3040 3045

Gly?Glu?Tyr?Asn?His?Val?Val?Ala?Ala?Asn?Ala?Leu?Leu?Phe?Leu

3050 3055 3060

Met?Ser?Phe?Thr?Ile?Leu?Cys?Leu?Val?Pro?Ala?Tyr?Ser?Phe?Leu

3065 3070 3075

Pro?Gly?Val?Tyr?Ser?Val?Phe?Tyr?Leu?Tyr?Leu?Thr?Phe?Tyr?Phe

3080 3085 3090

Thr?Asn?Asp?Val?Ser?Phe?Leu?Ala?His?Leu?Gln?Trp?Phe?Ala?Met

3095 3100 3105

Phe?Ser?Pro?Ile?Val?Pro?Phe?Trp?Ile?Thr?Ala?Ile?Tyr?Val?Phe

3110 3115 3120

Cys?Ile?Ser?Leu?Lys?His?Cys?His?Trp?Phe?Phe?Asn?Asn?Tyr?Leu

3125 3130 3135

Arg?Lys?Arg?Val?Met?Phe?Asn?Gly?Val?Thr?Phe?Ser?Thr?Phe?Glu

3140 3145 3150

Glu?Ala?Ala?Leu?Cys?Thr?Phe?Leu?Leu?Asn?Lys?Glu?Met?Tyr?Leu

3155 3160 3165

Lys?Leu?Arg?Ser?Glu?Thr?Leu?Leu?Pro?Leu?Thr?Gln?Tyr?Asn?Arg

3170 3175 3180

Tyr?Leu?Ala?Leu?Tyr?Asn?Lys?Tyr?Lys?Tyr?Phe?Ser?Gly?Ala?Leu

3185 3190 3195

Asp?Thr?Thr?Ser?Tyr?Arg?Glu?Ala?Ala?Cys?Cys?His?Leu?Ala?Lys

3200 3205 3210

Ala?Lau?Asn?Asp?Phe?Ser?Asn?Ser?Gly?Ala?Asp?Val?Leu?Tyr?Gln

3215 3220 3225

Pro?Pro?Gln?Thr?Ser?Ile?Thr?Ser?Ala?Val?Leu?Gln?Ser?Gly?Phe

3230 3235 3240

Arg?Lys?Met?Ala?Phe?Pro?Ser?Gly?Lys?Val?Glu?Gly?Cys?Met?Val

3245 3250 3255

Gln?Val?Thr?Cys?Gly?Thr?Thr?Thr?Leu?Asn?Gly?Leu?Trp?Leu?Asp

3260 3265 3270

Asp?Thr?Val?Tyr?Cys?Pro?Arg?His?Val?Ile?Cys?Thr?Ala?Glu?Asp

3275 3280 3285

Met?Leu?Asn?Pro?Asn?Tyr?Glu?Asp?Leu?Leu?Ile?Arg?Lys?Ser?Asn

3290 3295 3300

His?Ser?Phe?Leu?Val?Gln?Ala?Gly?Asn?Val?Gln?Leu?Arg?Val?Ile

3305 3310 3315

Gly?His?Ser?Met?Gln?Asn?Cys?Leu?Leu?Arg?Leu?Lys?Val?Asp?Thr

3320 3325 3330

Ser?Asn?Pro?Lys?Thr?Pro?Lys?Tyr?Lys?Phe?Val?Arg?Ile?Gln?Pro

3335 3340 3345

Gly?Gln?Thr?Phe?Ser?Val?Leu?Ala?Cys?Tyr?Asn?Gly?Ser?Pro?Ser

3350 3355 3360

Gly?Val?Tyr?Gln?Cys?Ala?Met?Arg?Pro?Asn?His?Thr?Ile?Lys?Gly

3365 3370 3375

Ser?Phe?Leu?Asn?Gly?Ser?Cys?Gly?Ser?Val?Gly?Phe?Asn?Ile?Asp

3380 3385 3390

Tyr?Asp?Cys?Val?Ser?Phe?Cys?Tyr?Met?His?His?Met?Glu?Leu?Pro

3395 3400 3405

Thr?Gly?Val?His?Ala?Gly?Thr?Asp?Leu?Glu?Gly?Lys?Phe?Tyr?Gly

3410 3415 3420

Pro?Phe?Val?Asp?Arg?Gln?Thr?Ala?Gln?Ala?Ala?Gly?Thr?Asp?Thr

3425 3430 3435

Thr?Ile?Thr?Leu?Asn?Val?Leu?Ala?Trp?Leu?Tyr?Ala?Ala?Val?Ile

3440 3445 3450

Asn?Gly?Asp?Arg?Trp?Phe?Leu?Asn?Arg?Phe?Thr?Thr?Thr?Leu?Asn

3455 3460 3465

Asp?Phe?Asn?Leu?Val?Ala?Met?Lys?Tyr?Asn?Tyr?Glu?Pro?Leu?Thr

3470 3475 3480

Gln?Asp?His?Val?Asp?Ile?Leu?Gly?Pro?Leu?Ser?Ala?Gln?Thr?Gly

3485 3490 3495

Ile?Ala?Val?Leu?Asp?Met?Cys?Ala?Ala?Leu?Lys?Glu?Leu?Leu?Gln

3500 3505 3510

Asn?Gly?Met?Asn?Gly?Arg?Thr?Ile?Leu?Gly?Ser?Thr?Ile?Leu?Glu

3515 3520 3525

Asp?Glu?Phe?Thr?Pro?Phe?Asp?Val?Val?Arg?Gln?Cys?Ser?Gly?Val

3530 3535 3540

Thr?Phe?Gln?Gly?Lys?Phe?Lys?Lys?Ile?Val?Lys?Gly?Thr?His?His

3545 3550 3555

Trp?Met?Leu?Leu?Thr?Phe?Leu?Thr?Ser?Leu?Leu?Ile?Leu?Val?Gln

3560 3565 3570

Ser?Thr?Gln?Trp?Ser?Leu?Phe?Phe?Phe?Val?Tyr?Glu?Asn?Ala?Phe

3575 3580 3585

Leu?Pro?Phe?Thr?Leu?Gly?Ile?Met?Ala?Ile?Ala?Ala?Cys?Ala?Met

3590 3595 3600

Leu?Leu?Val?Lys?His?Lys?His?Ala?Phe?Leu?Cys?Leu?Phe?Leu?Leu

3605 3610 3615

Pro?Ser?Leu?Ala?Thr?Val?Ala?Tyr?Phe?Asn?Met?Val?Tyr?Met?Pro

3620 3625 3630

Ala?Ser?Trp?Val?Met?Arg?Ile?Met?Thr?Trp?Leu?Glu?Leu?Ala?Asp

3635 3640 3645

Thr?Ser?Leu?Ser?Gly?Tyr?Arg?Leu?Lys?Asp?Cys?Val?Met?Tyr?Ala

3650 3655 3660

Ser?Ala?Leu?Val?Leu?Leu?Ile?Leu?Met?Thr?Ala?Arg?Thr?Val?Tyr

3665 3670 3675

Asp?Asp?Ala?Ala?Arg?Arg?Val?Trp?Thr?Leu?Met?Asn?Val?Ile?Thr

3680 3685 3690

Leu?Val?Tyr?Lys?Val?Tyr?Tyr?Gly?Asn?Ala?Leu?Asp?Gln?Ala?Ile

3695 3700 3705

Ser?Met?Trp?Ala?Leu?Val?Ile?Ser?Val?Thr?Ser?Asn?Tyr?Ser?Gly

3710 3715 3720

Val?Val?Thr?Thr?Ile?Met?Phe?Leu?Ala?Arg?Ala?Ile?Val?Phe?Val

3725 3730 3735

Cys?Val?Glu?Tyr?Tyr?Pro?Leu?Leu?Phe?Ile?Thr?Gly?Asn?Thr?Leu

3740 3745 3750

Gln?Cys?Ile?Met?Leu?Val?Tyr?Cys?Phe?Leu?Gly?Tyr?Cys?Cys?Cys

3755 3760 3765

Cys?Tyr?Phe?Gly?Leu?Phe?Cys?Leu?Leu?Asn?Arg?Tyr?Phe?Arg?Leu

3770 3775 3780

Thr?Leu?Gly?Val?Tyr?Asp?Tyr?Leu?Val?Ser?Thr?Gln?Glu?Phe?Arg

3785 3790 3795

Tyr?Met?Asn?Ser?Gln?Gly?Leu?Leu?Pro?Pro?Lys?Ser?Ser?Ile?Asp

3800 3805 3810

Ala?Phe?Lys?Leu?Asn?Ile?Lys?Leu?Leu?Gly?Ile?Gly?Gly?Lys?Pro

3815 3820 3825

Cys?Ile?Lys?Val?Ala?Thr?Val?Gln?Ser?Lys?Met?Ser?Asp?Val?Lys

3830 3835 3840

Cys?Thr?Ser?Val?Val?Leu?Leu?Ser?Val?Leu?Gln?Gln?Leu?Arg?Val

3845 3850 3855

Glu?Ser?Ser?Ser?Lys?Leu?Trp?Ala?Gln?Cys?Val?Gln?Leu?His?Asn

3860 3865 3870

Asp?Ile?Leu?Leu?Ala?Lys?Asp?Thr?Thr?Glu?Ala?Phe?Glu?Lys?Met

3875 3880 3885

Val?Ser?Leu?Leu?Ser?Val?Leu?Leu?Ser?Met?Gln?Gly?Ala?Val?Asp

3890 3895 3900

Ile?Asn?Arg?Leu?Cys?Glu?Glu?Met?Leu?Asp?Asn?Arg?Ala?Thr?Leu

3905 3910 3915

Gln?Ala?Ile?Ala?Ser?Glu?Phe?Ser?Ser?Leu?Pro?Ser?Tyr?Ala?Ala

3920 3925 3930

Tyr?Ala?Thr?Ala?Gln?Glu?Ala?Tyr?Glu?Gln?Ala?Val?Ala?Asn?Gly

3935 3940 3945

Asp?Ser?Glu?Val?Val?Leu?Lys?Lys?Leu?Lys?Lys?Ser?Leu?Asn?Val

3950 3955 3960

Ala?Lys?Ser?Glu?Phe?Asp?Arg?Asp?Ala?Ala?Met?Gln?Arg?Lys?Leu

3965 3970 3975

Glu?Lys?Met?Ala?Asp?Gln?Ala?Met?Thr?Gln?Met?Tyr?Lys?Gln?Ala

3980 3985 3990

Arg?Ser?Glu?Asp?Lys?Arg?Ala?Lys?Val?Thr?Ser?Ala?Met?Gln?Thr

3995 4000 4005

Met?Leu?Phe?Thr?Met?Leu?Arg?Lys?Leu?Asp?Asn?Asp?Ala?Leu?Asn

4010 4015 4020

Asn?Ile?Ile?Asn?Asn?Ala?Arg?Asp?Gly?Cys?Val?Pro?Leu?Asn?IIe

4025 4030 4035

Ile?Pro?Leu?Thr?Thr?Ala?Ala?Lys?Leu?Met?Val?Val?Val?Pro?Asp

4040 4045 4050

Tyr?Gly?Thr?Tyr?Lys?Asn?Thr?Cys?Asp?Gly?Asn?Thr?Phe?Thr?Tyr

4055 4060 4065

Ala?Ser?Ala?Leu?Trp?Glu?Ile?Gln?Gln?Val?Val?Asp?Ala?Asp?Ser

4070 4075 4080

Lys?Ile?Val?Gln?Leu?Ser?Glu?Ile?Asn?Met?Asp?Asn?Ser?Pro?Asn

4085 4090 4095

Leu?Ala?Trp?Pro?Leu?Ile?Val?Thr?Ala?Leu?Arg?Ala?Asn?Ser?Ala

4100 4105 4110

Val?Lys?Leu?Gln?Asn?Asn?Glu?Leu?Ser?Pro?Val?Ala?Leu?Arg?Gln

4115 4120 4125

Met?Ser?Cys?Ala?Ala?Gly?Thr?Thr?Gln?Thr?Ala?Cys?Thr?Asp?Asp

4130 4135 4140

Asn?Ala?Leu?Ala?Tyr?Tyr?Asn?Asn?Ser?Lys?Gly?Gly?Arg?Phe?Val

4145 4150 4155

Leu?Ala?Leu?Leu?Ser?Asp?His?Gln?Asp?Leu?Lys?Trp?Ala?Arg?Phe

4160 4165 4170

Pro?Lys?Ser?Asp?Gly?Thr?Gly?Thr?Ile?Tyr?Thr?Glu?Leu?Glu?Pro

4175 4180 4185

Pro?Cys?Arg?Phe?Val?Thr?Asp?Thr?Pro?Lys?Gly?Pro?Lys?Val?Lys

4190 4195 4200

Tyr?Leu?Tyr?Phe?Ile?Lys?Gly?Leu?Asn?Asn?Leu?Asn?Arg?Gly?Met

4205 4210 4215

Val?Leu?Gly?Ser?Leu?Ala?Ala?Thr?Val?Arg?Leu?Gln?Ala?Gly?Asn

4220 4225 4230

Ala?Thr?Glu?Val?Pro?Ala?Asn?Ser?Thr?Val?Leu?Ser?Phe?Cys?Ala

4235 4240 4245

Phe?Ala?Val?Asp?Pro?Ala?Lys?Ala?Tyr?Lys?Asp?Tyr?Leu?Ala?Ser

4250 4255 4260

Gly?Gly?Gln?Pro?Ile?Thr?Asn?Cys?Val?Lys?Met?Leu?Cys?Thr?His

4265 4270 4275

Thr?Gly?Thr?Gly?Gln?Ala?Ile?Thr?Val?Thr?Pro?Glu?Ala?Asn?Met

4280 4285 4290

Asp?Gln?Glu?Ser?Phe?Gly?Gly?Ala?Ser?Cys?Cys?Leu?Tyr?Cys?Arg

4295 4300 4305

Cys?His?Ile?Asp?His?Pro?Asn?Pro?Lys?Gly?Phe?Cys?Asp?Leu?Lys

4310 4315 4320

Gly?Lys?Tyr?Val?Gln?Ile?Pro?Thr?Thr?Cys?Ala?Asn?Asp?Pro?Val

4325 4330 4335

Gly?Phe?Thr?Leu?Arg?Asn?Thr?Val?Cys?Thr?Val?Cys?Gly?Met?Trp

4340 4345 4350

Lys?Gly?Tyr?Gly?Cys?Ser?Cys?Asp?Gln?Leu?Arg?Glu?Pro?Leu?Met

4355 4360 4365

Gln?Ser?Ala?Asp?Ala?Ser?Thr?Phe?Leu?Asn?Gly?Phe?Ala?Val

4370 4375 4380

<210>75

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＜213〉coronavirus

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Arg?Val?Cys?Gly?Val?Ser?Ala?Ala?Arg?Leu?Thr?Pro?Cys?Gly?Thr?Gly

1 5 10 15

Thr?Ser?Thr?Asp?Val?Val?Tyr?Arg?Ala?Phe?Asp?Ile?Tyr?Asn?Glu?Lys

20 25 30

Val?Ala?Gly?Phe?Ala?Lys?Phe?Leu?Lys?Thr?Asn?Cys?Cys?Arg?Phe?Gln

35 40 45

Glu?Lys?Asp?Glu?Glu?Gly?Asn?Leu?Leu?Asp?Ser?Tyr?Phe?Val?Val?Lys

50 55 60

Arg?His?Thr?Met?Ser?Asn?Tyr?Gln?His?Glu?Glu?Thr?Ile?Tyr?Asn?Leu

65 70 75 80

Val?Lys?Asp?Cys?Pro?Ala?Val?Ala?Val?His?Asp?Phe?Phe?Lys?Phe?Arg

85 90 95

Val?Asp?Gly?Asp?Met?Val?Pro?His?Ile?Ser?Arg?Gln?Arg?Leu?Thr?Lys

100 105 110

Tyr?Thr?Met?Ala?Asp?Leu?Val?Tyr?Ala?Leu?Arg?His?Phe?Asp?Glu?Gly

115 120 125

Asn?Cys?Asp?Thr?Leu?Lys?Glu?Ile?Leu?Val?Thr?Tyr?Asn?Cys?Cys?Asp

130 135 140

Asp?Asp?Tyr?Phe?Asn?Lys?Lys?Asp?Trp?Tyr?Asp?Phe?Val?Glu?Asn?Pro

145 150 155 160

Asp?Ile?Leu?Arg?Val?Tyr?Ala?Asn?Leu?Gly?Glu?Arg?Val?Arg?Gln?Ser

165 170 175

Leu?Leu?Lys?Thr?Val?Gln?Phe?Cys?Asp?Ala?Met?Arg?Asp?Ala?Gly?Ile

180 185 190

Val?Gly?Val?Leu?Thr?Leu?Asp?Asn?Gln?Asp?Leu?Asn?Gly?Asn?Trp?Tyr

195 200 205

Asp?Phe?Gly?Asp?Phe?Val?Gln?Val?Ala?Pro?Gly?Cys?Gly?Val?Pro?Ile

210 215 220

Val?Asp?Ser?Tyr?Tyr?Ser?Leu?Leu?Met?Pro?Ile?Leu?Thr?Leu?Thr?Ara

225 230 235 240

Ala?Leu?Ala?Ala?Glu?Ser?His?Met?Asp?Ala?Asp?Leu?Ala?Lys?Pro?Leu

245 250 255

Ile?Lys?Trp?Asp?Leu?Leu?Lys?Tyr?Asp?Phe?Thr?Glu?Glu?Arg?Leu?Cys

260 265 270

Leu?Phe?Asp?Arg?Tyr?Phe?Lys?Tyr?Trp?Asp?Gln?Thr?Tyr?His?Pro?Asn

275 280 285

Cys?Ile?Asn?Cys?Leu?Asp?Asp?Arg?Cys?Ile?Leu?His?Cys?Ala?Asn?Phe

290 295 300

Asn?Val?Leu?Phe?Ser?Thr?Val?Phe?Pro?Pro?Thr?Ser?Phe?Gly?Pro?Leu

305 310 315 320

Val?Arg?Lys?Ile?Phe?Val?Asp?Gly?Val?Pro?Phe?Val?Val?Ser?Thr?Gly

325 330 335

Tyr?His?Phe?Arg?Glu?Leu?Gly?Val?Val?His?Asn?Gln?Asp?Val?Asn?Leu

340 345 350

His?Ser?Ser?Arg?Leu?Ser?Phe?Lys?Glu?Leu?Leu?Val?Tyr?Ala?Ala?Asp

355 360 365

Pro?Ala?Met?His?Ala?Ala?Ser?Gly?Asn?Leu?Leu?Leu?Asp?Lys?Arg?Thr

370 375 380

Thr?Cys?Phe?Ser?Val?Ala?Ala?Leu?Thr?Asn?Asn?Val?Ala?Phe?Gln?Thr

385 390 395 400

Val?Lys?Pro?Gly?Asn?Phe?Asn?Lys?Asp?Phe?Tyr?Asp?Phe?Ala?Val?Ser

405 410 415

Lys?Gly?Phe?Phe?Lys?Glu?Gly?Ser?Ser?Val?Glu?Leu?Lys?His?Phe?Phe

420 425 430

Phe?Ala?Gln?Asp?Gly?Asn?Ala?Ala?Ile?Ser?Asp?Tyr?Asp?Tyr?Tyr?Arg

435 440 445

Tyr?Asn?Leu?Pro?Thr?Met?Cys?Asp?Ile?Arg?Gln?Leu?Leu?Phe?Val?Val

450 455 460

Glu?Val?Val?Asp?Lys?Tyr?Phe?Asp?Cys?Tyr?Asp?Gly?Gly?Cys?Ile?Asn

465 470 475 480

Ala?Asn?Gln?Val?Ile?Val?Asn?Asn?Leu?Asp?Lys?Ser?Ala?Gly?Phe?Pro

485 490 495

Phe?Asn?Lys?Trp?Gly?Lys?Ala?Arg?Leu?Tyr?Tyr?Asp?Ser?Met?Ser?Tyr

500 505 510

Glu?Asp?Gln?Asp?Ala?Leu?Phe?Ala?Tyr?Thr?Lys?Arg?Asn?Val?Ile?Pro

515 520 525

Thr?Ile?Thr?Gln?Met?Asn?Leu?Lys?Tyr?Ala?Ile?Ser?Ala?Lys?Asn?Arg

530 535 540

Ala?Arg?Thr?Val?Ala?Gly?Val?Ser?Ile?Cys?Ser?Thr?Met?Thr?ASn?Arg

545 550 555 560

Gln?Phe?His?Gln?Lys?Leu?Leu?Lys?Ser?Ile?Ala?Ala?Thr?Arg?Gly?Ala

565 570 575

Thr?Val?Val?Ile?Gly?Thr?Ser?Lys?Phe?Tyr?Gly?Gly?Trp?His?Asn?Met

580 585 590

Leu?Lys?Thr?Val?Tyr?Ser?Asp?Val?Glu?Thr?Pro?His?Leu?Met?Gly?Trp

595 600 605

Asp?Tyr?Pro?Lys?Cys?Asp?Arg?Ala?Met?Pro?Asn?Met?Leu?Arg?Ile?Met

610 615 620

Ala?Ser?Leu?Val?Leu?Ala?Arg?Lys?His?Asn?Thr?Cys?Cys?Asn?Leu?Ser

625 630 635 640

His?Arg?Phe?Tyr?Arg?Leu?Ala?Asn?Glu?Cys?Ala?Gln?Val?Leu?Ser?Glu

645 650 655

Met?Val?Met?Cys?Gly?Gly?Ser?Leu?Tyr?Val?Lys?Pro?Gly?Gly?Thr?Ser

660 665 670

Ser?Gly?Asp?Ala?Thr?Thr?Ala?Tyr?Ala?Asn?Ser?Val?Phe?Asn?Ile?Cys

675 680 685

Gln?Ala?Val?Thr?Ala?Asn?Val?Asn?Ala?Leu?Leu?Ser?Thr?Asp?Gly?Asn

690 695 700

Lys?Ile?Ala?Asp?Lys?Tyr?Val?Arg?Asn?Leu?Gln?His?Arg?Leu?Tyr?Glu

705 710 715 720

Cys?Leu?Tyr?Arg?Asn?Arg?Asp?Val?Asp?His?Glu?Phe?Val?Asp?Glu?Phe

725 730 735

Tyr?Ala?Tyr?Leu?Arg?Lys?His?Phe?Ser?Met?Met?Ile?Leu?Ser?Asp?Asp

740 745 750

Ala?Val?Val?Cys?Tyr?Asn?Ser?Asn?Tyr?Ala?Ala?Gln?Gly?Leu?Val?Ala

755 760 765

Ser?Ile?Lys?Asn?Phe?Lys?Ala?Val?Leu?Tyr?Tyr?Gln?Asn?Asn?Val?Phe

770 775 780

Met?Ser?Glu?Ala?Lys?Cys?Trp?Thr?Glu?Thr?Asp?Leu?Thr?Lys?Gly?Pro

785 790 795 800

His?Glu?Phe?Cys?Ser?Gln?His?Thr?Met?Leu?Val?Lys?Gln?Gly?Asp?Asp

805 810 815

Tyr?Val?Tyr?Leu?Pro?Tyr?Pro?Asp?Pro?Ser?Arg?Ile?Leu?Gly?Ala?Gly

820 825 830

Cys?Phe?Val?Asp?Asp?Ile?Val?Lys?Thr?Asp?Gly?Thr?Leu?Met?Ile?Glu

835 840 845

Arg?Phe?Val?Ser?Leu?Ala?Ile?Asp?Ala?Tyr?Pro?Leu?Thr?Lys?His?Pro

850 855 860

Asn?Gln?Glu?Tyr?Ala?Asp?Val?Phe?His?Leu?Tyr?Leu?Gln?Tyr?Ile?Arg

865 870 875 880

Lys?Leu?His?Asp?Glu?Leu?Thr?Gly?His?Met?Leu?Asp?Met?Tyr?Ser?Val

885 890 895

Met?Leu?Thr?Asn?Asp?Asn?Thr?Ser?Arg?Tyr?Trp?Glu?Pro?Glu?Phe?Tyr

900 905 910

Glu?Ala?Met?Tyr?Thr?Pro?His?Thr?Val?Leu?Gln?Ala?Val?Gly?Ala?Cys

915 920 925

Val?Leu?Cys?Asn?Ser?Gln?Thr?Ser?Leu?Arg?Cys?Gly?Ala?Cys?Ile?Arg

930 935 940

Arg?Pro?Phe?Leu?Cys?Cys?Lys?Cys?Cys?Tyr?Asp?His?Val?Ile?Ser?Thr

945 950 955 960

Ser?His?Lys?Leu?Val?Leu?Ser?Val?Asn?Pro?Tyr?Val?Cys?Asn?Ala?Pro

965 970 975

Gly?Cys?Asp?Val?Thr?Asp?Val?Thr?Gln?Leu?Tyr?Leu?Gly?Gly?Met?Ser

980 985 990

Tyr?Tyr?Cys?Lys?Ser?His?Lys?Pro?Pro?Ile?Ser?Phe?Pro?Leu?Cys?Ala

995 1000 1005

Asn?Gly?Gln?Val?Phe?Gly?Leu?Tyr?Lys?Asn?Thr?Cys?Val?Gly?Ser

1010 1015 1020

Asp?Asn?Val?Thr?Asp?Phe?Asn?Ala?Ile?Ala?Thr?Cys?Asp?Trp?Thr

1025 1030 1035

Ash?Ala?Gly?Asp?Tyr?Ile?Leu?Ala?Asn?Thr?Cys?Thr?Glu?Arg?Leu

1040 1045 1050

Lys?Leu?Phe?Ala?Ala?Glu?Thr?Leu?Lys?Ala?Thr?Glu?Glu?Thr?Phe

1055 1060 1065

Lys?Leu?Ser?Tyr?Gly?Ile?Ala?Thr?Val?Arg?Glu?Val?Leu?Ser?Asp

1070 1075 1080

Arg?Glu?Leu?His?Leu?Ser?Trp?Glu?Val?Gly?Lys?Pro?Arg?Pro?Pro

1085 1090 1095

Leu?Asn?Arg?Asn?Tyr?Val?Phe?Thr?Gly?Tyr?Arg?Val?Thr?Lys?Asn

1100 1105 1110

Ser?Lys?Val?Gln?Ile?Gly?Glu?Tyr?Thr?Phe?Glu?Lys?Gly?Asp?Tyr

1115 1120 1125

Gly?Asp?Ala?Val?Val?Tyr?Arg?Gly?Thr?Thr?Thr?Tyr?Lys?Leu?Asn

1130 1135 1140

Val?Gly?Asp?Tyr?Phe?Val?Leu?Thr?Ser?His?Thr?Val?Met?Pro?Leu

1145 1150 1155

Ser?Ala?Pro?Thr?Leu?Val?Pro?Gln?Glu?His?Tyr?Va Arg?Ile?Thr

1160 1165 1170

Gly?Leu?Tyr?Pro?Thr?Leu?Asn?Ile?Ser?Asp?Glu?Phe?Ser?Ser?Asn

1175 1180 1185

Val?Ala?Asn?Tyr?Gln?Lys?Val?Gly?Met?Gln?Lys?Tyr?Ser?Thr?Leu

1190 1195 1200

Gln?Gly?Pro?Pro?Gly?Thr?Gly?Lys?Ser?His?Phe?Ala?Ile?Gly?Leu

1205 1210 1215

Ala?Leu?Tyr?Tyr?Pro?Ser?Ala?Arg?Ile?Val?Tyr?Thr?Ala?Cys?Ser

1220 1225 1230

His?Ala?Ala?Val?Asp?Ala?Leu?Cys?Glu?Lys?Ala?Leu?Lys?Tyr?Leu

1235 1240 1245

Pro?Ile?Asp?Lys?Cys?Ser?Arg?Ile?Ile?Pro?Ala?Arg?Ala?Arg?Val

1250 1255 1260

Glu?Cys?Phe?Asp?Lys?Phe?Lys?Val?Asn?Ser?Thr?Leu?Glu?Gln?Tyr

1265 1270 1275

Val?Phe?Cys?Thr?Val?Asn?Ala?Leu?Pro?Glu?Thr?Thr?Ala?Asp?Ile

1280 1285 1290

Val?Val?Phe?Asp?Glu?Ile?Ser?Met?Ala?Thr?Asn?Tyr?Asp?Leu?Ser

1295 1300 1305

Val?Val?Asn?Ala?Arg?Leu?Arg?Ala?Lys?His?Tyr?Val?Tyr?Ile?Gly

1310 1315 1320

Asp?Pro?Ala?Gln?Leu?Pro?Ala?Pro?Arg?Thr?Leu?Leu?Thr?Lys?Gly

1325 1330 1335

Thr?Leu?Glu?Pro?Glu?Tyr?Phe?Asn?Ser?Val?Cys?Arg?Leu?Met?Lys

1340 1345 1350

Thr?Ile?Gly?Pro?Asp?Met?Phe?Leu?Gly?Thr?Cys?Arg?Arg?Cys?Pro

1355 1360 1365

Ala?Glu?Ile?Val?Asp?Thr?Val?Ser?Ala?Leu?Val?Tyr?Asp?Asn?Lys

1370 1375 1380

Leu?Lys?Ala?His?Lys?Asp?Lys?Ser?Ala?Gln?Cys?Phe?Lys?Met?Phe

1385 1390 1395

Tyr?Lys?Gly?Val?Ile?Thr?His?Asp?Val?Ser?Ser?Ala?Ile?Asn?Arg

1400 1405 1410

Pro?Gln?Ile?Gly?Val?Val?Arg?Glu?Phe?Leu?Thr?Arg?Asn?Pro?Ala

1415 1420 1425

Trp?Arg?Lys?Ala?Val?Phe?Ile?Ser?Pro?Tyr?Asn?Ser?Gln?Asn?Ala

1430 1435 1440

Val?Ala?Ser?Lys?Ile?Leu?Gly?Leu?Pro?Thr?Gln?Thr?Val?Asp?Ser

1445 1450 1455

Ser?Gln?Gly?Ser?Glu?Tyr?Asp?Tyr?Val?Ile?Phe?Thr?Gln?Thr?Thr

1460 1465 1470

Glu?Thr?Ala?His?Ser?Cys?Asn?Val?Asn?Arg?Phe?Asn?Val?Ala?Ile

1475 1480 1485

Thr?Arg?Ala?Lys?Ile?Gly?Ile?Leu?Cys?Ile?Met?Ser?Asp?Arg?Asp

1490 1495 1500

Leu?Tyr?Asp?Lys?Leu?Gln?Phe?Thr?Ser?Leu?Glu?Ile?Pro?Arg?Arg

1505 1510 1515

Asn?Val?Ala?Thr?Leu?Gln?Ala?Glu?Asn?Val?Thr?Gly?Leu?Phe?Lys

1520 1525 1530

Asp?Cys?Ser?Lys?Ile?Ile?Thr?Gly?Leu?His?Pro?Thr?Gln?Ala?Pro

1535 1540 1545

Thr?His?Leu?Ser?Val?Asp?Ile?Lys?Phe?Lys?Thr?Glu?Gly?Leu?Cys

1550 1555 1560

Val?Asp?Ile?Pro?Gly?Ile?Pro?Lys?Asp?Met?Thr?Tyr?Arg?Arg?Leu

1565 1570 1575

Ile?Ser?Met?Met?Gly?Phe?Lys?Met?Asn?Tyr?Gln?Val?Asn?Gly?Tyr

1580 1585 1590

Pro?Asn?Met?Phe?Ile?Thr?Arg?Glu?Glu?Ala?Ile?Arg?His?Val?Arg

1595 1600 1605

Ala?Trp?Ile?Gly?Phe?Asp?Val?Glu?Gly?Cys?His?Ala?Thr?Arg?Asp

1610 1615 1620

Ala?Val?Gly?Thr?Asn?Leu?Pro?Leu?Gln?Leu?Gly?Phe?Ser?Thr?Gly

1625 1630 1635

Val?Asn?Leu?Val?Ala?Val?Pro?Thr?Gly?Tyr?Val?Asp?Thr?Glu?Asn

1640 1645 1650

Asn?Thr?Glu?Phe?Thr?Arg?Val?Asn?Ala?Lys?Pro?Pro?Pro?Gly?Asp

1655 1660 1665

Gln?Phe?Lys?His?Leu?Ile?Pro?Leu?Met?Tyr?Lys?Gly?Leu?Pro?Trp

1670 1675 1680

Asn?Val?Val?Arg?Ile?Lys?Ile?Val?Gln?Met?Leu?Ser?Asp?Thr?Leu

1685 1690 1695

Lys?Gly?Leu?Ser?Asp?Arg?Val?Val?Phe?Val?Leu?Trp?Ala?His?Gly

1700 1705 1710

Phe?Glu?Leu?Thr?Ser?Met?Lys?Tyr?Phe?Val?Lys?IIe?Gly?Pro?Glu

1715 1720 1725

Arg?Thr?Cys?Cys?Leu?Cys?Asp?Lys?Arg?Ala?Thr?Cys?Phe?Ser?Thr

1730 1735 1740

Ser?Ser?Asp?Thr?Tyr?Ala?Cys?Trp?Asn?His?Ser?Val?Gly?Phe?Asp

1745 1750 1755

Tyr?Val?Tyr?Asn?Pro?Phe?Met?Ile?Asp?Val?Gln?Gln?Trp?Gly?Phe

1760 1765 1770

Thr?Gly?Asn?Leu?Gln?Ser?Asn?His?Asp?Gln?His?Cys?Gln?Val?His

1775 1780 1785

Gly?Asn?Ala?His?Val?Ala?Ser?Cys?Asp?Ala?Ile?Met?Thr?Arg?Cys

1790 1795 1800

Leu?Ala?Val?His?Glu?Cys?Phe?Val?Lys?Arg?Val?Asp?Trp?Ser?Val

1805 1810 1815

Glu?Tyr?Pro?Ile?Ile?Gly?Asp?Glu?Leu?Arg?Val?Asn?Ser?Ala?Cys

1820 1825 1830

Arg?Lys?Val?Gln?His?Met?Val?Val?Lys?Ser?Ala?Leu?Leu?Ala?Asp

1835 1840 1845

Lys?Phe?Pro?Val?Leu?His?Asp?Ile?Gly?Asn?Pro?Lys?Ala?Ile?Lys

1850 1855 1860

Cys?Val?Pro?Gln?Ala?Glu?Val?Glu?Trp?Lys?Phe?Tyr?Asp?Ala?Gln

1865 1870 1875

Pro?Cys?Ser?Asp?Lys?Ala?Tyr?Lys?Ile?Glu?Glu?Leu?Phe?Tyr?Ser

1880 1885 1890

Tyr?Ala?Thr?His?His?Asp?Lys?Phe?Thr?Asp?Gly?Val?Cys?Leu?Phe

1895 1900 1905

Trp?Asn?Cys?Asn?Val?Asp?Arg?Tyr?Pro?Ala?Asn?Ala?Ile?Val?Cys

1910 1915 1920

Arg?Phe?Asp?Thr?Arg?Val?Leu?Ser?Asn?Leu?Asn?Leu?Pro?Gly?Cys

1925 1930 1935

Asp?Gly?Gly?Ser?Leu?Tyr?Val?Asn?Lys?His?Ala?Phe?His?Thr?Pro

1940 1945 1950

Ala?Phe?Asp?Lys?Ser?Ala?Phe?Thr?Asn?Leu?Lys?Gln?Leu?Pro?Phe

1955 1960 1965

Phe?Tyr?Tyr?Ser?Asp?Ser?Pro?Cys?Glu?Ser?His?Gly?Lys?Gln?Val

1970 1975 1980

Val?Ser?Asp?Ile?Asp?Tyr?Val?Pro?Leu?Lys?Ser?Ala?Thr?Cys?Ile

1985 1990 1995

Thr?Arg?Cys?Asn?Leu?Gly?Gly?Ala?Val?Cys?Arg?His?His?Ala?Asn

2000 2005 2010

Glu?Tyr?Arg?Gln?Tyr?Leu?Asp?Ala?Tyr?Asn?Met?Met?Ile?Ser?Ala

2015 2020 2025

Gly?Phe?Ser?Leu?Trp?Ile?Tyr?Lys?Gln?Phe?Asp?Thr?Tyr?Asn?Leu

2030 2035 2040

Trp?Asn?Thr?Phe?Thr?Arg?Leu?Gln?Ser?Leu?Glu?Asn?Val?Ala?Tyr

2045 2050 2055

Asn?Val?Val?Asn?Lys?Gly?His?Phe?Asp?Gly?His?Ala?Gly?Glu?Ala

2060 2065 2070

Pro?Val?Ser?Ile?Ile?Asn?Asn?Ala?Val?Tyr?Thr?Lys?Val?Asp?Gly

2075 2080 2085

Ile?Asp?Val?Glu?Ile?Phe?Glu?Asn?Lys?Thr?Thr?Leu?Pro?Val?Asn

2090 2095 2100

Val?Ala?Phe?Glu?Leu?Trp?Ala?Lys?Arg?Asn?Ile?Lys?Pro?Val?Pro

2105 2110 2115

Glu?Ile?Lys?Ile?Leu?Asn?Asn?Leu?Gly?Val?Asp?Ile?Ala?Ala?Asn

2120 2125 2130

Thr?Val?Ile?Trp?Asp?Tyr?Lys?Arg?Glu?Ala?Pro?Ala?His?Val?Ser

2135 2140 2145

Thr?Ile?Gly?Val?Cys?Thr?Met?Thr?Asp?Ile?Ala?Lys?Lys?Pro?Thr

2150 2155 2160

Glu?Ser?Ala?Cys?Ser?Ser?Leu?Thr?Val?Leu?Phe?Asp?Gly?Arg?Val

2165 2170 2175

Glu?Gly?Gln?Val?Asp?Leu?Phe?Arg?Asn?Ala?Arg?Asn?Gly?Val?Leu

2180 2185 2190

Ile?Thr?Glu?Gly?Ser?Val?Lys?Gly?Leu?Thr?Pro?Ser?Lys?Gly?Pro

2195 2200 2205

Ala?Gln?Ala?Ser?Val?Asn?Gly?Val?Thr?Leu?Ile?Gly?Glu?Ser?Val

2210 2215 2220

Lys?Thr?Gln?Phe?Asn?Tyr?Phe?Lys?Lys?Val?Asp?Gly?Ile?Ile?Gln

2225 2230 2235

Gln?Leu?Pro?Glu?Thr?Tyr?Phe?Thr?Gln?Ser?Arg?Asp?Leu?Glu?Asp

2240 2245 2250

Phe?Lys?Pro?Arg?Ser?Gln?Met?Glu?Thr?Asp?Phe?Leu?Glu?Leu?Ala

2255 2260 2265

Met?Asp?Glu?Phe?Ile?Gln?Arg?Tyr?Lys?Leu?Glu?Gly?Tyr?Ala?Phe

2270 2275 2280

Glu?His?Ile?Val?Tyr?Gly?Asp?Phe?Ser?His?Gly?Gln?Leu?Gly?Gly

2285 2290 2295

Leu?His?Leu?Met?Ile?Gly?Leu?Ala?Lys?Arg?Ser?Gln?Asp?Ser?Pro

2300 2305 2310

Leu?Lys?Leu?Glu?Asp?Phe?Ile?Pro?Met?Asp?Ser?Thr?Val?Lys?Asn

2315 2320 2325

Tyr?Phe?Ile?Thr?Asp?Ala?Gln?Thr?Gly?Ser?Ser?Lys?Cys?Val?Cys

2330 2335 2340

Ser?Val?Ile?Asp?Leu?Leu?Leu?Asp?Asp?Phe?Val?Glu?Ile?Ile?Lys

2345 2350 2355

Ser?Gln?Asp?Leu?Ser?Val?Ile?Ser?Lys?Val?Val?Lys?Val?Thr?Ile

2360 2365 2370

Asp?Tyr?Ala?Glu?Ile?Ser?Phe?Met?Leu?Trp?Cys?Lys?Asp?Gly?His

2375 2380 2385

Val?Glu?Thr?Phe?Tyr?Pro?Lys?Leu?Gln?Ala?Ser?Gln?Ala?Trp?Gln

2390 2395 2400

Pro?Gly?Val?Ala?Met?Pro?Asn?Leu?Tyr?Lys?Met?Gln?Arg?Met?Leu

2405 2410 2415

Leu?Glu?Lys?Cys?Asp?Leu?Gln?Asn?Tyr?Gly?Glu?Asn?Ala?Val?Ile

2420 2425 2430

Pro?Lys?Gly?Ile?Met?Met?Asn?Val?Ala?Lys?Tyr?Thr?Gln?Leu?Cys

2435 2440 2445

Gln?Tyr?Leu?Asn?Thr?Leu?Thr?Leu?Ala?Val?Pro?Tyr?Asn?Met?Arg

2450 2455 2460

Val?Ile?His?Phe?Gly?Ala?Gly?Ser?Asp?Lys?Gly?Val?Ala?Pro?Gly

2465 2470 2475

Thr?Ala?Val?Leu?Arg?Gln?Trp?Leu?Pro?Thr?Gly?Thr?Leu?Leu?Val

2480 2485 2490

Asp?Ser?Asp?Leu?Asn?Asp?Phe?Val?Ser?Asp?Ala?Asp?Ser?Thr?Leu

2495 2500 2505

Ile?Gly?Asp?Cys?Ala?Thr?Val?His?Thr?Ala?Asn?Lys?Trp?Asp?Leu

2510 2515 2520

Ile?Ile?Ser?Asp?Met?Tyr?Asp?Pro?Arg?Thr?Lys?His?Val?Thr?Lys

2525 2530 2535

Glu?Asn?Asp?Ser?Lys?Glu?Gly?Phe?Phe?Thr?Tyr?Leu?Cys?Gly?Phe

2540 2545 2550

Ile?Lys?Gln?Lys?Leu?Ala?Leu?Gly?Gly?Ser?Ile?Ala?Val?Lys?Ile

2555 2560 2565

Thr?Glu?His?Ser?Trp?Asn?Ala?Asp?Leu?Tyr?Lys?Leu?Met?Gly?His

2570 2575 2580

Phe?Ser?Trp?Trp?Thr?Ala?Phe?Val?Thr?Asn?Val?Asn?Ala?Ser?Ser

2585 2590 2595

Ser?Glu?Ala?Phe?Leu?Ile?Gly?Ala?Asn?Tyr?Leu?Gly?Lys?Pro?Lys

2600 2605 2610

Glu?Gln?Ile?Asp?Gly?Tyr?Thr?Met?His?Ala?Asn?Tyr?Ile?Phe?Trp

2615 2620 2625

Arg?Asn?Thr?Asn?Pro?Ile?Gln?Leu?Ser?Ser?Tyr?Ser?Leu?Phe?Asp

2630 2635 2640

Met?Ser?Lys?Phe?Pro?Leu?Lys?Leu?Arg?Gly?Thr?Ala?Val?Met?Ser

2645 2650 2655

Leu?Lys?Glu?Asn?Gln?Ile?Asn?Asp?Met?Ile?Tyr?Ser?Leu?Leu?Glu

2660 2665 2670

Lys?Gly?Arg?Leu?Ile?Ile?Arg?Glu?Asn?Asn?Arg?Val?Val?Val?Ser

2675 2680 2685

Ser?Asp?Ile?Leu?Val?Asn?Asn

2690 2695

<210>76

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉S/L3/+/4932 primers

<400>76

ccacacacag?cttgtggata 20

<210>77

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉S/L4/+/6401 primers

<400>77

ccgaagttgt?aggcaatgtc 20

<210>78

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉S/L4/+/6964 primers

<400>78

tttggtgctc?cttcttattg 20

<210>79

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉S/L4/-/6817 primers

<400>79

ccggcatcca?aacataattt 20

<210>80

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉S/L5/-/7633 primers

<400>80

tggtcagtag?ggttgattgg 20

<210>81

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉S/L5/-/8127 primers

<400>81

catcctttgt?gtcaacatcg 20

<210>82

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉S/L5/-/8633 primers

<400>82

gtcacgagtg?acaccatcct 20

<210>83

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉S/L5/+/7839 primers

<400>83

atgcgacgag?tctgcttcta 20

<210>84

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉S/L5/+/8785 primers

<400>84

ttcatagtgc?ctggcttacc 20

<210>85

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉S/L5/+/8255 primers

<400>85

atcttggcgc?atgtattgac 20

<210>86

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉S/L6/-/9422 primers

<400>86

tgcattagca?gcaacaacat 20

<210>87

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉S/L6/-/9966 primers

<400>87

tctgcagaac?agcagaagtg 20

<210>88

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉S/L6/-/10542 primers

<400>88

cctgtgcagt?ttgtctgtca 20

<210>89

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉S/L6/+/10677 primers

<400>89

ccttgtggca?atgaagtaca 20

<210>90

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉S/L6/+/10106 primers

<400>90

atgtcatttg?cacagcagaa 20

<210>91

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉S/L6/+/9571 primers

<400>91

cttcaatggt?ttgccatgtt 20

<210>92

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉S/L7/-/11271 primers

<400>92

tgcgagctgt?catgagaata 20

<210>93

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉S/L7/-/11801 primers

<400>93

aaccgagagc?agtaccacag 20

<210>94

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉S/L7/-/12383 primers

<400>94

tttggctgct?gtagtcaatg 20

<210>95

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉S/L7/+/12640 primers

<400>95

ctacgacaga?tgtcctgtgc 20

<210>96

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉S/L7/+/12088 primers

<400>96

gagcaggctg?tagctaatgg 20

<210>97

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉S/L7/+/11551 primers

<400>97

ttaggctatt?gttgctgctg 20

<210>98

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉S/L8/-/13160 primers

<400>98

cagacaacat?gaagcaccac 20

<210>99

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉S/L8/-/13704 primers

<400>99

cgctgacgtg?atatatgtgg 20

<210>100

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉S/L8/-/14284 primers

<400>100

tgcacaatga?aggatacacc 20

<210>101

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉S/L8/+/14453 primers

<400>101

acatagctcg?cgtctcagtt 20

<210>102

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉S/L8/+/13968 primers

<400>102

ggcattgtag?gcgtactgac 20

<210>103

<211>19

<212>DNA

＜213〉artificial sequence

<220>

＜223〉S/L8/+/13401 primers

<400>103

gtttgcggtg?taagtgcag 19

<210>104

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉S/L9/-/15098 primers

<400>104

tagtggcggc?tattgacttc 20

<210>105

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉S/L9/-/15677 primers

<400>105

ctaaaccttg?agccgcatag 20

<210>106

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉S/L9/-/16247 primers

<400>106

catggtcata?gcagcacttg 20

<210>107

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉S/L9/+/16323 primers

<400>107

ccaggttgtg?atgtcactga?t 21

<210>108

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉S/L9/+/15858 primers

<400>108

ccttacccag?atccatcaag 20

<210>109

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉S/L9/+/15288 primers

<400>109

cgcaaacata?acacttgctg 20

<210>110

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉S/L10/-/16914 primers

<400>110

agtgttgggt?acaagccagt 20

<210>111

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉S/L10/-/17466 primers

<400>111

gttccaagga?acatgtctgg 20

<210>112

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉S/L10/-/18022 primers

<400>112

aggtgcctgt?gtaggatgaa 20

<210>113

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉S/L10/+/18245 primers

<400>113

gggctgtcat?gcaactagag 20

<210>114

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉S/L10/+/17663 primers

<400>114

tcttacacgc?aatcctgctt 20

<210>115

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉S/L10/+/17061 primers

<400>115

tacccatctg?ctcgcatagt 20

<210>116

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉S/L11/-/18877 primers

<400>116

gcaagcagaa?ttaaccctca 20

<210>117

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉S/L11/-/19396 primers

<400>117

agcaccacct?aaattgcatc 20

<210>118

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉S/L11/-/20002 primers

<400>118

tggtcccttt?gaaggtgtta 20

<210>119

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉S/L11/+/20245 primers

<400>119

tcgaacacat?cgtttatgga 20

<210>120

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉S/L11/+/19611 primers

<400>120

gaagcacctg?tttccatcat 20

<210>121

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉S/L11/+/19021 primers

<400>121

acgatgctca?gccatgtagt 20

<210>122

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉SARS/L1/F3/+/800 primers

<400>122

gaggtgcagt?cactcgctat 20

<210>123

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉SARS/L1/F4/+/1391 primers

<400>123

cagagattgg?acctgagcat 20

<210>124

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉SARS/L1/F5/+/1925 primers

<400>124

cagcaaacca?ctcaattcct 20

<210>125

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉SARS/L1/R3/-/1674 primers

<400>125

aaatgatggc?aacctcttca 20

<210>126

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉SARS/L1/R4/-/1107 primers

<400>126

cacgtggttg?aatgactttg 20

<210>127

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉SARS/L1/R5/-/520 primers

<400>127

atttctgcaa?ccagctcaac 20

<210>128

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉SARS/L2/F3/+/2664 primers

<400>128

cgcattgtct?cctggtttac 20

<210>129

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉SARS/L2/F4/+/3232 primers

<400>129

gagattgagc?cagaaccaga 20

<210>130

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉SARS/L2/F5/+/3746 primers

<400>130

atgagcaggt?tgtcatggat 20

<210>131

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉SARS/L2/R3/-/3579 primers

<400>131

ctgccttaag?aagctggatg 20

<210>132

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉SARS/L2/R4/-/2991 primers

<400>132

tttcttcacc?agcatcatca 20

<210>133

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉SARS/L2/R5/-/2529 primers

<400>133

caccgttctt?gagaacaacc 20

<210>134

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉SARS/L3/F3/+/4708 primers

<400>134

tctttggctg?gctcttacag 20

<210>135

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉SRAS/L3/F4/+/5305 primers

<400>135

gctggtgatg?ctgctaactt 20

<210>136

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉SARS/L3/F5/+/5822 primers

<400>136

ccatcaagcc?tgtgtcgtat 20

<210>137

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉SARS/L3/R3/-/5610 primers

<400>137

caggtggtgc?agacatcata 20

<210>138

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉SARS/L3/R4/-/4988 primers

<400>138

aacatcagca?ccatccaagt 20

<210>139

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉SARS/L3/R5/-/4437 primers

<400>139

atcggagacc?atagtcaacg 20

<210>140

<211>7788

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic S gene

<400>140

tcaatattgg?ccattagcca?tattattcat?tggttatata?gcataaatca?atattggcta 60

ttggccattg?catacgttgt?atctatatca?taatatgtac?atttatattg?gctcatgtcc 120

aatatgaccg?ccatgttggc?attgattatt?gactagttat?taatagtaat?caattacggg 180

gtcattagtt?catagcccat?atatggagtt?ccgcgttaca?taacttacgg?taaatggccc 240

gcctggctga?ccgcccaacg?acccccgccc?attgacgtca?ataatgacgt?atgttcccat 300

agtaacgcca?atagggactt?tccattgacg?tcaatgggtg?gagtatttac?ggtaaactgc 360

ccacttggca?gtacatcaag?tgtatcatat?gccaagtccg?ccccctattg?acgtcaatga 420

cggtaaatgg?cccgcctggc?attatgccca?gtacatgacc?ttacgggact?ttcctacttg 480

gcagtacatc?tacgtattag?tcatcgctat?taccatggtg?atgcggtttt?ggcagtacac 540

caatgggcgt?ggatagcggt?ttgactcacg?gggatttcca?agtctccacc?ccattgacgt 600

caatgggagt?ttgttttggc?accaaaatca?acgggacttt?ccaaaatgtc?gtaataaccc 660

cgccccgttg?acgcaaatgg?gcggtaggcg?tgtacggtgg?gaggtctata?taagcagagc 720

tcgtttagtg?aaccgtcaga?tcactagaag?ctttattgcg?gtagtttatc?acagttaaat 780

tgctaacgca?gtcagtgctt?ctgacacaac?agtctcgaac?ttaagctgca?gaagttggtc 840

gtgaggcact?gggcaggtaa?gtatcaaggt?tacaagacag?gtttaaggag?accaatagaa 900

actgggcttg?tcgagacaga?gaagactctt?gcgtttctga?taggcaccta?ttggtcttac 960

tgacatccac?tttgcctttc?tctccacagg?tgtccactcc?cagttcaatt?acagctctta 1020

aggctagagt?acttaatacg?actcactata?ggctagcgga?tccaccatgt?tcatcttcct 1080

gctgttcctg?accctgacca?gcggcagcga?cctggaccgg?tgcaccacct?tcgacgacgt 1140

gcaggccccc?aactacaccc?agcacaccag?cagcatgcgg?ggcgtgtact?accccgacga 1200

gatctttcgg?agcgacaccc?tgtacctgac?ccaggacctg?ttcctgccct?tctacagcaa 1260

cgtgaccggc?ttccacacca?tcaaccacac?cttcggcaac?cccgtgatcc?ccttcaagga 1320

cggcatctac?ttcgccgcca?ccgagaagag?caacgtggtg?cggggctggg?tgttcggcag 1380

caccatgaac?aacaagagcc?agagcgtgat?catcatcaac?aacagcacca?acgtggtgat 1440

ccgggcctgc?aacttcgagc?tgtgcgacaa?ccccttcttc?gccgtgtcca?aacccatggg 1500

cacccagacc?cacaccatga?tcttcgacaa?cgccttcaac?tgcaccttcg?agtacatcag 1560

cgacgccttc?agcctggacg?tgagcgagaa?gagcggcaac?ttcaagcacc?tgcgggagtt 1620

cgtgttcaag?aacaaggacg?gcttcctgta?cgtgtacaag?ggctaccagc?ccatcgacgt 1680

ggtgagagac?ctgcccagcg?gcttcaacac?cctgaagccc?atcttcaagc?tgcccctggg 1740

catcaacatc?accaacttcc?gggccatcct?gaccgccttt?agccctgccc?aggacatctg 1800

gggcaccagc?gccgccgcct?acttcgtggg?ctacctgaag?cctaccacct?tcatgctgaa 1860

gtacgacgag?aacggcacca?tcaccgacgc?cgtggactgc?agccagaacc?ccctggccga 1920

gctgaagtgc?agcgtgaaga?gcttcgagat?cgacaagggc?atctaccaga?ccagcaactt 1980

cagagtggtg?cctagcggcg?atgtggtgcg?gttccccaat?atcaccaacc?tgtgcccctt 2040

cggcgaagtg?ttcaacgcca?ccaagttccc?cagcgtgtac?gcctgggagc?ggaagaagat 2100

cagcaactgc?gtggccgact?acagcgtgct?gtacaactcc?accttcttca?gcaccttcaa 2160

gtgctacggc?gtgagcgcca?ccaagctgaa?cgacctgtgc?ttcagcaacg?tgtacgccga 2220

cagcttcgtg?gtgaagggcg?acgacgtgag?acagatcgcc?cctggccaga?ccggcgtgat 2280

cgccgactac?aactacaagc?tgcccgacga?cttcatgggc?tgcgtgctgg?cctggaacac 2340

ccggaacatc?gacgccacaa?gcaccggcaa?ctacaattac?aagtaccgct?acctgcggca 2400

cggcaagctg?cggcccttcg?agcgggacat?ctccaacgtg?cccttcagcc?ccgacggcaa 2460

gccctgcacc?ccccctgccc?tgaactgcta?ctggcccctg?aacgactacg?gcttctacac 2520

caccaccggc?atcggctatc?agccctacag?agtggtggtg?ctgagcttcg?agctgctgaa 2580

cgcccctgcc?accgtgtgcg?gccccaagct?gagcaccgac?ctgatcaaga?accagtgcgt 2640

gaacttcaac?ttcaacggcc?tgaccggcac?cggcgtgctg?acccccagca?gcaagcgctt 2700

ccagcccttc?cagcagttcg?gccgggatgt?gagcgacttc?accgacagcg?tgcgggaccc 2760

caagaccagc?gagatcctgg?acatcagccc?ctgcagcttc?ggcggcgtgt?ccgtgatcac 2820

ccccggcacc?aacgccagca?gcgaagtggc?cgtgctgtac?caggacgtga?actgcaccga 2880

cgtgagcacc?gccatccacg?ccgaccagct?gacccccgcc?tggcggatct?acagcaccgg 2940

gaacaacgtg?ttccagaccc?aggccggctg?cctgatcggc?gccgagcacg?tggacaccag 3000

ctacgagtgc?gacatcccca?ttggcgccgg?aatctgcgcc?agctaccaca?ccgtgagcct 3060

gctgcggagc?accagccaga?agtccatcgt?ggcctacacc?atgagcctgg?gcgccgacag 3120

cagcatcgcc?tacagcaaca?acaccatcgc?catccccacc?aacttcagca?tctccatcac 3180

caccgaagtg?atgcccgtga?gcatggccaa?gacaagcgtg?gattgcaaca?tgtacatctg 3240

cggcgacagc?accgagtgcg?ccaacctgct?gctgcagtac?ggcagcttct?gcacccagct 3300

gaaccgggcc?ctgagcggca?tcgccgccga?gcaggaccgg?aacaccagag?aagtgttcgc 3360

ccaagtgaag?cagatgtata?agacccccac?cctgaagtac?ttcgggggct?tcaacttctc 3420

tcagatcctg?cccgaccctc?tgaagcccac?caagcgctcc?ttcatcgagg?acctgctgtt 3480

caacaaagtg?accctggccg?acgccggctt?tatgaagcag?tacggcgagt?gcctgggcga 3540

catcaacgcc?cgggacctga?tctgcgccca?gaagtttaac?gggctgaccg?tgctgccccc 3600

cctgctgacc?gacgacatga?tcgccgccta?tacagccgcc?ctggtgagcg?gcaccgccac 3660

cgccggctgg?accttcggag?ccggagccgc?cctgcagatc?cccttcgcca?tgcagatggc 3720

ctaccggttc?aacggcatcg?gcgtgaccca?gaacgtgctg?tacgagaacc?agaagcagat 3780

cgccaaccag?ttcaacaagg?ccatcagcca?gatccaggag?agcctgacca?caaccagcac 3840

cgccctgggc?aagctgcagg?acgtggtgaa?ccagaacgcc?caggccctga?acaccctggt 3900

gaagcagctg?agcagcaact?tcggcgccat?cagctctgtg?ctgaacgaca?tcctgagcag 3960

gctggacaaa?gtggaggccg?aagtgcagat?cgaccggctg?atcaccggac?gcctgcagtc 4020

cctgcagacc?tacgtgaccc?agcagctgat?cagagccgcc?gagatccggg?ccagcgccaa 4080

tctggccgcc?accaagatga?gcgagtgcgt?gctgggccag?agcaagagag?tggacttctg 4140

cggcaagggc?tatcacctga?tgagcttccc?ccaggccgcc?ccccacggcg?tggtgttcct 4200

gcacgtgacc?tacgtgccta?gccaggagcg?gaacttcacc?accgccccag?ccatctgcca 4260

cgagggcaag?gcctacttcc?cccgggaggg?cgtgttcgtg?tttaacggca?ccagctggtt 4320

catcacccag?cgcaacttct?tcagccccca?gatcatcacc?acagacaaca?ccttcgtgtc 4380

cggcaactgt?gatgtggtga?tcggcatcat?caataacacc?gtgtacgacc?ccctgcagcc 4440

cgagctggac?agcttcaagg?aggagctgga?caaatacttc?aagaaccaca?cctcccccga 4500

cgtggacctg?ggcgatatca?gcggcatcaa?cgcctccgtg?gtgaacatcc?agaaggagat 4560

cgacagactg?aacgaagtgg?ccaagaacct?gaacgagagc?ctgatcgacc?tgcaggagct 4620

gggcaagtac?gagcagtaca?tcaagtggcc?ctggtacgtg?tggctgggct?tcatcgccgg 4680

cctgatcgcc?atcgtgatgg?tgaccatcct?gctgtgctgc?atgaccagct?gctgtagctg 4740

cctgaaaggc?gcctgcagct?gtggcagctg?ctgcaagttc?gacgaggacg?acagcgagcc 4800

cgtgctgaag?ggcgtgaagc?tgcactacac?ctgataactc?gagaattcac?gcgtggtacc 4860

tctagagtcg?acccgggcgg?ccgcttcgag?cagacatgat?aagatacatt?gatgagtttg 4920

gacaaaccac?aactagaatg?cagtgaaaaa?aatgctttat?ttgtgaaatt?tgtgatgcta 4980

ttgctttatt?tgtaaccatt?ataagctgca?ataaacaagt?taacaacaac?aattgcattc 5040

attttatgtt?tcaggttcag?ggggagatgt?gggaggtttt?ttaaagcaag?taaaacctct 5100

acaaatgtgg?taaaatcgat?aaggatccgg?gctggcgtaa?tagcgaagag?gcccgcaccg 5160

atcgcccttc?ccaacagttg?cgcagcctga?atggcgaatg?gacgcgccct?gtagcggcgc 5220

attaagcgcg?gcgggtgtgg?tggttacgcg?cagcgtgacc?gctacacttg?ccagcgccct 5280

agcgcccgct?cctttcgctt?tcttcccttc?ctttctcgcc?acgttcgccg?gctttccccg 5340

tcaagctcta?aatcgggggc?tccctttagg?gttccgattt?agagctttac?ggcacctcga 5400

ccgcaaaaaa?cttgatttgg?gtgatggttc?acgtagtggg?ccatcgccct?gatagacggt 5460

ttttcgccct?ttgacgttgg?agtccacgtt?ctttaatagt?ggactcttgt?tccaaactgg 5520

aacaacactc?aaccctatct?cggtctattc?ttttgattta?taagggattt?tgccgatttc 5580

ggcctattgg?ttaaaaaatg?agctgattta?acaaatattt?aacgcgaatt?ttaacaaaat 5640

attaacgttt?acaatttcgc?ctgatgcggt?attttctcct?tacgcatctg?tgcggtattt 5700

cacaccgcat?atggtgcact?ctcagtacaa?tctgctctga?tgccgcatag?ttaagccagc 5760

cccgacaccc?gccaacaccc?gctgacgcgc?cctgacgggc?ttgtctgctc?ccggcatccg 5820

cttacagaca?agctgtgacc?gtctccggga?gctgcatgtg?tcagaggttt?tcaccgtcat 5880

caccgaaacg?cgcgagacga?aagggcctcg?tgatacgcct?atttttatag?gttaatgtca 5940

tgataataat?ggtttcttag?acgtcaggtg?gcacttttcg?gggaaatgtg?cgcggaaccc 6000

ctatttgttt?atttttctaa?atacattcaa?atatgtatcc?gctcatgaga?caataaccct 6060

gataaatgct?tcaataatat?tgaaaaagga?agagtatgag?tattcaacat?ttccgtgtcg 6120

cccttattcc?cttttttgcg?gcattttgcc?ttcctgtttt?tgctcaccca?gaaacgctgg 6180

tgaaagtaaa?agatgctgaa?gatcagttgg?gtgcacgagt?gggttacatc?gaactggatc 6240

tcaacagcgg?taagatcctt?gagagttttc?gccccgaaga?acgttttcca?atgatgagca 6300

cttttaaagt?tctgctatgt?ggcgcggtat?tatcccgtat?tgacgccggg?caagagcaac 6360

tcggtcgccg?catacactat?tctcagaatg?acttggttga?gtactcacca?gtcacagaaa 6420

agcatcttac?ggatggcatg?acagtaagag?aattatgcag?tgctgccata?accatgagtg 6480

ataacactgc?ggccaactta?cttctgacaa?cgatcggagg?accgaaggag?ctaaccgctt 6540

ttttgcacaa?catgggggat?catgtaactc?gccttgatcg?ttgggaaccg?gagctgaatg 6600

aagccatacc?aaacgacgag?cgtgacacca?cgatgcctgt?agcaatggca?acaacgttgc 6660

gcaaactatt?aactggcgaa?ctacttactc?tagcttcccg?gcaacaatta?atagactgga 6720

tggaggcgga?taaagttgca?ggaccacttc?tgcgctcggc?ccttccggct?ggctggttta 6780

ttgctgataa?atctggagcc?ggtgagcgtg?ggtctcgcgg?tatcattgca?gcactggggc 6840

cagatggtaa?gccctcccgt?atcgtagtta?tctacacgac?ggggagtcag?gcaactatgg 6900

atgaacgaaa?tagacagatc?gctgagatag?gtgcctcact?gattaagcat?tggtaactgt 6960

cagaccaagt?ttactcatat?atactttaga?ttgatttaaa?acttcatttt?taatttaaaa 7020

ggatctaggt?gaagatcctt?tttgataatc?tcatgaccaa?aatcccttaa?cgtgagtttt 7080

cgttccactg?agcgtcagac?cccgtagaaa?agatcaaagg?atcttcttga?gatccttttt 7140

ttctgcgcgt?aatctgctgc?ttgcaaacaa?aaaaaccacc?gctaccagcg?gtggtttgtt 7200

tgccggatca?agagctacca?actctttttc?cgaaggtaac?tggcttcagc?agagcgcaga 7260

taccaaatac?tgtccttcta?gtgtagccgt?agttaggcca?ccacttcaag?aactctgtag 7320

caccgcctac?atacctcgct?ctgctaatcc?tgttaccagt?ggctgctgcc?agtggcgata 7380

agtcgtgtct?taccgggttg?gactcaagac?gatagttacc?ggataaggcg?cagcggtcgg 7440

gctgaacggg?gggttcgtgc?acacagccca?gcttggagcg?aacgacctac?accgaactga 7500

gatacctaca?gcgtgagcta?tgagaaagcg?ccacgcttcc?cgaagggaga?aaggcggaca 7560

ggtatccggt?aagcggcagg?gtcggaacag?gagagcgcac?gagggagctt?ccagggggaa 7620

acgcctggta?tctttatagt?cctgtcgggt?ttcgccacct?ctgacttgag?cgtcgatttt 7680

tgtgatgctc?gtcagggggg?cggagcctat?ggaaaaacgc?cagcaacgcg?gcctttttac 7740

ggttcctggc?cttttgctgg?ccttttgctc?acatggctcg?acagatct 7788

210>141

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉SNE-S1 primer

<400>141

ggttgggatt?atccaaaatg?tga 23

<210>142

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜223〉SNE-AS1 primer

<400>142

gcatcatcag?aaagaatcat?catg 24

<210>143

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉SAR1-S primer

<400>143

cctctcttgt?tcttgctcgc?a 21

<210>144

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉SAR1-AS primer

<400>144

tatagtgagc?cgccacacat?g 21

<210>145

<211>45

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>145

ataggatcca?ccatgtttat?tttcttatta?tttcttactc?tcact 45

<210>146

<211>37

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>146

atactcgagt?tatgtgtaat?gtaatttgac?acccttg 37

<210>147

<211>45

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>147

ataggatcca?ccatgtttat?tttcttatta?tttcttactc tcact 45

<210>148

<211>36

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>148

acctccggat ttaatatatt?gctcatattt tcccaa 36

<210>149

<211>13

<212>PRT

＜213〉artificial sequence

<220>

<223>N-terminal?end?of?SRAS-CoV?S?protein(amino?acids?1?to?13)

<400>149

Met?Phe?Ile?Phe?Leu?Leu?Phe?Leu?Thr?Leu?Thr?Ser?Gly

1 5 10

<210>150

<211>10

<212>PRT

＜213〉artificial sequence

<220>

＜223〉oligopeptides

<400>150

Ser?Gly?Asp?Tyr?Lys?Asp?Asp?Asp?Asp?Lys

1 5 10

<210>151

<211>34

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>151

actagctagc?ggatccacca?tgttcatctt?cctg 34

<210>152

<211>33

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>152

agtatccgga?cttgatgtac?tgctcgtact?tgc 33

<210>153

<211>59

<212>DNA

＜213〉artificial sequence

<220>

＜223〉oligonucleotide

<400>153

tatgagcttt?tttttttttt?tttttttggc?atataaatag?actcggcgcg?ccatctgca 59

<210>154

<211>53

<212>DNA

＜213〉artificial sequence

<220>

＜223〉oligonucleotide

<400>154

gatggcgcgc?cgagtctatt?tatatgccaa?aaaaaaaaaa?aaaaaaaagc?tca 53

<210>155

<211>45

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>155

atacgtacga?ccatgtttat?tttcttatta?tttcttactc?tcact 45

<210>156

<211>40

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>156

atagcgcgct?cattatgtgt?aatgtaattt?gacacccttg 40

<210>157

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>157

ccatttcaac?aatttggccg 20

<210>158

<211>45

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>158

ataggatccg?cgcgctcatt?atttatcgtc?gtcatcttta?taatc 45

Claims (29)

1. human coronary virus's strain that severe acute respiratory syndrome isolating or purifying is associated, it is characterized in that containing in its genome and occur: the codon glycine of the Serine codon of S protein gene 23220-23222 position or ORF3 gene 25298-25300 position with the complementary DNA form, and the Serine codon of the L-Ala codon of ORF1a 7918-7920 position or M protein gene 26857-26859 position, described reference by location Genbank sequence A Y274119.3 represents.

2. according to the coronavirus strain of the isolating or purifying of claim 1, it is characterized in that its genomic DNA Equivalent contains the sequence corresponding to sequence SEQ ID No:1.

3. an isolating or purified polynucleotides is characterized in that its sequence is the genome sequence according to the isolating coronavirus strain of claim 1 or 2.

4. according to the isolating or purified polynucleotides of claim 3, it is characterized in that its sequence is SEQ ID No:1.

5. the segmental primer of the sequence of the genome of the SARS associated coronavirus that can increase or its DNA Equivalent is right, it is characterized in that it is selected from:

-No.1 primer is right, corresponds respectively to 28507 to 28522 (sense primer, SEQ ID No:60) and 28774 to 28759 (antisense primer, SEQ ID No:61) according to the polynucleotide sequence of claim 3 or 4,

-No.2 primer is right, correspond respectively to according to 28375 to 28390 (sense primer, the SEQ ID No:62) and 28702 to 28687 (antisense primer, SEQ ID No:63) of the polynucleotide sequence of claim 3 or 4 and

-right by primer SEQ ID No:55 and 56 primers that constitute.

6. one kind can be detected the probe that SARS associated coronavirus genome or its fragment exist, and it is characterized in that it is selected from corresponding to the fragment according to the following position of the polynucleotide of claim 3 or 4: 28561 to 28586,28588 to 28608,28541 to 28563 and 28565 to 28589 (SEQ ID No:64 to 67).

7. recombinant clone and/or expression vector, it is characterized in that it comprises the inset with sequence SEQ IDNo:38, it is contained in a kind of bacterial isolates and on June 5th, 2003 and is preserved in French microbial preservation center with numbering No.I-3048,25 rue duDocteur Roux, 75724 Paris Cedex 15.

8. recombinant clone and/or expression vector is characterized in that it comprises to be selected from following cDNA fragment:

The C of-coding polyhistidine label and N albumen (SEQ ID No:37) hold fusions the cDNA fragment and

The cDNA fragment of the N end fusions of-coding polyhistidine label and N albumen (SEQ ID No:37).

9. the recombinant expression vector of claim 8 is characterized in that it is contained in to be preserved in French microbial preservation center on October 23rd, 2003 that 25 rue du Docteur Roux are in the No.I-3117 bacterial isolates of 75724Paris Cedex 15.

10. one kind by according to each the cell of carrier modification of claim 7 to 9.

11. a hybridoma of producing anti-N protein monoclonal antibody is characterized in that it is selected from following hybridoma:

The hybridoma of-manufacture order clonal antibody 87 is preserved in CNCM, preservation date on December 1st, 2004, numbering I-3328

The hybridoma of-manufacture order clonal antibody 86 is preserved in CNCM, preservation date on December 1st, 2004, numbering I-3329

The hybridoma of-manufacture order clonal antibody 57 is preserved in CNCM, preservation date on December 1st, 2004, numbering I-3330 and

The hybridoma of-manufacture order clonal antibody 156 is preserved in CNCM, preservation date on December 1st, 2004, numbering I-3331.

12. one kind at the proteic polyclonal antibody of N or monoclonal antibody or antibody fragment, it is characterized in that it is by the hybridoma production according to claim 11.

13. chip or filter is characterized in that it comprises antibody or antibody fragment according to claim 12.

14. one kind is intended to detect the immunocapture testing method that the SARS associated coronavirus infects, and it is characterized in that it has used the monoclonal antibody of specificity at natural viral nucleoprotein (N albumen).

15. according to the immunocapture testing method of claim 14, the antibody that it is characterized in that being used to catching natural viral nucleoprotein is special monoclonal antibody at central section and/or conformational epitope.

16. according to the immunocapture testing method of claim 14 or 15, it is characterized in that being used to catch the proteic antibody of N is monoclonal antibody mAb87, it is by the hybridoma production that was preserved in CNCM, numbering I-3328 on December 1st, 2004.

17. according to the immunocapture testing method of claim 14 or 15, it is characterized in that being used to catch the proteic antibody of N is monoclonal antibody mAb86, it is by the hybridoma production that was preserved in CNCM, numbering I-3329 on December 1st, 2004.

18., it is characterized in that monoclonal antibody mAb86 and mAb87 be used for that N is proteic to be caught according to the immunocapture testing method of claim 14 or 15.

19. according to each immunocapture testing method of claim 14 to 18, it is characterized in that being used for the visual antibody of N albumen is the mAb57 monoclonal antibody of being produced by the hybridoma of the numbering I-3330 that was preserved in CNCM on December 1st, 2004, and described antibody is puted together with visual molecule or particle.

20. according to each immunocapture testing method of claim 14 to 18, it is proteic visual that the combination that it is characterized in that the mAb57 that will put together with visual molecule or particle and mAb87 antibody is used for N.

21. one kind is used for the reagent that the SARS associated coronavirus detects, it is characterized in that it is selected from:

(a) right according to the primer of claim 5, or according to the probe of claim 6,

(b) according to claim 7 to 9 each recombinant vectors or according to the modified cell of claim 10,

(c) according to the isolating coronavirus strain of claim 1 or 2 or according to claim 3 and 4 each polynucleotide,

(d) according to the antibody or the antibody fragment of claim 12,

(e) antibody that comprises monoclonal antibody mAb86 and/or mAb87 and monoclonal antibody mAb57 makes up,

(f) according to the chip or the filter of claim 13.

22. a product is used for the detection of SARS associated coronavirus and randomly is used for the purposes of reagent of the gene type of SARS associated coronavirus in preparation, described product is selected from: according to the primer of claim 5 to, according to the probe of claim 6, according to claim 7 to 9 each recombinant vectors, according to the modified cell of claim 10, according to the isolating coronavirus strain of claim 1 or 2, according to the polynucleotide of claim 3 or 4.

23. a method that detects the SARS associated coronavirus from biological specimen, described method is characterised in that and which comprises at least:

(a) extract the nucleic acid that is present in the described biological specimen,

(b) use according to the primer of claim 5 to RT-PCR the ORF-N fragment is increased and

(c) use suitable means that the amplified production of gained in (b) is detected.

24. according to the method for claim 23, (b) step that it is characterized in that detection method uses at least one corresponding to probe of column position down: according to 28561 to 28586,28588 to 28608,28541 to 28563 and 28565 to 28589 positions of the polynucleotide sequence of claim 3 or 4.

25. one kind by using the proteic indirect IgG ELISA of N to detect the method that the SARS associated coronavirus infects from biological specimen, described method is characterised in that with N protein solution sensitization culture plate, the N protein concentration is between 0.5 and 4 μ g/ml, preferred 2 μ g/ml, be dissolved in contain 0.25ml/l phenol red, pH is in 7.2 the 10mM PBS damping fluid.

26. a method that detects the infection of SARS associated coronavirus by the two epi-position ELISA of use from biological specimen is characterized in that test serum mixes mutually with visual antigen, contacts described mixture then with the antigen that is attached to solid support.

27. an immunocomplex, it is by forming according to the polyclone of claim 11 or the protein or the peptide of monoclonal antibody or antibody fragment and SARS associated coronavirus.

28. a SARS associated coronavirus detection kit is characterized in that it comprises at least a following reagent: according to the primer of claim 5 to, according to the probe of claim 6, according to claim 7 to 9 each recombinant vectors, according to the modified cell of claim 10, according to the isolating coronavirus strain of claim 1 or 2 with according to the polynucleotide of claim 3 or 4.

29. the fragment according to the polynucleotide of claim 3 is characterized in that it comprises corresponding to two base or base pairs in groups that descend column position at least: 7919 and 23220,7919 and 25298,16622 and 23220,19064 and 23220,16622 and 25298,19064 and 25298,23220 and 24872,23220 and 26857,24872 and 25298,25298 and 26857.

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