CN100386343C - SARS virus S protein and N protein fusion protein, and preparation and use thereof - Google Patents

SARS virus S protein and N protein fusion protein, and preparation and use thereof Download PDF

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CN100386343C
CN100386343C CNB031321623A CN03132162A CN100386343C CN 100386343 C CN100386343 C CN 100386343C CN B031321623 A CNB031321623 A CN B031321623A CN 03132162 A CN03132162 A CN 03132162A CN 100386343 C CN100386343 C CN 100386343C
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李越希
陶开华
朱敏生
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Abstract

The present invention relates to a fusion protein of SARS virus S proteins and N proteins, and the preparation and the application thereof, which belongs to the fields of the gene engineering technology, vaccines and diagnostic reagents. An S protein fragment containing SARS virus with strong antigen epitope and totally comprising 299 amino acids from the 162nd amino acid to the 460th amino acid, and an N protein fragment containing SARS virus with strong antigen epitope from the first amino acid to the 258th amino acid are screened out by analyzing the amino acid sequence of SARS virus S proteins and N proteins with a computer. Codons partial by eukaryotic organisms and prokaryotic organisms are selected to chemically synthesize fire-new gene sequences of the SARS virus S protein fragment and the N protein fragment; the two gene fragments are connected in series. The fusion protein of the S protein fragment and the N protein fragment is expressed by the gene engineering technology; the two protein fragments are connected by glycin and serine; one glycin is inserted behind the first amino acid methionine of the N protein fragment; the full length of the fusion protein comprises 560 amino acids. The expressed fusion protein can be used for detecting vaccines and SARS virus antibodies or antigens and preparing a single antibody, multiple antibodies, etc. resisting SARS virus in an immune mode.

Description

Proteic fusion rotein of SARS virus S albumen and N and preparation thereof, application
Technical field
The present invention is connected the proteic brand-new gene fragment of the SARS virus S of chemosynthesis with the proteic gene fragment of N, utilize genetic engineering technique, preparation SARS virus S albumen and the proteic fusion rotein of N.By Computer Analysis, filter out the S protein fragments of the SARS virus that contains the strong antigen epi-position and the fusion rotein and the N protein fragments of N protein fragments, the codon of selecting eucaryon and prokaryotic organism all to have a preference for, S gene order that chemosynthesis is brand-new and N gene order, utilize genetic engineering technique to express both fusion roteins, the fusion rotein of expressing can be used for vaccine and SARS virus antibody or detection of antigens etc., the present invention relates to genetic engineering technique, vaccine and diagnostic reagent field.
Background technology
SARS (Severe Acute Respiratory Syndrome) also claims SARS (Severe Acute Respiratory Syndrome), is a kind of potent virus sexually transmitted disease.On November 16th, 2,003 first routine patient SARS occurred in China Guangzhou, after this involved more than 30 countries in the world in the some months, and people more than 8000 infects, dead people more than 800.Mortality ratio according to WHO (WorldHealth Organization) statistics SARS can be up to about 15%, and the elderly's mortality ratio that infects this disease can reach 40% to 50%.The people has 10 to 14 days latent period after infecting SARS virus, occur high heat, cough, general malaise and headache, diarrhoea during morbidity, and respiratory distress syndrome can take place severe person in the week of one after the morbidity, so that the forfeiture respiratory function.
SARS virus is a kind of novel coronavirus, because this virus had not infected human in the past, so the crowd is to the general susceptible of this virus, SARS virus is a kind of sub-thread positive chain RNA virus, at present after measured to its complete genome sequence, announced about 10 total length virus gene sequence in the Genebank, laid a good foundation for utilizing genetic engineering technique research diagnostic reagent, vaccine and screening antiviral.
SARS virus can vitro culture with Vero-E6, set up immunofluorescence method and the enzyme linked immunosorbent detection method that detects SARS virus antibody so utilize the SARS virus of cultivating, but make Detection of antigen antibody with the SARS virus of cultivating, be difficult to mass production, also can produce false positive, developing special gene recombinant antigens is to set up the developing direction of SARS virus specific antibody detection reagent.The most desirable approach that prevention SARS infects is a vaccinate, but does not prevent the vaccine of SARS at present, both at home and abroad all actively developing the development of vaccine, especially Inactivated Vaccine is made fast progress, and recombinant vaccine is final developing direction.
Summary of the invention
SARS can stimulate body to produce multiple antibody after infecting human body, but sometimes in the serum only at the proteic antibody of part SARS virus, the time that may produce with various antibody or relevant with modulation on immune status, so when detecting intraserous SARS virus antibody, will use multiple antigen, with raising antibody recall rate.
The outermost S albumen of SARS virus is mediation virus and host cell bonded major protein, seals this proteic binding site and can stop virus infected cell, so the S albumen of SARS virus is the first-selected albumen that develops vaccine.The N albumen of SARS virus has higher conservative property, and it is very little to make a variation between each strain SARS virus, so be the ideal protein as antigen development SARS virus antibody test reagent.By Computer Analysis SARS virus S albumen and the proteic aminoacid sequence of N, we filter out the S protein fragments and the N protein fragments of the SARS virus that contains the strong antigen epi-position, the codon of selecting eucaryon and prokaryotic organism all to have a preference for, the S protein fragments that chemosynthesis is brand-new and the gene order of N protein fragments, with two gene fragment series connection, utilize genetic engineering technique to express the fusion rotein of S protein fragments and N protein fragments, the fusion rotein of expression can be used for vaccine and SARS virus antibody or detection of antigens etc.
The present invention is that the SARS virus S albumen with chemosynthesis is connected with the proteic gene fragment of N, utilizes genetic engineering technique, the fusion rotein of preparation SARS virus S protein fragments and N protein fragments.By Computer Analysis SARS virus S albumen and the proteic aminoacid sequence of N, filter out the S protein fragments of the SARS virus that contains the strong antigen epi-position, i.e. 460 amino acid of the 162nd amino acid to the, totally 299 amino acid, with the N protein fragments of the SARS virus that contains the strong antigen epi-position, i.e. the 1st amino acid to 258 amino acid.The codon of selecting eucaryon and prokaryotic organism all to have a preference for, the SARS virus S protein fragments that chemosynthesis is brand-new and the gene order of N protein fragments, with two gene fragment series connection, utilize genetic engineering technique to express the fusion rotein of S protein fragments and N protein fragments, be connected with Serine with glycine between two protein fragments, behind first amino acid methionine of N protein fragments, insert a glycine, 560 amino acid of fusion rotein total length.The fusion rotein of expressing can be used for vaccine and SARS virus antibody or detection of antigens, and is used for immunity preparation anti-SARS virus monoclonal antibody and how anti-etc.
Proteic fusion rotein of SARS virus S albumen and N and preparation thereof, application take following steps to implement:
1.SARS the screening of virus S protein and N proteantigen epi-position and the chemosynthesis of gene fragment thereof:
Utilize softwares such as ANTHEWIN, by Computer Analysis SARS virus S albumen and the proteic aminoacid sequence of N, find that the proteic N of S holds 460 amino acid of the 162nd amino acid to the to contain stronger antigenic determinant, the proteic N of N holds 258 amino acid of the 1st amino acid to the to contain stronger antigenic determinant.The codon of selecting eucaryon and prokaryotic organism all to have a preference for, chemosynthesis comprises S albumen and the proteic gene order of N of above-mentioned antigenic determinant respectively.NcoI restriction enzyme site (following setting-out part) and 8 protection bases (GCGAGTCACC) have been increased at the segmental 5 ' end of synthetic N protein gene; and after initiator codon ATG, inserted a codon GGA, increased BamHI restriction enzyme site (following setting-out part) and 3 protection bases (GCG) at 3 ' end.Increased BamHI restriction enzyme site (following setting-out part) and two protection bases (GT) at the segmental 5 ' end of synthetic S protein gene, increased terminator codon (TAA) and EcoRI restriction enzyme site (following setting-out part) and two protection bases (AC) at 3 ' end.Making two gene fragments of synthetic be easy to series connection is cloned in plasmid pET28a (+) the interior NcoI and EcoRI restriction enzyme site.
Epitope aminoacid sequence (the 258th aa of the 1st aa-) in the SARS virus N albumen of screening:
Met?Ser?Asp?Asn?Gly?Pro?Gln?Ser?Asn?Gln?Arg?Ser?Ala?Pro?Arg?Ile?Thr?Phe?Gly
Gly?Pro?Thr?Asp?Ser?Thr?Asp?Asn?Asn?Gln?Asn?Gly?Gly?Arg?Asn?Gly?Ala?Arg?Pro
Lys?Gln?Arg?Arg?Pro?Gln?Gly?Leu?Pro?Asn?Asn?Thr?Ala?Ser?Trp?Phe?Thr?Ala?Leu
Thr?Gln?His?Gly?Lys?Glu?Glu?Leu?Arg?Phe?Pro?Arg?Gly?Gln?Gly?Val?Pro?Ile?Asn
Thr?Asn?Ser?Gly?Pro?Asp?Asp?Gln?Ile?Gly?Tyr?Tyr?Arg?Arg?Ala?Thr?Arg?Arg?Val
Arg?Gly?Gly?Asp?Gly?Lys?Met?Lys?Glu?Leu?Ser?Pro?Arg?Trp?Tyr?Phe?Tyr?Tyr?Leu
Gly?Thr?Gly?Pro?Glu?Ala?Ser?Leu?Pro?Tyr?Gly?Ala?Asn?Lys?Glu?Gly?Ile?Val?Trp
Val?Ala?Thr?Glu?Gly?Ala?Leu?Asn?Thr?Pro?Lys?Asp?His?Ile?Gly?Thr?Arg?Asn?Pro
Asn?Asn?Asn?Ala?Ala?Thr?Val?Leu?Gln?Leu?Pro?Gln?Gly?Thr?Thr?Leu?Pro?Lys?Gly
Phe?Tyr?Ala?Glu?Gly?Ser?Arg?Gly?Gly?Ser?Gln?Ala?Ser?Ser?Arg?Ser?Ser?Ser?Arg
Ser?Arg?Gly?Asn?Ser?Arg?Asn?Ser?Thr?Pro?Gly?Ser?Ser?Arg?Gly?Asn?Ser?Pro?Ala
Arg?Met?Ala?Ser?Gly?Gly?Gly?Glu?Thr?Ala?Leu?Ala?Leu?Leu?Leu?Leu?Asp?Arg?Leu
Asn?Gln?Leu?Glu?Ser?Lys?Val?Ser?Gly?Lys?Gly?Gln?Gln?Gln?Gln?Gly?Gln?Thr?Val
Thr?Lys?Lys?Ser?Ala?Ala?Glu?Ala?Ser?Lys?Lys
Epitope aminoacid sequence (the 460th aa of the 162nd aa-) in the SARS virus S albumen of screening:
Glu?Tyr?Ile?Ser?Asp?Ala?Phe?Ser?Leu?Asp?Val?Ser?Glu?Lys?Ser?Gly?Asn?Phe?Lys
His?Leu?Arg?Glu?Phe?Val?Phe?Lys?Asn?Lys?Asp?Gly?Phe?Leu?Tyr?Val?Tyr?Lys?Gly
Tyr?Gln?Pro?Ile?Asp?Val?Val?Arg?Asp?Leu?Pro?Ser?Gly?Phe?Asn?Thr?Leu?Lys?Pro
Ile?Phe?Lys?Leu?Pro?Leu?Gly?Ile?Asn?Ile?Thr?Asn?Phe?Arg?Ala?Ile?Leu?Thr?Ala
Phe?Ser?Pro?Ala?Gln?Asp?Thr?Trp?Gly?Thr?Ser?Ala?Ala?Ala?Tyr?Phe?Val?Gly?Tyr
Leu?Lys?Pro?Thr?Thr?Phe?Met?Leu?Lys?Tyr?Asp?Glu?Asn?Gly?Thr?Ile?Thr?Asp?Ala
Val?Asp?Cys?Ser?Gln?Asn?Pro?Leu?Ala?Glu?Leu?Lys?Cys?Ser?Val?Lys?Ser?Phe?Glu
Ile?Asp?Lys?Gly?Ile?Tyr?Gln?Thr?Ser?Asn?Phe?Arg?Val?Val?Pro?Ser?Gly?Asp?Val
Val?Arg?Phe?Pro?Asn?Ile?Thr?Asn?Leu?Cys?Pro?Phe?Gly?Glu?Val?Phe?Asn?Ala?Thr
Lys?Phe?Pro?Ser?Val?Tyr?Ala?Trp?Glu?Arg?Lys?Lys?Ile?Ser?Asn?Cys?Val?Ala?Asp
Tyr?Ser?Val?Leu?Tyr?Asn?Ser?Thr?Phe?Phe?Ser?Thr?Phe?Lys?Cys?Tyr?Gly?Val?Ser
Ala?Thr?Lys?Leu?Asn?Asp?Leu?Cys?Phe?Ser?Asn?Val?Tyr?Ala?Asp?Ser?Phe?Val?Val
Lys?Gly?Asp?Asp?Val?Arg?Gln?Ile?Ala?Pro?Gly?Gln?Thr?Gly?Val?Ile?Ala?Asp?Tyr
Asn?Tyr?Lys?Leu?Pro?Asp?Asp?Phe?Met?Gly?Cys?Val?Leu?Ala?Trp?Asn?Thr?Arg?Asn
Ile?Asp?Ala?Thr?Ser?Thr?Gly?Asn?Tyr?Asn?Tyr?Lys?Tyr?Arg?Tyr?Leu?Arg?His?Gly
Lys?Leu?Arg?Pro?Phe?Glu?Arg?Asp?Ile?Ser?Asn?Val?Pro?Phe
The dna sequence dna (796bp) that contains SARS virus N proteantigen epitope gene of chemosynthesis:
GCG?AGT?CA C?CAT?GGG?ATC?TGA?TAA?CGG?TCC?GCA?GTC?AAA?CCA?ACG?TAG?TGC?CCC?CCG
CAT?TAC?ATT?TGG?TGG?ACC?CAC?AGA?TTC?AAC?TGA?CAA?TAA?CCA?GAA?TGG?AGG?ACG?CAA
TGG?GGC?AAG?GCC?AAA?ACA?GCG?CCG?ACC?CCA?AGG?TTT?ACC?CAA?TAA?TAC?TGC?GTC?TTG
GTT?CAC?AGC?TCT?CAC?TCA?GCA?TGG?CAA?GGA?GGA?ACT?TAG?ATT?CCC?TCG?AGG?CCA?GGG
CGT?TCC?AAT?CAA?CAC?CAA?TAG?TGG?TCC?AGA?TGA?CCA?AAT?TGG?CTA?CTA?CCG?AAG?AGC
TAC?CCG?ACG?AGT?TCG?TGG?TGG?TGA?CGG?CAA?AAT?GAA?AGA?GCT?CAG?CCC?CAG?ATG?GTA
CTT?CTA?TTA?CCT?AGG?AAC?TGG?CCC?AGA?AGC?TTC?ACT?TCC?CTA?CGG?CGC?TAA?CAA?AGA
AGG?CAT?CGT?ATG?GGT?TGC?AAC?TGA?GGG?AGC?CTT?GAA?TAC?ACC?CAA?AGA?CCA?CAT?TGG
CAC?CCG?CAA?TCC?TAA?TAA?CAA?TGC?TGC?CAC?CGT?GCT?ACA?ACT?TCC?TCA?AGG?AAC?AAC
ATT?GCC?AAA?AGG?CTT?CTA?CGC?AGA?GGG?AAG?CAG?AGG?CGG?CAG?TCA?AGC?CTC?TTC?TCG
CTC?CTC?ATC?ACG?TAG?TCG?CGG?TAA?TTC?AAG?AAA?TTC?AAC?TCC?TGG?CAG?CAG?TAG?GGG
AAA?TTC?TCC?TGC?TCG?AAT?GGC?TAG?CGG?AGG?TGG?TGA?AAC?TGC?CCT?CGC?GCT?ATT?GCT
GCT?AGA?CAG?ATT?GAA?CCA?GCT?TGA?GAG?CAA?AGT?TTC?TGG?TAA?AGG?CCA?ACA?ACA?ACA
AGG?CCA?AAC?TGT?CAC?TAA?GAA?ATC?TGC?TGC?TGA?GGC?ATC?TAA?AAA?G GG?ATC?CGC?G
The dna sequence dna (916bp) that contains SARS virus S proteantigen epitope gene of chemosynthesis:
GT GGATCC?GAA?TAC?ATC?TCT?GAC?GCA?TTC?TCT?CTG?GAC?GTT?TCC?GAA?AAG?TCT?GGT?AAC
TTC?AAA?CAC?CTG?CGC?GAG?TTC?GTG?TTT?AAA?AAC?AAA?GAC?GGT?TTC?CTG?TAC?GTT?TAC
AAG?GGC?TAC?CAG?CCG?ATC?GAC?GTA?GTT?CGT?GAC?CTG?CCG?TCT?GGT?TTT?AAC?ACT?CTG
AAA?CCG?ATC?TTC?AAG?CTG?CCG?CTG?GGT?ATT?AAC?ATC?ACT?AAC?TTC?CGC?GCT?ATC?CTG
ACT?GCT?TTC?TCT?CCG?GCT?CAG?GAC?ACT?TGG?GGC?ACT?TCT?GCT?GCA?GCC?TAC?TTC?GTT
GGC?TAC?CTG?AAG?CCA?ACT?ACC?TTT?ATG?CTG?AAG?TAC?GAC?GAA?AAC?GGT?ACT?ATC?ACT
GAT?GCT?GTT?GAC?TGC?TCT?CAG?AAC?CCA?CTG?GCT?GAA?CTG?AAA?TGC?TCT?GTT?AAG?AGC
TTT?GAG?ATC?GAC?AAA?GGT?ATT?TAC?CAG?ACC?TCT?AAC?TTC?CGT?GTT?GTT?CCG?TCT?GGT
GAC?GTT?GTG?CGT?TTC?CCT?AAC?ATC?ACT?AAC?CTG?TGC?CCG?TTT?GGT?GAA?GTT?TTC?AAC
GCT?ACT?AAA?TTC?CCT?TCT?GTC?TAC?GCA?TGG?GAG?CGT?AAA?AAA?ATT?TCT?AAC?TGC?GTT
GCT?GAT?TAC?TCT?GTG?CTG?TAC?AAC?TCT?ACC?TTT?TTC?TCT?ACC?TTC?AAG?TGC?TAC?GGC
GTT?TCT?GCT?ACT?AAG?CTG?AAC?GAC?CTG?TGC?TTC?TCC?AAC?GTT?TAC?GCA?GAT?TCT?TTC
GTA?GTT?AAG?GGT?GAT?GAC?GTA?CGT?CAG?ATC?GCT?CCA?GGT?CAG?ACT?GGT?GTT?ATC?GCT
GAC?TAC?AAC?TAT?AAA?CTG?CCG?GAC?GAT?TTC?ATG?GGT?TGC?GTT?CTG?GCT?TGG?AAC?ACT
CGT?AAC?ATT?GAC?GCT?ACT?TCT?ACT?GGT?AAC?TAC?AAC?TAC?AAA?TAT?CGT?TAC?CTG?CGT
CAC?GGC?AAA?CTG?CGT?CCG?TTC?GAA?CGT?GAC?ATC?TCT?AAC?GTG?CCG?TTC?TAA? GAATTCAC
2. express the fusion rotein construction of recombinant plasmid of SARS virus S protein fragments and N protein fragments:
Extract plasmid pET28a (+) (U.S. Novagen company product),, reclaim the big fragment of plasmid that enzyme is cut behind the electrophoresis, be dissolved in the deionized water with Nco I and EcoR I double digestion.With the SARS virus N protein gene fragment of Nco I and the chemosynthesis of BamHI double digestion,, after electrophoresis reclaims respectively, be dissolved in the deionized water with the SARS virus S protein gene fragment of BamH I and the chemosynthesis of EcoR I double digestion.
Above-mentioned three kinds of enzymes of volumetric molar concentration such as get and cut the back dna fragmentation, in same centrifuge tube, connect with the T4 dna ligase, make SARS virus N protein gene fragment and S protein gene fragment after BamH I enzyme is connected, be inserted between the Nco I and EcoR I site in the carrier pET28a (+), express the fusion rotein of a SARS virus S protein fragments and N protein fragments.
3. the screening of recombinant plasmid and evaluation:
With recombinant plasmid transformed e. coli bl21 (DE3), coating contains kantlex (60 μ g/ml) LB flat board, puts 37 ℃ and spends the night.Next day, picking transformed a bacterium colony and a contrast bacterium (plasmid pET28a transformed bacteria) at random, extract plasmid respectively, plasmid with extraction is a template, right with the primer of primer of N protein gene fragment 5 ' end (5 '-GCGAGTCACCATGGGATCTGATAACGGTCCGCAGTCAAACCAACG-3 ') and S protein gene fragment 3 ' end (5 '-GTGAATTCTTAGAAAGGCACATTAGATATGT-3 ') composition primer, SARS virus N protein gene fragment that pcr amplification links together and S protein gene fragment, contain the positive recombinant plasmid of two gene fragments of connecting, should amplify the tandem gene fragment that is about 1701bp.The plasmid that will contain tandem gene carries out dna sequence analysis, and sequential analysis confirms that recombinant plasmid contains SARS virus N protein gene fragment and the S protein gene fragment that links together, and sequence is entirely true:
ATG?GGA?TCT?GAT?AAC?GGT?CCG?CAG?TCA?AAC?CAA?CGT?AGT?GCC?CCC?CGC?ATT?ACA?TTT
GGT?GGA?CCC?ACA?GAT?TCA?ACT?GAC?AAT?AAC?CAG?AAT?GGA?GGA?CGC?AAT?GGG?GCA?AGG
CCA?AAA?CAG?CGC?CGA?CCC?CAA?GGT?TTA?CCC?AAT?AAT?ACT?GCG?TCT?TGG?TTC?ACA?GCT
CTC?ACT?CAG?CAT?GGC?AAG?GAG?GAA?CTT?AGA?TTC?CCT?CGA?GGC?CAG?GGC?GTT?CCA?ATC
AAC?ACC?AAT?AGT?GGT?CCA?GAT?GAC?CAA?ATT?GGC?TAC?TAC?CGA?AGA?GCT?ACC?CGA?CGA
GTT?CGT?GGT?GGT?GAC?GGC?AAA?ATG?AAA?GAG?CTC?AGC?CCC?AGA?TGG?TAC?TTC?TAT?TAC
CTA?GGA?ACT?GGC?CCA?GAA?GCT?TCA?CTT?CCC?TAC?GGC?GCT?AAC?AAA?GAA?GGC?ATC?GTA
TGG?GTT?GCA?ACT?GAG?GGA?GCC?TTG?AAT?ACA?CCC?AAA?GAC?CAC?ATT?GGC?ACC?CGC?AAT
CCT?AAT?AAC?AAT?GCT?GCC?ACC?GTG?CTA?CAA?CTT?CCT?CAA?GGA?ACA?ACA?TTG?CCA?AAA
GGC?TTC?TAC?GCA?GAG?GGA?AGC?AGA?GGC?GGC?AGT?CAA?GCC?TCT?TCT?CGC?TCC?TCA?TCA
CGT?AGT?CGC?GGT?AAT?TCA?AGA?AAT?TCA?ACT?CCT?GGC?AGC?AGT?AGG?GGA?AAT?TCT?CCT
GCT?CGA?ATG?GCT?AGC?GGA?GGT?GGT?GAA?ACT?GCC?CTC?GCG?CTA?TTG?CTG?CTA?GAC?AGA
TTG?AAC?CAG?CTT?GAG?AGC?AAA?GTT?TCT?GGT?AAA?GGC?CAA?CAA?CAA?CAA?GGC?CAA?ACT
GTC?ACT?AAG?AAA?TCT?GCT?GCT?GAG?GCA?TCT?AAA?AAG? GGA?TCC?GAA?TAC?ATC?TCT?GAC
GCA?TTC?TCT?CTG?GAC?GTT?TCC?GAA?AAG?TCT?GGT?AAC?TTC?AAA?CAC?CTG?CGC?GAG?TTC
GTG?TTT?AAA?AAC?AAA?GAC?GGT?TTC?CTG?TAC?GTT?TAC?AAG?GGC?TAC?CAG?CCG?ATC?GAC
GTA?GTT?CGT?GAC?CTG?CCG?TCT?GGT?TTT?AAC?ACT?CTG?AAA?CCG?ATC?TTC?AAG?CTG?CCG
CTG?GGT?ATT?AAC?ATC?ACT?AAC?TTC?CGC?GCT?ATC?CTG?ACT?GCT?TTC?TCT?CCG?GCT?CAG
GAC?ACT?TGG?GGC?ACT?TCT?GCT?GCA?GCC?TAC?TTC?GTT?GGC?TAC?CTG?AAG?CCA?ACT?ACC
TTT?ATG?CTG?AAG?TAC?GAC?GAA?AAC?GGT?ACT?ATC?ACT?GAT?GCT?GTT?GAC?TGC?TCT?CAG
AAC?CCA?CTG?GCT?GAA?CTG?AAA?TGC?TCT?GTT?AAG?AGC?TTT?GAG?ATC?GAC?AAA?GGT?ATT
TAC?CAG?ACC?TCT?AAC?TTC?CGT?GTT?GTT?CCG?TCT?GGT?GAC?GTT?GTG?CGT?TTC?CCT?AAC
ATC?ACT?AAC?CTG?TGC?CCG?TTT?GGT?GAA?GTT?TTC?AAC?GCT?ACT?AAA?TTC?CCT?TCT?GTC
TAC?GCA?TGG?GAG?CGT?AAA?AAA?ATT?TCT?AAC?TGC?GTT?GCT?GAT?TAC?TCT?GTG?CTG?TAC
AAC?TCT?ACC?TTT?TTC?TCT?ACC?TTC?AAG?TGC?TAC?GGC?GTT?TCT?GCT?ACT?AAG?CTG?AAC
GAC?CTG?TGC?TTC?TCC?AAC?GTT?TAC?GCA?GAT?TCT?TTC?GTA?GTT?AAG?GGT?GAT?GAC?GTA
CGT?CAG?ATC?GCT?CCA?GGT?CAG?ACT?GGT?GTT?ATC?GCT?GAC?TAC?AAC?TAT?AAA?CTG?CCG
GAC?GAT?TTC?ATG?GGT?TGC?GTT?CTG?GCT?TGG?AAC?ACT?CGT?AAC?ATT?GAC?GCT?ACT?TCT
ACT?GGT?AAC?TAC?AAC?TAC?AAA?TAT?CGT?TAC?CTG?CGT?CAC?GGC?AAA?CTG?CGT?CCG?TTC
GAA?CGT?GAC?ATC?TCT?AAC?GTG?CCG?TTC?TAA
The virus S protein fragment of the expression of recombinant plasmid SARS that makes up and the fusion rotein of N protein fragments, 560 amino acid of total length, be the N protein fragments at its N end, long 259 amino acid, the C end is the S protein fragments, long 299 amino acid are connected by two amino acid of propylhomoserin and Serine between the two, and its aminoacid sequence is as follows:
Met?Gly?Ser?Asp?Asn?Gly?Pro?Gln?Ser?Asn?Gln?Arg?Ser?Ala?Pro?Arg?Ile?Thr?Phe
Gly?Gly?Pro?Thr?Asp?Ser?Thr?Asp?Asn?Asn?Gln?Asn?Gly?Gly?Arg?Asn?Gly?Ala?Arg
Pro?Lys?Gln?Arg?Arg?Pro?Gln?Gly?Leu?Pro?Asn?Asn?Thr?Ala?Ser?Trp?Phe?Thr?Ala
Leu?Thr?Gln?His?Gly?Lys?Glu?Glu?Leu?Arg?Phe?Pro?Arg?Gly?Gln?Gly?Val?Pro?Ile
Asn?Thr?Asn?Ser?Gly?Pro?Asp?Asp?Gln?Ile?Gly?Tyr?Tyr?Arg?Arg?Ala?Thr?Arg?Arg
Val?Arg?Gly?Gly?Asp?Gly?Lys?Met?Lys?Glu?Leu?Ser?Pro?Arg?Trp?Tyr?Phe?Tyr?Tyr
Leu?Gly?Thr?Gly?Pro?Glu?Ala?Ser?Leu?Pro?Tyr?Gly?Ala?Asn?Lys?Glu?Gly?Ile?Val
Trp?Val?Ala?Thr?Glu?Gly?Ala?Leu?Asn?Thr?Pro?Lys?Asp?His?Ile?Gly?Thr?Arg?Asn
Pro?Asn?Asn?Asn?Ala?Ala?Thr?Val?Leu?Gln?Leu?Pro?Gln?Gly?Thr?Thr?Leu?Pro?Lys
Gly?Phe?Tyr?Ala?Glu?Gly?Ser?Arg?Gly?Gly?Ser?Gln?Ala?Ser?Ser?Arg?Ser?Ser?Ser
Arg?Ser?Arg?Gly?Asn?Ser?Arg?Asn?Ser?Thr?Pro?Gly?Ser?Ser?Arg?Gly?Asn?Ser?Pro
Ala?Arg?Met?Ala?Ser?Gly?Gly?Gly?Glu?Thr?Ala?Leu?Ala?Leu?Leu?Leu?Leu?Asp?Arg
Leu?Asn?Gln?Leu?Glu?Ser?Lys?Val?Ser?Gly?Lys?Gly?Gln?Gln?Gln?Gln?Gly?Gln?Thr
Val?Thr?Lys?Lys?Ser?Ala?Ala?Glu?Ala?Ser?Lys?Lys?Gly?Ser?Glu?Tyr?Ile?Ser?Asp
Ala?Phe?Ser?Leu?Asp?Val?Ser?Glu?Lys?Ser?Gly?Asn?Phe?Lys?His?Leu?Arg?Glu?Phe
Val?Phe?Lys?Asn?Lys?Asp?Gly?Phe?Leu?Tyr?Val?Tyr?Lys?Gly?Tyr?Gln?Pro?Ile?Asp
Val?Val?Arg?Asp?Leu?Pro?Ser?Gly?Phe?Asn?Thr?Leu?Lys?Pro?Ile?Phe?Lys?Leu?Pro
Leu?Gly?Ile?Asn?Ile?Thr?Asn?Phe?Arg?Ala?Ile?Leu?Thr?Ala?Phe?Ser?Pro?Ala?Gln
Asp?Thr?Trp?Gly?Thr?Ser?Ala?Ala?Ala?Tyr?Phe?Val?Gly?Tyr?Leu?Lys?Pro?Thr?Thr
Phe?Met?Leu?Lys?Tyr?Asp?Glu?Asn?Gly?Thr?Ile?Thr?Asp?Ala?Val?Asp?Cys?Ser?Gln
Asn?Pro?Leu?Ala?Glu?Leu?Lys?Cys?Ser?Val?Lys?Ser?Phe?Glu?Ile?Asp?Lys?Gly?Ile
Tyr?Gln?Thr?Ser?Asn?Phe?Arg?Val?Val?Pro?Ser?Gly?Asp?Val?Val?Arg?Phe?Pro?Asn
Ile?Thr?Asn?Leu?Cys?Pro?Phe?Gly?Glu?Val?Phe?Asn?Ala?Thr?Lys?Phe?Pro?Ser?Val
Tyr?Ala?Trp?Glu?Arg?Lys?Lys?Ile?Ser?Asn?Cys?Val?Ala?Asp?Tyr?Ser?Val?Leu?Tyr
Asn?Ser?Thr?Phe?Phe?Ser?Thr?Phe?Lys?Cys?Tyr?Gly?Val?Ser?Ala?Thr?Lys?Leu?Asn
Asp?Leu?Cys?Phe?Ser?Asn?Val?Tyr?Ala?Asp?Ser?Phe?Val?Val?Lys?Gly?Asp?Asp?Val
Arg?Gln?Ile?Ala?Pro?Gly?Gln?Thr?Gly?Val?Ile?Ala?Asp?Tyr?Asn?Tyr?Lys?Leu?Pro
Asp?Asp?Phe?Met?Gly?Cys?Val?Leu?Ala?Trp?Asn?Thr?Arg?Asn?Ile?Asp?Ala?Thr?Ser
Thr?Gly?Asn?Tyr?Asn?Tyr?Lys?Tyr?Arg?Tyr?Leu?Arg?His?Gly?Lys?Leu?Arg?Pro?Phe
Glu?Arg?Asp?Ile?Ser?Asn?Val?Pro?Phe
4. the Screening and Identification of expressed fusion protein engineering bacteria:
The positive transformant that will contain recombinant plasmid, be seeded to and contain 3ml LB substratum (containing kanamycin 60 μ g/ml) in vitro, 37 ℃ of shaking culture 3h, add IPTG to final concentration 0.5mmol/L, continue shaking culture and induce 6h, centrifugal collection thalline carries out SDS-PAGE and detects, and recon is expressed SARS virus S albumen and the proteic fusion rotein of N that relative molecular weight is about 60kD, and contrast bacterium BL21 (DE3) does not have this protein band.
5. express the purifying of SARS virus S albumen and the proteic fusion rotein of N:
1) ultrasonic degradation of expression SARS virus S albumen and the proteic fusion rotein engineering bacteria of N
Centrifugal (8000rpm, 10min, 4 ℃) receives bacterium with the engineering bacteria of abduction delivering fusion rotein, thalline is resuspended in the bacterial lysate (20mmol/L PB pH7.4,10mmol/L EDTA, 1mmol/LDTT, 5% glycerine) of original fluid 1/10 volume, ice-bath ultrasonic is broken bacterium, centrifugal collection supernatant.
2) the sulfuric acid amine fractionation precipitation of expression SARS virus S albumen and the proteic fusion rotein of N
Accurately measure the supernatant volume, add an amount of saturated sulfuric acid amine aqueous solution in supernatant, the limit edged stirs, and making the final concentration of sulfuric acid amine is 30%, puts in the ice bath and spends the night.Centrifugal collection supernatant adds an amount of solid sulfur acid amide powder, and the limit edged stirs, and making the final concentration of sulfuric acid amine is 50%, puts in the ice bath and spends the night.Next day centrifugal collecting precipitation.With 100ml balance liquid (20mmol/L PB pH7.4,0.5mmol/L EDTA, 0.2mmol/L DTT) suspension precipitation, in the dialysis tubing of packing into,, change liquid 1 time to 500ml balance liquid dialysed overnight.Centrifugal collection supernatant is directly gone up S-Sepearse FF cation seperation column purifying purifying
3) S-Sepearse FF cation seperation column purifying
With balance liquid (20mmol/L PB pH7.4,0.5mmol/L EDTA, 0.2mmol/L DTT) flushing balance S-Sepharose FF cation seperation column, will go up the good direct upper prop of supernatant of step dialysis then, last sample flow velocity 1.5ml/min.Fully wash post with balance liquid liquid behind the end of the sample, again successively with contain 50,100,200, the balance liquid eluted protein of 1000mmol/L NaCl, collect each elution peak albumen, with each peak albumen of 12%SDS-PAGE electrophoresis detection, determine which elution peak contains the SARS virus S albumen and the proteic fusion rotein of N of expression.100mmol/LNaCl eluted protein peak is SARS virus S albumen and the proteic fusion rotein of N.
6. the SARS virus S albumen of purifying and the proteic fusion rotein of N are used as Detection of antigen SARS virus antibody:
With the SARS virus S albumen of renaturation and the proteic fusion rotein of N with bag behind carbonate (pH9.6) doubling dilution of 50mmol/L by elisa plate, the indirect enzyme-linked immunosorbent method detects known anti-SARS virus positive serum and normal human serum, the proteic fusion rotein of SARS virus S albumen and N can react with the anti-SARS virus positive serum, not with the normal human serum reaction, illustrate that the SARS virus S albumen and the proteic fusion rotein of N of expressing have better antigenicity and specificity.
7. with horseradish peroxidase (HRP) mark SARS virus S albumen and the proteic fusion rotein of N, detect anti-SARS virus IgM or IgG antibody with pouncing on the method for obtaining or sandwich assay.The proteic fusion egg of SARS virus S albumen and N detects anti-SARS virus antibody as antigen with golden mark method.
8. with the SARS virus S albumen and the proteic fusion rotein of N of expressing, be used for vaccine, SARS virus antibody or detection of antigens and be used for immunity preparation anti-SARS virus monoclonal antibody and how anti-etc.
9. SARS virus S protein gene fragment is connected with the N gene fragment, expresses, prepare with the form of fusion rotein.
10. by gene recombination technology, utilize bacterium, yeast cell, insect cell, mammalian cell and genetically modified animals and plants to carry out recombinant expressed, preparation SARS virus S albumen and the proteic fusion rotein of N.
English abbreviation explanation: SARS (Severe Acute Respiratory Syndrome): SARS (Severe Acute Respiratory Syndrome); EDTA: Tetramethyl Ethylene Diamine; IPTG: isopropylthiogalactoside; DTT: dithiothreitol (DTT); SDS: dodecyl sodium sulfonate is received; PAGE: polyacrylamide gel electrophoresis; SKL: t-inosinic acid is received; PB: phosphate buffered saline buffer; DNA: thymus nucleic acid; RNA: Yeast Nucleic Acid; KD: kilodalton; Triton: Triton.
The advantage that the present invention compared with prior art has
The S protein fragments of the SARS virus that we express and the fusion rotein of N protein fragments have more advantage:
1. the enzyme-linked immunologic detecting kit of the SARS virus antibody of using now, the antigen of its use is totivirus antigen, produce dangerous, cost is high, with other coronavirus shortcomings such as cross reaction are arranged.The S protein fragments of the SARS virus of expressing and the fusion rotein of N protein fragments can overcome above-mentioned shortcoming as antigen.
2. the fusion rotein of the S protein fragments of the SARS virus of Biao Daing and N protein fragments exists with soluble form, is easy to purifying, and does not need renaturation to handle.
3. with the S protein fragments and the N protein fragments amalgamation and expression of SARS virus, two independent protein fragments are more convenient, economical than using respectively.When making the Detection of antigen anti-SARS virus antibody, no longer need to prepare respectively this two kinds of albumen, also do not need to adjust two antigenic usage ratios, so application is convenient and cost is lower with these two albumen.When detecting anti-SARS virus antibody, only need this a kind of fusion rotein of enzyme labelling, no longer need two albumen of mark respectively with prize law or sandwich assay.
4. there is not the SARS virus vaccine at present.Inactivated Vaccine has obtained bigger progress, but production cost height, dangerous big.The outermost S albumen of SARS virus is mediation virus and host cell bonded major protein, seals this proteic binding site and can stop virus infected cell, so the S albumen of SARS virus is the first-selected albumen that develops vaccine.N albumen can stimulate body to produce stronger cellular immunization.We utilize genetic engineering technique to express the S protein fragments of preparation SARS virus and the fusion rotein of N protein fragments, for the development recombinant vaccine lays the foundation.Recombinant vaccine has safety, advantage that cost is low.
Description of drawings
The invention will be further described below with reference to accompanying drawing:
Fig. 1 is that molecular biology software carries out analytical results to the proteic epitope of SARS virus N.The result shows, from 258 amino acid of the 1st amino acid to the, contains strong wetting ability epitope at the N of SARS virus albumen n end, i.e. the position that arrow indicates in the figure.
Fig. 2 is that molecular biology software carries out analytical results to the proteic epitope of SARS virus S.The result shows, from 460 amino acid of the 162nd amino acid to the, contains strong wetting ability epitope at the S of SARS virus albumen n end, i.e. the position that arrow indicates in the figure.
Fig. 3 is the construction of recombinant plasmid schema of expressing the fusion rotein of the S protein fragments of SARS virus and N protein fragments.
Fig. 4 is the pcr amplification product with 8 transformants of Agarose gel detection of 1.2%.M: the low-molecular-weight dna standard (TaKaRa, DL2000); 2,3,4,5,6: the 2,3,4,5, No. 65 transformants amplify the target gene fragment of 1701bp, i.e. the position that arrow indicates in the figure, 1,7,8: 1st, 7, No. 83 transformants do not amplify the target gene fragment of 1701bp.
Fig. 5 is the SDS-PAGE analytical results of expressing the engineering bacteria of the S protein fragments of SARS virus and N protein fragments fusion rotein.M: low molecular weight protein (LMWP) standard (Pharmacia); 2,4,5,6: 2nd, 4,5, No. 64 recons are all expressed relative molecular weight and are about 60000 purpose fusion rotein, i.e. the position that arrow indicates in the figure.1,3,7,8: 1st, 3,7, No. 84 transformants are not expressed target protein.
Fig. 6 is the SDS-PAGE analytical results of expressing before and after the fusion rotein purifying of the S protein fragments of SARS virus and N protein fragments.M: low molecular weight protein (LMWP) standard (Pharmacia); 1: contrast bacterium BL21 (DE3); 2: the engineering bacteria of the N protein fragments of expression SARS virus and the fusion rotein of S protein fragments; Behind the 3:S-Sepharose FF cation seperation column purifying the S protein fragments of SARS virus and the fusion rotein of N protein fragments.
Embodiment
The detailed description of embodiment of the present invention:
The analysis of the S albumen of SARS virus and N proteantigen epi-position, synthetic the reaching of gene are expressed
By Computer Analysis SARS virus N albumen and the proteic aminoacid sequence of S, filter out the N protein fragments of the SARS virus that contains the strong antigen epi-position, i.e. the 1st amino acid to 258 amino acid, S protein fragments with the SARS virus that contains the strong antigen epi-position, i.e. 460 amino acid of the 162nd amino acid to the, totally 299 amino acid.The codon of selecting eucaryon and prokaryotic organism all to have a preference for, the SARS virus N protein fragments that chemosynthesis is brand-new and the gene order of S protein fragments, utilize genetic engineering technique that two gene fragments are connected, be cloned into the NcoI/EcoRI site in the plasmid pET28a (+), express the fusion rotein of S protein fragments and N protein fragments.With recombinant plasmid transformed e. coli bl21 (DE3), screening has obtained the engineering bacteria of highly effectively expressing SARS virus S protein fragment and N protein fragments fusion rotein, and the fusion rotein of the SARS virus of expression accounts for about 30% of tropina total amount.
Materials and methods
1. bacterial classification and plasmid: host bacterium BL21 (DE3) and expression vector pET28a (+) are U.S. Novagen company product.
2. molecular biology reagent: restriction enzyme NcoI, BamHI, EcoRI, and the T4DNA ligase enzyme be TaKaRa company product.Plasmid purification test kit and the test kit that reclaims dna fragmentation in the sepharose are TaKaRa company product.DTT and IPTG are TaKaRa company product.Other reagent is import or homemade analytical reagent.
3. gene fragment is synthetic: helped synthetic by Dalian TaKaRa company.
The enzyme of gene clone method: DNA cut, connection, electrophoresis; The extraction of plasmid, conversion; General molecular cloning methods such as proteic SDS-PAGE analysis carry out according to a conventional method.Other test kit by specification is operated.
5.DNA sequential analysis: with TaKaRa company plasmid purification test kit plasmid purification, with the full-automatic sequenator order-checking of DNA.
The result
1.SARS the screening of virus S protein and N proteantigen epi-position and the chemosynthesis of gene fragment thereof:
Utilize softwares such as ANTHEWIN, by Computer Analysis SARS virus S albumen and the proteic aminoacid sequence (GeneBank of N, closing number: AY278488), find that the proteic N of N holds 258 amino acid of the 1st amino acid to the to contain stronger antigenic determinant (Fig. 1), the proteic N of S holds 460 amino acid of the 162nd amino acid to the to contain stronger antigenic determinant (Fig. 2).The codon of selecting eucaryon and prokaryotic organism all to have a preference for, chemosynthesis comprises S albumen and the proteic gene order of N of above-mentioned antigenic determinant respectively.NcoI restriction enzyme site (following setting-out part) and 8 protection bases (GCGAGTCACC) have been increased at the segmental 5 ' end of synthetic N protein gene; and after initiator codon ATG, inserted a codon GGA, increased BamHI restriction enzyme site (following setting-out part) and 3 protection bases (GCG) at 3 ' end.Increased BamHI restriction enzyme site (following setting-out part) and two protection bases (GT) at the segmental 5 ' end of synthetic S protein gene, increased terminator codon (TAA) and EcoRI restriction enzyme site (following setting-out part) and two protection bases (AC) at 3 ' end.Making two gene fragments of synthetic be easy to series connection is cloned in plasmid pET28a (+) the interior NcoI and EcoRI restriction enzyme site.
Epitope aminoacid sequence (the 258th aa of the 1st aa-) in the SARS virus N albumen of screening:
Met?Ser?Asp?Asn?Gly?Pro?Gln?Ser?Asn?Gln?Arg?Ser?Ala?Pro?Arg?Ile?Thr?Phe?Gly
Gly?Pro?Thr?Asp?Ser?Thr?Asp?Asn?Asn?Gln?Asn?Gly?Gly?Arg?Asn?Gly?Ala?Arg?Pro
Lys?Gln?Arg?Arg?Pro?Gln?Gly?Leu?Pro?Asn?Asn?Thr?Ala?Ser?Trp?Phe?Thr?Ala?Leu
Thr?Gln?His?Gly?Lys?Glu?Glu?Leu?Arg?Phe?Pro?Arg?Gly?Gln?Gly?Val?Pro?Ile?Asn
Thr?Asn?Ser?Gly?Pro?Asp?Asp?Gln?Ile?Gly?Tyr?Tyr?Arg?Arg?Ala?Thr?Arg?Arg?Val
Arg?Gly?Gly?Asp?Gly?Lys?Met?Lys?Glu?Leu?Ser?Pro?Arg?Trp?Tyr?Phe?Tyr?Tyr?Leu
Gly?Thr?Gly?Pro?Glu?Ala?Ser?Leu?Pro?Tyr?Gly?Ala?Asn?Lys?Glu?Gly?Ile?Val?Trp
Val?Ala?Thr?Glu?Gly?Ala?Leu?Asn?Thr?Pro?Lys?Asp?His?Ile?Gly?Thr?Arg?Asn?Pro
Asn?Asn?Asn?Ala?Ala?Thr?Val?Leu?Gln?Leu?Pro?Gln?Gly?Thr?Thr?Leu?Pro?Lys?Gly
Phe?Tyr?Ala?Glu?Gly?Ser?Arg?Gly?Gly?Ser?Gln?Ala?Ser?Ser?Arg?Ser?Ser?Ser?Arg
Ser?Arg?Gly?Asn?Ser?Arg?Asn?Ser?Thr?Pro?Gly?Ser?Ser?Arg?Gly?Asn?Ser?Pro?Ala
Arg?Met?Ala?Ser?Gly?Gly?Gly?Glu?Thr?Ala?Leu?Ala?Leu?Leu?Leu?Leu?Asp?Arg?Leu
Asn?Gln?Leu?Glu?Ser?Lys?Val?Ser?Gly?Lys?Gly?Gln?Gln?Gln?Gln?Gly?Gln?Thr?Val
Thr?Lys?Lys?Ser?Ala?Ala?Glu?Ala?Ser?Lys?Lys
Epitope aminoacid sequence (the 460th aa of the 162nd aa-) in the SARS virus S albumen of screening:
Glu?Tyr?Ile?Ser?Asp?Ala?Phe?Ser?Leu?Asp?Val?Ser?Glu?Lys?Ser?Gly?Asn?Phe?Lys
His?Leu?Arg?Glu?Phe?Val?Phe?Lys?Asn?Lys?Asp?Gly?Phe?Leu?Tyr?Val?Tyr?Lys?Gly
Tyr?Gln?Pro?Ile?Asp?Val?Val?Arg?Asp?Leu?Pro?Ser?Gly?Phe?Asn?Thr?Leu?Lys?Pro
Ile?Phe?Lys?Leu?Pro?Leu?Gly?Ile?Asn?Ile?Thr?Asn?Phe?Arg?Ala?Ile?Leu?Thr?Ala
Phe?Ser?Pro?Ala?Gln?Asp?Thr?Trp?Gly?Thr?Ser?Ala?Ala?Ala?Tyr?Phe?Val?Gly?Tyr
Leu?Lys?Pro?Thr?Thr?Phe?Met?Leu?Lys?Tyr?Asp?Glu?Asn?Gly?Thr?Ile?Thr?Asp?Ala
Val?Asp?Cys?Ser?Gln?Asn?Pro?Leu?Ala?Glu?Leu?Lys?Cys?Ser?Val?Lys?Ser?Phe?Glu
Ile?Asp?Lys?Gly?Ile?Tyr?Gln?Thr?Ser?Asn?Phe?Arg?Val?Val?Pro?Ser?Gly?Asp?Val
Val?Arg?Phe?Pro?Asn?Ile?Thr?Asn?Leu?Cys?Pro?Phe?Gly?Glu?Val?Phe?Asn?Ala?Thr
Lys?Phe?Pro?Ser?Val?Tyr?Ala?Trp?Glu?Arg?Lys?Lys?Ile?Ser?Asn?Cys?Val?Ala?Asp
Tyr?Ser?Val?Leu?Tyr?Asn?Ser?Thr?Phe?Phe?Ser?Thr?Phe?Lys?Cys?Tyr?Gly?Val?Ser
Ala?Thr?Lys?Leu?Asn?Asp?Leu?Cys?Phe?Ser?Asn?Val?Tyr?Ala?Asp?Ser?Phe?Val?Val
Lys?Gly?Asp?Asp?Val?Arg?Gln?Ile?Ala?Pro?Gly?Gln?Thr?Gly?Val?Ile?Ala?Asp?Tyr
Asn?Tyr?Lys?Leu?Pro?Asp?Asp?Phe?Met?Gly?Cys?Val?Leu?Ala?Trp?Asn?Thr?Arg?Asn
Ile?Asp?Ala?Thr?Ser?Thr?Gly?Asn?Tyr?Asn?Tyr?Lys?Tyr?Arg?Tyr?Leu?Arg?His?Gly
Lys?Leu?Arg?Pro?Phe?Glu?Arg?Asp?Ile?Ser?Asn?Val?Pro?Phe
The dna sequence dna (796bp) that contains SARS virus N proteantigen epitope gene of chemosynthesis:
GCG?AGT?CA C?CAT?GGG?ATC?TGA?TAA?CGG?TCC?GCA?GTC?AAA?CCA?ACG?TAG?TGC?CCC?CCG
CAT?TAC?ATT?TGG?TGG?ACC?CAC?AGA?TTC?AAC?TGA?CAA?TAA?CCA?GAA?TGG?AGG?ACG?CAA
TGG?GGC?AAG?GCC?AAA?ACA?GCG?CCG?ACC?CCA?AGG?TTT?ACC?CAA?TAA?TAC?TGC?GTC?TTG
GTT?CAC?AGC?TCT?CAC?TCA?GCA?TGG?CAA?GGA?GGA?ACT?TAG?ATT?CCC?TCG?AGG?CCA?GGG
CGT?TCC?AAT?CAA?CAC?CAA?TAG?TGG?TCC?AGA?TGA?CCA?AAT?TGG?CTA?CTA?CCG?AAG?AGC
TAC?CCG?ACG?AGT?TCG?TGG?TGG?TGA?CGG?CAA?AAT?GAA?AGA?GCT?CAG?CCC?CAG?ATG?GTA
CTT?CTA?TTA?CCT?AGG?AAC?TGG?CCC?AGA?AGC?TTC?ACT?TCC?CTA?CGG?CGC?TAA?CAA?AGA
AGG?CAT?CGT?ATG?GGT?TGC?AAC?TGA?GGG?AGC?CTT?GAA?TAC?ACC?CAA?AGA?CCA?CAT?TGG
CAC?CCG?CAA?TCC?TAA?TAA?CAA?TGC?TGC?CAC?CGT?GCT?ACA?ACT?TCC?TCA?AGG?AAC?AAC
ATT?GCC?AAA?AGG?CTT?CTA?CGC?AGA?GGG?AAG?CAG?AGG?CGG?CAG?TCA?AGC?CTC?TTC?TCG
CTC?CTC?ATC?ACG?TAG?TCG?CGG?TAA?TTC?AAG?AAA?TTC?AAC?TCC?TGG?CAG?CAG?TAG?GGG
AAA?TTC?TCC?TGC?TCG?AAT?GGC?TAG?CGG?AGG?TGG?TGA?AAC?TGC?CCT?CGC?GCT?ATT?GCT
GCT?AGA?CAG?ATT?GAA?CCA?GCT?TGA?GAG?CAA?AGT?TTC?TGG?TAA?AGG?CCA?ACA?ACA?ACA
AGG?CCA?AAC?TGT?CAC?TAA?GAA?ATC?TGC?TGC?TGA?GGC?ATC?TAA?AAA?G GG?ATC?CGC?G
The dna sequence dna (916bp) that contains SARS virus S proteantigen epitope gene of chemosynthesis:
GT GGATCC?GAA?TAC?ATC?TCT?GAC?GCA?TTC?TCT?CTG?GAC?GTT?TCC?GAA?AAG?TCT?GGT?AAC
TTC?AAA?CAC?CTG?CGC?GAG?TTC?GTG?TTT?AAA?AAC?AAA?GAC?GGT?TTC?CTG?TAG?GTT?TAC
AAG?GGC?TAC?CAG?CCG?ATC?GAC?GTA?GTT?CGT?GAC?CTG?CCG?TCT?GGT?TTT?AAC?ACT?CTG
AAA?CCG?ATC?TTC?AAG?CTG?CCG?CTG?GGT?ATT?AAC?ATC?ACT?AAC?TTC?CGC?GCT?ATC?CTG
ACT?GCT?TTC?TCT?CCG?GCT?CAG?GAC?ACT?TGG?GGC?ACT?TCT?GCT?GCA?GCC?TAC?TTC?GTT
GGC?TAC?CTG?AAG?CCA?ACT?ACC?TTT?ATG?CTG?AAG?TAC?GAC?GAA?AAC?GGT?ACT?ATC?ACT
GAT?GCT?GTT?GAC?TGC?TCT?CAG?AAC?CCA?CTG?GCT?GAA?CTG?AAA?TGC?TCT?GTT?AAG?AGC
TTT?GAG?ATC?GAC?AAA?GGT?ATT?TAC?CAG?ACC?TCT?AAC?TTC?CGT?GTT?GTT?CCG?TCT?GGT
GAC?GTT?GTG?CGT?TTC?CCT?AAC?ATC?ACT?AAC?CTG?TGC?CCG?TTT?GGT?GAA?GTT?TTC?AAC
GCT?ACT?AAA?TTC?CCT?TCT?GTC?TAC?GCA?TGG?GAG?CGT?AAA?AAA?ATT?TCT?AAC?TGC?GTT
GCT?GAT?TAC?TCT?GTG?CTG?TAC?AAC?TCT?ACC?TTT?TTC?TCT?ACC?TTC?AAG?TGC?TAC?GGC
GTT?TCT?GCT?ACT?AAG?CTG?AAC?GAC?CTG?TGC?TTC?TCC?AAC?GTT?TAC?GCA?GAT?TCT?TTC
GTA?GTT?AAG?GGT?GAT?GAC?GTA?CGT?CAG?ATC?GCT?CCA?GGT?CAG?ACT?GGT?GTT?ATC?GCT
GAC?TAC?AAC?TAT?AAA?CTG?CCG?GAC?GAT?TTC?ATG?GGT?TGC?GTT?CTG?GCT?TGG?AAC?ACT
CGT?AAC?ATT?GAC?GCT?ACT?TCT?ACT?GGT?AAC?TAC?AAC?TAC?AAA?TAT?CGT?TAC?CTG?CGT
CAC?GGC?AAA?CTG?CGT?CCG?TTC?GAA?CGT?GAC?ATC?TCT?AAC?GTG?CCG?TTC?TAA? GAATTCAC
2. express the fusion rotein construction of recombinant plasmid of SARS virus S protein fragments and N protein fragments:
Extract plasmid pET28a (+) (U.S. Novagen company product),, reclaim the big fragment of plasmid that enzyme is cut behind the electrophoresis, be dissolved in the deionized water with Nco I and EcoR I double digestion.With the SARS virus N protein gene fragment of Nco I and the chemosynthesis of BamH I double digestion,, after electrophoresis reclaims respectively, be dissolved in the deionized water with the SARS virus S protein gene fragment of BamH I and the chemosynthesis of EcoR I double digestion.
Above-mentioned three kinds of enzymes of volumetric molar concentration such as get and cut the back dna fragmentation, in same centrifuge tube, connect with the T4 dna ligase, make SARS virus N protein gene fragment and S protein gene fragment after BamH I enzyme is connected, be inserted between the Nco I and EcoR I site in the carrier pET28a (+), express the fusion rotein of a SARS virus S protein fragments and N protein fragments.Make up flow process and see Fig. 3.
3. the screening of recombinant plasmid and evaluation:
The recombinant plasmid transformed that the last step was connected arrives e. coli bl21 (DE3), and the converted product coating is contained on the solid LB substratum of kantlex (60 μ g/ml), puts 37 ℃ of overnight incubation.8 transformant bacterium colonies of random choose next day (being labeled as respectively 1-8 number) are inoculated into respectively and contain 4ml liquid LB substratum (containing kantlex 60 μ g/ml) and in vitro, put 37 ℃ of shaking culture 6h, get bacterium liquid 1ml, centrifugal receipts bacterium.Use 50 μ l deionized water suspension thalline respectively, boiling water boils 5min, centrifugal (4 ℃, 12000rpm) 5min, get supernatant (in plasmid is arranged) 1 μ l as pcr template, right with the primer of primer of N protein gene fragment 5 ' end (5 '-GCGAGTCACCATGGGATCTGATAACGGTCCGCAGTCAAACCAACG-3 ') and S protein gene fragment 3 ' end (5 '-GTGAATTCTTAGAAAGGCACATTAGATATGT-3 ') composition primer, SARS virus N protein gene fragment that pcr amplification links together and S protein gene fragment, contain the positive recombinant plasmid of two gene fragments of connecting, should amplify the tandem gene fragment that is about 1701bp.The PCR reaction density is: plasmid template 1 μ l, upstream and downstream primer each 1 μ l, 10 * Buffer, 5.0 μ l, 2.5mmol/L dNTP 4.0 μ l, Taq archaeal dna polymerase 0.5 μ l (2.5U), deionized water 37.5 μ l, cumulative volume 50 μ l.Amplification condition is: 94 ℃ 30 seconds, 60 ℃ 30 seconds, 72 ℃ 60 seconds, 30 circulations; Last 72 ℃ were extended 7 minutes.Get pcr amplification product 5 μ l, Agarose gel detection with 1.2%, the result is in 8 transformants, 2nd, 3,4,5, No. 65 transformants amplify the target gene fragment (see figure 4) of 1701bp, and the 1st, 7, No. 83 transformants do not amplify this gene fragment.Tentative confirmation has 5 transformants to contain SARS virus N protein gene fragment and the segmental tandem gene of S protein gene.
Extract the plasmid of No. 5 recons, measure SARS virus N protein gene fragment and the segmental tandem gene sequence of S protein gene in the plasmid, dna sequence analysis confirms that recombinant plasmid contains placed in-line SARS virus S protein gene fragment and N protein gene fragment, and sequence is entirely true:
ATG?GGA?TCT?GAT?AAC?GGT?CCG?CAG?TCA?AAC?CA ACG?TAG?TGC?CCC?CCG?CAT?TAC?ATT
TGG?TGG?ACC?CAC?AGA?TTC?AAC?TGA?CAA?TAA?CCA?GAA?TGG?AGG?ACG?CAA?TGG?GGC?AAG
GCC?AAA?ACA?GCG?CCG?ACC?CCA?AGG?TTT?ACC?CAA?TAA?TAC?TGC?GTC?TTG?GTT?CAC?AGC
TCT?CAC?TCA?GCA?TGG?CAA?GGA?GGA?ACT?TAG?ATT?CCC?TCG?AGG?CCA?GGG?CGT?TCC?AAT
CAA?CAC?CAA?TAG?TGG?TCC?AGA?TGA?CCA?AAT?TGG?CTA?CTA?CCG?AAG?AGC?TAC?CCG?ACG
AGT?TCG?TGG?TGG?TGA?CGG?CAA?AAT?GAA?AGA?GCT?CAG?CCC?CAG?ATG?GTA?CTT?CTA?TTA
CCT?AGG?AAC?TGG?CCC?AGA?AGC?TTC?ACT?TCC?CTA?CGG?CGC?TAA?CAA?AGA?AGG?CAT?CGT
ATG?GGT?TGC?AAC?TGA?GGG?AGC?CTT?GAA?TAC?ACC?CAA?AGA?CCA?CAT?TGG?CAC?CCG?CAA
TCC?TAA?TAA?CAA?TGC?TGC?CAC?CGT?GCT?ACA?ACT?TCC?TCA?AGG?AAC?AAC?ATT?GCC?AAA
AGG?CTT?CTA?CGC?AGA?GGG?AAG?CAG?AGG?CGG?CAG?TCA?AGC?CTC?TTC?TCG?CTC?CTC?ATC
ACG?TAG?TCG?CGG?TAA?TTC?AAG?AAA?TTC?AAC?TCC?TGG?CAG?CAG?TAG?GGG?AAA?TTC?TCC
TGC?TCG?AAT?GGC?TAG?CGG?AGG?TGG?TGA?AAC?TGC?CCT?CGC?GCT?ATT?GCT?GCT?AGA?CAG
ATT?GAA?CCA?GCT?TGA?GAG?CAA?AGT?TTC?TGG?TAA?AGG?CCA?ACA?A?CAA?CAA?GGC?CAA?ACT
GTC?ACT?AAG?AAA?TCT?GCT?GCT?GAG?GCA?TCT?AAA?AAG? GGA?TCC?GAA?TAC?ATC?TCT?GAC
GCA?TTC?TCT?CTG?GAC?GTT?TCC?GAA?AAG?TCT?GGT?AAC?TTC?AAA?CAC?CTG?CGC?GAG?TTC
GTG?TTT?AAA?AAC?AAA?GAC?GGT?TTC?CTG?TAC?GTT?TAC?AAG?GGC?TAC?CAG?CCG?ATC?GAC
GTA?GTT?CGT?GAC?CTG?CCG?TCT?GGT?TTT?AAC?ACT?CTG?AAA?CCG?ATC?TTC?AAG?CTG?CCG
CTG?GGT?ATT?AAC?ATC?ACT?AAC?TTC?CGC?GCT?ATC?CTG?ACT?GCT?TTC?TCT?CCG?GCT?CAG
GAC?ACT?TGG?GGC?ACT?TCT?GCT?GCA?GCC?TAC?TTC?GTT?GGC?TAC?CTG?AAG?CCA?ACT?ACC
TTT?ATG?CTG?AAG?TAC?GAC?GAA?AAC?GGT?ACT?ATC?ACT?GAT?GCT?GTT?GAC?TGC?TCT?CAG
AAC?CCA?CTG?GCT?GAA?CTG?AAA?TGC?TCT?GTT?AAG?AGC?TTT?GAG?ATC?GAC?AAA?GGT?ATT
TAC?CAG?ACC?TCT?AAC?TTC?CGT?GTT?GTT?CCG?TCT?GGT?GAC?GTT?GTG?CGT?TTC?CCT?AAC
ATC?ACT?AAC?CTG?TGC?CCG?TTT?GGT?GAA?GTT?TTC?AAC?GCT?ACT?AAA?TTC?CCT?TCT?GTC
TAC?GCA?TGG?GAG?CGT?AAA?AAA?ATT?TCT?AAC?TGC?GTT?GCT?GAT?TAC?TCT?GTG?CTG?TAC
AAC?TCT?ACC?TTT?TTC?TCT?ACC?TTC?AAG?TGC?TAC?GGC?GTT?TCT?GCT?ACT?AAG?CTG?AAC
GAC?CTG?TGC?TTC?TCC?AAC?GTT?TAC?GCA?GAT?TCT?TTC?GTA?GTT?AAG?GGT?GAT?GAC?GTA
CGT?CAG?ATC?GCT?CCA?GGT?CAG?ACT?GGT?GTT?ATC?GCT?GAC?TAC?AAC?TAT?AAA?CTG?CCG
GAC?GAT?TTC?ATG?GGT?TGC?GTT?CTG?GCT?TGG?AAC?ACT?CGT?AAC?ATT?GAC?GCT?ACT?TCT
ACT?GGT?AAC?TAC?AAC?TAC?AAA?TAT?CGT?TAC?CTG?CGT?CAC?GGC?AAA?CTG?CGT?CCG?TTC
GAA?CGT?GAC?ATC?TCT?AAC?GTG?CCG?TTC?TAA
The virus S protein fragment of the expression of recombinant plasmid SARS that makes up and the fusion rotein of N protein fragments, 560 amino acid of total length, be the N protein fragments at its N end, long 259 amino acid, the C end is the S protein fragments, long 299 amino acid are connected by two amino acid of propylhomoserin and Serine between the two, and its aminoacid sequence is as follows:
Met?Gly?Ser?Asp?Asn?Gly?Pro?Gln?Ser?Asn?Gln?Arg?Ser?Ala?Pro?Arg?Ile?Thr?Phe
Gly?Gly?Pro?Thr?Asp?Ser?Thr?Asp?Asn?Asn?Gln?Asn?Gly?Gly?Arg?Asn?Gly?Ala?Arg
Pro?Lys?Gln?Arg?Arg?Pro?Gln?Gly?Leu?Pro?Asn?Asn?Thr?Ala?Ser?Trp?Phe?Thr?Ala
Leu?Thr?Gln?His?Gly?Lys?Glu?Glu?Leu?Arg?Phe?Pro?Arg?Gly?Gln?Gly?Val?Pro?Ile
Asn?Thr?Asn?Ser?Gly?Pro?Asp?Asp?Gln?Ile?Gly?Tyr?Tyr?Arg?Arg?Ala?Thr?Arg?Arg
Val?Arg?Gly?Gly?Asp?Gly?Lys?Met?Lys?Glu?Leu?Ser?Pro?Arg?Trp?Tyr?Phe?Tyr?Tyr
Leu?Gly?Thr?Gly?Pro?Glu?Ala?Ser?Leu?Pro?Tyr?Gly?Ala?Asn?Lys?Glu?Gly?Ile?Val
Trp?Val?Ala?Thr?Glu?Gly?Ala?Leu?Asn?Thr?Pro?Lys?Asp?His?Ile?Gly?Thr?Arg?Asn
Pro?Asn?Asn?Asn?Ala?Ala?Thr?Val?Leu?Gln?Leu?Pro?Gln?Gly?Thr?Thr?Leu?Pro?Lys
Gly?Phe?Tyr?Ala?Glu?Gly?Ser?Arg?Gly?Gly?Ser?Gln?Ala?Ser?Ser?Arg?Ser?Ser?Ser
Arg?Ser?Arg?Gly?Asn?Ser?Arg?Asn?Ser?Thr?Pro?Gly?Ser?Ser?Arg?Gly?Asn?Ser?Pro
Ala?Arg?Met?Ala?Ser?Gly?Gly?Gly?Glu?Thr?Ala?Leu?Ala?Leu?Leu?Leu?Leu?Asp?Arg
Leu?Asn?Gln?Leu?Glu?Ser?Lys?Val?Ser?Gly?Lys?Gly?Gln?Gln?Gln?Gln?Gly?Gln?Thr
Val?Thr?Lys?Lys?Ser?Ala?Ala?Glu?Ala?Ser?Lys?Lys?Gly?Ser?Glu?Tyr?Ile?Ser?Asp
Ala?Phe?Ser?Leu?Asp?Val?Ser?Glu?Lys?Ser?Gly?Asn?Phe?Lys?His?Leu?Arg?Glu?Phe
Val?Phe?Lys?Asn?Lys?Asp?Gly?Phe?Leu?Tyr?Val?Tyr?Lys?Gly?Tyr?Gln?Pro?Ile?Asp
Val?Val?Arg?Asp?Leu?Pro?Ser?Gly?Phe?Asn?Thr?Leu?Lys?Pro?Ile?Phe?Lys?Leu?Pro
Leu?Gly?Ile?Asn?Ile?Thr?Asn?Phe?Arg?Ala?Ile?Leu?Thr?Ala?Phe?Ser?Pro?Ala?Gln
Asp?Thr?Trp?Gly?Thr?Ser?Ala?Ala?Ala?Tyr?Phe?Val?Gly?Tyr?Leu?Lys?Pro?Thr?Thr
Phe?Met?Leu?Lys?Tyr?Asp?Glu?Asn?Gly?Thr?Ile?Thr?Asp?Ala?Val?Asp?Cys?Ser?Gln
Asn?Pro?Leu?Ala?Glu?Leu?Lys?Cys?Ser?Val?Lys?Ser?Phe?Glu?Ile?Asp?Lys?Gly?Ile
Tyr?Gln?Thr?Ser?Asn?Phe?Arg?Val?Val?Pro?Ser?Gly?Asp?Val?Val?Arg?Phe?Pro?Asn
Ile?Thr?Asn?Leu?Cys?Pro?Phe?Gly?Glu?Val?Phe?Asn?Ala?Thr?Lys?Phe?Pro?Ser?Val
Tyr?Ala?Trp?Glu?Arg?Lys?Lys?Ile?Ser?Asn?Cys?Val?Ala?Asp?Tyr?Ser?Val?Leu?Tyr
Asn?Ser?Thr?Phe?Phe?Ser?Thr?Phe?Lys?Cys?Tyr?Gly?Val?Ser?Ala?Thr?Lys?Leu?Asn
Asp?Leu?Cys?Phe?Ser?Asn?Val?Tyr?Ala?Asp?Ser?Phe?Val?Val?Lys?Gly?Asp?Asp?Val
Arg?Gln?Ile?Ala?Pro?Gly?Gln?Thr?Gly?Val?Ile?Ala?Asp?Tyr?Asn?Tyr?Lys?Leu?Pro
Asp?Asp?Phe?Met?Gly?Cys?Val?Leu?Ala?Trp?Asn?Thr?Arg?Asn?Ile?Asp?Ala?Thr?Ser
Thr?Gly?Asn?Tyr?Asn?Tyr?Lys?Tyr?Arg?Tyr?Leu?Arg?His?Gly?Lys?Leu?Arg?Pro?Phe
Glu?Arg?Asp?Ile?Ser?Asn?Val?Pro?Phe
4. the Screening and Identification of expressed fusion protein engineering bacteria:
Above-mentioned 8 transformants are seeded to contain 3ml LB substratum (containing kanamycin 60 μ g/ml) in vitro, 37 ℃ of shaking culture 3h, add IPTG to final concentration 0.5mmol/L, continue shaking culture and induce 6h, centrifugal collection thalline carries out SDS-PAGE and detects, 2nd, 4,5, No. 64 transformants are expressed SARS virus S albumen and the proteic fusion rotein of N that relative molecular weight is about 60kD, and the 1st, 3,7, No. 84 transformants do not have this protein band (Fig. 5).
Express the purifying of SARS virus S albumen and the proteic fusion rotein of N
According to the aminoacid sequence of expressing SARS virus S albumen and the proteic fusion rotein of N, analyze its physicochemical property, determine suitable purification process.The isoelectric pH 9.7 of SARS virus S albumen and the proteic fusion rotein of N is expressed in machine analysis as calculated, so our decision is in pH is 7.4 phosphate buffered saline buffer, with S-SepharoseFF positively charged ion purifying.Concrete steps are as follows:
Material and method
1. main agents:
S-SepharoseFF positively charged ion gel is a Pharmacia company product, and IPTG, DTT are TaTaRa company product.Other reagent is homemade or the import analytical reagent.
2. express the abduction delivering and the ultrasonic degradation of SARS virus S albumen and the proteic fusion rotein engineering bacteria of N
Have from inoculation on the LB flat board of No. 5 engineering bacterias, choose single bacterium colony, be inoculated in the Erlenmeyer flask that contains 200ml LB liquid nutrient medium, add kantlex, put overnight incubation in 37 ℃ of shaking tables to final concentration 60 μ g/ml with toothpick.Be inoculated into 4 Erlenmeyer flasks that respectively contain 200ml LB liquid nutrient medium with bacterium liquid next day, and every bottle graft kind bacterium liquid 50ml put the interior shaking culture of 37 ℃ of shaking tables 1 hour, added IPTG then to final concentration 0.1mmol/L, abduction delivering 4 hours.
Centrifugal (8000rpm, 10min, 4 ℃) receives bacterium with the 1000ml engineering bacteria of abduction delivering fusion rotein, thalline is resuspended in the bacterial lysate (20mmol/L PB pH7.4,10mmol/L EDTA, 1mmol/L DTT, 5% glycerine) of 100ml, ice-bath ultrasonic is broken bacterium 10min, centrifugal (12000rpm, 30min, 4 ℃) collects supernatant.
3. express the sulfuric acid amine fractionation precipitation of SARS virus S albumen and the proteic fusion rotein of N
Accurately measure the supernatant volume, add an amount of saturated sulfuric acid amine aqueous solution in supernatant, the limit edged stirs, and making the final concentration of sulfuric acid amine is 30%, puts in the ice bath and spends the night.Next day, centrifugal (12000rpm, 20min, 4 ℃) collected supernatant, added an amount of solid sulfur acid amide powder, and the limit edged stirs, and making the final concentration of sulfuric acid amine is 50%, puts in the ice bath and spends the night.Next day centrifugal (12000rpm, 20min, 4 ℃) collecting precipitation.With 100ml balance liquid (20mmol/L PB pH7.4,0.5mmol/L EDTA, 0.2mmol/L DTT) suspension precipitation, in the dialysis tubing of packing into,, change liquid 1 time to 500ml balance liquid dialysed overnight.Centrifugal (12000rpm, 20min, 4 ℃) collects supernatant, directly goes up S-Sepearse FF cation seperation column purifying purifying
4.S-Sepearse FF cation seperation column purifying
With balance liquid (20mmol/L PB pH7.4,0.5mmol/L EDTA, 0.2mmol/L DTT) flushing balance S-Sepharose FF cation seperation column, will go up the good direct upper prop of supernatant of step dialysis then, last sample flow velocity 1.5ml/min.Fully wash post with balance liquid behind the end of the sample, again successively with contain 50,100,200, the balance liquid eluted protein of 1000mmol/L NaCl, collect each elution peak albumen, with each peak albumen of 12%SDS-PAGE electrophoresis detection, determine which elution peak contains the SARS virus S albumen and the proteic fusion rotein of N of expression.
The result
Different concns NaCl albumen of wash-out from the S-Sepharose FF post is carried out SDS-PAGE to be analyzed, the result shows (see figure 6), electrophoresis result shows, 100mmol/L NaCl eluted protein peak is SARS virus S albumen and the proteic fusion rotein of N, the S albumen and the proteic fusion rotein of N (about 60kD place) of a dense SARS virus of colour developing, no obvious SARS virus S albumen and the proteic fusion rotein band of N in other elution peak.
The evaluation and the application of purifying SARS virus S albumen and the proteic fusion rotein of N
The reorganization SARS virus S albumen and the proteic fusion rotein of N of purifying are used as antigen, by the indirect ELISA test method, detect anti-SARS virus antibody (IgM or IgG) positive and negative serum, with the SARS virus S albumen of evaluation expression and the antigenicity and the specificity of the proteic fusion rotein of N.Experimental result shows that this recombination fusion protein has good antigenicity and specificity, can be used as Detection of antigen anti-SARS virus antibody etc.
Material and method
1. anti-SARS virus antibody positive serum and normal human serum: the positive control in the anti-SARS virus antibody ELISA test kit that GBI company produces, and the anti-SARS virus antibody positive serum of 2 parts of deactivations.Normal human serum is preserved by this laboratory.
2. integrated enzyme reaction material: elisa plate is that 96 orifice plates are produced in Shenzhen, and the mouse-anti people μ chain monoclonal antibody of horseradish peroxidase (HRP) mark is available from Sigma company.Other material is the conventional material of integrated enzyme reaction.
3.ELISA test: adopt the anti-SARS virus antibody (IgG or IgM) in the indirect ELISA detection serum.Basic step is: with the S albumen and the proteic fusion rotein of N of 50mmol/L carbonate solution (pH9.6) dilution SARS virus, wraps by elisa plate, and every hole 100 μ l, 4 ℃ are spent the night.Inferior daily confining liquid (10mmol/L phosphate buffered saline buffer, pH7.4,10% lowlenthal serum, 20% calf serum, 0.05% sulphur sulphur mercury, 5% sucrose) sealing, every hole 130 μ l, 37 ℃ of 1h (or 4 ℃ spend the night).With anti-SARS virus antibody to be measured (IgG or IgM) the moon, positive serum, with sample diluent (10mmol/L phosphate buffered saline buffer, pH7.4,10% lowlenthal serum, 20% calf serum, 0.05% sulphur sulphur mercury, 0.5mmol/L NaCl) dilution back (IgG dilution in 1: 20, IgM dilution in 1: 100), add to respectively in the enzyme linked plate holes after the sealing, every hole 100 μ l, each sample adds 2 holes, 37 ℃ of reaction 30min, with PBST washing lotion (10mmol/L phosphate buffered saline buffer, pH7.4,0.5% tween 20) wash 5 times after, add the anti-human IgG or the anti-people μ chain monoclonal antibody of horseradish peroxidase-labeled of dilution in 1: 5000, every hole 100 μ l, 37 ℃ of reaction 30min, PBST washes 5 times, adds substrate tetramethyl biphenyl ammonia (TMB) solution 100 μ l, 37 ℃ of lucifuge colour developing 10min, add 50 μ l 4N sulfuric acid mixing termination reactions, measure the A450 value with enzyme connection instrument.
The result
1. indirect ELISA detects anti-SARS virus antibody (IgG or IgM) in the serum
The SARS virus S albumen of purifying and bovine serum albumin (the 5th part) co-electrophoresis, the colour developing back of proteic fusion rotein of N and known different concns are compared, determine that the S albumen and the proteic fusion rotein concentration of N of the SARS virus of purifying is about 0.5mg/ml.By elisa plate, detect known 2 parts of anti-SARS virus IgG antibody positive serum and 1 part of anti-SARS virus IgM antibody positive serum with 1: 500 dilution back of 50mmol/L carbonate solution (pH9.6) bag.The result shows that color reaction all appears in 2 parts of anti-SARS virus antibody positive serums, and the A450 value is respectively 1.335 and 1.098.Color reaction also appears in 1 part of anti-SARS virus antibody (IgM) positive serum, and the A450 value is 0.872.Detected 180 portions of normal human serums simultaneously, their A450 value illustrates that all less than 0.05 the S albumen of SARS virus and the proteic fusion rotein of N have antigenicity and specificity preferably.
SARS virus S protein fragments and the sequence table of N protein fragments fusion rotein
＜110〉Li Yuexi
＜120〉SARS virus S protein fragments and N protein fragments fusion rotein and preparation thereof, application
<160>2
<210>1
<211>560
<212>PRT
＜213〉SARS virus S protein fragments and N protein fragments fusion rotein
<220>
＜223〉fusion rotein of SARS virus S protein fragments and N protein fragments, i.e. proteic the 162nd amino of the S of SARS virus
Acid is to the fusion rotein of proteic the 1st amino acid to 258 amino acid fragment of N of the 460th amino acid fragment and SARS virus,
The N protein fragments is at the N of fusion rotein end, and the S protein fragments is used glycine and silk at the C of fusion rotein end between two protein fragments
Propylhomoserin connects, and inserts a glycine, 560 ammonia of fusion rotein total length behind first amino acid methionine of N protein fragments
Base acid.
<400>1
Met?Gly?Ser?Asp?Asn?Gly?Pro?Gln?Ser?Asn?Gln?Arg?Ser?Ala?Pro?Arg
1 5 10 15
Ile?Thr?Phe?Gly?Gly?Pro?Thr?Asp?Ser?Thr?Asp?Asn?Asn?Gln?Asn?Gly
20 25 30
Gly?Arg?Asn?Gly?Ala?Arg?Pro?Lys?Gln?Arg?Arg?Pro?Gln?Gly?Leu?Pro
35 40 45
Asn?Asn?Thr?Ala?Ser?Trp?Phe?Thr?Ala?Leu?Thr?Gln?His?Gly?Lys?Glu
50 55 60
Glu?Leu?Arg?Phe?Pro?Arg?Gly?Gln?Gly?Val?Pro?Ile?Asn?Thr?Asn?Ser
65 70 75 80
Gly?Pro?Asp?Asp?Gln?Ile?Gly?Tyr?Tyr?Arg?Arg?Ala?Thr?Arg?Arg?Val
85 90 95
Arg?Gly?Gly?Asp?Gly?Lys?Met?Lys?Glu?Leu?Ser?Pro?Arg?Trp?Tyr?Phe
100 105 110
Tyr?Tyr?Leu?Gly?Thr?Gly?Pro?Glu?Ala?Ser?Leu?Pro?Tyr?Gly?Ala?Asn
115 120 125
Lys?Glu?Gly?Ile?Val?Trp?Val?Ala?Thr?Glu?Gly?Ala?Leu?Asn?Thr?Pro
130 135 140
Lys?Asp?His?Ile?Gly?Thr?Arg?Asn?Pro?Asn?Asn?Asn?Ala?Ala?Thr?Val
145 150 155 160
Leu?Gln?Leu?Pro?Gln?Gly?Thr?Thr?Leu?Pro?Lys?Gly?Phe?Tyr?Ala?Glu
165 170 175
Gly?Ser?Arg?Gly?Gly?Ser?Gln?Ala?Ser?Ser?Arg?Ser?Ser?Ser?Arg?Ser
180 185 190
Arg?Gly?Asn?Ser?Arg?Asn?Ser?Thr?Pro?Gly?Ser?Ser?Arg?Gly?Asn?Ser
195 200 205
Pro?Ala?Arg?Met?Ala?Ser?Gly?Gly?Gly?Glu?Thr?Ala?Leu?Ala?Leu?Leu
210 215 220
Leu?Leu?Asp?Arg?Leu?Asn?Gln?Leu?Glu?Ser?Lys?Val?Ser?Gly?Lys?Gly
225 230 235 240
Gln?Gln?Gln?Gln?Gly?Gln?Thr?Val?Thr?Lys?Lys?Ser?Ala?Ala?Glu?Ala
245 250 255
Ser?Lys?Lys?Gly?Ser?Glu?Tyr?Ile?Ser?Asp?Ala?Phe?Ser?Leu?Asp?Val
260 265 270
Ser?Glu?Lys?Ser?Gly?Asn?Phe?Lys?His?Leu?Arg?Glu?Phe?Val?Phe?Lys
275 280 285
Asn?Lys?Asp?Gly?Phe?Leu?Tyr?Val?Tyr?Lys?Gly?Tyr?Gln?Pro?Ile?Asp
290 295 300
Val?Val?Arg?Asp?Leu?Pro?Ser?Gly?Phe?Asn?Thr?Leu?Lys?Pro?Ile?Phe
305 310 315 320
Lys?Leu?Pro?Leu?Gly?Ile?Asn?Ile?Thr?Asn?Phe?Arg?Ala?Ile?Leu?Thr
325 330 335
Ala?Phe?Ser?Pro?Ala?Gln?Asp?Thr?Trp?Gly?Thr?Ser?Ala?Ala?Ala?Tyr
340 345 350
Phe?Val?Gly?Tyr?Leu?Lys?Pro?Thr?Thr?Phe?Met?Leu?Lys?Tyr?Asp?Glu
355 360 365
Asn?Gly?Thr?Ile?Thr?Asp?Ala?Val?Asp?Cys?Ser?Gln?Asn?Pro?Leu?Ala
370 375 380
Glu?Leu?Lys?Cys?Ser?Val?Lys?Ser?Phe?Glu?Ile?Asp?Lys?Gly?Ile?Tyr
385 390 395 400
Gln?Thr?Ser?Asn?Phe?Arg?Val?Val?Pro?Ser?Gly?Asp?Val?Val?Arg?Phe
405 410 415
Pro?Asn?Ile?Thr?Asn?Leu?Cys?Pro?Phe?Gly?Glu?Val?Phe?Asn?Ala?Thr
420 425 430
Lys?Phe?Pro?Ser?Val?Tyr?Ala?Trp?Glu?Arg?Lys?Lys?Ile?Ser?Asn?Cys
435 440 445
Val?Ala?Asp?Tyr?Ser?Val?Leu?Tyr?Asn?Ser?Thr?Phe?Phe?Ser?Thr?Phe
450 455 460
Lys?Cys?Tyr?Gly?Val?Ser?Ala?Thr?Lys?Leu?Asn?Asp?Leu?Cys?Phe?Ser
465 470 475 480
Asn?Val?Tyr?Ala?Asp?Ser?Phe?Val?Val?Lys?Gly?Asp?Asp?Val?Arg?Gln
485 490 495
Ile?Ala?Pro?Gly?Gln?Thr?Gly?Val?Ile?Ala?Asp?Tyr?Asn?Tyr?Lys?Leu
500 505 510
Pro?Asp?Asp?Phe?Met?Gly?Cys?Val?Leu?Ala?Trp?Asn?Thr?Arg?Asn?Ile
515 520 525
Asp?Ala?Thr?Ser?Thr?Gly?Asn?Tyr?Asn?Tyr?Lys?Tyr?Arg?Tyr?Leu?Arg
530 535 540
His?Gly?Lys?Leu?Arg?Pro?Phe?Glu?Arg?Asp?Ile?Ser?Asn?Val?Pro?Phe
545 550 555 560
<210>2
<211>1683
<212>DNA
＜213〉artificial sequence
<220>
<221>CDS
<222>(1)...(1683)
＜223〉gene fragment of synthetic, the fusion rotein of encoding SARS virus S protein fragment and N protein fragments.
<220>
<221>mis-feature
<222>(1)...(777)
＜223〉gene fragment of synthetic, proteic the 1st amino acid to 258 amino acid fragment of encoding SARS virus N,
Behind the sub-ATG of first amino acid code of N albumen, insert a codon glycine GGA.
<220>
<221>mis-feature
<222>(778)...(783)
＜223〉connect SARS virus S protein gene fragment and the segmental BamHI restriction enzyme site of N protein gene.
<220>
<221>mis-feature
<222>(784)...(1680)
＜223〉gene fragment of synthetic, the 162nd amino acid to 460 amino acid fragment of encoding SARS virus S protein.
<220>
<221>mis-feature
<222>(1681)...(1683)
＜223〉terminator codon that increases during synthetic gene.
<400>2
ATG?GGA?TCT?GAT?AAC?GGT?CCG?CAG?TCA?AAC?CAA?CGT?AGT?GCC?CCC?CGC?ATT?ACA?TTT?GGT 60
Met?Gly?Ser?Asp?Asn?Gly?Pro?Gln?Ser?Asn?Gln?Arg?Ser?Ala?Pro?Arg?Ile?Thr?Phe?Gly
1 5 10 15 20
GGA?CCC?ACA?GAT?TCA?ACT?GAC?AAT?AAC?CAG?AAT?GGA?GGA?CGC?AAT?GGG?GCA?AGG?CCA?AAA 120
Gly?Pro?Thr?Asp?Ser?Thr?Asp?Asn?Asn?Gln?Asn?Gly?Gly?Arg?Asn?Gly?Ala?Arg?Pro?Lys
25 30 35 40
CAG?CGC?CGA?CCC?CAA?GGT?TTA?CCC?AAT?AAT?ACT?GCG?TCT?TGG?TTC?ACA?GCT?CTC?ACT?CAG?180
Gln?Arg?Arg?Pro?Gln?Gly?Leu?Pro?Asn?Asn?Thr?Ala?Ser?Trp?Phe?Thr?Ala?Leu?Thr?Gln
45 50 55 60
CAT?GGC?AAG?GAG?GAA?CTT?AGA?TTC?CCT?CGA?GGC?CAG?GGC?GTT?CCA?ATC?AAC?ACC?AAT?AGT?240
His?Gly?Lys?Glu?Glu?Leu?Arg?Phe?Pro?Arg?Gly?Gln?Gly?Val?Pro?Ile?Asn?Thr?Asn?Ser
65 70 75 80
GGT?CCA?GAT?GAC?CAA?ATT?GGC?TAC?TAC?CGA?AGA?GCT?ACC?CGA?CGA?GTT?CGT?GGT?GGT?GAC?300
Gly?Pro?Asp?Asp?Gln?Ile?Gly?Tyr?Tyr?Arg?Arg?Ala?Thr?Arg?Arg?Val?Arg?Gly?Gly?Asp
85 90 95 100
GGC?AAA?ATG?AAA?GAG?CTC?AGC?CCC?AGA?TGG?TAC?TTC?TAT?TAC?CTA?GGA?ACT?GGC?CCA?GAA?360
Gly?Lys?Met?Lys?Glu?Leu?Ser?Pro?Arg?Trp?Tyr?Phe?Tyr?Tyr?Leu?Gly?Thr?Gly?Pro?Glu
105 110 115 120
GCT?TCA?CTT?CCC?TAC?GGC?GCT?AAC?AAA?GAA?GGC?ATC?GTA?TGG?GTT?GCA?ACT?GAG?GGA?GCC?420
Ala?Ser?Leu?Pro?Tyr?Gly?Ala?Asn?Lys?Glu?Gly?Ile?Val?Trp?Val?Ala?Thr?Glu?Gly?Ala
125 130 135 140
TTG?AAT?ACA?CCC?AAA?GAC?CAC?ATT?GGC?ACC?CGC?AAT?CCT?AAT?AAC?AAT?GCT?GCC?ACC?GTG?480
Leu?Asn?Thr?Pro?Lys?Asp?His?Ile?Gly?Thr?Arg?Asn?Pro?Asn?Asn?Asn?Ala?Ala?Thr?Val
145 150 155 160
CTA?CAA?CTT?CCT?CAA?GGA?ACA?ACA?TTG?CCA?AAA?GGC?TTC?TAC?GCA?GAG?GGA?AGC?AGA?GGC?540
Leu?Gln?Leu?Pro?Gln?Gly?Thr?Thr?Leu?Pro?Lys?Gly?Phe?Tyr?Ala?Glu?Gly?Ser?Arg?Gly
165 170 175 180
GGC?AGT?CAA?GCC?TCT?TCT?CGC?TCC?TCA?TCA?CGT?AGT?CGC?GGT?AAT?TCA?AGA?AAT?TCA?ACT?600
Gly?Ser?Gln?Ala?Ser?Ser?Arg?Ser?Ser?Ser?Arg?Ser?Arg?Gly?Asn?Ser?Arg?Asn?Ser?Thr
185 190 195 200
CCT?GGC?AGC?AGT?AGG?GGA?AAT?TCT?CCT?GCT?CGA?ATG?GCT?AGC?GGA?GGT?GGT?GAA?ACT?GCC?660
Pro?Gly?Ser?Ser?Arg?Gly?Asn?Ser?Pro?Ala?Arg?Met?Ala?Ser?Gly?Gly?Gly?Glu?Thr?Ala
205 210 215 220
CTC?GCG?CTA?TTG?CTG?CTA?GAC?AGA?TTG?AAC?CAG?CTT?GAG?AGC?AAA?GTT?TCT?GGT?AAA?GGC?720
Leu?Ala?Leu?Leu?Leu?Leu?Asp?Arg?Leu?Asn?Gln?Leu?Glu?Ser?Lys?Val?Ser?Gly?Lys?Gly
225 230 235 240
CAA?CAA?CAA?CAA?GGC?CAA?ACT?GTC?ACT?AAG?AAA?TCT?GCT?GCT?GAG?GCA?TCT?AAA?AAG?GGA?780
Gln?Gln?Gln?Gln?Gly?Gln?Thr?Val?Thr?Lys?Lys?Ser?Ala?Ala?Glu?Ala?Ser?Lys?Lys?Gly
245 250 255 260
TCC?GAA?TAC?ATC?TCT?GAC?GCA?TTC?TCT?CTG?GAC?GTT?TCC?GAA?AAG?TCT?GGT?AAC?TTC?AAA?840
Ser?Glu?Tyr?Ile?Ser?Asp?Ala?Phe?Ser?Leu?Asp?Val?Ser?Glu?Lys?Ser?Gly?Asn?Phe?Lys
265 270 275 280
CAC?CTG?CGC?GAG?TTC?GTG?TTT?AAA?AAC?AAA?GAC?GGT?TTC?CTG?TAC?GTT?TAC?AAG?GGC?TAC?900
His?Leu?Arg?Glu?Phe?Val?Phe?Lys?Asn?Lys?Asp?Gly?Phe?Leu?Tyr?Val?Tyr?Lys?Gly?Tyr
285 290 295 300
CAG?CCG?ATC?GAC?GTA?GTT?CGT?GAC?CTG?CCG?TCT?GGT?TTT?AAC?ACT?CTG?AAA?CCG?ATC?TTC?960
Gln?Pro?Ile?Asp?Val?Val?Arg?Asp?Leu?Pro?Ser?Gly?Phe?Asn?Thr?Leu?Lys?Pro?Ile?Phe
305 310 315 320
AAG?CTG?CCG?CTG?GGT?ATT?AAC?ATC?ACT?AAC?TTC?CGC?GCT?ATC?CTG?ACT?GCT?TTC?TCT?CCG1020
Lys?Leu?Pro?Leu?Gly?Ile?Asn?Ile?Thr?Asn?Phe?Arg?Ala?Ile?Leu?Thr?Ala?Phe?Ser?Pro
325 330 335 340
GCT?CAG?GAC?ACT?TGG?GGC?ACT?TCT?GCT?GCA?GCC?TAC?TTC?GTT?GGC?TAC?CTG?AAG?CCA?ACT?1080
Ala?Gln?Asp?Thr?Trp?Gly?Thr?Ser?Ala?Ala?Ala?Tyr?Phe?Val?Gly?Tyr?Leu?Lys?Pro?Thr
345 350 355 360
ACC?TTT?ATG?CTG?AAG?TAC?GAC?GAA?AAC?GGT?ACT?ATC?ACT?GAT?GCT?GTT?GAC?TGC?TCT?CAG?1140
Thr?Phe?Met?Leu?Lys?Tyr?Asp?Glu?Asn?Gly?Thr?Ile?Thr?Asp?Ala?Val?Asp?Cys?Ser?Gln
365 370 375 380
AAC?CCA?CTG?GCT?GAA?CTG?AAA?TGC?TCT?GTT?AAG?AGC?TTT?GAG?ATC?GAC?AAA?GGT?ATT?TAC?1200
Asn?Pro?Leu?Ala?Glu?Leu?Lys?Cys?Ser?Val?Lys?Ser?Phe?Glu?Ile?Asp?Lys?Gly?Ile?Tyr
385 390 395 400
CAG?ACC?TCT?AAC?TTC?CGT?GTT?GTT?CCG?TCT?GGT?GAC?GTT?GTG?CGT?TTC?CCT?AAC?ATC?ACT?1260
Gln?Thr?Ser?Asn?Phe?Arg?Val?Val?Pro?Ser?Gly?Asp?Val?Val?Arg?Phe?Pro?Asn?Ile?Thr
405 410 415 420
AAC?CTG?TGC?CCG?TTT?GGT?GAA?GTT?TTC?AAC?GCT?ACT?AAA?TTC?CCT?TCT?GTC?TAC?GCA?TGG?1320
Asn?Leu?Cys?Pro?Phe?Gly?Glu?Val?Phe?Asn?Ala?Thr?Lys?Phe?Pro?Ser?Val?Tyr?Ala?Trp
425 430 435 440
GAG?CGT?AAA?AAA?ATT?TCT?AAC?TGC?GTT?GCT?GAT?TAC?TCT?GTG?CTG?TAC?AAC?TCT?ACC?TTT?1380
Glu?Arg?Lys?Lys?Ile?Ser?Asn?Cys?Val?Ala?Asp?Tyr?Ser?Val?Leu?Tyr?Asn?Ser?Thr?Phe
445 450 455 460
TTC?TCT?ACC?TTC?AAG?TGC?TAC?GGC?GTT?TCT?GCT?ACT?AAG?CTG?AAC?GAC?CTG?TGC?TTC?TCC?1440
Phe?Ser?Thr?Phe?Lys?Cys?Tyr?Gly?Val?Ser?Ala?Thr?Lys?Leu?Asn?Asp?Leu?Cys?Phe?Ser
465 470 475 480
AAC?GTT?TAC?GCA?GAT?TCT?TTC?GTA?GTT?AAG?GGT?GAT?GAC?GTA?CGT?CAG?ATC?GCT?CCA?GGT?1500
Asn?Val?Tyr?Ala?Asp?Ser?Phe?Val?Val?Lys?Gly?Asp?Asp?Val?Arg?Gln?Ile?Ala?Pro?Gly
485 490 495 500
CAG?ACT?GGT?GTT?ATC?GCT?GAC?TAC?AAC?TAT?AAA?CTG?CCG?GAC?GAT?TTC?ATG?GGT?TGC?GTT?1560
Gln?Thr?Gly?Val?Ile?Ala?Asp?Tyr?Asn?Tyr?Lys?Leu?Pro?Asp?Asp?Phe?Met?Gly?Cys?Val
505 510 515 520
CTG?GCT?TGG?AAC?ACT?CGT?AAC?ATT?GAC?GCT?ACT?TCT?ACT?GGT?AAC?TAC?AAC?TAC?AAA?TAT?1620
Leu?Ala?Trp?Asn?Thr?Arg?Asn?Ile?Asp?Ala?Thr?Ser?Thr?Gly?Asn?Tyr?Asn?Tyr?Lys?Tyr
525 530 535 540
CGT?TAC?CTG?CGT?CAC?GGC?AAA?CTG?CGT?CCG?TTC?GAA?CGT?GAC?ATC?TCT?AAC?GTG?CCG?TTC?1680
Arg?Tyr?Leu?Arg?His?Gly?Lys?Leu?Arg?Pro?Phe?Glu?Arg?Asp?Ile?Ser?Asn?Val?Pro?Phe
545 550 555 560
TAA 1683

Claims (2)

1. the fusion rotein of SARS virus S protein fragments and N protein fragments, it is the fusion rotein of proteic the 1st amino acid to 258 amino acid fragment of N of proteic 460 amino acid fragments of the 162nd amino acid to the of S of SARS virus and SARS virus, the N protein fragments is at the N of fusion rotein end, the S protein fragments is at the C of fusion rotein end, be connected with Serine with glycine between two protein fragments, behind first amino acid methionine of N protein fragments, insert a glycine, 560 amino acid of fusion rotein total length, aminoacid sequence is as follows:
Met?Gly?Ser?Asp?Asn?Gly?Pro?Gln?Ser?Asn?Gln?Arg?Ser?Ala?Pro?Arg
1 5 10 15
Ile?Thr?Phe?Gly?Gly?Pro?Thr?Asp?Ser?Thr?Asp?Asn?Asn?Gln?Asn?Gly
20 25 30
Gly?Arg?Asn?Gly?Ala?Arg?Pro?Lys?Gln?Arg?Arg?Pro?Gln?Gly?Leu?Pro
35 40 45
Asn?Asn?Thr?Ala?Ser?Trp?Phe?Thr?Ala?Leu?Thr?Gln?His?Gly?Lys?Glu
50 55 60
Glu?Leu?Arg?Phe?Pro?Arg?Gly?Gln?Gly?Val?Pro?Ile?Asn?Thr?Asn?Ser
65 70 75 80
Gly?Pro?Asp?Asp?Gln?Ile?Gly?Tyr?Tyr?Arg?Arg?Ala?Thr?Arg?Arg?Val
85 90 95
Arg?Gly?Gly?Asp?Gly?Lys?Met?Lys?Glu?Leu?Ser?Pro?Arg?Trp?Tyr?Phe
100 105 110
Tyr?Tyr?Leu?Gly?Thr?Gly?Pro?Glu?Ala?Ser?Leu?Pro?Tyr?Gly?Ala?Asn
115 120 125
Lys?Glu?Gly?Ile?Val?Trp?Val?Ala?Thr?Glu?Gly?Ala?Leu?Asn?Thr?Pro
130 135 140
Lys?Asp?His?Ile?Gly?Thr?Arg?Asn?Pro?Asn?Asn?Asn?Ala?Ala?Thr?Val
145 150 155 160
Leu?Gln?Leu?Pro?Gln?Gly?Thr?Thr?Leu?Pro?Lys?Gly?Phe?Tyr?Ala?Glu
165 170 175
Gly?Ser?Arg?Gly?Gly?Ser?Gln?Ala?Ser?Ser?Arg?Ser?Ser?Ser?Arg?Ser
180 185 190
Arg?Gly?Asn?Ser?Arg?Asn?Ser?Thr?Pro?Gly?Ser?Ser?Arg?Gly?Asn?Ser
195 200 205
Pro?Ala?Arg?Met?Ala?Ser?Gly?Gly?Gly?Glu?Thr?Ala?Leu?Ala?Leu?Leu
210 215 220
Leu?Leu?Asp?Arg?Leu?Asn?Gln?Leu?Glu?Ser?Lys?Val?Ser?Gly?Lys?Gly
225 230 235 240
Gln?Gln?Gln?Gln?Gly?Gln?Thr?Val?Thr?Lys?Lys?Ser?Ala?Ala?Glu?Ala
245 250 255
Ser?Lys?Lys?Gly?Ser?Glu?Tyr?Ile?Ser?Asp?Ala?Phe?Ser?Leu?Asp?Val
260 265 270
Ser?Glu?Lys?Ser?Gly?Asn?Phe?Lys?His?Leu?Arg?Glu?Phe?Val?Phe?Lys
275 280 285
Asn?Lys?Asp?Gly?Phe?Leu?Tyr?Val?Tyr?Lys?Gly?Tyr?Gln?Pro?Ile?Asp
290 295 300
Val?Val?Arg?Asp?Leu?Pro?Ser?Gly?Phe?Asn?Thr?Leu?Lys?Pro?Ile?Phe
305 310 315 320
Lys?Leu?Pro?Leu?Gly?Ile?Asn?Ile?Thr?Asn?Phe?Arg?Ala?Ile?Leu?Thr
325 330 335
Ala?Phe?Ser?Pro?Ala?Gln?Asp?Thr?Trp?Gly?Thr?Ser?Ala?Ala?Ala?Tyr
340 345 350
Phe?Val?Gly?Tyr?Leu?Lys?Pro?Thr?Thr?Phe?Met?Leu?Lys?Tyr?Asp?Glu
355 360 365
Asn?Gly?Thr?Ile?Thr?Asp?Ala?Val?Asp?Cys?Ser?Gln?Asn?Pro?Leu?Ala
370 375 380
Glu?Leu?Lys?Cys?Ser?Val?Lys?Ser?Phe?Glu?Ile?Asp?Lys?Gly?Ile?Tyr
385 390 395 400
Gln?Thr?Ser?Asn?Phe?Arg?Val?Val?Pro?Ser?Gly?Asp?Val?Val?Arg?Phe
405 410 415
Pro?Asn?Ile?Thr?Asn?Leu?Cys?Pro?Phe?Gly?Glu?Val?Phe?Asn?Ala?Thr
420 425 430
Lys?Phe?Pro?Ser?Val?Tyr?Ala?Trp?Glu?Arg?Lys?Lys?Ile?Ser?Asn?Cys
435 440 445
Val?Ala?Asp?Tyr?Ser?Val?Leu?Tyr?Asn?Ser?Thr?Phe?Phe?Ser?Thr?Phe
450 455 460
Lys?Cys?Tyr?Gly?Val?Ser?Ala?Thr?Lys?Leu?Asn?Asp?Leu?Cys?Phe?Ser
465 470 475 480
Asn?Val?Tyr?Ala?Asp?Ser?Phe?Val?Val?Lys?Gly?Asp?Asp?Val?Arg?Gln
485 490 495
Ile?Ala?Pro?Gly?Gln?Thr?Gly?Val?Ile?Ala?Asp?Tyr?Asn?Tyr?Lys?Leu
500 505 510
Pro?Asp?Asp?Phe?Met?Gly?Cys?Val?Leu?Ala?Trp?Asn?Thr?Arg?Asn?Ile
515 520 525
Asp?Ala?Thr?Ser?Thr?Gly?Asn?Tyr?Asn?Tyr?Lys?Tyr?Arg?Tyr?Leu?Arg
530 535 540
His?Gly?Lys?Leu?Arg?Pro?Phe?Glu?Arg?Asp?Ile?Ser?Asn?Val?Pro?Phe。
2. the fusion rotein of described SARS virus S albumen of claim 1 and N protein fragments is used for the method for SARS virus antibody or detection of antigens.
CNB031321623A 2003-07-03 2003-07-03 SARS virus S protein and N protein fusion protein, and preparation and use thereof Expired - Fee Related CN100386343C (en)

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