CN101173290A

CN101173290A - Chemically synthesized HSV1 virus gB glucoprotein extracellular region gene fragment, representation and application of the same

Info

Publication number: CN101173290A
Application number: CNA2007101338941A
Authority: CN
Inventors: 李越希; 吕敏; 潘明洁; 杨华凤; 戚菁; 王正茂
Original assignee: Individual
Current assignee: Individual
Priority date: 2007-10-24
Filing date: 2007-10-24
Publication date: 2008-05-07
Anticipated expiration: 2027-10-24
Also published as: CN101173290B

Abstract

The invention discloses the gene fragment of the extracellular region in the virus HSV1 gB glycoprotein of chemosynthesis, expression and application, belonging to the technical field of genetic engineering, vaccine and diagnostic reagent. Through computer analysis, the invention screens out strong epitope, from the first amino acid to the 696th amino acid, 696 amino acids in all, chooses codon favored both by eukaryote and prokaryote, synthesizes chemically a brand new gene sequence of the epitope, and utilizes the genetic engineering technology to express the gene fragment and prepare the strong epitope of the virus HSV1 gB glycoprotein. The strong epitope of the virus HSV1 gB glycoprotein expressed can be used for the vaccine, the virus HSV1 antigen and antibody detection, immunization preparation of mono-and poly-clonal antibodies of anti-HSV1.

Description

The HSV1 virus gB glucoprotein extracellular region gene fragment of chemosynthesis and expression thereof, application

Technical field

That the present invention relates to is a kind of herpes simplex virus type 1 (Herpes simplex virus 1 of chemosynthesis, HSV1) Glycoprotein B (glycoproteinB, gB glycoprotein) the brand-new gene fragment of extracellular region is utilized genetic engineering technique, preparation reorganization HSV1 virus gB glycoprotein.By Computer Analysis, filter out the gB glucoprotein extracellular region fragment of the HSV1 virus that contains the strong antigen epi-position, the codon of selecting eucaryon and prokaryotic organism all to have a preference for, the gene order that chemosynthesis is brand-new, utilize genetic engineering technique to express, expressed proteins can be used for vaccine and HSV1 antiviral antibody or detection of antigens etc., the present invention relates to genetic engineering technique, vaccine and diagnostic reagent field.

Background technology

(Herpes simplex virus is the common disease substance of harm humans health HSV) to hsv, causes recurrent infection and latent infection easily, and is serious to human body harm, is divided into HSV-1 and HSV-2 amphitypy.Herpes simplex types 1 virus mainly causes the infection of actinal surface portion, ocular infection and herpes simplex encephalitis etc.; And 2 types mainly cause genital infection, and closely related with the generation of women's cervical cancer.In recent years the infection of HSV1 significantly increases, and accounts for 10%～40% of this disease.Pharmacological agent HSV infects and occurs corresponding resistance often at present, is practicable effective ways so develop vaccine, and it can make body in anti-HSV infection immunity, and performance humoral immunization and cellular immune function are eliminated HSV and infected.So far developed multiple HSV vaccine; existing two kinds of HSV glucoprotein vaccines enter the III clinical trial phase; it is the vaccine that the reorganization gD2 glycoprotein developed of the vaccine that forms of reorganization gD2/gB2 glycoprotein and the MF59 adjuvant compatibility of Chiron company development and Glaxo SmithKline (GSK) company and another adjuvant (3-de-O-acidylate monophosphoryl lipid A and alum) compatibility form; these two kinds of vaccines all have the certain protection effect, but clinical effectiveness is limited.Domestic at present to the research of HSV vaccine, mainly concentrate on the exploratory development aspect of DNA nucleic acid vaccine.

The HSV envelope glycoprotein has critical role in absorption, invasion and the stimulation body generation immunne response and the vaccine development of virus.The HSV envelope glycoprotein of definite designation at present has 12 kinds, wherein gB glycoprotein content in the cell that infects is maximum, be the conservative albumen of simplexvirus family camber, each strain differences is less, and bigger homology is arranged with all herpes-like virus subgroups, gB albumen has stronger immunogenicity simultaneously, can induce body to produce neutralizing antibody and cell immune response, so HSV1gB glycoprotein is to make up HSV vaccine ideal goal gene.

HSV1gB glycoprotein gene total length 2712bp, the signal peptide of 29 aminoacid sequences of coding, the extracellular region of 696 aminoacid sequences, the intracellular region of striding film district, 109 aminoacid sequences of 69 aminoacid sequences.Remove the part signal peptide sequence and do not influence its secreting, expressing in prokaryotic cell prokaryocyte.Studies show that,, can remove its partial function encoding sequence, the host is had under the situation of toxicity or immunosuppressive action at complete antigen protein especially, antigen protein is carried out shorten expression just be even more important for strengthening the proteic immunogenicity of coding for antigens.And the gene constructed vaccine of the gB after the brachymemma, with virus attack has identical provide protection to HSV-1 with the gene constructed vaccine of complete gB.The present enzyme-linked immunologic detecting kit of the HSV1 antiviral antibody of using, the antigen of its use is totivirus antigen, has that production is dangerous, cost is high, with other virus shortcomings such as cross reaction are arranged.Lack the HSV1 virus vaccines at present.Inactivated Vaccine has obtained bigger progress, but production cost height, dangerous big.

Summary of the invention

The present invention seeks to provide a kind of brand-new gene fragment of HSV1 virus gB glucoprotein extracellular region of chemosynthesis, utilize genetic engineering technique, preparation reorganization HSV1 virus gB glucoprotein extracellular region fragment at above-mentioned weak point.By Computer Analysis, filter out the HSV1 virus gB glucoprotein extracellular region fragment that contains the strong antigen epi-position, 696 amino acid of the 1st amino acid-Di, totally 696 amino acid, the codon of selecting eucaryon and prokaryotic organism all to have a preference for, the gene order that chemosynthesis is brand-new utilizes genetic engineering technique to express this gene.Expressed proteins can be used for vaccine, HSV1 antiviral antibody or detection of antigens and is used for the anti-HSV1 virus monoclonal antibody of immunity preparation and how anti-etc.

The HSV1 virus gB glucoprotein extracellular region gene fragment of chemosynthesis and expression thereof, application take following scheme to realize:

1.HSV1 the screening of viral gB glucoprotein extracellular region epitope and the chemosynthesis of gene fragment thereof:

Utilize softwares such as ANTHEWIN, by the aminoacid sequence of Computer Analysis HSV1 virus gB glycoprotein, the N end (the 1st amino acid-the 696th amino acid) that filters out gB glycoprotein contains stronger antigenic determinant.The codon of selecting eucaryon and prokaryotic organism all to have a preference for; the gene order that chemosynthesis is brand-new; and BamHI restriction enzyme site (following setting-out part) and two protection bases (GC) have been increased at 5 ' end; increased terminator codon (TGA) and EcoRI restriction enzyme site (following setting-out part) and two protection bases (GC) at 3 ' end, made this gene fragment be easy to be cloned in plasmid the pGEX4T-2 interior BamHI and EcoRI restriction enzyme site.

Epitope aminoacid sequence (the 696th aa of the 1st aa-) in the HSV1 virus gB glycoprotein of screening:

Pro Ser Ser Pro Gly Thr Pro Gly Val Ala Ala Ala Thr Gln Ala Ala Asn Gly

Gly Pro Ala Thr Pro Ala Pro Pro Ala Leu Gly Ala Ala Pro Thr Gly Asp Pro

Lys Pro Lys Lys Asn Lys Lys Pro Lys Asn Pro Thr Pro Pro Arg Pro Ala Gly

Asp Asn Ala Thr Val Ala Ala Gly His Ala Thr Leu Arg Glu His Leu Arg Asp

Ile Lys Ala Glu Asn Thr Asp Ala Asn Phe Tyr Val Cys Pro Pro Pro Thr Gly

Ala Thr Val Val Gln Phe Glu Gln Pro Arg Arg Cys Pro Thr Arg Pro Glu Gly

Gln Asn Tyr Thr Glu Gly Ile Ala Val Val Phe Lys Glu Asn Ile Ala Pro Tyr

Lys Phe Lys Ala Thr Met Tyr Tyr Lys Asp Val Thr Val Ser Gln Val Trp Phe

Gly His Arg Tyr Ser Gln Phe Met Gly Ile Phe Glu Asp Arg Ala Pro Val Pro

Phe Glu Glu Val Ile Asp Lys Ile Asn Ala Lys Gly Val Cys Arg Ser Thr Ala

Lys Tyr Val Arg Asn Asn Leu Glu Thr Thr Ala Phe His Arg Asp Asp His Glu

Thr Asp Met Glu Leu Lys Pro Ala Asn Ala Ala Thr Arg Thr Ser Arg Gly Trp

His Thr Thr Asp Leu Lys Tyr Asn Pro Ser Arg Val Glu Ala Phe His Arg Tyr

Gly Thr Thr Val Asn Cys Ile Val Glu Glu Val Asp Ala Arg Ser Val Tyr Pro

Tyr Asp Glu Phe Val Leu Ala Thr Gly Asp Phe Val Tyr Met Ser Pro Phe Tyr

Gly Tyr Arg Glu Gly Ser His Thr Glu His Thr Ser Tyr Ala Ala Asp Arg Phe

Lys Gln Val Asp Gly Phe Tyr Ala Arg Asp Leu Thr Thr Lys Ala Arg Ala Thr

Ala Pro Thr Thr Arg Asn Leu Leu Thr Thr Pro Lys Phe Thr Val Ala Trp Asp

Trp Val Pro Lys Arg Pro Ser Val Cys Thr Met Thr Lys Trp Gln Glu Val Asp

Glu Met Leu Arg Ser Glu Tyr Gly Gly Ser Phe Arg Phe Ser Ser Asp Ala Ile

Ser Thr Thr Phe Thr Thr Asn Leu Thr Glu Tyr Pro Leu Ser Arg Val Asp Leu

Gly Asp Cys Ile Gly Lys Asp Ala Arg Asp Ala Met Asp Arg Ile Phe Ala Arg

Arg Tyr Asn Ala Thr His Ile Lys Val Gly Gln Pro Gln Tyr Tyr Leu Ala Asn

Gly Gly Phe Leu Ile Ala Tyr Gln Pro Leu Leu Ser Asn Thr Leu Ala Glu Leu

Tyr Val Arg Glu His Leu Arg Glu Gln Ser Pro Lys Pro Pro Asn Pro Thr Pro

Pro Pro Pro Gly Ala Ser Ala Asn Ala Ser Val Glu Arg Ile Lys Thr Thr Ser

Ser Ile Glu Phe Ala Arg Leu Gln Phe Thr Tyr Asn His Ile Gln Asn His Val

Asn Asp Met Leu Gly Arg Val Ala Ile Ala Trp Cys Glu Leu Gln Asn His Glu

Leu Thr Leu Trp Asn Glu Ala Arg Lys Leu Asn Pro Asn Ala Ile Ala Ser Ala

Thr Val Gly Arg Arg Val Ser Ala Arg Met Leu Gly Asp Val Met Ala Val Ser

Thr Cys Val Pro Val Ala Ala Asp Asn Val Ile Val Gln Asn Ser Met Arg Ile

Ser Ser Arg Pro Gly Ala Cys Tyr Ser Arg Pro Leu Val Ser Phe Arg Tyr Glu

Asp Gln Gly Pro Leu Val Glu Gly Gln Leu Gly Glu Asn Asn Glu Leu Arg Leu

Thr Arg Asp Ala Ile Glu Pro Cys Thr Val Gly His Arg Arg Tyr Phe Thr Phe

Gly Gly GlY Tyr Val Tyr Phe Glu Glu Ser Ala Tyr Ser His Gln Leu Ser Arg

Ala Asp Ile Thr Thr Val Ser Thr Phe Ile Asp Leu Asn Ile Thr Met Leu Glu

Asp His Glu Phe Val Pro Leu Glu Val Tyr Thr Arg His Glu Ile Lys Asp Ser

Gly Leu Leu Asp Tyr Thr Glu Val Gln Arg Arg Asn Gln Leu His Asp Leu Arg

Phe Ala Asp Ile Asp Thr Val Ile His Ala Asp Ala

The dna sequence dna (2107bp) that contains HSV1 virus gB glucoprotein extracellular region antigen epitope genes of chemosynthesis:

GC GGATCC CCT ACC TCT CCT GGT ACT CCT GGT GTG GCT GCC GCT ACC CAG GCC GCT AAC

GGT GGT CCT GCC ACT CCT GCT CCT CCT CCT CTG GGT GCC GCT CCT ACT GGT GAC CCT

AAG CCT AAG AAG AAC AAG AAG CCT AAG AAC CCT ACT CCT CCT AGG CCT GCT GGT GAC

AAC GCC ACC GTG GCC GCT GGC CAC GCC ACT CTG AGA GAG CAC CTG AGA GAC ATC AAG

GCT GAG AAC ACC GAC GCT AAC TTC TAC GTG TGT CCT CCT CCT ACT GGT GCC ACC GTG

GTG CAG TTC GAG CAG CCT CGC AGG TGC CCT ACC AGG CCT GAA GGT CAG AAC TAC ACC

GAA GGC ATC GCC GTG GTG TTC AAG GAG AAC ATC GCT CCT TAC AAG TTC AAG GCC ACC

ATG TAC TAC AAG GAC GTG ACC GTG AGC CAG GTG TGG TTC GGC CAC CGC TAC TCC CAG

TTC ATG GGC ATC TTC GAG GAC CGC GCT CCT GTG CCT TTC GAG GAG GTG ATC GAC AAG

ATC AAC GCC AAG GGT GTG TGT AGG TCC ACC GCT AAG TAC GTG CGC AAC AAC CTG GAG

ACC ACT GCT TTC CAC AGA GAC GAT CAC GAG ACC GAC ATG GAG CTG AAG CCT GCC AAC

GCC GCT ACT CGC ACC AGC AGA GGC TGG CAC ACC ACT GAC CTG AAG TAC AAC CCT AGC

AGA GTG GAG GCC TTC CAC AGG TAC GGC ACC ACC GTG AAC TGC ATC GTG GAG GAG GTG

GAC GCT CGC AGC GTG TAC CCT TAC GAC GAG TTC GTG CTG GCC ACT GGT GAC TTC GTG

TAC ATG TCT CCT TTC TAC GGC TAC AGA GAA GGT AGC CAC ACC GAG CAC ACT ACC TAC

GCT GCT GAC AGG TTC AAG CAG GTG GAC GGC TTC TAC GCT CGC GAC CTG ACC ACC AAG

GCT AGA GCC ACT GCT CCT ACC ACT AGG AAC CTG CTG ACC ACT CCT AAG TTC ACC GTG

GCT TGG GAC TGG GTG CCT AAG CGC CCT AGC GTG TGC ACC ATG ACC AAG TGG CAG GAG

GTG GAC GAG ATG CTG CGC TCC GAG TAC GGC GGT TCC TTC AGG TTC TCC TCT GAC GCT

ATC TCC ACT ACC TTC ACT ACC AAC CTG ACC GAG TAC CCT CTG TCC AGA GTG GAC CTG

GGT GAC TGC ATC GGT AAG GAC GCT CGC GAC GCC ATG GAC CGC ATC TTC GCT CGC AGG

TAC AAC GCT ACT CAC ATC AAG GTG GGC CAG CCT CAG TAC TAC CAG GCC AAC GGT GGT

TTC CTG ATC GCC TAC CAG CCT CTG CTG AGC AAC ACT CTG GCT GAG CTG TAC GTG AGA

GAG CAC CTG AGA GAG CAG AGC CGC AAG CCT CCT AAC CCT ACG CCT CCT CCT CCC GGT

GCT AGC GCC AAC GCT TCC GTG GAG CGC ATC AAG ACT ACC TCT AGC ATC GAG TTC GCC

AGG CTG CAG TTC ACC TAC AAC CAC ATC CAG CGC CAC GTG AAC GAC ATG CTG GGT CGC

GTG GCT ATC GCT TGG TGC GAG CTG CAG AAC CAC GAG CTG ACT CTG TGG AAC GAG GCT

CGC AAG CTG AAC CCT AAC GCT ATC GCC AGC GTG ACC GTG GGC AGG AGA GTG AGC GCT

AGA ATG CTG GGC GAC GTG ATG GCC GTG TCC ACC TGC GTG CCT GTG GCT GCT GAC AAC

GTG ATC GTG CAG AAC AGC ATG CGC ATC AGC TCC AGA CCT GGT GCC TGC TAC AGC AGA

CCT CTG GTG AGC TTC AGG TAC GAG GAC CAA GGT CCT CTG GTG GAA GGT CAG CTG GGT

GAG AAC AAC GAG CTG AGG CTG ACT CGC GAC GCT ATC GAG CCT TGC ACC GTC GGT CAC

AGA CGC TAC TTC ACC TTC GGT GGC GGT TAC GTG TAC TTC GAG GAG TAC GCT TAC TCT

CAC CAG CTG AGC CGC GCT GAC ATC ACT ACC GTG AGC ACC TTC ATC GAC CTG AAC ATC

ACC ATG CTG GAG GAC CAC GAG TTC GTG CCT CTG GAG GTG TAC ACC CGC CAC GAG ATC

AAG GAC AGC GGC CTG CTG GAC TAC ACC GAG GTG CAG CGC CGC AAC CAG CTG CAC GAC

CTG CGC TTC GCT GAC ATC GAC ACC GTG ATC CAC GCC GAC GCC TAA GAATTCGC

2. express HSV1 virus gB glucoprotein extracellular region fragment construction of recombinant plasmid:

Extract plasmid pGEX4T-2,, reclaim the big fragment of plasmid that enzyme is cut behind the electrophoresis, be dissolved in the deionized water with Bam H I and EcoRI double digestion.Same HSV1 virus gB glycoprotein gene fragment with BamHI and the chemosynthesis of EcoR I double digestion, electrophoresis is dissolved in the deionized water after reclaiming.

Above-mentioned two 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, HSV1 virus gB glycoprotein gene fragment is inserted between carrier pGEX4T-2 interior the BamH I and EcoR I site, consistent with the initiator codon translation framework on the carrier, express a fusion rotein.

3. the screening of recombinant plasmid and evaluation:

With the recombinant plasmid transformed e. coli tg1, coating contains penbritin (100 μ g/ml) LB flat board, puts 37 ℃ and spends the night.Next day, picking transformed bacterium colony and 1 contrast bacterium (plasmid pGEX4T-2 transformed bacteria) at random, extract plasmid respectively, plasmid with extraction is a template, pcr amplification HSV1 virus gB glucoprotein extracellular region gene fragment, contain the positive recombinant plasmid of HSV1 virus gB glucoprotein extracellular region gene fragment, should amplify the gene fragment that is about 2107bp.The plasmid that will contain foreign gene carries out dna sequence analysis, and sequential analysis confirms that recombinant plasmid contains synthetic HSV1 virus gB glycoprotein gene fragment, and sequence is entirely true:

CCT ACC TCT CCT GGT ACT CCT GGT GTG GCT GCC GCT ACC CAG GCC GCT AAC GGT GGT

CCT GCC ACT CCT GCT CCT CCT CCT CTG GGT GCC GCT CCT ACT GGT GAC CCT AAG CCT

AAG AAG AAC AAG AAG CCT AAG AAC CCT ACT CCT CCT AGG CCT GCT GGT GAC AAC GCC

ACC GTG GCC GCT GGC CAC GCC ACT CTG AGA GAG CAC CTG AGA GAC ATC AAG GCT GAG

AAC ACC GAC GCT AAC TTC TAC GTG TGT CCT CCT CCT ACT GGT GCC ACC GTG GTG CAG

TTC GAG CAG CCT CGC AGG TGC CCT ACC AGG CCT GAA GGT CAG AAC TAC ACC GAA GGC

ATC GCC GTG GTG TTC AAG GAG AAC ATC GCT CCT TAC AAG TTC AAG GCC ACC ATG TAC

TAC AAG GAC GTG ACC GTG AGC CAG GTG TGG TTC GGC CAC CGC TAC TCC CAG TTC ATG

GGC ATC TTC GAG GAC CGC GCT CCT GTG CCT TTC GAG GAG GTG ATC GAC AAG ATC AAC

GCC AAG GGT GTG TGT AGG TCC ACC GCT AAG TAC GTG CGC AAC AAC CTG GAG ACC ACT

GCT TTC CAC AGA GAC GAT CAC GAG ACC GAC ATG GAG CTG AAG CCT GCC AAC GCC GCT

ACT CGC ACC AGC AGA GGC TGG CAC ACC ACT GAC CTG AAG TAC AAC CCT AGC AGA GTG

GAG GCC TTC CAC AGG TAC GGC ACC ACC GTG AAC TGC ATC GTG GAG GAG GTG GAC GCT

CGC AGC GTG TAC CCT TAC GAC GAG TTC GTG CTG GCC ACT GGT GAC TTC GTG TAC ATG

TCT CCT TTC TAC GGC TAC AGA GAA GGT AGC CAC ACC GAG CAC ACT ACC TAC GCT GCT

GAC AGG TTC AAG CAG GTG GAC GGC TTC TAC GCT CGC GAC CTG ACC ACC AAG GCT AGA

GCC ACT GCT CCT ACC ACT AGG AAC CTG CTG ACC ACT CCT AAG TTC ACC GTG GCT TGG

GAC TGG GTG CCT AAG CGC CCT AGC GTG TGC ACC ATG ACC AAG TGG CAG GAG GTG GAC

GAG ATG CTG CGC TCC GAG TAC GGC GGT TCC TTC AGG TTC TCC TCT GAC GCT ATC TCC

ACT ACC TTC ACT ACC AAC CTG ACC GAG TAC CCT CTG TCC AGA GTG GAC CTG GGT GAC

TGC ATC GGT AAG GAC GCT CGC GAC GCC ATG GAC CGC ATC TTC GCT CGC AGG TAC AAC

GCT ACT CAC ATC AAG GTG GGC CAG CCT CAG TAC TAC CAG GCC AAC GGT GGT TTC CTG

ATC GCC TAC CAG CCT CTG CTG AGC AAC ACT CTG GCT GAG CTG TAC GTG AGA GAG CAC

CTG AGA GAG CAG AGC CGC AAG CCT CCT AAC CCT ACG CCT CCT CCT CCC GGT GCT AGC

GCC AAC GCT TCC GTG GAG CGC ATC AAG ACT ACC TCT AGC ATC GAG TTC GCC AGG CTG

CAG TTC ACC TAC AAC CAC ATC CAG CGC CAC GTG AAC GAC ATG CTG GGT CGC GTG GCT

ATC GCT TGG TGC GAG CTG CAG AAC CAC GAG CTG ACT CTG TGG AAC GAG GCT CGC AAG

CTG AAC CCT AAC GCT ATC GCC AGC GTG ACC GTG GGC AGG AGA GTG AGC GCT AGA ATG

CTG GGC GAC GTG ATG GCC GTG TCC ACC TGC GTG CCT GTG GCT GCT GAC AAC GTG ATC

GTG CAG AAC AGC ATG CGC ATC AGC TCC AGA CCT GGT GCC TGC TAC AGC AGA CCT CTG

GTG AGC TTC AGG TAC GAG GAC CAA GGT CCT CTG GTG GAA GGT CAG CTG GGT GAG AAC

AAC GAG CTG AGG CTG ACT CGC GAC GCT ATC GAG CCT TGC ACC GTC GGT CAC AGA CGC

TAC TTC ACC TTC GGT GGC GGT TAC GTG TAC TTC GAG GAG TAC GCT TAC TCT CAC CAG

CTG AGC CGC GCT GAC ATC ACT ACC GTG AGC ACC TTC ATC GAC CTG AAC ATC ACC ATG

CTG GAG GAC CAC GAG TTC GTG CCT CTG GAG GTG TAC ACC CGC CAC GAG ATC AAG GAC

AGC GGC CTG CTG GAC TAC ACC GAG GTG CAG CGC CGC AAC CAG CTG CAC GAC CTG CGC

TTC GCT GAC ATC GAC ACC GTG ATC CAC GCC GAC GCC TGA

The viral gB glucoprotein extracellular region fragment (696 amino acid) of the expression of recombinant plasmid HSV1 that makes up has merged 238 amino acid on the carrier at its N end, 934 amino acid of total length, and its aminoacid sequence is as follows:

Met Pro Met Ile Leu Gly Tyr Trp Asp Ile Arg Gly Leu Ala His Ala Ile Arg

Leu Leu Leu Glu Tyr Thr Asp Ser Ser Tyr Glu Glu Lys Lys Tyr Thr Met Gly

Gly Ala Pro Asp Tyr Asp Arg Ser Gln Trp Leu Asn Glu Lys Phe Lys Leu Gly

Leu Asp Phe Pro Asn Leu Pro Tyr Leu Ile Asp Gly Ala His Lys Ile Thr Gln

Ser Asn Ala Ile Leu Cys Tyr Ile Ala Arg Lys His Asn Leu Cys Gly Glu Thr

Glu Glu Glu Lys Ile Arg Val Asp Ile Leu Glu Asn Gln Ala Met Asp Val Ser

Asn Gln Leu Ala Arg Val Cys Tyr Ser Pro Asp Phe Glu Lys Leu Lys Pro Glu

Tyr Leu Glu Glu Leu Pro Thr Met Met Gln His Phe Ser Gln Phe Leu Gly Lys

Arg Pro Trp Phe Val Gly Asp Lys Ile Thr Phe Val Asp Phe Leu Ala Tyr Asp

Val Leu Asp Leu His Arg Ile Phe Glu Pro Asn Cys Leu Asp Ala Phe Pro Asn

Leu Lys Asp Phe Ile Ser Arg Phe Glu Gly Leu Glu Lys Ile Ser Ala Tyr Met

Lys Ser Ser Arg Phe Leu Pro Lys Pro Leu Tyr Thr Arg Met Ala Val Trp Gly

Asn Lys Pro Ser Ser Pro Gly Thr Pro Gly Val Ala Ala Ala Thr Gln Ala Ala

Asn Gly Gly Pro Ala Thr Pro Ala Pro Pro Ala Leu Gly Ala Ala Pro Thr Gly

Asp Pro Lys Pro Lys Lys Asn Lys Lys Pro Lys Asn Pro Thr Pro Pro Arg Pro

Ala Gly Asp Asn Ala Thr Val Ala Ala Gly His Ala Thr Leu Arg Glu His Leu

Arg Asp Ile Lys Ala Glu Asn Thr Asp Ala Asn Phe Tyr Val Cys Pro Pro Pro

Thr Gly Ala Thr Val Val Gln Phe Glu Gln Pro Arg Arg Cys Pro Thr Arg Pro

Glu Gly Gln Asn Tyr Thr Glu Gly Ile Ala Val Val Phe Lys Glu Asn Ile Ala

Pro Tyr Lys Phe Lys Ala Thr Met Tyr Tyr Lys Asp Val Thr Val Ser Gln Val

Trp Phe Gly His Arg Tyr Ser Gln Phe Met Gly Ile Phe Glu Asp Arg Ala Pro

Val Pro Phe Glu Glu Val Ile Asp Lys Ile Asn Ala Lys Gly Val Cys Arg Ser

Thr Ala Lys Tyr Val Arg Asn Asn Leu Glu Thr Thr Ala Phe His Arg Asp Asp

His Glu Thr Asp Met Glu Leu Lys Pro Ala Asn Ala Ala Thr Arg Thr Ser Arg

Gly Trp His Thr Thr Asp Leu Lys Tyr Asn Pro Ser Arg Val Glu Ala Phe His

Arg Tyr Gly Thr Thr Val Asn Cys Ile Val Glu Glu Val Asp Ala Arg Ser Val

Tyr Pro Tyr Asp Glu Phe Val Leu Ala Thr GlY Asp Phe Val Tyr Met Ser Pro

Phe Tyr Gly Tyr Arg Glu Gly Ser His Thr Glu His Thr Ser Tyr Ala Ala Asp

Arg Phe Lys Gln Val Asp Gly Phe Tyr Ala Arg Asp Leu Thr Thr Lys Ala Arg

Ala Thr Ala Pro Thr Thr Arg Asn Leu Leu Thr Thr Pro Lys Phe Thr Val Ala

Trp Asp Trp Val Pro Lys Arg Pro Ser Val Cys Thr Met Thr Lys Trp Gln Glu

Val Asp Glu Met Leu Arg Ser Glu Tyr Gly Gly Ser Phe Arg Phe Ser Ser Asp

Ala Ile Ser Thr Thr Phe Thr Thr Asn Leu Thr Glu Tyr Pro Leu Ser Arg Val

Asp Leu Gly Asp Cys Ile Gly Lys Asp Ala Arg Asp Ala Met Asp Arg Ile Phe

Ala Arg Arg Tyr Asn Ala Thr His Ile Lys Val Gly Gln Pro Gln Tyr Tyr Leu

Ala Asn Gly Gly Phe Leu Ile Ala Tyr Gln Pro Leu Leu Ser Asn Thr Leu Ala

Glu Leu Tyr Val Arg Glu His Leu Arg Glu Gln Ser Pro Lys Pro Pro Asn Pro

Thr Pro Pro Pro Pro Gly Ala Ser Ala Asn Ala Ser Val Glu Arg Ile Lys Thr

Thr Ser Ser Ile Glu Phe Ala Arg Leu Gln Phe Thr Tyr Asn His Ile Gln Asn

His Val Asn Asp Met Leu Gly Arg Val Ala Ile Ala Trp Cys Glu Leu Gln Asn

His Glu Leu Thr Leu Trp Asn Glu Ala Arg Lys Leu Asn Pro Asn Ala Ile Ala

Ser Ala Thr Val Gly Arg Arg Val Ser Ala Arg Met Leu Gly Asp Val Met Ala

Val Ser Thr Cys Val Pro Val Ala Ala Asp Asn Val Ile Val Gln Asn Ser Met

Arg Ile Ser Ser Arg Pro Gly Ala Cys Tyr Ser Arg Pro Leu Val Ser Phe Arg

Tyr Glu Asp Gln Gly Pro Leu Val Glu Gly Gln Leu Gly Glu Asn Asn Glu Leu

Arg Leu Thr Arg Asp Ala Ile Glu Pro Cys Thr Val GlY His Arg Arg Tyr Phe

Thr Phe Gly Gly Gly Tyr Val Tyr Phe Glu Glu Ser Ala Tyr Ser His Gln Leu

Ser Arg Ala Asp Ile Thr Thr Val Ser Thr Phe Ile Asp Leu Asn Ile Thr Met

Leu Glu Asp His Glu Phe Val Pro Leu Glu Val Tyr Thr Arg His Glu Ile Lys

Asp Ser Gly Leu Leu Asp Tyr Thr Glu Val Gln Arg Arg Asn Gln Leu His Asp

Leu Arg Phe Ala Asp Ile Asp Thr Val Ile His Ala Asp Ala

4. the Screening and Identification of expressed fusion protein engineering bacteria:

With the recombinant plasmid transformed e. coli tg1, coating contains the LB flat board of 100 μ g/ml penbritins, putting 37 ℃ spends the night, next day, picking transformed bacterium colony and the contrast bacterium that contains plasmid pGEX4T-2 at random, extract plasmid, plasmid with extraction is a template, the gB glucoprotein extracellular region gene fragment of pcr amplification HSV1 virus, the positive recombinant plasmid that contains the gB glucoprotein extracellular region gene fragment of HSV1 virus, should amplify the gene fragment that is about 2107bp, the positive transformant that will contain recombinant plasmid, be seeded in the LB substratum that contains penbritin 100 μ g/mL, 37 ℃ of shaking culture 3h add IPTG to final concentration 0.5～1.0mmol/L, continue shaking culture and induce 4～6h, centrifugal collection thalline carries out SDS-PAGE and detects, recon is expressed the HSV virus gB glycoprotein that relative molecular weight is about 96kD, and expression amount is about 30%, and contrast bacterium TG1 does not have this protein band;

5. express the purifying of HSV1 virus gB glycoprotein:

1) ultrasonic degradation of expression HSV1 virus GB glycoprotein engineering bacteria

Centrifugal (8000rpm, 20min, 4 ℃) receives bacterium with the engineering bacteria of abduction delivering fusion rotein, thalline is resuspended in the lysate of original fluid 1/10 volume, lysate is 50mmol/L Tris-HCl pH8.0,10mmol/LEDTA, 10mmol/L DTT, ice-bath ultrasonic is broken bacterium 10min, centrifugal (8000rpm, 20min, 4 ℃) collects supernatant, abandons precipitation.The supernatant of collecting is used for next step affinitive layer purification.

2) purifying of expression HSV1 virus gB glycoprotein

Supernatant solution adds equilibrated Glutathione Sepharose 4B gel 3ml, and room temperature is in conjunction with 60min, and last sample is collected and penetrated liquid.With 1 * PBS washing pillar of ten times of column volumes, then with the GSH elutriant of 15ml high density, elutriant is 50mmol/LTris-HCL pH8.0+10mmol/LGST, divides three times the wash-out target protein, is the HSV1 virus gB glucoprotein extracellular region fragment of purifying.

6. the ELISA of the HSV1 of purifying virus gB glycoprotein detects:

The fusion rotein that preliminary purification is obtained is by 1: 1000～1: 32000 doubling dilution, and with negative control with same concentration doubling dilution, with the antigenicity (concrete operation method is seen the test kit specification sheets) of Human HSV-1 ELISA test kit detection expressing protein.The result shows that (table 1) this amalgamation and expression albumen has antigenicity and specificity preferably.

7. with the HSV1 virus gB glycoprotein fragment of expressing, be used for vaccine, HSV1 antiviral antibody or detection of antigens and be used for the anti-HSV1 virus monoclonal antibody of immunity preparation and how anti-etc.

8. synthetic HSV1 virus gB glycoprotein gene fragment is connected with other gene fragment, expresses, prepare with the form of fusion rotein.

The application of HSV1 virus gB glucoprotein extracellular region gene fragment in preparation HSV1 viral sub-units vaccine of method for preparing.

The HSV1 virus gB glucoprotein extracellular region gene fragment of chemosynthesis can utilize bacterium, yeast cell, insect cell, mammalian cell and genetically modified animals and plants to carry out recombinant expressed, preparation.

The advantage that the present invention compared with prior art has:

The gB glycoprotein fragment of the HSV1 virus that the present invention expresses has more advantage:

1. the HSV1 virus gB glucoprotein extracellular region fragment expressed of the present invention is as the enzyme-linked immunologic detecting kit of antigen prepd HSV1 antiviral antibody, have that production is safer, cost is low, with advantages such as other viral cross reaction is few.

2.HSV1 the gB glycoprotein of virus content in the cell that infects is maximum, be the conservative albumen of simplexvirus family camber, each strain differences is less, and bigger homology is arranged with all herpes-like virus subgroups, gB albumen has stronger immunogenicity simultaneously, can induce body to produce neutralizing antibody and cell immune response, so HSV1gB glycoprotein is to make up HSV vaccine ideal goal gene.The present invention has selected its strong antigen epi-position, utilizes genetic engineering technique to express preparation, for the development recombinant vaccine lays the foundation.Recombinant vaccine safety, cost are low.

3. the present invention is according to the gB glucoprotein extracellular region fragment aminoacid sequence of the HSV1 virus that filters out, the codon of selecting eucaryon and prokaryotic organism all to have a preference for, the gene order that chemosynthesis is brand-new, this gene high expression level in eucaryon and prokaryotic cell prokaryocyte that suits.

4. the engineering bacteria of the gB glycoprotein of the expression HSV1 virus that makes up of the present invention, expression amount can reach 30% of tropina, is easy to purifying, but large-scale purification prepares this albumen.

Description of drawings

The invention will be further described below with reference to accompanying drawing.

Fig. 1 is the construction of recombinant plasmid schema that the present invention expresses the gB glycoprotein of HSV1 virus.

Fig. 2 is the electrophorogram of pcr amplification HSV1 virus gB glucoprotein extracellular region gene segment of the present invention.

Fig. 3 is the present invention with the pcr amplification product of 5 recons of Agarose gel detection of 1.0%.M in the accompanying drawing 3: the low-molecular-weight dna standard (TaKaRa, 500bp); 1～5: 5 transformants all amplify the target gene fragment of 2107bp.

Fig. 4 is the SDS-PAGE analytical results that the present invention expresses the gB glycoprotein reorganization bacterium of HSV1 virus.M in the accompanying drawing 4: low molecular weight protein (LMWP) standard (Pharmacia); 7: contrast bacterium E.coli TG1; 1～No. 6 reorganization of 1～6 expression bacterium, No. 2, No. 52 recons are expressed relative molecular weight and are about 96000 fusion rotein, and 1,3,4, No. 64 transformants do not have this protein band.

Fig. 5 is the SDS-PAGE analytical results that the present invention expresses the gB glycoprotein purifying front and back of HSV1 virus.M in the accompanying drawing 5: low molecular weight protein (LMWP) standard (Pharmacia); 1: contrast bacterium TG1; 2: the engineering bacteria of expressing the gB glycoprotein of HSV1 virus; The inclusion body of the gB glycoprotein of 3:HSV1 virus; Pass the peak in the purge process of the gB glycoprotein of 4:HSV1 virus; Behind the 5:Glutathione Sepharose 4B affinity chromatography column purification the gB glycoprotein of HSV1 virus; Supernatant in the purge process of the gB glycoprotein of 6:HSV1 virus.

Embodiment

The detailed description of embodiment of the present invention:

The analysis of the gB glycoprotein antigen epi-position of HSV1 virus, synthetic the reaching of gene are expressed

Whole aminoacid sequences of the gB glycoprotein by Computer Analysis HSV1 virus, filter out the interior strong antigen epi-position of gB glycoprotein of HSV1 virus, select the codon of bacterium preference for use, the brand-new gene fragment of the gB glycoprotein strong antigen epi-position of chemosynthesis HSV1 virus.With the BamHI/EcoRI site of gene fragment clone to the plasmid pGEX4T-2, consistent with the translation framework of initiator codon on the carrier, can express a fusion rotein.With the recombinant plasmid transformed e. coli tg1, screening has obtained to efficiently express the engineering bacteria of the gB glycoprotein of HSV1 virus, and the gB glycoprotein of the HSV1 virus of expression accounts for about 30% of tropina total amount.

Materials and methods

1. bacterial classification and plasmid: host bacterium TG1 and expression vector pGEX4T-2 are that preserve in the laboratory.

2. molecular biology reagent: restriction enzyme BamHI, EcoRI, and the T4 dna ligase be commercially available TaKaRa company product.Plasmid purification test kit and the test kit that reclaims dna fragmentation in the sepharose are commercially available TaKaRa company product.DTT and IPTG are commercially available Promega company product.Other reagent is import or homemade analytical reagent.

3. gene fragment is synthetic: synthetic by design by 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 QIAGEN company plasmid purification test kit plasmid purification, with the full-automatic sequenator order-checking of DNA.

Embodiment

1.HSV1 screening of the gB glycoprotein antigen epi-position of virus and gene fragment is synthetic:

Utilize softwares such as ANTHEWIN, whole aminoacid sequence (GeneBank of the gB glycoprotein by Computer Analysis HSV1 virus, closing number: AY278488), filter out the interior strong antigen epi-position (Fig. 1) of gB glycoprotein of HSV1 virus, promptly from 696 amino acid of the 1st amino acid to the, its aminoacid sequence is as follows:

Pro Ser Ser Pro Gly Thr Pro GIY Val Ala Ala Ala Thr Gln Ala Ala Asn Gly

Gly Pro Ala Thr Pro Ala Pro Pro Ala Leu GlY Ala Ala Pro Thr GlY Asp Pro

Lys Pro Lys Lys Asn Lys Lys Pro Lys Asn Pro Thr Pro Pro Arg Pro Ala Gly

Asp Asn Ala Thr Val Ala Ala Gly His Ala Thr Leu Arg Glu His Leu Arg Asp

Ile Lys Ala Glu Asn Thr Asp Ala Asn Phe Tyr Val Cys Pro Pro Pro Thr Gly

Ala Thr Val Val Gln Phe Glu Gln Pro Arg Arg Cys Pro Thr Arg Pro Glu Gly

Gln Asn Tyr Thr Glu Gly Ile Ala Val Val Phe Lys Glu Asn Ile Ala Pro Tyr

Lys Phe Lys Ala Thr Met Tyr Tyr Lys Asp Val Thr Val Ser Gln Val Trp Phe

Gly His Arg Tyr Ser Gln Phe Met Gly Ile Phe Glu Asp Arg Ala Pro Val Pro

Phe Glu Glu Val Ile Asp LYs Ile Asn Ala Lys Gly Val Cys Arg Ser Thr Ala

Lys Tyr Val Arg Asn Asn Leu Glu Thr Thr Ala Phe His Arg Asp Asp His Glu

Thr Asp Met Glu Leu Lys Pro Ala Asn Ala Ala Thr Arg Thr Ser Arg Gly Trp

His Thr Thr Asp Leu Lys Tyr Asn Pro Ser Arg Val Glu Ala Phe His Arg Tyr

Gly Thr Thr Val Asn Cys Ile Val Glu Glu Val Asp Ala Arg Ser Val Tyr Pro

Tyr Asp Glu Phe Val Leu Ala Thr Gly Asp Phe Val Tyr Met Ser Pro Phe Tyr

Gly Tyr Arg Glu Gly Ser His Thr Glu His Thr Ser Tyr Ala Ala Asp Arg Phe

Lys Gln Val Asp Gly Phe Tyr Ala Arg Asp Leu Thr Thr Lys Ala Arg Ala Thr

Ala Pro Thr Thr Arg Asn Leu Leu Thr Thr Pro Lys Phe Thr Val Ala Trp Asp

Trp Val Pro Lys Arg Pro Ser Val Cys Thr Met Thr Lys Trp Gln Glu Val Asp

Glu Met Leu Arg Ser Glu Tyr Gly Gly Ser Phe Arg Phe Ser Ser Asp Ala Ile

Ser Thr Thr Phe Thr Thr Asn Leu Thr Glu Tyr Pro Leu Ser Arg Val Asp Leu

Gly Asp Cys Ile Gly Lys Asp Ala Arg Asp Ala Met Asp Arg Ile Phe Ala Arg

Arg Tyr Asn Ala Thr His Ile Lys Val Gly Gln Pro Gln Tyr Tyr Leu Ala Asn

Gly Gly Phe Leu Ile Ala Tyr Gln Pro Leu Leu Ser Asn Thr Leu Ala Glu Leu

Tyr Val Arg Glu His Leu Arg Glu Gln Ser Pro Lys Pro Pro Asn Pro Thr Pro

Pro Pro Pro Gly Ala Ser Ala Asn Ala Ser Val Glu Arg Ile Lys Thr Thr Ser

Ser Ile Glu Phe Ala Arg Leu Gln Phe Thr Tyr Asn His Ile Gln Asn His Val

Asn Asp Met Leu Gly Arg Val Ala Ile Ala Trp Cys Glu Leu Gln Asn His Glu

Leu Thr Leu Trp Asn Glu Ala Arg Lys Leu Asn Pro Asn Ala Ile Ala Ser Ala

Thr Val Gly Arg Arg Val Ser Ala Arg Met Leu Gly Asp Val Met Ala Val Ser

Thr Cys Val Pro Val Ala Ala Asp Asn Val Ile Val Gln Asn Ser Met Arg Ile

Ser Ser Arg Pro Gly Ala Cys Tyr Ser Arg Pro Leu Val Ser Phe Arg Tyr Glu

Asp Gln Gly Pro Leu Val Glu Gly Gln Leu Gly Glu Asn Asn Glu Leu Arg Leu

Thr Arg Asp Ala Ile Glu Pro Cys Thr Val Gly His Arg Arg Tyr Phe Thr Phe

Gly Gly Gly Tyr Val Tyr Phe Glu Glu Ser Ala Tyr Ser His Gln Leu Ser Arg

Ala Asp Ile Thr Thr Val Ser Thr Phe Ile Asp Leu Asn Ile Thr Met Leu Glu

Asp His Glu Phe Val Pro Leu Glu Val Tyr Thr Arg His Glu Ile Lys Asp Ser

Gly Leu Leu Asp Tyr Thr Glu Val Gln Arg Arg Asn Gln Leu His Asp Leu Arg

Phe Ala Asp Ile Asp Thr Val Ile His Ala Asp Ala

According to the epitope aminoacid sequence in the HSV1 virus gB glycoprotein of screening; the codon of selecting eucaryon and prokaryotic organism all to have a preference for; the gene order that chemosynthesis is brand-new; and BamHI restriction enzyme site (following setting-out part) and two protection bases (GC) have been increased at 5 ' end; increased terminator codon (TGA) and EcoRI restriction enzyme site (following setting-out part) and two protection bases (GC) at 3 ' end, made this gene fragment be easy to be cloned in plasmid the pGEX4T-2 interior BamHI and EcoRI restriction enzyme site.The dna sequence dna (2107bp) that contains HSV1 virus gB glycoprotein antigen epitope gene of chemosynthesis is as follows:

GC GGATCC CCT ACC TCT CCT GGT ACT CCT GGT GTG GCT GCC GCT ACC CAG GCC GCT AAC

GGT GGT CCT GCC ACT CCT GCT CCT CCT CCT CTG GGT GCC GCT CCT ACT GGT GAC CCT

AAG CCT AAG AAG AAC AAG AAG CCT AAG AAC CCT ACT CCT CCT AGG CCT GCT GGT GAC

AAC GCC ACC GTG GCC GCT GGC CAC GCC ACT CTG AGA GAG CAC CTG AGA GAC ATC AAG

GCT GAG AAC ACC GAC GCT AAC TTC TAC GTG TGT CCT CCT CCT ACT GGT GCC ACC GTG

GTG CAG TTC GAG CAG CCT CGC AGG TGC CCT ACC AGG CCT GAA GGT CAG AAC TAC ACC

GAA GGC ATC GCC GTG GTG TTC AAG GAG AAC ATC GCT CCT TAC AAG TTC AAG GCC ACC

ATG TAC TAC AAG GAC GTG ACC GTG AGC CAG GTG TGG TTC GGC CAC CGC TAC TCC CAG

TTC ATG GGC ATC TTC GAG GAC CGC GCT CCT GTG CCT TTC GAG GAG GTG ATC GAC AAG

ATC AAC GCC AAG GGT GTG TGT AGG TCC ACC GCT AAG TAC GTG CGC AAC AAC CTG GAG

ACC ACT GCT TTC CAC AGA GAC GAT CAC GAG ACC GAC ATG GAG CTG AAG CCT GCC AAC

GCC GCT ACT CGC ACC AGC AGA GGC TGG CAC ACC ACT GAC CTG AAG TAC AAC CCT AGC

AGA GTG GAG GCC TTC CAC AGG TAC GGC ACC ACC GTG AAC TGC ATC GTG GAG GAG GTG

GAC GCT CGC AGC GTG TAC CCT TAC GAC GAG TTC GTG CTG GCC ACT GGT GAC TTC GTG

TAC ATG TCT CCT TTC TAC GGC TAC AGA GAA GGT AGC CAC ACC GAG CAC ACT ACC TAC

GCT GCT GAC AGG TTC AAG CAG GTG GAC GGC TTC TAC GCT CGC GAC CTG ACC ACC AAG

GCT AGA GCC ACT GCT CCT ACC ACT AGG AAC CTG CTG ACC ACT CCT AAG TTC ACC GTG

GCT TGG GAC TGG GTG CCT AAG CGC CCT AGC GTG TGC ACC ATG ACC AAG TGG CAG GAG

GTG GAC GAG ATG CTG CGC TCC GAG TAC GGC GGT TCC TTC AGG TTC TCC TCT GAC GCT

ATC TCC ACT ACC TTC ACT ACC AAC CTG ACC GAG TAC CCT CTG TCC AGA GTG GAC CTG

GGT GAC TGC ATC GGT AAG GAC GCT CGC GAC GCC ATG GAC CGC ATC TTC GCT CGC AGG

TAC AAC GCT ACT CAC ATC AAG GTG GGC CAG CCT CAG TAC TAC CAG GCC AAC GGT GGT

TTC CTG ATC GCC TAC CAG CCT CTG CTG AGC AAC ACT CTG GCT GAG CTG TAC GTG AGA

GAG CAC CTG AGA GAG CAG AGC CGC AAG CCT CCT AAC CCT ACG CCT CCT CCT CCC GGT

GCT AGC GCC AAC GCT TCC GTG GAG CGC ATC AAG ACT ACC TCT AGC ATC GAG TTC GCC

AGG CTG CAG TTC ACC TAC AAC CAC ATC CAG CGC CAC GTG AAC GAC ATG CTG GGT CGC

GTG GCT ATC GCT TGG TGC GAG CTG CAG AAC CAC GAG CTG ACT CTG TGG AAC GAG GCT

CGC AAG CTG AAC CCT AAC GCT ATC GCC AGC GTG ACC GTG GGC AGG AGA GTG AGC GCT

AGA ATG CTG GGC GAC GTG ATG GCC GTG TCC ACC TGC GTG CCT GTG GCT GCT GAC AAC

GTG ATC GTG CAG AAC AGC ATG CGC ATC AGC TCC AGA CCT GGT GCC TGC TAC AGC AGA

CCT CTG GTG AGC TTC AGG TAC GAG GAC CAA GGT CCT CTG GTG GAA GGT CAG CTG GGT

GAG AAC AAC GAG CTG AGG CTG ACT CGC GAC GCT ATC GAG CCT TGC ACC GTC GGT CAC

AGA CGC TAC TTC ACC TTC GGT GGC GGT TAC GTG TAC TTC GAG GAG TAC GCT TAC TCT

CAC CAG CTG AGC CGC GCT GAC ATC ACT ACC GTG AGC ACC TTC ATC GAC CTG AAC ATC

ACC ATG CTG GAG GAC CAC GAG TTC GTG CCT CTG GAG GTG TAC ACC CGC CAC GAG ATC

AAG GAC AGC GGC CTG CTG GAC TAC ACC GAG GTG CAG CGC CGC AAC CAG CTG CAC GAC

CTG CGC TTC GCT GAC ATC GAC ACC GTG ATC CAC GCC GAC GCC TGA GAATTCGC

Extract plasmid pGEX4T-2,, reclaim the big fragment of plasmid that enzyme is cut behind the electrophoresis, be dissolved in the deionized water with Bam H I and EcoRI double digestion.Same HSV1 virus gB glucoprotein extracellular region gene fragment with BamH I and the chemosynthesis of EcoR I double digestion, electrophoresis is dissolved in the deionized water after reclaiming.

Above-mentioned two 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, HSV1 virus gB glucoprotein extracellular region gene fragment is inserted between carrier pGEX4T-2 interior the BamH I and EcoRI site, consistent with the initiator codon translation framework on the carrier, express a fusion rotein (make up flow process and see Fig. 2).

3. the screening of recombinant plasmid and evaluation:

The recombinant plasmid transformed that the last step was connected arrives intestinal bacteria TGl, and the converted product coating is contained on the solid LB substratum of penbritin (100 μ g/ml), puts 37 ℃ of overnight incubation.5 transformant bacterium colonies of random choose next day (being labeled as respectively 1-5 number) and 1 contrast bacterium (plasmid pGEX4T-2 transformed bacteria), be inoculated into respectively and contain 3ml liquid LB substratum (containing penbritin 100 μ g/ml) in vitro, put 37 ℃ of shaking culture 5h, 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) 2 μ l as pcr template, pcr amplification inserts and carries intravital HSV1 virus gB glucoprotein extracellular region gene fragment, and the PCR reaction density is: the normal chain P1 (GC of plasmid template 2 μ l, HSV1 virus gB glucoprotein extracellular region gene fragment GGATCCCCTACCTCTCCTGGTACTC) and minus strand primer P2 (GC GAATTCTTAGGCGTCGGC) each 1 μ l, 10 * pyrobest buffer5.0 μ l, 2.5mmol/L dNTP4.0 μ l, Pyrobest DNA Taq enzyme 0.5 μ l (2.5U), deionized water 36.5 μ l, cumulative volume 50 μ l.Amplification condition is: 94 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ 4 minutes, 35 circulations; Last 72 ℃ were extended 7 minutes.Get pcr amplification product 5 μ l, the Agarose gel detection with 1.0%, the result, 5 transformants all amplify the target gene fragment (see figure 3) of 2107bp, and the contrast bacterium that contains plasmid pGEX4T-2 does not amplify this gene fragment.Tentative confirmation, these 5 transformants all contain HSV1 virus gB glycoprotein gene fragment.

Extract the plasmid of No. 1 recon, measure the HSV1 virus gB glycoprotein gene sequence in the plasmid, dna sequence analysis confirms that recombinant plasmid contains synthetic HSV1 virus gB glycoprotein gene fragment, and sequence is entirely true:

CCT ACC TCT CCT GGT ACT CCT GGT GTG GCT GCC GCT ACC CAG GCC GCT AAC GGT GGT

CCT GCC ACT CCT GCT CCT CCT CCT CTG GGT GCC GCT CCT ACT GGT GAC CCT AAG CCT

AAG AAG AAC AAG AAG CCT AAG AAC CCT ACT CCT CCT AGG CCT GCT GGT GAC AAC GCC

ACC GTG GCC GCT GGC CAC GCC ACT CTG AGA GAG CAC CTG AGA GAC ATC AAG GCT GAG

AAC ACC GAC GCT AAC TTC TAC GTG TGT CCT CCT CCT ACT GGT GCC ACC GTG GTG CAG

TTC GAG CAG CCT CGC AGG TGC CCT ACC AGG CCT GAA GGT CAG AAC TAC ACC GAA GGC

ATC GCC GTG GTG TTC AAG GAG AAC ATC GCT CCT TAC AAG TTC AAG GCC ACC ATG TAC

TAC AAG GAC GTG ACC GTG AGC CAG GTG TGG TTC GGC CAC CGC TAC TCC CAG TTC ATG

GGC ATC TTC GAG GAC CGC GCT CCT GTG CCT TTC GAG GAG GTG ATC GAC AAG ATC AAC

GCC AAG GGT GTG TGT AGG TCC ACC GCT AAG TAC GTG CGC AAC AAC CTG GAG ACC ACT

GCT TTC CAC AGA GAC GAT CAC GAG ACC GAC ATG GAG CTG AAG CCT GCC AAC GCC GCT

ACT CGC ACC AGC AGA GGC TGG CAC ACC ACT GAC CTG AAG TAC AAC CCT AGC AGA GTG

GAG GCC TTC CAC AGG TAC GGC ACC ACC GTG AAC TGC ATC GTG GAG GAG GTG GAC GCT

CGC AGC GTG TAC CCT TAC GAC GAG TTC GTG CTG GCC ACT GGT GAC TTC GTG TAC ATG

TCT CCT TTC TAC GGC TAC AGA GAA GGT AGC CAC ACC GAG CAC ACT ACC TAC GCT GCT

GAC AGG TTC AAG CAG GTG GAC GGC TTC TAC GCT CGC GAC CTG ACC ACC AAG GCT AGA

GCC ACT GCT CCT ACC ACT AGG AAC CTG CTG ACC ACT CCT AAG TTC ACC GTG GCT TGG

GAC TGG GTG CCT AAG CGC CCT AGC GTG TGC ACC ATG ACC AAG TGG CAG GAG GTG GAC

GAG ATG CTG CGC TCC GAG TAC GGC GGT TCC TTC AGG TTC TCC TCT GAC GCT ATC TCC

ACT ACC TTC ACT ACC AAC CTG ACC GAG TAC CCT CTG TCC AGA GTG GAC CTG GGT GAC

TGC ATC GGT AAG GAC GCT CGC GAC GCC ATG GAC CGC ATC TTC GCT CGC AGG TAC AAC

GCT ACT CAC ATC AAG GTG GGC CAG CCT CAG TAC TAC CAG GCC AAC GGT GGT TTC CTG

ATC GCC TAC CAG CCT CTG CTG AGC AAC ACT CTG GCT GAG CTG TAC GTG AGA GAG CAC

CTG AGA GAG CAG AGC CGC AAG CCT CCT AAC CCT ACG CCT CCT CCT CCC GGT GCT AGC

GCC AAC GCT TCC GTG GAG CGC ATC AAG ACT ACC TCT AGC ATC GAG TTC GCC AGG CTG

CAG TTC ACC TAC AAC CAC ATC CAG CGC CAC GTG AAC GAC ATG CTG GGT CGC GTG GCT

ATC GCT TGG TGC GAG CTG CAG AAC CAC GAG CTG ACT CTG TGG AAC GAG GCT CGC AAG

CTG AAC CCT AAC GCT ATC GCC AGC GTG ACC GTG GGC AGG AGA GTG AGC GCT AGA ATG

CTG GGC GAC GTG ATG GCC GTG TCC ACC TGC GTG CCT GTG GCT GCT GAC AAC GTG ATC

GTG CAG AAC AGC ATG CGC ATC AGC TCC AGA CCT GGT GCC TGC TAC AGC AGA CCT CTG

GTG AGC TTC AGG TAC GAG GAC CAA GGT CCT CTG GTG GAA GGT CAG CTG GGT GAG AAC

AAC GAG CTG AGG CTG ACT CGC GAC GCT ATC GAG CCT TGC ACC GTC GGT CAC AGA CGC

TAC TTC ACC TTC GGT GGC GGT TAC GTG TAC TTC GAG GAG TAC GCT TAC TCT CAC CAG

CTG AGC CGC GCT GAC ATC ACT ACC GTG AGC ACC TTC ATC GAC CTG AAC ATC ACC ATG

CTG GAG GAC CAC GAG TTC GTG CCT CTG GAG GTG TAC ACC CGC CAC GAG ATC AAG GAC

AGC GGC CTG CTG GAC TAC ACC GAG GTG CAG CGC CGC AAC CAG CTG CAC GAC CTG CGC

TTC GCT GAC ATC GAC ACC GTG ATC CAC GCC GAC GCC TGA

The viral gB glycoprotein fragment (696 amino acid) of the expression of recombinant plasmid HSV1 that makes up has merged 238 amino acid on the carrier at its N end, 934 amino acid of total length, and its aminoacid sequence is as follows:

Met Pro Met Ile Leu Gly Tyr Trp Asp Ile Arg Gly Leu Ala His Ala Ile Arg

Leu Leu Leu Glu Tyr Thr Asp Ser Ser Tyr Glu Glu Lys Lys Tyr Thr Met Gly

Gly Ala Pro Asp Tyr Asp Arg Ser Gln Trp Leu Asn Glu Lys Phe Lys Leu Gly

Leu Asp Phe Pro Asn Leu Pro Tyr Leu Ile Asp Gly Ala His Lys Ile Thr Gln

Ser Asn Ala Ile Leu Cys Tyr Ile Ala Arg Lys His Asn Leu Cys Gly Glu Thr

Glu Glu Glu Lys Ile Arg Val Asp Ile Leu Glu Asn Gln Ala Met Asp Val Ser

Asn Gln Leu Ala Arg Val Cys Tyr Ser Pro Asp Phe Glu Lys Leu Lys Pro Glu

Tyr Leu Glu Glu Leu Pro Thr Met Met Gln His Phe Ser Gln Phe Leu Gly Lys

Arg Pro Trp Phe Val Gly Asp Lys Ile Thr Phe Val Asp Phe Leu Ala Tyr Asp

Val Leu Asp Leu His Arg Ile Phe Glu Pro Asn Cys Leu Asp Ala Phe Pro Asn

Leu Lys Asp Phe Ile Ser Arg Phe Glu Gly Leu Glu Lys Ile Ser Ala Tyr Met

Lys Ser Ser Arg Phe Leu Pro Lys Pro Leu Tyr Thr Arg Met Ala Val Trp Gly

Asn Lys Pro Ser Ser Pro Gly Thr Pro Gly Val Ala Ala Ala Thr Gln Ala Ala

Asn Gly Gly Pro Ala Thr Pro Ala Pro Pro Ala Leu Gly Ala Ala Pro Thr Gly

Asp Pro Lys Pro Lys Lys Asn Lys Lys Pro Lys Asn Pro Thr Pro Pro Arg Pro

Ala Gly Asp Asn Ala Thr Val Ala Ala Gly His Ala Thr Leu Arg Glu His Leu

Arg Asp Ile Lys Ala Glu Asn Thr Asp Ala Asn Phe Tyr Val Cys Pro Pro Pro

Thr Gly Ala Thr Val Val Gln Phe Glu Gln Pro Arg Arg Cys Pro Thr Arg Pro

Glu Gly Gln Asn Tyr Thr Glu Gly Ile Ala Val Val Phe Lys Glu Asn Ile Ala

Pro Tyr Lys Phe Lys Ala Thr Met Tyr Tyr Lys Asp Val Thr Val Ser Gln Val

Trp Phe Gly His Arg Tyr Ser Gln Phe Met Gly Ile Phe Glu Asp Arg Ala Pro

Val Pro Phe Glu Glu Val Ile Asp Lys Ile Asn Ala Lys Gly Val Cys Arg Ser

Thr Ala Lys Tyr Val Arg Asn Asn Leu Glu Thr Thr Ala Phe His Arg Asp Asp

His Glu Thr Asp Met Glu Leu Lys Pro Ala Asn Ala Ala Thr Arg Thr Ser Arg

Gly Trp His Thr Thr Asp Leu Lys Tyr Asn Pro Ser Arg Val Glu Ala Phe His

Arg Tyr Gly Thr Thr Val Asn Cys Ile Val Glu Glu Val Asp Ala Arg Ser Val

Tyr Pro Tyr Asp Glu Phe Val Leu Ala Thr Gly Asp Phe Val Tyr Met Ser Pro

Phe Tyr Gly Tyr Arg Glu Gly Ser His Thr Glu His Thr Ser Tyr Ala Ala Asp

Arg Phe Lys Gln Val Asp Gly Phe Tyr Ala Arg Asp Leu Thr Thr Lys Ala Arg

Ala Thr Ala Pro Thr Thr Arg Asn Leu Leu Thr Thr Pro Lys Phe Thr Val Ala

Trp Asp Trp Val Pro Lys Arg Pro Ser Val Cys Thr Met Thr Lys Trp Gln Glu

Val Asp Glu Met Leu Arg Ser Glu Tyr Gly Gly Ser Phe Arg Phe Ser Ser Asp

Ala Ile Ser Thr Thr Phe Thr Thr Asn Leu Thr Glu Tyr Pro Leu Ser Arg Val

Asp Leu Gly Asp Cys Ile Gly Lys Asp Ala Arg Asp Ala Met Asp Arg Ile Phe

Ala Arg Arg Tyr Asn Ala Thr His Ile Lys Val Gly Gln Pro Gln Tyr Tyr Leu

Ala Asn Gly Gly Phe Leu Ile Ala Tyr Gln Pro Leu Leu Ser Asn Thr Leu Ala

Glu Leu Tyr Val Arg Glu His Leu Arg Glu Gln Ser Pro Lys Pro Pro Asn Pro

Thr Pro Pro Pro Pro Gly Ala Ser Ala Asn Ala Ser Val Glu Arg Ile Lys Thr

Thr Ser Ser Ile Glu Phe Ala Arg Leu Gln Phe Thr Tyr Asn His Ile Gln Asn

His Val Asn Asp Met Leu Gly Arg Val Ala Ile Ala Trp Cys Glu Leu Gln Asn

His Glu Leu Thr Leu Trp Asn Glu Ala Arg Lys Leu Asn Pro Asn Ala Ile Ala

Ser Ala Thr Val Gly Arg Arg Val Ser Ala Arg Met Leu Gly Asp Val Met Ala

Val Ser Thr Cys Val Pro Val Ala Ala Asp Asn Val Ile Val Gln Asn Ser Met

Arg Ile Ser Ser Arg Pro Gly Ala Cys Tyr Ser Arg Pro Leu Val Ser Phe Arg

Tyr Glu Asp Gln Gly Pro Leu Val Glu Gly Gln Leu Gly Glu Asn Asn Glu Leu

Arg Leu Thr Arg Asp Ala Ile Glu Pro Cys Thr Val Gly His Arg Arg Tyr Phe

Thr Phe Gly Gly Gly Tyr Val Tyr Phe Glu Glu Ser Ala Tyr Ser His Gln Leu

Ser Arg Ala Asp Ile Thr Thr Val Ser Thr Phe Ile Asp Leu Asn Ile Thr Met

Leu Glu Asp His Glu Phe Val Pro Leu Glu Val Tyr Thr Arg His Glu Ile Lys

Asp Ser Gly Leu Leu Asp Tyr Thr Glu Val Gln Arg Arg Asn Gln Leu His Asp

Leu Arg Phe Ala Asp Ile Asp Thr Val Ile His Ala Asp Ala

4. express the Screening and Identification of HSV1 virus gB glycoprotein engineering bacteria:

5 positive transformants and 1 contrast bacterium (plasmid pGEX4T-2 transformed bacteria) that will contain recombinant plasmid, be seeded to and contain 3ml LB substratum (containing penbritin 100 μ g/ml) in vitro, 37 ℃ of shaking culture 3h, add IPTG to final concentration 1.0mmol/L, continue shaking culture and induce 5h, centrifugal collection thalline carries out SDS-PAGE and detects, and recon is expressed the HSV1 virus gB glycoprotein that relative molecular weight is about 96kD, expression amount is about 30%, and contrast bacterium TG1 does not have this protein band (Fig. 4).

Express the purifying of HSV1 virus gB glycoprotein

According to the aminoacid sequence of expressing HSV1 virus gB glycoprotein, analyze its physicochemical property, determine suitable purification process.Our expressed HSV1gB glycoprotein merges the GST albumen that has on the carrier, gst fusion protein has been expressed glutathione s simultaneously when expressing change enzyme, can easily separate with GST sepharose FF, therefore we determine to adopt affinity chromatography, carry out purifying with Glutathione Sepharose 4B gel.Concrete steps are as follows:

Material and method

1. main agents:

Glutathione Sepharose 4B gel is a GE Heathcare company product, and IPTG, DTT are Promega company product.Other reagent is homemade or the import analytical reagent.

2. express the ultrasonic degradation of the gB glycoprotein of HSV1 virus:

Centrifugal (the 8000rpm of engineering bacteria with the gB glycoprotein of the expression HSV1 virus of cultivating, 20mins, 4 ℃), abandon supernatant, thalline is resuspended in the lysate (50mmol/L Tris-HCl pH8.0,10mmol/LEDTA, 10mmol/L DTT) of original fluid 1/10 volume, and ice-bath ultrasonic is broken bacterium 10mins, centrifugal (8000rpm, 20mins, 4 ℃) collect supernatant, abandon precipitation.The supernatant of collecting is used for next step affinitive layer purification.

3. express the purifying of HSV1 virus gB glycoprotein:

Supernatant solution adds equilibrated Glutathione Sepharose 4B gel 3ml room temperature in conjunction with 60min, and last sample is collected and penetrated liquid.With 1 * PBS washing pillar of ten times of column volumes, then the GSH elutriant (50mmol/LTris-HCL pH8.0+10mmol/LGST) with the 15ml high density divides three wash-out target proteins, is the HSV1 virus gB glucoprotein extracellular region fragment of purifying.

The result:

To carry out SDS-PAGE from the albumen of wash-out on the Glutathione Sepharose 4B gel column and analyze, the result shows (see figure 5), obviously gives expression to the HSV1gB/GST fusion rotein through inducing, and expression product mainly is present in the supernatant liquor.SDS-PAGE shows the about 96kDa of expression product.

The gB identification of glycoproteins and the application of purifying HSV1 virus

Reorganization HSV1 virus gB glycoprotein antigen with purifying detects by the double-antibody sandwich elisa test method, to identify the antigenicity and the specificity of the HSV1 virus gB glycoprotein of expressing.Experimental result shows that this recombinant protein has good antigenicity and specificity.

Material and method

1. integrated enzyme reaction test kit: Human HSV-1 ELISA test kit is a U.S. Uscnlife company product.

2.ELISA test: the fusion rotein that preliminary purification obtains is pressed 1: 1000～1: 32000 doubling dilution, and with negative control with same concentration doubling dilution, detect the antigenicity (concrete operation method is seen the test kit specification sheets) of expressing protein with Human HSV-1 ELISA test kit.

The result

The proteic ELISA of HSV1gB detects

The fusion rotein that preliminary purification is obtained is by 1: 1000～1: 32000 doubling dilution, and with negative control with same concentration doubling dilution, detect the antigenicity of expressing protein with Human HSV-1 ELISA test kit.The result shows that (table 1) this amalgamation and expression albumen has antigenicity and specificity preferably.

The ELISA experimental result of table 1 expressing protein

Table1.ELISA results of the expressed protein

The albumen weaker concn	1∶1000	1∶2000	1∶4000	1∶8000	1∶16000	1∶32000
The albumen weaker concn	1∶1000	1∶2000	1∶4000	1∶8000	1∶16000	1∶32000	HSVlgB Control	0.814 0.021	0.539 0.019	0.428 0.025	0.400 0.011	0.396 0.003	0.129 0.007

The HSV virus gB protein extracellular gene fragment order table of chemosynthesis

＜110〉Li Yuexi

＜120〉HSV of chemosynthesis virus gB protein extracellular gene fragment and expression thereof, application

<160>2

<210>1

<211>696

<212>PRT

＜213〉HSV virus gB protein extracellular fragment

<220>

＜223〉contain the HSV virus gB protein extracellular fragment of strong antigen epi-position, 696 amino acid of the 1st amino acid-Di, totally 696 amino acid.

<400>1

Pro Ser Ser Pro Gly Thr Pro Gly Val Ala Ala Ala Thr Gln Ala Ala

1 5 10 15

Asn Gly Gly Pro Ala Thr Pro Ala Pro Pro Ala Leu Gly Ala Ala Pro

20 25 30

Thr Gly Asp Pro Lys Pro Lys Lys Asn Lys Lys Pro Lys Asn Pro Thr

35 40 45

Pro Pro Arg Pro Ala Gly Asp Asn Ala Thr Val Ala Ala Gly His Ala

50 55 60

Thr Leu Arg Glu His Leu Arg Asp Ile Lys Ala Glu Asn Thr Asp Ala

65 70 75 80

Asn Phe Tyr Val Cys Pro Pro Pro Thr Gly Ala Thr Val Val Gln Phe

85 90 95

Glu Gln Pro Arg Arg Cys Pro Thr Arg Pro Glu Gly Gln Asn Tyr Thr

100 105 110

Glu Gly Ile Ala Val Val Phe Lys Glu Asn Ile Ala Pro Tyr Lys Phe

115 120 125

Lys Ala Thr Met Tyr Tyr Lys Asp Val Thr Val Ser Gln Val Trp Phe

130 135 140

Gly His Arg Tyr Ser Gln Phe Met Gly Ile Phe Glu Asp Arg Ala Pro

145 150 155 160

Val Pro Phe Glu Glu Val Ile Asp Lys Ile Asn Ala Lys Gly Val Cys

165 170 175

Arg Ser Thr Ala Lys Tyr Val Arg Asn Asn Leu Glu Thr Thr Ala Phe

180 185 190

His Arg Asp Asp His Glu Thr Asp Met Glu Leu Lys Pro Ala Asn Ala

195 200 205

Ala Thr Arg Thr Ser Arg Gly Trp His Thr Thr Asp Leu Lys Tyr Asn

210 215 220

Pro Ser Arg Val Glu Ala Phe His Arg Tyr Gly Thr Thr Val Asn Cys

225 230 235 240

Ile Val Glu Glu Val Asp Ala Arg Ser Val Tyr Pro Tyr Asp Glu Phe

245 250 255

Val Leu Ala Thr Gly Asp Phe Val Tyr Met Ser Pro Phe Tyr Gly Tyr

260 265 270

Arg Glu Gly Ser His Thr Glu His Thr Ser Tyr Ala Ala Asp Arg Phe

275 280 285

Lys Gln Val Asp Gly Phe Tyr Ala Arg Asp Leu Thr Thr Lys Ala Arg

290 295 300

Ala Thr Ala Pro Thr Thr Arg Asn Leu Leu Thr Thr Pro Lys Phe Thr

305 310 315 320

Val Ala Trp Asp Trp Val Pro Lys Arg Pro Ser Val Cys Thr Met Thr

325 330 335

Lys Trp Gln Glu Val Asp Glu Met Leu Arg Ser Glu Tyr Gly Gly Ser

340 345 350

Phe Arg Phe Ser Ser Asp Ala Ile Ser Thr Thr Phe Thr Thr Asn Leu

355 360 365

Thr Glu Tyr Pro Leu Ser Arg Val Asp Leu Gly Asp Cys Ile Gly Lys

370 375 380

Asp Ala Arg Asp Ala Met Asp Arg Ile Phe Ala Arg Arg Tyr Asn Ala

385 390 395 400

Thr His Ile Lys Val Gly Gln Pro Gln Tyr Tyr Leu Ala Asn Gly Gly

405 410 415

Phe Leu Ile Ala Tyr Gln Pro Leu Leu Ser Asn Thr Leu Ala Glu Leu

420 425 430

Tyr Val Arg Glu His Leu Arg Glu Gln Ser Pro Lys Pro Pro Asn Pro

435 440 445

Thr Pro Pro Pro Pro Gly Ala Ser Ala Asn Ala Ser Val Glu Arg Ile

450 455 460

Lys Thr Thr Ser Ser Ile Glu Phe Ala Arg Leu Gln Phe Thr Tyr Asn

465 470 475 480

His Ile Gln Asn His Val Asn Asp Met Leu Gly Arg Val Ala Ile Ala

485 490 495

Trp Cys Glu Leu Gln Asn His Glu Leu Thr Leu Trp Asn Glu Ala Arg

500 505 510

Lys Leu Asn Pro Asn Ala Ile Ala Ser Ala Thr Val Gly Arg Arg Val

515 520 525

Ser Ala Arg Met Leu Gly Asp Val Met Ala Val Ser Thr Cys Val Pro

530 535 540

Val Ala Ala Asp Asn Val Ile Val Gln Asn Ser Met Arg Ile Ser Ser

545 550 555 560

Arg Pro Gly Ala Cys Tyr Ser Arg Pro Leu Val Ser Phe Arg Tyr Glu

565 570 575

Asp Gln Gly Pro Leu Val Glu Gly Gln Leu Gly Glu Asn Asn Glu Leu

580 585 590

Arg Leu Thr Arg Asp Ala Ile Glu Pro Cys Thr Val Gly His Arg Arg

595 600 605

Tyr Phe Thr Phe Gly Gly Gly Tyr Val Tyr Phe Glu Glu Ser Ala Tyr

610 615 620

Ser His Gln Leu Ser Arg Ala Asp Ile Thr Thr Val Ser Thr Phe Ile

625 630 635 640

Asp Leu Asn Ile Thr Met Leu Glu Asp His Glu Phe Val Pro Leu Glu

645 650 655

Val Tyr Thr Arg His Glu Ile Lys Asp Ser Gly Leu Leu Asp Tyr Thr

660 665 670

Glu Val Gln Arg Arg Asn Gln Leu His Asp Leu Arg Phe Ala Asp Ile

675 680 685

Asp Thr Val Ile His Ala Asp Ala

690 695

<210>2

<211>2107

<212>DNA

＜213〉artificial sequence

<220>

<221>CDS

<222>(1)...(2104)

＜223〉the brand-new gene fragment of synthetic, coding HSV virus gB protein extracellular fragment.

<220>

<221>mis-feature

<222>(2105)...(2107)

＜223〉terminator codon that increases during synthetic gene.

<400>2

CCT ACC TCT CCT GGT ACT CCT GGT GTG GCT GCC GCT ACC CAG GCC GCT AAC GGT GGT CCT 60

Pro Ser Ser Pro Gly Thr Pro Gly Val Ala Ala Ala Thr Gln Ala Ala Asn Gly Gly Pro

1 5 10 15 20

GCC ACT CCT GCT CCT CCT CCT CTG GGT GCC GCT CCT ACT GGT GAC CCT AAG CCT AAG AAG 120

Ala Thr Pro Ala Pro Pro Ala Leu Gly Ala Ala Pro Thr Gly Asp Pro Lys Pro Lys Lys

25 30 35 40

AAC AAG AAG CCT AAG AAC CCT ACT CCT CCT AGG CCT GCT GGT GAC AAC GCC ACC GTG GCC 180

Asn Lys Lys Pro Lys Asn Pro Thr Pro Pro Arg Pro Ala Gly Asp Asn Ala Thr Val Ala

45 50 55 60

GCT GGC CAC GCC ACT CTG AGA GAG CAC CTG AGA GAC ATC AAG GCT GAG AAC ACC GAC GCT 240

Ala Gly His Ala Thr Leu Arg Glu His Leu Arg Asp Ile Lys Ala Glu Asn Thr Asp Ala

65 70 75 80

AAC TTC TAC GTG TGT CCT CCT CCT ACT GGT GCC ACC GTG GTG CAG TTC GAG CAG CCT CGC 300

Asn Phe Tyr Val Cys Pro Pro Pro Thr Gly Ala Thr Val Val Gln Phe Glu Gln Pro Arg

85 90 95 100

AGG TGC CCT ACC AGG CCT GAA GGT CAG AAC TAC ACC GAA GGC ATC GCC GTG GTG TTC AAG 360

Arg Cys Pro Thr Arg Pro Glu Gly Gln Asn Tyr Thr Glu Gly Ile Ala Val Val Phe Lys

105 110 115 120

GAG AAC ATC GCT CCT TAC AAG TTC AAG GCC ACC ATG TAC TAC AAG GAC GTG ACC GTG AGC 420

Glu Asn Ile Ala Pro Tyr Lys Phe Lys Ala Thr Met Tyr Tyr Lys Asp Val Thr Val Ser

125 130 135 140

CAG GTG TGG TTC GGC CAC CGC TAC TCC CAG TTC ATG GGC ATC TTC GAG GAC CGC GCT CCT 480

Gln Val Trp Phe Gly His Arg Tyr Ser Gln Phe Met Gly Ile Phe Glu Asp Arg Ala Pro

145 150 155 160

GTG CCT TTC GAG GAG GTG ATC GAC AAG ATC AAC GCC AAG GGT GTG TGT AGG TCC ACC GCT 540

Val Pro Phe Glu Glu Val Ile Asp Lys Ile Asn Ala Lys Gly Val Cys Arg Ser Thr Ala

165 170 175 180

AAG TAC GTG CGC AAC AAC CTG GAG ACC ACT GCT TTC CAC AGA GAC GAT CAC GAG ACC GAC 600

Lys Tyr Val Arg Asn Asn Leu Glu Thr Thr Ala Phe His Arg Asp Asp His Glu Thr Asp

185 190 195 200

ATG GAG CTG AAG CCT GCC AAC GCC GCT ACT CGC ACC AGC AGA GGC TGG CAC ACC ACT GAC 660

Met Glu Leu Lys Pro Ala Asn Ala Ala Thr Arg Thr Ser Arg Gly Trp His Thr Thr Asp

205 210 215 220

CTG AAG TAC AAC CCT AGC AGA GTG GAG GCC TTC CAC AGG TAC GGC ACC ACC GTG AAC TGC 720

Leu Lys Tyr Asn Pro Ser Arg Val Glu Ala Phe His Arg Tyr Gly Thr Thr Val Asn Cys

225 230 235 240

ATC GTG GAG GAG GTG GAC GCT CGC AGC GTG TAC CCT TAC GAC GAG TTC GTG CTG GCC ACT 780

Ile Val Glu Glu Val Asp Ala Arg Ser Val Tyr Pro Tyr Asp Glu Phe Val Leu Ala Thr

245 250 255 260

GGT GAC TTC GTG TAC ATG TCT CCT TTC TAC GGC TAC AGA GAA GGT AGC CAC ACC GAG CAC 840

Gly Asp Phe Val Tyr Met Ser Pro Phe Tyr GlY Tyr Arg Glu Gly Ser His Thr Glu His

265 270 275 280

ACT ACC TAC GCT GCT GAC AGG TTC AAG CAG GTG GAC GGC TTC TAC GCT CGC GAC CTG ACC 900

Thr Ser Tyr Ala Ala Asp Arg Phe Lys Gln Val Asp Gly Phe Tyr Ala Arg Asp Leu Thr

285 290 295 300

ACC AAG GCT AGA GCC ACT GCT CCT ACC ACT AGG AAC CTG CTG ACC ACT CCT AAG TTC ACC 960

Thr Lys Ala Arg Ala Thr Ala Pro Thr Thr Arg Asn Leu Leu Thr Thr Pro Lys Phe Thr

305 310 315 320

GTG GCT TGG GAC TGG GTG CCT AAG CGC CCT AGC GTG TGC ACC ATG ACC AAG TGG CAG GAG 1020

Val Ala Trp Asp Trp Val Pro Lys Arg Pro Ser Val Cys Thr Met Thr Lys Trp Gln Glu

325 330 335 340

GTG GAC GAG ATG CTG CGC TCC GAG TAC GGC GGT TCC TTC AGG TTC TCC TCT GAC GCT ATC 1080

Val Asp Glu Met Leu Arg Ser Glu Tyr Gly Gly Ser Phe Arg Phe Ser Ser Asp Ala Ile

345 350 355 360

TCC ACT ACC TTC ACT ACC AAC CTG ACC GAG TAC CCT CTG TCC AGA GTG GAC CTG GGT GAC 1140

Ser Thr Thr Phe Thr Thr Asn Leu Thr Glu Tyr Pro Leu Ser Arg Val Asp Leu Gly Asp

365 370 375 380

TGC ATC GGT AAG GAC GCT CGC GAC GCC ATG GAC CGC ATC TTC GCT CGC AGG TAC AAC GCT 1200

Cys Ile Gly Lys Asp Ala Arg Asp Ala Met Asp Arg Ile Phe Ala Arg Arg Tyr Ash Ala

385 390 395 400

ACT CAC ATC AAG GTG GGC CAG CCT CAG TAC TAC CAG GCC AAC GGT GGT TTC CTG ATC GCC 1260

Thr His Ile Lys Val Gly Gln Pro Gln Tyr Tyr Leu Ala Asn GlY Gly Phe Leu Ile Ala

405 410 415 420

TAC CAG CCT CTG CTG AGC AAC ACT CTG GCT GAG CTG TAC GTG AGA GAG CAC CTG AGA GAG 1320

Tyr Gln Pro Leu Leu Ser Asn Thr Leu Ala Glu Leu Tyr Val Arg Glu His Leu Arg Glu

425 430 435 440

CAG AGC CGC AAG CCT CCT AAC CCT ACG CCT CCT CCT CCC GGT GCT AGC GCC AAC GCT TCC 1380

Gln Ser Pro Lys Pro Pro Asn Pro Thr Pro Pro Pro Pro Gly Ala Ser Ala Asn Ala Ser

445 450 455 460

GTG GAG CGC ATC AAG ACT ACC TCT AGC ATC GAG TTC GCC AGG CTG CAG TTC ACC TAC AAC 1440

Val Glu Arg Ile Lys Thr Thr Ser Ser Ile Glu Phe Ala Arg Leu Gln Phe Thr Tyr Asn

465 470 475 480

CAC ATC CAG CGC CAC GTG AAC GAC ATG CTG GGT CGC GTG GCT ATC GCT TGG TGC GAG CTG 1500

His Ile Gln Asn His Val Asn Asp Met Leu Gly Arg Val Ala Ile Ala Trp Cys Glu Leu

485 490 495 500

CAG AAC CAC GAG CTG ACT CTG TGG AAC GAG GCT CGC AAG CTG AAC CCT AAC GCT ATC GCC 1560

Gln Asn His Glu Leu Thr Leu Trp Asn Glu Ala Arg Lys Leu Asn Pro Asn Ala Ile Ala

505 510 515 520

AGC GTG ACC GTG GGC AGG AGA GTG AGC GCT AGA ATG CTG GGC GAC GTG ATG GCC GTG TCC 1620

Ser Ala Thr Val Gly Arg Arg Val Ser Ala Arg Met Leu Gly Asp Val Met Ala Val Ser

525 530 535 540

ACC TGC GTG CCT GTG GCT GCT GAC AAC GTG ATC GTG CAG AAC AGC ATG CGC ATC AGC TCC 1680

Thr Cys Val Pro Val Ala Ala Asp Asn Val Ile Val Gln Asn Ser Met Arg Ile Ser Ser

545 550 555 560

AGA CCT GGT GCC TGC TAC AGC AGA CCT CTG GTG AGC TTC AGG TAC GAG GAC CAA GGT CCT 1740

Arg Pro Gly Ala Cys Tyr Ser Arg Pro Leu Val Ser Phe Arg Tyr Glu Asp Gln Gly Pro

565 570 575 580

CTG GTG GAA GGT CAG CTG GGT GAG AAC AAC GAG CTG AGG CTG ACT CGC GAC GCT ATC GAG 1800

Leu Val Glu Gly Gln Leu Gly Glu Asn Asn Glu Leu Arg Leu Thr Arg Asp Ala Ile Glu

585 590 595 600

CCT TGC ACC GTC GGT CAC AGA CGC TAC TTC ACC TTC GGT GGC GGT TAC GTG TAC TTC GAG 1860

Pro Cys Thr Val Gly His Arg Arg Tyr Phe Thr Phe Gly Gly Gly Tyr Val Tyr Phe Glu

605 610 615 620

GAG TAC GCT TAC TCT CAC CAG CTG AGC CGC GCT GAC ATC ACT ACC GTG AGC ACC TTC ATC 1920

Glu Ser Ala Tyr Ser His Gln Leu Ser Arg Ala Asp Ile Thr Thr Val Ser Thr Phe Ile

625 630 635 640

GAC CTG AAC ATC ACC ATG CTG GAG GAC CAC GAG TTC GTG CCT CTG GAG GTG TAC ACC CGC 1980

Asp Leu Asn Ile Thr Met Leu Glu Asp His Glu Phe Val Pro Leu Glu Val Tyr Thr Arg

645 650 655 660

CAC GAG ATC AAG GAC AGC GGC CTG CTG GAC TAC ACC GAG GTG CAG CGC CGC AAC CAG CTG 2040

His Glu Ile Lys Asp Ser Gly Leu Leu Asp Tyr Thr Glu Val Gln Arg Arg Asn Gln Leu

665 670 675 680

CAC GAC CTG CGC TTC GCT GAC ATC GAC ACC GTG ATC CAC GCC GAC GCC TAA 2100

His Asp Leu Arg Phe Ala Asp Ile Asp Thr Val Ile His Ala Asp Ala

685 690 695

Claims

1. the extracellular region gene fragment of the HSV1 of chemosynthesis virus gB glycoprotein; this gene fragment coding contains the gB glucoprotein extracellular region gene fragment of the HSV1 virus of strong antigen epi-position; i.e. 696 amino acid of the 1st amino acid to the; totally 696 amino acid; 5 ' end in this gene fragment has increased BamHI restriction enzyme site and two protection bases G C; terminator codon TGA and EcoRI restriction enzyme site and two protection bases G C have been increased at 3 ' end; the gene order total length 2107bp of chemosynthesis, sequence is as follows:

GC GGATCC CCT ACC TCT CCT GGT ACT CCT GGT GTG GCT GCC GCT ACC CAG GCC GCT

AAC GGT GGT CCT GCC ACT CCT GCT CCT CCT CCT CTG GGT GCC GCT CCT ACT GGT

GAC CCT AAG CCT AAG AAG AAC AAG AAG CCT AAG AAC CCT ACT CCT CCT AGG CCT

GCT GGT GAC AAC GCC ACC GTG GCC GCT GGC CAC GCC ACT CTG AGA GAG CAC CTG

AGA GAC ATC AAG GCT GAG AAC ACC GAC GCT AAC TTC TAC GTG TGT CCT CCT CCT

ACT GGT GCC ACC GTG GTG CAG TTC GAG CAG CCT CGC AGG TGC CCT ACC AGG CCT

GAA GGT CAG AAC TAC ACC GAA GGC ATC GCC GTG GTG TTC AAG GAG AAC ATC GCT

CCT TAC AAG TTC AAG GCC ACC ATG TAC TAC AAG GAC GTG ACC GTG AGC CAG GTG

TGG TTC GGC CAC CGC TAC TCC CAG TTC ATG GGC ATC TTC GAG GAC CGC GCT CCT

GTG CCT TTC GAG GAG GTG ATC GAC AAG ATC AAC GCC AAG GGT GTG TGT AGG TCC

ACC GCT AAG TAC GTG CGC AAC AAC CTG GAG ACC ACT GCT TTC CAC AGA GAC GAT

CAC GAG ACC GAC ATG GAG CTG AAG CCT GCC AAC GCC GCT ACT CGC ACC AGC AGA

GGC TGG CAC ACC ACT GAC CTG AAG TAC AAC CCT AGC AGA GTG GAG GCC TTC CAC

AGG TAC GGC ACC ACC GTG AAC TGC ATC GTG GAG GAG GTG GAC GCT CGC AGC GTG

TAC CCT TAC GAC GAG TTC GTG CTG GCC ACT GGT GAC TTC GTG TAC ATG TCT CCT

TTC TAC GGC TAC AGA GAA GGT AGC CAC ACC GAG CAC ACT ACC TAC GCT GCT GAC

AGG TTC AAG CAG GTG GAC GGC TTC TAC GCT CGC GAC CTG ACC ACC AAG GCT AGA

GCC ACT GCT CCT ACC ACT AGG AAC CTG CTG ACC ACT CCT AAG TTC ACC GTG GCT

TGG GAC TGG GTG CCT AAG CGC CCT AGC GTG TGC ACC ATG ACC AAG TGG CAG GAG

GTG GAC GAG ATG CTG CGC TCC GAG TAC GGC GGT TCC TTC AGG TTC TCC TCT GAC

GCT ATC TCC ACT ACC TTC ACT ACC AAC CTG ACC GAG TAC CCT CTG TCC AGA GTG

GAC CTG GGT GAC TGC ATC GGT AAG GAC GCT CGC GAC GCC ATG GAC CGC ATC TTC

GCT CGC AGG TAC AAC GCT ACT CAC ATC AAG GTG GGC CAG CCT CAG TAC TAC CAG

GCC AAC GGT GGT TTC CTG ATC GCC TAC CAG CCT CTG CTG AGC AAC ACT CTG GCT

GAG CTG TAC GTG AGA GAG CAC CTG AGA GAG CAG AGC CGC AAG CCT CCT AAC CCT

ACG CCT CCT CCT CCC GGT GCT AGC GCC AAC GCT TCC GTG GAG CGC ATC AAG ACT

ACC TCT AGC ATC GAG TTC GCC AGG CTG CAG TTC ACC TAC AAC CAC ATC CAG CGC

CAC GTG AAC GAC ATG CTG GGT CGC GTG GCT ATC GCT TGG TGC GAG CTG CAG AAC

CAC GAG CTG ACT CTG TGG AAC GAG GCT CGC AAG CTG AAC CCT AAC GCT ATC GCC

AGC GTG ACC GTG GGC AGG AGA GTG AGC GCT AGA ATG CTG GGC GAC GTG ATG GCC

GTG TCC ACC TGC GTG CCT GTG GCT GCT GAC AAC GTG ATC GTG CAG AAC AGC ATG

CGC ATC AGC TCC AGA CCT GGT GCC TGC TAC AGC AGA CCT CTG GTG AGC TTC AGG

TAC GAG GAC CAA GGT CCT CTG GTG GAA GGT CAG CTG GGT GAG AAC AAC GAG CTG

AGG CTG ACT CGC GAC GCT ATC GAG CCT TGC ACC GTC GGT CAC AGA CGC TAC TTC

ACC TTC GGT GGC GGT TAC GTG TAC TTC GAG GAG TAC GCT TAC TCT CAC CAG CTG

AGC CGC GCT GAC ATC ACT ACC GTG AGC ACC TTC ATC GAC CTG AAC ATC ACC ATG

CTG GAG GAC CAC GAG TTC GTG CCT CTG GAG GTG TAC ACC CGC CAC GAG ATC AAG

GAC AGC GGC CTG CTG GAC TAC ACC GAG GTG CAG CGC CGC AAC CAG CTG CAC GAC

CTG CGC TTC GCT GAC ATC GAC ACC GTG ATC CAC GCC GAC GCC TGA GAATTCGC

2. the gene fragment of the described chemosynthesis HSV1 of claim 1 virus gB glucoprotein extracellular region, it is characterized in that adopting genetic engineering technique to express the gB glucoprotein extracellular region gene fragment of the HSV1 virus of this gene order coding, purifying expressed proteins fragment, concrete grammar is as follows:

Express the gB glucoprotein extracellular region gene fragment construction of recombinant plasmid of HSV1 virus:

GB glucoprotein extracellular region gene fragment with the HSV1 virus of BamH I and EcoR I double digestion plasmid pGEX4T-2 and chemosynthesis, after electrophoresis reclaims, connect with the T4 dna ligase, the gB glucoprotein extracellular region gene fragment of HSV1 virus is inserted between carrier the pGEX4T-2 interior BamHI and EcoRI site, consistent with the initiator codon translation framework on the carrier, express a fusion rotein, 934 amino acid of total length, this fusion rotein N end has merged 238 amino acid on the carrier, the C end comprises interior 696 amino acid of the 1st amino acid to the of gB glycoprotein of HSV1 virus, and full length amino acid sequence is as follows:

Met Pro Met Ile Leu Gly Tyr Trp Asp Ile Arg Gly Leu Ala His Ala Ile Arg

Leu Leu Leu Glu Tyr Thr Asp Ser Ser Tyr Glu Glu Lys Lys Tyr Thr Met Gly

Gly Ala Pro Asp Tyr Asp Arg Ser Gln Trp Leu Asn Glu Lys Phe Lys Leu Gly

Leu Asp Phe Pro Asn Leu Pro Tyr Leu Ile Asp Gly Ala His Lys Ile Thr Gln

Ser Asn Ala Ile Leu Cys Tyr Ile Ala Arg Lys His Asn Leu Cys Gly Glu Thr

Glu Glu Glu Lys Ile Arg Val Asp Ile Leu Glu Asn Gln Ala Met Asp Val Ser

Asn Gln Leu Ala Arg Val Cys Tyr Ser Pro Asp Phe Glu Lys Leu Lys Pro Glu

Tyr Leu Glu Glu Leu Pro Thr Met Met Gln His Phe Ser Gln Phe Leu Gly Lys

Arg Pro Trp Phe Val Gly Asp Lys Ile Thr Phe Val Asp Phe Leu Ala Tyr Asp

Val Leu Asp Leu His Arg Ile Phe Glu Pro Asn Cys Leu Asp Ala Phe Pro Asn

Leu Lys Asp Phe Ile Ser Arg Phe Glu Gly Leu Glu Lys Ile Ser Ala Tyr Met

Lys Ser Ser Arg Phe Leu Pro Lys Pro Leu Tyr Thr Arg Met Ala Val Trp Gly

Asn Lys Pro Ser Ser Pro Gly Thr Pro Gly Val Ala Ala Ala Thr Gln Ala Ala

Asn Gly Gly Pro Ala Thr Pro Ala Pro Pro Ala Leu Gly Ala Ala Pro Thr Gly

Asp Pro Lys Pro Lys Lys Asn Lys Lys Pro Lys Asn Pro Thr Pro Pro Arg Pro

Ala Gly Asp Asn Ala Thr Val Ala Ala Gly His Ala Thr Leu Arg Glu His Leu

Arg Asp Ile Lys Ala Glu Asn Thr Asp Ala Asn Phe Tyr Val Cys Pro Pro Pro

Thr Gly Ala Thr Val Val Gln Phe Glu Gln Pro Arg Arg Cys Pro Thr Arg Pro

Glu Gly Gln Asn Tyr Thr Glu Gly Ile Ala Val Val Phe Lys Glu Asn Ile Ala

Pro Tyr Lys Phe Lys Ala Thr Met Tyr Tyr Lys Asp Val Thr Val Ser Gln Val

Trp Phe Gly His Arg Tyr Ser Gln Phe Met Gly Ile Phe Glu Asp Arg Ala Pro

Val Pro Phe Glu Glu Val Ile Asp Lys Ile Asn Ala Lys Gly Val Cys Arg Ser

Thr Ala Lys Tyr Val Arg Asn Asn Leu Glu Thr Thr Ala Phe His Arg Asp Asp

His Glu Thr Asp Met Glu Leu Lys Pro Ala Asn Ala Ala Thr Arg Thr Ser Arg

Gly Trp His Thr Thr Asp Leu Lys Tyr Asn Pro Ser Arg Val Glu Ala Phe His

Arg Tyr Gly Thr Thr Val Asn Cys Ile Val Glu Glu Val Asp Ala Arg Ser Val

Tyr Pro Tyr Asp Glu Phe Val Leu Ala Thr Gly Asp Phe Val Tyr Met Ser Pro

Phe Tyr Gly Tyr Arg Glu Gly Ser His Thr Glu His Thr Ser Tyr Ala Ala Asp

Arg Phe Lys Gln Val Asp GlY Phe Tyr Ala Arg Asp Leu Thr Thr Lys Ala Arg

Ala Thr Ala Pro Thr Thr Arg Asn Leu Leu Thr Thr Pro Lys Phe Thr Val Ala

Trp Asp Trp Val Pro Lys Arg Pro Ser Val Cys Thr Met Thr Lys Trp Gln Glu

Val Asp Glu Met Leu Arg Ser Glu Tyr Gly Gly Ser Phe Arg Phe Ser Ser Asp

Ala Ile Ser Thr Thr Phe Thr Thr Asn Leu Thr Glu Tyr Pro Leu Ser Arg Val

Asp Leu Gly Asp Cys Ile Gly Lys Asp Ala Arg Asp Ala Met Asp Arg Ile Phe

Ala Arg Arg Tyr Asn Ala Thr His Ile Lys Val Gly Gln Pro Gln Tyr Tyr Leu

Ala Asn Gly Gly Phe Leu Ile Ala Tyr Gln Pro Leu Leu Ser Asn Thr Leu Ala

Glu Leu Tyr Val Arg Glu His Leu Arg Glu Gln Ser Pro Lys Pro Pro Asn Pro

Thr Pro Pro Pro Pro Gly Ala Ser Ala Asn Ala Ser Val Glu Arg Ile Lys Thr

Thr Ser Ser Ile Glu Phe Ala Arg Leu Gln Phe Thr Tyr Asn His Ile Gln Asn

His Val Asn Asp Met Leu Gly Arg Val Ala Ile Ala Trp Cys Glu Leu Gln Asn

His Glu Leu Thr Leu Trp Asn Glu Ala Arg Lys Leu Asn Pro Asn Ala Ile Ala

Ser Ala Thr Val Gly Arg Arg Val Ser Ala Arg Met Leu Gly Asp Val Met Ala

Val Ser Thr Cys Val Pro Val Ala Ala Asp Asn Val Ile Val Gln Asn Ser Met

Arg Ile Ser Ser Arg Pro Gly Ala Cys Tyr Ser Arg Pro Leu Val Ser Phe Arg

Tyr Glu Asp Gln Gly Pro Leu Val Glu Gly Gln Leu Gly Glu Asn Asn Glu Leu

Arg Leu Thr Arg Asp Ala Ile Glu Pro Cys Thr Val Gly His Arg Arg Tyr Phe

Thr Phe Gly Gly Gly Tyr Val Tyr Phe Glu Glu Ser Ala Tyr Ser His Gln Leu

Ser Arg Ala Asp Ile Thr Thr Val Ser Thr Phe Ile Asp Leu Asn Ile Thr Met

Leu Glu Asp His Glu Phe Val Pro Leu Glu Val Tyr Thr Arg His Glu Ile Lys

Asp Ser Gly Leu Leu Asp Tyr Thr Glu Val Gln Arg Arg Asn Gln Leu His Asp

Leu Arg Phe Ala Asp Ile Asp Thr Val Ile His Ala Asp Ala

The Screening and Identification of expressed fusion protein engineering bacteria:

Express the purifying of HSV1 virus gB glycoprotein:

With the centrifugal receipts of the engineering bacteria of abduction delivering fusion rotein bacterium, thalline is resuspended in the lysate, lysate is 50mmol/L Tris-HCl pH8.0,10mmol/L EDTA, 10mmol/L DTT, carrying out ultrasonic bacteria breaking 10min, centrifugal collection supernatant, supernatant solution add equilibrated Glutathione Sepharose 4B gel 3ml, and room temperature is in conjunction with 60min, last sample is collected and is penetrated liquid; 1 * PBS washing pillar with ten times of column volumes, then use the GSH elutriant of 15ml high density, elutriant is 50mmol/LTris-HCL pH8.0+10mmol/LGST, divides three times the wash-out target protein, is the HSV1 virus gB glucoprotein extracellular region gene fragment of purifying.

3. the HSV1 of the described chemosynthesis of claim 1 virus gB glucoprotein extracellular region gene fragment is characterized in that utilizing bacterium, yeast cell, insect cell, mammalian cell and genetically modified animals and plants to carry out recombinant expressed, preparation.

4. the application of HSV1 virus gB glucoprotein extracellular region gene fragment in preparation HSV1 viral sub-units vaccine of claim 2 or 3 described method preparations.