CN101173290B - Chemically synthesized HSV1 virus gB glucoprotein extracellular region gene fragment, representation and application of the same - Google Patents
Chemically synthesized HSV1 virus gB glucoprotein extracellular region gene fragment, representation and application of the same Download PDFInfo
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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
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
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 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 |
1∶1000 | 1∶2000 | 1∶4000 | 1∶8000 | 1∶16000 | 1∶32000 |
| HSVlgBControl | 0.8140.021 | 0.5390.019 | 0.428 0.025 | 0.4000.011 | 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 (4)
1. the extracellular region gene fragment of the HSV1 of chemosynthesis virus gB glycoprotein; this gene fragment coding contains the gB glucoprotein extracellular region 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 segmental purifying expression method of gB glucoprotein extracellular region of the HSV1 virus of the described gene fragment of claim 1 coding, it is characterized in that adopting genetic engineering technique to express the gB glucoprotein extracellular region 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 Bam HI and EcoR I double digestion plasmid pGEX4T-2 and chemosynthesis, after electrophoresis reclaims, connect with the T4DNA ligase enzyme, the gB glucoprotein extracellular region gene fragment of HSV1 virus 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, 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:
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;
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; 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.
3. the segmental purifying expression method of gB glucoprotein extracellular region of the HSV1 virus of the described gene fragment coding of claim 1 is characterized in that utilizing bacterium to carry out recombinant expressed, preparation.
4. the application of HSV1 virus gB glucoprotein extracellular region fragment in preparation HSV1 viral sub-units vaccine of claim 2 or 3 described method preparations.
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| CN110551187B (en) * | 2019-09-23 | 2022-09-16 | 新乡学院 | Chemically synthesized H7N9 avian influenza virus NA protein extracellular region antigen segment, preparation method and application |
| CN113755526A (en) * | 2021-09-29 | 2021-12-07 | 中国科学院武汉病毒研究所 | Application of HSV-2 envelope glycoprotein gJ in improving expression level of exogenous gene in mammalian cell |
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| CN1472321A (en) * | 2003-06-13 | 2004-02-04 | 李越希 | Chemosynthesized SARS virus S gene segement, its expression and application |
| CN1651081A (en) * | 2004-12-01 | 2005-08-10 | 广州军区广州总医院 | Herpes simplex virus II type DNA vaccine |
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| CN1472321A (en) * | 2003-06-13 | 2004-02-04 | 李越希 | Chemosynthesized SARS virus S gene segement, its expression and application |
| CN1651081A (en) * | 2004-12-01 | 2005-08-10 | 广州军区广州总医院 | Herpes simplex virus II type DNA vaccine |
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| SANCHEZPESCADOR L.EPITOPES OF HERPES-SIMPLEX VIRUS TYPE-1 GLYCOPROTEIN-B THAT BIND TYPE-COMMON NEUTRALIZING ANTIBODIES ELICIT TYPE-SPECIFIC ANTIBODY-DEPENDENT CELLULAR CYTOTOXICITY.《JOURNAL OF INFECTIOUS DISEASES》.1992,第166卷(第3期),全文. * |
| 冯非.单纯疱疹病毒1型糖蛋白B截短基因疫苗的构建及初步免疫学研究.《吉林大学硕士学位论文》.2005,全文. * |
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