CN103740736A - Chemically synthesized gene segment of gE (glycoprotein E) extracellular domain of HSV2 (herpes simplex virus) as well as expression method and application thereof - Google Patents
Chemically synthesized gene segment of gE (glycoprotein E) extracellular domain of HSV2 (herpes simplex virus) as well as expression method and application thereof Download PDFInfo
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Abstract
The invention provides a chemically synthesized gene segment of a gE (glycoprotein E) extracellular domain of an HSV2 (herpes simplex virus) as well as an expression method and application thereof, relating to the fields of gene engineering technology, vaccines and diagnostic reagents. A strong epitope in gE of the HSV2 is screened through computer analysis, namely the 21st amino acid to the 414th amino acid, totally 394 amino acids, and codons favored by both eukaryotes and prokaryotes are selected to chemically synthesize a brand-new gene sequence of the epitope. The gene segment is expressed and the strong epitope segment of gE of the HSV2 is prepared by utilizing the gene engineering technology. The expressed strong epitope segment of gE of the HSV2 can be used for HSV2 vaccine development, antibody detection and immunologic preparation of HSV2 monoclonal antibodies, multi-antibodies and the like.
Description
Technical field
What the present invention relates to is the brand-new gene fragment of the HSV2 virus gE glucoprotein extracellular region of chemosynthesis, utilizes genetic engineering technique, preparation restructuring HSV2 virus gE glycoprotein.By Computer Analysis, filter out the gE glucoprotein extracellular region fragment containing the HSV2 virus of 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, the albumen of expressing can be used for the detection of vaccine and HSV2 antiviral antibody or antigen etc., the present invention relates to genetic engineering technique, vaccine and diagnostic reagent field.
Background technology
Hsv (Herpers simplex virus, HSV) belongs to herpetoviridae α subfamily, is one of common virus of human infection, according to serotype, can be divided into two kinds, I and II type.Wherein, I type is mainly by a mouthful facial infection, and also existence is propagated, and the main trafficability characteristic of II type is propagated, and closely related with the generation of women's cervical cancer.Increasing research shows in the recent period, and HSV I is relevant to the generation of Alzheimer's disease, and the infection of HIV and HSV II are also related.HSV is generally popular in the whole world, and positive rate is up to more than 85% in HAS for HSV I type, and HSV II type whole world seroprevalence is about 20%, and number of the infected increases with annual 25% speed.
HSV can infected tissue or near position breeding form primary lesion, and set up lifelong latent infection at nuclei quintus spinal nerves, when body is subject to external stimulus or immunity degradation is, HSV virus comes downwards to epidermis breeds in a large number, forms recurrent infection.HSV shows effect relevant to HSV immunologic escape repeatedly, and HSV realizes latent infection and recurrent infection by hiding human immune system's attack, once therefore HSV infects, is difficult to be eliminated, and patient is subject to prolonged sickness and torments.
Viral glycoprotein (glycoproteins, gp) be the antigenic determinant on viral virion surface, found that also the envelope glycoprotein of definite designation has 12 (gB, gC, gD, gE, gG, gH, gI, gK, gL and gM etc.) at present.GB and gD glycoprotein participate in absorption and the fusion of virus and host cell, have been selected as candidate vaccine, more to its research.Research in recent years discovery, gE2 is the envelope glycoprotein that immunologic escape is relevant.HSV virus envelope protein gE and IgG Fc hold combination, thereby block the immunization route of multiple complement-mediated, make HSV virus successfully hide the immune system attack of body.Therefore he is one of protein molecular causing virus lays dormant infection.Choose gE glycoprotein for immune body, the latent infection of blocking virus, promises to be desirable candidate vaccine.
The present invention is by Computer Analysis, filter out the gE glucoprotein extracellular region fragment containing the HSV2 virus of 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, expression product has good antigenicity and specificity, and the albumen of expression can be used for the detection of vaccine and HSV2 antiviral antibody or antigen etc.
Summary of the invention
The present invention is the brand-new gene fragment of the HSV2 virus gE glucoprotein extracellular region of chemosynthesis, utilizes genetic engineering technique, preparation restructuring HSV2 virus gE glucoprotein extracellular region fragment.By Computer Analysis, filter out the HSV2 virus gE glucoprotein extracellular region fragment containing strong antigen epi-position, the 21st amino acid-414th amino acid, totally 394 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.The albumen of expressing can be used for the detection of vaccine, HSV2 antiviral antibody or antigen and prepares anti-HSV2 virus monoclonal antibody and how anti-etc. for immunity.
The present invention is by Computer Analysis, filter out the gE glucoprotein extracellular region fragment containing the HSV2 virus of 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, expression product has good antigenicity and specificity, and the albumen of expression can be used for the detection of vaccine and HSV2 antiviral antibody or antigen etc.
the HSV2 virus gE glucoprotein extracellular region gene fragment of chemosynthesis and expression thereof, application take following steps to realize:
The screening of 1.HSV2 virus gE glucoprotein extracellular region epitope and the chemosynthesis of gene fragment thereof:
Utilize the softwares such as ANTHEWIN, by the aminoacid sequence of Computer Analysis HSV2 virus gE glycoprotein, find that the N end (the 21st amino acid-414th amino acid) of gE 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 increased at 5' end
ecorI restriction enzyme site (underscore part), initiator codon (ATG), signal peptide (GGCTGGAGCTGTATTATCCTGTTCCTGGTCGCCACTGCCACTGGAGTTCATTCTGC C), 3' end increased His mark (CAC CAC CAC CAC CAC CAC), terminator codon (TGA) and
himdiII restriction enzyme site (underscore part), makes this gene fragment be easy to be cloned in plasmid pMCE5
ecorI and
himdin III restriction enzyme site.
Epitope aminoacid sequence (414 aa of the 21st aa-) in the HSV2 virus gE glycoprotein of screening:
Ala?Ala?Pro?Arg?Thr?Ser?Trp?Lys?Arg?Val?Thr?Ser?Gly?Glu?Asp?Val?Val?Leu?Leu?Pro?Ala?Pro?Ala?Glu?Arg?Thr?Arg?Ala?His?Lys?Leu?Leu?Trp?Ala?Ala?Glu?Pro?Leu?Asp?Ala?Cys?Gly?Pro?Leu?Arg?Pro?Ser?Trp?Val?Ala?Leu?Trp?Pro?Pro?Arg?Arg?Val?Leu?Glu?Thr?Val?Val?Asp?Ala?Ala?Cys?Met?Arg?Ala?Pro?Glu?Pro?Leu?Ala?Ile?Ala?Tyr?Ser?Pro?Pro?Phe?Pro?Ala?Gly?Asp?Glu?Gly?Leu?Tyr?Ser?Glu?Leu?Ala?Trp?Arg?Asp?Arg?Val?Ala?Val?Val?Asn?Glu?Ser?Leu?Val?Ile?Tyr?Gly?Ala?Leu?Glu?Thr?Asp?Ser?Gly?Leu?Tyr?Thr?Leu?Ser?Val?Val?Gly?Leu?Ser?Asp?Glu?Ala?Arg?Gln?Val?Ala?Ser?Val?Val?Leu?Val?Val?Glu?Pro?Ala?Pro?Val?Pro?Thr?Pro?Thr?Pro?Asp?Asp?Tyr?Asp?Glu?Glu?Asp?Asp?Ala?Gly?Val?Thr?Asn?Ala?Arg?Arg?Ser?Ala?Phe?Pro?Pro?Gln?Pro?Pro?Pro?Arg?Arg?Pro?Pro?Val?Ala?Pro?Pro?Thr?His?Pro?Arg?Val?Ile?Pro?Glu?Val?Ser?His?Val?Arg?Gly?Val?Thr?Val?His?Met?Glu?Thr?Leu?Glu?Ala?Ile?Leu?Phe?Ala?Pro?Gly?Glu?Thr?Phe?Gly?Thr?Asn?Val?Ser?Ile?His?Ala?Ile?Ala?His?Asp?Asp?Gly?Pro?Tyr?Ala?Met?Asp?Val?Val?Trp?Met?Arg?Phe?Asp?Val?Pro?Ser?Ser?Cys?Ala?Asp?Met?Arg?Ile?Tyr?Glu?Ala?Cys?Leu?Tyr?His?Pro?Gln?Leu?Pro?Glu?Cys?Leu?Ser?Pro?Ala?Asp?Ala?Pro?Cys?Ala?Val?Ser?Ser?Trp?Ala?Tyr?Arg?Leu?Ala?Val?Arg?Ser?Tyr?Ala?Gly?Cys?Ser?Arg?Thr?Thr?Pro?Pro?Pro?Arg?Cys?Phe?Ala?Glu?Ala?Arg?Met?Glu?Pro?Val?Pro?Gly?Leu?Ala?Trp?Leu?Ala?Ser?Thr?Val?Asn?Leu?Glu?Phe?Gln?His?Ala?Ser?Pro?Gln?His?Ala?Gly?Leu?Tyr?Leu?Cys?Val?Val?Tyr?Val?Asp?Asp?His?Ile?His?Ala?Trp?Gly?His?Met?Thr?Ile?Ser?Thr?Ala?Ala?Gln?Tyr?Arg?Asn?Ala?Val?Val?Glu?Gln?His?Leu?Pro?Gln?Arg?Gln?Pro?Glu?Pro?Val?Glu?Pro?Thr?Arg?Pro?His?Val?Arg?Ala?Pro?His?Pro?Ala?Pro?Ser?Ala?Arg?Gly?Pro?Leu?Arg
The DNA sequence (1282 bp) containing HSV2 virus gE glucoprotein extracellular region antigen epitope genes of chemosynthesis:
GAATTC?GCC?GCC?ACC?ATG?GGC?TGG?AGC?TGT?ATT?ATC?CTG?TTC?CTG?GTC?GCC?ACT?GCC?ACT?GGA?GTT?CAT?TCT?GCC?GCC?CCT?CGG?ACA?TCT?TGG?AAA?AGG?GTG?ACC?AGC?GGA?GAG?GAT?GTG?GTC?CTG?CTG?CCT?GCA?CCA?GCT?GAA?AGG?ACA?CGA?GCA?CAC?AAG?CTG?CTG?TGG?GCA?GCT?GAG?CCT?CTG?GAC?GCA?TGC?GGT?CCA?CTG?CGA?CCA?TCT?TGG?GTG?GCC?CTG?TGG?CCA?CCT?AGG?CGG?GTC?CTG?GAG?ACC?GTG?GTC?GAT?GCA?GCC?TGT?ATG?AGA?GCT?CCC?GAA?CCT?CTG?GCA?ATC?GCC?TAC?TCT?CCA?CCC?TTC?CCA?GCA?GGC?GAT?GAG?GGA?CTG?TAT?AGT?GAA?CTG?GCT?TGG?AGA?GAC?CGC?GTG?GCA?GTG?GTC?AAC?GAG?TCC?CTG?GTC?ATC?TAC?GGC?GCC?CTG?GAA?ACA?GAT?AGC?GGA?CTG?TAT?ACT?CTG?TCA?GTG?GTC?GGA?CTG?TCC?GAC?GAG?GCA?CGA?CAG?GTG?GCT?TCC?GTG?GTC?CTG?GTG?GTC?GAA?CCA?GCA?CCA?GTG?CCT?ACT?CCA?ACC?CCA?GAC?GAT?TAC?GAC?GAG?GAA?GAC?GAT?GCT?GGC?GTG?ACT?AAC?GCA?AGA?CGC?TCA?GCC?TTC?CCT?CCA?CAG?CCA?CCT?CCA?AGG?AGG?CCC?CCT?GTG?GCA?CCA?CCA?ACC?CAC?CCA?CGC?GTC?ATC?CCA?GAG?GTG?TCC?CAC?GTC?CGA?GGA?GTG?ACT?GTC?CAT?ATG?GAG?ACC?CTG?GAA?GCT?ATT?CTG?TTC?GCA?CCT?GGG?GAA?ACA?TTT?GGT?ACT?AAT?GTG?AGT?ATC?CAC?GCT?ATT?GCA?CAT?GAC?GAT?GGA?CCC?TAT?GCT?ATG?GAC?GTG?GTC?TGG?ATG?CGA?TTT?GAT?GTG?CCT?TCC?AGC?TGC?GCC?GAC?ATG?AGG?ATC?TAC?GAG?GCT?TGT?CTG?TAT?CAT?CCT?CAG?CTG?CCA?GAA?TGC?CTG?TCC?CCT?GCC?GAT?GCT?CCA?TGT?GCC?GTG?TCT?AGT?TGG?GCC?TAC?CGA?CTG?GCT?GTG?CGT?AGC?TAT?GCT?GGC?TGC?TCT?AGG?ACC?ACA?CCT?CCA?CCC?CGG?TGT?TTC?GCA?GAG?GCC?AGA?ATG?GAA?CCT?GTG?CCA?GGA?CTG?GCT?TGG?CTG?GCA?AGT?ACA?GTG?AAC?CTG?GAG?TTT?CAG?CAC?GCA?TCA?CCA?CAG?CAT?GCC?GGG?CTG?TAC?CTG?TGC?GTG?GTC?TAT?GTG?GAC?GAT?CAC?ATC?CAT?GCC?TGG?GGT?CAT?ATG?ACC?ATT?AGC?ACA?GCT?GCA?CAG?TAC?AGG?AAT?GCT?GTG?GTC?GAG?CAG?CAC?CTG?CCA?CAG?CGT?CAG?CCA?GAG?CCT?GTG?GAA?CCA?ACC?CGC?CCT?CAT?GTC?AGA?GCA?CCC?CAC?CCC?GCC?CCT?TCA?GCC?AGA?GGA?CCC?CTG?CGT?CAC?CAC?CAC?CAC?CAC?CAT?TGA?T
AAGCTT
2. express the structure of HSV2 virus gE glucoprotein extracellular region fragment recombinant plasmid:
Extract plasmid pMCE5, use
ecor I and
hindiII double digestion, reclaims the plasmid large fragment that enzyme is cut after electrophoresis, be dissolved in deionized water.Same using
ecor I and
hindthe HSV2 virus gE glycoprotein gene fragment of III double digestion chemosynthesis, electrophoresis is dissolved in deionized water after reclaiming.
Above-mentioned two kinds of enzymes of volumetric molar concentration such as get and cut rear DNA fragmentation, in same centrifuge tube, with T4 DNA ligase enzyme, connect, HSV2 virus gE glycoprotein gene fragment is inserted in carrier pMCE5
ecor I and
hindbetween III site, consistent with the initiator codon translation framework on carrier, express a fusion rotein.
3. the screening of recombinant plasmid and evaluation:
By recombinant plasmid transformed bacillus coli DH 5 alpha, coating, containing the LB flat board of 100 μ g/ml penbritins, is put 37 ℃ and is spent the night.Next day, random picking transformed bacterium colony, containing plasmid pMCE5 transformed bacteria, make negative control, containing plasmid pUC57-gC21 transformed bacteria, make positive control, bacterium liquid pcr amplification HSV2 virus gE glucoprotein extracellular region gene fragment, containing the positive recombinant plasmid of HSV2 virus gE glucoprotein extracellular region gene fragment, should amplify the gene fragment that is about 1282bp.Extracting positive single bacterium colony plasmid, to carry out double digestion evaluation and the plasmid that contains foreign gene is carried out to DNA sequence analysis, sequential analysis confirms that recombinant plasmid contains synthetic HSV2 virus gE glycoprotein gene fragment, sequence is entirely true:
GCC?GCC?ACC?ATG?GGC?TGG?AGC?TGT?ATT?ATC?CTG?TTC?CTG?GTC?GCC?ACT?GCC?ACT?GGA?GTT?CAT?TCT?GCC?GCC?CCT?CGG?ACA?TCT?TGG?AAA?AGG?GTG?ACC?AGC?GGA?GAG?GAT?GTG?GTC?CTG?CTG?CCT?GCA?CCA?GCT?GAA?AGG?ACA?CGA?GCA?CAC?AAG?CTG?CTG?TGG?GCA?GCT?GAG?CCT?CTG?GAC?GCA?TGC?GGT?CCA?CTG?CGA?CCA?TCT?TGG?GTG?GCC?CTG?TGG?CCA?CCT?AGG?CGG?GTC?CTG?GAG?ACC?GTG?GTC?GAT?GCA?GCC?TGT?ATG?AGA?GCT?CCC?GAA?CCT?CTG?GCA?ATC?GCC?TAC?TCT?CCA?CCC?TTC?CCA?GCA?GGC?GAT?GAG?GGA?CTG?TAT?AGT?GAA?CTG?GCT?TGG?AGA?GAC?CGC?GTG?GCA?GTG?GTC?AAC?GAG?TCC?CTG?GTC?ATC?TAC?GGC?GCC?CTG?GAA?ACA?GAT?AGC?GGA?CTG?TAT?ACT?CTG?TCA?GTG?GTC?GGA?CTG?TCC?GAC?GAG?GCA?CGA?CAG?GTG?GCT?TCC?GTG?GTC?CTG?GTG?GTC?GAA?CCA?GCA?CCA?GTG?CCT?ACT?CCA?ACC?CCA?GAC?GAT?TAC?GAC?GAG?GAA?GAC?GAT?GCT?GGC?GTG?ACT?AAC?GCA?AGA?CGC?TCA?GCC?TTC?CCT?CCA?CAG?CCA?CCT?CCA?AGG?AGG?CCC?CCT?GTG?GCA?CCA?CCA?ACC?CAC?CCA?CGC?GTC?ATC?CCA?GAG?GTG?TCC?CAC?GTC?CGA?GGA?GTG?ACT?GTC?CAT?ATG?GAG?ACC?CTG?GAA?GCT?ATT?CTG?TTC?GCA?CCT?GGG?GAA?ACA?TTT?GGT?ACT?AAT?GTG?AGT?ATC?CAC?GCT?ATT?GCA?CAT?GAC?GAT?GGA?CCC?TAT?GCT?ATG?GAC?GTG?GTC?TGG?ATG?CGA?TTT?GAT?GTG?CCT?TCC?AGC?TGC?GCC?GAC?ATG?AGG?ATC?TAC?GAG?GCT?TGT?CTG?TAT?CAT?CCT?CAG?CTG?CCA?GAA?TGC?CTG?TCC?CCT?GCC?GAT?GCT?CCA?TGT?GCC?GTG?TCT?AGT?TGG?GCC?TAC?CGA?CTG?GCT?GTG?CGT?AGC?TAT?GCT?GGC?TGC?TCT?AGG?ACC?ACA?CCT?CCA?CCC?CGG?TGT?TTC?GCA?GAG?GCC?AGA?ATG?GAA?CCT?GTG?CCA?GGA?CTG?GCT?TGG?CTG?GCA?AGT?ACA?GTG?AAC?CTG?GAG?TTT?CAG?CAC?GCA?TCA?CCA?CAG?CAT?GCC?GGG?CTG?TAC?CTG?TGC?GTG?GTC?TAT?GTG?GAC?GAT?CAC?ATC?CAT?GCC?TGG?GGT?CAT?ATG?ACC?ATT?AGC?ACA?GCT?GCA?CAG?TAC?AGG?AAT?GCT?GTG?GTC?GAG?CAG?CAC?CTG?CCA?CAG?CGT?CAG?CCA?GAG?CCT?GTG?GAA?CCA?ACC?CGC?CCT?CAT?GTC?AGA?GCA?CCC?CAC?CCC?GCC?CCT?TCA?GCC?AGA?GGA?CCC?CTG?CGT?CAC?CAC?CAC?CAC?CAC?CAT?TGA?T
The viral gE glucoprotein extracellular region fragment (394 amino acid) of the expression of recombinant plasmid HSV2 building, has merged 19 amino acid on carrier at its N end, at its C end, has merged 6 amino acid on carrier, 419 amino acid of total length, and its aminoacid sequence is as follows:
N end: Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly Val His Ser
C end: His His His His His His
4. the expression identification of recombinant plasmid in Chinese hamster ovary celI:
Chinese hamster ovary celI shakes cultivation, 37 ℃, 5% CO at the cell expressing substratum that does not contain serum in Erlenmeyer flask
2.Transfection the day before yesterday, with suitable concentration, Chinese hamster ovary celI is inoculated in not containing in the cell expressing substratum of serum to concussion suspension culture, 37 ℃, 5% CO
2.With DNA and PEI mixture transfection recombinant plasmid.The 5th day collecting cell supernatant after transfection, detects protein expression level, within the 6th day, collects all supernatants for purification of recombinant proteins.
5. the purifying of recombinant protein, evaluation and concentration detect:
Collecting cell supernatant 100ml, adds His Trap by supernatant with the speed of 1ml/min after centrifugal
tMin FF crude column.10 times of volumetric balance liquid (1 × PBS, 0.5mol/L NaCl, 20mmol/L imidazole, pH7.4) wash-out foreign protein, then add 1ml elutriant (1 × PBS, 0.5mol/L NaCl, 500mmol/L imidazole, pH7.4) wash-out target protein, wash-out after standing 20 minutes, collect elutriant, with dialyzate (1 × PBS, 0.5mol/L NaCl, pH7.4) dialysed overnight, be the target protein of purifying, SDS-PAGE, Western Blot detect, and measure protein concentration.
6. the sero-fast preparation of immune mouse and gE2:
10 male mouse of kunming are divided into 2 groups at random, 5 every group.Experimental group is mixed with freund's adjuvant equal-volume with the restructuring gE2 glycoprotein of purifying, fully emulsified, respectively at the 0th, and 2,4 weeks, abdominal injection immune mouse, 2 ug//times, only, control group is with PBS equal-volume mixing freund's adjuvant, fully emulsified for 0.1ml/, and 0.1ml/ is only.Eye socket blood sampling in 2 weeks after each immunity.37 ℃ of blood are placed after 1 hour 4 ℃ and are spent the night, and centrifugal 10 minutes of 4000rpm, gets supernatant and obtain recombinant protein gE2 antiserum(antisera).
7. the immunogenicity of recombinant protein gE2 detects:
With 1 × CBS, the restructuring gE2 glycoprotein of purifying being diluted is 1 ug/ml, coated elisa plate, and every hole 100ul, 4 ℃ are spent the night.Next day 5%FBS sealase yoke plate, every hole 130ul, room temperature 2 hours.The antiserum(antisera) of preparation is pressed to 1:500 with sample diluent, and 1:2500, after 1:12500 stepwise dilution, add to respectively in the enzyme linked plate holes after sealing, every hole 100ul, 37 ℃ are reacted 1 hour, wash after 5 times with PBST, add the goat anti-mouse igg-HRP of 1:5000 dilution, every hole 100ul, 37 ℃ are reacted 30 minutes, PBST washes 5 times, adds every hole 100 ul of substrate TMB solution, and 37 ℃ of lucifuges develop the color 10 minutes, add 50ul 1N hydrochloric acid termination reaction, with enzyme connection instrument mensuration A
450value.Result is judged, with testing sample A value and the ratio of negative control A value, is more than or equal to 2.1(P/N >=2.1) and A value >=0.15 positive, the serum antibody titer that the antibody dilution of demonstration positive findings maximum is immune mouse.Mean ± standard deviation for data (
) represent, carry out statistical analysis, carry out statistical analysis with GraphPad Prism 5.0, SPSS software, think that P < 0.05 is for there being significant difference.
The gene fragment order of the HSV2 virus gE glucoprotein extracellular region of described chemosynthesis, utilizes bacterium, yeast cell, insect cell, mammalian cell and genetically modified animals and plants to carry out recombinant expressed, preparation.
HSV2 virus gE glucoprotein extracellular region fragment prepared by method described above, for development and the antibody test of HSV2 viral sub-units vaccine.
the present invention compared with prior art has advantages of
The gE glycoprotein fragment of the HSV2 virus that the present invention expresses, has more advantages:
1. the current enzyme-linked immunologic detecting kit of the HSV2 antiviral antibody of application, the antigen of its use is totivirus antigen, produces more dangerous, cost is high, and other virus have the shortcomings such as cross reaction.The gE glucoprotein extracellular region fragment of the HSV2 virus of expressing can overcome above-mentioned shortcoming as antigen.
2. lack at present HSV2 virus vaccines.The research of inactivated vaccine has obtained greater advance, but production cost is high, danger large.The gE glycoprotein of HSV2 virus content in the cell infecting is maximum, the conservative albumen of simplexvirus family camber, each malicious strain difference is less, gE albumen has stronger immunogenicity simultaneously, can induce body to produce neutralizing antibody and cell immune response, and relevant with viral escape mechanism, therefore HSV2-gE glycoprotein is to build the desirable goal gene of HSV vaccine.We have selected its strong antigen epi-position, utilize genetic engineering technique to express preparation, for development recombinant vaccine lays the foundation.Recombinant vaccine safety, cost are low.
3. according to the gE glucoprotein extracellular region fragment aminoacid sequence of the HSV2 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 suitable in eucaryon and prokaryotic cell prokaryocyte high expression level.
4. the Chinese hamster ovary celI strain of the gE glycoprotein of the expression HSV2 virus that the present invention builds, can prepare this albumen by large-scale purification, is easy to purifying, and has great immunogenicity.Have no at present the report of expressing this section of albumen.
Accompanying drawing explanation
Below with reference to accompanying drawing, the invention will be further described:
Fig. 1 is the pcr amplification product that bacterium liquid PCR detects 8 recons.1:2000 DL DNA(TaKaRa); 2:gE2 gene PCR product; 3-10:1-8 picking list bacterium colony bacterium liquid PCR product; 11: empty pMCE5/DH5 α bacterium liquid PCR product.
Fig. 2 is double digestion qualification result.1:5000 DL DNA(TaKaRa); 2: recombinant plasmid warp
ecorI and
hindiII double digestion product; 3: recombinant plasmid warp
ecorI single endonuclease digestion product; 4:gE2 gene.
Fig. 3 is the SDS-PAGE analytical results of expressing the gE glycoprotein recombinant bacterium of HSV2 virus.M:Protein Marker; 1: electrophoresis under reductive condition; 2: non-reduced condition electrophoresis
Fig. 4 is the Western-Blots analytical results of expressing the gE glycoprotein purification front and back of HSV2 virus.M:Protein Marker; 1: electrophoresis under reductive condition; 2: non-reduced condition electrophoresis; P:Multiple-tag joins as sun.
Fig. 5 is that ELISA detects recombinant protein gE2 immunogenicity.
Embodiment
The detailed description of embodiment of the present invention:
the analysis of the gE glycoprotein antigen epi-position of HSV2 virus, gene synthesize and express
Whole aminoacid sequences of the gE glycoprotein by Computer Analysis HSV2 virus, filter out the strong antigen epi-position in the gE glycoprotein of HSV2 virus, select the codon of eucaryon preference, the brand-new gene fragment of the gE glycoprotein strong antigen epi-position of chemosynthesis HSV2 virus.By gene fragment clone in plasmid pMCE5
ecorI/
hindiII site, consistent with the translation framework of the initiator codon on carrier, can express a fusion rotein.The eukaryon expression plasmid pMCE5-gE2 of structure transfection to Chinese hamster ovary celI are carried out to eukaryotic expression, and screening has obtained the cell strain of high expression level gE glycoprotein.
materials and methods
1. bacterial classification and plasmid: Host Strains DH5 α and expression vector pMCE5 are for preserving in this laboratory.
2. molecular biology reagent: restriction enzyme
ecorI,
hindiII and T4 DNA ligase are TaKaRa company product.Plasmid purification test kit and be TaKaRa company product from the test kit that reclaims DNA fragmentation in sepharose. other reagent is import or domestic analytical reagent.
3. gene fragment is synthetic: by Dalian TaKaRa company, helped synthetic.
4. the enzyme of gene clone method: DNA is cut, connection, electrophoresis; The extraction of plasmid, conversion.Other test kit by specification operates.
5. DNA sequence analysis: with TAKARA company plasmid purification test kit plasmid purification, with the full-automatic sequenator order-checking of DNA.
result
1. the screening of the gE glycoprotein antigen epi-position of HSV2 virus and gene fragment is synthetic:
Utilize the softwares such as ANTHEWIN, whole aminoacid sequences (UniProtKB:P89475.1) of the gE glycoprotein by Computer Analysis HSV2 virus, filter out the strong antigen epi-position in the gE glycoprotein of HSV2 virus, from 414 amino acid of the 21st amino acid to the, its aminoacid sequence is as follows:
Ala?Ala?Pro?Arg?Thr?Ser?Trp?Lys?Arg?Val?Thr?Ser?Gly?Glu?Asp?Val?Val?Leu?Leu?Pro?Ala?Pro?Ala?Glu?Arg?Thr?Arg?Ala?His?Lys?Leu?Leu?Trp?Ala?Ala?Glu?Pro?Leu?Asp?Ala?Cys?Gly?Pro?Leu?Arg?Pro?Ser?Trp?Val?Ala?Leu?Trp?Pro?Pro?Arg?Arg?Val?Leu?Glu?Thr?Val?Val?Asp?Ala?Ala?Cys?Met?Arg?Ala?Pro?Glu?Pro?Leu?Ala?Ile?Ala?Tyr?Ser?Pro?Pro?Phe?Pro?Ala?Gly?Asp?Glu?Gly?Leu?Tyr?Ser?Glu?Leu?Ala?Trp?Arg?Asp?Arg?Val?Ala?Val?Val?Asn?Glu?Ser?Leu?Val?Ile?Tyr?Gly?Ala?Leu?Glu?Thr?Asp?Ser?Gly?Leu?Tyr?Thr?Leu?Ser?Val?Val?Gly?Leu?Ser?Asp?Glu?Ala?Arg?Gln?Val?Ala?Ser?Val?Val?Leu?Val?Val?Glu?Pro?Ala?Pro?Val?Pro?Thr?Pro?Thr?Pro?Asp?Asp?Tyr?Asp?Glu?Glu?Asp?Asp?Ala?Gly?Val?Thr?Asn?Ala?Arg?Arg?Ser?Ala?Phe?Pro?Pro?Gln?Pro?Pro?Pro?Arg?Arg?Pro?Pro?Val?Ala?Pro?Pro?Thr?His?Pro?Arg?Val?Ile?Pro?Glu?Val?Ser?His?Val?Arg?Gly?Val?Thr?Val?His?Met?Glu?Thr?Leu?Glu?Ala?Ile?Leu?Phe?Ala?Pro?Gly?Glu?Thr?Phe?Gly?Thr?Asn?Val?Ser?Ile?His?Ala?Ile?Ala?His?Asp?Asp?Gly?Pro?Tyr?Ala?Met?Asp?Val?Val?Trp?Met?Arg?Phe?Asp?Val?Pro?Ser?Ser?Cys?Ala?Asp?Met?Arg?Ile?Tyr?Glu?Ala?Cys?Leu?Tyr?His?Pro?Gln?Leu?Pro?Glu?Cys?Leu?Ser?Pro?Ala?Asp?Ala?Pro?Cys?Ala?Val?Ser?Ser?Trp?Ala?Tyr?Arg?Leu?Ala?Val?Arg?Ser?Tyr?Ala?Gly?Cys?Ser?Arg?Thr?Thr?Pro?Pro?Pro?Arg?Cys?Phe?Ala?Glu?Ala?Arg?Met?Glu?Pro?Val?Pro?Gly?Leu?Ala?Trp?Leu?Ala?Ser?Thr?Val?Asn?Leu?Glu?Phe?Gln?His?Ala?Ser?Pro?Gln?His?Ala?Gly?Leu?Tyr?Leu?Cys?Val?Val?Tyr?Val?Asp?Asp?His?Ile?His?Ala?Trp?Gly?His?Met?Thr?Ile?Ser?Thr?Ala?Ala?Gln?Tyr?Arg?Asn?Ala?Val?Val?Glu?Gln?His?Leu?Pro?Gln?Arg?Gln?Pro?Glu?Pro?Val?Glu?Pro?Thr?Arg?Pro?His?Val?Arg?Ala?Pro?His?Pro?Ala?Pro?Ser?Ala?Arg?Gly?Pro?Leu?Arg
According to the epitope aminoacid sequence in the HSV2 virus gE glycoprotein of screening, select the codon of eucaryon, the gene order that chemosynthesis is brand-new, and increased at 5' end
ecorI restriction enzyme site (underscore part), initiator codon (ATG), signal peptide (GGCTGGAGCTGTATTATCCTGTTCCTGGTCGCCACTGCCACTGGAGTTCATTC TGCC), 3' end increased His mark (CAC CAC CAC CAC CAC CAC), terminator codon (TGA) and
himdiII restriction enzyme site (underscore part), makes this gene fragment be easy to be cloned in plasmid pMCE5
ecorI and
hindin III restriction enzyme site.The DNA sequence (1282 bp) containing HSV2 virus gE glycoprotein antigen epitope gene of chemosynthesis is as follows:
GAATTC?GCC?GCC?ACC?ATG?GGC?TGG?AGC?TGT?ATT?ATC?CTG?TTC?CTG?GTC?GCC?ACT?GCC?ACT?GGA?GTT?CAT?TCT?GCC?GCC?CCT?CGG?ACA?TCT?TGG?AAA?AGG?GTG?ACC?AGC?GGA?GAG?GAT?GTG?GTC?CTG?CTG?CCT?GCA?CCA?GCT?GAA?AGG?ACA?CGA?GCA?CAC?AAG?CTG?CTG?TGG?GCA?GCT?GAG?CCT?CTG?GAC?GCA?TGC?GGT?CCA?CTG?CGA?CCA?TCT?TGG?GTG?GCC?CTG?TGG?CCA?CCT?AGG?CGG?GTC?CTG?GAG?ACC?GTG?GTC?GAT?GCA?GCC?TGT?ATG?AGA?GCT?CCC?GAA?CCT?CTG?GCA?ATC?GCC?TAC?TCT?CCA?CCC?TTC?CCA?GCA?GGC?GAT?GAG?GGA?CTG?TAT?AGT?GAA?CTG?GCT?TGG?AGA?GAC?CGC?GTG?GCA?GTG?GTC?AAC?GAG?TCC?CTG?GTC?ATC?TAC?GGC?GCC?CTG?GAA?ACA?GAT?AGC?GGA?CTG?TAT?ACT?CTG?TCA?GTG?GTC?GGA?CTG?TCC?GAC?GAG?GCA?CGA?CAG?GTG?GCT?TCC?GTG?GTC?CTG?GTG?GTC?GAA?CCA?GCA?CCA?GTG?CCT?ACT?CCA?ACC?CCA?GAC?GAT?TAC?GAC?GAG?GAA?GAC?GAT?GCT?GGC?GTG?ACT?AAC?GCA?AGA?CGC?TCA?GCC?TTC?CCT?CCA?CAG?CCA?CCT?CCA?AGG?AGG?CCC?CCT?GTG?GCA?CCA?CCA?ACC?CAC?CCA?CGC?GTC?ATC?CCA?GAG?GTG?TCC?CAC?GTC?CGA?GGA?GTG?ACT?GTC?CAT?ATG?GAG?ACC?CTG?GAA?GCT?ATT?CTG?TTC?GCA?CCT?GGG?GAA?ACA?TTT?GGT?ACT?AAT?GTG?AGT?ATC?CAC?GCT?ATT?GCA?CAT?GAC?GAT?GGA?CCC?TAT?GCT?ATG?GAC?GTG?GTC?TGG?ATG?CGA?TTT?GAT?GTG?CCT?TCC?AGC?TGC?GCC?GAC?ATG?AGG?ATC?TAC?GAG?GCT?TGT?CTG?TAT?CAT?CCT?CAG?CTG?CCA?GAA?TGC?CTG?TCC?CCT?GCC?GAT?GCT?CCA?TGT?GCC?GTG?TCT?AGT?TGG?GCC?TAC?CGA?CTG?GCT?GTG?CGT?AGC?TAT?GCT?GGC?TGC?TCT?AGG?ACC?ACA?CCT?CCA?CCC?CGG?TGT?TTC?GCA?GAG?GCC?AGA?ATG?GAA?CCT?GTG?CCA?GGA?CTG?GCT?TGG?CTG?GCA?AGT?ACA?GTG?AAC?CTG?GAG?TTT?CAG?CAC?GCA?TCA?CCA?CAG?CAT?GCC?GGG?CTG?TAC?CTG?TGC?GTG?GTC?TAT?GTG?GAC?GAT?CAC?ATC?CAT?GCC?TGG?GGT?CAT?ATG?ACC?ATT?AGC?ACA?GCT?GCA?CAG?TAC?AGG?AAT?GCT?GTG?GTC?GAG?CAG?CAC?CTG?CCA?CAG?CGT?CAG?CCA?GAG?CCT?GTG?GAA?CCA?ACC?CGC?CCT?CAT?GTC?AGA?GCA?CCC?CAC?CCC?GCC?CCT?TCA?GCC?AGA?GGA?CCC?CTG?CGT?CAC?CAC?CAC?CAC?CAC?CAT?TGA?T
AAGCTT
2. express the structure of HSV2 virus gE glucoprotein extracellular region fragment recombinant plasmid:
Extract plasmid pMCE5, use
ecorI and
hindiII double digestion, reclaims the plasmid large fragment that enzyme is cut after electrophoresis, be dissolved in deionized water.Same using
ecorI and
hindthe HSV2 virus gE glucoprotein extracellular region gene fragment of III double digestion chemosynthesis, electrophoresis is dissolved in deionized water after reclaiming.
Above-mentioned two kinds of enzymes of volumetric molar concentration such as get and cut rear DNA fragmentation, in same centrifuge tube, with T4 DNA ligase enzyme, connect, HSV2 virus gE glucoprotein extracellular region gene fragment is inserted in carrier pMCE5
ecorI and
hindbetween III site, consistent with the initiator codon translation framework on carrier, express a fusion rotein.
3. the screening of recombinant plasmid and evaluation:
The recombinant plasmid transformed that upper step is connected, to bacillus coli DH 5 alpha, containing on the solid LB substratum of 100 μ g/ml penbritins, is put 37 ℃ of overnight incubation by converted product coating.Next day random choose 1-8 transformant bacterium colony, one containing plasmid pMCE5 transformed bacteria, do negative control and one and make positive control containing plasmid pUC57-gE2 transformed bacteria, be inoculated into respectively the liquid LB substratum of 3 ml containing penbritin 100 μ g/ml, 37 ℃ of shaking culture 5h, get bacterium liquid 1ml, centrifugal receipts bacterium.Use respectively 50 μ l deionized water suspension thalline, boiling water boiling 5min, centrifugal (4 ℃, 12000rpm) 5min, get supernatant 2 μ l as pcr template, HSV2 virus gE glucoprotein extracellular region gene fragment in pcr amplification insertion vector, PCR reaction density is: plasmid template 2 μ l, the normal chain P1(CCGGAATTCGCCGCCACCATGGG of HSV2 virus gE glucoprotein extracellular region gene fragment) and minus strand primer P2(AAATGCGGCCGCTGACCATG ATTACGCCAAGCT) each 1 μ l, 10 × pyrobest buffer, 5.0 μ l, 2.5 mmol/L dNTP 4.0 μ l, Pyrobest DNA Taq enzyme 0.5 μ l (2.5 U), 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 ℃ are extended 7 minutes.Get pcr amplification product 5 μ l, by 1.0% Agarose gel detection, result, No. 1 transformant amplifies the goal gene fragment (see figure 1) of 1282bp, and does not amplify this gene fragment containing the contrast bacterium of plasmid pMCE5.Tentative confirmation, this No. 1 transformant contains HSV2 virus gE glycoprotein gene fragment.
Extract the plasmid of No. 3 recons, double digestion is identified (see figure 2) and is measured the HSV2 virus gE glycoprotein gene sequence in plasmid, DNA sequence analysis confirmation, and recombinant plasmid contains synthetic HSV2 virus gE glycoprotein gene fragment, and sequence is entirely true:
GCC?GCC?ACC?ATG?GGC?TGG?AGC?TGT?ATT?ATC?CTG?TTC?CTG?GTC?GCC?ACT?GCC?ACT?GGA?GTT?CAT?TCT?GCC?GCC?CCT?CGG?ACA?TCT?TGG?AAA?AGG?GTG?ACC?AGC?GGA?GAG?GAT?GTG?GTC?CTG?CTG?CCT?GCA?CCA?GCT?GAA?AGG?ACA?CGA?GCA?CAC?AAG?CTG?CTG?TGG?GCA?GCT?GAG?CCT?CTG?GAC?GCA?TGC?GGT?CCA?CTG?CGA?CCA?TCT?TGG?GTG?GCC?CTG?TGG?CCA?CCT?AGG?CGG?GTC?CTG?GAG?ACC?GTG?GTC?GAT?GCA?GCC?TGT?ATG?AGA?GCT?CCC?GAA?CCT?CTG?GCA?ATC?GCC?TAC?TCT?CCA?CCC?TTC?CCA?GCA?GGC?GAT?GAG?GGA?CTG?TAT?AGT?GAA?CTG?GCT?TGG?AGA?GAC?CGC?GTG?GCA?GTG?GTC?AAC?GAG?TCC?CTG?GTC?ATC?TAC?GGC?GCC?CTG?GAA?ACA?GAT?AGC?GGA?CTG?TAT?ACT?CTG?TCA?GTG?GTC?GGA?CTG?TCC?GAC?GAG?GCA?CGA?CAG?GTG?GCT?TCC?GTG?GTC?CTG?GTG?GTC?GAA?CCA?GCA?CCA?GTG?CCT?ACT?CCA?ACC?CCA?GAC?GAT?TAC?GAC?GAG?GAA?GAC?GAT?GCT?GGC?GTG?ACT?AAC?GCA?AGA?CGC?TCA?GCC?TTC?CCT?CCA?CAG?CCA?CCT?CCA?AGG?AGG?CCC?CCT?GTG?GCA?CCA?CCA?ACC?CAC?CCA?CGC?GTC?ATC?CCA?GAG?GTG?TCC?CAC?GTC?CGA?GGA?GTG?ACT?GTC?CAT?ATG?GAG?ACC?CTG?GAA?GCT?ATT?CTG?TTC?GCA?CCT?GGG?GAA?ACA?TTT?GGT?ACT?AAT?GTG?AGT?ATC?CAC?GCT?ATT?GCA?CAT?GAC?GAT?GGA?CCC?TAT?GCT?ATG?GAC?GTG?GTC?TGG?ATG?CGA?TTT?GAT?GTG?CCT?TCC?AGC?TGC?GCC?GAC?ATG?AGG?ATC?TAC?GAG?GCT?TGT?CTG?TAT?CAT?CCT?CAG?CTG?CCA?GAA?TGC?CTG?TCC?CCT?GCC?GAT?GCT?CCA?TGT?GCC?GTG?TCT?AGT?TGG?GCC?TAC?CGA?CTG?GCT?GTG?CGT?AGC?TAT?GCT?GGC?TGC?TCT?AGG?ACC?ACA?CCT?CCA?CCC?CGG?TGT?TTC?GCA?GAG?GCC?AGA?ATG?GAA?CCT?GTG?CCA?GGA?CTG?GCT?TGG?CTG?GCA?AGT?ACA?GTG?AAC?CTG?GAG?TTT?CAG?CAC?GCA?TCA?CCA?CAG?CAT?GCC?GGG?CTG?TAC?CTG?TGC?GTG?GTC?TAT?GTG?GAC?GAT?CAC?ATC?CAT?GCC?TGG?GGT?CAT?ATG?ACC?ATT?AGC?ACA?GCT?GCA?CAG?TAC?AGG?AAT?GCT?GTG?GTC?GAG?CAG?CAC?CTG?CCA?CAG?CGT?CAG?CCA?GAG?CCT?GTG?GAA?CCA?ACC?CGC?CCT?CAT?GTC?AGA?GCA?CCC?CAC?CCC?GCC?CCT?TCA?GCC?AGA?GGA?CCC?CTG?CGT?CAC?CAC?CAC?CAC?CAC?CAT?TGA?T
The viral gE glycoprotein fragment (394 amino acid) of the expression of recombinant plasmid HSV2 building, has merged 19 amino acid on carrier at its N end, at its C end, has merged 6 amino acid on carrier, 419 amino acid of total length, and its aminoacid sequence is as follows:
N end: Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly Val His Ser
C end: His His His His His His
4. the expression identification of recombinant plasmid in Chinese hamster ovary celI:
Chinese hamster ovary celI shakes cultivation, 37 ℃, 5% CO at the cell expressing substratum that does not contain serum in Erlenmeyer flask
2.Transfection the day before yesterday, with suitable concentration, Chinese hamster ovary celI is inoculated in not containing in the cell expressing substratum of serum to concussion suspension culture, 37 ℃, 5% CO
2.With DNA and PEI mixture transfection recombinant plasmid.The 5th day collecting cell supernatant after transfection, detects protein expression level, within the 6th day, collects all supernatants for purification of recombinant proteins.
express the purifying of HSV2 virus gE glycoprotein
According to the aminoacid sequence of expressing HSV2 virus gE glycoprotein, analyze its physicochemical property, determine suitable purification process.The HSV2 gE glycoprotein of expressing cho cell merges the His albumen having on carrier, and therefore we determine to adopt affinity chromatography, use His Trap
tMfF crude column carries out purifying.Purifying has obtained the gE glycoprotein of expressing, and concrete steps are as follows:
Materials and methods
1. main agents: His Trap
tMfF crude column is GE Heathcare company product, and other reagent is domestic or Import Analysis pure reagent.
2. the purifying of recombinant protein: collecting cell supernatant 100ml, adds His Trap by supernatant with the speed of 1ml/min after centrifugal
tMin FF crude column.10 times of volumetric balance liquid (1 × PBS, 0.5mol/L NaCl, 20mmol/L imidazole, pH7.4) wash-out foreign protein, then add 1ml elutriant (1 × PBS, 0.5mol/L NaCl, 500mmol/L imidazole, pH7.4) wash-out target protein, wash-out after standing 20 minutes, collect elutriant, with dialyzate (1 × PBS, 0.5mol/L NaCl, pH7.4) dialysed overnight, be the target protein of purifying, SDS-PAGE, Western Blot detect, and measure protein concentration.
Result
Will be from His Trap
tMon FF crude column, the albumen of wash-out carries out SDS-PAGE, Western Blot analysis, and result shows (seeing respectively Fig. 3,4), the about 55kDa of expression product, and protein concentration is 27 μ g/ml.Obtained the gE glycoprotein of expressing.
the Analysis of Immunogenicity of the gE glycoprotein of purifying HSV2 virus
the gE2 recombinant protein obtaining take purifying, as antigen, mixes freund's adjuvant immune mouse, prepares antiserum(antisera), and indirect elisa method detects and tires, the immunogenicity of the gE2 recombinant protein that evaluation and test obtains.
materials and methods
1. male mouse of kunming, age in 6-8 week, purchased from Chinese military medicine academy of sciences Experimental Animal Center.
2. complete Freund's adjuvant and incomplete Freund's adjuvant are Sigma company product.The anti-His monoclonal antibody of mouse, goat anti-mouse igg-HRP are Jin Site company product, and other reagent is domestic or Import Analysis pure reagent.
3. animal immune method and antiserum(antisera) preparation: 10 male mouse of kunming are divided into 2 groups at random, 5 every group.Experimental group is mixed with freund's adjuvant equal-volume with the restructuring gE2 glycoprotein of purifying, fully emulsified, respectively at the 0th, and 2,4 weeks, abdominal injection immune mouse, 2 ug//times, only, control group is with PBS equal-volume mixing freund's adjuvant, fully emulsified for 0.1ml/, and 0.1ml/ is only.Eye socket blood sampling in 2 weeks after each immunity.37 ℃ of blood are placed after 1 hour 4 ℃ and are spent the night, and centrifugal 10 minutes of 4000rpm, gets supernatant and obtain recombinant protein gE2 antiserum(antisera).Indirect ELISA detects antiserum(antisera) titre.
4. ELISA test: with 1 × CBS, the restructuring gE2 glycoprotein of purifying being diluted is 1 ug/ml, coated elisa plate, every hole 100ul, 4 ℃ are spent the night.Next day 5%FBS sealase yoke plate, every hole 130ul, room temperature 2 hours.The antiserum(antisera) of preparation is pressed to 1:500 with sample diluent, and 1:2500, after 1:12500 stepwise dilution, add to respectively in the enzyme linked plate holes after sealing, every hole 100ul, 37 ℃ are reacted 1 hour, wash after 5 times with PBST, add the goat anti-mouse igg-HRP of 1:5000 dilution, every hole 100ul, 37 ℃ are reacted 30 minutes, PBST washes 5 times, adds every hole 100 ul of substrate TMB solution, and 37 ℃ of lucifuges develop the color 10 minutes, add 50ul 1N hydrochloric acid termination reaction, with enzyme connection instrument mensuration A
450value.
result
After each immunity, 2 weeks mouse eyeground vein clumps are got blood, prepare antiserum(antisera), and indirect elisa method detects serum Serum Antibody titre.Result shows, the anti-gE2 serum of mouse average titer before immunity, after immunity for the first time, after immunity for the second time, the antiserum(antisera) titre after immunity is for the third time respectively 0.00 ± 0.00,4.10 ± 0.28,5.49 ± 0.28,6.89 ± 0.28(is as Fig. 5), and the anti-gE2 serum titer of PBS control group is always 0.00.Mouse produces the antibody of higher level after immunity for the first time, and by for the second time, after immunity for the third time, in Mice Body, antibody horizontal still slowly raises, and produces high titre level.Use SPSS 13.0 softwares to carry out one-way analysis of variance and Q check, P < 0.05, the raising of mouse resisting anteserum titre all has remarkable statistical significance before immunity and after three immunity.The recombinant protein gE2 expressing can produce good Humoral by stimulating immune system.
The HSV virus gE protein extracellular gene fragment order table of chemosynthesis sees appendix document: Nucleotide or the readable carrier of aminoacid sequence list machine.
Claims (4)
1. the extracellular region gene fragment of the HSV2 of chemosynthesis virus gE glycoprotein, this gene fragment coding is containing the gE glucoprotein extracellular region gene fragment of the HSV2 virus of strong antigen epi-position, i.e. 414 amino acid of the 21st amino acid to the, totally 419 amino acid, have increased at 5' end
ecorI restriction enzyme site (underscore part), initiator codon (ATG), signal peptide (GGCTGGAGCTGTATTATCCTGTTCCTGGTCGCCACTGCCACTGGAGTTCA TTCTGCC), 3' end increased His mark (CAC CAC CAC CAC CAC CAC), terminator codon (TGA) and
himdiII restriction enzyme site (underscore part), gene order total length 1282 bp of chemosynthesis, sequence is as follows:
GAATTC?GCC?GCC?ACC?ATG?GGC?TGG?AGC?TGT?ATT?ATC?CTG?TTC?CTG?GTC?GCC?ACT?GCC?ACT?GGA?GTT?CAT?TCT?GCC?GCC?CCT?CGG?ACA?TCT?TGG?AAA?AGG?GTG?ACC?AGC?GGA?GAG?GAT?GTG?GTC?CTG?CTG?CCT?GCA?CCA?GCT?GAA?AGG?ACA?CGA?GCA?CAC?AAG?CTG?CTG?TGG?GCA?GCT?GAG?CCT?CTG?GAC?GCA?TGC?GGT?CCA?CTG?CGA?CCA?TCT?TGG?GTG?GCC?CTG?TGG?CCA?CCT?AGG?CGG?GTC?CTG?GAG?ACC?GTG?GTC?GAT?GCA?GCC?TGT?ATG?AGA?GCT?CCC?GAA?CCT?CTG?GCA?ATC?GCC?TAC?TCT?CCA?CCC?TTC?CCA?GCA?GGC?GAT?GAG?GGA?CTG?TAT?AGT?GAA?CTG?GCT?TGG?AGA?GAC?CGC?GTG?GCA?GTG?GTC?AAC?GAG?TCC?CTG?GTC?ATC?TAC?GGC?GCC?CTG?GAA?ACA?GAT?AGC?GGA?CTG?TAT?ACT?CTG?TCA?GTG?GTC?GGA?CTG?TCC?GAC?GAG?GCA?CGA?CAG?GTG?GCT?TCC?GTG?GTC?CTG?GTG?GTC?GAA?CCA?GCA?CCA?GTG?CCT?ACT?CCA?ACC?CCA?GAC?GAT?TAC?GAC?GAG?GAA?GAC?GAT?GCT?GGC?GTG?ACT?AAC?GCA?AGA?CGC?TCA?GCC?TTC?CCT?CCA?CAG?CCA?CCT?CCA?AGG?AGG?CCC?CCT?GTG?GCA?CCA?CCA?ACC?CAC?CCA?CGC?GTC?ATC?CCA?GAG?GTG?TCC?CAC?GTC?CGA?GGA?GTG?ACT?GTC?CAT?ATG?GAG?ACC?CTG?GAA?GCT?ATT?CTG?TTC?GCA?CCT?GGG?GAA?ACA?TTT?GGT?ACT?AAT?GTG?AGT?ATC?CAC?GCT?ATT?GCA?CAT?GAC?GAT?GGA?CCC?TAT?GCT?ATG?GAC?GTG?GTC?TGG?ATG?CGA?TTT?GAT?GTG?CCT?TCC?AGC?TGC?GCC?GAC?ATG?AGG?ATC?TAC?GAG?GCT?TGT?CTG?TAT?CAT?CCT?CAG?CTG?CCA?GAA?TGC?CTG?TCC?CCT?GCC?GAT?GCT?CCA?TGT?GCC?GTG?TCT?AGT?TGG?GCC?TAC?CGA?CTG?GCT?GTG?CGT?AGC?TAT?GCT?GGC?TGC?TCT?AGG?ACC?ACA?CCT?CCA?CCC?CGG?TGT?TTC?GCA?GAG?GCC?AGA?ATG?GAA?CCT?GTG?CCA?GGA?CTG?GCT?TGG?CTG?GCA?AGT?ACA?GTG?AAC?CTG?GAG?TTT?CAG?CAC?GCA?TCA?CCA?CAG?CAT?GCC?GGG?CTG?TAC?CTG?TGC?GTG?GTC?TAT?GTG?GAC?GAT?CAC?ATC?CAT?GCC?TGG?GGT?CAT?ATG?ACC?ATT?AGC?ACA?GCT?GCA?CAG?TAC?AGG?AAT?GCT?GTG?GTC?GAG?CAG?CAC?CTG?CCA?CAG?CGT?CAG?CCA?GAG?CCT?GTG?GAA?CCA?ACC?CGC?CCT?CAT?GTC?AGA?GCA?CCC?CAC?CCC?GCC?CCT?TCA?GCC?AGA?GGA?CCC?CTG?CGT?CAC?CAC?CAC?CAC?CAC?CAT?TGA?T
AAGCTT?。
2. the gene fragment of chemosynthesis claimed in claim 1, adopts genetic engineering technique to express the gE glucoprotein extracellular region gene fragment of the HSV2 virus of this gene order coding, the protein fragments that purifying is expressed, and concrete grammar is as follows:
Express the structure of the gE glucoprotein extracellular region gene fragment recombinant plasmid of HSV2 virus:
With
ecor I and
himdthe gE glucoprotein extracellular region gene fragment of the HSV2 virus of III double digestion plasmid pMCE5 and chemosynthesis, after electrophoresis reclaims, with the connection of T4 DNA ligase enzyme, is inserted in carrier pMCE5 the gE glucoprotein extracellular region gene fragment of HSV2 virus
ecor I and
himdbetween III site, consistent with the initiator codon translation framework on carrier, express a fusion rotein, 419 amino acid of total length, this fusion rotein N end has merged 19 amino acid on carrier, C holds 414 amino acid of the 21st amino acid to the and the His label (6 amino acid) in the gE glycoprotein that comprises HSV2 virus, and total length 419 aminoacid sequences are as follows:
Ala?Ala?Pro?Arg?Thr?Ser?Trp?Lys?Arg?Val?Thr?Ser?Gly?Glu?Asp?Val?Val?Leu?Leu?Pro?Ala?Pro?Ala?Glu?Arg?Thr?Arg?Ala?His?Lys?Leu?Leu?Trp?Ala?Ala?Glu?Pro?Leu?Asp?Ala?Cys?Gly?Pro?Leu?Arg?Pro?Ser?Trp?Val?Ala?Leu?Trp?Pro?Pro?Arg?Arg?Val?Leu?Glu?Thr?Val?Val?Asp?Ala?Ala?Cys?Met?Arg?Ala?Pro?Glu?Pro?Leu?Ala?Ile?Ala?Tyr?Ser?Pro?Pro?Phe?Pro?Ala?Gly?Asp?Glu?Gly?Leu?Tyr?Ser?Glu?Leu?Ala?Trp?Arg?Asp?Arg?Val?Ala?Val?Val?Asn?Glu?Ser?Leu?Val?Ile?Tyr?Gly?Ala?Leu?Glu?Thr?Asp?Ser?Gly?Leu?Tyr?Thr?Leu?Ser?Val?Val?Gly?Leu?Ser?Asp?Glu?Ala?Arg?Gln?Val?Ala?Ser?Val?Val?Leu?Val?Val?Glu?Pro?Ala?Pro?Val?Pro?Thr?Pro?Thr?Pro?Asp?Asp?Tyr?Asp?Glu?Glu?Asp?Asp?Ala?Gly?Val?Thr?Asn?Ala?Arg?Arg?Ser?Ala?Phe?Pro?Pro?Gln?Pro?Pro?Pro?Arg?Arg?Pro?Pro?Val?Ala?Pro?Pro?Thr?His?Pro?Arg?Val?Ile?Pro?Glu?Val?Ser?His?Val?Arg?Gly?Val?Thr?Val?His?Met?Glu?Thr?Leu?Glu?Ala?Ile?Leu?Phe?Ala?Pro?Gly?Glu?Thr?Phe?Gly?Thr?Asn?Val?Ser?Ile?His?Ala?Ile?Ala?His?Asp?Asp?Gly?Pro?Tyr?Ala?Met?Asp?Val?Val?Trp?Met?Arg?Phe?Asp?Val?Pro?Ser?Ser?Cys?Ala?Asp?Met?Arg?Ile?Tyr?Glu?Ala?Cys?Leu?Tyr?His?Pro?Gln?Leu?Pro?Glu?Cys?Leu?Ser?Pro?Ala?Asp?Ala?Pro?Cys?Ala?Val?Ser?Ser?Trp?Ala?Tyr?Arg?Leu?Ala?Val?Arg?Ser?Tyr?Ala?Gly?Cys?Ser?Arg?Thr?Thr?Pro?Pro?Pro?Arg?Cys?Phe?Ala?Glu?Ala?Arg?Met?Glu?Pro?Val?Pro?Gly?Leu?Ala?Trp?Leu?Ala?Ser?Thr?Val?Asn?Leu?Glu?Phe?Gln?His?Ala?Ser?Pro?Gln?His?Ala?Gly?Leu?Tyr?Leu?Cys?Val?Val?Tyr?Val?Asp?Asp?His?Ile?His?Ala?Trp?Gly?His?Met?Thr?Ile?Ser?Thr?Ala?Ala?Gln?Tyr?Arg?Asn?Ala?Val?Val?Glu?Gln?His?Leu?Pro?Gln?Arg?Gln?Pro?Glu?Pro?Val?Glu?Pro?Thr?Arg?Pro?His?Val?Arg?Ala?Pro?His?Pro?Ala?Pro?Ser?Ala?Arg?Gly?Pro?Leu?Arg
The screening of recombinant plasmid and evaluation:
By recombinant plasmid transformed bacillus coli DH 5 alpha, coating, containing penbritin (100 μ g/ml) LB flat board, is put 37 ℃ and is spent the night; Next day, random picking transformed bacterium colony, 1 positive control bacterium (pUC57-gE2) of 1 contrast bacterium (plasmid pMCE5 transformed bacteria), bacterium liquid pcr amplification HSV2 virus gE glucoprotein extracellular region gene fragment, containing the positive recombinant plasmid of HSV2 virus gE glucoprotein extracellular region gene fragment, should amplify the gene fragment that is about 1282bp; Extracting positive single bacterium colony plasmid, carry out double digestion evaluation and order-checking;
The expression identification of recombinant plasmid in Chinese hamster ovary celI:
Chinese hamster ovary celI shakes cultivation, 37 ℃, 5% CO at the cell expressing substratum that does not contain serum in Erlenmeyer flask
2, transfection the day before yesterday, with suitable concentration, Chinese hamster ovary celI is inoculated in not containing in the cell expressing substratum of serum to concussion suspension culture, 37 ℃, 5% CO
2; With DNA and PEI mixture transfection recombinant plasmid, the 5th day collecting cell supernatant after transfection, detect protein expression level, within the 6th day, collect all supernatants for purification of recombinant proteins;
Purifying, evaluation and the concentration of recombinant protein detect:
Collecting cell supernatant 100ml, adds His Trap by supernatant with the speed of 1ml/min after centrifugal
tMin FF crude column; 10 times of volumetric balance liquid (1 × PBS, 0.5mol/L NaCl, 20mmol/L imidazole, pH7.4) wash-out foreign protein, then add 1ml elutriant (1 × PBS, 0.5mol/L NaCl, 500mmol/L imidazole, pH7.4) wash-out target protein, wash-out after standing 20 minutes, collect elutriant, with dialyzate (1 × PBS, 0.5mol/L NaCl, pH7.4) dialysed overnight, be the target protein of purifying, SDS-PAGE, Western Blot detect, and measure protein concentration; The gE2 molecular weight of albumen that purifying obtains is about 55kD.
3. the gene fragment order of the HSV2 of chemosynthesis claimed in claim 1 virus gE glucoprotein extracellular region, utilizes bacterium, yeast cell, insect cell, mammalian cell and genetically modified animals and plants to carry out recombinant expressed, preparation.
4. the HSV2 virus gE glucoprotein extracellular region fragment that prepared by claim 2 or method claimed in claim 3, for development and the antibody test of HSV2 viral sub-units vaccine.
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| CN101583381A (en) * | 2006-09-08 | 2009-11-18 | 宾夕法尼亚大学董事会 | HSV-1 and HSV-2 vaccines and methods of use thereof |
| CN101616688B (en) * | 2006-12-28 | 2013-12-11 | 宾夕法尼亚大学理事会 | Herpes simplex virus combined subunit vaccines and methods of use thereof |
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| CN101583381A (en) * | 2006-09-08 | 2009-11-18 | 宾夕法尼亚大学董事会 | HSV-1 and HSV-2 vaccines and methods of use thereof |
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| CN110551187A (en) * | 2019-09-23 | 2019-12-10 | 新乡学院 | Chemically synthesized H7N9 avian influenza virus NA protein extracellular region antigen segment, preparation method and application |
| 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 |
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