CN1301749C - Polyepitope DNA vaccine of anti-simple herpes virus-2 infection and its preparing method - Google Patents
Polyepitope DNA vaccine of anti-simple herpes virus-2 infection and its preparing method Download PDFInfo
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- CN1301749C CN1301749C CNB2005100284841A CN200510028484A CN1301749C CN 1301749 C CN1301749 C CN 1301749C CN B2005100284841 A CNB2005100284841 A CN B2005100284841A CN 200510028484 A CN200510028484 A CN 200510028484A CN 1301749 C CN1301749 C CN 1301749C
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Abstract
The present invention relates to a multi-epitope DNA vaccine for resisting herpes simplex viruses-2 and a preparation method thereof, which belongs to the technical field of medicine and gene engineering. The DNA vaccine of the present invention comprises: gB, gC and gD of HSV-2 and the immediate early protein ICP27 of the main antigen epitope region added with intervening sequences are connected in series to a eukaryotic expression vector pcDNA3.1 so as to obtain HV-pcDNA3.1. The DNA vaccine can be used for preventing and treating diseases caused by the infection of HSV-2, such as genital herpes, etc. The vaccine has the advantages of high specificity, good protection and simple preparation method.
Description
Technical field
The invention belongs to medicine and gene engineering technology field, be specifically related to a kind of new anti-HSV-2 Polyepitope DNA vaccine and application
Background technology
It is human more common infectious disease that herpes simplex virus (HSV) infects.HSV-1 mainly causes non-genital infection such as herpes labialis, gingivostomatitis, encephalitis, keratoconjunctivitis, and HSV-2 causes genital infection.Most of genital herpess are caused by HSV-2, can be chronic recurrence process, and the good medicine of radical cure is not arranged at present as yet.This disease is fairly obvious in the popularity in the whole world, has accounted for about 10% of sexually transmitted disease (STD), infects the public health problem that causes by HSV-2 and impels people to develop the vaccine that can prevent with control infection.There are some researches show, dna vaccination is compared with inactivated vaccine, attenuation Seedling, the subunit vaccine of routine, but dna vaccination is the inducing producing specificity immunoglobulin not only, but and the cell immune response of simultaneous excitation body (especially CTL immunity), more effective to the disease prevention of dependent cells immune clearance cause of diseases such as disease of viral infection.Another outstanding advantage of dna vaccination is immunogenic unicity.Have only the required antigenic gene of coding to be imported into cell and obtain expressing, itself does not have immunogenicity carrier.And the live recombinant vectors vaccine is except that the expression of genes of interest, and itself also has huge and complicated immunogen protein.Dna vaccination not only can be used for prevention, and can be used for treatment.
The present invention is from the mechanism and the whole life process angle of HSV invasion and breeding, HSV-2 glycoprotein D, B, C gene (the gD immunogenicity is strong, gB and gC and the absorption of corresponding receptors bind energy fixed virus at cell surface) and immediate early protein ICP27 gene (this gene can start viral DNA to enter cell after transcript and expression) contacted as target gene is inserted into pCDNA3.1, has made up the novel Polyepitope DNA vaccine of HSV-2 first.
Summary of the invention
The purpose of this invention is to provide a kind of can be effectively anti-the Polyepitope DNA vaccine that infects of HSV-2 and preparation method thereof.
Technology path of the present invention is, at first cultivate amplification HSV-2 virus, and its genomic DNA of extracting, use the immunogenicity analysis of bioinformatics method simultaneously, choose required epitope, design corresponding primer according to glycoprotein, then with the required genes of interest of PCR method segmentation amplification, at last six fragment genes are coupled together and be cloned into earlier on the pGEM-T carrier, the enzyme action pcDNA3.1 that packs into obtains the Polyepitope DNA vaccine (HV-pcDNA3.1 plasmid) that anti-HSV-2 infects again.
The Polyepitope DNA vaccine that the effective anti-HSV-2 that the present invention proposes infects, it is composed as follows: with the major antigen epitope regions of the immediate early protein ICP27 of strong immunogen gD, gB, gC and the participation virus replication of HSV-2 virus after birth glycoprotein, with main protection antigen gD is the center, selected 6 sections antigen genes add intervening sequence AAY and are connected in series to carrier for expression of eukaryon pcDNA3.1, obtain the HV-pcDNA3.1 plasmid; Wherein putting in order of 6 sections antigen genes is followed successively by ICP27 the last the 377th～459 amino acid whose one section antigenic peptides, gD the last the 146th～179, the 223rd～306 amino acid whose two-stage nitration antigenic peptides, gB the last the 529th～606 amino acid whose one section antigenic peptides, gC the last the 247th～282 amino acid whose one section antigenic peptides, gD the last the 1st～77 amino acid whose one section antigenic peptides.
The preparation method of the Polyepitope DNA vaccine that the anti-HSV-2 that the present invention proposes infects, concrete steps are as follows:
1. design primer: analyze according to the target antigen immunogenicity, with people HSV-2 totivirus genomic DNA is (SEQ ID NO.7) template, design 6 pairs of primers (containing restriction enzyme site), be respectively applied for three kinds of envelope glycoprotein gD of amplification, it is 1 listed that main immunodominance section and the ICP27 fragment of gB and gC, primer sequence see Table:
Table 1.DNA vaccine primer sequence table
| The epi-position name | Primer sequence |
| ICP27 | ICP27-F：5’CAG GAATTCGAAATGGACCCTATCATCGGAACG 3’(EcoR I) |
| ICP27-R：5’CTA CTCTTCAGGCAGCCGTCTCGCCGGCCCCAAC 3’(Ear I) | |
| gD2 | PgD2-F：5’CAG CTCTTCTGCCTACAGCGAGGATAACCTGGGATT 3’(Ear I) |
| PgD2-R：5’GTC CTCTTCGCGTTGTGTGATCTCCGTCCAGTC 3’(Ear I) | |
| gD3 | PgD3-F：5’CCA CTCTTCAACGCTTTATCCCCGAAAACCA 3’(Ear I) |
| PgD3-R：5’CTA CTCTTCTGGCAGCGTGCGGCGCGACGTCCT 3’(Ear I) | |
| gB4 | PgB4-F：5’TAC CTCTTCTGCCTACCAGAACCACGAGCTGACTC 3’(Ear I) |
| PgB4-R：5’CTA CTCTTCTAAGCGGCCTGGTCTTCGTACCGAAAG 3’ (Ear I) | |
| gC5 | PgC5-F：5’TAG CTCTTCGCTTACGAGGGCCAGCCGTTTAAGG 3’(Ear I) |
| PgC5-R：5’GCA CTCTTCTGGCAGCGTGTATCTGGGCCGGATCG 3’(Ear I) | |
| gD6 | PgD6-F：5’CCA CTCTTCAGCCTACGCCAAATACGCCTTAGCAG 3’(Ear I) |
| PgF6-R：5’CGA GGTACCAATCATCACTCCGATGGGGCATGTAGGA 3’ (Kpn I) |
2. the segmentation of antigen target gene clone reclaims: get the HSV employing virus cracking liquid, respectively 6 sections selected antigen genes are carried out pcr amplification with the Taqplus high-fidelity DNA polymerase.These 6 sections antigen genes are respectively: ICP27 the last the 377th～459 amino acid whose one section antigenic peptides (SEQ ID NO.1), last the 1st～77 (the SEQ ID NO.2) of gD, the amino acid whose three sections antigenic peptides of the 146th～179 (SEQ IDNO.3), the 223rd～306 (SEQ ID NO.4), last amino acid whose one section antigenic peptides of the 529th～606 (SEQ ID NO.5) of gB and the amino acid whose one section antigenic peptides of last the 247th～282 (the SEQ ID NO.6) of gC;
Get pcr amplification product, observe with agarose gel electrophoresis whether the genes of interest band is arranged, the target gene fragment rubber tapping recovery that obtains, quantitative;
With the enzyme action of six sections PCR products be connected: each section HSV antigen fragment that purification is obtained becomes sticky end with Ear I enzyme action respectively, then with T4 ligase them according to following being linked in sequence (seeing shown in Figure 1):
On the ICP27 377～459,146～179 on the gD, 223～306 on the gD, 529～606 on the gB, 247～282 on the gC; On the gD 1～77 obtains complete epitope, and the intervening sequence that is used to connect is AAY;
4. the genes of interest after will connecting makes up plasmid: with above-mentioned complete epitope that obtains and carrier for expression of eukaryon pcDNA3.1 while double digestion, couple together then, constitute required dna vaccination: HV-pcDNA3.1.
By the dna vaccination that the present invention prepares, process is to mouse immuning test, and the result shows that this vaccine can effectively cause specific humoral immunization of mice and cellullar immunologic response.Carry out immune mouse toxicity challenge trial then; Measure its relative rate index, show the effectiveness of this vaccine immunity, and have specificity height, advantage that lethality is strong.
Description of drawings
Fig. 1 is a Polyepitope DNA combination vaccine antigen sketch map.
Fig. 2 is the route diagram of clone's antigene fragment.
The specific embodiment
Further describe embodiments of the present invention below.
1. design of primers: with reference to 6 pairs of primers of [Fig. 1] design, be template with people HSV-2 totivirus genomic DNA, design primer (containing restriction enzyme site) the three kinds of envelope glycoprotein gD that increase respectively, main immunodominance section and the ICP27 fragment of gB and gC.Primer sequence is referring to [table 1]:
2. the segmentation of antigen target gene clone reclaims: get 2ul HSV employing virus cracking liquid, respectively 6 sections selected antigen genes are carried out pcr amplification with the Taqplus high-fidelity DNA polymerase.Condition is as follows:
50 μ l pcr amplification reaction system: ddH
2O 39.5 μ l
10×Buffer 5.0μl
4×dNTP(10mM) 1.0μl
Primer F(10μM) 1.0μl
Primer R(10μM) 1.0μl
Template 2.0μl
Taqplus(5U/μl) 0.5μl
The pcr amplification reaction condition:
Get 2 μ l pcr amplification products, whether 2.0% agarose gel electrophoresis is observed the genes of interest band.The target gene fragment rubber tapping recovery that obtains, quantitative.
The enzyme action of six sections PCR products be connected: each section HSV antigen fragment that purification obtains becomes sticky end with Ear I enzyme action respectively, with T4 ligase they is successively connected in order then.With reference to [Fig. 1]
Ear I enzyme action condition:
2ul Buffer；
1ul Ear I；
The PCR fragment of 10ul purification;
7.5ul ddH
2O；
Reaction Mix is in 37 ℃ of insulation 3h.
T4 ligase each section enzyme action product that is linked in sequence, 4 ℃ of connections are spent the night, and connect as follows respectively:
(1)2ul 10×T4 ligase buffer
1ul T4 ligase
8.5ul ICP1+8.5ul D2
(2)2ul 10×T4 ligase buffer
1ul T4 ligase
8.5ul D3+8.5ul B4
(3)2ul 10×T4 ligase buffer
1ul T4 ligase
8.5ul C5+8.5ul D6
Connect products to 3 sections with 1.5% sepharose electrophoresis observed result.Downcut 3 sections junction fragments, the purpose band is 377bp (A) in (1) number connection product, and the purpose band is 503bp (B) in (2) number connection product, and the purpose band is 376bp (II) in (3) number connection product.Reclaim these purpose fragments.
Connect A and the B fragment that reclaims with T4 ligase, 4 ℃ of connections are spent the night, and Mix is as follows:
2ul 10×T4 ligase buffer
1ul T4 ligase
8.5ul A+8.5ul B
A and B be connected product with 1.5% sepharose electrophoresis observed result.Downcut the position of 600～1000bp band correspondence, reclaim DNA, use pcr amplification.
50 μ l pcr amplification reaction system: ddH
2O 39.5 μ l
10×Buffer 5.0μl
4×dNTP(10mM) 1.0μl
Primer ICP-F(10μM) 1.0μl
Primer B4-R(10μM) 1.0μl
Template 2.0μl
Taqplus(5U/μl) 0.5μl
The pcr amplification reaction condition:
Get 100 μ l pcr amplification products, 2.0% agarose gel electrophoresis downcuts 880bp band (I), reclaims the purpose fragment, reuse Ear I endonuclease bamhi I, and condition is the same.
The fragment II of T4 ligase junction fragment I enzyme action product and recovery, 4 ℃ of connections are spent the night, and Mix is as follows:
2ul 10×T4 ligase buffer
1ul T4 ligase
8.5ul I+8.5ul II
Connect product 1.4% sepharose electrophoresis, downcut 1100～1300bp band (HV), reclaim the purpose fragment.Whether the HV fragment that the PCR check is reclaimed has the connection product of 6 fragment genes:
50 μ l pcr amplification reaction system: ddH
2O 39.5 μ l
10×Buffer 5.0μl
4×dNTP(10mM) 1.0μl
Primer ICP-F(10μM) 1.0μl
Primer D6-R(10μM) 1.0μl
Template 1.0μl
Taqplus(5U/μl) 0.5μl
The pcr amplification reaction condition:
Have 6 sections antigenic peptides epi-positions, be respectively ICP27 the last the 377th～459 amino acid whose one section antigenic peptides, gD the last the the 1st～77, the 146th～179, the 223rd～306 amino acid whose three sections antigenic peptides, gB the last the 529th～606 amino acid whose one section antigenic peptides and gC the last the 247th～284 amino acid whose one section antigenic peptides.Couple together with intervening sequence (AAY) between the wherein dissimilar glycoproteins,, shield to improve immunocompetence and to prevent that the N end is destroyed.
4.HV the cloning and sequencing of junction fragment: the HV fragment of above-mentioned recovery is connected in the pGEM-T carrier, and 16 ℃ of connections are spent the night:
1ul Progema T4 ligase
1ul pGEMT-vector
5ul 2×Progema T4 ligase Buffer
3ul HV refined solution
HV-pGEMT-vector connects product, is transformed into E.coli DH 5 α competent cells, selects positive transformant and identifies.To positive transformant and pcDNA3.1 empty carrier EcoR I and Kpn I double digestion, EcoR I singly cuts plasmid respectively, 37 ℃ of insulation 3h:
3.0ul Buffer H
2.0ul EcoR I
20.0ul plasmid
5.0ul ddH
2O
37 ℃ of the EcoR I digested plasmid reuse Kpn I enzyme action that purification obtains, 3h:
3.0ul Buffer 1
0.3ul 100×BSA
2.0ul Kpn I
24.7ul plasmid
Cut glue 1200bp band for HV-pGEMT-vector double digestion product; PcDNA3.1 empty plasmid double digestion product is then cut the band of the about 5.4kb of glue, respectively it is reclaimed quantitatively.The HV fragment is linked the pcDNA3.1 carrier, and 16 ℃ are spent the night:
1ul 10×T4 ligase buffer
1ul T4 ligase
The two pcDNA3.1 that cut of 1ul reclaim fragment
The two fragments that cut back to close of 3ul HV
HV, pcDNA3.1 connect product and are transformed into DH 5 α competent cells, select positive recombinant, get HV-pcDNA3.1, and sequence verification.
Immune mouse: C57/BL6 female 6 age in week 10 of mices, be divided into 2 groups (the negative group of empty carrier pcDNA3.1 and HV-pcDNA3.1+IL2-pcDNA3.1 experimental grouies) at random, 5 every group.Method is that initial immunity is that 10ug plasmid and 1ug liposome merge transfection C57/BL6 mice mononuclearcell in subcutaneous injection; Secondary immunity is that 100ug plasmid and 10ug liposome mix in the direct injection C57/BL6 mouse muscle of back after 14 days; 50ug plasmid of booster immunization and 5ug liposome mix in the direct injection C57/BL6 mouse muscle of back after 14 days.After 14 days, detect.
6. carrying out the immunogenicity of HSV-2 Polyepitope DNA vaccine by the following method identifies:
(1) the negative group of OD value during 400 times of specific IgG antibodies ELISA serum dilutions as a result is 0.113 ± 0.035; Experimental group is 0.288 ± 0.091, and tiring is about 500.
(2) killer T cell poison analysis result is measured CTL kill rate (%) with the LDH method, and when effect target ratio was 50: 1, negative group was 8.301 ± 2.906; Experimental group is 47.913 ± 15.086.
(3) cytokines measurement as a result ELISA record the content (ng/L) of mIL2 and mIFN-γ in the serum, negative group mIL2 is 685.21 ± 104.20, mIFN-γ is 547.71 ± 189.33; Experimental group mIL2 is 1421.16 ± 220.98, and mIFN-γ is 1956.19 ± 219.60.
(4) the mice spleen T lymphocyte stimulation indices (SI) that records of T cellular antigens specificity multiplication reaction measurement result mtt assay, negative group is 0.709 ± 0.035; Experimental group is 2.751 ± 0.259.
Conclusion: the HSV-2 Polyepitope DNA vaccine can effectively cause specific humoral immunization of mice and cellullar immunologic response.
7. concrete the application:
According to the measurement result of a series of mouse immune indexs, show that it is successful that this dna vaccination makes up design.Directly carry out HSV-2 Polyepitope DNA vaccine immune mouse virus attack experiment then,, observe mice prevention HSV infected animals test effect, further reacted the effectiveness of this vaccine by measuring the survival rate index.
Clinical practice: after the mice counteracting toxic substances Success in Experiment, test, can be used as the Novel DNA vaccine of anti-HSV-2 virus, be used to prevent and treat the disease that infects HSV-2, as diseases such as genital herpes, cervical cancer, neonate herpess through clinical I, II, III phase.
Sequence table
SEQ ID NO.1：
DPIGTAAAVLENLATRLRPFLQCYLKARGLCGLDDLCSRRRLSDIKDIASFVLVILARLANRVERGVSE
IDYTTVGVGAGET
SEQ ID NO.2：
AKYALADPSLKMADPNRFRGKNLPVLDQLTDPPGVKRVYHIQPSLEDPFQPPSIPITVYYAVLERACRS
VLLHAPSE
SEQ ID NO.3：
SEDNLGFLMHAPAFETAGTYLRLVKINDWTEITQ
SEQ ID NO.4：
RFIPENQRTVALYSLKIAGWHGPKPPYTSTLLPPELSDTTNATQPELVPEDPEDSALLEDPAGTVSSQIPP
NWHIPSIQDVAPH
SEQ ID NO.5：
QNHELTLWNEARKLNPNAIASATVGRRVSARMLGDVMAVSTCVPVAPDNVIVQNSMRVSSRPGTCYS
RPLVSFRYEDQ
SEQ ID NO.6：
EGQPFKATCTAATYYPGNRAEFVWFEDGRRVFDPAQ
SEQ ID NO.7
ATGGACCCTATCATCGGAACGGCGGCCGCCGTGCTGGAAAACCTCGCCACGCGCCTGCGCCCCTT
TCTGCAGTGCTACCTGAAGGCCCGAGGCCTGTGCGGGCTGGACGACCTGTGCTCGCGGCGACGCC
TGTCGGACATTAAGGATATTGCCTCCTTTGTGTTGGTCATCCTGGCCCGCCTCGCCAACCGCGTCGA
GCGCGGCGTGTCGGAGATCGACTACACGACCGTGGGGGTTGGGGCCGGCGAGACGGCCGCTTAC
AGCGAGGATAACCTGGGATTCCTGATGCACGCCCCCGCCTTCGAGACCGCGGGTACGTACCTGCG
GCTAGTGAAGATAAACGACTGGACGGAGATCACACAACGCTTTATCCCCGAAAACCAGCGCACCG
TCGCCCTATACAGCTTAAAAATCGCCGGGTGGCACGGCCCCAAGCCCCCGTACACCAGCACCCTG
CTGCCGCCGGAGCTGTCCGACACCACCAACGCCACGCAACCCGAACTCGTTCCGGAAGACCCCG
AGGACTCGGCCCTCTTAGAGGATCCCGCCGGGACGGTGTCTTCGCAGATCCCCCCAAACTGGCAC
ATCCCGTCGATCCAGGACGTCGCGCCGCACGCTGCCTACCAGAACCACGAGCTGACTCTCTGGAA
CGAGGCCCGCAAGCTCAACCCCAACGCCATCGCCTCCGCCACCGTCGGCCGGCGGGTGAGCGCG
CGCATGCTCGGAGACGTCATGGCCGTCTCCACGTGCGTGCCCGTCGCCCCGGACAACGTGATCGT
GCAGAACTCGATGCGCGTCAGCTCGCGGCCGGGGACGTGCTACAGCCGCCCCCTGGTCAGCTTTC
GGTACGAAGACCAGGCCGCTTACGAGGGCCAGCCGTTTAAGGCGACGTGCACGGCCGCCACCTAC
TACCCGGGCAACCGCGCGGAGTTCGTCTGGTTCGAGGACGGTCGCCGGGTATTCGATCCGGCCCA
GGCTGCCTACGCCAAATACGCCTTAGCAGACCCCTCGCTTAAGATGGCCGATCCCAATCGATTTCG
CGGGAAGAACCTTCCGGTTTTGGACCAGCTGACCGACCCCCCCGGGGTGAAGCGTGTTTACCACA
TTCAGCCGAGCCTGGAGGACCCGTTCCAGCCCCCCAGCATCCCGATCACTGTGTACTACGCAGTGC
TGGAACGTGCCTGCCGCAGCGTGCTCCTACATGCCCCATCGGAGTGATGA
Claims (2)
- One kind can be effectively anti-the Polyepitope DNA vaccine that infects of HSV-2; it is characterized in that composed as follows: with the strong immunogen gD of HSV-2 virus after birth glycoprotein; gB; the epitope zone of the immediate early protein ICP27 of gC and participation virus replication; with protective antigen gD is the center; selected 6 sections antigen genes add intervening sequence AAY and are connected in series to carrier for expression of eukaryon pcDNA3.1; obtain the HV-pcDNA3.1 plasmid: wherein putting in order of 6 sections antigen genes is followed successively by ICP27 the last the 377th～459 amino acid whose one section antigenic peptides; gD the last the 146th～179; the 223rd～306 amino acid whose two-stage nitration antigenic peptides; gB the last the 529th～606 amino acid whose one section antigenic peptides, gC the last the 247th～282 amino acid whose one section antigenic peptides and the amino acid whose one section antigenic peptides of the last 1-77 of gD.
- 2, a kind of can be effectively anti-the preparation method of the Polyepitope DNA vaccine that infects of HSV-2, it is characterized in that concrete steps are as follows:(1) design primer: with people HSV-2 totivirus genomic DNA is template, design 6 pairs of primers, is respectively applied for the three kinds of after birth glycoprotein gds that increase, the immunodominance section of gB and gC and ICP27 fragment, and it is 1 listed that primer sequence sees Table:Table 1.DNA vaccine primer sequence table
The epi-position name Primer sequence Restriction enzyme site ICP27 ICP27-F：5’CAG GAATTCGAAATGGACCCTATCATCGGAACG3’， EcoR I ICP27-R：5’CTA CTCTTCAGGCAGCCGTCTCGCCGGCCCCAAC3’， Ear I gD2 PgD2-F：5’CAG CTCTTCTGCCTACAGCGAGGATAACCTGGGATT3’， Ear I PgD2-R：5’GTC CTCTTCGCGTTGTGTGATCTCCGTCCAGTC3’， Ear I gD3 PgD3-F：5’CCA CTCTTCAACGCTTTATCCCCGAAAACCA3’， Ear I PgD3-R：5’CTA CTCTTCTGGCAGCGTGCGGCGCGACGTCCT3’， Ear I gB4 PgB4-F：5’TAC CTCTTCTGCCTACCAGAACCACGAGCTGACTC3’， Ear I PgB4-R：5’CTA CTCTTCTAAGCGGCCTGGTCTTCGTACCGAAAG3’， Ear I gC5 PgC5-F：5’TAG CTCTTCGCTTACGAGGGCCAGCCGTTTAAGG3’， Ear I PgC5-R：5’GCA CTCTTCTGGCAGCGTGTATCTGGGCCGGATCG3’， Ear I gD6 PgD6-F：5’CCA CTCTTCAGCCTACGCCAAATACGCCTTAGCAG3’， Ear I PgD6-R：5’CGA GGTACCAATCATCACTCCGATGGGGCATGTAGGA3’， Kpn I (2) segmentation of antigen target gene clone reclaims: get the HSV employing virus cracking liquid, respectively 6 sections selected antigen genes are carried out pcr amplification with the Taqplus high-fidelity DNA polymerase, these 6 sections antigen genes are respectively: ICP27 the last the 377th～459 amino acid whose one section antigenic peptides, gD the last the the 1st～77, the 146th～179, the 223rd～306 amino acid whose three sections antigenic peptides, gB the last the 529th～606 amino acid whose one section antigenic peptides and gC the last the 247th～282 amino acid whose one section antigenic peptides;Get pcr amplification product, observe with agarose gel electrophoresis whether the genes of interest band is arranged, the target gene fragment rubber tapping recovery that obtains, quantitative;The enzyme action of (3) six sections PCR products be connected: each section HSV antigen fragment that purification is obtained becomes sticky end with Ear I enzyme action respectively, with T4ligase they is linked in sequence according to following then:On the ICP27 377～459,146～179 on the gD, 223～306 on the gD, 529～606 on the gB, 247～282 on the gC; On the gD 1～77 obtains complete epitope, and the intervening sequence that is used to connect is AAY;(4) genes of interest after will connecting makes up plasmid: will above-mentioned complete epitope that obtains and carrier for expression of eukaryon pcDNA3.1 while double digestion, couple together then, and constitute required dna vaccination: HV-pcDNA3.1.
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| CN104940923B (en) * | 2015-06-26 | 2017-10-10 | 东南大学 | A kind of herpes simplex keratitis vaccine and preparation method thereof |
| CN106267182B (en) * | 2015-06-29 | 2020-02-07 | 普莱柯生物工程股份有限公司 | Preparation method of porcine pseudorabies virus subunit vaccine, vaccine composition and application |
| KR101962683B1 (en) * | 2016-09-28 | 2019-03-27 | 주식회사 에스엘백시젠 | A DNA vaccine for preventing and treating HSV-2 infection |
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|---|---|---|---|---|
| US6193984B1 (en) * | 1992-02-03 | 2001-02-27 | Cedars-Sinai Medical Center | Pharmaceutical composition of herpes simplex virus typ-1 (HSV-1) glycoproteins |
| CN1299287A (en) * | 1998-02-12 | 2001-06-13 | 美国氰胺公司 | Vaccines comprising interleukin-12 and herpes simplex viral antigen |
| CN1299868A (en) * | 2001-01-16 | 2001-06-20 | 本元正阳基因技术股份有限公司 | Construction and use of recombinant herpes simplex virus |
| US6572860B1 (en) * | 1997-09-30 | 2003-06-03 | Cel-Sci Corporation | Immunogenic conjugated polypeptide for treatment of herpes simplex virus |
| CN1651081A (en) * | 2004-12-01 | 2005-08-10 | 广州军区广州总医院 | Herpes simplex virus II type DNA vaccine |
-
2005
- 2005-12-09 CN CNB2005100284841A patent/CN1301749C/en not_active Expired - Fee Related
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6193984B1 (en) * | 1992-02-03 | 2001-02-27 | Cedars-Sinai Medical Center | Pharmaceutical composition of herpes simplex virus typ-1 (HSV-1) glycoproteins |
| US6572860B1 (en) * | 1997-09-30 | 2003-06-03 | Cel-Sci Corporation | Immunogenic conjugated polypeptide for treatment of herpes simplex virus |
| CN1299287A (en) * | 1998-02-12 | 2001-06-13 | 美国氰胺公司 | Vaccines comprising interleukin-12 and herpes simplex viral antigen |
| CN1299868A (en) * | 2001-01-16 | 2001-06-20 | 本元正阳基因技术股份有限公司 | Construction and use of recombinant herpes simplex virus |
| 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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