Molecluar Cloning,Prokaryotic Expression And Immune Efficiency Of DNA Vaccine Of The GD Gene Of Pseudorabies Virus Fa Strain

In this research,gD gene of Pseudorabies Virus Strain Fa was amplified and subcloned into the prokaryotic and eukaryotic expression Vectors.The gD gene was expressed in E.coli BL21,and the immune efficiency of gD gene contained in the eukaryotic expression vectors were evaluated.The content of this study are as follows:1.Cloning and sequence analysis of gD geneBased on the sequence of gD gene of Pseudorabies Virus Strain Fa registered in GenBank,(register No.AY217094),a pair of primers was designed.Cultured in vero cells, the genome of PRV was extracted and used as template to amplify gD gene.Then the amplified product was subcloned into pMD18-T-simple vector,confirmed positive by enzyme digestion,the recombinant plasmid was named pMD-gD and sequenced by Takara Company.The sequence is 1245bp,including a 1209bp ORF which codes 402 amino acids, and shared 98.2%～99.6%nucleotide sequences identity and 96.3～98.8%the amino acid sequence homology among 12 virus stains.The results showed that the gD gene was highly conserved between different PRV stains.2.Construction of gD Prokaryotic Expression plasmid and its activity detectionA pair of primers was designed to amplify the de-signal-peptide of gD gene(gD-n). Using the pMD-gD plasmid as template,the objective gD gene was amplified by PCR and cloned into the pMD18-T-simple vector and named pMD-gD-n.Then the recombinant plasmid pMD-gD-n was sequenced,the sequence of gD-n was identical with that of the de-signal-peptide of gD.The pMD-gD-n plasmid was digested with EcoRⅠand HindⅢto obtain gD-n gene fragment.Then the fragment of gD-n was cloned into the pET32a(+),after confirmed positive,the recombination prokaryotic expression plasmid was named pET-gD-n and transformed into E.coli BL21(DE3).Induced by IPTG,the fusion protein was expressed and its molecular weight 63.8KDa was determined by SDS-PAGE.Detected by Western Blot,the pET-gD-n possesses specific reactionogenicity3.Construction of gD gene DNA vaccine and the study of its immune efficiencyThe pMD-gD plasmid was digested with EcoRⅠand XbaⅠto obtain gD gene fragment,then the fragment was subcloned into the the eukaryotic expression vector pcDNA3.1(+),pCI-neo respectively.The recombinant eukaryotic expression plasmids were successfully constructed and named pcD-gD,pCI-gD.BALB/c mice serologically negative to PRV were intramuscularly immunized twice with pcD-gD,pCI-gD DNA vaccines and both using IL-15 eukaryotic expression plasmids as adjuvant.Blood samples were collected at 2,4,6,8 weeks after the first immunization. The serum was used to detect IgG and neutralization antibody;The anti-coagulated blood was used to detect CD4~+ and CD8~+ T lymphocytes.In contrast with the control groups,the results demonstrated that pcD-gD,pCI-gD DNA vaccines could induce mice producing specific antibody IgG and neutralization antibody.After the second immunization,the titer of antibody was measured higher than that of before.The antibody titer level of the experimental groups always were lower than that of the inactived PRV groups at the whole experiment time,but antibody titers of DNA vaccine groups fell slowly after the hightest level than inactived PRV groups which suggested that the antibody induced by DNA vaccine could sustain longer.The recombination eukaryotic expression plasmids can stimulate significant CD4~+ and CD8~+ T lymphocytes production in vivo almost without any conspicuous difference to the positive control group.The challenge experiment confirmed that immune responses of mice can resist lethal dose PRV.The results demonstrated that pcD-gD,pCI-gD DNA vaccines could induce good humoral and cellular immune responses.The experiment groups vaccined with cytokine IL-15 eukaryotic expression plasmids and pcD-gD,pCI-gD DNA vaccine co-injection showed higher humoral and cellular immune responses than that of vaccined with DNA vaccine alone,especially the level of CD8~+ T lymphocytes.The results demonstrated that IL-15 eukaryotic expression plasmid was a good adjuvant which could significantly enhance the immunogenicity of pcD-gD,pCI-gD DNA vaccine.