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

In this research,gL gene of Pseudorabies Virus Strain Fa was amplified and subcloned into the prokaryotic and eukaryotic expression Vectors.The gL gene was expressed in E.coli BL21,and the immune efficiency of gL gene contained in the eukaryotic expression vectors were evaluated.The content of this study are as follows:1.Cloning and sequence analysis of gL geneBased on the sequence of gL gene of Pseudorabies Virus Strain Fa registered in GenBank,(register No.NC006151),a pair of primers was designed.Cultured in vero cells, the genome of PRV was extracted and used as template to amplify gL gene.Then the amplified product was subcloned into pMD18-T-simple vector,confirmed positive by enzyme digestion,the recombinant plasmid was named pMD-gL and sequenced by Takara Company.The sequence is 503bp,including a 471bp ORF which codes 127 amino acids, and shared 96%～100%%nucleotide sequences identity and 96.3%～98.8%%the amino acid sequence homology among 5 virus stains.The results showed that the gL gene was highly conserved between different PRV stains.2.Construction of gL Prokaryotic Expression plasmid and its activity detectionA pair of primers was designed to amplify the non-signal-peptide of gL gene(gL-n). Using the pMD-gL plasmid as template,the gL gene was amplified by PCR and cloned into the pMD18-T-simple vector and named pMD-gL-n.Then the recombinant plasmid pMD-gL-n was sequenced,the sequence of gL-n was identical with gL which cloned on former stage.The pMD-gL-n plasmid was digested with EcoRⅠand SalⅠto obtain gL-n gene fragment.Then the fragment of gL-n was cloned into the pET32a(+),after identification, the recombination prokaryotic expression plasmid was named pET-gL-n and transformed into E.coli BL21(DE3).Induced by IPTG,the fusion protein was expressed and its molecular weight 39KDa was determined by SDS-PAGE.Detected by Western Blot, the pET-gL-n possesses specific reactionogenicity3.Construction of gL gene DNA vaccine and the study of its immune efficiencyThe pMD-gL plasmid was digested with EcoRⅠand SalⅠto obtain gL gene fragment,then the fragment was subcloned into the the eukaryotic expression vector pCI-neo and named pCI-gL.Then the plasmid was electrotransformed into attenuated Salmonella choleraesuis S.C500 and designated S.C500/pCI-gL.30 BALB/c mice serologically negative to PRV were randomly divided into 3 groups:negative control, S.C500/pCI-neo and S.C500/pCI-IE gL.Intragastric administration was performed 3 times at the 2 weeks interval for the mice immunization.The negative control group mice were administrated with 0.2mL 10%sodium bicarbonate,while the other 2 groups were administrated with 10~9 recombinant bacteria suspended in 0.2mL 10%sodium bicarbonate. Blood samples were collected at 2,4,6weeks after the first immunization,indirect ELISA method was used to detect the antibody level.After the last blood collection,0.1mL of PRV diluted(200TCID50/0.1 ml) was intraperitoneally injected into each mouse.The ELISA result showed that the gL gene eukaryotic expression vector as DNA vaccine delivered by Salmonella choleraesuis SC500 had immunogenicity which could induce a relatively high level of antibody in mice.Challenged by PRV,the vaccine could protect about 70%of the mice from death,when all the mice in bland dead.