Cloning, Prokaryotic Expression Of The Pseudorabies Virus IE180 Gene And The Immune Efficiency Study Of Its DNA Vaccine Delivered By Salmonella Choleraesuis C500

In this research, IE180 gene of Pseudorabies Virus Strain Fa was amplified and subcloned into the prokaryotic and eukaryotic expression Vectors. The IE180 gene was expressed in E.coli Rosetta (DE3), and the immune efficiency of IE180 gene contained in the eukaryotic expression vector as DNA Vaccine delivered by Salmonella choleraesuis C500 was evaluated. The content of this study are as follows:1. Cloning and sequence analysis of IE180 geneBased on the sequence of IE180 gene of Pseudorabies Virus Strain TNL registered in GenBank, (register No. AF352564), utilizing the unique single enzyme digestion site Nco I within IE180 gene, 2 pairs of primers were designed. Both 2 segments of IE180 gene were amplified and subcloned into pMD18-T Simple vector which were named pMD-1 and pMD-â…¡. Then the 2 vectors were double digested using EcoRâ… , Salâ… and the 1.8kb fragment of pMD-1 (the upper half of IE180) together with the 5.2kb fragment of pMD-â…¡(the lower half of IE180 linked with pMD18-T Simple vector) were recovered. Then the 2 fragments were linked and the entire IE180 gene in vector pMD18-T Simple was obtained and designated pMD-IE180. Enzyme digestion and sequencing showed that the cloning of IE180 gene was successful. An ORF of 4389bp was cloned with 79.9% content of G+C, which encoded 1462 amino acid residue with putative molecular weight of 150,117Da.2. Construction of IE180 gene prokaryotic expression plasmid, IE180 protein expression and detection of its activityCloning vector pMD-IE180 was double digested using EcoRâ… and Salâ… , 4.4kb fragment of IE180 gene was recovered. Then the fragment was cloned into the prokaryotic expression vector pET-32a (+) and named pET-IE180. After confirmed positive by enzyme digestion, the recombinant plasmid was transformed into E.coli Rosetta (DE3), induced using IPTG for the expression of IE180 protein. The expressed fusion protein, which comes up to a small quantity of total protein, was determined by SDS-PAGE to have a molecular weight of 200 kD. Optimized expression condition was inducing at 37℃for 4h with IPTG concentration of 0.2mmol/L. The expressed protein was detected in the form of inclusion body and secretory protein. Detected by Western Blot, the IE180 protein possesses specific reactionogenicity.3. Construction of IE180 gene eukaryotic expression vector, and the evaluation of its immune efficiency as DNA vaccine delivered by Salmonella choleraesuis C500The pMD-IE180 plasmid was digested with EcoRâ… and Salâ… to obtain IE180 gene fragment, then the fragment was subcloned into the the eukaryotic expression vector pCI-neo and named pCI-IE180. Then the plasmid was electrotransformed into attenuated Salmonella choleraesuis C500 and designated S.C500/pCI-IE180. 30 BALB/c mice serologically negative to PRV were randomly divided into 3 groups: blank, S.C500/pCI-neo and S.C500/pCI-IE180. Intragastric administration was performed 4 times at the interval of 2 weeks for the mice immunization. The blank group mice were administrated with 0.2mL 10% sodium bicarbonate, while the other 2 groups were administrated with 10⁹ recombinant bacteria suspended in 0.2mL 10% sodium bicarbonate. Blood samples were collected at 2,4,6.8 weeks after the first immunization, indirect ELISA method was used to detect the antibody level. After the last blood collection, 0.1 mL of PRV diluted for 10^2.3 (5 X LD₅₀) was intraperitoneally injected into each mouse. The ELISA result showed that the IE180 gene eukaryotic expression vector as DNA vaccine delivered by Salmonella choleraesuis C500 had immunogenicity which could induce a relatively high level of antibody (1:1600) in mice. Challenged by PRV, the vaccine could protect about 30% of the mice from death, when all the mice in bland group were dead.