Cloning And Fusion Expression Of Porcine Pseudorabies Virus Glycoprotein B-cell Epitope Coding Sequences

Porcine pseudorabies(PR) is caused by pseudorabies virus(PRV), which one of the major infectious diseases causing serious damage to the healthy development of swine industry in our city. The PRV belongs to the herpes virus family herpes virus subfamily, linear double-stranded DNA virus, genomic DNA G+C content as high as 72%, coding protein 70-100. At present there are 11 species of known coding glycoprotein, including g B, gC, gD glycoprotein, there play an important function between the virus and host cell interaction.gB, g C, gD glycoproteins are capsule membrane proteins, which widely distributed in capsule membrane surface of virus particles and different herpesvirus g B, gC, gD glycoprotein basically consistent, highly conserved. gB, gC, g D glycoprotein with a multiple of spaces epitopes and linear epitopes, antibodies induced by each protein alone can neutralize the virus, preventing the virus from infected cells. The advantage of gB, gC, g D glycoprotein spatial and linear epitopes can induce the body to produce high levels of neutralizing antibodies. gB, g C or gD protein sub-vaccine can protect mice or pigs from PRV infection, having a good immunogenicity and reactogenicity and constructing recombinant DNA vaccine and protein vaccine of first choice detection.In this study, clinical isolates of PRV strain epidemics as research material, the virus extracted DNA after PRV virus in cell culture. Based on the PRV in Genbank, g B, g C, g D protein coding sequence of B cell epitope and using fusion PCR technology, designed epitope amplification primer B cell of g B, gC, gD fusion protein coding sequence; gB, gC, gD protein coding sequence of B cell epitope for g B organic fusion gB-C-M-C-N, gB-D epitope coding sequence. The gBCM, g B-CN, g B-D epitope coding sequences were cloned in PMD19-T carrier, constructing plasmid PMD19-T-gB-C-M, PMD19-T-gB-C-N, PMD19-T-g BD.Application BamHI and HindIII restriction enzymes enzyme cutting the cloning vector, agarose electrophoresis recycling enzyme products and cloned in the PET-28 a expression vector, the recombinant vector restriction enzyme digestion and the insert was analyzed and sequenced, which successfully constructed p ET-28-gB-CM, pET-28-gB-CN, pET-28-g B-D expression vector. Positive clones were picked containing the expression vector, positive clones were induced and optimize the conditions for its expression and successfully expressed the PRV fusion glycoprotein B cell epitope coding sequence. The expressed protein by SDS-PAGE electrophoresis and Westernblot blot test, preliminary identification of the biological activity of the expression product, the results showed that the protein can be PRV-positive serum identification, with good biological activity, the level of swine immunity and build subunit vaccine testing foundation, and the expression of protein SDS-PAGE electrophoresis and Western blot Western blot test, preliminary identification of the expression product of biological activity, the results showed that the expressed protein could be PRV positive serum identification, have good biological activities, for swine immune level detection and the construction of a subunit vaccine laid a foundation.