Construction Of Recombinant Virus Expressing The Major Immunogenic Genes Of Japanese Encephalitis Virus And Study On Its Biological Characteristics

Japanese encephalitis is a serious enzootic disease caused by Japanese encephalitis virus. JEV infection in the human body can damage the central nervous system seriously It’s also serious damage to human health. And JE is one of the major epidemic diseases which are serious harm to the pig industry, mainly causing sow abortion, stillbirths, mummified and boar orchitis reproductive failure.At present, vaccination is the most effective prophylactic measures to prevent and control the disease. Inactivated vaccine (P3 strain) and attenuated vaccine (SA14-14-2 strain) which are commonly used are both developed based on the JEV genotypeⅢ strain, while emergence of genotype I of JEV as the dominant genotype.There are some problems for now used JE vaccine to prevent and control these diseases. Faced with this question, it is important to develop the JE vaccine based on the JEV genotype I strain, In this study, recombinant psedorabies virus which expressing JEV PrM-E was constructed to achieve the purpose for simultaneous controlling of JE and pseudorabies.1.Theconstructionofrecombinant PRV virus expression JEV dominant immunogenic genes and study on its immunogenicity.The gene sequence of the structural proteins PrM-E of Japanese encephalitis virus was amplified by PCR using JE genome sequence as the template.It was cloned into the vector pCAG by restriction sites(EcoRI BglⅡ) and we got the vector pCA-PrM-E. The gene sequence was digested from the vector pCA-PrM-E by restriction sites(EcoRI bGL/Ⅱ) and linked to the vector pIE-CMV-BGH-CMV-SV40. Finally, we got the transfer vector plasmid pIE-pCA-PrM-E. Recombinant transfer vector was co-transfected PK-15 cells with the genome DNA of PRV Ea TK-/gE-/1acZ+ stain. With the methods of homologous recombination, the target gene PrM-E in the transfer vector was inserted into the PRV TK-/gE-/LacZ+. Recombinant virus TK-/gE-/PrM-E+ were obtained after the final screening by PCR and plaque purification. JEV PrM-E expression in the recombinant virus was proved by western blot. The heterologous PRM-E sequence stably existing in recombinant virus was proved by genetic stability experiments. Virus growth curve showed that JEV PrM-E can not affect the growth of pseudorabies virus. The animal experiments in mouse showed the recombinant virus could induce the high level of neutralizing and ELISA antibody against PRV in mouse? the recombinant virus also could induce ELISA antibody against JEV in mouse, but combinant virus had only 50% immune protection2.The construction of recombinant PRV virus expression JEV double copies domina imunogenic genes and study on its immunogenicity.Recombinant transfer vectors were co-transfected PK-15 cells with the genome DNA of TK-/gG-/PrM-E+/gE-/eGFP+ stain. Withthemethods of homologous recombination, the target genes PrM-E in the transfer vector were inserted into the recombinant virus TK-/gG-/PrM-E+/gE-/eGFP+ with taking the place of eGFP. Recombinant virus was selected by fluorescent method. Recombinant virus TK-/gG-/PrM-E+/gE-/PrM-E+ was obtained after the final screening by PCR and plaque purification. The PrM-E expression in the recombinant viruse was proved by western blot. The animal experiments in mouse showed the recombinant virus could induce the neutralizing and ELISA antibody against PRV in mouse and the recombinant virus could induce ELISA antibody against JEV in mouse,and recombinant virus had also only 50% immune protection...