Japanese Encephalitis Virus Isolation, Identification And Full-length Gene Cloning Research

Japanese encephalitis virus (JEV), being a member of the family Flaviviridae, is usually infected to the human body through mosquitoes and then may cause acute viral encephalitic and neurologic disease.Therefore,it is commonly regarded as a serious public health problem, with JEV being the infectious agent in many domestic and wild animals, especially with swine (viral associated abortions). Therefore, swine can be,and, actually,is a carrier or host of viremia-amplification. Hence, it is essential to prevent swine JE, which is usefel for swine production as well as humans health.The JEV genome contains a single-stranded, positive-sense RNA of approximately 11 kb in length . The single open reading frame contained in the genome is translated into a polyprotein which is cleaved co- and posttranslationally into structural and nonstructural proteins. The JEV virion contains three structural proteins: an envelope protein (E), a membrane protein M (precursor M [preM]) , and a capsid protein (C). At least seven nonstructural proteins: NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5, can be identified in JEV infected cells. Among them, M protein takes part in construe envelope, it is very important to keep E protein structure. E protein plays an important role in inducing protective immunity against virus infection. E protein can provide protection against JEV infection). The research of gene construction of Japanese encephalitis Virus have laid a firm theoretic foundation for further study of the genetic engineering vaccine against Japanese encephalitis Virus. This dissertation research results can be summarized as follows:1. The isolation and identification of Japanese encephalitis virus (JEV)Isolation and identification of Japanese encephalitis virus (JEV) was isolated from a porcine fetus and the mosquito. Isolated virus was injected into rat brain and passed to isolate JEV. The virus isolate was characterized as JEV by homologous genes assay, indirect fluorescent antibody assay, electron microscopy, serum neutralization assay and RT-PCR. The result is shown that both CQRC-1 and SCNJ-1 are JEV. Cloning and sequencing of PrM/E gene of the Japanese Encephalitis Virus(JEV) CQRC-1 strain by RT-PCR and analysis of the gene sequence. A comparison of the gene sequence with that of the JEV SA14 strain and SA14-14-2 strain demonstrates 98% and 97% sequence homology respectively.2.The construction of the infectious Japanese Encephalitis Virus cDNA clones of CQRC-1 strain , the measuring sequence and the acquirement of the recovered virusAccording to the published sequence of JEV SAI4 from GeneBank, six pairs of primers were designed for amplifying six overlapped sequences of the virus, obtain RNA of CQRC-1 strain by using Trizol Reagrnt Kit as template strand with One Step RT - PCR Kit of TaKaRa company, when they were ligated into a full- length sequence, and then cloned into pET-32a(+) vectors. The full - length cDNA clone was transfected into BHK-21 cells and CPE being observed in the fourth day. Its infectivity was verified byobservation of CPE, indirect immunofluorescene assay and RT-PCR. The construction of a stable Japanese encephalitis virus infectious cDNA Clone made the further research more easier.3. Cloning, sequencing and biological information analysis of PrM/E genes of Japanese encephalitis Virus (JEV) CQRC-1 strainA pair of primers P1 and P2 were designed and synthesized according to the PrM/E sequence of SAM strain of Japanese encephalitis virus. A 2000bp of target fragment amplified by RT-PCR from extracted RNA of CQRC-1, and then cloned into cloning vector pMD18-T. When the PrM/E gene in the pMD18-T-PrM/E was cut by BamHI and Sail, it was cloned into the expression plasmid pET-32a (+) which was treated with the same enzymes, resulting in a recombinant plasmid pET-PrM/E. The pET-PrM/E was identified by PCR, the restriction digestion and sequencing. The pET-PrM/E was used to transform Escherichia coli BL21 (DE3), In Vitro Expression of PrM/E gene of Japanese Encephalitis Virus Graduate, High-level ex...