| Japanes encephalitis(JE) caused by Japanes encephalitis virus (JEV)is a zoonotic severe acute infectious disease .The fatality rate is about 30%; and as high as 50% of patients suffer from neurological sequelae, and the cases in China is up to 80% of the total cases of the world. JEV transmission occurs in an enzootic cycle, invoving primarily Culex mosquitoes and swine as vectors and amplifiers, repectively; infected swines may suffer from abortion, stillborn delivery or mummified fetuses. Inactivated JEV vaccines prepared from infected mouse brain (BIKEN or JEV AX) or primary hamster kidney cells and a live attenuated JEV vaccine are effective against JEV infection. However, allergic reactions of the inactivated vaccine, high cost of production, need for multiple immunizations and lack of long term immunity necessitate the need to develop safer and less expensive vaccines for JE.In order to elucidating the pathogenesis of JEV/WHe strain and developing novel vaccine, the following work has been done:1.Characterization of the full-length genome of the JEV strain WHe.The complete nucleotide sequence of JEV/WHe strain was determined and its amino acid sequence was deduced. The results display that the genome of WHe strain contained a single open reading frame of 10,296 nucleotides (nts) which was flanked by un-translated region (UTR) containing 95 bases at the 5'-end and 585 base at the 3'-end, respectively. Comparison of sequences showed that the overall amino acid sequence of WHe strain was similar to P3 strain and the 3'UTR secondary structure was similar to Vellore P20778 strain. However, a similar RNA secondary structure of the last 84nts of 3'UTR was observed among every member of the genus flavivirus of Flaviviridae . Some significant amino acid differences of viral envelope (E) protein were observed among WHe,P3 and 19 other strains; the amino acid sequence of E protein in WHe and P3 strain possessed M(76), G(306), L(408) instead of T(76), E(306), S(408) in other stain.These data suggested that WHe strain and P3 strain shared the same evolutionary process. RGD(387-389) tripeptide were found in WHe strain and other attenuated JEV strains, these show that strain-specific determinant of JVE can be different among each virulence phenotype. In addition, phylogenetic relationship based on the full length genome are highly similar to those based on the Egene, indicating that phylogenetic analysis of the E gene will be useful for studying the genetic relationships among JEV isolates. A more extensive E gene-based phylogenetic analysis was done on a selection of 140 JEV isolates available from GenBank, which reprent a temporally and geographically wide variety of JEV strain.2.Amplification of full-length cDNAof JEV/WHe strain by long RT-PCR.The full-length cDNA was amplified by long reverse transcription-polymerase chain reaction (long RT-PCR) . The full-length cDNA was identified by PCR and the fragments which contain the 3'and 5'untranslation regions were cloned into pGEM-T vector, and the sequences of inserted fragments were determined by Invitrogen Corporation. The results showed that the approximate 11kb full-length cDNAs of WHe strain were amplified by long RT-PCR and it's correctness were proved by partial nucleotide sequences analysis.3.Construction and identification of DNA vaccine vector of JEV/WHe strain.The prM/E and NS1 gene of JEV/WHe strain were cloned and sequenced before cloned to the pAVX1 vector respectively. The recombinant plasmid pAVXl-prM/E and pAVXl-NSl were identified by restricted enzyme digestion with Kpn I and Not I .4.The expression of NS1 gene of JEV/WHe strain in E.coli BL21(DE3).The NS1 was cloned into the expression vector pET28b(+) to constructed a recombinant prokaryotic expression plasmid, pET28b(+)NSl, the recombinant plasmid was then transfected into E.coli BL21(DE3). SDS PAGE electrophoresis showed that the molecular weight of the expressed protein was about 43kD. These work provided the foundation for the development of subunit vaccine and DNA vaccine.
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