| The Japanese encephalitis is spreaded by mosquito and caused by Japanese encephalitis virus (JEV), damaged the central nervous system of animals and human being. The rate of the death and the mutilation are very high, and it is especially harmful to children. The disease mainly outbreaks in the summer and fall, and the area is closely correlated with the media. Our country is the epidemic area. In the 60s and 70s of the 20th century there is an outbreak. With inoculating the vaccine, the morbidity of JE remarkably reduced after the 70s, recent years the morbidity keeps lower level. There is 5000~10000 cases of JE every year, but somewhere occasionally outbreaks.JEV is member of the flavivirus genus (family Flaviviridae), is small enveloped particles with a single-stranded, positive polarity RNA genome of approximately 11 kb. The viral genome is capped at its 5'end and lacks a 3'terminal poly (A) tail. The unique open reading frame of the genomic RNA, flanked by two untranslated regions, encodes for a polyprotein in the order of NH2–C-prM-E-NS1-NS2A-NS- 2B-NS3-NS4-NS5—COOH in which C-prM-E are structural proteins and NS are nonstructural proteins. The polyprotein is co- and post-stranslationlly processed by a host signal peptidase associated with the endoplasmic reticulum and by a viral protease consisting of NS3. The envelope protein (E), the main structural protein of 53 kDa, is a glycoprotein embedded in the viral membrane. Since E protein is exposed on the surface of the viral membrane, it contains structural and functional elements that participate in the virus-host cell receptor interaction and it is hence known as the viral attachment protein. And E protein correlated with the toxicity, the pathogenicity, the cellular and tissular tropism of the virus, it contains the neutral epitopes of the virus. The three-dimensional structure of the envelope of JEV has three structural domains on E protein, and domainⅢ, located in the carboxy-terminal, is responsible for virus attachment to its receptor.The E protein is very important to research the receptors of JEV, but the structures of the E protein and the domainⅢprotein is not understanded.In order to analysis the biological characteristics of the E protein and the E domainⅢ, we amplificated the E protein and the E protein domainⅢof the gene, ligated them into expression vector pET28a and pET32a, and expressed the proteins in Ecoli using IPTG. By SDS-PAGE, analysed the molecular weight of the protein, the recombinant E protein's MWT is about 53 kDa, and the recombinant domainⅢprotein's MWT is 20 kDa. By Western blot assay detected the recombinant proteins using a mouse monoclonal antibody of the JEV and the anti-6His antibody. The result of the ELISA indicated the N'of the E protein can be recognized by the monoclonal antibody 2F2 and 2H4, also displayed the N'of the protein contained the neutral epitopes.JEV E protein gene fragment were amplified by PCR from vector pMD18-T-E, and cloned into pUC19 for DNA sequencing. After verified by DNA sequencing, they were subcloned into the bait vector pGBKT7 of yeast two-hybrid system (GAL4), and the recombinant plasmid were subsequently transferred into yeast cell AH109, and its expression products were tested whether it could activate the reporter genes in the yeast two-hybrid system. The results displayed the yeast cells AH109 having the recombinant plasmid could not grow in BD medium lacking histidine, this indicated the expression products of the recombinant plasmid could not activate the His3 gene. Byβ-Galactosidase clonal-laft-filter-analysis and plate process, we detected the expression products had not the activation to LacZ gene, and were nontoxic to yeast cells AH109.The research successfully expressed the E protein and E protein E domainⅢin Ecoli. , and obtained the yeast two-hybrid system having the bait vector with JEV E protein gene. The research provided the base for structural and functional research and interaction with the receptor proteins of E protein.
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