Prokaryotic Expression Of West Nile Virus E Protein Domain Ⅲ And Preparation Of Monoclonal Antibodies Against E Protein Domain Ⅲ

West Nile Virus (WNV) is a member of the Japanese encephalitis virus group of the genus Flavivirus family Flaviviridaecan, which can cause an acute disease of livestock and human, it was first isolated in 1937 from the blood of a febrile female patient in the West Nile district of Uganda. WNV is maintained in nature in a bird-mosquito-bird transmission cycle primarily involving Culex species mosquitoes. Human or equine infection results primarily from mosquito bites, and West Nile fever or West Nile encephalitis can be caused.According to published sequence of the New York strain of West Nile virus, one pair of unique primers for WNV E protein domain III were designed, and the domain III gene of WNV was amplified by polymerase chain reaction (PCR). The product of PCR, which is about 360bp was cloned into pGEM-T vector, then the recombinant plasmid was digested by restriction endonucleases BamHI and HindIII to get DIII gene. The fragment was inserted into BamHI and HindIII multiple cloning site of pET-32a expression vector, and the recombinant vector was transformed into E.coli BL21(DE3). Expression of the domain III was induced by IPTG and confirmed by SDS-PAGE and Western Blot, and its relative molecular mass of the recombinant domain III fusion protein was approximately 30.3×103. The fusion protein was purified by metal-ligand affinity chromatography, which should be used as diagnostic reagent of West Nile fever.BALB/c mice were immunized with recombinant envelope protein domain III of West Nile virus and its spleen cells were used to prepare the monoclonal antibodies (McAb) by hybridoma technique. Three hybridoma cell strains secreting McAbs against WNV envelope protein domain III, designated as 4F7, 6H3 and 8E4 respectively, were obtained by indirect enzyme linked immunosorbent assay (ELISA), and these three McAbs 4F7, 6H3 and 8E4, belonging to IgG1,IgG1,IgG2a respectively, can identified two epitopes of envelope protein domain III, among them, 4F7 and 6H3 against the same epitope and 8E4 to another one. The results of indirect ELISA, Western Blot and indirect immunofluorescence experiment indicated that these three McAbs were specific for West Nile Virus envelope protein domain III and didn't react with Japanese encephalitis virus and other viruses, so they can be used for specific detection of West Nile Virus.