| WNV and JEV are the members of the Japanese encephalitis virus group of the genus Flavivirus family Flaviviridae. These viruses are mosquito-borne flavivirus.and maintained in bird-mosquito-bird cycle in nature,with humans and other vertebrate animals like horse as accidental hosts. WNV was first isolated in1937from the blood of a febrile female patient in the West Nile district of Uganda, then it outbroke in New York in1999which caused people panic and great economic losses. Office International Des Epizooties(OIE) classified this disease as notifible disease. China was JEV epidemic district and JEV has Rology cross reaction with WNV, in order to exclude suspected cases of WNV. so we are necessary to establish a differential diagnosis methods as technical reserves.Envolope protein is the most important structural protein of WNV,its domain III contains antigen epitopes which specific to WNV and its main neutralizing epitopes are located in the E307、E330、E332residues,this area has always been considered as receptor area.Through the recognition of neutralization antibody of this area,we can distinguish between Japanese encephalitis virus (JEV), yellow fever virus, dengue virus and Murray valley encephalitis virus (MVEV).According to the published sequence of the WNV NY99strain and JEV SA14-14-2strain, one pair of specific primers for WNV E protein domain Ⅲ and JEV E protein domain Ⅲ were designed respectively.and the domain Ⅲ gene of WNV and JEV were amplified by polymerase chain reaction(PCR) and reverse transcription-polymerase chain reaction (RT-PCR).The product of PCR and RT-PCR,which is about378bp and481bp were cloned into pET-32a(+) vector respectively.then the recombinant plasmid were digested by restriction endonucleases EcoR I and HindⅢ.Domain Ⅲ gene were cloned into pET-32a(+) vector in right ORF were confirmed by sequence analysis. The recombinant vectors were transformed into E.coli BL21(DE3) and induced by IPTG.The expression product of the protein were confirmed by SDS-PAGE,and its relative molecular mass of the recombinant domain Ⅲ fusion protein were approximately30kDa and35kDa. The fusion protein were purified from bacterial inclusion bodies by His-Bind affinity chromatography which should be used as diagnostic reagent of West Nile fever. Use the purified WNV EⅢ and JEV EⅢ protein,3strain monoclony antibody were indentified by Western blot and indirect ELISA,the results showed that McAbs8F4A4,6H3B1were specific to WNV and didn’t react with JEV, so they can be used for specific detection of West Nile Virus.This study used purified WNV EⅢ protein as antigen coating the ELISA plate and monoclony antibody8F4A4which was specific to WNV as competitive detection antibody to establish a c-ELISA for detecting WNV antibody. We compared with different conditions to choose an optimum concentration of coating antigen, dilution rate of serum, the blocking solution choice, serum reaction time, as well as the substrate. The results showed that the concentration of coating antigen was530ng/mL the best dilution of McAb8F4A4was1:8000and the best dilution of sesum was1:10. A total number of56clinically confirmed negative sera were tested by the c-ELISA. Through analysis, we concluded that the cut-off value was30%. A set of711clinical sera were tested by the c-ELISA and all of them were negative. Contrast to the commercial kits, this method could identify the JEV and WNV antibody, which provide an efficient antibody detection method for the West Nile virus disease epidemiology investigation in our country. |
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