| West Nile virus (WNV) is an arbovirus that can lead to West Nile fever (WNF), fatal West Nile encephalitis (WNE) and West Nile meningitis (WNM) to human,horses and many other animals. Since it was discovered, the disease had already outbroken in many countries. The eisease is a great threat tohuman life and the development of animalhusbandry. At present, although our countryhave not yet found the west Nile virus, it is necessary to establish a quick and easy diagnostic method and make the disease prevention and treatment of immune technical reserves.Detection of WNV mainly includes the etiology diagnosis and serodiagnosis. The former highly depends on laboratory equipments and this method is easy to cause the virus diffusion. Cause this virus does not exist in our country, it is difficuLt to obtain the specimen. These leads to serodiagnosis is more in line with the actual conditions of our country.In recent years, recombinant antigens such as WNV E protein and E protein domainⅢ which based on Gene Engineering technologyhave been widely used in serodiagnosis. E protein is the best envelope protein of WNV, which can stimμlate the immune system to produce a lot of neutralization antibodies, and also mediate the combination of virus and the cell receptors and cell fusion. E proteinhas three structural domains and domain Ⅲ shows immunoglobuLin like fold which mediates virus adsorption on thehost cell. David W.C. Beasley expressed E protein structural domainⅢ in vitro and then took it as antigen, which is used to detect WNV antibodies of the serum sample in indirect ELISA test. Apart from this, these scientists also through experiments proved that the recombinant protein can avoid cross reaction between WNV and other members of the group.Take the Restriction Enzyme cutting site sequence and protect sequence in two sides of WNV NY99strain E protein structural domainⅢ gene sequences published on GeneBank and then sent it to biotechnology company. After synthesis, the target gene was combined to PET-28a express vector, then carry out the screening cuLture and select the positive recombinant plasmid. The correct positive restructured plasmids was got and named PET-DⅢ after enzyme cut experiment and sequencing appraisal. The restructured expression vector was transformated into E.coli BL21competence cells and the expression was induced by IPTG. After SDS-PAGE analysis and Western-Blot appraisal, it was determined that the express products already expressed recombinant protein, relative molecμLar weight is17000. Use the protein concentration assay kit to determine the purified recombinant protein and the concentration is124.03ug/mL.Through the checkerboard titration to determine the antigen best coating concentration is3.75ug/mL, the best serum dilution is1:1600, the concentration of HRP-Goat anti-rabbit IgG at1:6000. Use the specificity test, sensitivity test and repeatability test to evaluate the established indirect ELISA method and resμlts are satisfactory. In this study, we use the prokaryotic expression system to express the recombinant protein of E protein domain Ⅲ in vitro and establish indirect ELISA system to determine WNV antibodies. It provides valuable information for the diagnosis and prevention and control of West Nile virus disease of our country. |
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