Generation And Characterization Of A Mammalian Cell Linecontinuously Expressing PrM-E Protein Of WNV

West Nile virus(WNV) is a mosquito transmitted arbovirus which belonging to genus Flavivirus in the family Flaviviridae. Many kinds of birds and mammals are susceptive to WNV, in which horse suffer the most. Although 80% of the infections are asymptomatic, patients may develop clinical signs. Some infected patients develop severe neuroinvasive disease with about 10% of WNV cases being fatal. By now, there is no effective therapeutic treatment, detection and vaccination are good way to prevent virus spreading and infection.Vaccination is an efficient way to prevent virus infection. So far, several vaccines for animals are commercialized in America. Among which inactivated vaccine and attenuated live vaccine show good immunogenicity, however they are limited by several disadvantages such as side effects and high cost. Thus more and more researchers make efforts to develop novel vaccine which is more efficient and safe.It has been shown that prM and E protein of WNV expressed in eukaryotic cells could assemble into virus-like particles(VLPs). Mouse experiments and antigenic analysis revealed that the VLPs is similar with the WNV virions neutralizing antibody and protective immunity. We had successfully generated a mammalian cell line continuously expressing VLPs of JEV in previous study. Thus, establishment of eukaryotic cell lines that could continuously expressing VLPs would be an good way to develop an effective subunit vaccine against WNV.For identification of the cell line we generated a monoclonal antibody(MAb) against the WNV prM protein. Western blot analysis indicated that the MAb could reacted with WNV prM specifically. IFA assays demonstrated that the MAb recognized native prM protein in transfected BHK-21 cells. Dilution titers of MAb 10F7 in supernatants and ascites were 1:2048 and over:1:409,600, respectively. Isotype investigations revealed that the MAb produced by this clone was id- entified as the IgG2 a subtype, and the light chains were k type.BHK-21 cells were transfected with the recombinant plasmids pCAG-WVV-prME encoding WNV prM and E protein. Cells were then grew in medium containing G418 and then identified by IFA and Ag-ELISA. The cells with high-level protein expression were then purified by limiting dilution cloning. After several identification and subclone a cell line containing a high percentage of antigen-expressing cells were obtained, designated the W-92 cells. IFA and Flow cytometry analysis indicated that the W-92 cell line subclones could maintain high percentage of antigen expressing cells and high level of E antigen synthesis and release during 30 passages. Electron microscopy observation of purified secretion products of W-92 cells reveals that prM and E protein could assemble into virus-like particles(VLPs). Virus neutralizing antibody level against WNV in mouse were about:1:40 while the level against JEV is 1:20 to 1:80. Virus neutralizing antibody level against JEV in goose were about:1:20 to 1:40. The prME-VLPs could provide 50%-70% protection against lethal challenge of JEV P3 virusIn this study, a prokaryotic expression system was used to generate a recombinant protein for use as an antigen to immunize mice. A WNV-prM protein-specific MAb(10F7) was generated using hybridoma technology. The MAb reported here may provide a valuable tool for the further exploration of functions of the prM protein and the biological properties. And it may also be developed for potential clinical applications. A cell line continuously expressing prM-E protein of WNV was generated and it would appear to be a useful source of antigen for WNV subunit vaccines and diagnostics.