Construction Of Recombinant Baculovirus And Lentivirus Of West Nile Virus

West Nile Virus, a member of the genus Flavivirus, family Flaviviridae, is a mosquito-borne, neurotropic pathogen that has caused substantial morbidity and mortality in animals, often including birds, horses and humans. West Nile Virus had spread across North America within the next couple of years, since the initial introduction in New York in 1999. New Outbreak West Nile Virus isolates are highly virulent and which are causing severe neuro-invasive disease in many species, including humans. In view of no antiviral drugs available for treatment of West Nile disease, vaccines are proposed an effective alternative to prevent the disease. The West Nile virus has not been found in China, but China has suitable conditions for prevalence and propagation of the disease. Therefore, the developments of preliminary research (vaccines, diagnosises, and et.) have its strategic significance for strengthening technical reserves in the field and coping with possible outbreaks in the future. The main contents of the study are:1. Construction of recombinant baculovirus of West Nile Virus prMETo construct the transfer plasmid (pFast-VSV/G-prME) of eukaryotic expression, the plasmid DNA chain containing prME gene expression cassette (CMV-prME-poly A) was originated from plasmid prME and inserted into pFastBacl-G, which the G gene is in charged of the polyhedron promoter of pFastBacl. Transfer plasmid was transformed into E. coli DH10Bac which containing baculovirus shuttle plasmid and helper plasmid. The G-prME gene in E.coli DH10Bac was transposed into the bacmid. The colone of E.coli containing recombinant bacmid-G-prME was screened by blue-white screening. After isolation and purification, recombinant bacmid DNA was transfected into sf9 cells to acquire the recombinant baculovirus rBV-G-prME. To demonstrate the transduction efficiency of the recombinant baculovirus and the level of expression of prME gene within mammalian cells, BHK-21 cells were transduced with recombinant baculovirus rBV-G-prME. The truth and effectivity of prME gene expression was confirmed by Indirect Immunofluorescent Assay and Western Blotting.2. Lentiviral vector-mediated expression of West Nile Virus prME geneWNV/prME was inserted into transfer plasmid pHR-IRES-EGFP.293T cells were cotransfected with pHR-WNV/prME, pΔ8.2, and pVSV/G; the supernatant of 293T cell was harvested after 48 h post cotransfection. Normal 293T cells were transduced with the supernatant. Expressed protein was confirmed by Indirect Immunofluorescence. The result showed that:using Lentiviral Packaging System, pseudotyped viruses can efficiently transduce 293T cells. Expression of WNV/prME gene can be implemented.