| West nile virus is a member of the Flaviviridae family, genus Flavivirus with positive strand RNA genome. It is infectious to human, horse and birds. West nile virus has three structural proteins:capsid protein (C), envelope protein (E) and premembrane protein (prM).It was reported that the co-expression of premembrane protein and envelope protein led to the formation of VLP in specified conditions.In the first part of the research, the prm+e gene based on the published sequence of WNV strain NY99was synthesized. Restrict enzyme sites and His-tag were introduced into the prM+E coading fragment by PCR. The target DNA fragment was inserted into pFast-Bacl and then the correct recombinant plasmids were transformed into DHIO-Bac in which transposition happened and galactosidase gene was interrupted by the target DNA fragment. The recombinant bacmids were screened by blue-white selection and verified by PCR. After extraction and purification, recombinant bacmids were transfected into sf9cells to generate infectious recombinant baculovirus. These new recombinant baculovirus were used to infect normal sf9and then the cells and upper medium at different time were harvested. RT-PCR, SDS-PAGE and Western Blot were used to detect target mRNA or protein.The Pfast-Bacl-W was transformed to DH10-Bac. After amplification with couples of primers, the results showed that the recombinant bacmids were successfully constructed. Cytopathic effect(CPE) was not obvious after the recombinant bacmid was transfected into sf9cells, however, the upper medium could cause new normal cells to produce obvious CPE. The titer of the second generation venom was verified to be7.6×107pfu/mL. The second generation venom was used to infect sf9cells with Multiplicity of Infection(MOI) five. The prm+e gene was found to be transcripted24hours after the infection; at the same time, recombinant baculovirus release was detected. The SDS-PAGE showed that the infected cells have one more strap which was located between43.0kD and66.0kD compaired the nomal cells. This extra strap could react with WNV polyclonal antibody of rabbit source and it seems to be the complete E proten judging by its molecular weight.In the second part of the research, the prm+e gene was inserted into the expression vector pET-30a(+) and pGEX-4T-1to form two recombinant plasmids:pET-30-W, pGEX-4T-1-W. After sequenced, these two recombined plasmids were transformed into BL21and induced for different time or by different concentration of isopropyl-1-thio-β-galactopyranoside (IPTG). Samples were harvested and assayed by SDS-PAGE and Western Blot.It was disappointed that the prM+E protein did not express in BL21,and expanding the inducing time or changing concentration of IPTG made no difference.It was the first time at home and abroad to express the complete prM+E protein in baculovirus expression system successfully. Although the prM/M was not detected by Western Blot, the prm/m gene must have been transcripted and translated because its location was at the N terminal of e gene. The existing form of the prM+E and whether it was secreted still needs to be further researched. After purification, the E protein from the sf9cells could be used as the antigen in serological assay or subunit vaccine. The prM+E protein form needs to be futher researched to verify the VLP’s existence. The method and result present in this theses will offer some to the futher research which aims to buiding up a complete sensitive and specific testing method.If the VLP’s existence could be verified in the medium,then it could be used in the MAC-ELISA and other methods after being concentrated. In summary, this research built a foundation of the future construction of a complete, sensitive and specific testing method. |
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