登革2型病毒非结构蛋白NS4B及其突变体的真核表达与鉴定

目的：构建登革2型病毒非结构蛋白NS4B及其突变体Δ2K-NS4B基因的真核载体,并观察二者在哺乳动物细胞内的定位情况。方法：从登革2型病毒43株的全长cDNA克隆载体上扩增获得编码NS4B及缺失2K片段的NS4B突变体Δ2K-NS4B的基因;通过基因重组的方法分别将2段基因克隆入真核表达载体pcDNA6/V5-HisA,获得重组真核表达载体pc/D2-NS4B和pc/D2-Δ2K-NS4B;经脂质体法转染BHK-21细胞后,用RT-PCR、间接免疫荧光和Western印迹鉴定表达的蛋白。结果：重组蛋白D2-NS4B和D2-Δ2K-NS4B可在BHK-21细胞中表达,二者均定位于细胞质中,并具有较好的抗原性,能够被抗登革2型病毒NS4B的多克隆抗体特异识别。结论：重组蛋白D2-NS4B和D2-Δ2K-NS4B在哺乳动物细胞胞质中的正确表达,为深入了解NS4B在登革病毒致病过程中的生物学功能奠定了基础。

Expression and Identification of Dengue 2 Virus Nonstruetural Protein 4B and its Mutant in Mammalian Cells

Objective：To construct the recombinant enkaryotic plasmid expressing the nonstructural protein 4B（NS4B） of dengue 2 virus and its mutant Δ2K-NS4B lacking the 2K segment,and to obverse the location of both the recombinant proteins in the mammalian cells.Methods：The gene fragments of NS4B and Δ2K-NS4B were amplified by PCR from full length cDNA cloned vector of dengue 2 virus 43 strain,and then were cloned into enkaryotic expression vector pcDNA6/V5-HisA to generate the desired recombinant plasmid pc/D2-NS4B and pc/D2-Δ2K-NS4B.After transfected into the mammalian BHK-21 cells by lipofectamine,the expression of recombinant proteins were identified by RT-PCR,indirect immunofluorescence and Western blot.Results：Both the recombinant proteins D2-NS4B and D2-Δ2K-NS4B exist mainly in the cytoplasm,and they could react with the polyclonal antibody again the protein NS4B of dengue 2 virus.Conclusion：The correct expression of both the recombinant proteins in the cytoplasm of the mammalian cells has laid the foundation of interests on the biological function of the protein NS4B during the life cycle of dengue virus.