我国登革2型病毒43株PrM-E基因的构建及其在哺乳动物细胞中的表达

Construction and expression of the PrM E gene of dengue 2 virus 43 strain in mammalian cells

目的：构建我国登革２ 型病毒４３ 株（Ｄ２－４３）的ＰｒＭ－Ｅ基因片段，观察其在哺乳动物细胞中的表达。方法：采用ＲＴ－ＰＣＲ和分子克隆技术构建ＰｒＭ－Ｅ基因片段，包括编码ＰｒＭ 的信号肽序列、完整的ＰｒＭ 蛋白和去除羧基端跨膜疏水区部分氨基酸的９７．８％ Ｅ蛋白的基因片段。用电穿孔法将构建的含有ＰｒＭ－Ｅ基因的ｐＣＭＶ－ＭＥ真核重组质粒导入哺乳动物细胞中进行表达。采用蛋白印迹和间接免疫荧光法对表达产物进行特异性鉴定。结果与结论：构建的ＰｒＭ－Ｅ基因全长２ ０１９个核苷酸，序列分析证明其核苷酸序列是正确的。ＰｒＭ－Ｅ基因在ＢＨＫ－２１ 细胞中获得高效表达，表达蛋白可与Ｄ２－４３ 多克隆抗体起特异反应，且表达蛋白主要位于细胞浆中。

Objective: To construct the PrM E gene fragment of dengue 2 virus strain 43(D 2 43)isolated in China and to observe its expression in mammalian cells. Methods: The PrM E gene, containing the PrM signal sequence, whole PrM and 97.8% E, was constructed by RT PCR and molecular cloning technologies. The recombinant eukaryotic expression plasmid pCMV ME, containing the PrM E gene fragment, was transfected into BHK 21 cell by electroporation to express the PrM E gene. The specificity of the expressed products was identified by Western blotting and the location of the expressed products in the cell was confirmed by indirect immunofluorescence analysis. Results and Conclusion: The constructed PrM E gene was 2 019 nts in length and consistent with the reported sequence,which was proved by DNA sequencing analysis. The SDS PAGE and indirect immunofluorescence analyses showed that the gene was expressed highly in BHK 21 cell and the expressed products mostly located at the cytoplasm. Western blot analysis further confirmed that the expressed protein was capable of reacting with the polyclonal antibody against the D 2 43 virus.