我国登革2 型病毒株 N S1 基因的克隆与表达

Cloning and expression of the NS1 gene of the Chinese strain of dengue 2 virus

目的：通过对登革病毒 Ｎ Ｓ１ 基因的表达，研究表达的 Ｎ Ｓ１ 蛋白的抗原性，为研制新型登革疫苗提供依据。方法：采用 Ｒ Ｔ Ｐ Ｃ Ｒ 和分子克隆技术将扩增的 Ｎ Ｓ１ 基因插入到ｐ Ｂ Ｖ２２０ 载体中，通过温控诱导进行外源蛋白的表达，用 Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ 和蛋白质印迹法分别测定表达产物的相对分子质量及特异性。结果：获得正向插入的 Ｎ Ｓ１ｐ Ｂ Ｖ２２０ 重组质粒，表达产物的相对分子质量约为５１ ０００，与预期大小一致，并且可与登革 ２ 型病毒的腹水抗体起特异反应。结论：构建的 Ｎ Ｓ１ 基因ｐ Ｂ Ｖ２２０ 重组质粒所表达的蛋白为我国登革 ２ 型病毒株所特有。

Objective: To investigate the antigenicity of the NS1 protein of dengue 2 virus by expression of the NS1 gene in orde to provide the basis for developing new vaccine against dengue infection. Methods: The NS1 gene fragment was amplified and cloned into the plasmid vector pBV220 by using RT PCR and molecular cloning technique. The protein NS1 was expressed in E.coli containing recombinant NS1 pBV220 vector by the temperature induction. SDS PAGE and Western immunoblotting were used to analyze the molecular weight and specificity of the expressed protein, respectively. Results: The positive recombinant plasmids harbouring the right inserted NS1 fragment were obtained. The molecular weight of protein expressed in E.coli containing the recombinant plasmid was about 51 000, corresponding to the estimated molecular size of the NS1 protein, and the protein could react with the DEN 2 Chinese strain hyperimmune mouse ascitic fluid. Conclusion: The expressed NS1 protein was specific for the Chinese strain of dengue 2 virus.