摘要
目的克隆登革2型病毒(DEN2)NS3基因并用酵母真核表达,获得全长的重组NS3蛋白质,为其结构与功能的研究提供条件。方法从感染DEN2(NGC株)后病变的C6/36细胞上清液中提取RNA,通过RTPCR扩增NS3基因片段,与载体pPICZαB连接筛选得到重组质粒,电穿孔法将重组体整合入酵母菌P.pastoris,经抗生素Zeocin筛选产生的转化子进行表型鉴定,取Mut+菌诱导表达,SDSPAGE检测表达产物。结果RTPCR得到1.85kb的NS3基因片段,酶切鉴定与测序显示重组质粒含DEN2全长NS3基因;Mut+酵母转化菌可分泌表达Mr约78000的蛋白质,与DEN2多抗和登革2型病毒患者恢复期血清的免疫印迹证实它为型特异的重组NS3蛋白质。结论成功将克隆的全长DEN2NS3基因在酵母菌中表达,获得的重组NS3蛋白质具有免疫反应性。
Objective To clone and express the non-structure protein 3 （NS3） gene of dengue type 2 virus （DEN2） in P/ch/a pastor/s for studying the structure and function of NS3 protein and providing a potential source of developing dengue subunit vaccine. Methods The full-length gene NS3 of DEN2 was cloned from the DEN2-（NGC strain） infected C6/36 cells by RT-PCR, and then inserted into multi-cloning site of the pPICZaB vector. The recombinant plasmid was integrated into P. pastoris strain X-33 by electroporation, and then selected by Zeocin. Mut phenotype determination was performed on the yeast transformants and the expression of Mut^＋ colonies was induced by methanol, The expressed products were analyzed by SDS-PAGE and Western blotting. Results Full-length NS3 gene with the size of 1,85 kb was detected in the recombinant plasmid pPICZαB-NS3 by sequencing and restriction analysis. The recombinant NS3, whose relative molecular weight was approximately 78000, was expressed in yeast transformants and the secret expression was performed by screening Mut^＋ colonies in the yeast transformants. The specific recombinant NS3 was able to react with mouse/human polyclonal anti-DEN2 antibody. Conclusion Full-length DEN2 NS3 gene is cloned and expressed in P.pastoris successfully, and the recombinant NS3 glycoprotein could react with anti-DEN2 antibodies specifically.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2005年第6期482-484,488,共4页
Immunological Journal
