摘要
目的 构建恙虫病东方体56kDa表面抗原基因(sta56)片段在不同载体的重组表达质粒,在E.coli中表达sta56重组抗原并比较不同表达系统对sta56的表达效果。方法 从含有恙虫病东方体Karp株sta56基因的重组克隆,扩增出不同长度的截短的sta56片段,定向插入pPROEX HTb及pET30a载体,转化大肠埃希菌DH5a或BL21(DE3),IPTG诱导表达,应用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)观察重组蛋白表达情况,应用蛋白印迹(WB)方法分析重组抗原的活性。结果 成功构建含sta56不同长度片段的重组表达质粒pHTbOt957、pHTbOt498、pHTbOt342和pETOt957、pETOt498、pETOt342,各重组子均可在E.coli中以融合蛋白的形式有效表达,SDS-PAGE显示各表达重组子表达不同分子量的重组蛋白,WB证实各重组蛋白均能被恙虫病患者阳性血清所识别。结论 恙虫病东方体Karp株sta56基因可在大肠埃希菌获得高效表达,pET30a对sta56的表达效果优于pPROEX HTb;重组蛋白具有免疫反应性,纯化后可用作免疫诊断抗原。
Objective To construct recombinant plasmids containing the truncated gene of the major surface antigen sta56 of Orientia tsutsugarnushi(Ot.) Karp strain for expression antigen in E. coli so as to compare the expression efficiency in different systems. Methods From the recombinant plasmid TOPO-sta56 containing sta56 of Orientia tsutsugarnushi Karp strain, several truncated genes of sta56 with different length were amplified and subcloned into the expression vectors pPROEX HTb and pET30a. These genes were expressed in E. coli DH5α and BL21(DE3) respectively when induced by IPTG. The expressed recombinant proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel elecrophoresis (SDS-PAGE)and Western blot. Results Six recombinant plamids containing truncated sta56 genes of different length were constructed as follow: pHTbOt957, pHTbOt498, pHTbOt342 and pETOt957, pETOt498, pETOt342. The recombinant sta56 proteins were highly expressed as 6×His fusion proteins in E. coli DH5α and BL21(DE3)respectively. The fusion proteins showed as different bands of different molecular weight respectively when analyzed with SDS-PAGE. Western blot demonstrated that the recombinant proteins were recognized by the positive serum of Ot. patients. Conclusion The sta56 gene of Orientia tsutsugamushi Karp strain could be highly expressed in E. coli and its expression showed better efficiency in pET30a than in pPROEX HTb. The recombinant sta56 antigen with immunoreactivity could be used as diagnostic reagent for Ot. infection.
出处
《中华流行病学杂志》
CAS
CSCD
北大核心
2004年第11期973-977,共5页
Chinese Journal of Epidemiology
基金
福建省卫生厅青年科研基金(2001-1-25)
福建省自然科学基金(C0310032)
