摘要
目的构建基于大肠杆菌α-溶血素(HlyA)分泌机制的原核胞外分泌表达载体系统,将重组人白介素6(rhIL-6)分泌至胞外培养基中。方法利用分子克隆手段,从引起人泌尿道感染的大肠杆菌UPECJ96菌株染色体上获得了α-溶血素(HlyA)系统的分泌功能基因,与外源基因片段hIL-6一起克隆至pQE30骨架载体,构建了胞外分泌表达质粒pISQ,转化E.coliTop10,诱导表达后通过Western blot检测培养上清中hIL-6的表达。结果获得了与设计完全一致的pISU载体,Western blot结果显示,可以在pISQ/E.coli Top10培养液上清中检测到与His-hIL6-HlyAs融合蛋白相对分子质量大小一致的特异条带。结论大肠杆菌HlyA分泌系统操纵子能够在染色体和质粒间传递,rhIL-6能够通过该系统被特异地分泌至胞外培养基中,以可溶形式存在,为后续生物学研究奠定了基础。
Objective To develop a novel prokaryotic extracellular secretion system utilizing E. coli co-hemolysin （HlyA） system to export rhIL-6 extracellularly. Methods E. coli HlyA secretion elements and foreign hIL-6 gene were cloned into pQE30 vector for generating the secretion vector, named pISQ. The secretion plasmid and the control plasmid were transformed into the host strain E. coli Top10, respectively. Western blotting was conducted for assessing the expression product of the hlyAs-fused rhIL-6 gene in the culture supematant of E. coli Top10 harboring the newly-developed vector. Results The premeditated vector pISU was constructed successfully. The expression of the recombinant hIL-6 protein in the culture supematant of pISQ/E, coli Top10 was detected by Western blotting. Conclusion The hly operon could be transferred from chromosome to plasmid. The recombinant hIL-6 is secreted to the culture medium specifically by HlyA-based system, which facilitates the further study on hIL6.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2008年第4期380-384,共5页
Immunological Journal
基金
重庆自然科学基金重点项目(2006BA5024)
