摘要
目的:构建结核分枝杆菌Rv3872的原核重组表达质粒pET-3872并对其进行表达及鉴定。构建表达结核分枝杆菌抗原蛋白Rv3872即PE35的重组卡介苗。方法:应用PCR技术扩增Rv3872基因,定向克隆入pET32a(+)并转化E.coli BL21(DE3)菌株,测序后用IPTG诱导蛋白表达,通过SDS-PAGE和Western blot对目的蛋白进行检测及鉴定。用纯化蛋白免疫新西兰大白兔制备多克隆抗体。构建重组穿梭表达质粒pMV-3872,将重组质粒电穿孔进入卡介苗,对重组卡介苗进行诱导表达用SDS-PAGE和Western blot检测和鉴定目的蛋白。结果:表达融合蛋白的pET-3872质粒构建成功,重组蛋白经Western blot检测出特异性阳性信号。重组卡介苗BCG-3872构建成功,热诱导表达后经SDS-PAGE和Western blot,在培养上清中检测到目的蛋白。结论:成功构建了重组质粒pET32a(+),并在大肠杆菌中表达了PE35蛋白,有利于进一步研究Rv3872基因功能。本研究还对表达结核分枝杆菌蛋白PE35的重组卡介苗进行了鉴定,为进一步研究该重组卡介苗的免疫功能奠定了基础。
AIM:To construct, express and identify a recombinant prokaryotic expression vector pET-3872 carrying Rv3872 of Mycobacterium tuberculosis H37Rv strain, and to construct the recombinant Bacillus Calmette-Guerin (rBCG) strain expressing Rv3872 (PE35)of Mycobacterium tuberculosis H37Rv strain. METHODS: The Rv3872 gene was amplified by PCR from Mycobacterium tuberculosis H37Rv strain and cloned into prokaryotic expression vector pET32a(+). The recombinant plasmid pET-3872 was sequenced and transformed into E.coli BL21(DE3), and was induced with IPTG to express a 35 kD fusion protein, which was confirmed as His-Rv3872 by Western blot. The expression product was purified and the new Zealand rabbits were immunized. Rv3872 was amplified by PCR and cloned into E.coli-Mycobacteria shuttle vector pMV361. The recombinant vector was named as pMV-3872, and then pMV-3872 was transformed the to BCG via electroporation, the recombinant BCG-3872 strain. The PE35 protein expression of BCG-3872 was induced with heat shock reaction. Then BCG-3872 culture supernatant and bacterial precipitation were collected respectively and analyzed by Western blot. RESULTS: A recombinant fused expression vector pET-3872 was constructed and His-3872 protein was confirmed by Western blot. BCG-3872 strain was constructed and Western blot confirmed the presenle of the PE35 protein in the supernatant of BCG-3872 strain culture. CONCLUSION: The prokaryotic expression vector pET-3872 was constructed, and the 35 kD fusion protein His-3872 was expressed and purified successfully, which provides a tool for further functional study of the Rv3872 . The recombinant BCG strain expressing PE35 protein, BCG-3872 strain, was constructed successfully, which facilitates further study on BCG-3872 immune function.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2010年第6期539-542,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金资助项目(30872257)
国家传染病重大专项(2008ZX10003-013)
关键词
结核分枝杆菌
Rv3872基因
表达
重组卡介苗
Mycobacterium tuberculosis
Rv3872
expression
recombinant Bacillus Calmette-Guerin (rBCG)