摘要
目的建立稳定表达丙型肝炎病毒(HCV)核心蛋白的原核表达系统,获得高产量的纯化核心蛋白。方法以HCV株全长cDNA序列为模板,PCR扩增获得核心蛋白基因,构建原核表达载体pET-32a(+)-HCV-Core。转化大肠埃希菌DH5α,提取质粒,验证正确后,再次转化大肠埃希菌BL21中,IPTG诱导表达,SDS-PAGE、Westernblot验证融合蛋白的表达。超声破碎表达菌,Ni+-亲和柱对表达蛋白进行纯化及柱上复性。结果成功构建了原核表达载体pET-32a(+)-HCV-Core,并表达了预期分子量大小的目的蛋白。结论成功表达、纯化HCV核心融合蛋白,为进一步研究其生物学功能奠定了坚实的基础。
Objective To construct a prokaryotic expression vector of HCV core protein and to express and purify it.Methods DNA of HCV core protein gene was amplified by polymerase chain reaction(PCR) using cDNA of HCV as template.After sequencing,the correct DNA fragment was inserted into inducible prokaryotic expression vector pET-32a(+) and the vector was transformed into the competent E.coli BL21.The expression of HCV was induced by IPTG,and cell lysate was analyzed by SDS-PAGE and Western blot.Expressed bacteria were quassationed by supersound and analyzed by SDS-PAGE.The expressed product was purified and renatured by Ni+-affinity column chromatography.Results The prokaryotic expression vector was constructed successfully.HCV core fusion protein was expressed and purified with expected molecular weight.Conclusions The successful expression and purification of HCV core fusion protein will be useful for further study on its biological function.
出处
《中华实验和临床感染病杂志（电子版）》
CAS
2008年第2期21-26,共6页
Chinese Journal of Experimental and Clinical Infectious Diseases(Electronic Edition)
关键词
核心蛋白
原核表达
蛋白纯化
Core protein
Prokaryotic expression
Protein purification
