丙型肝炎病毒截短型核心基因表达载体的构建与表达

Construction and expression of prokaryotic expression vector for truncated HCV-core gene

目的构建重组丙型肝炎病毒核心基因表达载体pVec-core,并进行原核表达。方法人工合成丙型肝炎病毒截短型核心基因至pUC57载体上,经双酶切后,将其克隆至原核表达质粒pVec中,测序正确后将重组质粒pVec-core转化入表达宿主菌BL21,经IPTG诱导表达核心蛋白,SDS-PAGE检测目的蛋白的表达情况。采用GST亲和层析方法纯化融合蛋白并进行Western印迹法验证。结果丙型肝炎病毒核心基因表达载体构建成功,重组菌表达出相对分子质量约为40.6 kD重组融合蛋白,并以可溶形式存在于菌体中。蛋白经谷胱甘肽琼脂糖凝胶纯化,得到纯度较高的目的蛋白。经Western印迹分析,该蛋白可与HCV患者阳性血清发生特异性结合反应,具有良好的抗原活性。结论核心抗原表达载体在原核表达系统中高效表达,为利用其作为诊断抗原及研究核心蛋白的其他生物学功能奠定了基础。

Objective To construct the recombinant plasmid expression system for human hepatitis C virus core （HCV core） gene, and to detect its prokaryotic expression. Methods Truncated HCV core gene was artificially synthesized onto pUC57 vector and the resulted vector was digested by BamH I/EcoR I. The fragment containing core gene was then cloned into expression vector pVec. After sequencing, the recombinant plasmid （named pVec-core） was transfected into BL21 host cells, followed by induction with IPTG. The core protein produced was detected by SDS-PAGE, purified with Glutathione Sepharose 4B, and confirmed with Western immunoblotting. Results pVec-core vector was successfully constructed, and it produced a recombinant dissoluble protein of 40.6 kD. Thus, the protein was purified on GST resin column and a highly pure protein was obtained. The purified protein could react with the positive serum from HCV patient in Western blot test, indicating that the expressed protein had satisfactory antigenicity. Conclusion The highly efficient expression in prokaryotic system establishes a foundation of diagnostic antigen with biological functions of HCV protein core.