HCV核心区全基因片段的表达及产物抗原活性分析

Expression of Full-length Gene Fragment at HCV Core Region and Antige-nicity of Expressed Product

目的 表达并纯化核心基因全片段,以得到良好特异性的核心蛋白。方法 自克隆载体pUC19/HCV-C中切下核心基因全片段,将其插入带有His 6纯化标签的融合表达质粒pTrcHisA中,转化入大肠杆菌,经IPTG诱导,通过SDS-PAGE、中和抑制ELISA和Western blot对占菌体总蛋白15％以上的表达产物进行鉴定,用IMAC一步法纯化,纯化产物检测HCV阴阳性血清标本,并与日本东燃公司核心抗原C_(11)检测结果进行比较。结果 核心基因全片段插入pTrcHisA载体,转化入大肠杆菌TOP10后构建成功稳定的表达株PTrcHisA/HCV-C/TDP10,用纯化产物检测血清标本,与日本东燃公司核心抗原C_(11)检测结果的阳性均值与阴性均值之比及A值分布基本相同。结论 表达抗原具有良好的免疫反应性和特异性。

Objective To express and purify the full-length gene fragment at HCV core region and obtain the core protein with good specificity. Methods The full-length core gene fragment derived from cloning vector pUC19/HCV-C was inserted into expression vector pTrcHisA and fused to the six histidine downstream , then transformed to E, coli and expressed under induction of IPTG. The expressed product was analyzed by SDS-PAGE,neutralization inhibiting EOSA and Western blot, and purified by one-step immobilized metal affinity chromatography, then used for detecting HCV-positive and negative sera.The detection result was compared with that using the C11 antigen from a Japanese manufacturer. Results A recombinant strain pTrcHis/ HCV-C/TOP10 for stable expression was constructed.No significant difference was observed in the ratios of A values of positive to negative sera,as well as A value distribution detected by the purified goal protein and C11 antigen. Conclusion The expressed product showed good immunoreactivity and specificity.