摘要
目的 在大肠杆菌中表达HGVNS5抗原 ,以建立HGV抗体血清学检测方法。方法 利用反转录PCR(RT PCR)法从人血浆中分离庚型肝炎病毒NS5部分基因 ,克隆到pRSETA载体 ,经酶切初步鉴定后进行DNA序列分析 ,结果表明克隆序列正确。以pPROEX 1为表达载体 ,转化DH5α ,诱导表达后进行SDS PAGE和Westernblot分析。结果 克隆的基因片段在大肠杆菌中表达出相对分子质量 (Mr)约 2 0× 10 3 的可溶性蛋白 ,其氨基端含有 6个组胺酸肽段 ,可经Ni NTA亲和层析纯化。免疫印迹分析表明 ,该表达产物可与HGV感染患者体内的HGV抗体特异性结合。结论 克隆了HGVNS5部分基因并获得初步表达 ,表达的HGVNS5蛋白具有特异抗体结合活性 。
Objective To clone and express HGV NS5 gene, an EIA method was established to detect anti HGV NS5 antibody. Methods Partial NS5 gene of HGV was obtained from a healthy plasma donor by RT PCR, then inserted into pRSET A vector, and identified by restriction enzyme digestion and DNA sequencing. The gene was cloned into expression vector pPROEX 1. E.coli DH5α was transformed by the recombinant pPROEX 1 and induced by IPTG. Results A molecular weight of 20×10 3 soluble protein was expressed, which had a polyhistidine (6×His) “tag” at the N terminal, resulted in simple purification by Ni NTA(Ni 2+ nitrilo tri acetic acid) affinity chromatography. Western blot and ELISA demonstrated that the purified protein had specific HGV antibody binding activity. Conclusion The result indicated that the expressed protein can be used to establish an EIA method for detection of anti HGV NS5.
出处
《中华微生物学和免疫学杂志》
CSCD
北大核心
2000年第5期469-472,共4页
Chinese Journal of Microbiology and Immunology
