论文部分内容阅读
目的 建立一种检测人类免疫缺陷病毒 1型 (HIV - 1 )整合酶的酶联免疫吸附试验(ELISA)方法 ,用于筛选和研究HIV - 1整合酶抑制剂。方法 将质粒F1 85K C2 80SIN1 2 88转化到大肠埃希菌中 ,经IPTG诱导表达 ,柱亲和层析纯化 ,获得HIV 1整合酶融合蛋白。建立酶联免疫吸附试验方法测定其生物学活性 ,与3 2 P同位素标记方法比较 ,并用ELISA方法筛选HIV 1整合酶的抑制剂。结果 SDS PAGE电泳分析显示 ,相对分子质量 30 0 0 0上方有HIV 1整合酶融合蛋白条带出现。ELISA及3 2 P同位素标记法证实 ,此融合蛋白对于特异底物具有 3′切割和链转移活性。ELISA反应的平均P N值为 2 836± 0 1 61 ,批内及批间变异系数 (CV)分别为 4 63 %和 5 89%。检测到中药丹参提取物CEH等有抑制整合酶的活性 ,CEH的大孔树脂洗脱物CEHL活性提高。结论 ELISA法检测HIV 1整合酶活性技术简单 ,快速 ,重复性好 ,无同位素污染 ,可用于HIV 1整合酶为靶点的抑制剂的筛选及抗 HIV药物作用机理的研究。
Objective To establish an enzyme - linked immunosorbent assay (ELISA) for the detection of human immunodeficiency virus type 1 (HIV - 1) integrase for screening and studying HIV - 1 integrase inhibitors. Methods The plasmid F1 85K C2 80SIN1 2 88 was transformed into Escherichia coli. The recombinant plasmid was induced by IPTG and purified by column affinity chromatography to obtain HIV 1 integrase fusion protein. The establishment of enzyme-linked immunosorbent assay for the determination of biological activity, compared with the 3 2 P isotope labeling method, and ELISA method for screening HIV 1 integrase inhibitors. Results SDS PAGE electrophoresis showed that a band of HIV 1 integrase fusion protein appeared above the molecular weight of 30 000. ELISA and 3 2 P isotope labeling confirmed that this fusion protein has specific 3 ’cleavage and strand transfer activity. The average P N value of ELISA was 2836 ± 0 1 61, and the intra-assay and inter-assay CVs were 463% and 59%, respectively. It was detected that the activity of integrase, such as CEH of traditional Chinese medicine Salvia miltiorrhiza extract, inhibited the CEHL activity of macroporous resin eluate of CEH. Conclusion The ELISA method for detecting integrase activity of HIV-1 is simple, rapid, reproducible and isotope-free and can be used to screen inhibitors of HIV-1 integrase and to study the anti-HIV drug mechanism.