HIV-1整合酶酶联免疫吸附试验及其抑制剂的研究

HIV-1 integrase enzymelinked immunosorbent assay and inhibitors

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 ELISA method for detecting the activity of HIV 1 integrase and screening of inhibitors.Methods HIV recombinant plasmid F185K/C280S IN1 288 was transformed into the E.coli strain BL21(DE3) integrase with a his tag was induced by adding isopropyl β D thiogalactopyranoside (IPTG)and purified by nickel affinity chromatography.The synthesized donor substrate oligonucleotide representing the HIV 1 U5LTR was immobilized onto covalink polystyrene microtiter plates,and a synthesized biotinlated 20 bp oligonucleotide was used as the target substrate.The products were detected and quantified using a colorimetic avidin linked alkaline phosphatase reporter system,and identified by 32 P autoradiography.Some natural products and chemically synthesized compounds were screened for HIV 1 integrase inhibitors.Results The purified integrase was identified by SDS PAGE and showed integration activity by ELISA and 32 P radioisotopic assay. Coefficients of variation ( CV )of ELISA in a lot and among the lots were 4 63% and 5 89% respectively,the mean of P/N was 2 836±0 161 under the optimal experimental condition.Some plant extracts were found as potent integrase inhibitors.The IC 50 s for CEH and CEHL were (20 41±5 68)μg/ml and (7 56±1 86) μg/ml respectively.Conclusion The authors have established a simple and rapid ELISA method with stable repeatability for detecting integrase activity,which can be used for screening and studying of specific inhibitors of HIV 1 integrase.