HIV逆转录酶抑制剂酶联免疫评估方法的研究

Study on evaluation of human immunodeficiency virus reverse transcriptase inhibitor by ELISA

目的:建立检测逆转录酶活性的酶联免疫吸附实验方法,用于筛选人类免疫缺陷病毒(HIV-1)逆转录酶抑制剂。方法:利用DNA的固相固定技术将引物固定于96孔微量滴定板上,以poly(rA).Oligo(dT)15为模板,在逆转录酶的作用下,将生物素标记的dUTP掺入,用碱性磷酯酶反应系统检测酶活性。结果:建立了检测逆转录酶活性的酶联免疫(ELISA)法,并用ELISA法验证了已知阳性药物对逆转录酶的抑制作用,对ELISA法在动力学研究的应用做了初步探讨,评估ELISA法的重复性、稳定性、特异性和敏感性。结论:ELISA法检测HIV-1逆转录酶活性具有简单、快速、特异性强、重复性好、污染小等特点,适用于以HIV-1逆转录酶为靶点的抑制剂的筛选及相关机制的研究。

Objective：to establish an ELISA method for detecting the activity of reverse transcriptase.Methods：Oligo（dT）15 was immobilized via its 5＇-terminal phosphate to Covalink-NH microtiter plates,The biotin-dUTP was incorporated by reverse transcriptase.The products were detected and quantified using a colorimetric streptavidin-alkaline phosphatase reporter system.Results：We established an ELISA RT assay method and tested the inhibition activity of PFA and NVP respectively.Using this method we also studied the kinetic mechanism of AMV-RT and HIV-1 RT.At the same time,we evaluated the stability,repeatability,specificity and sensitivity of this ELISA method.Conclusion：This ELISA method has particular advantags,such as simplicity,rapidity,specificity and little pollution,and is suitable for screening and studying of specific inhibitors of HIV-1 reverse transcriptase and other RTs.