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用于耐药性HIV突变体表型分化的荧光测定法。

Fluorometric assay for phenotypic differentiation of drug-resistant HIV mutants.

作者信息

Zhu Qinchang, Yu Zhiqiang, Kabashima Tsutomu, Yin Sheng, Dragusha Shpend, El-Mahdy Ahmed F M, Ejupi Valon, Shibata Takayuki, Kai Masaaki

机构信息

Faculty of Pharmaceutical Sciences, Graduate School of Biomedical Sciences, Nagasaki University, 1-14, Bunkyo-Machi, Nagasaki 852-8521, Japan.

出版信息

Sci Rep. 2015 May 19;5:10323. doi: 10.1038/srep10323.

DOI:10.1038/srep10323
PMID:25988960
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC4437315/
Abstract

Convenient drug-resistance testing of viral mutants is indispensable to effective treatment of viral infection. We developed a novel fluorometric assay for phenotypic differentiation of drug-resistant mutants of human immunodeficiency virus-I protease (HIV-PR) which uses enzymatic and peptide-specific fluorescence (FL) reactions and high-performance liquid chromatography (HPLC) of three HIV-PR substrates. This assay protocol enables use of non-purified enzyme sources and multiple substrates for the enzymatic reaction. In this study, susceptibility of HIV mutations to drugs was evaluated by selective formation of three FL products after the enzymatic HIV-PR reaction. This proof-of-concept study indicates that the present HPLC-FL method could be an alternative to current phenotypic assays for the evaluation of HIV drug resistance.

摘要

对病毒突变体进行便捷的耐药性检测对于有效治疗病毒感染至关重要。我们开发了一种新型荧光测定法，用于对人免疫缺陷病毒I型蛋白酶（HIV-PR）的耐药突变体进行表型分化，该方法利用酶促反应和肽特异性荧光（FL）反应以及三种HIV-PR底物的高效液相色谱（HPLC）。该测定方案允许使用非纯化的酶源和多种底物进行酶促反应。在本研究中，通过HIV-PR酶促反应后三种FL产物的选择性形成来评估HIV突变对药物的敏感性。这项概念验证研究表明，目前的HPLC-FL方法可作为评估HIV耐药性的现有表型测定方法的替代方法。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a36f/4437315/a9ca02f426fa/srep10323-f6.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a36f/4437315/4ed892efa4a7/srep10323-f1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a36f/4437315/695593ca74dc/srep10323-f2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a36f/4437315/2441af0107b1/srep10323-f3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a36f/4437315/62b820b73ad6/srep10323-f4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a36f/4437315/eee3e4b8c674/srep10323-f5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a36f/4437315/a9ca02f426fa/srep10323-f6.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a36f/4437315/4ed892efa4a7/srep10323-f1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a36f/4437315/695593ca74dc/srep10323-f2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a36f/4437315/2441af0107b1/srep10323-f3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a36f/4437315/62b820b73ad6/srep10323-f4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a36f/4437315/eee3e4b8c674/srep10323-f5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a36f/4437315/a9ca02f426fa/srep10323-f6.jpg

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