E92A是HIV-1整合酶耐药突变N155S的活性回复突变

发布时间：2018-06-05 10:33

本文选题：人免疫缺陷病毒(HIV) + 整合酶　；参考：《生物化学与生物物理进展》2014年05期

【摘要】：HIV-1整合酶是目前抗艾滋病药物研发的重要靶点之一,整合酶的耐药突变是导致整合酶抑制剂类药物治疗失败的主要原因,但突变产生耐药性的机理仍不清楚.本工作通过人工构建突变型整合酶,测试其活性和耐药性,对整合酶的耐药机理进行初步探索.构建整合酶的突变型包括E92A、N155S两种单突变及E92A/N155S双突变.通过基因工程操作引入突变、构建质粒、表达纯化得到整合酶蛋白.用基于磁珠的整合酶链转移ELISA测试整合酶的链转移活性,用S-1360和Raltegravir两种抑制剂测试整合酶的耐药性.另外,用Autodock软件做了S-1360和整合酶核心区(包括野生型和突变型)的分子对接.结果表明,N155S突变使整合酶链转移活性下降约80%,而E92A/N155S双突变仅使活性下降约42%,这表明N155S突变基础上的E92A突变可使整合酶的活性大幅回复.E92A和E92A/N155S对不同的抑制剂可产生不同的耐药性,它们对Raltegravir的耐药性强于对S-1360.突变对整合酶活性和耐药性的影响主要是通过改变整合酶活性中心结构实现的,E92A突变可能导致其与周围残基静电相互作用减弱,间接影响到D64和D116残基,产生活性回复作用.
[Abstract]:HIV-1 integrase is one of the important targets in the research and development of anti-AIDS drugs. The drug resistance mutation of integrase is the main cause of the failure of integrase inhibitors, but the mechanism of drug resistance is still unclear. In this work, mutant integrase was constructed, its activity and drug resistance were tested, and the mechanism of drug resistance of integrase was preliminarily explored. The mutants of integrase include E92An155S and E92A / N155S double mutations. The plasmids were constructed by introducing mutagenesis into gene engineering, and the integrase protein was obtained by expression and purification. The chain transfer activity of integrase was tested by magnetic bead based integrase chain transfer Elisa, and the resistance of integrase was tested by S-1360 and Raltegravir inhibitors. In addition, the molecular docking between S-1360 and integrase core (including wild type and mutant type) was performed with Autodock software. The results showed that the N155S mutation reduced the activity of integrase chain transfer by about 80%, while the double mutation of E92A / N155S decreased the activity only about 42%, which indicated that the E92A mutation based on the N155S mutation could significantly restore the activity of integrase. E92A and E92A / N155S could produce different inhibitors. Different drug resistance, They are more resistant to Raltegravir than to S-1360. The effect of mutagenesis on integrase activity and drug resistance is mainly due to the change of the structure of integrase active center, which may lead to the weakening of electrostatic interaction between the mutant and the surrounding residues, which indirectly affects the D64 and D116 residues and produces an active recovery effect.
【作者单位】：北京工业大学生命科学与生物工程学院;
【基金】：国家自然科学基金资助项目(31100523,21173014,31171267) 国家重点基础研究发展计划(973)资助项目(2009CB930203) 科技部国际合作资助项目(2010DFA31710) 北京市教委面上项目(KM201310005030)~~
【分类号】：R373

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