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HIV蛋白酶(protease,PR)耐药突变的大量出现严重地影响了AIDS的治疗.应用突变PR对展示HIVPR靶序列随机文库的噬菌体进行切割筛选,可获得突变PR的敏感噬菌体,该噬菌体可用于针对HIVPR耐药突变株的蛋白酶抑制剂(protease inhibitor,PI)新药筛选.为了探索这一可能性,将包含HIVPR靶位点P2/NC序列的Gag蛋白CAP2NC片段展示于噬菌体表面,并在该片段的N端连接一可与人免疫球蛋白分子特异结合的固相化标签序列LD3,将该噬菌体固定于人免疫球蛋白包被的酶标板上,用HIVSF2PR进行切割,用抗M13噬菌体酶标抗体ELISA法检测未被切割的剩余噬菌体以反映切割效果.结果表明,所构建的噬菌体能被HIVPR有效切割,最大切割效应可达80%以上,其ELISA检测值明显下降,并且该切割效应与HIVPR呈明显的量效关系,能被PI类药物Indinavir(IDV)特异抑制.首次成功构建了展示HIV Gag CAP2NC片段的噬菌体蛋白酶切割模型,不仅可为研究HIVPR的耐药性变异及其靶序列的适应性变异提供一新的研究平台,同时也为构建一种全新的PI类药物,尤其是针对耐药的PI类药物大规模体外噬菌体筛选模型打下基础.
The emergence of a large number of HIV-resistant protease (PR) mutations has seriously affected the treatment of AIDS.Selection and screening of phages displaying a random library of HIVPR target sequences using a mutant PR results in a sensitive phage mutant PR that can be used against In order to explore this possibility, a Gag protein CAP2NC fragment containing the P2 / NC sequence of HIVPR site was displayed on the surface of the phage, and a new protease inhibitor (PI) N-terminal is connected with a human immunoglobulin molecule-specific binding of the immobilized tag sequence LD3, the phage immobilized on a human immunoglobulin coated ELISA plate, with HIVSF2PR cut with anti-M13 phage enzyme-labeled antibody The results showed that the constructed phages could be effectively cut by HIVPR and the maximal cleavage effect could reach more than 80%, and the ELISA detection value decreased obviously, and the cleavage effect was not correlated with HIVPR Obvious dose-effect relationship, which can be specifically inhibited by Indinavir (IDV), a PI-type drug.The cleavage model of phage protease displaying HIV Gag CAP2NC fragment was successfully constructed for the first time , Not only can provide a new research platform for studying the variation of drug resistance of HIVPR and the adaptive mutation of its target sequence, but also for building a brand new PI drug, especially for large-scale in vitro drug-resistant PIs Phage screening model to lay the foundation.