中文摘要：

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HIV PR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HIV PR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HIV PR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HIV SF2 PR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HIV PR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HIV PR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 GagCAP2NC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.

英文摘要：

The emergence of drug-resistant protease （PR） has seriously affected the anti AIDS therapy. Using mutated PR to screen phage library displaying randomized HIV PR target sites, phages susceptible to mutated PR can be obtained ,and used in drug screening of protease inhibitor（PD against to drug-resistant HIV PR. In order to validate the feasibility of this suppose, a designed LD3-CAP2NC peptide composing ofHIV CAP2NC with P2/NC target site and LD3 peptide locating in the NH2 terminus of the CAP2NC which could immobilise the phage to the plate through binding to human IgG was displayed on the surface of phage. This phage was fixed on plate, and suffered to the cleaving by HIV SF2 PR. The uncleaved phage leaving on the plate was measured using HRP/Anti-M13 conjugate, which reflects the cleavage efficiency. The results showed the phage can be cleaved effectively in a dose-dependent manner to PR concentration, with the most cleavage effect reached to more than 80%.Comparing with the control, the ELISA value of the cleaved phage decreased obviously. Furthermore, this cleavage was specifically inhibited by HIV protease inhibitor durg Indinavire. The data proved that a novel HIV protease cleavage model of the phage displaying Gag CAP2NC was firstly successfully established, which can not only be used as a new research platform to investigate the relationship between PR drug-resistant mutation and PR target sequence adapted mutation, but also lay a foundation to establish the new phage screening model for PIs especially against to drug-resistant PR.