人免疫缺陷病毒1型蛋白酶抑制剂的微生物筛选模型的建立

Establishment of A Microbial Assay for Screening Anti Human Immunodeficiency Virus Type 1 Protease Inhibitors

目的采用基因工程方法，建立人免疫缺陷病毒１型蛋白酶（ＨＩＶ－１ＰＲ）的微生物检测法，用以筛选抗ＨＩＶＰＲ抑制剂。方法合成编码ＨＩＶ－１ＰＲ识别序列２４ｂｐ低聚核苷酸片段，并将其插入质粒ｐＢＲ３２２四环素耐药基因ｔｅｔｒ，经亚克隆构建含突变之四环素耐药基因ｍｔｅｔｒ的ｐＡＣＹＣ１８４Ｍ。以后者转染含ＨＩＶ－１ＰＲ表达载体（ｐＰＯＬＯ）的大肠杆菌，构建能同时表达四环素耐药蛋白及ＨＩＶ－１ＰＲ之工程菌（ＴＬＶ３３１）。结果在选择培养基内，ＴＬＶ３３１生长受ＨＩＶ－１ＰＲ控制，ＨＩＶ－１ＰＲ表达时可破坏ＴＬＶ３３１四环素抗性；ＨＩＶ－１ＰＲ活性受到抑制时，其四环素抗性则恢复。此特性经ＨＩＶ－１ＰＲ的已知抑制剂验证并以该法对３１个化学合成样品进行筛选。结论通过基因工程方法建立了ＨＩＶ－１ＰＲ的微生物检测法，该法可作为抗ＨＩＶＰＲ抑制剂之初筛模型。

Objective This study was to establish a microbial assay of human immunodeficiency virus type 1 protease (HIV 1 PR) activity for screening anti HIV PR inhibitors.Methods A 24 bp synthetic oligonucleotide fragment that encodes the HIV 1 PR recognition sequence was inserted into the tet r gene of pBR322 (mtet r). Escherichia coli containing HIV 1 PR expression vector pPOLO was transformed with pACYC184M containing modified mtet r gene. The transformant could express both HIV 1 PR and the modified Tet protein.Results The growth of engineered E.coli was prevented in the presence of tetracycline because the resistance Tet protein was degraded by HIV 1 PR.However inhibition of the HIV 1 PR restored tetracycline resistance. 31 chemical synthetic compounds were tested by the microbial assay.Conclusions A microbial assay method of HIV 1 PR activity was established through a engineered E.coli . 5 μmol/L saqunavir a special HIV 1 PR inhibitor showed inhibitory effect on the engineered E.coli. That means this model could be used as a initial screening model for anti HIV PR agents.