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广西HIV-1 B亚型假病毒的构建

Construction of HIV-1 B subtype pseudovirus system in Guangxi
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摘要 目的针对广西HIV-1 B亚型毒株构建一种优化的假病毒耐药检测模型,为表型耐药检测提供成本较低、简便易行的研究工具。方法从质粒p SV-EGFP中扩增出EGFP基因,采用双酶切法将EGFP基因克隆至骨架质粒p NL4-3.Luc.E-R-,以此构建假病毒重组骨架质粒;采用RT-PCR方法从HIV-1 B亚型慢性感染者RNA中获得包膜蛋白基因,采用双酶切法将env基因克隆至真核表达质粒pc DNATM3.1(+),以此构建重组包膜质粒;单转染重组包膜质粒至293t细胞验证表达;共转染重组质粒至293t细胞获得假病毒;采用与TZM-b1细胞共培养法,通过荧光表达检测验证假病毒感染性。结果两种质粒以质量比2:1(pc DNA3.1-env:p NL4-3.EGFP.E-R-)共转染至293t细胞,48 h后收集细胞上清获得假病毒。将假病毒加入到TZM-bl细胞共培养48 h后,可以观察到荧光表达。结论带有EGFP基因重组假病毒的系统构建成功。 Objective To establish a pseudovirus system for phenotypic drug-resistance detection andprovide a relatively cheap and easy method for drug-resistance testing. Methods EGFP gene was amplified fromplasmid p SV-EGFP and then cloned to backbone plasmid p NL4-3.Luc. E-R-by double enzyme digestion;env genewas amplified from RNA isolated from HIV-1-infected persons and cloned to eukaryotic expression plasmid cellsand EGFP or ENV expression. Pseudovirus was produced by co-transfection of two recombinant plasmids to 293 tcells. Infection of pseudovirus was determined by co-cultured with TZM-b1 cells and immunofluorescent test.Results Two recombinant plasmids(mass ratio,pc DNA3.1-env:p NL4-3.EGFP.E-R-. = 2:1)were co-transfectedto 293 t cells. Cultured supernatants containing pseudovirus were harvested at 48 h post-transfection. Fluorescencewas observed in TZM-b1 cells after TZM-b1 cells were infected with pseudovirus at 48 h post-infection. Conclusion The recombinant pseudovirus carrying EGFP gene is constructed successfully and it could be used for phenotypicdrug-resistance detection.
作者 黄春湲 王洪 梁浩 叶力 梁冰玉 蒋俊俊 陈荣凤 宁传艺 廖艳研 余军 黄颉刚 HUANG Chunyuan, WANG Hong, LIANG Hao, YE Li, LIANG Bingyu, JIANG Junjun, CHEN Rongfeng, NING Chuanyi, LIAO Yanyan, YU Jun, HUANG Jiegang.(School of Public Health of Guangxi Medical University & Guangxi Key Laboratory of AIDS Prevention and Control , Nanning 530021, Chin)
出处 《实用医学杂志》 CAS 北大核心 2018年第12期1942-1946,共5页 The Journal of Practical Medicine
基金 国家自然科学基金项目(编号:81360259 81660334) 广西自然科学基金项目(编号:2017GXNSFAA198190)
关键词 HIV-1 假病毒 耐药检测 HIV-1 pseudovirus drug-resistance detection
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