HIV-1假病毒的构建及对人原发性渗出性淋巴瘤细胞系的感染研究

Production of pseudotyped human immunodeficiency virus-1 and analysis on its ability to infect the primary effusion lymphoma cell line

目的:分离、克隆水泡性口炎病毒(vesicular stomatitis virus,VSV)的包膜糖蛋白(VSV-GP)编码基因,包装人类免疫缺陷病毒1型(human immunodeficiency virus-1,HIV-1)假病毒,并研究其对人原发性渗出性淋巴瘤细胞系BCBL-1细胞的感染状况。方法:根据GenBank登记的VSV-GP基因核苷酸序列设计1对PCR引物,其5′端分别引入HindⅢ和BamHⅠ酶切位点。以质粒pHCMV-G为模板,PCR扩增VSV-GP基因,经双酶切后定向克隆进真核表达载体pcDNA3.1(+)中,构建重组真核表达质粒pVSV-GP。重组质粒经酶切鉴定、核酸序列测定和分析后与含包装和复制功能缺陷的HIV-1基因组重组质粒pNL4-3.Luc.R-E-共转染人胚肾HEK293T细胞,收获细胞进行Western blot,检测HIV-1p24蛋白的表达;收获HIV-1假病毒感染BCBL-1细胞,进行虫荧光素酶(Luciferase)报告基因活性检测以及用ELISA的方法检测感染后细胞上清中HIV-1p24蛋白的含量。结果:PCR分离、克隆的VSV-GP序列全长1536bp,序列经过核酸分析证实;Western blot在HIV-1p24蛋白预期位置检测到特异性条带;HIV-1假病毒感染BCBL-1细胞后上清可以检测到HIV-1p24蛋白,并且测得Luciferase活性值较对照组显著增高(P<0.05)。结论:成功包装了HIV-1假病毒,并且该病毒可以感染BCBL-1细胞。

Objective：To isolate and clone the vesicular stomatitis virus envelope glycoprotein （VSV-GP） gene and produce the VSV-GP pseudotyped Human Immunodeficiency Virus-1 （HIV-1）,investigating its ability to infect the BCBL-1 cells. Methods：A pair of PCR primers for VSV-GP gene with Hindm and BamH I restriction enzyme sites was designed according to the sequence in GenBank. Then the VSV-GP gene was amplified from the plasmid pHCMV-G using PCR. Subsequently,amplified gene fragments were digested and cloned into pcDNA3.1 （＋） vector to create recombinant eukaryotic expression plasmid pVSV-GP. BCBL-1 cells were infected by VSV-GP pseudotyped HIV-l,which was harvested from HEK 293T cells cotransfected by pVSV-GP and pNL4-3.Luc.R-E-.Then BCBL-1 cells were lysed to calculate the relative luciferase unit （RLU）, and the concentration of HIV-1 p24 antigen in the supernatant was determined by using a HIV-1 p24 antigen ELISA kit. Meanwhile,HEK 293T cells were lysed to detect the expression of HIV-1 p24 antigen by Western blott. Results：The isolated and cloned pVSV-GP sequence was 1 536 bp and was confirmed by sequencing. HIV-1 p24 antigen could be detected in the supematant of VSV-GP pseudotyped HIV-1 infected BCBL-1 cells. The RLU of BCBL-1 cells infected by VSV-GP pseudotyped HIV-1 increased significantly compared with the negative control （P 〈 0.05）, and the band of HIV-1 p24 antigen was detected by Western blott. Conclusion：VSV-GP pseudotyped HIV-1 was successfully produced and had the ability of infecting BCBL-1 cells.