文章摘要
朱晓蕾,卢 春,吕志刚,秦 娣.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[J].南京医科大学学报,2008,28(5):565~569
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
投稿时间:2008-01-10  
DOI:10.7655
中文关键词: 水泡性口炎病毒包膜糖蛋白  人类免疫缺陷病毒1型假病毒  卡波济肉瘤相关疱疹病毒  感染
英文关键词: vesicular stomatitis virus envelope glycoprotein  pseudotyped HIV-1  Kaposi’s Sarcoma-associated herpesvirus  infection
基金项目:霍英东青年教师基金资助(101038)
作者单位
朱晓蕾 南京医科大学微生物学与免疫学系,江苏 南京 210029 
卢 春  
吕志刚  
秦 娣  
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中文摘要:
      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HEK 293T细胞,收获细胞进行Western blot,检测HIV-1 p24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-1 p24蛋白的含量?结果:PCR 分离?克隆的VSV-GP序列全长1 536 bp,序列经过核酸分析证实;Western blot在HIV-1 p24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-1 p24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?
英文摘要:
      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 HindⅢ and BamHⅠ 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-1,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 supernatant 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.
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