文章摘要
方 欣,卢 春,曹 戌,李久明,秦 娣,吕志刚.HIV-I编码病毒蛋白R基因在真核细胞中的表达及其表达蛋白对KSHV溶解性周期复制的影响[J].南京医科大学学报,2008,28(9):1089~1094
HIV-I编码病毒蛋白R基因在真核细胞中的表达及其表达蛋白对KSHV溶解性周期复制的影响
The eukaryotic expression of HIV-1 encoded viral protein regulatory gene and its effect on the lytic cycle replication of KSHV
投稿时间:2008-04-19  
DOI:10.7655
中文关键词: 人类免疫缺陷病毒1型  病毒蛋白R  卡波济肉瘤相关疱疹病毒  真核表达  复制
英文关键词: human immunodeficiency virus-1  viral protein regulatory  KSHV  eukaryotic expression  replication
基金项目:霍英东青年教师基金资助(101038)
作者单位
方 欣 南京医科大学微生物学与免疫学系,江苏 南京 210029 
卢 春  
曹 戌  
李久明  
秦 娣  
吕志刚  
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中文摘要:
      目的:构建含人类免疫缺陷病毒1型(HIV-1)编码病毒蛋白R(Vpr)基因的重组真核表达质粒并初步探索Vpr基因编码蛋白对卡波济肉瘤相关疱疹病毒(KSHV)溶解性周期复制的影响?方法:构建pVpr-Flag重组质粒并进行酶切鉴定和序列测定;将pVpr-Flag重组质粒临时转染BCBL-1和NIH3T3细胞,采用RT-PCR?IFA和Western blot分别从mRNA和蛋白水平检测Vpr基因的表达情况;提取临时转染pVpr-Flag重组质粒的BCBL-1细胞总RNA,进行RT-PCR检测KSHV次要衣壳蛋白编码基因ORF26(仅在病毒裂解期表达)mRNA转录水平,初步探索HIV-1 Vpr基因编码蛋白对KSHV复制的影响?结果:克隆的Vpr基因序列与GenBank中已登记的Vpr序列100%同源?RT-PCR?IFA和Western blot结果显示Vpr基因mRNA及其编码蛋白在真核细胞中得到了转录和表达?RT-PCR结果进一步显示,Vpr基因编码蛋白能够降低KSHV ORF26 mRNA转录水平?结论:成功构建含Vpr基因序列的重组质粒并在真核细胞中获得正确表达;初步探索表明Vpr蛋白能够抑制KSHV溶解性周期复制?
英文摘要:
       Objective:To investigate the expression of the recombinant plasmid of HIV-1 viral protein regulatory(Vpr) in the eukaryotic cells, and evaluate the effect on Kaposi’s sarcoma-associated herpesvirus(KSHV) lytic cycle replication by Vpr protein in BCBL-1 cells. Methods:After identification with enzyme digestion and nucleotide sequences analysis,the recombinant plasmid pVpr-Flag was transiently transfected into BCBL-1 cells and NIH3T3 cells. The mRNA and protein expression of pVpr-Flag in both cells was detected by RT-PCR, IFA and Western blot,respectively. Subsequently, RNA was obtained from BCBL-1 cells transfected with pVpr-Flag plasmid. RT-PCR was carried out to evaluate the expression of ORF26 mRNA(encoding the minor capsid protein) of KSHV. Results:Nucleotide sequences analysis indicated that the cloned Vpr sequence was 100% homology with Vpr gene registered in GenBank previously. The specific band was detected at the expected place both by RT-PCR and Western blot. And the expression of Vpr protein was also detected in BCBL-1 cells by IFA. In addition,the expression of ORF26 mRNA in BCBL-1 cells transfected with plasmid pVpr-Flag was weaker than that in the control. Conclusion:Vpr gene was correctly expressed in BCBL-1 and NIH3T3 cells,and Vpr protein might inhibit KSHV lytic cycle replication
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