HIV-1Rev基因编码蛋白在PEL细胞系中的表达及其表达蛋白抑制HHV-8溶解性周期复制

The eukaryotic expression of HIV-1 Rev and its effect on the lytic cycle replication of KSHV

目的:构建含人类免疫缺陷病毒1型(HIV-1)病毒颗粒蛋白表达调节因子(regulator of virion protein expression,Rev)编码基因的重组真核表达质粒并初步探索Rev基因编码蛋白对人类疱疹病毒8型(HHV-8)溶解性周期复制的影响。方法:构建pRev-Flag重组质粒并进行酶切鉴定和序列测定;将pRev-Flag重组质粒瞬时转染原发性渗出性淋巴瘤细胞系(PEL)BCBL-1细胞和小鼠胚胎成纤维细胞NIH/3T3,采用RT-PCR、Western blot分别从mRNA和蛋白水平检测Rev基因的表达情况;提取瞬时转染pRev-Flag重组质粒的BCBL-1细胞总RNA,进行RT-PCR检测HHV-8次要衣壳蛋白编码基因ORF26 mRNA转录水平。结果:核酸序列分析结果表明,克隆的Rev基因序列与GenBank中已登记的Rev序列100%同源,RT-PCR和Western blot都在Rev预期位置检测到特异性条带。RT-PCR检测显示,Rev基因编码蛋白能够降低HHV-8 ORF26 mRNA转录水平。结论:成功构建含Rev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Rev蛋白能够抑制HHV-8溶解性周期复制。

Objective：To investigate the expression of the recombinant plasmid of regulator of HIV-1 virion protein expression（Rev） in the eukaryotic cells,and to evaluate the effect on human herpesvirus 8（HHV-8） lytic cycle replication by Rev protein in BCBL-1 cells. Methods：After identification with enzyme digestion and nucleotide sequences analysis,the recombinant plasmid pRev-Flag was transiently transfected into BCBL-1 cells and NIH/3T3 cells. The expression of pRev-Flag mRNA and protein in both cells was detected by RT-PCR and Western blot,respectively. Subsequently,RNA was obtained from BCBL-1 cells transfected with pRev-Flag. RT-PCR was carried out to evaluate the expression of ORF26 mRNA（encoding the minor capsid protein） of HHV-8. Results：Nucleotide sequences analysis indicated that the cloned Rev sequence was 100% homology with Rev gene previously registered in GenBank. The specific band was detected at the expected place by both RT-PCR and Western blot. In addition,the expression of ORF26 mRNA in BCBL-1 cells transfected with pRev-Flag was weaker than the control. Conclusion：Rev gene was correctly expressed in BCBL-1 cells and NIH/3T3 cells,and Rev gene might inhibit KSHV lytic cycle replication.