文章摘要
李久明,华立新,冯宁翰,曹 戌,吕志刚,秦 娣,卢 春.HIV-1 Rev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[J].南京医科大学学报,2008,28(6):702~706
HIV-1 Rev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
投稿时间:2007-11-20  
DOI:10.7655
中文关键词: 人类免疫缺陷病毒1型  病毒颗粒蛋白表达调节因子  人类疱疹病毒8型  复制
英文关键词: regulator of HIV-1 virion protein expression  human herpesvirus 8  replication
基金项目:江苏省高校自然科学基金资助(02KJD320019),霍英东青年教师基金资助(101038)
作者单位
李久明 南京医科大学第一附属医院泌尿外科,江苏 南京 210029 
华立新 南京医科大学第一附属医院泌尿外科,江苏 南京 210029 
冯宁翰 南京医科大学第一附属医院泌尿外科,江苏 南京 210029 
曹 戌 南京医科大学第一附属医院泌尿外科,江苏 南京 210029 
吕志刚 南京医科大学微生物学与免疫学系,江苏 南京 210029 
秦 娣 南京医科大学微生物学与免疫学系,江苏 南京 210029 
卢 春 南京医科大学微生物学与免疫学系,江苏 南京 210029 
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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)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?
英文摘要:
      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.
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