文章摘要
王〓平,黄〓敏,卢〓春.含HIV-1 Vif基因重组慢病毒表达载体的构建及其对KSHV裂解性周期复制影响的初探[J].南京医科大学学报,2010,(3):286~290355
含HIV-1 Vif基因重组慢病毒表达载体的构建及其对KSHV裂解性周期复制影响的初探
Construction of the recombinant expression lentivirus vector carrying HIV-1 Vif gene and effect of its protein expression on the lytic cycle replication of KSHV
投稿时间:2009-10-13  
DOI:10.7655
中文关键词: 慢病毒  Vif  KSHV  病毒复制
英文关键词: lentivirus  Vif  KSHV  replication
基金项目:教育部新世纪优秀人才支持计划(NCET-05-0506)
作者单位
王〓平 南京医科大学微生物学与免疫学系,江苏 南京〓210029 
黄〓敏  
卢〓春  
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中文摘要:
      目的:利用慢病毒表达载体将HIV-1 Vif基因导入原发性渗出性淋巴瘤(PEL)细胞BCBL-1中使之持续表达目的蛋白Vif,并检测Vif对其中潜伏KSHV裂解性周期复制的影响?方法:自表达质粒pCI-neo-Vif中扩增出Vif基因,插入到pHAGE-CMV-MCS-IzsGreen中构建成慢病毒载体pHAGE-Vif,利用脂质体将其与包装质粒psPAX2和包膜质粒pMD2.G共转染293T细胞?荧光显微镜观察293T细胞中绿色荧光蛋白(GFP)表达情况;收集培养上清经0.45 μm滤器过滤后即获得病毒悬液?梯度稀释法测定病毒滴度,感染293T细胞,48 h或72 h后进行RT-PCR和Western blot检测Vif基因的转录和表达情况?以MOI为1.0的病毒量感染靶细胞BCBL-1,利用Western blot技术检测Vif蛋白的表达,同时检测KSHV vIL-6和Rta的蛋白表达水平,初步探讨Vif对KSHV复制的影响?结果:限制性内切酶检测和基因测序证实成功构建了携带Vif基因的慢病毒表达载体,滴度为4×107 efu/ml?以MOI为1.0的重组慢病毒感染靶细胞BCBL-1 72 h后,能够检测到外源基因Vif的蛋白表达?Western blot结果初步显示,Vif能够下调vIL-6和Rta的蛋白表达水平?结论:成功构建了含Vif基因的慢病毒表达载体,获得的病毒能够有效感染BCBL-1细胞,并在其中大量表达目的蛋白?初步结果提示,Vif能够下调vIL-6和Rta的蛋白表达水平,对KSHV裂解性周期复制可能起到抑制作用?
英文摘要:
      Objective:To construct the recombinant lentivirus containing HIV-1 Vif gene and detect the effect of Vif protein expression on the lytic cycle replication of KSHV. Methods:The fragment of Vif gene from expression vector pCI-neo-Vif was cloned into the lentivirus vector pHAGE-CMV-MCS-IzsGreen,then the recombinant plasmid pHAGE-Vif,packaging vector psPAX2 and envelope vector pMD2.G were cotransfected into the 293T cells. Culture media were harvested and filtered through a 0.45 μm filter to remove the cells. The viral titer was checked by observing the expression of green fluorescent protein(GFP). After infected with the recombinant lentivirus,the mRNA transcription and protein expression of Vif in 293T cells were detected by RT-PCR and Western blot,respectively. Then,BCBL-1 cells were infected by the recombinant lentivirus with the MOI of 1.0,and the protein expression of Vif,vIL-6 and Rta were detected by Western blot. Results:The recombinant lentivirus vector carrying Vif was constructed successfully with the viral titer of 4×107 efu/ml. BCBL-1 cells could be efficiently infected by the recombinant lentivirus with the MOI of 1.0,and Vif protein was readily expressed in these cells. Moreover,the expression of Rta and vIL-6 proteins of KSHV were significantly downregulated by Vif protein. Conclusion:Recombinant lentivirus with high titer and efficient infection of BCBL-1 cells could be obtained quickly and simply by using the lentivirus vectors system,and Vif expression could be observed in BCBL-1 cells infected with lentivirus-Vif. Furthermore,the Vif protein may inhibit the lytic cycle replication of KSHV. The results of this study lay the foundation for further studying on the molecular mechanisms and signaling pathways involved.
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