文章摘要
朱小飞,秦 娣,周 峰,卫冰冰,卢 春.重组慢病毒载体介导HIV-1 Nef蛋白对原发性渗出性淋巴瘤细胞系和血管内皮细胞增殖作用影响的初探[J].南京医科大学学报,2011,(2):131~137
重组慢病毒载体介导HIV-1 Nef蛋白对原发性渗出性淋巴瘤细胞系和血管内皮细胞增殖作用影响的初探
Role of HIV-1 Nef protein carried by recombinant lentivirus in proliferation of primary effusion lymphoma cell line and vascular endothelial cells
投稿时间:2010-09-15  
DOI:10.7655
中文关键词: 慢病毒  Nef  卡波济肉瘤  卡波济肉瘤相关疱诊病毒
英文关键词: recombinant lentivirus  Nef  KS  KSHV
基金项目:国家自然科学基金(30900064);教育部高等学校博士点专项科研基金新教师基金(20093234120004);江苏省高校自然科学基金(09KJB310007)
作者单位
朱小飞 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 
周 峰 南京医科大学微生物学与免疫学系,江苏 南京 210029 
卫冰冰 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: 构建含有HIV-1负调控因子(Nef)基因的重组慢病毒表达载体,并检测Nef蛋白在人胚肾293T细胞?体腔渗出性淋巴瘤细胞BCBL-1和人血管内皮细胞EA.hy926中的表达情况及其对BCBL-1和EA.hy926细胞增殖的影响?方法:从本实验室已构建好的含Nef基因的重组真核表达质粒pCI-neo-Nef中扩增出Nef基因,插入到慢病毒载体pHAGE-CMV-MCS-IzsGreen中构建成pHAGE-Nef,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?荧光显微镜下观察293T细胞中绿色荧光蛋白(GFP)的表达并收集培养上清,此上清经0.45 μm滤器过滤后即获得慢病毒悬液?梯度稀释法测定慢病毒滴度?将重组慢病毒分别感染293T?BCBL-1和EA.hy926细胞,用Western blot检测Nef蛋白的表达?通过细胞增殖实验检测Nef蛋白对BCBL-1和EA.hy926细胞增殖的影响?结果:限制性内切酶鉴定和核酸序列测序证实成功构建了含HIV-1 Nef基因的重组慢病毒表达载体,病毒滴度为1×107 TU/ml?以重组慢病毒分别感染293T?BCBL-1和EA.hy926细胞,均能检测到Nef蛋白的表达,且Nef蛋白能够抑制BCBL-1和EA.hy926细胞的增殖作用?结论:成功构建了含HIV-1 Nef基因的慢病毒表达载体,获得的慢病毒不仅能够有效感染293T?BCBL-1和EA.hy926细胞,而且能够介导Nef蛋白在这些细胞中表达?重组慢病毒载体介导的Nef蛋白能够抑制BCBL-1和EA.hy926细胞的增殖?
英文摘要:
      Objective: To construct the recombinant lentivirus containing HIV-1 Nef gene and detect the protein expression in its target cell lines, such as 293T, BCBL-1 and EA.hy926, and explore the effect of Nef on cell proliferation. Methods:The fragment of Nef gene from expression vector pCI-neo-Nef was cloned into the lentivirus vector pHAGE-CMV-MCS-Izs-Green, then the recombinant vector pHAGE-Nef, 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 infection of the recombinant lentivirus, the protein expression of Nef in 293T, BCBL-1 and EA.hy926 cells were detected by Western blot. The effect of Nef on cell proliferation was investigated by Cell Counting Kit-8 assay. Results:The recombinant lentivirus vector carrying HIV-1 Nef was constructed successfully. The viral titer was 1×107 TU/ml. 293T, BCBL-1 and EA.hy926 cells could be efficiently infected by it. The expression of Nef in these cell lines could be detected by Western blot. In addition, Nef protein could inhibit the proliferation of BCBL-1 and EA.hy926 cells. Conclusion:Recombinant lentivirus with high titer and efficient infection of 293T, BCBL-1 and EA.hy926 cells, could be obtained quickly and simply by using the lentivirus vectors system, and Nef expression could be detected in 293T, BCBL-1 and EA.hy926 cells infected by lentivirus-Nef. Furthermore, our study suggested that Nef protein could inhibit the proliferation of BCBL-1 and EA.hy926 cells.
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