目的通过RNA干扰HIV-1vpr基因的表达后观察凋亡相关蛋白c-凋亡抑制蛋白（inhibitor of apoptosis protein, IAP）2的表达情况，同时检测各组Jurkat细胞的凋亡情况。方法将空载体（NC组）、HIV-1 vpr质粒（vpr组）、vpr＋磷酸化RNAT-U6.1...目的通过RNA干扰HIV-1vpr基因的表达后观察凋亡相关蛋白c-凋亡抑制蛋白（inhibitor of apoptosis protein, IAP）2的表达情况，同时检测各组Jurkat细胞的凋亡情况。方法将空载体（NC组）、HIV-1 vpr质粒（vpr组）、vpr＋磷酸化RNAT-U6.1/Neo-vpr-56质粒（Si56组）、vpr＋磷酸化RNAT-U6.1/Neo-vpr-160质粒（Si160组）分别转染Jurkat细胞并培养48 h，分别进行总RNA、蛋白提取，利用实时PCR检测vpr基因的表达，证实质粒转染成功，以实时PCR和蛋白质印迹法检测凋亡相关蛋白人类内源性抗凋亡蛋白（inhibitor of apoptosis protein 2 c-IAP2）的表达情况，流式细胞仪检测各组Jurkat细胞的凋亡情况。
结果vpr组、Si56组、Si160组均可检测到vpr基因的表达，与vpr组比较，Si56组、Si160组HIV-1vpr基因表达的mRNA水平均出现明显降低，分别下降82.2%、87.2%，差异均有统计学意义（均P〈0.05）。与NC组比较，vpr组、Si56组及Si160组c-IAP2基因表达的mRNA水平明显升高，分别为NC组的3.75、2.49、2.65倍；但Si56组、Si160组c-IAP2基因表达的mRNA水平均低于vpr组，分别下降33.7%和29.5% ；蛋白质印迹法检测显示，c-IAP2蛋白表达水平与mRNA结果一致，其中Si56组、Si160组c-IAP2蛋白表达水平比vpr组分别下降42.2%和46.8% （均P〈0.05）。NC组、vpr组、Si56组和Si160组均可检测到Jurkat细胞凋亡，与NC组比较，vpr组细胞凋亡率明显升高，为NC组的1.76倍，而Si56组和Si160组细胞凋亡率与NC组比较差异无统计学意义（均P〉0.05）；与vpr组比较，Si56组和Si160组细胞凋亡率明显降低，分别为19.26%和18.05%（P〈0.05）。结论通过沉默HIV-1vpr基因可下调Jurkat细胞c-IAP2基因的转录和蛋白表达水平，抑制Jurkat细胞的凋亡。展开更多
The recombinant suicide plasmid pYYvpr contained a Kan resistant cassette and a 778bp internal piece of the vpr gene. It was transformed into the wild type E.coli strain, MC 1061,which possessed the full-length vpr ge...The recombinant suicide plasmid pYYvpr contained a Kan resistant cassette and a 778bp internal piece of the vpr gene. It was transformed into the wild type E.coli strain, MC 1061,which possessed the full-length vpr gene and did not contain the λ pir gene. The recombinant suicide plasmid could not replicate in MC 1061. Colonies grew on the plates of Kan were analysed by PCR and Southern blot with two different Dig-probes,vpr and Kan R probes. One colony was detected vpr and Kan R gene by PCR and Southern blot showed the mutant had the different vpr map with the wild type. The isogenic knockout mutant, named MC1061 Δvpr,was confirmed.The mutant supported lysogenic infection of VT2 recombinant phage, but did not support lytic infection. All results showed that the vpr gene should be related to the lytic infection of VT2 phage.展开更多
文摘目的通过RNA干扰HIV-1vpr基因的表达后观察凋亡相关蛋白c-凋亡抑制蛋白（inhibitor of apoptosis protein, IAP）2的表达情况，同时检测各组Jurkat细胞的凋亡情况。方法将空载体（NC组）、HIV-1 vpr质粒（vpr组）、vpr＋磷酸化RNAT-U6.1/Neo-vpr-56质粒（Si56组）、vpr＋磷酸化RNAT-U6.1/Neo-vpr-160质粒（Si160组）分别转染Jurkat细胞并培养48 h，分别进行总RNA、蛋白提取，利用实时PCR检测vpr基因的表达，证实质粒转染成功，以实时PCR和蛋白质印迹法检测凋亡相关蛋白人类内源性抗凋亡蛋白（inhibitor of apoptosis protein 2 c-IAP2）的表达情况，流式细胞仪检测各组Jurkat细胞的凋亡情况。
结果vpr组、Si56组、Si160组均可检测到vpr基因的表达，与vpr组比较，Si56组、Si160组HIV-1vpr基因表达的mRNA水平均出现明显降低，分别下降82.2%、87.2%，差异均有统计学意义（均P〈0.05）。与NC组比较，vpr组、Si56组及Si160组c-IAP2基因表达的mRNA水平明显升高，分别为NC组的3.75、2.49、2.65倍；但Si56组、Si160组c-IAP2基因表达的mRNA水平均低于vpr组，分别下降33.7%和29.5% ；蛋白质印迹法检测显示，c-IAP2蛋白表达水平与mRNA结果一致，其中Si56组、Si160组c-IAP2蛋白表达水平比vpr组分别下降42.2%和46.8% （均P〈0.05）。NC组、vpr组、Si56组和Si160组均可检测到Jurkat细胞凋亡，与NC组比较，vpr组细胞凋亡率明显升高，为NC组的1.76倍，而Si56组和Si160组细胞凋亡率与NC组比较差异无统计学意义（均P〉0.05）；与vpr组比较，Si56组和Si160组细胞凋亡率明显降低，分别为19.26%和18.05%（P〈0.05）。结论通过沉默HIV-1vpr基因可下调Jurkat细胞c-IAP2基因的转录和蛋白表达水平，抑制Jurkat细胞的凋亡。
文摘The recombinant suicide plasmid pYYvpr contained a Kan resistant cassette and a 778bp internal piece of the vpr gene. It was transformed into the wild type E.coli strain, MC 1061,which possessed the full-length vpr gene and did not contain the λ pir gene. The recombinant suicide plasmid could not replicate in MC 1061. Colonies grew on the plates of Kan were analysed by PCR and Southern blot with two different Dig-probes,vpr and Kan R probes. One colony was detected vpr and Kan R gene by PCR and Southern blot showed the mutant had the different vpr map with the wild type. The isogenic knockout mutant, named MC1061 Δvpr,was confirmed.The mutant supported lysogenic infection of VT2 recombinant phage, but did not support lytic infection. All results showed that the vpr gene should be related to the lytic infection of VT2 phage.
