中文摘要：

目的:构建含卡波氏肉瘤病毒(Kaposi’s sarcoma-associated herpesvirus,KSHV)分子开关蛋白,即复制和转录激活蛋白(replication and transcriptional activator,Rta)基因的重组慢病毒载体。方法:从真核表达质粒pcDNA3.1-Rta中扩增出Rta基因,插入pHAGE-CMV-MCS-IzsGreen中,构建重组慢病毒载体pHAGE-Rta。将重组慢病毒骨架质粒pHAGE-Rta与包装质粒psPAX2和包膜质粒pMD2.G共转染293 T细胞,通过荧光显微镜观察表达绿色荧光蛋白的293 T细胞占总数的百分比。收集病毒悬液,采用梯度稀释法测定病毒滴度。以不同感染复数(MOI)的Lentivirus-Rta病毒量感染BCBL-1细胞,72 h后检测Rta基因的表达,同时通过蛋白质印迹和病毒颗粒释放实验检测KSHV裂解期蛋白病毒白细胞介素-6(vIL-6)的表达及子代病毒颗粒的产出。结果:从真核表达质粒pcDNA3.1-Rta中扩增出Rta基因,插入pHAGE-CMV-MCS-IzsGreen后,酶切鉴定和核酸序列测定证实重组慢病毒骨架质粒pHAGE-Rta构建成功。通过慢病毒包装3质粒表达系统获得了表达Rta基因的重组慢病毒,滴度约为2×107efu/mL。以不同MOI的重组慢病毒感染BCBL-1细胞,72 h后可检测到外源基因编码Rta蛋白的表达,且其表达能够上调KSHV vIL-6的表达水平及促进子代病毒颗粒的释放。结论:成功构建了含Rta基因的慢病毒表达载体,获得的重组病毒能够有效地感染BCBL-1细胞,并发挥激活KSHV裂解期复制的生物学功能。

英文摘要：

Objective:To construct the recombinant lentivirus carrying Kaposi's sarcoma-associated herpesvirus (KSHV) replication and transcriptional activator(Rta) gene.Methods:The fragment of Rta gene from expression vector pcDNA3.1-Rta was cloned into the lentivirus vector pHAGE-CMV-MCS-IzsGreen,then the recombinant vector pHAGE-Rta,packaging vector psPAX2 and envelope vector pMD2.G were cotransfected into the 293 T cells.Filtered culture media were harvested and the viral titer was checked by observing the expression of green fluorescent protein (GFP).BCBL-1 cells were infected with the LentivirusRta of which multiplicity of infection (MOI) was 1.0,5.0 and 10.0,respectively,and Rta protein was detected by western blotting; meanwhile,the expression of KSHV lytic protein viral interleukin-6 (vIL-6) and the production of KSHV virion were examined by western blotting and virion release assay,respectively.Results:Recombinant lentivirus with high titer and efficient infection were obtained by the lentivirus vectorssystem.BCBL-1 cells could be efficiently infected,and the expression of Rta protein in BCBL-1 cells infected with Lentivirus-Rta was detectable.The expression of Rta could promote KSHV lytic replication,upregulate the expression of vIL-6 protein and the production of KSHV virion.Conclusion:The recombinant lentivirus vector carrying Rta gene was constructed successfully; and the expression of Rta in BCBL-1 cells infected with Lentivirus-Rta could reactivate KSHV from latency successfully.