卡波西肉瘤相关疱疹病毒编码vFLIP基因真核表达载体的构建

Construction of a eukaryotic recombination vector encoding the viral FLICE inhibitory protein of Kaposi's sarcoma associated herpesvirus

目的构建卡波西肉瘤相关疱疹病毒(Kaposi′s sarcoma-associated herpesvirus,KSHV)编码的病毒FLICE抑制蛋白(viral FLICE inhibitory protein,vFLIP)真核表达载体,并转染入293T细胞中进行表达鉴定。方法据GenBank中KSHV编码vFLIP基因核苷酸序列人工化学合成"vFLIP"的碱基序列,在5′端下添加XhoI酶切位点,3′端添加BamHI酶切位点,合成的碱基序列经酶切克隆进真核载体GV144,经双酶切和测序验证,成功构建G-vFLIP真核表达载体,将该质粒转染293T细胞,通过转染后细胞内荧光蛋白的表达和实时荧光定量PCR鉴定转染后KSHV-vFLIP在胞内的表达。结果通过对构建质粒限制性内切酶产物进行基因测序证实成功构建了携带vFLIP基因的真核表达载体G-vFLIP,G-vFLIP真核表达载体转染293T细胞,24h后在荧光显微镜可观察到细胞表达绿色荧光,提取细胞总RNA进行逆转录,293T细胞中G-vFLIP组vFLIP mRNA的表达水平是GV144组的9 355 080.705倍(P<0.05)。结论成功构建了KSHV-vFLIP真核表达载体,转染后在293T细胞中初步获得表达。

Objective To construct a eukaryotic recombination vector encoding the viral FLICE inhibitory protein（vFLIP）of Kaposi′s sarcoma associated herpesvirus（KSHV）. Methods Chemical synthesis of the sequences of vFLIP was done based on the nucleotide sequences of KSHV encoding vFLIP gene in GenBank.The XhoI restriction site was added at the 5′end and BamHI cleavage site at the 3′end.The sequences was cloned into eukaryotic vector GV144.The eukaryotic expression vector of G-vFLIP was successfully constructed and verified by double enzyme digestion and sequencing.The plasmid was transfected into 293 Tcells and the expression of KSHV-vFLIP was identified by the expression of fluorescent protein and the real-time fluorescent quantitative PCR. Results The eukaryotic expression vector（G-vFLIP）carrying vFLIP gene was successfully constructed and confirmed by DNA sequencing.After transfecting into 293 Tcells for 24 hours,the fluorescence was observed by fluorescence microscope.Total RNA was extracted for reverse transcription.Expression level of vFLIP mRNA in the G-vFLIP group was 9 355 080.705 times higher than that in the GV144 control group（P〈0.01）. Conclusions The eukaryotic expression vector of KSHV-vFLIP was successfully constructed and expressed in 293 Tcells.