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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 the eukaryotic expression vector of virus FLICE inhibitory protein (FLFLP) encoded by Kaposi’s sarcoma-associated herpesvirus (KSHV) and transfect it into 293T cells for expression identification. Methods The base sequence of “vFLIP” was synthesized by the nucleotide sequence of vFLIP gene encoded by KSHV in GenBank. XhoI restriction site was added at 5 ’end and BamHI restriction site was added at 3’ end. The synthetic base The base sequence was cloned into eukaryotic vector GV144 by restriction enzyme digestion and sequencing. The eukaryotic expression vector of G-vFLIP was successfully constructed and transfected into 293T cells. The expression of intracellular fluorescent protein and real-time PCR Fluorescence quantitative PCR was used to identify the intracellular expression of KSHV-vFLIP after transfection. Results The recombinant plasmids containing the vFLIP gene, G-vFLIP, were successfully constructed by gene sequencing of plasmid restriction endonucleases. 293T cells were transfected with G-vFLIP eukaryotic expression vector and observed by fluorescence microscopy after 24h The expression of vFLIP mRNA in G-vFLIP group was 9355080.705 times that of GV144 group (P <0.05) after green fluorescence was detected and total cellular RNA was extracted for reverse transcription. Conclusion The recombinant eukaryotic expression vector KSHV-vFLIP was successfully constructed and transfected into 293T cells.