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目的构建促凋亡基因Bad真核表达载体并鉴定其在人食管鳞癌细胞ECA109和KYSE450细胞中的表达。方法设计Bad基因引物,通过PCR扩增获得其cDNA片段,经双酶切后,将此片段克隆至真核表达载体GV142中,转入DH5α,获得重组质粒GV142-Bad;PCR扩增、酶切及测序验证后,将其及对照空载体转染人食管鳞癌ECA109和KYSE450细胞,转染后24h,通过荧光倒置显微镜观察细胞中绿色荧光信号的表达情况,提取总RNA,使用qPCR检测Bad在转录水平的表达。结果成功构建Bad真核表达载体,PCR扩增出长为545bp的特异性片段,经克隆至真核表达载体GV142后酶切及测序鉴定准确。重组质粒GV142-Bad成功转染食管鳞癌细胞并在荧光倒置显微镜下观察到不同强度的绿色荧光信号。应用qPCR检测重组质粒GV142-Bad转染ECA109和KYSE450细胞的Bad基因mRNA表达水平上调,与空载体转染组和空白组比较差异有统计学意义(P<0.05)。结论本研究成功构建Bad真核表达载体,用电转染法成功转染食管鳞癌ECA109和KYSE-450细胞,获得高表达并上调Bad基因mRNA的水平,为进一步研究Bad基因的功能奠定了基础。
Objective To construct the Bad eukaryotic expression vector of apoptosis gene and identify its expression in human esophageal squamous carcinoma cell lines ECA109 and KYSE450. Methods Bad gene primers were designed and their cDNA fragments were amplified by PCR. After double digestion, the fragment was cloned into eukaryotic expression vector GV142 and transformed into DH5α to obtain recombinant plasmid GV142-Bad. After transfected into human esophageal squamous cell carcinoma ECA109 and KYSE450 cells, the expression of green fluorescence signal was observed by fluorescence inverted microscope. The total RNA was extracted and detected by qPCR. Transcriptional level of expression. Results The Bad eukaryotic expression vector was successfully constructed. The specific fragment of 545bp was amplified by PCR and cloned into eukaryotic expression vector GV142. The recombinant plasmid GV142-Bad was successfully transfected into esophageal squamous carcinoma cells and different intensity of green fluorescence signal was observed under fluorescence inverted microscope. Using qPCR to detect the mRNA expression of Bad gene in ECA109 and KYSE450 cells transfected with recombinant plasmid GV142-Bad, the difference was statistically significant (P <0.05) compared with the empty vector transfected group and the blank group. Conclusions This study successfully constructed Bad eukaryotic expression vector and successfully transfected esophageal squamous cell carcinoma ECA109 and KYSE-450 cells by electroporation to obtain high expression and up-regulate Bad gene mRNA level, which laid the foundation for further study on the function of Bad gene .