半定量逆转录-聚合酶链反应检测肺癌组织S100C基因的表达及其原核表达载体的构建和鉴定

Detection of the expression level of S100C gene in lung cancer tissue by RT-PCR and construction and identification ofprokaryotic expression vector

目的分析S100C基因在肺癌组织中的表达情况,并构建S100C蛋白的原核表达载体。方法以β-actin为内参,运用半定量RT-PCR技术检测26例肺癌组织和配对癌旁组织S100C基因mRNA的表达水平;运用RT-PCR技术克隆S100C基因的cDNA全长片段,与PET30a连接构建原核表达载体,转化大肠杆菌JM109感受态细胞,进行阳性克隆筛选鉴定。结果肺癌组织中S100C mRNA表达水平低于癌旁组织(P<0.05)。成功构建了S100C原核表达载体,序列同源性达100%。结论S100C基因表达下调在肺癌的发生中可能起重要作用;成功构建了S100C原核表达载体。

Objective To analyze the expression level of S100C gene in lung cancer tissue and to construct prokaryotic expression vector pET30a-S100C in order to select fusion antibody in further study. Methods mRNA expression level of S100C gene in lung cancer tissue and ambient tissue in 26 cases was detected by RT-PCR with β-actin as references, The full-length cDNA of S100C was cloned by RT-PCR. The amplified DNA fragments were ligated into pET30a vector and then transformed into E. coil stain JM109. The positive clones were screened out and indentified, Results The expression level of S100C in lung cancer tissue was lower than that in ambient tissue. The difference between the two groups was statistically significant （ P 〈 0.05 ）. The prokaryotic expression victor of S100C was successfully constructed, The sequence homogenuity was up to 100%. Conclusion The down-regulation expression of S100C gene probably takes important part in the carcinogenisis of lung cancer.