小鼠beta-防御素2真核表达载体的构建及表达

Molecular Cloning and Expression of Murie beta-Defensin 2 in Siha Cell

构建小鼠beta-防御素2(murie beta-defensin 2,mBD2)的真核表达载体pcDNA-mBD2,并观察其在真核细胞中的表达。从小鼠肝组织中提取mRNA通过逆转录聚合酶链反应扩增得到mBD2基因,定向克隆至真核表达载体pcDNA3.1(+)获得pcDNA-mBD2;经酶切和测序鉴定构建正确,应用脂质体法转染siha细胞,并通过蛋白免疫印迹法检测细胞内mBD2的表达。结果显示,构建了小鼠mBD2的真核表达载体,转染siha细胞培养并采用G418稳定筛选后,获得稳定转染细胞,收集并裂解转染细胞,蛋白免疫印迹法检测到转染细胞内mBD2的表达。成功构建了真核表达载体pcDNA-mBD2,为研究mBD2在宫颈癌发生发展过程中的作用及作用机制奠定基础。

The research was to construct a recombinant eukaryotic expression vector.Total RNA was extracted from mouse liver tissue,and reverse transcription polymerase chain reaction（RT-PCR）,then the murie beta-defensin 2（mBD2）DNA was inserted into the pcDNA3.1（＋）vector by EcoR I and Xho I cloning sites.The construct of pcDNA/mBD2 was further conformed by restricted enzyme digestion analysis and DNA sequencing.The expression of mBD2 was detected by Western blotting assay.Results showed that the eukaryotic expression vector pcDNA-mBD2 was constructed correctly,and significant expression was detected in siha cells aftert ransfection constantly.This result would facilitate the future study on the f unction and mechanism of mBD2 in uterine cervix cancer.