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

作　　者：魏晓丽[1] 温浩[2] 金一帮[2] 蒋中华[3] 李明远[3] 

机构地区：[1]新疆医科大学基础医学院免疫学教研室临床医学博士后流动站,乌鲁木齐830011 [2]新疆医科大学包虫病重点实验室,乌鲁木齐830011 [3]四川大学华西医学中心微生物教研室,成都610041

出　　处：《生物技术通报》2011年第5期98-101,共4页Biotechnology Bulletin

基　　金：自治区高校科研计划项目(XIEDU2009I20);新疆包虫病基础医学重点实验室开放课题(XJDXO0202-2008-04)

摘　　要：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.

关 键 词：beta-防御素2 真核表达 逆转录聚合酶链反应 蛋白免疫印迹 

分 类 号：Q78[生物学—分子生物学]