Id1真核表达载体的构建及胃黏膜上皮细胞系的转染

Construct the eukaryocytic expression vector of Id1 and transfection into immortal gastric mucosa epithelial cell

目的 :扩增Id1基因 ,构建Id1真核表达载体pcD NA3.1 /V5HisA Id1 .方法 :以胃癌细胞SGC790 1cDNA为模板 ,以Id1基因编码区外的两段特异性序列为引物 ,获得Id1基因的全长 .将目的基因插入载体pUCm T ,经序列测定证实后 ,亚克隆至pcDNA3.1 /V5HisA并经酶切鉴定 .采用脂质体转染的方法将重组质粒稳定转染至胃粘膜上皮永生化细胞系GES 1中 .结果 :经RT PCR方法扩增出大小为 6 0 8bp的基因片断 ,序列测定其编码序列及读框正确 .亚克隆经酶切鉴定正确 .经过 8wkG4 1 8筛选后 ,获得稳定表达Id1的细胞亚系 .经Westernblotting证实 ,Id1蛋白表达水平高于对照组 .结论 :成功地构建了Id1真核表达载体 。

AIM: To amplify the Id1 gene and to construct a eukaryocytic expression vector of Id1. METHODS: RT PCR was performed to amplify the Id1 gene containing the complete CDS by using the total cDNA of gastric cancer cell line SGC7901 as template. Id1 gene was inserted into the pUCm T vector and sequenced. The correct Id1 cDNA was subcloned into eukaryocytic expression vector pcDNA3.1/V5HisA and transfected into GES 1 by lipofectamine mediated transfection. RESULTS: RT PCR yielded a fragment of 608 bp and Id1 was verified by sequence analysis. The subclone was identified by restriction enzyme digestion. Western blotting confirmed the transfetant in which Id1 was over expressed. CONCLUSION: Eukaryocytic expression vector of Id1 has been successfully constructed, which provides a premise for further investigation of its function in the gastric cancer.