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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 ,为进一步研究其在胃癌细胞中的作用奠定了基础
OBJECTIVE: To amplify Id1 gene and construct Id1 eukaryotic expression vector pcDNA3.1 / V5HisA Id1.Methods: The gastric cancer cell SGC7901 cDNA was used as a template and two specific sequences of Id1 gene coding region were used as primers to obtain the Id1 gene The full length of the target gene was inserted into the vector pUCm T, confirmed by sequence analysis, subcloned into pcDNA3.1 / V5HisA and identified by restriction endonucleases using liposome transfection method recombinant plasmid was stably transfected into the gastric mucosa epithelium immortalized Cell line GES 1. RESULTS: The gene fragment of 680 bp in size was amplified by RT-PCR, and its coding sequence and reading frame were determined correctly.The subclones were identified by restriction enzyme digestion.After 8wkG418 screening, Obtained stably expressing Id1 cell subline.It was confirmed by Western blotting that the expression level of Id1 protein was higher than that of the control group.Conclusion: Id1 eukaryotic expression vector was successfully constructed, which laid the foundation for the further study of its role in gastric cancer cells