摘要
目的构建尾型同源盒转录因子2(CDX2)基因真核表达质粒PEGFP-N1-CDX2,并观察其在胃癌细胞中的表达。方法采用RT-PCR技术从胃癌细胞中扩增CDX2,并将PCR产物双酶切后定向克隆入PEGFP-N1的多克隆位点,构建PEGFP-N1-CDX2重组质粒。经过双酶切及测序验证后,采用阳离子聚合物转染介导将PEGFP-N1-CDX2重组质粒转染胃癌SGC-7901细胞(C组),随后实时荧光定量PCR(qRT-PCR)及Western blot检测未转染组(A组)、转染PEGFP-N1组(B组)和C组细胞CDX2的表达情况。结果重组质粒经XhoⅠ和HindⅢ双酶切和测序与CDX2基因序列一致,利用阳离子聚合物转染试剂介导将PEGFP-N1-CDX2重组质粒成功转染到胃癌SGC-7901细胞中,并获得了CDX2基因的高表达。结论成功构建PEGFP-N1-CDX2真核表达质粒,并在SGC-7901细胞内高表达。
Objective To construct the eukaryotic expression vector PEGFP-N1-CDX2 and induce the vector expression in human gastric carcinoma cells. Methods CDX2 was amplified from gastric carcinoma cell AGS by RT-PCR. The PCR products were cloned into the eukaryotic expression vector PEGFP-N1 after Xho I and Hind ]]1 digestion. Recombinant plasmid PEGFP-N1-CDX2 was identified by double enzyme digestion and DNA sequencing, and transfected into SCK;-7901 cells with cationic polymer. The expression of CDX2 in S（K;-7901 cells was identified by qRT-PCR and Western blot techniques, respectively. Results Correct construction of PEGFP-N1-CDX2 was identified by double enzyme digestion and DNA sequencing. CDX2 gene expressed by the transfected cells was testified by qRT-PCR and Western blot techniques. Conclusion The recombinant eukaryotic expression vector has been constructed successfully, which is expressed effectively in SCK2-7901 cells.
出处
《江苏医药》
CAS
北大核心
2013年第17期1996-1999,共4页
Jiangsu Medical Journal
