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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 eukaryotic expression plasmid PEGFP-N1-CDX2 of tail-type homologous box transcription factor 2 (CDX2) gene and observe its expression in gastric cancer cells. Methods CDX2 was amplified from gastric cancer cells by RT-PCR. The double-digested PCR products were cloned into multiple cloning sites of PEGFP-N1 to construct recombinant plasmid PEGFP-N1-CDX2. After double enzyme digestion and sequencing, the recombinant plasmid of PEGFP-N1-CDX2 was transfected into SGC-7901 cells (C group) by cationic polymer mediated transfection, followed by real-time qRT-PCR and Western blot The expression of CDX2 in untransfected group (A group), PEGFP-N1 group (B group) and C group were detected. Results The recombinant plasmid was digested with XhoⅠ and Hind Ⅲ and sequenced. The recombinant plasmid was successfully transfected into gastric cancer cell line SGC-7901 by using cationic polymer transfection reagent and CDX2 High gene expression. Conclusion The eukaryotic expression plasmid PEGFP-N1-CDX2 was successfully constructed and was highly expressed in SGC-7901 cells.