摘要
构建真核表达载体pcDNA3.1（-）-Smad4,通过稳定转染建立高效表达Smad4的人胃癌细胞系.利用PCR扩增出人Smad4全长基因的cDNA编码区序列,使用基因重组技术将其克隆到真核表达载体pcDNA3.1中,得到重组表达质粒pcDNA3.1-Smad4.双酶切及测序鉴定后,利用脂质体法将其转染入人胃癌MKN28细胞,G418加压筛选后得到阳性克隆.通过流式细胞术、免疫荧光及Western blot检测Smad4在细胞系中的表达情况.经测序及双酶切鉴定,pcDNA3.1-Smad4真核表达载体pcDNA3.1（-）-Smad4构建重组质粒构建正确,稳定转染人Smad4的MKN28细胞系.结论：建立的稳定转染人Smad4的MKN28细胞系能够高效表达Smad4基因,为进一步研究Smad4在人胃癌中的作用机制奠定了基础,为胃癌的基因治疗提供了潜在靶点.
Objective： To construct the eukaryotic expressing plasmid pcDNA3. 1- Smad4 and establish a MKN28 cell line with stably expressing Smad4. Method： The full length of Smad4 eDNA fragment was amplified by PCR and inserted into eukaryotic expression vector peDNA3.1. The recombi- nant plasmid pcDNA3.1--Smad4 was identified by double restriction enzyme digestion and DNA sequencing. Then the plasmid pcDNA3.1--Smad4 was transfeeted into MKN28 cells by lipofection. After screening by G418, a stably transfected with Smad4 MKN28 cell line was established. The expression of Smad4 gene was identified by Rt--PCR and Western blot. Result： The eukaryotic plasmid peDNA3.1--Smad4 was successfully constructed. A MKN28 cell line with stably expressing Smad4 was established. Conclusion： The established MKN28 cell line can highly express Smad4 gene, which provided a basic experiment foundation for further studies on the function of the Smad4 and a potential target site for gene therapy in gastric cancer.
