GDDR位点突变载体的构建和稳转细胞系的建立

GDDR^(mut) vector construction and establishment of SGC-7901-GDD^(mut) stable cell lines

目的构建GDDR位点突变载体,使GDDR翻译蛋白无法通过二硫键与TFF1结合,建立稳定转染GDDR位点突变载体的人胃癌SGC-7901稳转细胞系。方法设计GDDR Cys38位点突变引物,构建GDDR位点突变的真核表达载体;脂质体法转染重组质粒至人胃癌细胞系SGC-7901;G418筛选阳性细胞并扩大培养;实时定量PCR检测突变GDDR mRNA在人胃癌细胞系SGC-7901的表达。结果经限制性内切酶酶切和DNA测序证实质粒中插入的为所需序列;实时定量PCR证实突变GDDR在人胃癌SGC-7901稳转细胞系高表达。结论 GDDR定点突变载体构建成功,建立了稳定转染GDDR位点突变载体的SGC-7901细胞系,为进一步研究GDDR的结构和功能提供了条件。

Objective To construct the eukaryotic vector of the human GDDR（mut） gene and to establish SGC-7901 cell lines that can express GDDR（mut） stably. Methods The DNA sequences encoding GDDRmut were designed. Using pcDNA3.1/myc-his（-）-GDDR as a template, the recombinant plasmid pcDNA3.1/myc-his（-）-GDDRmut was constructed by site-directed mutagenesis and transfected into SGC-7901 cells with lipofectamin. The G418 resistant clones were selected and cultured. The mRNA level of GDDRmut was detected by real-time PCR in positive clones. Results Restriction enzyme and sequencing indicated the GDDRmut sequence was correct. Over-expression of GDDRmut mRNA was detected successfully in SGC-7901 cells by real-time PCR. Conclusion The eukaryotic vector of the human GDDRmut gene and SGC-7901 cell lines stably expressing GDDR-mutant were constructed successfully in this study.