摘要
目的人ArgBP2基因真核表达载体的构建及其在胃癌细胞系中的表达。方法从人胃癌细胞系SCG7901中提取RNA,反转录为cDNA,并以此为模板,扩增ArgBP2的全长编码基因,并构建到真核表达载体pcDNA3.1/HisC中,同时应用Western blot方法检测其表达,然后利用RT-PCR方法检测在各种胃癌细胞系内源性ArgBP2的表达。结果人ArgBP2编码序列已被克隆至pcDNA3.1/HisC表达载体,双酶切鉴定片段大小为1935bp,Western blot验证其表达成功,大小为70kDa,并在RNA水平上检测ArgBP2在胃癌细胞系中的表达。结论成功构建了人ArgBP2基因真核表达载体,在胃癌细胞系表达,为进一步研究ArgBP2的生物学特性及其信号转导通路奠定了基础。
Objective To construct the expression plasmid of human Arg-binding protein 2（hArgBP2） gene and identify the expression in gastric cancer cells.Methods Total mRNA was extracted from SCG7901 cells,and cDNA was formed by reverse transcription.The hArgBP2 coding sequence was amplified by polymerase chain reaction（PCR） and cloned into pcDNA3.1/HisC plasmid.After the hArgBP2 gene was identified by enzyme digestion and sequencing,the plasmid was transfected into SCG7901 cells.The expression of the recombinant plasmid in SCG7901 cells was detected by Western blot assay.The endogenous ArgBP2 in different gastric cancer cells was examined by RT-PCR assay.Results hArgBP2 was successfully constructed into the pcDNA3.1/HisC expressing vector,the length of the fragment identified by restriction enzyme digestion was 1935bp.The expression of His-hArgBP2 fusion protein with a molecular weight of 70kDa was detected by Western blot.The endogenous ArgBP2 in gastric cancer cells was identified.Conclusion The recombinant plasmid of hArgBP2 gene was successfully constructed,the expression of extraneous and endogenous ArgBP2 was identified,which provides the basis for research on biological features and signal transduction pathway of ArgBP2.
出处
《解剖科学进展》
CAS
2012年第2期106-108,112,共4页
Progress of Anatomical Sciences
基金
国家自然科学基金资助项目(No.30800415)
