中南医学科学杂志2016，Vol.44Issue(1)：5-10,6.DOI:10.15972/j.cnki.43-1509/r.2016.01.002
稳定高表达 RORα基因的人胃癌 MGC803细胞系的构建与鉴定
Construction and Identification of MGC803 Cell Transfacted by RORα Gene
摘要
Abstract
Objective To construct the eukaryotic expression vector inserted by RORα gene,and establish human gastric cancer MGC803 cell lines that express higher level RORα gene to lay the foundation for investigating the function of RORα. Methods Based on the complete cDNA sequence of RORα isoform,to design a pair of primers with restriction sites. To extract the total mRNA from human gastric cancer MGC803 cells as a template for synthesize first strand cDNA of RORα,and amplify the complete sequences of target gene by the above primers. Then,after double enzyme digestion,the amplified sequences was inserted into the eukaryotic expression vector pcDNA3. 1( + ) for transforming into E. coli DH5α. The positive strains containing pcDNA3. 1( + )-RORα were screened by ampicillin,and were identified by restriction analy-sis and sequencing. MGC803 cells was transfected by the identified recombinant plasmid pcDNA3. 1( + )-RORα,and was screened by G418 for obtaining stable passage RORα/ MGC803 cell lines. RORα expression of the cell lines was certified by Real-time PCR and Western blot. Results By restriction analysis,the constructed eukaryotic expression vector pcD-NA3. 1( + )-RORα contains about 1640bp insert fragment,which is consistent with the expected size. By sequencing,the insert fragment’s base sequence of the expression vector was exactly the same cDNA sequence of RORα1 gene. To transfect MGC803 cells by the constructed eukaryotic expression vector pcDNA3. 1( + )-RORα,we obtained stable passage RORα/MGC803 cell lines that express higher level RORα mRNA and protein. Conclusion We successfully constructed eu-karyotic expression vector pcDNA3. 1( + )-RORα and RORα/ MGC803 cell lines which express higher level RORα gene.关键词
维甲酸相关孤核受体 α/真核表达/转染/人胃癌 MGC803 细胞Key words
RORα/eukaryotic expression/transfection/human gastric cancer MGC803 cells分类
医药卫生引用本文复制引用
赵晓红,向姝霖,刘芳,夏红,曾希,苏波,凌晖,苏琦..稳定高表达 RORα基因的人胃癌 MGC803细胞系的构建与鉴定[J].中南医学科学杂志,2016,44(1):5-10,6.基金项目
国家自然科学基金(81374013,81102854),湖南省高校创新平台开放基金(09K074),湖南省卫计委科研课题(B2015-182) （81374013,81102854）
