稳定高表达 RORα基因的人胃癌 MGC803细胞系的构建与鉴定

摘要

目的构建维甲酸相关孤核受体α(RORα)基因的真核表达载体,并建立稳定高表达 RORα的人胃癌MGC803细胞,为研究 RORα的功能奠定基础。方法根据 RORα基因的 cDNA 全序列,设计一对带有限制性酶切位点的引物。从人胃癌 MGC803细胞中提取 mRNA 作为模板合成 RORα cDNA 第一链,并用上述引物扩增目的基因全序列,然后经双酶切插入到真核表达载体 pcDNA3.1(+),转化大肠杆菌 DH5α；用氨苄青霉素筛选 pcDNA3.1(+)-RORα的阳性菌株,双酶切和测序进行鉴定。用经鉴定的重组质粒 pcDNA3.1(+)-RORα转染 MGC803细胞,G418筛选获得 RORα/MGC803细胞株；Real-time PCR 和 Western blot 检测该细胞株 RORα的表达情况。结果经双酶切鉴定,构建的真核表达载体 pcDNA3.1(+)-RORα包含有大约1640 bp 的插入片段,与预期大小一致；表达载体经测序鉴定,其插入片段碱基序列与 RORα基因的 cDNA 序列完全一致。用构建的真核表达载体 pcDNA3.1(+)-RORα转染 MGC803细胞后,筛选到可稳定传代的 RORα/ MGC803细胞株。 Real-time PCR 与 Western blot 检测显示,RORα/ MGC803细胞 RORα mRNA 和蛋白表达增高。结论成功构建真核表达载体 pcDNA3.1(+)-RORα和建立高表达 RORα基因的 RORα/ MGC803细胞。%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.