摘要
目的建立稳定过表达N-cadherin的人类胃癌MGC-803细胞系。方法从含有N-cadherin的质粒中利用PCR技术克隆N-cadherin基因编码序列。制备并浓缩慢病毒颗粒,将构建好的过表达N-cadherin慢病毒载体感染MGC-803细胞,并稳定遗传,嘌呤霉素筛选的阳性克隆传代培养5代后鉴定细胞内N-cadherin的表达。结果荧光显微镜下过表达N-cadherin组细胞有绿色荧光表达;PCR扩增得到含N-cadherin基因编码序列的特异性条带;重组的过表达N-cadherin载体中,过表达N-cadherin编码序列与目标序列几乎一致;Western blotting结果显示,在分子量为98 k D处有相应条带。结论成功建立稳定过表达N-cadherin的人类胃癌MGC-803细胞,此研究为进一步探讨N-cadherin基因的功能提供了良好的研究基础。
Objective To establish a stable human gastric cancer MGC-803 cell line overexpressing N-cadherin. Methods N-cadherin gene coding sequences were cloned from plasmids containing N-cadherin by PCR. Lentiviral particles were preparated and concentrated, overexpression of N-cadherin lentiviral vectors transfected MGC-803 cells. The positive clones screened by puromycin were subcultured for 5 generations, the expression of N-cadherin in cells was identified. Results Overexpression of N-cadherin group under fluorescence microscope showed green fluorescence. Specific bands containing N-cadherin gene coding sequence were amplificated by PCR. In overexpression of recombinant N-cadherin, overexpression of N-cadherin coding sequence was almost coincident with the target sequence. Western blotting showed that the molecular weight was 98 kD with a corresponding strip. Conclusion Stable overexpression of N-cadherin human gastric cancer MGC-803 cells are established successfully, this study provides a good basis for further research on the function of N-cadherin gene.
出处
《胃肠病学和肝病学杂志》
CAS
2017年第12期1370-1372,共3页
Chinese Journal of Gastroenterology and Hepatology
