摘要
目的构建含ERGIC3基因真核表达载体,并转染至支气管上皮细胞株BEAS-2B,筛选并建立ERGIC3稳定表达的细胞株,为进一步研究ERGIC3在肺癌中的作用奠定基础。方法采用RT-PCR方法获取人类ERGIC3基因的ORF框的DNA序列,构建逆转录病毒载体pLXSN-ERGIC3,用PT-67细胞包装后获得病毒颗粒;用病毒颗粒感染BEAS-2B细胞,通过G418筛选获取整合了外源基因ERGIC3的单克隆化细胞;采用定量RT-PCR和Western blot对ERGIC3稳定表达的细胞株进行鉴定,并用划痕实验分析稳定转染细胞株的迁移能力和形态学变化。结果获得4个ERGIC3稳定表达的BEAS-2B细胞株,ERGIC3的表达量均远高于对照组细胞,分别为对照组细胞的45.3、48.8、41.5和28.2倍,且稳定转染细胞株的迁移能力也显著增强(P<0.05),但其形态学没有明显变化。结论成功构建了含ERGIC3基因真核表达载体pLXSNERGIC3;筛选出多个ERGIC3基因稳定转染的BEAS-2B细胞株,且迁移能力明显提高。
Objective To construct a eukaryotic expression vector carrying human gene ERGIC3 and to obtain human bronchial epithelial cell lines stably transfected with ERGIC3, providing the foundation of ERGIC3 in lung cancer. Methods The pLXSN retroviral vector was inserted the DNA fragment of open reading frame sequence （ORF） of gene ERGIC3 by PCR method. After the constructed pLXSN vector was transfected into the packaging cells, the PT67 cell line, for 48 hours, the replication -incompetent retroviral particles were harvested and infected the BEAS -2B cells. Screened with G418 ,the single cell clones were sought out and cultured to obtain stable transfection cell lines. Then the migration of the cell lines was tested by wound healing scratch assay. Results The vector carrying ERGIC3 was successfully constructed and 4 stable transfection cell lines were obtained. The ERGIC3 expressions were higher in transfected cells than in control cells （45.3, 48.8, 41.5 and 28.2 times） by real -time PCR and Western blot. The migration ability of the stably transfected cell line with ERGIC3 was significantly faster than the control cells （ P 〈 0.05）. Conclusion The stable cell lines with expression ERGIC3 have been successfully constructed and the migration ability of these cells increased significantly.
出处
《遵义医学院学报》
2014年第4期400-403,408,共5页
Journal of Zunyi Medical University
基金
贵州省科学技术基金项目(NO:黔科合J字[2013]2319)
遵义医学院博士启动基金项目(NO:F-655)
关键词
ERGIC3
稳定转染
真核表达
肺癌
ERGIC3
stable transfection
eukaryotic expression
lung cancer
