pGL3/EGR1-Bax重组载体的构建及对非小细胞肺癌细胞的转染

Construction of recombinant vector pGL3/EGR1-Bax and its transfection into cells of non-small-cell lung carcinoma

目的构建早期生长反应基因1(EGR1)启动子与Bax基因共表达载体pGL3/EGR1-Bax并转染非小细胞肺癌细胞。方法以小鼠脑组织基因组DNA为模板,采用PCR法分别扩增EGR1和Bax基因全长编码序列,分别克隆入载体pTA2,构建重组质粒pTA2/EGR1和pTA2/Bax,测序鉴定;再将EGR1与Bax基因亚克隆至pGL3-Basic载体中,构建双基因共表达载体pGL3/EGR1-Bax,测序鉴定。采用基因转染技术将基因导入非小细胞肺癌NCI-H460细胞,分别以未转染及空pGL3-Basic载体转染的NCI-H460细胞株作为空白对照组和阴性对照组,采用RT-PCR技术和Western blotting方法检测NCI-H460细胞EGR1和BaxmRNA和蛋白的表达。结果测序鉴定证实,构建的双基因共表达载体pGL3/EGR1-Bax的EGR1、Bax序列与GenBank发布基因序列完全一致。与对照组和空载体转染组NCI-H460细胞比较,pGL3/EGR1-Bax转染组NCI-H460细胞中EGR1和Bax的mRNA表达以及Bax的蛋白表达明显增强。结论成功构建双基因共表达载体pGL3/EGR1-Bax,并实现了EGR1和Bax基因在NCI-H460细胞中的有效表达。

Objective To clone early growth response-1 （EGR1） gene and BCL2-associated X protein （Bax） gene, construct their co-expression vector pGL3/EGR1-Bax, and transfect it into cells of non-small-cell lung carcinoma. Methods Full-length coded sequence of EGR1 and Bax genes was amplified by PCR with the genome DNA isolated from mouse brain tissue as template respectively. PCR amplified fragment of EGR1 and Bax genes were first cloned into pTA2 vector, and then subcloned into pGL3-Basic vector to construct pGL3/EGR1-Bax recombinant vector after sequencing. EGR1 and Bax genes were transfected into non-small-cell lung carcinoma NCI-H460 cells with the technology of gene transfection. NCI-H460 cells without transfection and those transfected with blank pGL3-Basic were served as blank control group and negative control group respectively. RT-PCR and Western blotting were employed to detect the expression of EGR1 and Bax mRNA and protein in NCI-H460 cells. Results It was identified that the sequence of EGR1 and Bax in the constructed pGL3/EGR1-Bax was in line with that of the GenBank. The expression of EGR1 mRNA and Bax mRNA and protein in NCI-H460 cells in pGL3/ EGR1-Bax transfection group was significantly higher than that in blank control group and negative control group. Conclusion Co-expression vector pGL3/EGR1-Bax has been successfully constructed, and the efficient expression of EGR1 and Bax genes in NCI-H460 cells has been realized.