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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 construct the co-expression vector pGL3 / EGR1-Bax of Bcl-2 gene and the promoter of early growth-responsive gene 1 (EGR1) and transfect it into non-small cell lung cancer cells. Methods The full-length coding sequence of EGR1 and Bax gene was amplified by PCR from genomic DNA of mouse brain and cloned into pTA2 vector respectively. The recombinant plasmids pTA2 / EGR1 and pTA2 / Bax were constructed and sequenced. Bax gene was subcloned into pGL3-Basic vector to construct a double gene co-expression vector pGL3 / EGR1-Bax, which was sequenced and identified. NCI-H460 cells were transfected with NCI-H460 cells transfected with untransfected and empty pGL3-Basic vector as control group and negative control group by RT-PCR Western blotting was used to detect the expression of EGR1 and Bax mRNA and protein in NCI-H460 cells. Results Sequencing and identification confirmed that the constructed co-expression vector pGL3 / EGR1-Bax of EGR1, Bax sequence and GenBank published gene sequence exactly the same. Compared with NCI-H460 cells in NCI-H460 cells transfected with pGL3 / EGR1-Bax, the expression of EGR1 and Bax and the expression of Bax in pGL3 / EGR1-Bax transfected NCI-H460 cells were significantly increased. Conclusion The double gene coexpression vector pGL3 / EGR1-Bax was successfully constructed and the expression of EGR1 and Bax gene in NCI-H460 cells was achieved.