Abstract:

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.

Key words: early growth response gene 1, Bax gene, clone, transfection, gene expression