Abstract:

Objective To construct human Oct3/4 eukaryotic expression vectors and transfect human ovarian cancer cell line SKOV3, and observe the expression of Oct3/4 in SKOV3 cells. Methods Destination gene (Oct3/4) cDNA products were obtained by amplification with RT-PCR. Human recombinant Oct3/4 eukaryotic expression vectors pcDNA3.1-Oct3/4 were constructed by pcDNA3.1 vector plasmid, and were identified by restriction enzyme digestion and DNA sequence analysis. Recombinant plasmid was transfected into SKOV3 cells with Lipofectamine 2000, and SKOV3 cells transfected with empty vectors and those without transfection were served as transfection control group and negative control group, respectively. Stable transfection cells were screened by G418 (neomycin), and Western blotting was employed to detect the relative expression of Oct3/4 protein in cells. Results Destination gene amplified by PCR was about 1 000 bp in length analysed by 12 g/L agarose gel electrophoresis, which met the expectancy. Human recombinant Oct3/4 eukaryotic expression plasmid vectors pcDNA3.1-Oct3/4 were constructed, and the result of DNA sequencing was consistent with that of GenBank. Western blotting analysis revealed that the relative expression of Oct3/4 protein in stable pcDNA3.1-Oct3/4 transfected cells (10.225±0.987) was significantly higher than that in transfection control group (1.713±0.896) and negative control group (1 for calibration value) (P<0.01). Conclusion Human recombinant Oct3/4 eukaryotic expression vectors were successfully constructed, SKOV3 was transfected, and Oct3/4 protein is stably overexpressed in stable transfected cells, which lays a foundation for the further study of roles of Oct3/4 gene in ovarian cancer development and drug-resistance to chemotherapy.

Key words: Oct3/4 gene, vector, protein expression, ovarian cancer