Construction of survivin short hairpin RNA eukaryotic expression vector and its influence on the survivin expression in human cervical cancer cells / 医学研究生学报

ABSTRACT

Objective:To construct a short hairpin RNA(shRNA) eukaryotic expression vector of the survivin gene,and investigate its inhibitory effect on the survivin expression in human cervical cancer cells.Methods:We designed and synthesized 2 pairs of survivin-specific small interfering RNA primers(s1 and s2),cloned them into the eukaryotic expression vector pSilencer 2.1-U6 neo by DNA recombined techniques after annealing connection reaction,and transfected them respectively into human cervical cancer cell line HeLa using LipofectAMINE2000 after identificationby restrict endonuclease digestion and DNA sequencing.Then we selected the positive clones by G418 and detected the expression of survivin mRNA by semi-quantitative RT-PCR and that of the survivin protein by Western blot.Results:The survivin shRNA eukaryotic expression vectors pSilencer2.1-s1 and pSilencer2.1-s2 were successfully constructed.Positive clones were obtained by screening with G418 for 24 days,the survivin expression in HeLa cells decreased in different degrees after transfected with pSilencer2.1-s1 and pSilencer2.1-s2,and the latter showed a better interference efficiency.Conclusion:The shRNA eukaryotic expression vector of the survivin gene we constructed,with its improved interfering efficiency,has paved the way for further research on its role in regulating the biological behavior of cervical cancer cells.