Construction Of Survivin SiRNA Expression Vector And Its Effects On The Transfected Glioma Cells

Background:Survivin specifically overexpressed in tumor tissues, especially in glioma.Many reports have shown that the inhibition of its function was useful for tumor therapy. RNA interference is a new technique proved to be effective for suppressing gene expression. It has become a hot spot in the tumor gene therapy field at present.Objective:To construct a siRNA expression plasmid transcripted by RNA interference, using gene survivin as a clone target. To identify the recombinant plasmid nucleic acid sequence by sequencing on the standard sequencer, and to evaluate the plasmid impact to the survivin gene expression in transfected glioma. These results lay a foundation for the further research on the function of survivin in the development and therapy of glioma.Methods:1. Two DNA sequences with small hairpin structure were designed and synthesized. The complement form was obtained by annealing and cloned into vector pSilencer 3.1-H1-hygro. Recombinant plasmid was transformed into strain DH5α, identified by using bacterial colonies PCR, restriction enzyme and sequencing of nucleic acid.2. The siRNA expression plasmid was transfected into U87 cells by lipofectamine. The expression of the survivin gene mRNA and protein on the transfected cells was measured by RT-PCR and western blot.Results:1. The strain DH5αtransformed by recombinant plasmid could clony grow by ampicillin-resistant screen.2. There was obvious objective band in recombinant plasmid by bacterial colonies PCR.3. The recombinant plasmid could be cut by BglⅡ.