Construction and identification of lentiviral vector encoding human survivin gene / 中国组织工程研究

ABSTRACT

BACKGROUND:Inhibiting the apoptosis of intervertebral disc cel s can postpone the degenerative process of intervertebral disc. Survivin has a strong function of regulating cel proliferation and anti-apoptosis. OBJECTIVE:To construct and identify the lentiviral vector encoding survivin gene of human. METHODS:The survivin gene of human (BIRC5) was synthesized through the gene synthesis technology, amplified by PCR and analyzed by electrophoresis. The target gene was cloned into lentiviral expression plasmid to obtain the recombinant lentiviral vector Lenti-BIRC5. After transformation into competent E. coli cel s, the candidate clones were identified by PCR firstly. The positive clones were identified by gene sequencing. The lentivirus plasmid containing target gene was transfected into 293T cel s, and the expression of recombinant lentiviral vector Flag-Survivin fusion protein was detected through western blot analysis. RESULTS AND CONCLUSION:The PCR results of electrophoresis and DNA sequencing showed that lentiviral vector containing human survivin gene was constructed successful y. Western blot analysis results showed that the target gene was transfected successful y and over-expressed in cultured cel s. The lentiviral expression vector of human survivin gene Lenti-BIRC5 was constructed successful y, which lays a foundation for the study addressing the anti-apoptotic effects of survivin on human nucleus pulposus cel s.