论文部分内容阅读
背景:抑制椎间盘细胞的凋亡可以延缓椎间盘的退变,而生存素具有调节细胞增殖和抗凋亡功能。目的:构建人生存素基因的慢病毒载体。方法:应用全基因合成技术合成人生存素基因(BIRC5),通过PCR扩增目的基因并对PCR结果进行电泳分析。将目的基因克隆到慢病毒表达质粒构建重组慢病毒质粒Lenti-BIRC5。将重组的慢病毒质粒转化细菌感受态细胞,PCR鉴定阳性的克隆进行基因测序。将含有目的基因的慢病毒质粒转染293T细胞,应用Western blot技术对重组慢病毒载体Flag-Survivin融合蛋白的表达进行检测。结果与结论:PCR鉴定及基因测序结果显示成功构建了含有人Survivin基因的慢病毒表达载体,Western blot检测结果显示目的基因在体外培养细胞中转染成功并且过表达。说明慢病毒表达载体Lenti-BIRC5构建成功,这为下一步研究Survivin在人髓核细胞中的抗凋亡作用提供了载体。
BACKGROUND: Inhibition of apoptosis in disc cells delays the degeneration of discs, whereas survivin regulates cell proliferation and anti-apoptotic functions. Objective: To construct a lentiviral vector for human survivin gene. Methods: Human survivin gene (BIRC5) was synthesized by whole genome synthesis. The target gene was amplified by PCR and the PCR results were analyzed by electrophoresis. The target gene was cloned into lentiviral expression plasmid to construct recombinant lentiviral plasmid Lenti-BIRC5. Recombinant lentiviral plasmids were transformed into bacterial competent cells, and positive clones were identified by PCR for gene sequencing. The lentiviral plasmid containing the target gene was transfected into 293T cells, and the expression of recombinant lentiviral vector Flag-Survivin fusion protein was detected by Western blot. RESULTS AND CONCLUSION: The results of PCR and gene sequencing showed that the lentiviral vector containing human Survivin gene was successfully constructed. The result of Western blot showed that the target gene was successfully transfected and overexpressed in cultured cells. This indicated that the Lenti-BIRC5 lentiviral vector was successfully constructed, which provided the carrier for the further study on the anti-apoptotic effect of Survivin in human nucleus pulposus cells.