构建及鉴定人生存素基因的慢病毒表达载体（英文）  

Construction and identification of lentiviral vector encoding human survivin gene

作　　者：赵亮[1] 张国庆[1] 马学晓[1] 杨堃[2] 胡有谷[1] 陈伯华[1] 

机构地区：[1]青岛大学医学院附属医院脊柱外科,山东省青岛市266003 [2]青岛大学医学院附属医院中心实验室,山东省青岛市266003

出　　处：《中国组织工程研究》2014年第11期1755-1760,共6页Chinese Journal of Tissue Engineering Research

基　　金：the National Natural Science Foundation of China,No.81171758~~

摘　　要：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：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 cells.

关 键 词：组织构建 组织工程 生存素 慢病毒 髓核细胞 细胞凋亡 基因转染 国家自然科学基金 

分 类 号：R318[医药卫生—生物医学工程]