BMI-1干扰基因重组腺病毒质粒的构建及鉴定

Construction and identification of recombinant adenoviruses carrying small interference RNA of BMI-1 gene

背景:BMI-1的表达对于维持干细胞特别是造血干细胞、神经干细胞、肿瘤干细胞的自我更新能力是不可缺少的。目的:以细菌内同源重组法构建携带BMI-1干扰基因的重组腺病毒载体,制备重组腺病毒pShuttle-H1-AdEasy-1-P1P2/P3P4。设计、时间及地点:开放性实验,于2006-02/2007-08在江苏大学医学技术研究所完成。材料:限制性内切酶由NewEngland BioLabs提供,腺病毒骨架质粒pAdEasy-1、大肠杆菌BJ5183、Ad293细胞由南京师范大学生科院分子医学生物技术江苏省重点实验室惠赠。方法:采用细菌内同源重组法构建RNA干扰腺病毒载体pShuttle-H1,PmeⅠ线性化质粒,通过同源重组腺病毒骨架蛋白pAdeasy-1,得到重组的腺病毒干扰质粒;经PacⅠ线性化后用脂质体转染方法转染293A细胞,收获腺病毒重组病毒子,以未转入小干扰RNA片段的重组质粒为对照,包装后收集细胞,反复裂解细胞获得腺病毒。主要观察指标:腺病毒质粒重组构建及鉴定情况。结果:经酶切鉴定,已成功构建siRNA的重组腺病毒表达载体pShuttle-H1-AdEasy-1-P1P2/P3P4,获得了重组病毒子。结论:利用新型腺病毒载体pAdeasy-1系统可在短期内制备BMI-1干扰基因的腺病毒表达载体,并制备出重组腺病毒P1P2/P3P4。

BACKGROUND： Expression of BMI-1 gene is necessary to maintain the self-renewal ability of stem cells, especially haemopoietic stem cells, nerve stem cells and tumor stem cells. OBJECTIVE： To construct the recombinant adenoviruses carrying small interference RNA of BMI-1 gene using homologous recombination, and to prepare the expression vector of pShuttle-H1-AdEasy-1-P1P2/P3P4. DESIGN, TIME AND SETTING： An open study was carried out in the School of Medical Technology, Jiangsu University （Zhenjiang, Jiangsu, China） between February 2006 and August 2007. MATERIALS： Restriction enzyme was offered by NewEngland BioLabs. Adenovirus backbone vector pAdEasy-1, E. coli B J5183 and Ad 293 cells were provided by Jiangsu Provincial Key Laboratory for Molecular Medicine and Biotechnology in Biochemistry College of Nanjing Normal University （China）. METHODS： The synthesized oligos aiming at BMI-1 gene by chemical methods in vitro were inserted into the RNA interference adenoviral vector of pShuttle-H1, then the linearized shuttle plasmids by Pine I were co-transformed into bacteria containing pAdEasy-1 backbone vector for homologous recombination and obtaining the recombinant adenoviral plasmid pShuttle-H1-AdEasy-1-P1P2/P3P4. The recombined adenovirus DNA was linearized by Pac I and transfected into 293A cells by Lipofectamine. The packaged cells were collected after the package and lyzed for adenovirus. MAIN OUTCOME MEASURES： Construction and identification of recombinant adenovirus plasmid. RESULTS：The recombinant plasmid pShuttle-H1-AdEasy-1-P1P2/P3P4 carrying small interference RNA was successfully constructed and confirmed by restriction endonuclease digestion, and the recombinant virus could be observed after the package of linearized plasmids. CONCLUSION： The recombinant adenovirus vector of BMI-1 gene can be generated in a short time by using the AdEasy system, and recombinant adenovirus P1P2/P3P4 is also constructed.