基于T7RNA聚合酶／启动子的细胞融合报导系统载体的构建  

Construction of reporting system based on T7 RNA polymerase and T7 promoter in cell-cell fusion

作　　者：刘利东[1] 朱蔚云[2] 黄金水[3] 李冰[4] 

机构地区：[1]广州医学院第一附属医院检验科,广东广州510120 [2]广州医学院遗传学教研室,广东广州510182 [3]中山大学附属第二医院输血科,广东广州510120 [4]广州医学院医学实验中心,广东广州510182

出　　处：《广州医学院学报》2010年第2期20-23,共4页Academic Journal of Guangzhou Medical College

基　　金：国家自然科学基金主任基金分题（No.30340029）.

摘　　要：目的：建立T7RNA聚合酶／T7启动子载体系统，作为观察细胞融合是否成功的报导系统。方法：设计并合成引物从BL21细菌中扩增T7RNA聚合酶序列，并将其克隆进入真核表达载体pRc／CMV2中，作为RNA聚合酶的供体。T7RNA聚合酶特异性识别的r17启动子人工合成，构建于荧光素酶的表达载体pGL3中，利用其荧光素酶基因作为报导基因。两质粒共转染A549细胞和VeroE6细胞，检测荧光素酶的表达。结果：共转染pRc／CMV2。T7RNAP和pGL3-T7的A549细胞和VeroE6细胞的荧光强度分别为57745±7747和43679±8616，转染质粒pGL3-T7的细胞空白对照荧光强度1034±201和1091±157，分别与空白对照组比较差异有统计学意义（P〈0．01）。结论：成功构建了T7RNA聚合酶/T7启动子载体的细胞融合报导系统，可应用于细胞融合的相关实验研究中。Objective：To construct a system including T7 RNA polymerase and T7 promoter as a reporting system in cell-cell fusion. Methods： The gene of T7 RNA polymerase was amplified from BL21 bacteria with the specific primers and recombined into the vector pRc/CMV2 as the donor of RNA polymerase. T7 promoter was synthesized and recombined into the vector pGL-3. The luciferase gene in the vector pGL-3 was used as a reporting gene. The two vectors were co-transfected into A549 cell and Vero E6 cell and the level of luciferase were detected. Results：The light intensity of A549 cell and Veto E6 cotransfected pRc/CMV2-T7RNAP and pGL-T7 was 57 745 ±7 747 and 43 679 ± 8 616 respectively,compared with 1 034 ±201 and 1 091 -± 157 of the control cell. There was a significant difference between test and control groups （P 〈 0.01 ）. Conclusion：The system of T7 RNA polymerase and T7 promoter was successfully constructed, can be used in many researches about cell-cell fusion.

关 键 词：细胞融合 TTRNA聚合酶 T7启动子 荧光素酶 报告系统 

分 类 号：Q813[生物学—生物工程]