HproTα／hIL-2融合蛋白真核载体的构建及鉴定  

Construction and Identification of Eukaryotic Expression Vector of Human Prothymosin α and Interleukin-2

作　　者：周飞燕[1] 黄明 韦剑 丘力功 卢柳燕[1] 孙希海 

机构地区：[1]广西师范大学生命科学学院,广西桂林541004 [2]广州拜迪生物医药有限公司,广东广州511495

出　　处：《广西师范大学学报（自然科学版）》2008年第2期76-80,共5页Journal of Guangxi Normal University:Natural Science Edition

基　　金：广东省自然科学基金资助项目(04006313)

摘　　要：利用基因重组技术构建了含信号肽的人源胸腺素α原和白介素2(proTα/IL-2)融合基因的真核表达载体pVAX-PI。用脂质体转染试剂转染COS-7细胞,转染后24、48、72、96 h采用ELISA法检测细胞培养上清中IL-2的表达情况,分别用CTLL-2依赖细胞株/MTT法和脾细胞增殖实验测定融合蛋白IL-2和proTα活性,采用ECL western blotting分析和鉴定目的蛋白的相对分子质量大小。结果表明,转染上清在31 kDa处有特异性阳性反应条带,质粒在转染COS-7细胞后不同时间均可进行分泌表达,且于转染后48 h目的蛋白的表达量达峰值。细胞转染上清中的IL-2活性可达58 IU/mL,proTα具有促进小鼠脾脏细胞增殖的作用,可作为基因治疗药物的开发之用。The eukaryotic expression vector pVAX-PI with the full length of human prothymosin a and interleukin2 gene including GM-CSF signal sequence was constructed by DNA recombinant technique and its functional activity was detected in vitro. After identification by restriction analysis and DNA sequencing,the recombinant plasmid was transfected into COS-7 cells in vitro with transfection reagent Lipofectamine. IL-2 fusion protein was analyzed by ELISA at 24 h,48 h,72 h and 96 h,and the biological activities of IL-2 and proTα were detected by CTLL-2 cell line proliferation and splenocytes proliferation respectively. The expect molecular weight of proTα and IL-2 fusion protein was 31 kDa by ECL western blott assay. The results indicated that the recombinant plasmid could express in eukaryotic cell at anytime after COS-7 transfection and expressed fusion protein reached the peak after 48 h transfection. The IL-2 activities in the culture, supernatant of COS-7 cells transfected with pVAX-PI were 58 IU/mL and proTα obviously stimulated the proliferation of splenocytes,which can be laid a good foundation to further use for gene therapy.

关 键 词：胸腺素Α原 白介素如融合蛋白 转染 

分 类 号：R979.1[医药卫生—药品]