VEGF165-PE38重组免疫毒素真核表达载体的构建及表达  

Construction of eukaryotic expression vector of recombinant immunotoxin human VEGF165-PE38 and its expression

作　　者：胡昌辰[1] 柯以铨[1] 王斌全[2] 周丽媛[2] 吕俊[1] 张发兵[1] 卢建侃[1] 蔡颖谦[1] 秦玲莎[1] 

机构地区：[1]南方医科大学珠江医院神经外科,广州510282 [2]山西医科大学第一医院耳鼻咽喉-头颈外科

出　　处：《肿瘤研究与临床》2009年第4期222-225,共4页Cancer Research and Clinic

基　　金：基金项目：广东省自然科学基金（06024442）;广东省科技计划（20088030301152）

摘　　要：目的构建血管内皮细胞生长因子（VEGF）基因和铜绿假单胞菌外毒素衍生物（PE38）基因的真核融合表达载体，研究其在HEK293细胞中的表达情况。方法通过PCR获得实验需要的VEGF165基因片段，通过酶切pRB391质粒获得铜绿假单胞菌外毒素衍生物PE38基因，再通过适当的酶切及连接反应，构建VEGF165与PE38融合基因的真核表达载体pIRES2-VEGF165-PE38-EGFP，重组质粒经限制性内切酶及DNA序列测定证实构建成功后，用Lipofectamine2000脂质体转染HEK293细胞，然后用RT—PCR及ELISA法检测VEGF165-PE38融合蛋白在HEK293细胞的表达。结果酶切分析及DNA序列测定证实，VEGF165-PE38融合基因被成功克隆入真核表达质粒载体pIRES2-EGFP，RT—PCR及ELISA法检测证实重组基因可在HEK293细胞表达。结论VEGF165-PE38融合基因可在HEK293细胞中表达，为进-步研究其对肿瘤血管内皮细胞的靶向毒性及临床应用奠定基础。Objective To construct a new recombinant immunotoxin expression vector by using human VEGF165 and a truncated pseudomonas exotoxin A ramification （PE38） gene, and explore the expression of the VEGF165-PE38 fusion protein in HEK293 cells. Methods VEGF165 was cloned by polymerase chain reaction （PCR）. PE38 gene was gained from an vector plasmid pRB391 by restriction endonuclease digestion, and then inserted to the eukaryotic expression vector pIRES2-EGFP. After the eukaryotic recombinant vector plRES2-VEGF165-PE38-EGFP was identified by restriction endonuclease digestion and sequence analysis, the vector was transfected into HEK293 cells by liposome protocol. RT-PCR and ELISA method was used to confirm the expression of the fusion gene in the HEK293 cells. Results Restriction endonuclease digestion and sequence analysis revealed the VEGF165-PE38 fusion gene was cloned into the eukaryotic expression plasmid vector pIRES2-EGFP successfully. The pIRES2-VEGF165- PE38-EGFP fusion gene could express in the HEK293 cells. Conclusion The result provide the basis for search of the targeted cytotoxic activity to tumor vascular endothelial cells and may have some potential value in clinical application.

关 键 词：血管内皮生长因子 PE38 重组 免疫毒素 HEK293细胞 真核表达 

分 类 号：R73-3[医药卫生—肿瘤]