重组免疫毒素GnRH-PTD-PE39KDEL的构建及表达  

Construction and expression of a recombinant immunotoxin GnRH-PTD-PE39KDEL

作　　者：扈进冬[1] 魏艳丽[1] 杨合同[1,2] 李纪顺[1] 张广志[1] 

机构地区：[1]山东省科学院生物技术研究中心,山东省应用微生物重点实验室,山东济南250014 [2]山东理工大学生命科学学院,山东淄博255049

出　　处：《山东科学》2013年第3期5-10,14,共7页Shandong Science

基　　金：国家"重大新药创制"科技重大专项(2009ZX09102-214)

摘　　要：通过引入蛋白质转导域(PTD),提高免疫毒素人促黄体激素释放激素-绿脓杆菌外毒素A衍生物(GnRH-PE39KDEL)对肿瘤细胞的杀伤活性。设计了3条引物,通过2轮PCR在GnRH-PE39KDEL基因的人促黄体激素释放激素C端和绿脓杆菌外毒素A衍生物N端引入PTD,获得GnRH-PTD-PE39KDEL基因经酶切后插入pET-His载体,并转化至BL21(DE3)中。采用镍离子螯合层析法纯化诱导表达样品,并进行了生物活性测定。成功构建了免疫毒素表达载体pET-His-GnRH-PTD-PE39KDEL;诱导产物可以实现可溶性表达;表达产物占菌体总蛋白的20%,靶向融合蛋白引入PTD后对人乳腺癌MCF-7细胞的IC50为0.860μg/mL,表明较GnRH-PE39KDEL生物活性增强。成功地表达了融合蛋白GnRH-PTD-PE39KDEL,为其进一步大规模表达、纯化和功能研究奠定了基础。We improve killing activity to tumor cells of recombinant immunotoxin composed by a human gonadotropin-releasing hormone gene and Pseudomonas exotoxin a derivative（GnRH-PE39KDEL） by introducing the protein transduction domain（PTD）.We design three primers,and then obtain the gene GnRH-PTD-PE39KDEL by two rounds PCR and PTD induced at the C end of gonadotropin-releasing hormone and the N end of Pseudomonas exotoxin a derivative.The gene is through endonucleases digestion,inserted into a pET-His carrier and then transformed into E.coli BL21（DE3）.We also purify resolvable protein by Ni-NTA,and determine biological activity.We successfully construct the expression carrier pET-His-GnRH-PTD-PE39KDEL.Fusion protein has good solubility.The quantity of expressed fusion protein is 20% of total bacterial proteins.IC50 of human breast cancer cell MCF-7 is 0.860 μg/mL.It indicates that GnRH-PTD-PE39KDEL has stronger biological activity than GnRH-PE39KDEL.The GnRH-PTD-PE39KDEL is successfully expressed.It will lay the foundation for its further large-scale expression,purification and functionality research.

关 键 词：蛋白质转导域 人促黄体激素释放激素 绿脓杆菌外毒素A衍生物 免疫毒素 

分 类 号：Q78[生物学—分子生物学] R73[医药卫生—肿瘤]