重组免疫毒素GnRH-PTD-PE39KDEL的构建及表达

摘要

We improve killing activity to tumor cells of recombinant immunotoxin composed by a human gonadotropinreleasing 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 GnRHPTD-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.%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.