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

Abstract:

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.

Key words: PTD, GnRH, PE39KDEL, immunotoxin