重组人促性腺激素-铜绿假单胞菌外毒素A蛋白的可溶性表达、纯化和对肿瘤细胞的抑制活性  

Soluble expression and purification of GnRH-PE39KDEL and the inhibition to the activity of a tumor cell

作　　者：郭凯[1] 赵晓燕[1] 周红姿[1] 陈凯[1] 李纪顺[1] 陈泉[1] 杨合同[1] 

机构地区：[1]山东省科学院中日友好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

出　　处：《山东科学》2013年第2期41-47,共7页Shandong Science

基　　金：国家"重大新药创制"科技重大专项(2009ZX09102-214);山东省科技发展计划(2012GSF12114);山东省科学院博士基金(201207-18)

摘　　要：以人促性腺激素-铜绿假单胞菌外毒素A衍生物(GnRH-PE39KDEL)为研究对象,根据大肠杆菌密码子偏好性优化,通过基因合成的方法获得重组蛋白核酸序列,连接至pET-24b载体中,并转化入大肠杆菌BL21(DE3)及BL21(DE3)plyS中进行诱导表达。结果显示,在含有2 mg/mL葡萄糖的LB培养基中37℃培养至OD600约为0.6 h,加入0.2 mmol/L IPTG在30℃诱导2 h后,重组蛋白可以实现可溶性高表达。工程菌经超声波破碎、高速离心后,经镍柱纯化和脱盐后得到重组蛋白,蛋白纯度达到95%以上,蛋白最终得率在2.6 mg/g菌体。重组蛋白经IC50检测,可以较好地抑制肿瘤细胞株的生长。We obtained full-length nucleic acid sequence of an recombinant immunotoxin of GnRH and PE39KDEL by gene synthesis based on the E. coil codon bias. Factor Xa restriction site was inserted at the C termanol of GnRH-PE39KDEL to obtion pET-24b-GnRH-PE39KDEL recombinated plasmid which was then converted into E. coil BL21 （DE3） and BL21 （DE3） plyS. Results show that recombinant immunotoxin exhibits high-level soluble expression .after 0.2 mmol/L IPTG induction at 30 %3 for 2 h in LB media with 0.2 % glucose till OD600 is acquired at 37 ℃ for about 0. 6 h. The purity of fusion protein is above 95% and the protein yield is about 2.6 mg/g after sonication, high-speed centrifugation, HisTrap column purification and desalt. IC50 detection indicates that the recombinant protein can significantly inhibit the growth of tumor celt strains.

关 键 词：人促黄体激素释放激素 绿脓杆菌外毒素A 可溶性表达 表达菌株 

分 类 号：Q753[生物学—分子生物学]