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

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.