小分子GnRH-luffinS重组嵌合毒素的制备及其细胞毒性检测

Preparation and cytotoxic activity assay of micromolecule recombinant chimeric toxin GnRH-luffinS

目的:制备以小分子丝瓜素(luffin)S2为毒性部分,以Ⅰ型促性腺激素释放激素(GnRH)为导向部分的重组嵌合毒素GnRH-luffinS,体外检测其对肿瘤细胞的杀伤作用。方法:重叠PCR方法扩增GnRH-luffinS的基因,克隆至表达载体pET32a中,转化大肠杆菌BL21(DE3),挑取阳性克隆诱导表达,产物用Ni-NTA亲和层析柱纯化。纯化蛋白经重组肠激酶(rEK)切割去除Trx融合蛋白,XTT法检测重组毒素对HeLa,A549,HepG-2,SP2/0和鸡胚成纤维细胞(CEF)的体外细胞毒性。结果:成功构建了GnRH-luffinS的表达质粒,并在大肠杆菌中获得表达,纯化后的纯度为93%。GnRH-luffinS对HeLa,A549,HepG-2和SP2/0的IC50分别为67.19,70.42,84.44和106.25μg/ml,而对CEF无作用。结论:重组毒素GnRH-luffinS对肿瘤细胞有一定的杀伤作用。

Objectives： To prepare micromolecule recombinant chimeric toxins containing luffinS2 and gonadotropin releasing hormone type Ⅰ （GnRH）, in which luffinS is toxic moiety and GnRH is target moiety, and to analyze their lethal effect on some tumour cells in vitro. Methods ： The gene of GnRH-luffinS was amplified by overlapping PCR technique and cloned into pET32a vector, and then the recombinant plasmid was transformed into E. coli BL21 （DE3）. Positive clones were induced to express recombinant proteins which were purified by Ni-NTA affinity column. Purified protein was cleaved to remove the Trx fusion protein by rEK and then its cytotoxicity to HeLa, A549, HepG-2, sp2/0 and CEF was analyzed by XTT method. Results： The recombinant expression plasmid was constructed and was expressed in E. coli and purified with purity of 93%. The IC50 of GnRH-luffinS for HeLa, A549, HepG-2 and SP2/0 was 67. 19 μg/ml, 70.42 μg/ml, 84.44 μg/ml and 106.25 μg/ml,respectively,and the recombinant protein had no toxic effect on CEF. Conclusion： GnRH-luffinS has cytotoxicity to cancer cell lines.