中国生物工程杂志

Objective: To prepare recombinant chimeric toxins containing luffinS2, Ⅰ type of GnRH and domain Ⅱ of Pseudomonas exotin, in which luffinS2 is toxic moiety, GnRH is target moiety and PEⅡ is translocation moiety, and analyze its lethal effect to some tumour cells in vitro. Methods: The gene of GnRH-PEⅡ-luffinS was amplified by overlapping PCR techniques and cloned into pET32a vector, and then the recombinant plasmid was transformed into E. coli BL21(DE3). Positive strains were induced and expressed recombinant proteins were purified by Ni-NTA affinity column. Purified protein which was cleaved to remove the Trx fusion protein by rEK and then its cytotoxicity to Hela, A549, HepG-2, SP2/0 and CEF were analyzed by XTT method. Results: We constructed the recombinant expression plasmid which succeeded to express in E. coli and be purified with purity of 94％. The IC50 of GnRH-PEⅡ-luffinS were 13.50μg/ml, 13.74μg/ml, 16.79μg/ml and 26.07μg/ml respectively and the recombinant protein had shown no toxic effect to CEF. Conclusion: GnRH-PEⅡ-luffinS have cytotoxicity to cancer cell lines.