CAJ | 학술논문

为在蛋白水平认识半滑舌鳎类胰岛素生长因子Ⅱ(IGF-Ⅱ)的生理功能，将 IGF-Ⅱ成熟肽序列克隆到原核表达载体 pET-28a 中，成功构建了重组半滑舌鳎 IGF-Ⅱ/pET28a 质粒，导入到 E. coli BL21(DE3)菌株后经 IPTG 诱导，获得了大小为11.4 kDa 的重组 IGF-Ⅱ蛋白，N 端含6个组氨酸，可特异性地被6×His 抗体识别。重组 IGF-Ⅱ蛋白在最优诱导条件37℃诱导2 h，目的蛋白表达量占重组表达菌总蛋白的43.7%，重组蛋白主要以包涵体存在。将获得的重组蛋白包涵体经变性、纯化和复性后，获得了纯化的 IGF-Ⅱ重组蛋白，其可在体外显著促进人乳腺癌 MDA231细胞的增殖，表明 IGF-Ⅱ重组蛋白具有体外细胞水平的生物活性。本研究结果可为认识鱼类 IGF-Ⅱ生理功能及半滑舌鳎生长调控机制提供理论支撑。
위재단백수평인식반활설탑류이도소생장인자Ⅱ(IGF-Ⅱ)적생리공능，장 IGF-Ⅱ성숙태서렬극륭도원핵표체재체 pET-28a 중，성공구건료중조반활설탑 IGF-Ⅱ/pET28a 질립，도입도 E. coli BL21(DE3)균주후경 IPTG 유도，획득료대소위11.4 kDa 적중조 IGF-Ⅱ단백，N 단함6개조안산，가특이성지피6×His 항체식별。중조 IGF-Ⅱ단백재최우유도조건37℃유도2 h，목적단백표체량점중조표체균총단백적43.7%，중조단백주요이포함체존재。장획득적중조단백포함체경변성、순화화복성후，획득료순화적 IGF-Ⅱ중조단백，기가재체외현저촉진인유선암 MDA231세포적증식，표명 IGF-Ⅱ중조단백구유체외세포수평적생물활성。본연구결과가위인식어류 IGF-Ⅱ생리공능급반활설탑생장조공궤제제공이론지탱。
To explore the role of insulin-like factor Ⅱ(IGF-Ⅱ) in growth regulation of Cynoglossus semilaevis Günther, the IGF-Ⅱ gene was expressed in vitro and the bioactivity was determined by methyl thiazolyl tetrazolium (MTT method). The mature peptide domain of IGF-Ⅱ gene of C. semilaevis Günther was cloned by PCR amplification and sequenced for verification. The obtained mature peptide fragment was then subcloned into the prokaryotic expression vector pET-28a (IGF-Ⅱ/pET28a). The recombinant plasmid was expressed in E.coli BL21 (DE3) cells and the recombinant IGF-Ⅱ protein containing 6×His tag at N-terminus was induced by IPTG. SDS-PAGE analysis indicated that the obtained IGF-Ⅱ protein was found in the form of inclusion bodies with molecular weight of 11.4 kDa, which accounted for 43.7% of the whole bacterial protein post 2-hour induction with IPTG. Western blotting analysis indicated that the recombinant IGF-Ⅱ protein had the antigenicity to 6×His antibody. The inclusion bodies containing recombinant protein were denaturalized, purified and annealed. The recombinant IGF-Ⅱ protein significantly promoted the proliferation of human breast cancer cells MDA231. Results could provide basic information on the role of IGF-Ⅱ in fish and be helpful to better understand the endocrine mechanism of sex-based dimorphic growth performance of C. semilaevis Günther.