梅花鹿IGF-1的原核表达与纯化

Expression and purification of IGF-1 from Cervus nippon

【目的】克隆梅花鹿(Cervus nippon)胰岛素样生长因子1(Insulin-like growth factor 1,IGF-1)的成熟肽基因,并对其进行原核表达及纯化,以获得具有生物学活性的蛋白,为研究IGF-1在梅花鹿鹿茸生长发育中的调控作用及机理提供参考。【方法】以GenBank中的梅花鹿IGF-1基因序列(登录号:HQ890468)设计1对引物,以构建的梅花鹿pMD-18T-IGF-1重组质粒为模板,克隆IGF-1成熟肽的基因序列,构建重组质粒pMD-IGF-1,将其插入pET-32a表达载体后,构建pET-32a-IGF-1质粒,鉴定后,将pET-32a-IGF-1转入Rosetta大肠杆菌进行诱导表达(0.6mmol/L IPTG,37℃,4h)后,对其表达产物进行SDS-PAGE和Western blotting检测。用Ni-Agarose柱亲和层析试剂盒分离、羟胺裂解纯化目标蛋白,并利用四唑盐比色法(MTT法)和流式细胞仪检测目标蛋白对NIH3T3细胞增殖及不同细胞周期下NIH3T3细胞比例的影响。【结果】获得了梅花鹿IGF-1成熟肽基因序列(234bp);经鉴定,原核表达载体pET-32a-IGF-1构建成功;SDS-PAGE和Western blotting检测结果表明,在Rosetta中成功诱导表达了融合蛋白,且羟胺裂解纯化后的目的蛋白纯度较高;MTT法和细胞周期检测结果表明,复性后的IGF-1重组蛋白能促进细胞增殖。【结论】成功克隆了梅花鹿IGF-1成熟肽基因序列,获得了具有生物学活性的IGF-1蛋白。

【Objective】 The research was conducted to obtain the biologic activity protein which was renaturated after cloning and expression IGF-1 mature peptide gene from Cervus nippon and provided reference to study IGF-1 regulation effect and mechanism in Cervus nippon.【Method】 A pair of primers was designed by Cervus nippon IGF-1 gene sequence from GenBank（Accession number：HQ890468）.The IGF-1 mature peptide sequence was amplified from pMD-18T-IGF-1 and recombinant plasmid pMD-IGF-1 was gained.And the production was inserted to expression vector pET-32a to obtain recombinant plasmid pET-32a-IGF-1 and expressed（0.6 mmol/L IPTG for 4 h at 37 ℃） in the host Rosetta cells.The results were analysed by SDS-PAGE and Western blotting.Target protein was purified by Ni-Agarose affinity column and cut by hydroxylamine.Target protein on the proliferation and cell cycle of NIH3T3 cell were analysed by MTT and FCM.【Result】 The Cervus nippon IGF-1 mature peptide gene was 234 bp.Restriction enzyme mapping and sequencing showed that pET-32a-IGF-1 expression vector was constructed successfully.SDS-PAGE and Western blotting showed that fusion protein was induced in Rosetta host cells and the purity of interest protein was very high.The analysis of MTT and cell cycle showed that the growth rate of NIH3T3 cell increased significantly with the renaturated recombinant protein,and the cell population in S phases increased significantly too.【Conclusion】 IGF-1 mature peptide gene was cloned and expressed,and biologic activity protein of IGF-1 was obtained in this study.