力生长因子在大肠杆菌中的表达及活性分析

Expression of Mechano-growth Factor in Escherichia coli and Activity Analysis

MGF(Mechano-growth factor)是一种IGF-1变体形式,研究发现该因子具有应力敏感性,并且具有促进肌肉肥大、再生以及神经损伤修复的功能。通过RT-PCR从拉伸刺激的人成骨细胞中克隆MGF cDNA序列,并去除5'端9bp的序列,使N端缺少对肠激酶(Enterokinase,EK)具有抑制作用的脯氨酸,将截短型MGF(des(1-3)MGF)cDNA序列克隆入pET32a(+)质粒,构建重组表达质粒。重组质粒转化E.coli BL21(DE3),在30oC培养下以可溶形式表达融合蛋白Trx/des(1-3)MGF,采用离子交换层析和Ni2+金属亲和层析,获得纯度95%以上的融合蛋白。再对融合蛋白EK酶切,rpHPLC分离获得纯度达98%的des(1-3)MGF,SDS-PAGE及质谱分析蛋白分子量与理论值相符。生物活性实验显示,所制备的des(1-3)MGF比des(1-3)IGF-1更显著的促进MC3T3-E1细胞的增值和迁移。

Mechano-growth factor （MGF） is one of IGF-1 isoforms. MGF is mechanosensitive and has important functions in muscle hypertrophy, regeneration and nerve injury recovery. In this study, MGF cDNA （330 bp） was cloned from stretched osteoblasts by RT-PCR. In order to avoid prolin residue inhibiting enterokinase cleavage, 9bp of MGF cDNA 5＇ end sequence was truncated by primer, then the obtained truncated MGF （des（1-3）MGF） cDNA （321 bp） was subcloned in pET32a（＋） vector to construct a prokaryotic recombination expression plasmid. Trx/des（1-3）MGF fusion protein, existing in forms of solution, was expressed in transformed Escherichia coli strain BL21（DE3） by IPTG induction at 30℃. The supernatant of cell lysates was subjected to ion exchange chromatography and Ni2＋ metal affinity chromatography, and the fusion protein was obtained with the purity over 95%. After the fusion protein was cleaved by enterokinase, Trx and des（1-3）MGF was isolated by reverse-phase HPLC. Through these procedures, des（l-3） MGF was obtained with the purity of 98%. The protein molecular mass was conformity to the theoretical value by SDS-PAGE and mass spectrometry analysis. The purified des（1-3）MGF was incubated with MC3T3-E1 for cell proliferation and migration assays. The results show that des（1-3）MGF exhibited more facilitative effects on proliferation and migration of MC3T3-E 1 than that of des（1-3）IGF- 1.