骨形态发生蛋白10在中国仓鼠卵巢细胞中的表达、纯化及其生物活性

Expression of bone morphogenetic protein 10 in Chinese hamster ovary cells and purification and biological activity of expressed product

目的在中国仓鼠卵巢细胞(Chinese hamster ovary,CHO)中表达改造后的骨形态发生蛋白10(bone morphogenetic protein 10,BMP10)全长基因,纯化后检测其生物活性,以期获得具有生物活性的BMP10分子。方法于rh BMP10的propeptide与mature chain基因片段之间插入6×his-tag标签及DDDDK(肠激酶识别位点),构建p MH3-rh BMP10-histag-DDDDK质粒,电转入CHO细胞,用500μg/ml G418从单克隆中筛选出稳定表达目的蛋白的重组细胞,悬浮驯化表达8 d后,收集培养液上清,阴离子交换柱和镍柱纯化目的蛋白。采用q-PCR法检测纯化蛋白的体外细胞活性。结果目的蛋白纯化液经SDS-PAGE检测到的条带相对分子质量与目的蛋白理论值相符,Western blot分析可见相对分子质量约48 000、96 000和190 000的3个条带,与目的蛋白单体及多聚体相对分子质量一致。酶切后的目的蛋白可有效刺激P19细胞的标志蛋白(Smad6)表达上调。结论 BMP10 propeptide的存在对BMP10的蛋白活性具有重要作用,改造后的BMP10在CHO细胞中表达具有一定的生物活性。本研究为全长BMP10活性二聚体的制备提供了新的思路。

Objective To express the full-length gene of bone morphogenetic protein 10 （BMP10） in Chinese hamster ovary （CHO） cells, purify the expressed product and determine its biological activity in order to obtain bioactive BMP10 molecules. Methods A 6 x his-tag and DDDDK （enterokinase recognition site） were inserted into the part between propeptide and mature chain gene segment of recombinant human BMP10 （rhBMP10）, and the constructed recombinant plasmid pMH3-rhBMP10-histag-DDDDK was transformed to CHO cells by electroporation. The recombinant cells for stable expression of target protein were screened from single clones with 500 txg / ml G418 for suspension domestication expression for 8 d, of which the culture superuatant was collected and purified by anion exchange column and nickel column chromatography. The cell activity in vitro of the purified protein was determined by q-PCR. Results Western blot showed three bands with relative molecular masses of about 48 000, 96 000 and 190 000 respectively, which were consistent with those of monomer and polymers of target protein. The target protein after digestion with restriction enzyme stimulated the up-regulation of Smad6 expression in P19 cells effectively. Conclusion The presence of propeptide plays an important role in the activity of BMP10. The modified BMP10 was expressed in CHO cells, which showed a certain biological activity. The study provided a novel route to preparation of active full-length BMP10 dimer.