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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 BMP10 gene in Chinese hamster ovary (CHO) and to examine its biological activity after purification, in order to obtain the bioactive BMP10 molecule . Methods 6 × his-tag and DDDDK (enterokinase recognition site) were inserted between the propeptide of rh BMP10 and the mature chain gene fragment to construct p MH3-rh BMP10-histag-DDDDK plasmid. / ml G418 The recombinant cells stably expressing the target protein were screened from the monoclonal antibodies. After being cultured in suspension for 8 days, the culture supernatants, the anion exchange column and the nickel column were purified. In vitro cell activity of purified protein was detected by q-PCR. Results The relative molecular mass of target protein purified by SDS-PAGE was consistent with the theoretical value of the target protein. Western blot analysis showed three bands with relative molecular mass of about 48 000, 96 000 and 190 000, Body and polymer relative molecular mass consistent. The digested protein can effectively stimulate the expression of Smad6 in P19 cells. Conclusion The presence of BMP10 propeptide plays an important role in the activity of BMP10 protein. The modified BMP10 has certain biological activity in CHO cells. This study provides a new idea for the preparation of full-length BMP10 dimer.