重组人HBNF的表达和提纯及其生物活性的测定 |
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引用本文: | 周强,彭裕文,顾银良,汪洋,宋后燕. 重组人HBNF的表达和提纯及其生物活性的测定[J]. 复旦学报(医学版), 2003, 30(5): 422-425,F002 |
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作者姓名: | 周强 彭裕文 顾银良 汪洋 宋后燕 |
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作者单位: | 1. 复旦大学附属金山医院骨科,上海,200540 2. 上海医学院人体解剖与组织胚胎学系,上海,200032 3. 上海医学院,细胞与遗传学系,上海,200032 |
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摘 要: | 目的 通过酵母表达系统表达重组人亲肝素性促轴突生长因子(heparin—binding neuritepromoting factor,HBNF)蛋白并检测其生物活性。方法 将重组表达质粒pPIC9k/HBNf通过电穿孔法转入酵母表达系统GSI15,利用G418筛选高拷贝子用于大量表达HBNF、蛋白,再检测其生物活性。结果 通过G418筛选出抗性大于2mg/mL的高拷贝子,大量表达、提纯后获得相对分子质量约18000蛋白,Western—blot证实为HBNF蛋白,生物活性检测显示提纯HBNF有显著的神经营养作用。结论 通过酵母表达系统获得的重组人HBNF有一定的生物活性。
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关 键 词: | 重组人HBNF 基因表达 提纯 生物活性 测定 酵母表达系统 |
Expression, Purification and Bio-activity Analysis of Recombined Human HBNF |
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Abstract: | Purpose To express recombined human heparin-binding neuritepromoting factor (HBNF) by yeast expression system and analyse its bio-activity. Methods Transforming the linearized pPIC9k/HBNF DNA into the competenced GS115 by electroporation,multi-copy clones were screened by G418 for scale-up expression,the bio-activity of the HBNF was analyzed. Results Multi-copy clones were screened out at a final G418 resistant concentration of 2 mg/mL,after scale-up expression,the 18 000 protein was purified,which proved to be HBNF by Western-blot,and the bio-activity analysis showed its obvious neurotrophic effects. Conclusions The rh?HBNF expressed by yeast expression system had wanted bio-activity. |
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Keywords: | heparin-binding neuritepromoting factor yeast expression system bio-activity |
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