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目的 研究酵母系统表达人亲肝素性促轴突生长因子 (heparin -bindingneurite -pro motingfactor,HBNF)的活性 ,并观察其对体外培养细胞的促轴突生长作用。 方法 从 18周流产的人胎脑组织提取总RNA ,利用逆转录聚合酶链反应 (RT -PCR)获得HBNF的编码基因 ,扩增后与表达载体pPIC9K重组 ,鉴定筛选得到含HBNFcDNA基因的阳性克隆。用此重组表达载体通过电穿孔法转化毕赤酵母菌株GS115 ,在不含组氨酸的选择性含糖基础培养基 (MD)平板上筛选His +表型 ,利用G418抗药性筛选多拷贝酵母菌重组子 ,PCR鉴定含有HBNFcDNA基因与酵母染色体基因组整合的菌株。阳性克隆经甲醇诱导后获得重组人HBNF(hrHBNF)在培养液中的分泌表达。将层析纯化的表达产物加入体外培养的大鼠嗜铬细胞瘤 (PC12 )细胞株 ,观察其促轴突生长作用。结果 目的基因序列与基因库资料完全一致 ,目的基因在该载体中位于α因子分泌信号序列的下游 ,与α因子开放阅读框相连。SDS -PAGE蛋白电泳证实诱导表达的产物相对分子质量约为180 0 0 ,它对培养的PC12细胞株具有促轴突生长作用。 结论 通过毕赤酵母菌可以稳定地表达hrHBNF ,而且表达产物hrHBNF具有促进神经轴突生长的生物学活性。
OBJECTIVE To study the activity of human heparin-binding protein-induced motoneurin (HBNF) in yeast and to observe its effect on the growth of cultured cells in vitro. Methods Total RNA was extracted from fetal brain tissue of 18 weeks abortion. The HBNF coding gene was obtained by RT-PCR. After amplification, it was recombined with the expression vector pPIC9K. The positive clones containing HBNF gene were identified by screening . The Pichia pastoris strain GS115 was transformed by electroporation using this recombinant expression vector, His + phenotype was screened on histidine-free selective sugar-containing basal medium (MD) and the multi-copy yeast was screened by G418 resistance Recombinant, PCR identification of strains containing the HBNF cDNA gene and yeast genome chromosomal integration. After positive clones were induced by methanol, the secreted expression of recombinant human HBNF (hrHBNF) in culture medium was obtained. The purified product was added to the cultured rat pheochromocytoma (PC12) cell line to observe its role in promoting axonal growth. Results The sequence of the target gene was completely consistent with that of the gene bank. The target gene was located in the vector downstream of the secretion signal sequence of α-factor and linked to the α-factor open reading frame. SDS-PAGE electrophoresis confirmed that the relative molecular mass of the induced product was about 180 0 0, which had a promoting axonal growth effect on the cultured PC12 cell line. Conclusion hrHBNF can be stably expressed by Pichia pastoris, and the expression product hrHBNF has the biological activity of promoting axonal growth.