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目的 亲肝素性促轴突生长因子 (heparin bindingneurite promotingactor,HBNF)最早在脑组织中发现 ,能促进体外培养的神经细胞的轴突生长。本研究利用酵母系统表达人亲肝素性促轴突生长因子 ,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式反应 (RT PCR)获得人亲肝素性促轴突生长因子 (HBNF)的编码基因 ,扩增后与穿梭表达载体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酵母菌株GS115 ,在不含组氨酸的MD平板上筛选His+表型 ,利用G4 18抗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亲肝素性促轴突生长因子 (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 )细胞观察其促轴突生长作用。结果 对重组表达载体的序列分析表明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分子量约为 18kDa ,与文献报道一致。该蛋白产物对于培养的大鼠嗜铬细胞瘤 (PC12 )细胞具有促轴突生长作用。结论
Objective Heparin binding neurotrophic promoting growth factor (hNFF) was first found in brain tissue, which can promote axon growth of cultured nerve cells in vitro. In this study, yeast-based human heparin-induced axon growth factor was expressed and its effects on axonal growth in vitro were observed. Methods Total RNA was extracted from fetal brain tissue of 18 weeks abortion, and the gene encoding human heparin-promoting growth factor (HBNF) was obtained by RT-PCR. After amplification and shuttle of expression vector pPIC9K recombinant, identification and screening to obtain HBNFcDNA positive clones. The recombinant expression vector was used to transform yeast strain GS115 by electroporation, His + phenotype was screened on histidine-free MD plates, multi-copy yeast recombinants were screened by G418 resistance, and PCR was used to identify the presence of HBNFcDNA and yeast chromosome Genome-integrated strains. After positive clones were induced by methanol, the secretory expression of recombinant human proheparin-promoting axonal growth factor (hrHBNF) in culture medium was obtained. Chromatographic purification of the expression product was added to cultured rat pheochromocytoma (PC12) cells observed axonal growth promoting effect. Results The sequence analysis of the recombinant expression vector showed that the sequence of the target gene was exactly the same as that of the gene bank. The target gene was located in the vector downstream of the signal peptide of α - factor secretion and linked to the α - factor open reading frame. The molecular weight of the product induced by SDS PAGE electrophoresis was about 18kDa, which was consistent with that reported in the literature. This protein product has axonal growth promoting effects on cultured rat pheochromocytoma (PC12) cells. in conclusion