摘要
OBJECTIVES: Heparin-binding neurite-promoting factor (HBNF) is a heparin-binding protein primarily found in the brain, which can stimulate neurite outgrowth in vitro. We expressed recombinant human heparin-binding neurite-promoting factor (hrHBNF) using a yeast system, and observed its activity in stimulating neurite outgrowth in vitro. METHODS: cDNA encoding mature human HBNF was amplified from total RNA isolated from an 18-week aborted human fetal brain by RT-PCR method. After amplification, the HBNF cDNA gene was cloned into pPIC9K, a shuttle expression vector for yeast system. The positive clone of expression vector bearing HBNF cDNA gene was obtained by screening. Verified recombinant vector was then used to transform Pichia strain GS115 by electroporation. His(+) transformants were selected on minimal dextrose medium (MD) plates which were histidine free. His(+) yeast recombinants with multi-copy inserts were screened in vivo by their resistance to G418. PCR analysis was used to confirm the integration of the HBNF cDNA gene into the Pichia genome. Secreted expression of hrHBNF protein in culture medium was obtained when the positive clone containing the HBNF cDNA gene was induced by methanol. The hrHBNF product purified by gel chromatography was added to cultured rat pheochromocytoma (PC12) cells to observe its ability to stimulate neurite outgrowth. RESULTS: In the recombinant expression vector, the insert was sequenced to show exactly the sequence encoding human HBNF according to Genbank data. The HBNF cDNA gene was cloned downstream to the alpha-factor, and its open reading frame was in frame with the alpha-factor signal sequence in pPIC9K. SDS-PAGE showed that the molecular weight of the induced expression product was about 18 kDa, consistent with that of human HBNF reported in the literature. The protein product did promote neurite outgrowth in cultured rat pheochromocytoma (PC12) cells. CONCLUSION: Recombinant human heparin-binding neurite-promoting factor can be expressed with a yeast system, and its product possesses the biological activity to promote neurite outgrowth.
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。
基金
ThisworkwasconductedinpartwithfundsfromtheShanghaiHealthBureau
