人脑源性神经营养因子cDNA克隆及在酵母中的表达

Cloning of human brainderived neurotrophic factor cDNA and its expression in Saccharomyces cerevisiae

目的：克隆和表达人脑源性神经营养因子（ｈＢＤＮＦ）。方法和结果：从原代培养人雪旺细胞中提取总ＲＮＡ，用ＲＴ－ＰＣＲ法获取了编码带前导肽的和成熟的ｈＢＤＮＦ２个ｃＤＮＡ片段，经ＤＮＡ测序表明其顺序与文献报道一致。将这２个ｃＤＮＡ片段分别插入到大肠－酵母穿梭质粒ｐＶＴ１０２Ｕ／α上。用酵母整体细胞法将重组质粒转化酵母宿主细胞，经筛选后，获得分泌性表达。表达产物经ＳＤＳ－ＰＡＧＥ电泳鉴定，大小都在１５０００左右。将这２种部分纯化的ＢＤＮＦ样品经鼠胚背根神经节生物活性分析，表明具有明显的促进细胞存活和突起生长的作用。

Objective:To clone and express human brainderived neurotrophic factor (hBDNF) cDNAs. Methods and Results: hBDNF cDNA fragments encoding proBDNF and mature BDNF were obtained by using RTPCR method with total RNA extracted from the human cultured Schwann cells. Sequences of the hBDNF cDNAs obtained were the same as those reported. The verified hBDNF cDNAs were inserted into the Ecoliyeast shuttle vector of pVT102 U/α. After transformed with the intact yeast cell method , engineered strain habouring recombinant vector of pVT102 U/αproBDNF or pVT102 U/αmBDNF were selected respectively.The expressed and secreted products were isolated and purified. The expressed products of the two genes appeared to be identical with the similar electrophoretic mobility around 15 000 indicated by SDSPAGE. Bioactivity of the partially purified products was found to increase cell survival and neurite growth in the primary cultures of rat embryonic dorsal root ganglion neurons as compared to control. Conclusion: The results indicate that hBDNF cDNA can be expressed in Saccharomyces cerevisiae with high bioactivity.