神经营养因子-3 cDNA的克隆以及在酿酒酵母中的分泌性表达

从原代培养人许旺细胞申提取总RNA，用RT－PCR方法获取了编码带向导肽和成熟的NT-3两个cDNA片段，经DNA序列测定表明其顺序与文献报道一致。将这两个cDNA片段克隆到大肠-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。这两种cDNA片段的表达产物经SDS-PAGE电泳鉴定，大小都在15ku左右，说明带前导肽的NT-3在酵母细胞中获得了正确的加工。将这两种部分纯化的NT－3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。人神经营子-3％在酿酒酵母系统获得有生物活性的表达。

CLONING OF HUMAN NEUROTROPHIN-3 (hNT-3)cDNA AND THE SECRETED EXPRESSION OF hNT-3 IN SACCHAROMYCES CEREVISIAE

Human neurotropbin-3 cDNA fragments coding for pro-NT-3 and mature NT-3 were obtamed by RT-PCR method with total RNA extracted from the human cultured Sckwann cells. The cloned gene fragments checked by the dideoxynucleotide sequencing were the same as those reported. The verified hNT-3cDNA were inserted into the E. coli/yeast shuttle vector of pVT 102U/. Arter transformed with the intact yeast cell method, the yeast engineered strain containing recombinant vector of pVT102U/Q-Pro-NT-3 or PVT 102U/-mNT-3 were selected respectively. The expressed and secreted products were isolated and purified. The expressed products of the two genes appeared to identical with the similar electrophoretic mobility around 15 ku indicated by SDS-PAGE. It was implicated that the precuror was correctly processed. And bioactivity of the partially purified samples were shown to promote cell survival and neurite overgrowth in the primary cultures of rat embryonic dorsal root ganglion(DRG)neurons as compared with the control. These results Indicate that recombinant products of human neurotropkin-3 expressed in Saccharomyces cercvisiae show high activity.