人神经生长因子基因在大肠杆菌中的克隆、表达及产物纯化

从人胎盘组织中提取总DNA， 经PCR扩增编码人β神经生长因子（β-NGF）成熟肽的基因，并克隆到大肠杆菌表达载体pET15b中。重组质粒pET15b-NGF经测序与报道的完全一致。重组质粒转化大肠杆菌BL21(DE3)pLysS，经IPTG诱导表达得到16kD的目的蛋白带，与预期的大小一致，NGF表达量约占全菌总蛋白的25%.经过亲和层析柱（Ni2+-charged IDA his-bind column）纯化后得到了单一的NGF蛋白条带，蛋白纯度可达90%以上，从每升表达菌液中可以得到4.56mgNGF。表达产物的Western 印迹鉴定结果显示：重组人神经生长因子能与兔抗人β-NGF的多克隆抗体发生特异性结合反应，在16kD处出现单一的条带，表明诱导表达的重组NGF具有免疫学活性。鸡胚背根神经节感觉神经元鉴定试验表明，本实验表达的重组NGF具有良好的生物学活性。

Cloning, Expression and Purification of Gene Encoding

Nerve growth factor is one of the most importment factors playing an importment role in regulating the growth, development and survival of the neuron. The purified NGF from human placenta has been reported, the tissue from which can be isolated the NGF is very limited. It is important for basic research and clinic application to expression hNGF by genetic engineering. By polymerase chain reaction，gene fragment encoding the mature part of β-NGF was amplified using the DNA of human placenta as template. The fragment was sequenced and inserted into expression vector pET-15b, and the recombinant expression vector pET15b-NGF was transformed into E.coli BL21(DE3)pLysS. After inducing with IPTG the NGF was higher expressed up to 25% of the total cell proteins. The expression product was purified with metal chelate affinity chromatography on Ni-IDA agarose under denaturing condition. The purity of rNGF was higher than 90% and yield of rNGF was 4.56mg/L expressing bacteria. SDS-PAGE revealed the NGF expression product had a Mr 16kD. Western-blot displayed the recombinant product had strong immunological activity with rabbit anti-human β-NGF polyclonic antibodies. The expression products were dealed with solubilizing inclusion bodies and refolding protein. The test of nerve fiber growth of chicken embryo DRG neurons displayed rNGF had biological activity.