重组人脑源性神经营养因子的纯化和鉴定

在确定培养条件和发酵参数后 ,工程菌 E.coli BL2 1 ( DE3) /PVBN6在 5 L发酵罐中稳定表达。获得的菌体经超声破碎 ,离心收集包含体。 6mol/L盐酸胍缓冲液溶解包含体 ,用透析法将盐酸胍替换成脲后 ,经过 CM Sepharose F.F.阳离子交换色谱和 C8反相色谱 ,可得到纯度达 95 %以上的rh BDNF。Western- blot表明 ,rh BDNF与抗 - h BDNF多克隆抗体有结合特异性。用 9日龄鸡胚背根神经节测定生物活性 ,rh BDNF活性为 5 0 ng/ml。N-末端氨基酸序列测定表明 rh BDNF N-末端为Met,其后 1 6个氨基酸残基与天然 h BDNF N-末端氨基酸残基序列一致

Purification and identification of recombinant human brain-derived neurotrophic facter

Recombinant human brain derived neurotrophic factor was expressed in E.coli BL21(DE3)/p VBN6 which cultivated in 5 liter fermentor. The cell suspension was disrupted by sonication, and the inclusion bodies were isolated from cells. First, the inclusion bodies dissolved in guanidine hydrochloride buffer, the supernatant obtained after centrifugation was dialyzed in urea solution. Then, the protein solution went through CM Sepharose Fast Flow ion exchange column and C 8 reverse phase chromatography column. The purity of rhBDNF could reach above 95%. The resulting protein had biological activity in promoting the neurite outgrowth of cultured dorsal root ganglion neurons from E9 chicken in concentration of 50 ng/ml. Except that the first amino acid was Met, the NH 2 terminal amino acid sequence of the rhBDNF was that of natural BDNF by the sequence analysis.