人睫状神经营养因子的原核表达、纯化及其生物效应

EXPRESSION,PURIFICATION AND BIOLOGICAL EFFECT OF RECOMBINANT HUMAN CILIARY NEUROTROPHIC FACTOR

人睫状神经营养因子（ｈＣＮＴＦ）克隆入ｐＢＶ２２０中，在ＤＨ５α菌株中表达，重组蛋白以包含体的形式存在，表达量为菌体总蛋白的５０％左右。经比较发现用２ｍｏｌ／Ｌ脲洗涤包含体可溶解大量可溶性细菌蛋白，且包含体损失较小。在高浓度变性剂条件下进行ｓｅｐｈａｒｃｙｌＳ－２００凝胶过滤，解决了纯化中ｈＣＮＴＦ易聚合的问题，在低浓度变性剂条件下进行ＤＥＡＥ离子交换，有利于蛋白活性的保持。经两步纯化后得到均一性ｈＣＮＴＦ，纯度达９５％以上。在自然状态下使ｈＣＮＴＦ复性。纯化复性后的ｈＣＮＴＦ对无血清培养的鸡胚背根节神经元和脊髓腹角运动神经元有明显的维持存活和促进生长发育的生物效应。

Human ciliary neurotrophic factor(hCNTF)was expressed and purified from E.coli.Thecoding sequence of hCNTF gene was cloned in the expression vector pBV220.The E.coli strainDH 5α transformed with pBV220/hCNTF expressed high level(about 50 % by gel scanning)ofhCNTF,which was insoluble in E.coli and was contained within inclusion bodies.It was showedthat the best condition for washing inclusion bodies was 2 mol/L urea.hCNTF waschromatographed on Sephacryl S-200 column at high concentration of detergent,then on DEAE-Sepharose column at low concentration of detergent.After these two chromatographicsteps,the purity of prepared hCNTF is about 98%.hCNTF was biologically active after refold-ing in air.The promoting effects of hCNTF on the survival and development of cultured E10chick DRG neurons and spinal motor neurons were also significantly demonstrated.