人心肌营养素在大肠杆菌中的表达纯化和活性鉴定

Expression and purification of human cardiotrophin-1 in E.coli and identification of its biological activity

目的 利用大肠杆菌高效表达可溶性具有生物学活性的重组huCT 1蛋白。方法 采用PCR及T A克隆将huCT 1开放阅读框基因克隆到pMD 18T载体上 ,再构建原核表达载体pGEX2T huCT1。在不同温度不同时间 ,通过IPTG诱导pGEX2T huCT1在DH5a大肠杆菌中表达 ,SDS PAGE分析表达量和可溶性蛋白比例。GST亲和柱纯化huCT 1融合蛋白 ,凝血酶酶切融合蛋白并纯化。坐骨神经切断模型观察重组huCT 1对脊髓前角神经元的存活作用。结果 构建了huCT 1的融合表达载体pGEX2T huCT1。通过IPTG诱导 ,发现 2 9℃诱导 4h ,目的蛋白表达量占全菌的 1 5 ,可溶性蛋白与包涵体比为 2 5。对其进行亲和柱纯化 ,GST huCT 1融合蛋白纯度达到 85 %以上。融合蛋白酶切后 ,重组huCT 1蛋白纯度达到80 %以上。发现原核表达重组huCT 1蛋白能挽救 5 5 %成年大鼠脊髓运动神经元的存活。结论 成功地获取了具有生物活性的huCT

Objective To express recombinant human cardiotrophin 1 (huCT 1) protein with biological activity in E coli Methods huCT 1 open reading frame gene from plasmid pBSSK huCT1 was cloned into plasmid pMD18 T by PCR and T A cloning, and then cloned into prokaryotic GST fusion expression vector pGEX 2T to give pGEX2T huCT1 After pGEX2T huCT1 expression was induced by IPTG in E coli at different temperature and different time, the expression level and the proportion of the soluble protein were analyzed by SDA PAGE Then the soluble GST/huCT1 was purified by immobilized glutathione columns The GST fusion protein was cleaved by thrombin and purified again The recombinant huCT 1 with biological activity was identified according to the survival of axotomized sciatic motoneurons Results After the induction of pGEX2T huCT1 DH5α cells by IPTG at 29 ℃ for 4 h, the highest expression level of the recombinant protein was about 1/5 of total cell proteins, and the soluble portion was about 2/5 of fusion protein Purification of the soluble portion and thrombin cleaved fusion protein resulted in 85% and 80% purified recombinant GST fusion protein and huCT 1 protein, respectively Recombinant huCT 1 protected 55% motoneurons in spinal cord against sciatic axotomy in vivo in adult rats Conclusion Recombinant huCT 1 has biological activity in rat neurons