人神经生长因子的A-T克隆及在Pichia Pastoris酵母中的表达

目的对人含前导肽的β-NGF基因序列进行体外扩增、克隆及分析鉴定,并将其插入Pichiapastoris酵母分泌型表达载体pPIC9K中进行表达及鉴定.方法采用PCR技术,从人胎盘DNA基因组中扩增出含前导及信号肰的β-NGF基因序列;用A-r克隆法,与pGEM-T载体连接,经筛选得到重组质粒pGEM-Tβ-NGF,用酶切、PCR法及序列分析进行鉴定;将β-NGF插入酵母表达载体pPIC9K,用脂质体法导入CS115中,甲醇诱导分泌蛋白.结果亚克隆中,1.2%的琼脂糖凝胶电泳显示在748bp位置出现阳性条带;发酵液中蛋白SIS-PAGE电泳在14.OKD附近有特异表达带;蛋白表达量占菌体蛋白的30%;Westernblotting检测表明此带有特异活性.结论重组质粒pGEM-Tβ-NGF蛋白产量高,具有免疫活性.

A-T cloning and expression of human β-nerve growth factor gene

Objective:To analyse and identify subcloning of pro-β-nerve growth factor(β-NGF)gene and expression in Pichia pastoris stain cells.Methods:The pro-β-NGF gene vas amplified by PCR.The recombined plasmid pGEM-Tβ-NGF was obtained by A-T cloning and was detected by enzymolysis,PCR and sequence analysis.The β-NGF gene was inserted into pPIC9K vector,and the β-NGF protein was induced by methanol.Results:The agarose gel electropboresis results of PCR showed the pro-β-NGF gene appeared at the zone of 748 bp.The 10% SDS-PAGE electrophonesis results of expression protein showed a single band corresponding with monomeric(14.0 KD)form of human β-NGF protein,which represented 30% of the total Pichia pastoris secretive protein approximately.The result of Western Blot showed the immune activity of the expression protein.Conclusions:Recombined plasmid pGEM-Tβ-NGF was successful;the expression protein of β-NGF has the immune activity.