神经营养因子GDNF编码顺序的克隆及表达产物的纯化

Cloning of GDNF Coding Sequence and Purificationof Recombinant Protein

根据基因库中的顺序，设计了胶质细胞源神经营养因子（ＧＤＮＦ）基因的ＰＣＲ引物，以此从人基因组ＤＮＡ中扩增并克隆了ＧＤＮＦ的编码序列，经ＤＮＡ测序确认后，该片段克隆到表达质粒ｐＥＴ－３ａ中，转化大肠杆菌ＢＬ２１（ＤＥ３）．培养的重组菌经ＩＰＴＧ诱导，在Ｔ７启动子调控下表达出ｈＧＤＮＦ蛋白．经电泳分析表明ＧＤＮＦ主要存在于细菌包涵体中．从培养菌中制备包涵体，经充分洗涤，溶解于含８ｍｏｌ／Ｌ尿素的变性缓冲液中．经ＳＰ－Ｓｅｐｈａｒｏｓｅ柱层析分离，梯度洗脱，以１５％ＳＤＳ－ＰＡＧＥ检查含ＧＤＮＦ的部分．将含单体ＧＤＮＦ部分进行复性，再次用ＳＰ－Ｓｅｐｈａｒｏｓｅ离子柱分离同源二体ＧＤＮＦ．最后经ＳＤＳ－ＰＡＧＥ制备电泳纯化，纯度大于９５％．经Ｎ端测序表明序列正确．经测定，每升培养菌可得约１０ｍｇ纯化的ＧＤＮＦ．

GDNF coding sequence was amplified by PCR from human genomic DNA with primers designed according to the data in GenBank.After proved by gene sequencing,this fragment was cloned in plasmid pET3a,and transferred into E.coli BL21(DE3) strain.The expression of GDNF protein in transformant was controled by T7 promotor with IPTG induction.Electrophoresis analysis showed that GDNF mainly existed in inclusion bodies.Prepared inclusion bodies were fully washed and then solubilized in 8 mol/L urea denaturing buffer.The denatured protein was applied to a SPSepharose column and eluted by NaCl gradient.GDNF monomer in pooled fractions was refolded into GDNF dimer.Another SPSepharose column was used to separate the GDNF dimer,which was further purified by SDSPAGE.The yield of GDNF was about 10 mg per litre culture,and the purity of GDNF was up to 95%.The product was also proved by Nterminal sequencing.