基础医学与临床 ›› 2007, Vol. 27 ›› Issue (3): 324-328.

• 技术与方法 • 上一篇    下一篇

大鼠脑组织NT3基因的融合表达、纯化及抗体制备

李志全 胡蕴玉 朱庆生 朱锦宇 刘方   

  1. 第四军医大学西京医院全军骨科研究所 第四军医大学西京医院全军骨科研究所 第四军医大学西京医院全军骨科研究所
  • 收稿日期:2006-01-17 修回日期:2006-08-04 出版日期:2007-03-25 发布日期:2007-03-25
  • 通讯作者: 朱庆生

Expression and purification of fusion protein of normal rat brain NT3 geme and antibody preparation

  

  • Received:2006-01-17 Revised:2006-08-04 Online:2007-03-25 Published:2007-03-25

摘要: 目的 克隆大鼠脑组织神经营养素3(NT-3)基因,原核诱导表达并纯化,制备高效价抗体,为研究其对周围神经损伤修复的影响奠定基础。方法 用RT-PCR 法扩增目的基因,依次克隆入pMD-18T载体及亚克隆入pRSET-A原核表达载体。导入大肠杆菌BL21 经 IPTG 诱导表达,SDS-PAGE电泳鉴定后用镍柱纯化。免疫家兔制备特异性抗体。结果 RT-PCR 得到777bp的片断,酶切鉴定及测序证实与GeneBank中大鼠NT-3 序列一致,成功构建了原核表达载体pRSET-A-NT-3。 导入 BL21 诱导得到一条约34kD 的新蛋白带。免疫家兔获得1∶64000 的高效价特异性抗大鼠NT-3 血清。结论 成功克隆了大鼠脑组织NT-3 基因,使其编码的蛋白质在原核细胞中顺利表达并纯化。制备了特异性抗体,为进一步研究其在周围神经损伤修复中的作用奠定了基础。

Abstract: Objective To clone NT-3 gene from normal rat brain and purify its fusion protein and prepare specific high titer antibody so that to provide foundation for further study for peripheral nerve injury.Methods We amplified target gene by RT-PCR and cloned it into the vector of pMD-18T , then analyzed its sequence and compared it with the sequence in GenBank.We subcloned it into pRSET-A vector and introduced it into Escherichia coli BL21.The expression was induced by IPTG,and identified by SDS-PAGE.The fusion protein was purified by niccolum purify kit.We immuned rabbits with immunological adjuvant for specificity antibody preparation. Results We got a 777bp gene segment by RT-PCR.The DNA sequence was identical to rat NT-3 gene sequence in GenBank. It suggested that the target gene was correctly inserted into the vector.A new protein band of about 34 kD appeared on SDS-PAGE after induction of IPTG.A specific high titer antibody of 1:64000 was gained by immuning rabbits with immunological adjuvant.Conclusions The fusion protein encoded by NT-3 gene which was successfully derived from normal rats has been expressed in Escherichia coli and been purified.A specific high titer antibody has been prepared , all this make it possible to do further investigation in reparation of peripheral nerve injury.