Basic & Clinical Medicine ›› 2007, Vol. 27 ›› Issue (3): 324-328.

• 技术与方法 • Previous Articles     Next Articles

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

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.