Expression and purification of rat brain NT-3 fusion protein and its antibody preparation
- VernacularTitle:大鼠脑组织NT-3融合蛋白的表达、纯化及抗体制备
- Author:
Zhiquan LI
;
Yunyu HU
;
Qingsheng ZHU
;
Jinyu ZHU
;
Fang LIU
- Publication Type:Journal Article
- Keywords:
rat;
NT-3 gene;
gene expression;
protein purification
- From:
Basic & Clinical Medicine
2006;0(03):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To clone NT-3 gene from normal rat brain and to purify its fusion protein and to prepare specific high titer antibody so that to provide a foundation for further study for peripheral nerve injury.MethodsWe 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 from 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 777 bp gene segment by RT-PCR.The DNA sequence was identical to rat NT-3 gene sequence in GenBank.It proved that the target gene was correctly inserted into the vector.A new protein band of about 34 ku appeared on SDS-PAGE after induction of IPTG.A specific high titer antibody of 1∶64000 was gained by immunizing rabbits with adjuvant.
