Expression and purification of rat brain NT-3 fusion protein and its antibody preparation / 基础医学与临床
Basic & Clinical Medicine
;
(12)2006.
Article
in Chinese
| WPRIM
| ID: wpr-588731
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.
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Index:
WPRIM (Western Pacific)
Language:
Chinese
Journal:
Basic & Clinical Medicine
Year:
2006
Type:
Article
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