Expression,purification of rat ANT1 protein and preparation of its polyclonal antibody
AIM:To express and purify the adenine nucleotide transporte protein(ANT1)and to prepare its polyclonal antibody.METHODS:The CDS of ANT1 was amplified by PCR from rat,and then subcloned into prokaryotic expression vector pET28a,after transformed with the vector,the BL21(DE3)stains were induced by IPTG.The expression product was purified by Ni2+-NTA ion exchange resin.The purified protein was analyzed by SDS-PAGE,and then immuned the New Zealand white rabbits to prepare antibody.The polyclonal antibody was received by Affinity purification from rabbit serum.The prepare antibody was used in sample assay by Western Blot.RESULTS:The expression plasmid pET28a-ANT1 was constructed successfully,and rat ANT1 with 32 000 molecular weight was expressed in E.coli.The purity of the ANT1 protein was purification with Ni~(2+)-NTA ion exchange resin and then immuned the New Zealand white rabbit to prepare antibody.It was comfirmed by Western Blot technique that antibody specifically recognized the 32 000 ANT1 protein.CONCLUSION:We had successfully obtained prokaryotic fusion protein of ANT1,got high sensitivity and specification anti-ANT1 polyclonal antibody.That lays a solid foundation for the studies of ANT1 in pathogenesis of epilepsy.
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