1.Department of Parasitology, Basic Medical College, Xinjiang Medical University; 2.Hydatid Disease Key Laboratory, the First Affiliated Hospital, Xinjiang Medical University, Urumqi 830011, China
Abstract：The objective was to establish expression vector pTWIN1-AgB8/3 of a self-splicing prokaryotic expression system and obtain pure recombinant antigen of Echinococcus granulosus AgB8/3 (rEgAgB8/3). The specific primers were designed according to the published nucleotide sequence of EgAgB8/3 deposited in the GenBank database(AF362442). The nucleotide sequence corresponding to the secreted form of EgAgB8/3 was amplified by PCR, and the product was directionally ligated into Prokaryotic Expression Vector pTWIN1. The ligation was transferred into the competent cell ER2566, the fusion protein CBD-intein1-EgAgB8/3 was expressed by inducing with IPTG, and the target protein rEgAgB8/3 was purified by chitin binding affinity purification column. The expression product and the target protein were analyzed by SDS-PAGE and western blot. Results showed that the EgAgB8/3 gene was successfully cloned and ligated into pTWIN1 to form protein self-splicing prokaryotic recombimant expression vector pTWIN1-EgAgB8/3. The SDS-PAGE and western blot analysis showed that the fusion protein CBD-intein1-EgAgB8/3 was expressed as soluble form. The chitin binding domain (CBD) and intein1 were removed from target protein rEgAgB8/3 at one step during the chitin column affinity purification. It suggested that the high level of soluble target protein rEgAgB8/3 with few additional amino acid residue was successfully purified by simple treatment after expression, which may allow us to keep the original amino acid sequence and possible activity of the target protein and facilitate the process of production of anti-rEgAgB8/3 monoclonal antibodies.
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2013, Vol. 29 
