Cloning and expression of cyclophilin A gene from Echinoccocus granulosus

Abstract

Objective In order to assess the value of Echinoccocus granulosus cyclophilin A (EgypA) in immune diagnosis, this novel gene was cloned and expressed. Methods By screening the EST library, the coding region of EgCypA was identified, and the PCR primers were designed based on this sequence.Bioinformatic tools were used to deduce the amino acid sequence of EgCypA and analyzed its biological characteristics.EgCypA was amplified from E.granulosus cDNA library by using PCR.Then, it was cloned into the prokaryotic expression vector pET28a and transformed into E.coli BL21.The recombinant protein was expressed in E.coli BL21 after IPTG induction.The immunogenicity of EgCypA was evaluated by Western blot using the Echinoccocus granulosus and other parasites infected animals′ sera. Results Results of SDS-PAGE electrophoresis show that the recombinant BL21 expressed 18 400-25 000 protein, which was identical with the molecular weight calculated by bioinformatic analysis.Western blot shows that the recombinant protein only reacted with its immune serum and E.granulosus cystic fluid immune serum, and EgCypA immune serum could react with the excretion and secretion antigen from E.granulosus protoscolexes, and no cross-reaction between EgCypA and sera from other parasites infected animals. Conclusions Cloning and expression of EgCypA are successful.EgCypA has good immunologic activity and could be a candidate molecular for immune diagnosis of echinococcosis in early stage.（Chin J Lab Med,2014,37:56-59） Key words: Echinococcus granulosus; Cyclophilin A; Sequence analysis; Recombinant proteins; Blotting, western