Cloning,Expression and Immunogenicity Analysis of TPx Gene of Coenurus Cerebralis
To study the immunogenicity of Thioredoxin peroxidase(TPx)for Coenurus cerebralis(Cc),the protoscoleces of Coenurus cerebralis were collected in sheep brain,and total RNA was extracted.Specific primers were designed according to the Taenia asiatica TaHc2-D11 mRNA sequences in GenBank,the Cc TPx gene was amplified by RT-PCR,and the recombinant plasmid pMD18-T-CcTPx was constructed,then cloned into the prokaryotic expression vector pGEX-4T-1.The recombinant expression plasmid pGEX-4T-1-CcTPx was correctly identified and transformed into E.coli BL21(DE3)for induction of expression,and the Cc TPx recombinant protein was detected by sodium dodecyl sulphate-polyacrylamide gel electrophoresis(SDS-PAGE).The purified Cc TPx recombinant protein was immunized in sheep by subcutaneous multi-point injection,and the antibodies in serum were detected with ELISA.The result showed that the Cc TPx gene was 614 bp in length and comprised an open reading frame(ORF)of 591 bp.The molecular mass of Cc TPx recombinant protein was 47 kD and it existed mainly in the form of inclusion bodies.The recombinant protein could stimulate sheep to produce antibodies,and the antibody level gradually increased after the second or third immunization,and the antibody level was significantly higher than that of the control group in sheep,which were immunized with the protoscoleces of Coenurus cerebralis at the same time.In summary,the recombinant protein of Cc TPx has strong immunogenicity and can be used as a candidate antigen for the Coenurus cerebralis vaccine.
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