Abstract: The aim of this study was to express Clostridium chauvoei toxin A (CctA) gene in prokaryotic system and establish indirect ELISA detection method using purified recombinant protein,and to verify the immune effect of gangraena emphysematosa inactivated vaccine.The sequence of CctA gene was cloned into prokaryotic expression vector pET-28a(+) according to the codon bias of Escherichia coli (E.coli).The prokaryotic expression plasmid pET28a-CctA was identified by double enzyme digestion and sequencing.The high expression of recombinant CctA inclusion body protein was induced by IPTG after recombinant plasmid was transferred into E.coli.The inclusion body protein was purified by Ni column after denaturation and its purification effect was tested by SDS-PAGE.Western blotting was used to detect the reactivity of recombinant CctA protein.Establishment of indirect ELISA was accompanied by the checkerboard titration method.The recombinant CctA protein was used as detected antigen,and the concentration of the coated antigen,the type and concentration of the blocking solution,the optimal dilution of the antibody and the reaction conditions were explored.After the guinea pigs were immunized with 3 batches of gangraena emphysematosa inactivated vaccine,the serum was collected.The immune effect of the vaccine was verified both by challenge and indirect ELISA.The results of double digestion of plasmid showed that the band about 853 bp was in line with expectation,and the sequencing results was correct.SDS-PAGE results indicated that 35 ku recombinant CctA protein was successfully expressed and purified.The results of Western blotting showed that the antiserum of guinea pigs against Clostridium chauvoei had good reactivity with recombinant CctA protein.The optimal conditions of indirect ELISA method were as follows.The coating concentration of antigen was 0.5 μg/mL overnight at 4 ℃,10% fetal bovine serum was selected for blocking solution and incubated at 37 ℃ for 2 h,the dilution of the secondary antibody was 1:8 000,the reaction was stopped after 10 min at room temperature and D_{450 nm} was determined.When P/N>4.6,the results of indirect ELISA fitted well with the guinea pig challenge test.In this study,CctA gene was successfully expressed and recombinant CctA protein was purified,and indirect ELISA was established with recombinant CctA protein as detected antigen,which was expected to be an alternative method to verify the immune effect of gangraena emphysematosa inactivated vaccine.

Key words: gangraena emphysematosa; CctA gene; prokaryotic expression; ELISA detection; efficacy test