Prokaryotic expression of cathepsin Z gene from Angiostrongylus cantonensis and the immunological analysis of the fusion protein

In order to clone and express cathepsin Z gene of Angiostrongylus cantonensis(A.cantonensis) to evaluate its prospects in immunodiagnosis,bioinformatics analysis tools were applied to analyze physicochemical properties,structural,and functional characteristics of gene encoded protein.Target gene was cloned into prokaryotic expression vector pET30a(+) and the recombinant was identified by PCR and double enzyme digestion respectively.When the recombinant was expressed in E.coli BL21 by IPTG induction,the expressed product was identified by SDS-PAGE and the fusion protein was purified by Ni-IDA affinity chromatography.As fusion protein was used as diagnostic antigen,the sensitivity and specificity of that were tested by ELISA.The protein contained a secretory signal peptide with stable physicochemical property and three key amino acids for cysteine proteinase,which contained Cys84,His232 and Asn253 to form the catalytic sites.The recombined plasmid was successfully constructed and expressed in E.coli BL21 effectively.Purified protein was obtained by affinity chromatography and could react with serum of mouse that infected with A.cantonensis or immunized with the fusion protein.ELISA results showed that the sensitivity and specificity were 100% in testing serum of mouse by using fusion protein as the coating antigen,and has no difference compared with crude antigen.But in the test for human serum,its specificity was higher than that of crude antigen.In conclusion,cathepsin Z of A.cantonensis is homologous with cathepsin Z from other species and contained a secretory signal peptide.It is probably a crucial component of excretory-secretory antigen and might be a promising candidate for immunodiagnosis of Angiostrongyliasis.