Cloning, Expression And Characterization Of Cathepsin B Gene Of Schistosoma Japonicum

Objective:Isolation of the individual schistosome cathepsin-like protease has proved difficult because of the paucity of material that can be obtained. In order to obtain purified and active recombinant cathepsin B for the further studies on their biological function and development of the novel anti-schistosomal drugs, the prokaryotic expression systems was established in an atempt to efficiently express cathepsin B gene in our study.Methods:1.Gene cloning:Extract totol RNA of Schistosoma japonicum .The Sj31 cDNA was amplified by RT-PCR employing primers designed based on their own reporteds equences.The cDNAs of th Sj 31 gene was cloned into the prokaryotic expression vector pET-30. Then the recombinant construct was transformed into Ecoli BL21 to establish the engineering strains for expression of Sj31.The correct recon was identifyied by antibiotics,PCR,enzyme digestion and sequecing.2.Protein expression: Recombination protein was detected by Mouce Anti His polyantibody with SDS-PAGE after induced by IPTG.. Optimize the time and IPTG density of the induction. The antigenicity of the recombination protion was determined with Western-Blot by pre-treated rabbit anti-schistosome sera.Results:1.It was confirmed by PCR, restriction endonucleas digestion and sequencing that the recombinant prokaryotic expression plasmid pET30-Sj31 was constructed successful.2.The fusion protein could induced by IPTG .The expressionfrom the engineering strain was analyzed by SDS-PAGE in which the molecular weight of the interest protein expressed in expression system was consistent with the deduced one. The expression was maximum when induced with 1.0mmol/L IPTG for 4hours.Western-blot analysis indicated that the recombinant proteases fused with His from prokaryotic expression systems could be recognized by the anti-His antibody and it also could react with the pre-treated rabbit anti-schistosome sera.Conclusion: Prokaryotic expression system of Sj31 gene were successfully constructed in our study.The recombination protein peptide showed fine antigenicity.