Construction And Expression Of Recombinant Protein Of SIEA26-28kDa ScFv Of Schistosoma Japonicum And Human IL18

Objective To lay the work foundation for exploring the possibility of applying the specific single-chain Fv antibody combined with human IL-18 to targeting immunotherapy of schistosomiasis japonica through construction and expression of the recombinant plasmid of single-chain Fv antibody against Schistosoma japonicum immature egg 26-28kDa soluble antigen and human IL-18,and determination of the biological characteristics of the plasmid and the given fusion proteins.Method Based on the constructed plasmid of pET32a/ SIEA26-28kDa-EGFP-scFv and pGEM-T-CMV/IL18,PCR was performed to amplify the full length IL-18 gene fragment with special restriction enzyme site.The corresponding restriction enzymes then were used to digest this fragment and the pET32a/SIEA26-28kDa-EGFP-scFv plasmid.After isolation and purification of the desired IL-18 fragment and the backbone of pET32a/SIEA 26-28kDa-scFv,DNA ligase was employed to produce the recombinant construction.Through transforming the ligation product to competent cells,identifying the recombinant colon, purifying the aim plasmid and transforming this plasmid to Escherichia coli strain BL21,the expressing of the IL18-scFv recombinant protein under induction of IPTG,and the EGFP-scFv was conducted.These recombinant proteins were successfully verified by Western-Blot. Result1.Restriction enzymes digestion verified that the preserved plasmid is pET32a/SIEA26-28 kDa-EGFP-scFv;2.IL-18 gene fragment was successfully amplified by PCR;3.After restriction enzymes digestion,ligation by DNA ligase,and transformation to competent cells,pET32a/SIEA26-28kDa-IL18-scFv plasmid was successfully constructed;4.Induced by IPTG at different time points,the recombinant protein IL18-scFv and EGFP-scFv were expressed in Escherichia coli strain BL21,which were identified by Coomassie brilliant blue staining and Western Blot.Conclusion1.pET32a/SIEA26-28kDa-IL18-scFv plasmid was successfully constructed;2.The recombinant proteins were expressed after induced by IPTG.