Preparation Of Anti-IL-4R Single-chain Antibody And Construction, Expression And Purification Of Fusion Protein With Melittin

Objective:Build anti-IL-4R scFv expression vector and express recombinant protein gene;Build anti-IL-4R scFv banding MLT gene expression vector and express recombinant fusion protein.Methods:1)Anti-IL-4R scFv recombinant protein was expressed and detected by phage surface display technique.2)The pET-32a-scFv expression vector of anti-IL-4R scFv gene was constructed,and was tranformed into E.coli BL21(DE3)and expressed,the anti-IL-4R scFv fusion protein expression was improved.3)Anti-IL-4R scFv gene and MLT gene were connected by the SOE-PCR technology,built the pET-32a-scFv-MLT recombinant plasmid,then transformed it into E.coli BL21(DE3)for inducible expression recombinant protein.4)The molecular weight and specificity of this two scFv recombinant proteins and scFv-MLT recombinant protein were detected by SDS-PAGE and Western Blot,then via purification and dialysis-dilution composite renaturation the recombinant protein,finallly gained the purificated objective protein.Results:1)The recombinant protein of anti-IL-4R scFv was expressed by phage surface display system in soluble protein type,its molecular mass was approximately 28KD,but the expression quantity was too low to purification.The recombinant protein of anti-IL-4R scFv was high expressed in the form of inclusion body by prokaryotic expression system of pET-32a vector and E.coli BL21(DE3).After purification and dialysis-dilution composite renaturation.The soluble protein of anti-IL-4R scFv was gained,its molecular mass was approximately 45KD.2)The connection of anti-IL-4R scFv gene and melittin(MLT)gene was successfully built,and the recombinant plasmid of pET-32a-scFv-MLT was constructed,then turned the recombinant plasmid into E.coli BL21(DE3)to express recombinant fusion protein.The results of SDS-PAGE and Western Blot detection showed that a high expression level of scFv-MLT protein which was observed in a form of inclusion body.After purification and dialysis-dilution composite renaturation gained the purificated objective protein,its molecular mass was approximately 52KD.Conclusion:1)The anti-IL-4R scFv recombinant protein was expressed by the phage surface display technique,but the expression quantity was too low to purification.2)The recombinant protein of anti-IL-4R scFv was expressed by prokaryotic expression system.3)The recombinant fusion protein of anti-IL-4R scFv-MLT was expressed by prokaryotic expression system.4)This study would be lay the foundation for the deeper research on anti-IL-4R and MLT biological target therapy.