Cloning, Expression And Activity Analysis Of Single Chain Variable Fragment (scFv) Against Carbaryl

Variable regions of light (324bp) chains (VL) and heavy (360bp) chains (VH) of antibody genes were cloned from hybridoma that secreting specific antibody against carbaryl using generate primers. The VL and VH were cloned into pGEM-T-Easy and sequenced. Specific primers synthesized based on the sequenced fragments were used to amplified single chain variable fragment (scFv) which was composed of729bp. The scFv was subcloned into pET30a and transformed into E. coli BL21(DE3) for recombinant expression. SDS-PAGE results indicated that the recombinant expressed scFv formed inclusion bodies and the molecular weight was about33kDa. Dialysis was chosen to renature scFv activity and different renaturation conditions were selected to obtain more soluble proteins and the results demonstrated that48h,200μg/mL initiative protein concentration and renaturation reagent containing arginine were the optimal conditions for renaturation. His-Bind resin was used to purify scFv. The purified recombinant antibodies presented good affinity and specificity base on competitive ELISA.In order to obtain soluble recombinant scFv with high affinity and specificity, another vector and signal peptides of other gene were introduced to construct recombinant plasmids. scFv was amplified from recombinant plasmid scFv-pET30a and cloned into pET26b. The new recombinant vector was transformed into BL21(DE3) for recombinant expression. Small amount of soluble scFv was expressed under conditions of20℃and0.05mmol/L IPTG and the molecular weight was30kDa. Affinity and specificity of purified soluble antibodies by His-Bind resin was not better than renatured scFv based on results of competitive ELISA. Signal peptides of organophosphate hydrolase gene were introduced to construct recombinant vector OPH-scFv-pET26b and the plasmid was transformed into BL21(DE3) for recombinant expression. But the fusion expressed protein also formed inclusion body which molecular weight was about40kDa.