Construction, Expression And Characterization Of Anti-idiotype Scfv Specific To Anti-PRRSV GP5 Antibody

Porcine reproductive and respiratory syndrome (PRRS) is an acute infectious disease caused by PRRS virus (PRRSV) infection.Our previous studies demonstrated that pigs infected with PRRSV produced auto-anti-idiotypic antibodies (auto-Ab2s) against idiotypic antibodies to M and GP5 protein, which may up- or down-regulate the immune responses against PRRSV infection. Anti-idiotype plays important roles in the immune responses against PRRSV. Our previous study has shown that the monoclonal anti-idiotype antibody (Mab2-5G2) specific to anti-PRRSV GP5 antibody can recognize and bind to Marc-145 cells. ScFv still keeps the binding specificity and affinity comparable to that of its parent antibody. The scFv strategy has become one of the popular methods in antibody engineering because of it's less immunogencity. Furthermore, it's small molecular size endows scFv with better tissue penetration and shorter half life in plasma, there, at present scFv has a wide range of application in research, diagnosis and therapy. To further understand the functional domain of Mab2-5G2 for binging to Marc-145 cells, we need to generate scFv of Mab2-5G2 containing the fragment of variable domain (Fv) . Variable light (VL) and variable heavy (VH) chain genes of Mab2-5G2 were generated from the hybridoma cells secreting Mab2-5G2. Primers were designed and synthesized according to amino acid sequence loaded in framework region and complementarity-determining region of mouse immunoglobulins. VL and VH gene were amplified and linked with a linker to form VH-linker-VL by overlapping-PCR. Sequence analysis showed that the VH and VL genes were spliced by 45 bases coding a (G4S)3 flexible linker. The length of scFv fragment was 762bp, VH fragment was 360bp encoding 120 amino acids and VL fragment was 357bp encoding 119 amino acids. The scFv gene was cloned into pEasy- E1 vector and constructed prokaryotic expressing plasmid pEasy-scFv. The plasmid was transformed into E.coli BL21(rosetta) and induced by IPTG.. The scFv antibody was expressed in form of inclusion body. The molecular weight of the fusion protein was about 30KD, which was equivalent to that estimated. After purification by Ni-resin, the activity of the pEasy-scFv protein was detected by western-blotting. The result of detection revealed that pEasy-scFv fusion protein had the activity of binding anti-His antibody and Ab3. The expressed scFv fusion proteins were finally renatured by PH(6～8.5)and Urea gradient(8mol/L～2 mol/L) at 16℃for 12h every gradient. The result revealed that refolded scFv conserved the same characteristics of specific recognition and binding to specific protein on Marc-145 cell. To induce Ab3 production in the serum, rabbits were immunized with Mab2-5G2 or scFv. Mab2-5G2 and scFv an react with each other's antibody in serum.