Preparation of a single-chain variable fragment against the IBV nucleocapsid protein.

A single chain variable fragment (scFv) was amplified from the hybridoma cell line 6H3 specific to IBV nucleocapsid protein by using splice-overlap extension polymerase chain reaction (SOE-PCR) through the flexible (Gly(4)Ser) (3) linker.The scFv gene was clone into the prokaryotic expression vector pET-30a,transformed into E.coli BL21 (DE3),and protein expression was induced by IPTG.The recombinant protein was purified using ProBondTM Purification System and was refolded by urea dialysis.Sandwich enzyme-linked immunosorbent assay (ELISA) and western blotting were performed to detect the reactivity between the scFv and IBV.The results indicated that scFv could react specifically with IBV nucleocapsid protein.