Cloning and expression of single chain Fv gene against human colorectal carcinoma

Background Objective:Single chain Fv(scFv)has been employed as a favorable targ eting carrier in the therapy and diagnosis of tumors d ue to its advantages in relatively lo w immunogenity and stronger penetra nce to tumor tissues over intact mAb.This study was designed to recombine the genes fr om the variable regions of light chain and heavy chain of ND-1,a monoclonal antibody against human colorectal carcinoma ,by a short peptide(Gly 4 Ser) 3 to construct the ND-1scFv gene.The ND-1scFv protein was expressed in Escherichia coli.Methods:V H and V L gene were amplified from hybridoma cell IC-2,secreting monoclonal antibody ND-1,by RT-PCR,and then were connected to each other by a linker peptide using extension overlap spl icing PCR to obtain the ND-1scFv gene.The latter was cloned into the expre ssion vector PET-28a(+)and induced by IPTG to express a fusio n protein scFv and His-tag in E.coli BL-21.The expressed product was purified by affinity chromatography using Ni-NTA resin and its immun oactivity was analyzed using ELISA.Results:Sequence analysis showed that scFv g ene consisted of 732bp,among them,354bp for heavy chain gene,located up stream of scFv gene,and 330bp for the light chain gene,located donwstream.SDS-PA GE analysis showed that the relative molecular weight of fusion protein is 30kDa whi ch was consistent with the theoretic ally predicted value.scFv expression was in the form of an inclusion body,and SDS-PA GE analysis of the purified scFv show ed 94%purity.ELISA analysis revealed that scFv had equal immunoreactivity to the parent ND-1antibody.Conclusions:ND-1scFv gene against human colorectal carcinoma was successfu lly constructed,and functionally expressed in E.coli.