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

BACKGROUND & OBJECTIVE: Single chain Fv(scFv) has been employed as a favorable targeting carrier in the therapy and diagnosis of tumors due to its advantages in relatively low immunogenity and stronger penetrance to tumor tissues over intact mAb. This study was designed to recombine the genes from the variable regions of light chain and heavy chain of ND-1, a monoclonal antibody against human colorectal carcinoma, by a short peptide (Gly4Ser)3 to construct the ND-1scFv gene. The ND-1scFv protein was expressed in Escherichia coli.

METHODS

VH and VL 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 splicing PCR to obtain the ND-1scFv gene. The latter was cloned into the expression vector PET-28a(+) and induced by IPTG to express a fusion protein scFv and His-tag in E. coli BL-21. The expressed product was purified by affinity chromatography using Ni-NTA resin and its immunoactivity was analyzed using ELISA.

RESULTS

Sequence analysis showed that scFv gene consisted of 732 bp, among them, 354 bp for heavy chain gene, located upstream of scFv gene, and 330 bp for the light chain gene, located donstream. SDS-PAGE analysis showed that the relative molecular weight of fusion protein is 30 kDa which was consistent with the theoretically predicted value. scFv expression was in the form of an inclusion body, and SDS-PAGE analysis of the purified scFv showed 94% purity. ELISA analysis revealed that scFv had equal immunoreactivity to the parent ND-1 antibody.

CONCLUSIONS

ND-1scFv gene against human colorectal carcinoma was successfully constructed, and functionally expressed in E. coli.