[Construction and expression of single-chain antibody of neutralizing monoclonal antibody 13D8 against hepatitis E virus]

Abstract

Aim: To weaken the immunogenicity of the neutralizing monoclonal antibody (mAb) 13D8 against hepatitis E virus and express its scFv.

Methods: The V(L) and V(H) genes were cloned by RT-PCR from hybridoma cells producing mouse mAb. And then V(H)-linker-V(L) fragment (scFv) was constructed and cloned into vector pTO-T7. The scFv protein was expressed in E.coli. The activity of expressed scFv was detected by ELISA and Western blot.

Results: SDS-PAGE analysis showed that the scFv was highly expressed mostly in the form of inclusion body in E.coli, and the yield was up to 26.8% of the total bacteria protein. The results of indirect ELISA and Western blot showed that the expressed scFv could bind specifically to a recombinant protein in OFR2 region of HEV (NE2). The result of competitive ELISA demonstrated that the epitope recognized by the scFv was the same as that by mAb 13D8.

Conclusion: The scFv constructed from V(H) and V(L) genes of mAb 13D8 with immunological activity was successfully expressed.