[Construction and expression of single-chain antibody of neutralizing monoclonal antibody 13D8 against hepatitis E virus].Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2004 Sep; 20(5):556-9.XB
To weaken the immunogenicity of the neutralizing monoclonal antibody (mAb) 13D8 against hepatitis E virus and express its scFv.
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.
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.
The scFv constructed from V(H) and V(L) genes of mAb 13D8 with immunological activity was successfully expressed.