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


CONCLUSIONS
The scFv constructed from V(H) and V(L) genes of mAb 13D8 with immunological activity was successfully expressed.
Li
Li-feng
LF
The Key Laboratory of the Ministry of Education for Cell Biology and Tumor Cell Engineering, Xiamen University, Xiamen 361005, China.
Luo
Wen-xin
WX
Gu
Ying
Y
Zhu
Zi-heng
ZH
Wang
Ying-bin
YB
Zhang
Jun
J
Xia
Ning-shao
NS
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics* / Q000378:metabolism; D000918:Antibody Specificity; D001483:Base Sequence; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000235:genetics; D000939:Epitopes; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D050438:Genes, Immunoglobulin Heavy Chain; D050439:Genes, Immunoglobulin Light Chain; D005822:Genetic Vectors; D016752:Hepatitis E virus / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D006825:Hybridomas; D007128:Immunoglobulin Fragments / Q000235:genetics* / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000235:genetics* / Q000378:metabolism; D002479:Inclusion Bodies / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D014162:Transfection
D000911:Antibodies, Monoclonal; D004279:DNA, Viral; D000939:Epitopes; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; C055966:immunoglobulin Fv