[Cloning of the variable region genes from hybridoma against HAAH and then construction and expression of anti-HAAH scFv].
OBJECTIVE
Construction and expression of anti-HAAH single chain variable fragment (scFv) by cloning of the variable region genes from anti-HAAH hybridoma cells G3/F11.


METHODS
Total RNA was extracted from hybridoma cells G3/F11. By RT-PCR, murine V(H) and V(L) genes of mAb were amplified respectively. Then, They were assembled into V(H)-linker-V(L) scFv template by SOE-PCR and anti-HAAH scFv was express in E.coli by constructed pHEN 1-anti-HAAH vector. The expression of anti-HAAH scFv were detected by SDS-PAGE and Western blotting and the binding activity were demonstrated by ELISA.


RESULTS
The analysis of DNA sequencing shown that the full-length of constructed scFv gene was 744 bp, encoding 248 amino acids. Moreover, the V(H) and V(L) genes were functional antibody variable region genes, as there were four FRs and three CDRs in both of them. By SDS-PAGE and Western blotting, the expression level of anti-HAAH scFv were detected. The expression level of pHEN 1-anti-HAAH scFv, which was expressed in E.coli HB2151, was 7.8% in total E.coli protein and were existed in soluble protein mainly. By indirect ELISA detection with HAAH protein, the binding activity of soluble anti-HAAH scFv was very well.


CONCLUSIONS
The murine V(H) and V(L) genes of mAb against HAAH have been cloned successfully and anti-HAAH scFv have been constructed and expressed. Besides, the scFv could be further studied about their biological activity and application, due to their high affinity shown in preliminary detection.
Wang
Hua
H
Faculty of Life Sciences, Northwestern Polytechnical University, Xi'an 710072, China.
Xue
Xiao-ping
XP
Lei
Ying-feng
YF
Song
Kai
K
Hu
Yan-ting
YT
Wang
Wei
W
Yang
Hui
H
D000818:Animals; D015153:Blotting, Western; D003001:Cloning, Molecular / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D006825:Hybridomas; D051379:Mice; D006899:Mixed Function Oxygenases / Q000276:immunology*; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D057127:Single-Chain Antibodies / Q000032:analysis / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification
D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; D006899:Mixed Function Oxygenases; C058291:aspartic acid 2-oxoglutarate-dependent dioxygenase