[Construction and expression of scFv phage antibody from natural anti-keratin monoclonal antibody 3B4].
OBJECTIVE
To construct and express the scFv phage antibody from natural anti-keratin monoclonal antibody 3B4 in E.coli and then compare its antigenic specificity with that of the parental antibody 3B4.


METHODS
The V(H) and V(L) of 3B4 were amplified by PCR from pMD-18T- mAb 3B4 V(H) and pMD-18T- mAb 3B4 V(L), and then cloned into the phagemid pscMH. E.coli was transfected by recombined pscMH and superinfected by the helper phage VCSM13. Then the antigenic specificity of the scFv concentrated from the supernatant of the transfected E.coli was detected by indirect ELISA.


RESULTS
DNA sequence analysis showed that the scFv sequence was the same as the original sequence except that there was one base mutation in the FR1 of V(L). Indirect ELISA showed that the scFv phage antibody was polyreactive as mAb 3B4, but its antigenic specificity was not the same with that of mAb 3B4.


CONCLUSIONS
The scFv phage antibody from natural anti-keratin monoclonal antibody 3B4 was successfully constructed and expressed. Its antigenic specificity was different from that of its parental antibody 3B4.
Guan
Hai-hong
HH
Department of Dermatology, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, China.
Fu
Meng
M
Li
Wei
W
Xia
Ru-shan
RS
Wang
Gang
G
Liu
Yu-feng
YF
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D000918:Antibody Specificity; D001435:Bacteriophages / Q000235:genetics*; D001483:Base Sequence; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics* / Q000821:virology; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D007633:Keratins / Q000276:immunology*; D008969:Molecular Sequence Data; D009154:Mutation; D019151:Peptide Library; D016133:Polymerase Chain Reaction
D000906:Antibodies; D000911:Antibodies, Monoclonal; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D007633:Keratins