Construction and characterization of phage display library: recognition of mouse serologically detected male (SDM) antigen.
Improvement of animal embryo sexing depends upon high-titer serologically detected male (SDM) antibody fragments. SDM sera collected from isogenic C57BL/7 female mice after inoculation with male spleen cells were characterized and used for construction of a recombinant Fab antibody library against SDM antigen, and used for analysis of the binding capacity and specificity to SDM antigen. The heavy-chain Fd and full-length light-chain kappa were amplified by RT-PCR from a mouse (#6) that'ed high-titer antiserum. The amplified product was inserted into the pComb3 vector followed by co-infections with the help phage VCSM 13 for construction of the phage library, which gave 1.5x10(7) colonies with the titer of 3.2x10(11) pfu/ml by a recombination rate of 80%. Sequence analysis of the PCR products of plasmid DNA of E5 clones showed that V(H) and V(kappa) had common characteristics shared by other known variable region of antibodies. The Fab antibody libraries against SDM antigen were enriched by three cycles of affinity enrichment with male spleen cells, and two cycles of non-specific absorption with female spleen cells. The ELISA results showed that 9 of 15 clones had binding capacity to the SDM antigen. This is the first report on a phage display library of SDM antigen. The mouse Fab antibody library could be used for identifying SDM antigen, and for the development of sex determination of early embryos in mammals.
Wang
Naidong
N
College of Veterinary Medicine, Hunan Agricultural University, Changsha, Hunan Province 410128, PR China.
Xu
Daojun
D
Yuan
Anwen
A
Deng
Zhibang
Z
Xue
Liqun
L
Cui
Shuliang
S
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000097:blood; D000918:Antibody Specificity; D000941:Antigens / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D001483:Base Sequence; D004247:DNA; D005260:Female; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry; D007135:Immunoglobulin Variable Region / Q000737:chemistry; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D019151:Peptide Library; D013154:Spleen
D000906:Antibodies; D000941:Antigens; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D004247:DNA