A method for constructing reshaping single-domain antibody.
The aim of this research was to demonstrate a novel and practical method for constructing reshaping Single-domain antibodies. Different from other methods, our method does not need to model the configuration of antibodies with specific sequences to determine the sequences of human acceptor FRs and then determine which amino acid residues in human acceptor FRs should be substituted. Most importantly, reshaping and enhancing the antigen binding affinity shared one procedure at the same time. Using this method, the reshaping anti-CD28 single-domain antibodies were constructed. According to the amino acid sequence of a mouse anti-human CD28 monoclonal antibody VH, two most homologous sequences of human antibodies were selected from GenBank and one of them was used as a main framework region for constructing the reshaping antibody. Before the original mouse antibody CDRs were inserted into the human acceptor FRs, some amino acid residues which were different from those of the original mouse antibody in the corresponding positions of the human acceptor FRs were determined or alternatively mutated by their conservative properties in Kabat classification. When the synthesized nucleotide fragments in different length were spliced by overlap PCR into the entire reshaping genes, Taq DNA polymerase and high Mg2+ concentration were used to introduce more mutation in FRs and CDRs randomly. A phage library was constructed using these PCR products and several reshaping Single-domain antibodies with high antigen binding affinity were selected after three rounds of panning. Two of them were expressed in E. coli BL21 (DE3). The antigen-binding affinity of refolded proteins was still in a high level measured by ELISA. These results suggested that this method was feasible and efficient for constructing reshaping Single-domain antibodies.
Cheng
Ju-Long
JL
Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China.
Wang
Xiang-Bin
XB
Zhang
Zhong
Z
Liu
Jing
J
Yao
Xin-Sheng
XS
Huang
Hua-Liang
HL
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000235:genetics*; D000915:Antibody Affinity; D018106:CD28 Antigens / Q000276:immunology*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D019151:Peptide Library
D000911:Antibodies, Monoclonal; D018106:CD28 Antigens; D019151:Peptide Library