[Construction of anti-B7-H4-scFv library and screening and identification of anti-B7-H4-scFv].
Objective To construct the ribosome display library of anti-B7-H4 extracellular domain, and select the antibody with high specificity. Methods The cDNA of B7-H4 extracellular domain was amplified from A549 cells by reverse transcription PCR (RT-PCR). To express ectodomains of B7-H4, the sequence of B7-H4 gene, which encodes the B7-H4 extracellular domains, was inserted into plasmid pET-28a(+). The purified recombinant protein of B7-H4 extracellular domain was used to immunize BALB/c mice. The total RNA was extracted from the spleen of BALB/c mice which had been immunized with B7-H4 recombinant protein. The genes of VH, Vκ and VH/Vκ were amplified separately by RT-PCR and splicing by overlap extension PCR (SOE-PCR). The gene of VH/ Vκ was ligated into pUM19-T vector and the ligated sample was transformed into competent E.coli DH5α. The resulting plasmid was isolated and then subjected to sequencing to verify the gene sequence. TNT(R)T7 Quick for PCR DNA kit was used to translate and screen the anti-B7-H4-scFv in vitro from the ribosome display library. Western blotting and an indirect ELISA were performed to detect the specificity of anti-B7-H4-scFv. Results The right sequences of VH, Vκ and VH/Vκ were acquired, which were 439, 680 and 1098 bp in length, respectively. The analysis of specificity demonstrated that the anti-B7-H4-scFv screened from the ribosome display library had a high specific combining ability with B7-H4. Conclusion The experiment has successively constructed the ribosome display library of anti-B7-H4 extracellular domain, and selected the anti-B7-H4-scFv which has a high specific binding ability with recombinant protein of B7-H4 extracelluar domain.
College of Life Scienses, Nanjing Normal University, Jiangsu Key Laboratory of Molecular and Biotechnology, Nanjing 210046, China.
Luanluan
L
Shao
College of Life Scienses, Nanjing Normal University, Jiangsu Key Laboratory of Molecular and Biotechnology, Nanjing 210046, China.
Chaochao
C
Xu
College of Life Scienses, Nanjing Normal University, Jiangsu Key Laboratory of Molecular and Biotechnology, Nanjing 210046, China.
Hongshuai
H
Ji
College of Life Scienses, Nanjing Normal University, Jiangsu Key Laboratory of Molecular and Biotechnology, Nanjing 210046, China. *Corresponding author, E-mail: maoweiping@njnu.edu.cn.
Weiping
W
Mao
College of Life Scienses, Nanjing Normal University, Jiangsu Key Laboratory for Supramolecular Medicine Materials and Applications, Nanjing 210046, China.
Yingying
Y
Wang
College of Life Scienses, Nanjing Normal University, Jiangsu Key Laboratory of Molecular and Biotechnology, Nanjing 210046, China.
Xiaoqian
X
Liu
College of Life Scienses, Nanjing Normal University, Jiangsu Key Laboratory of Molecular and Biotechnology, Nanjing 210046, China.
Yanyan
Y
Zhu
D000818:Animals; D000915:Antibody Affinity; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D015723:Gene Library; D007147:Immunoglobulin Light Chains / Q000235:genetics / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D057127:Single-Chain Antibodies / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D060907:V-Set Domain-Containing T-Cell Activation Inhibitor 1 / Q000235:genetics* / Q000276:immunology
D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; D060907:V-Set Domain-Containing T-Cell Activation Inhibitor 1; C475826:VTCN1 protein, human