title: Construction of scFv that bind both fibronectin-binding protein A and clumping factor A of Stapylococcus aureus.
abstract: Bovine mastitis (BM) causes significant losses to the dairy industry. Vaccines against the causative agent of BM, Staphylococcus aureus, do not confer adequate protection. Because passive immunization with antibodies permits disease prevention, we constructed a recombinant single-chain antibody (scFv) against fibronectin-binding protein A (FnBPA) and clumping factor A (ClfA), two important virulence factors in S. aureus infection. The DNA coding sequences of the variable heavy (VH) and variable light (VL) domains of antibodies produced in the peripheral blood lymphocytes of cows with S. aureus-induced mastitis were obtained using reverse transcription and polymerase chain reaction, and the VH and VL cDNAs were assembled in-tandem using a DNA sequence encoding a (Gly4Ser)3 peptide linker. The scFv cDNAs were cloned into the pOPE101 plasmid for the expression of soluble scFv protein in Escherichia coli. The binding of the scFvs to both FnBPA and ClfA was confirmed using an indirect ELISA and Western blotting. The DNA sequences of the framework regions of the VH and VL domains were highly conserved, and the complementarity-determining regions displayed significant diversity, especially in CDR3 of the VH domain. These novel bovine antibody fragments may be useful as a therapeutic candidate for the prevention and treatment of S. aureus-induced bovine mastitis.
authors.affiliation: Shanghai Key Laboratory of Veterinary Biotechnology, School of Agriculture and Biology, Shanghai JiaoTong University, 800 Dongchuan Road, Shanghai 200240, China.
authors.forename: Man
authors.initials: M
authors.lastname: Wang
authors.affiliation: Shanghai Key Laboratory of Veterinary Biotechnology, School of Agriculture and Biology, Shanghai JiaoTong University, 800 Dongchuan Road, Shanghai 200240, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Shanghai Key Laboratory of Veterinary Biotechnology, School of Agriculture and Biology, Shanghai JiaoTong University, 800 Dongchuan Road, Shanghai 200240, China.
authors.forename: Benqiang
authors.initials: B
authors.lastname: Li
authors.affiliation: Shanghai Key Laboratory of Veterinary Biotechnology, School of Agriculture and Biology, Shanghai JiaoTong University, 800 Dongchuan Road, Shanghai 200240, China. Electronic address: zhu_jg@sjtu.edu.cn.
authors.forename: Jianguo
authors.initials: J
authors.lastname: Zhu
mesh_terms: D018829:Adhesins, Bacterial / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D002417:Cattle; D003030:Coagulase / Q000235:genetics* / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D007963:Leukocytes, Mononuclear / Q000276:immunology; D008414:Mastitis, Bovine / Q000276:immunology* / Q000382:microbiology; D030781:Organisms, Genetically Modified / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013203:Staphylococcal Infections / Q000276:immunology / Q000382:microbiology / Q000662:veterinary*; D013211:Staphylococcus aureus / Q000235:genetics* / Q000378:metabolism
chemical_list: D018829:Adhesins, Bacterial; C515944:ClfA protein, Staphylococcus aureus; D003030:Coagulase; D018076:DNA, Complementary; D011994:Recombinant Proteins; C078635:fibronectin-binding proteins, bacterial
keywords: Bovine mastitis (BM); Clumping factor A (ClfA); Fibronectin-binding protein A (FnBPA); Single-chain antibody (scFv); Stapylococcus aureus; Western blotting