Wang Man, Zhang Yan, Li Benqiang, Zhu Jianguo
Shanghai Key Laboratory of Veterinary Biotechnology, School of Agriculture and Biology, Shanghai JiaoTong University, 800 Dongchuan Road, Shanghai 200240, China.
Shanghai Key Laboratory of Veterinary Biotechnology, School of Agriculture and Biology, Shanghai JiaoTong University, 800 Dongchuan Road, Shanghai 200240, China.
Res Vet Sci. 2015 Jun;100:109-14. doi: 10.1016/j.rvsc.2015.02.012. Epub 2015 Apr 7.
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.
牛乳腺炎（BM）给乳制品行业造成了巨大损失。针对BM病原体金黄色葡萄球菌的疫苗并不能提供足够的保护。由于抗体被动免疫可预防疾病，我们构建了一种针对纤连蛋白结合蛋白A（FnBPA）和凝聚因子A（ClfA）的重组单链抗体（scFv），这两种是金黄色葡萄球菌感染中的重要毒力因子。使用逆转录和聚合酶链反应获得了由金黄色葡萄球菌诱导的乳腺炎奶牛外周血淋巴细胞产生的抗体可变重链（VH）和可变轻链（VL）结构域的DNA编码序列，并使用编码（Gly4Ser）3肽接头的DNA序列将VH和VL cDNA串联组装。将scFv cDNA克隆到pOPE101质粒中，以便在大肠杆菌中表达可溶性scFv蛋白。使用间接ELISA和蛋白质印迹法确认了scFv与FnBPA和ClfA的结合。VH和VL结构域框架区的DNA序列高度保守，互补决定区表现出显著的多样性，尤其是在VH结构域的CDR3中。这些新型牛抗体片段可能作为预防和治疗金黄色葡萄球菌诱导的牛乳腺炎的治疗候选物。
