牛源抗金黄色葡萄球菌弹性纤维结合蛋白N端蛋白单链抗体的筛选及其体外抑菌的研究

Screening of Bovine-Derived Single-Chain Antibodies Against N-terminal Protein of Staphylococcus aureus Elastic Fiber Binding Protein and Their in vitro Antibacterial Properties

由金黄色葡萄球菌(S.aureus)引发的奶牛乳腺炎是造成国内外奶牛产业经济损失的重要疾病之一。为了获得牛源抗S.aureus弹性纤维结合蛋白N端蛋白(nEbpS)的单链抗体(scFv),本研究利用噬菌体展示技术,将原核表达的nEbpS包被到固相载体酶标板上,以实验室前期构建好的抗牛源S.aureus噬菌体库为原始文库进行筛选,得到1株靶向nEbpS的高亲和力scFv,将得到的scFv构建原核质粒进行原核表达,获得了高纯度scFv蛋白,将其与S.aureus ATCC25923在含有Zn2+的Luria-Bertani培养基中共同孵育后发现,50μg/mL scFv组菌液浓度显著小于生理盐水组(P<0.05),结果表明,针对nEbpS的牛源scFv具有良好的抑菌能力,浓度为50μg/mL时即可显著抑制S.aureus ATCC25923的生长。本研究为S.aureus所导致的奶牛乳腺炎提供了一种新型免疫制剂,也为进一步研究nEbpS抗原的功能奠定了基础。

Mastitis in dairy cows caused by Staphylococcus aureus is one of the important diseases that cause economic losses in the dairy industry at home and abroad.In order to obtain the bovine anti-S.aureus elastic fibronectin N-terminal protein(nEbpS)scFv,this study used phage display technology to coat the prokaryotic expression of nEbpS onto a solid-phase vector ELISA plate,which was constructed in the laboratory.The anti-bovine S.aureus phage library was the original library for screening,and a high-affinity scFv targeting nEbpS was obtained.The obtained scFv was constructed into a prokaryotic plasmid for prokaryotic expression,and a high-purity scFv protein was obtained.After co-incubation of S.aureus in Luria-Bertani medium containing Zn2+,it was found that the concentration of the 50μg/mL scFv group was significantly lower than that of the normal saline group(P<0.05),which indicated that the bovine scFv against nEbpS had good antibacterial ability,scFv significantly inhibited the growth of S.aureus ATCC25923 at a concentration of 50μg/mL.This study provided a new type of immune preparation for dairy cow mastitis caused by S.aureus,and also laid a foundation for further research on the function of nEbpS antigen.