Mihklepp K, Kivirand K, Nikopensius M, Peedel D, Utt M, Rinken T
Institute of Chemistry, University of Tartu, Ravila 14a, Tartu, Estonia.
Institute of Biomedicine and Translational Medicine, University of Tartu, Ravila 19, Tartu, Estonia.
Enzyme Microb Technol. 2017 Jan;96:135-142. doi: 10.1016/j.enzmictec.2016.10.009. Epub 2016 Oct 17.
Streptococcus uberis (S. uberis) is an important environmental pathogen causing mastitis in dairy cattle. Mastitis or mammary gland infection is the most common disease in milking cows and cause significant economic burden to farmers due to the reduction of the amount of milk and its quality. The development of rapid analytical methods for the determination of mastitis-causing pathogens in milk is of utmost importance for the early identification of the causes of mastitis and the beginning of timely treatment of cows. Combining in silico bioinformatic analysis and solid phase peptide synthesis using Fmoc chemistry, we have made two different peptides to mimic the adhesion protein of S. uberis, which is promoting the attachment of bacteria to epithelial cells. After purification with RP-HPLC, the peptides were conjugated with a larger carrier protein (KLH) and used for immunization of rabbits to produce specific antibodies. The separation of anti-S. uberis antibodies from rabbit blood antisera was carried out with affinity chromatography, using the synthetic peptides as affinity ligands. The purified antibodies showed high affinity and specificity towards S. uberis and were used for rapid bio-recognition and identification of S. uberis with an immunobiosensing system.
乳房链球菌是引起奶牛乳腺炎的一种重要环境病原体。乳腺炎或乳腺感染是泌乳奶牛中最常见的疾病，由于牛奶产量和质量下降，给养殖户造成了巨大的经济负担。开发快速分析方法来测定牛奶中引起乳腺炎的病原体，对于早期确定乳腺炎病因并及时开始治疗奶牛至关重要。结合计算机生物信息学分析和使用Fmoc化学的固相肽合成，我们制备了两种不同的肽来模拟乳房链球菌的粘附蛋白，该蛋白促进细菌与上皮细胞的附着。经反相高效液相色谱（RP-HPLC）纯化后，将这些肽与一种更大的载体蛋白（钥孔血蓝蛋白）偶联，并用于免疫兔子以产生特异性抗体。使用合成肽作为亲和配体，通过亲和色谱法从兔血抗血清中分离抗乳房链球菌抗体。纯化后的抗体对乳房链球菌表现出高亲和力和特异性，并用于通过免疫生物传感系统对乳房链球菌进行快速生物识别和鉴定。
