Penicillin-binding protein 3 of Streptococcus pneumoniae and its application in screening of β-lactams in milk.
Penicillin-binding protein 3 of Streptococcus pneumoniae and its application in screening of β-lactams in milk.
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DOI：
10.1016/j.ab.2013.07.042
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发表时间：
2013-11
影响因子：
2.9
通讯作者：
Jing Zhang;Zhanhui Wang;K. Wen;Xiao Liang;Jianzhong Shen
中科院分区：
文献类型：
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作者：
Jing Zhang;Zhanhui Wang;K. Wen;Xiao Liang;Jianzhong Shen
肺炎链球菌青霉素结合蛋白3（sPBP 3）可溶性形式在大肠杆菌中表达为六组氨酸融合蛋白。该蛋白质被纯化，并用于开发直接竞争形式的检测牛奶中的青霉素和头孢菌素的微孔板测定法。该测定基于牛奶中游离β-内酰胺抗生素对辣根过氧化物酶标记的氨苄青霉素（HRP-HRP）与sPBP 3结合的竞争性抑制。在优化条件下，大多数β-内酰胺类抗生素（11种青霉素类和16种头孢菌素类）可在欧盟规定的最大残留限量（MRL）浓度下检出。添加牛奶样品的分析表明，可接受的回收率范围为74.06%至106.31%，脱脂牛奶和全脂牛奶从63.97%至107.26%，变异系数（CV）小于16%。基于sPBP 3酶的检测方法对青霉素类和头孢菌素类抗生素具有较高的灵敏度和较宽的检测范围，有望成为牛奶中β-内酰胺类抗生素的快速检测方法。
The soluble form of penicillin-binding protein 3 (sPBP3∗) fromStreptococcus pneumoniaewas expressed inEscherichia colias a six-histidine fusion protein. The protein was purified and used to develop a microplate assay in direct competitive format for the detection of penicillins and cephalosporins in milk. The assay was based on competitive inhibition of the binding of horseradish peroxidase-labeled ampicillin (HRP–Amp) to the sPBP3∗by free β-lactam antibiotics in milk. Under optimized conditions, most of the β-lactam antibiotics (11 penicillins and 16 cephalosporins) could be detected at concentrations corresponding to the maximum residue limits (MRLs) set by the European Union. Analysis of spiked milk samples showed that acceptable recoveries ranged from 74.06 to 106.31% in skimmed milk and from 63.97 to 107.26% in whole milk, with coefficients of variation (CVs) less than 16%. With the high sensitivity and wide-range affinities to penicillins and cephalosporins, the developed assay based on sPBP3∗exhibited the potential to be a screening assay for fast detection of β-lactam antibiotics in milk.