| 牛源耐药性金黄色葡萄球菌的分离鉴定及黏附因子基因FnBP和clfA的表达分析 |
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| 引用本文：孙志华，刘 君，张 辉，刘 娟，许长萌，张华雷，陈创夫.牛源耐药性金黄色葡萄球菌的分离鉴定及黏附因子基因FnBP和clfA的表达分析[J].西北农业学报,2014,23(6):22~28 |
| DOI:10.7606/j.issn.1004-1389.2014.06.004 |
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| 基金项目:兵团博士基金(2011BB013)。 |
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| 中文摘要:为探究致奶牛乳房炎主要致病菌金黄色葡萄球菌（Staphylococcus aureus，S.aureus）的生物学特性，对新疆垦区部分奶牛场乳房炎奶样进行病原学分析，并对病原菌进行耐药性、毒力因子的表达分析。从乳房炎奶样中分离得到 S.aureus，采用血浆凝固酶试验、KB法、琼脂筛选法、生化鉴定法（VITEK32）、聚合酶链式反应等法进行鉴定。针对毒力基因 clfA（凝集因子A）和 FnBP（纤维结合素结合蛋白）进行特异性PCR扩增，用T/A克隆法将其插入pMD18-T载体，并构建原核表达载体pET-28a(+)-FnBP和pET-28a(+)-clfA。转化BL21（DE3）菌，经IPTG诱导表达，通过SDS-PAGE和Western blot分析鉴定表达产物。常规鉴定出葡萄球菌105株，其中金黄色葡萄球菌（S.aureus）62株，从62株S.aureus中检出耐药性金黄色葡萄球菌（MRSA) 13 株，检出率为20.10%，2 株未确检。经测序证实与 GenBank 中 FnBP 基因（J04151）和 clfA 基因（AB245457）序列同源性分别为100％和95.69%。SDS-PAGE显示，clfA和FnBP蛋白相对分子质量分别为45 ku和58 ku。Western blot 分析显示，clfA蛋白和 FnBP蛋白均可与金黄色葡萄球菌多克隆抗血清发生特异性反应。病畜奶样中MRSA检出率较高，新疆垦区部分牛场呈蔓延趋势，成功表达了clfA、FnBP蛋白，具有一定的免疫保护效果。 |
| 中文关键词:奶牛乳房炎 耐甲氧西林金黄色葡萄球菌 凝集因子A基因 纤维结合素结合蛋白基因 |
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| Isolation, Identification of Bovine S.aureus and Expression Analysis of FnBP and ClfA Gene |
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| Abstract:To research the resistance of Staphylococcus aureus of mastitis in dairy cows and expression of the gene encoding FnBP, clfA of Staphylococcus aureus, in this study, isolated S.aureus from the samples of in Xinjiang dairy cattle mastitis, and systematic researched the methicillin-resistant Staphylococcus aureus (MRSA). With coagulase test re-verification SA was isolated from mastitis in dairy cows, with KB, agar screening method, apparatus Identification Act (VITEK32), the polymerase chain reaction method re-verification MRSA, further amplifying the resistance genes clfA (agglutination factor A) and FnBP (fiber combined hormone binding protein), inserting into pMD18 -T vector by T/A cloning, and transforming the constructed recombinant plasmid pET-28a(+)-FnBP and pET-28a(+)-clfA into BL21(DE3)/pET system. The expressed product was identified by SDS-PAGE and Western blot. 62 strains of Staphylococcus aureus (SA) were identified from 105 strains, including MRSA 13 strains with the detection rate of 20.1%. The sequence of amplified FnBP and clfA gene showed 100% and 95.69% homology to FnBP of Staphylococcus aureus (J04151) and (AB245457) reported in GenBank, coding proteins with mass of molecule of 58 ku and 45 ku, respectively. The activity of recombinant protein was analyzed by Western blot. The highly detection rate of MRSA in milk samples of the sick livestock indicated that Staphylococcus aureus has a spreading trend in Xinjiang dairy cattle mastitis of the reclamation area. Staphylococcus aureus FnBP fusion protein and clfA fusion protein were successful expressed. This study laid a foundation for exploring the role of FnBP and clfA in pathopoiesis of bacteria and development of relevant vaccine. |
| keywords:Dairy cow mastitis MRSA clfA FnBP |
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