奶牛源金黄色葡萄球菌的分离鉴定及氟苯尼考耐药基因fexA的克隆

发布时间：2018-04-08 07:13

本文选题：金黄色葡萄球菌　切入点：氟苯尼考　出处：《动物医学进展》2017年12期

【摘要】：从100个奶牛场环境及脓汁样品中分离、鉴定了9株奶牛源耐氟苯尼考金黄色葡萄球菌,分别提取质粒DNA和基因组DNA,通过特异性引物进行PCR扩增氟苯尼考耐药基因fexA,以质粒DNA为模板的PCR分别扩增出了1 446bp特异性目的片段,而以基因组DNA为模板的PCR未扩增出该目的片段。将该目的片段克隆到pET-32(a)载体上,成功构建了pET-fexA质粒载体,将质粒载体转入BL21中,药物敏感性试验显示构建的pET-fexA/BL21基因工程菌对氯霉素和氟苯尼考高度耐药。同时,fexA阳性金黄色葡萄球菌分离株对氯霉素和氟苯尼考的耐药性显著高于质控菌株,说明fexA基因贡献细菌对氯霉素和氟苯尼考的交叉耐药。成功构建了一个基因背景清楚且对氟苯尼考耐药的细菌模型,排除了细菌中其他氟苯尼考耐药基因或泵出蛋白对fexA基因贡献细菌耐药的影响,为fexA基因编码的蛋白定位研究奠定了基础。
[Abstract]:Nine strains of staphylococcus aureus resistant to florfenicol were isolated from 100 dairy farm environment and pus samples.Plasmid DNA and genomic DNA were extracted, and florfenicol resistance gene fexA was amplified by PCR with specific primers. The 1 446bp specific target fragment was amplified by PCR using plasmid DNA as template.However, the PCR with genomic DNA as template did not amplify the target fragment.The target fragment was cloned into pET-32a) vector and pET-fexA plasmid vector was successfully constructed. The plasmid vector was transferred into BL21. The drug sensitivity test showed that the constructed pET-fexA/BL21 gene engineering strain was highly resistant to chloramphenicol and florfenicol.The resistance to chloramphenicol and florfenicol of Staphylococcus aureus isolates was significantly higher than that of quality control strains, indicating that fexA gene contributed to the cross resistance of bacteria to chloramphenicol and florfenicol.A bacterial model with a clear genetic background and resistance to florfenicol was successfully constructed, which excluded the effect of other florfenicol resistance genes or pump proteins on the drug resistance of bacteria contributing to the fexA gene.It lays a foundation for the study of protein localization of fexA gene.
【作者单位】：四川省畜牧科学研究院;动物遗传育种四川省重点实验室;
【基金】：四川省畜牧科学研究院基本科研业务专项(SASA2014A14)
【分类号】：S852.611

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