金黄色葡萄球菌PV-杀白细胞素的表达及其对奶牛乳腺上皮细胞的损伤作用  

Expression of Panton-Valenine Leukocidin from Staphylococcus aureus and Its Effects on Bovine Mammary Epithelial Cells

作　　者：马维武 周学章 MA Weiwu;ZHOU Xuezhang(Key Lab of Ministry of Education for Protection and Utilization of Special Biological Resourcesin Western China,Ningxia University,Yinchuan 750021,China)

机构地区：[1]宁夏大学西部特色生物资源保护与利用教育部重点实验室

出　　处：《畜牧兽医学报》2019年第10期2105-2112,共8页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金(31660728;31460676)

摘　　要：研究产PV-杀白细胞素(PVL)金黄色葡萄球菌ATCC49775、pvl缺陷株△pvl 49775和相应的回补株C-△pvl 49775以及体外重组的PVL(rPVL)对奶牛乳腺上皮细胞的损伤作用,为系统研究金黄色葡萄球菌感染奶牛乳腺上皮细胞的机制提供依据。采用原核表达的方法构建金黄色葡萄球菌LukS-PV和LukF-PV重组质粒并分别转化大肠杆菌BL21,诱导表达纯化后对其进行Western blot鉴定;用Live/Dead细胞荧光染色和Annexin V-FITC/PI双染法检测ATCC49775、△pvl 49775、C-△pvl 49775和rPVL对奶牛乳腺上皮细胞凋亡的影响。成功构建pET28a-LukS-PV、pET28a-LukF-PV重组表达载体,经诱导表达纯化后获得的LukS-PV浓度为0.6 mg·mL^-1,LukF-PV浓度为1.15 mg·mL^-1。rPVL主要诱导奶牛乳腺上皮细胞凋亡(P<0.01),金黄色葡萄球菌ATCC49775、△pvl 49775和C-△pvl 49775均能诱导奶牛乳腺上皮细胞明显凋亡和坏死,但pvl阳性菌株ATCC49775和C-△pvl 49775诱导的奶牛乳腺上皮细胞凋亡率和坏死率明显高于△pvl 49775(P<0.05)。结果表明原核表达的rPVL对奶牛乳腺上皮细胞表现出较强的毒性;pvl缺陷株△pvl 49775诱导奶牛乳腺上皮细胞凋亡和坏死的能力比pvl阳性菌株ATCC49775和C-△pvl 49775低,说明PVL在金黄色葡萄球菌感染奶牛乳腺上皮细胞过程中有重要作用。In order to provide a basis for systematically studying the mechanism of Staphylococcus aureus infection of bovine mammary epithelial cells,the damages of producing panton-valenine leukocidin(PVL)S.aureus ATCC49775,pvl-knockout mutant△pvl 49775,complemented mutant C-△pvl 49775 and recombinant PVL(rPVL)in vitro on bovine mammary epithelial cells were studied.Recombinant plasmids of S.aureus LukS-PV and LukF-PV were transformed into E.coli BL21,respectively.After expression and purification,they were identified by Western blot.The effects of ATCC49775,△pvl 49775,C-△pvl 49775 and rPVL on the apoptosis of bovine mammary epithelial cells were detected by Live/Dead cell fluorescence staining and Annexin V-FITC/PI double staining.The recombinant expression vectors pET28a-LukS-PV and pET28a-LukF-PV were successfully constructed.The concentration of LukS-PV obtained after induction and purification was 0.6 mg·mL^-1,and the concentration of LukF-PV was 1.15 mg·mL^-1.rPVL mainly induced apoptosis(P<0.01).Both S.aureus ATCC49775,△pvl 49775 and C-△pvl 49775 induced significant apoptosis and necrosis on bovine mammary epithelial cells,but ATCC49775 and C-△pvl 49775 induced worse(P<0.05).The results showed that rPVL of prokaryotic expression had a strong toxicity on bovine mammary epithelial cells.The pvl-knockout mutant△pvl 49775 induced the apoptosis and necrosis of the bovine mammary epithelial cells low than the pvl positive strain ATCC49775 and C-△pvl 49775.Our study indicates that the PVL plays an important role in S.aureus infected bovine mammary epithelial cells.

关 键 词：金黄色葡萄球菌 PV-杀白细胞素 毒力因子 奶牛乳腺上皮细胞 

分 类 号：S852.611[农业科学—基础兽医学]