摘要
为探究奶牛乳房炎源性肺炎克雷伯菌(K.pn)黏附素MrkD蛋白对K.pn黏附奶牛乳腺上皮细胞的影响以及黏附素抗血清对阻断K.pn黏附奶牛乳腺上皮细胞的效果,本研究利用PCR扩增了K.pn MrkD基因,构建重组质粒pColdI-MrkD,将其转化至大肠杆菌后诱导表达,采用SDS-PAGE和western blot鉴定。结果显示,MrkD重组蛋白以可溶性形式表达,并具有较好的反应原性。将牛乳腺上皮细胞与1×10^(8)cfu/mL K.pn稀释液共同孵育后,利用间接免疫荧光试验检测K.pn对奶牛乳腺上皮细胞的黏附,结果显示K.pn可以黏附奶牛乳腺上皮细胞。利用K.pn稀释液孵育经不同浓度MrkD重组蛋白作用的奶牛乳腺上皮细胞后,通过对黏附细菌数的统计和分析,结果表明MrkD重组蛋白可以显著抑制K.pn黏附奶牛乳腺上皮细胞。利用MrkD重组蛋白抗血清和K.pn抗血清分别与K.pn作用后计数黏附的奶牛乳腺上皮细胞,结果显示,MrkD重组蛋白抗血清与K.pn抗血清均能有效阻断K.pn的黏附,且重组蛋白抗血清效果较好。本研究获得了MrkD重组蛋白,首次检测了其在K.pn黏附奶牛乳腺上皮细胞中的影响以及该重组蛋白抗血清在阻断K.pn黏附细胞中的作用,为探究阻断细菌黏附以及奶牛乳房炎亚单位疫苗的研究奠定基础。
The purpose of this study was to investigate the effect of adhesin MrkD protein of Klebsiella pneumoniae(K.pn)on the adhesion of K.pn to bovine mammary epithelial cells and the effect of the antiserum against adhesion factor on blocking K.pn adhesion to bovine mammary epithelial cells.In this study,the MrkD gene of Klebsiella pneumoniae was amplified by PCR,which was cloned into the pColdI vector.The positive recombinant plasmid pColdI-MrkD was identified by double enzyme digestion.Subsequently,the pColdI-MrkD was transformed into Escherichia coli for protein expression,which was confirmed by SDS-PAGE and western blot.The results showed that the recombinant protein MrkD was expressed in soluble form with good reactivity.After co-incubating bovine mammary epithelial cells with 1×10^(8)cfu/mL diluent of Klebsiella pneumoniae,an indirect immunofluorescence assay was used to detect the adhesion of K.pn to bovine mammary epithelial cells.It turned out that K.pn could adhere to bovine mammary epithelial cells.Bovine mammary epithelial cells treated with different concentrations of MrkD recombinant protein were incubated with K.pn diluent.Statistical analysis of the number of adherent bacteria showed that MrkD recombinant protein could significantly inhibit the adhesion of K.pn to bovine mammary epithelial cells.In addition,antiserum against MrkD recombinant protein and anti-K.pn serum were used to adhere to bovine mammary epithelial cells after treatment with K.pn,respectively.Both MrkD antiserum against recombinant protein and anti-K.pn serum could effectively block the adhesion of K.pn,and the blocking effect of antiserum against recombinant protein was better.In this study,MrkD recombinant protein was obtained,and its effect on K.pn adhesion to bovine mammary epithelial cells and the effect of recombinant protein antiserum on blocking adhesion were verified for the first time,which laid a foundation for the study of blocking bacterial adhesion and bovine mastitis subunit vaccine.
作者
崔琦
周雅坪
郭婷
武春霞
王宇琛
田广原
思汗
孙衍立
郝永清
CUI Qi;ZHOU Ya-ping;GUO Ting;WU Chun-xia;WANG Yu-chen;TIAN Guang-yuan;SI Han;SUN Yan-li;HAO Yong-qing(Laboratory of Veterinary Microbiology and Immunology,Inner Mongolia Agricultural University,Hohhot 010018,China;Hulunbuir Animal Disease Prevention and Control Center,Hulunbuir 021000,China;Linhe District Bureau of Agriculture and Animal Husbandry,Bayannaoer 015000,China)
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2021年第12期1312-1317,共6页
Chinese Journal of Preventive Veterinary Medicine
基金
自治区科技计划项目(31460664)。
