摘要
为分析原核表达的金黄色葡萄球菌ClfA部分基因表达产物的免疫原性,作者采用PCR方法从奶牛乳房炎病例中分离的金黄色葡萄球菌中克隆出ClfA基因的部分片段,测序后用DNAman软件进行抗原位点分析。构建原核表达质粒ClfA-pET-28a+,转化BL21(DE3)菌,经IPTG诱导表达。用His-bind柱纯化表达蛋白,加弗氏佐剂混匀后免疫小鼠。小鼠分娩后乳腺内攻菌,检查乳腺组织病理变化,乳腺组织中金葡菌数量和TNF-α水平来评价原核表达蛋白的免疫保护效果。结果显示转化BL21(DE3)菌,经IPTG诱导可以表达ClfA蛋白。原核表达蛋白抗原免疫小鼠可以显著降低乳腺组织内金葡菌数量,TNF-α水平也显著低于对照组和全菌蛋白抗原组,提示原核表达的ClfA蛋白免疫小鼠可以减少乳腺内金葡菌定植的数量,减轻乳腺内感染造成炎症的严重程度。本研究表明原核表达金黄色葡萄球菌ClfA蛋白有一定的免疫保护效果,为奶牛乳房炎疫苗的研究提供科学依据。
This experiment was conducted to study the immunogenicity of partial ClfA gene product of Staphylococcus aureus in mice.The region of clumping factor A（ClfA） gene was amplified by PCR from Staphylococcus aureus causing mastitis in dairy cows,and sequenced.The epitopes were analyzed by DNAman software.Then the fragment was inserted into the vector pET-28a（＋） constructed the recombinant plasmid ClfA-pET-28a＋.The plasmid was transformed into the host cell BL21（DE3）,and the protein was expressed in BL21（DE3） after being induced by IPTG.The target protein was purified by His-bind chromatography.Mice were immunized with the purified protein emulsified with Freund＇s adjuvant.Challenge test was performed by injecting S.aureus via the mammal glands to mice after parturition.And the immunogenicity was evaluated by detecting the pathological change,the concentration of TNF-α,the amounts of S.aureus in per-gram mammal gland tissues and the antibody levels.The results showed that the expressed protein was ClfA protein of S.aureus,and the protein had significant effects on improving the pathological change,reducing the concentration of TNF-α and the amounts of S.aureus in mammal tissues in mice.These demonstrated that the prokaryotic expressed protein of ClfA have a better immunoprotection effect against S.aureus,and provide a reference for the study on cows.
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2010年第12期1584-1591,共8页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
兵团博士资金(2008JC08)
