【摘要】 优化并合成牛支原体的p48基因,将其克隆到pET-32a(+)表达载体上后,转化大肠杆菌BL21(DE3),16℃低温诱导表达,获得可溶性表达的大小约66ku的重组P48蛋白。将纯化后的重组P48蛋白致敏戊二醛和鞣酸处理过的绵羊红细胞,建立了检测牛支原体抗体的间接血凝试验。重组P48蛋白致敏红细胞的最佳质量浓度是20~40μg/mL,牛支原体超免疫血清抗体效价达1∶2 048~1∶4 096。该方法对牛肺疫、牛巴氏杆菌病、牛病毒性腹泻病、布氏杆菌病、结核病、口蹄疫、牛生殖道支原体感染、里奇氏支原体感染、大肠杆菌病阳性血清的检测结果均为阴性。该方法的特异性为100%,敏感性为93.33%。与国外商品化的牛支原体ELISA试剂盒相比,平均符合率为96.15%。对833份田间血清和1 084份乳清进行检测,阳性率分别是15.37%和19.56%。结果表明,该方法敏感性高、特异性强和重复性好,可用于牛支原体抗体水平检测、牛支原体病的辅助诊断和流行病学调查。更多还原

【Abstract】 The p48 gene of Mycoplasma bovis was optimized,synthesized and cloned into the expression plasmid pET-32a(+).The recombinant plasmid pET-32a(+)-p48 was transformed into Escherichia coli BL21(DE3)and was then induced to express at 16℃.A soluble recombinant P48 protein with molecular weight of 66 ku was expressed and purified.To establish an indirect hemagglutination assay(IHA)for detection of antibodies against M.bovis,the purified P48 protein was injected into the sheep red blood cells(SRBC)treated with the glutaraldehyde and tannic acid.The titer of hyperimmune serum against M.bovis was up to 1∶2 048 to 1∶4 096.The concentration of P48 protein sensitizing SRBC was from 20 to 40μg/mL.IHA was employed to detect the antisera to contagious bovine pleuropneumonia,bovine pasteurellosis,bovine viral diarrhea,brucellosis,bovine tuberculosis,foot-and-mouth disease,M.bovigenitalium infection,M.leachii infection and E.coli infection,but all were negative.The analytical specificity and sensitivity of IHA were 100%and 93.33%,respectively.The coincidence rate of IHA was 96.15% when compared with the commercialized M.bovis ELISA Kit(BioX).Detection of 833 field serum samples and 1 084 milk samples with IHA revealed that the positive rates of antibodies to M.bovis was 15.37%and 19.56%,respectively.These results indicated that the established IHA is a robust tool for detection of antibodies to M.bovis,which will be also helpful for the diagnosis and sero-epidemiological investigation of M.bovisinfection.更多还原