【摘要】 为制备牛结核分枝杆菌（MB）感染早期分泌性蛋白MPB₇₀₊₈₃的胶体金标记免疫层析试剂条,本研究将构建的重组大肠杆菌p ET30a（+）-MPB₇₀₊₈₃/BL21诱导表达,经纯化后免疫小鼠,制备MPB₇₀₊₈₃融合蛋白的单克隆抗体（MAb）,对MAb标记胶体金后制备检测试纸,并确定其特异性、灵敏度等技术指标。结果显示,获得分子量为39 ku的MPB₇₀₊₈₃融合蛋白,经纯化后重组蛋白的纯度>95%,经4次免疫BALB/c小鼠后,最终获得3株阳性杂交瘤细胞株,分别命名为4H6、5A2、4D11,小鼠腹水MAb效价分别达到1∶12 800,1∶12 800,1∶25 600。制备的腹水MAb均经纯化后浓度分别为2.1 mg/m L、1.8 mg/mL、2.5 mg/mL。对杂交瘤细胞经15代培养,MAb均具有良好的稳定性。利用4D11 MAb标记金颗粒,4H6 MAb包被检测线,并对不同培养物人工污染健康牛血清样品进行检测,结果显示,胶体金试纸对人工污染MB培养物的健康牛血清出现阳性反应,对无乳链球菌、金黄色葡萄球菌、大肠杆菌、沙门氏菌、副结核杆菌培养物人工污染的健康牛血清的检测均为阴性,最低可以检测到1∶2 500倍稀释的MB培养上清。本研究为牛结核分枝杆菌感染的快速检测提供可行技术手段。更多还原

【Abstract】 In this study, the colloidal gold immunochromatographictest strip was developed to detect secretory protein MPB₇₀+MPB₈₃ in early stage of Mycobacterium bovis（MB） infection. The prokaryotic fusion protein MPB₇₀₊₈₃ was expressed and purified in BL21 E.coli cells. Monoclonal antibody（MAb） against MPB₇₀₊₈₃ was prepared in the ascites of MPB₇₀₊₈₃ immunized mice. After MAb was labeled with colloidal gold, the test strip was prepared and its specificity, sensitivity and repeatability were evaluated. The results showed that MB₇₀₊₈₃ fusion protein had a molecular weight of 39 ku, the purity was more than 95%. Three positive hybridoma cell lines were obtained from MPB₇₀₊₈₃ immunized mice, namely 4 H6, 5 A2 and 4 D11 respectively. The MAb titers of the cell culture supernatants of the three hybridoma cells were 1 ∶1,024, 1 ∶1,024, 1 ∶2,048, respectively. MAb titers in ascites of mice reached 1 ∶12,800, 1 ∶12,800 and 1 ∶25,600, respectively. The concentration of MAb in purified ascites was 2.1 mg/mL, 1.8 mg/m L and 2.5 mg/m L, respectively. MAb production maintains stable after fifteen serial passage of hybridoma cells.To develop the test strip, MAb 4 D11 was used to label gold particles and MAb 4 H6 was used to coat the detection line. Results showed that colloidal gold test strip successfully detected the MB antigen in bovine serum with the lower detection limit of 1 ∶2,500. While the test strip was negative for Streptococcus agalactis, Staphylococcus aureus, Escherichia coli, Salmonella and Paratuberculosis antigen containing bovine serum. Our results provide a useful tool for rapid detection of Mycobacterium tuberculosisearly infection in cattle.更多还原