基于TbpA蛋白的副猪嗜血杆菌抗体间接酶联免疫吸附试验检测方法的建立及应用

Indirect enzyme-linked immuno sorbent assay (ELISA) detection method for Haemophilus parasuis antibody based on TbpA protein

摘要:

【背景】副猪嗜血杆菌(Haemophilus parasuis，HPS)是猪革拉瑟氏病(Gl?sser’s Disease)的病原体，抗生素治疗和疫苗接种对于防控该病效果不明显，建立快速、准确的抗体检测方法尤为重要。【目的】利用表达纯化的HPS转铁结合蛋白A (TbpA)建立检测HPS抗体的间接酶联免疫吸附试验(ELISA)方法。【方法】PCR扩增HPS的tbpA基因并与原核表达载体pET-SUMO连接，通过PCR、双酶切及测序鉴定，将阳性重组质粒转入Escherichia coli Rosetta(DE3)，IPTG诱导表达，SDS-PAGE和Western Blot鉴定产物。以纯化的TbpA蛋白作包被抗原，通过各种反应条件优化，建立检测HPS抗体的间接ELISA方法，并进行临床应用与评价。【结果】该方法的最佳条件：包被抗原浓度为5 μg/mL，4 °C过夜；5%脱脂奶粉于37 °C封闭2 h；血清稀释度为1:1 600，37 °C孵育45 min；酶标二抗稀释度为1:5 000，37 °C作用30 min；显色时间为37 °C 5 min。该方法可特异性检测HPS抗体，阳性血清稀释至1:12 800仍可检出，与其他猪病原阳性血清均无交叉反应，批内及批间变异系数均小于10%，与全菌体包被间接ELISA、商品化ELISA试剂盒及Western Blot比较，该方法总符合率分别为90.00%、86.67%和90.00%，其中阳性符合率分别为90.38%、88.46%和92.00%，阴性符合率分别为87.50%、75.00%和80.00%。该方法检测免疫抗体阳性率为80.00%，感染抗体阳性率为19.50%。【结论】基于TbpA蛋白建立的HPS抗体间接ELISA检测方法，具有良好的特异性、敏感性和重复性以及临床应用的可靠性，为HPS的免疫监测和流行病学调查提供了技术手段。

Abstract:

[Background] Haemophilus parasuis (HPS) is the pathogen of Gl?sser’s disease. Antibiotic therapy and vaccination are not obvious for the prevention and control of the disease, it is particularly important to establish a rapid and accurate antibody detection method. [Objective] Using the expression and purified transferrin-binding protein (TbpA) of HPS to establish an indirect enzyme-linked immuno sorbent assay (ELISA) method for detecting HPS antibodies. [Methods] The tbpA gene of HPS was cloned and connected pET-SUMO prokaryotic expression vector. After identification by PCR, double enzyme digestion and sequencing, the positive recombinant plasmid was transformed into the receptor bacteria Escherichia coli Rosetta(DE3), and the expression was induced by IPTG, and the expression products were identified by SDS-PAGE and Western Blot. Using purified TbpA as coating antigen, the indirect ELISA method for detecting HPS antibody was established through the optimization of a series of reaction conditions, and its clinical application and evaluation were carried out. [Results] The optimum reaction conditions of this method were as follows: 5 μg/mL, 4 °C overnight concentration of coated antigen; 5% skim milk powder sealed at 37 °C for 2 h; The dilution of serum was 1:1 600, 37 °C incubation 45 min; The dilution of HRP was 1:5 000, 37 °C action 30 min; The optimal reaction time of TMB was 5 min. This method can specifically detect the HPS antibody. The positive serum is still positive after 1:12 800 dilution, but no cross-reactivity with other porcine pathogen positive serum. the coefficient of variation between in the batch and batches all less than 6%. The total coincidence rates of this method were 90.00%, 86.67% and 90.00% compared with the commercial ELISA kit and Western Blot, whole bacterial for indirect ELISA, among which the positive coincidence rates were 90.38%, 88.46%, 92.00%, and the negative coincidence rates were 87.50%, 75.00% and 80.00%, respectively. The positive rate of immune antibody was 80%, and the positive rate of infection antibody was 19.50%. [Conclusion] This study based on the recombinant TbpA established indirect ELISA detection method for HPS antibodies with good specificity, sensitivity and repeatability, as well as the reliability of clinical applications, provides a technical means for HPS immune surveillance and epidemiological investigation.