基于奶牛乳腺炎无乳链球菌PI-2a菌毛岛屿rAP1-BP-AP2重组蛋白的间接ELISA方法的建立
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摘要
为建立一种快速准确高效的检测奶牛乳腺炎无乳链球菌PI-2a菌毛岛屿抗原相应抗体的检测方法,本研究以牛乳腺炎无乳链球菌Ia型PI-2a菌毛岛屿辅助蛋白AP1、AP2及骨架蛋白BP 3基因串联表达的重组蛋白为ELISA包被抗原,通过方阵滴定法优化反应条件,建立了奶牛乳腺炎无乳链球菌抗体的间接ELISA检测方法。优化后抗原的最佳包被量为5.0μg/孔,血清样品最佳稀释倍数为1∶40,酶标二抗最佳稀释倍数为1∶5 000。对S.agalactiae、S.pyogens、E.coli、S.aureus、S.epidermidis阳性血清进行检测结果显示,后4种阳性对照血清未出现阳性反应,表明该方法具有良好的特异性;对已知牛无乳链球菌阳性血清倍比稀释后进行检测时,当稀释倍数达到1:12 800时仍出现阳性结果,表明该方法具有较高的敏感度;重复性试验显示批内变异系数为2.41%~8.89%,批间试验变异系数为7.53%~10.46%;用该方法对426份临床样品进行奶牛无乳链球菌抗体检测,结果显示,其样品阳性检出率为45.07%。本研究首次基于乳腺炎无乳链球菌PI-2a菌毛岛屿三联重组蛋白建立的间接ELISA方法为奶牛无乳链球菌的快速检测提供了一种准确、可靠的检测方法。
To establish a rapid and specific method for detection of antibodies against Streptococcus agalactiea of dairy cow mastitis, an indirect ELISA was developed with recombinant fusion protein of fimbriae protein AP1, AP2 and auxiliary island skeleton protein BP(rAP1-BP-AP2) as coating antigen. The optimized reaction conditions of the assay included 5.0μg/well for coating antigen, the 1:40 for sample serum dilution, and 1:5,000 for the HRP conjugated antibody dilution. The assay showed no cross-reactivity with the reference sera of S.pyogens, E.coli, S.aureus and S.epidermidis, so it showed that the assay was specificity. The results of the sensitivity test showed that a positive result could be obtained even if the serum dilution reached 1:128,000, indicting the high sensitivity of the method. Repeatability tests showed that the coefficient of variation of serum samples within and among runs were 2.41% to 8.89% and 7.53% to 10.46%, respectively. The detection rate for 426 clinical samples was 45.07%. The indirect ELISA method established in this study could provide a more reliable diagnostic method for S.agalactiae induced dairy mastitis.
To establish a rapid and specific method for detection of antibodies against Streptococcus agalactiea of dairy cow mastitis, an indirect ELISA was developed with recombinant fusion protein of fimbriae protein AP1, AP2 and auxiliary island skeleton protein BP(rAP1-BP-AP2) as coating antigen. The optimized reaction conditions of the assay included 5.0μg/well for coating antigen, the 1:40 for sample serum dilution, and 1:5,000 for the HRP conjugated antibody dilution. The assay showed no cross-reactivity with the reference sera of S.pyogens, E.coli, S.aureus and S.epidermidis, so it showed that the assay was specificity. The results of the sensitivity test showed that a positive result could be obtained even if the serum dilution reached 1:128,000, indicting the high sensitivity of the method. Repeatability tests showed that the coefficient of variation of serum samples within and among runs were 2.41% to 8.89% and 7.53% to 10.46%, respectively. The detection rate for 426 clinical samples was 45.07%. The indirect ELISA method established in this study could provide a more reliable diagnostic method for S.agalactiae induced dairy mastitis.
引文
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[5]刘洋.BMSA菌毛岛屿PI-2a亚基AP1抗原表位序列原核表达产物的抗原性研究[D].通辽:内蒙古民族大学,2013.
[6]杜长智.牛乳腺炎无乳链球菌PI-2A菌毛岛屿AP2亚基基因B细胞表位序列的克隆与原核表达产物的抗原性研究[D].通辽:内蒙古民族大学,2016.
[7]白文丽.BMSA菌毛岛屿PI-2a BP亚基基因抗原表位的克隆及原核表达产物的抗原性研究[D].通辽:内蒙古民族大学,2017.
[8]Perichon B,Szili N,du Merle L,et al.Regulation of PI-2b pilus expression in hypervirulent Streptococcus agalactiae ST-17BM110[J].PLo S One,2017,12(1):e0169840
[9]吴金花,布日额,王金良,等.基于奶牛乳腺炎无乳链球菌rS ip-pgk-FbsA融合蛋白的间接ELISA方法的建立[J].中国预防兽医学报,2016,3(38):230-234.
[2]Rosini R,Rinaudo C D,Soriani M,et al.Identification of novel genomic islands coding for antigenic pilus-like structures in Streptococcus agalactiae[J].Mol Microbiol,2006,61(1):126-141.
[3]Konto-Ghiorghi Y,Mairey E,Mallet A,et al.Dual role for pilus in adherence to epithelial cells and biofilm formation in Streptococcus agalactiae[J].PLo S Pathog,2009,5(5):e1000422.
[4]布日额,王金良,吴金花,等.牛乳腺炎无乳链球菌PI-2a菌毛岛辅助蛋白AP1、BP主要抗原域串联表达及其免疫活性鉴定[J].中国病原生物学杂志,2017,(12)7:609-613.
[5]刘洋.BMSA菌毛岛屿PI-2a亚基AP1抗原表位序列原核表达产物的抗原性研究[D].通辽:内蒙古民族大学,2013.
[6]杜长智.牛乳腺炎无乳链球菌PI-2A菌毛岛屿AP2亚基基因B细胞表位序列的克隆与原核表达产物的抗原性研究[D].通辽:内蒙古民族大学,2016.
[7]白文丽.BMSA菌毛岛屿PI-2a BP亚基基因抗原表位的克隆及原核表达产物的抗原性研究[D].通辽:内蒙古民族大学,2017.
[8]Perichon B,Szili N,du Merle L,et al.Regulation of PI-2b pilus expression in hypervirulent Streptococcus agalactiae ST-17BM110[J].PLo S One,2017,12(1):e0169840
[9]吴金花,布日额,王金良,等.基于奶牛乳腺炎无乳链球菌rS ip-pgk-FbsA融合蛋白的间接ELISA方法的建立[J].中国预防兽医学报,2016,3(38):230-234.