地方株马驽巴贝斯虫Bc48截短体单克隆抗体的制备及应用  

Preparation and Application of Monoclonal Antibody against Truncated Bc48 of Babesia caballi Local Strains

作　　者：王盼举 樊新丽[1] 张梦圆[1] 宋晶晶 李敏 吾力江 巴音查汗[1] WANG Panju;FAN Xinli;ZHANG Mengyuan;SONG Jingjing;LI Min;WU Lijiang;Bayin Chahan(College of Animal Medicine, Xinjiang Agricultural University, Urumqi 830052, China)

机构地区：[1]新疆农业大学动物医学学院

出　　处：《畜牧兽医学报》2019年第9期1684-1689,共6页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金地区科学基金项目(31660711)

摘　　要：本研究旨在建立马驽巴贝斯虫抗体的快速、准确检测方法。以纯化的马驽巴贝斯虫Bc48基因片段的原核表达产物免疫6周龄雌性BALB/c小鼠制备单克隆抗体,并利用重组抗原与单克隆抗体建立间接竞争ELISA(CI-ELISA)方法检测马驽巴贝斯虫抗体。结果显示:制备出3株能稳定分泌单克隆抗体的杂交瘤细胞株,命名为1H2、7F4、11F4,通过对CI-ELISA条件筛选得出,抗原最佳包被浓度为0.19μg·mL^-1,单克隆抗体11F4的最佳工作浓度为1∶3.2×10^5,通过检测30份马驽巴贝斯虫阴性血清及20份阳性血清,确定该检测方法临界值为45%;特异性试验发现,该CI-ELISA方法不与感染马泰勒虫病的阳性血清发生反应,具有特异性;用所建立的CI-ELISA检测临床血清90份,与标准c-ELISA试剂盒总符合率为92.2%、阳性符合率92.1%、阴性符合率94.1%。试验结果表明,该CI-ELISA方法特异性强,敏感性高,稳定性和重复性好,操作简便。基于单克隆抗体(11F4)与重组蛋白(HIS-Bc48)所建立的CI-ELISA特异性、重复性好,可为新疆马驽巴贝斯虫的检测、监控提供有效手段。This study aimed to establish a rapid and accurate detection method for Babesia caballi. Six-week-old female BALB/c mice were immunized with purified Bc48 recombinant protein to prepare monoclonal antibodies, and a CI-ELISA method was established by Bc48 recombinant antigen and monoclonal antibody. The results showed that three hybridoma cell lines stably secreting monoclonal antibodies were prepared and named as 1 H2, 7 F4 and 11 F4. By screening for CI-ELISA conditions, the optimal coating concentration of the antigen was 0.19 μg·mL^-1, and the optimal working concentration of monoclonal antibody 11 F4 was 1:3.2×10^5. The CI-ELISA was determined to have a critical value of 45% by detecting 30 Babesia caballi negative sera and 20 positive sera. Specificity test showed that the CI-ELISA does not reacted with positive sera from Theileria equi infected horses. Using the established CI-ELISA to detected 90 clinical sera, the total coincidence rate with the standard c-ELISA kit was 92.2%, the positive coincidence rate was 92.1%, and the negative coincidence rate was 94.1%. This results indicated that CI-ELISA method has characteristics of strong specificity, high sensitivity, good stability and repeatability, and simple operation. These results suggested that CI-ELISA established by monoclonal antibody(11 F4) and recombinant protein(His-Bc48) was specific and reproducible, which could provide an effective means for the detection and monitoring of Babesia caballi infection in XinJiang, China.

关 键 词：马驽巴贝斯虫 Bc48基因 单克隆抗体 CI-ELISA 

分 类 号：S852.723[农业科学—基础兽医学]