梅花鹿结核病间接ELISA方法的建立  

Establishment of an indirect ELISA method for tuberculosis in Sika deer

作　　者：包福祥[1,2] 张磊 徐文博[1] 敖可心 蒋强 吴玘瑶 张慧敏 程艺 白文成 杜雅楠[1,2] BAO Fuxiang;ZHANG Lei;XU Wenbo;AO Kexin;JIANG Qiang;WU Qiyao;ZHANG Huimin;CHENG Yi;BAI Wencheng;DU Yanan(College of Veterinary Medicine,Inner Mongolia Agricultural University,Hohhot 010018,China;Ministry of Agriculture and Rural Affairs Key Laboratory of Clinical Diagnosis and Treatment Techniques for Animal Diseases,Huhhot 010018,China)

机构地区：[1]内蒙古农业大学兽医学院,呼和浩特010018 [2]农业农村部动物疾病临床诊疗技术重点实验室,呼和浩特010018

出　　处：《黑龙江畜牧兽医》2022年第21期69-74,79,共7页Heilongjiang Animal Science And veterinary Medicine

基　　金：国家自然科学基金项目(81660297);内蒙古农业大学兽医学院青年创新基金项目(2015QNJJ01)。

摘　　要：为了建立鹿结核病的快速检测方法,试验采用亲和层析方法纯化梅花鹿血清IgG,并免疫兔,制备兔抗梅花鹿IgG抗体,辣根过氧化物酶(HRP)进行标记,通过ELISA和Western-blot对HRP标记的兔抗梅花鹿IgG抗体进行鉴定。同时合成结核分枝杆菌38 ku基因,并与pET-22b(+)质粒连接,转化到大肠杆菌BL21(DE3)感受态细胞中,诱导表达和纯化38 ku蛋白,以38 ku蛋白作为包被抗原,以HRP标记的兔抗梅花鹿IgG抗体作为二抗,建立间接ELISA方法。利用建立的间接ELISA方法对结核菌素皮肤试验检出的20份阴性血清和10份阳性血清进行验证。结果表明:对梅花鹿血清IgG进行纯化,得到大小分别约为50 ku和25 ku的蛋白质;制备的HRP标记的兔抗梅花鹿IgG抗体可特异性识别梅花鹿血清IgG,在抗体浓度稀释比例为1∶20000时仍有很好的结合效果;38 ku蛋白作为抗原的最佳包被浓度为1μg/mL,被检梅花鹿血清的最佳稀释比例为1∶100,HRP标记的兔抗梅花鹿IgG抗体的最佳稀释比例为1∶10000;间接ELISA方法能够准确区分结核菌素皮肤试验确定的阳性和阴性梅花鹿血清。说明试验建立的间接ELISA方法能够作为鹿结核病血清学快速检测的有效方法,弥补了传统方法的不足。In order to establish a rapid detection method for deer tuberculosis, in the experiment the method of affinity chromatography was used to purify Sika deer serum IgG, which was immunized to rabbits to prepare rabbit anti-Sika deer IgG antibody;it was labeled with horseradish peroxidase(HRP);the HRP-labeled rabbit anti-Sika IgG antibody was identified by ELISA and Western-blot. At the same time, the 38 ku gene of Mycobacterium tuberculosis was synthesized, which was ligated to the pET-22 b(+) plasmid and transformed into E. coli BL21(DE3) competent cells to induce the expression and purification of the 38 ku protein. Using it as the coating antigen and HRP-labeled rabbit anti-Sika deer IgG antibody as the secondary antibody, an indirect ELISA detection method was established. 20 negative sera and 10 positive sera detected by Sika deer tuberculin test were verified by the established indirect ELISA method. The results showed that the Sika deer serum IgG was purified, and the proteins with sizes of about 50 ku and 25 ku were obtained. The prepared HRP-labeled rabbit anti-Sika deer IgG antibody could specifically recognize the Sika deer serum IgG, and still had a good effect when the antibody concentration dilution ratio was 1∶20 000. The optimal coating concentration of 38 ku protein as antigen was 1 μg/mL, the optimal dilution ratio of tested Sika deer serum was 1∶100, and the optimal dilution ratio of HRP-labeled rabbit anti-Sika deer IgG antibody was 1∶10 000. The indirect ELISA method could accurately distinguish tuberculin skintested positive and negative sera. The results suggested that the established indirect ELISA method could be used as an effective method for the rapid detection of deer tuberculosis serology, which made up for the deficiency of traditional methods.

关 键 词：牛结核分枝杆菌 梅花鹿 38 ku蛋白 兔抗梅花鹿IgG抗体 间接ELISA 

分 类 号：S858.25[农业科学—临床兽医学] S855.2[农业科学—兽医学]