基于TPO18基因蛋白的兔豆状囊尾蚴感染间接ELISA检测方法的建立  

Establishment of an indirect ELISA for detection of antibodies against Cysticercus pisiformis infection based on TPO18 protein

作　　者：王泽祥 罗永禄 薛萍 车亮 王由森 苟惠天[1] 孙晓林[1] WANG Zexiang;LUO Yonglu;XUE Ping;CHE Liang;WANG Yousen;GOU Huitian;SUN Xiaolin(College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,China;Animal Disease Prevention and Control Center of Yongdeng County,Lanzhou 730030,China;Animal Disease Prevention and Control Center of Pingliang City,Pingliang,Gansu 744000,China)

机构地区：[1]甘肃农业大学动物医学院,甘肃兰州730070 [2]兰州市永登县动物疫病预防控制中心,甘肃兰州730300 [3]平凉市动物疫病预防控制中心,甘肃平凉744000

出　　处：《中国兽医学报》2024年第6期1213-1222,共10页Chinese Journal of Veterinary Science

基　　金：甘肃省自然科学基金资助项目(21JR7RA823);国家自然科学基金资助项目(32160843,31460659);甘肃省国际科技合作专项资助项目(144WCGA169);甘肃省重点研发计划-社会发展类资助项目(18YF1FA080)。

摘　　要：豆状囊尾蚴病是由豆状带绦虫(Taenia pisiformis)的幼虫——豆状囊尾蚴(Cysticercus pisiformis)寄生于家兔等啮齿类动物所引起的一种寄生虫病,该病威胁养兔业发展和兔肉食品安全。为了鉴定TPO18抗原在豆状囊尾蚴和豆状带绦虫的分布,并建立兔豆状囊尾蚴病抗体间接ELISA检测方法,本研究原核表达兔豆状囊尾蚴18 kDa抗原,经蛋白纯化获得了可溶性的TPO18蛋白。TPO18蛋白免疫家兔制备了效价达1∶51200多克隆抗体。蛋白免疫印迹鉴定多克隆抗体与兔豆状囊尾蚴总蛋白、豆状带绦虫总蛋白及重组蛋白TPO18的反应原性,免疫组织化学法鉴定兔豆状囊尾蚴和豆状带绦虫虫体TPO18抗原分布,多克隆抗体与兔豆状囊尾蚴总蛋白、豆状带绦虫总蛋白及重组蛋白TPO18具有良好的反应原性,兔豆状囊尾蚴TPO18抗原主要分布于生发层和绒毛层,豆状带绦虫TPO18抗原主要分布在吸盘及吸盘周围和集合管上层细胞及绒毛层。以TPO18重组抗原包被酶标板,优化确定间接ELISA各项技术参数,建立基于TPO18抗原的间接ELISA抗体检测方法。经优化后的ELISA检测方法的检测条件为血清稀释度1∶100,抗原包被质量浓度为5 mg/L,抗原包被条件37℃1 h+4℃过夜,封闭条件1%BSA 37℃封闭60 min,血清反应时间60 min,酶标二抗稀释度1∶1000,酶标二抗作用时间60 min,底物反应时间15 min,临界值0.295。ELISA方法的灵敏性、特异性和重复性检测结果表明,该方法灵敏度较高,特异性较好,不与兔瘟、兔肝片吸虫、兔球虫、兔弓形虫等的阳性血清发生交叉反应;批内和批间重复性试验的变异系数均小于7%,重复性良好。运用该ELISA方法与剖检检测试验分别对86份临床血清样品检测,两种方法检测结果符合率为97.7%。综上所述,本研究成功建立了兔感染豆状囊尾蚴抗体的间接ELISA方法,为兔感染豆状囊尾蚴后感染情况监测提供了新的监测方法。Cysticercosis,caused by the larval stage of the tapeworm Taenia pisiformis,known as Cysticercus pi siformis,is a parasitic ailment affecting lagomorphs,particularly domestic rabbits,posing a threat to the rabbit industry and the safety of rabbit meat products.This study aims to identify the distribution of the TPO18 antigen in Cysticercus pisiformis and Taenia pisiformis and establish an indirect enzyme-linked immunosorbent assay(ELISA)for detecting antibodies against rabbit cysticercosis.The research involved the prokaryotic expression of the 18 kDa antigen of rabbit Cysticercus pisiformis and the isolation of soluble TPO18 protein post-purification.Immunizing rabbits with the TPO18 protein resulted in the production of polyclonal antibodies with a titer of up to 1,51200.Western blot analysis validated the antigenicity of the polyclonal antibodies against total proteins from rabbit Cysticercus pisiformis,Taenia pisiformis and the recombinant TPO18 protein.Immunohistochemistry revealed the distribution of the TPO18 antigen in rabbit Cysticercus pisiformis and Taenia pisiformis,indicating the effective reactivity of the polyclonal antibodies with total proteins from both parasites and the recombinant TPO18 protein.TPO18 antigen in rabbit Cysticercus pisiformis predominantly localized in the germinal layer and the parenchyma,while in Taenia pisiformis,it was mainly present in the suckers,sucker peripheries,collecting duct upper cells,and parenchyma.An indirect ELISA based on the TPO18 antigen was developed using the recombinant antigen,and its technical parameters were optimized.The optimized ELISA conditions included a serum dilution of 1,100,antigen coating concentration of 5 mg/L,coating for 1 h at 37℃followed by overnight incubation at 4℃,blocking with 1%BSA for 60 min at 37℃,serum reaction for 60 min,secondary antibody dilution at 1:1000,secondary antibody incubation for 60 min,substrate reacting for 15 min,with a cutoff value of 0.295.Sensitivity,specificity,and repeatability tests of the ELISA demonstr

关 键 词：兔豆状囊尾蚴 18 kDa抗原 免疫组织化学 酶联免疫吸附试验 特异性 敏感性 

分 类 号：S852.723[农业科学—基础兽医学]