应用免疫沉淀-质谱联用技术筛选细粒棘球绦虫抗体识别分子  

Application of immunoprecipitation-mass spectrometry for screening of antibody-recognizing molecules against Echinococcus granulosus

作　　者：吴小霞 丁静 金雪敏 张壮志[2] 张藜潇 刘明远 WU Xiaoxia;DING Jing;JIN Xuemin;ZHANG Zhuangzhi;ZHANG Lixiao;LIU Mingyuan(State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases/Key Laboratory for Zoonosis Research of Ministry of Education,Institute of Zoonosis,College of Veterinary Medicine,Jilin University,Changchun 130062,China;National Animal Echinococcosis Reference Laboratory,Veterinary Research Institute of Xinjiang Academy of Animal Husbandry,Urumqi 830010,China)

机构地区：[1]吉林大学动物医学学院/人畜共患传染病重症诊治全国重点实验室/人兽共患病研究教育部重点实验室,吉林长春130062 [2]新疆畜牧科学院兽医研究所/国家动物包虫病参考实验室,新疆乌鲁木齐830010

出　　处：《中国兽医学报》2025年第3期519-526,共8页Chinese Journal of Veterinary Science

基　　金：国家重点研发计划资助项目(2022YFE0114600)。

摘　　要：收集免疫反应较强的中间宿主羊细粒棘球绦虫感染阳性血清,以健康血清为阴性对照,纯化该血清与靶标蛋白经免疫沉淀捕获和富集相应的抗原,获得靶标蛋白-抗体-目的蛋白复合物。联合质谱策略筛选与鉴定细粒棘球绦虫相关特异性抗原,利用在线预测软件对肽段覆盖率最高的蛋白进行生物信息学分析。结果显示,IP-MS鉴定得到133种细粒棘球绦虫相关蛋白,其中肽段覆盖率≥70%的有1种蛋白,为肌动蛋白;肽段覆盖率为30%~40%的有3种蛋白,分别为含Ton B结构域蛋白、NADH脱氢酶(泛醌)1α亚复合物2、乳酸脱氢酶;肽段覆盖率为20%~30%的有6种蛋白,分别为剪接因子3b亚基5、肿瘤蛋白D52、表达的保守蛋白、NADH脱氢酶(泛醌)1α亚复合物7、肌苷-5′-单磷酸脱氢酶和醛酮还原酶家族1成员B4。生物信息学分析显示,肌动蛋白无信号肽,可能为非分泌型蛋白,亚细胞定位在细胞骨架,存在6个最佳潜在抗原表位,二级结构和三级结构一致以α螺旋和无规则卷曲为主。结果表明,免疫沉淀-质普联用技术是筛选和鉴定细粒棘球绦虫抗原的一种高通量、简便、快速有效的方法,可为筛选中间宿主羊血清诊断标识的特异分子及包虫病新型诊断技术的研发提供依据。We collected the positive serum of Echinococcus granulosus infection in sheep,an intermediate host with strong immune response,and used healthy serum as negative control,purified the serum and target protein to capture and enrich the corresponding antigen by immunoprecipitation,and obtained target protein-antibody-target protein complex.Mass spectrometry strategies were combined to screen and identify specific antigens associated with Echinococcus granulosus,and the proteins with the highest peptide coverage were analyzed bioinformatically using online prediction software.The results showed that 133 Echinococcus granulosus related proteins were identified by IP-MS.Among them,one protein with peptide coverage≥70%was actinⅡ,and three proteins with peptide coverage between 30%to 40%were Ton B box domain containing protein,NADH dehydrogenase(ubiquinone)1α-subcomplex 2(NADH dehydrogenase[ubiquinone])and lactic dehydrogenase.There were six proteins with 20%to 30%peptide coverage,namely,splicing factor 3B subunit 5,tumor protein D52,expressed conserved protein,NADH dehydrogenase(ubiquinone)1 alpha subcomplex 7,inosine-5'-monophosphate dehydrogenase,and aldo keto reductase family 1 member B4.Bioinformatics analysis revealed that actin protein has no signal peptide,it is probably a non-secretory protein and is subcellularly localized to the cytoskeleton,six optimal potential antigenic epitopes are present,and the secondary and tertiary structures are consistently dominated byα-helices and irregular convolutions.The results indicate that immunoprecipitation-mass spectrometry is a high-throughput,simple,rapid and effective method for screening and identifying fine-grained Echinococcus granulosus antigens,which can provide a basis for screening specific molecules for serodiagnostic markers in intermediate host sheep and for the development of novel diagnostic techniques for hydatid diseases.

关 键 词：包虫病 细粒棘球绦虫 免疫沉淀-质谱联用技术 抗体识别分子 生物信息学分析 

分 类 号：S852.4[农业科学—基础兽医学] S852.7[农业科学—兽医学]