本课题以当前危害严重的猪囊虫为研究对象，以突破猪生前诊断及主动防控与阻断关键技术为核心，围绕适宜于猪早期活体诊断标识及疫苗候选分子筛选、适宜于养殖现场的高通量快速诊断与鉴别技术、安全高效的抗寄生虫病疫苗的创制及产业化为关键技术开展研究。针对业已接近成熟的猪囊虫TSOL18 疫苗，重点探索抗原大量制备工艺、建立质控检测技术，完成临床试验研究，为实现从源头阻断猪囊虫病的传播提供技术支撑；完成业已建立的ELISA 及免疫层析诊断方法的中试试验研；挖掘新型诊断标识分子，研制高通量核酸诊断技术；筛选针对特异性抗原的单克隆抗体，研发检测猪囊虫抗体或循环抗原的上转发光免疫层析试纸条，建立适宜于猪囊尾蚴病生前诊断的快速检测技术。该课题的实施不仅可以减少生猪因猪囊虫感染而造成的经济损失，而且可以有效保障食品安全和人类健康。This research subject to the current serious harm of cysticercosis as the research object,in order to break through the diagnosis of pigs before life and active prevention and control.The key technology is the core,centering on suitable for early in vivo diagnosis of pig and vaccine candidate molecular screening,suitable High throughput rapid diagnosis and identification technology in aquaculture field,the development of safe and efficient anti-parasitic vaccine.In view of the nearly mature cysticercosis TSOL18 vaccine,focus on exploration antigen preparation technology in large quantities,the establishment of quality control and detection technology,the completion of clinical trials,in order to achieve the source block the cysticersosis.To complete the established ELISA and immunochromatographic diagnostic methods experimental study,exploring new diagnostic marker molecules and developing high-throughput nucleic acid diagnostic techniques.Development of up-forward photoimmunochromatographic test strip for detection of cysticercosis antibody or circulating antigen.To establish a rapid detection technique suitable for the prenatal diagnosis of cysticercosis in pigs.The implementation of the subject can not only be reduced the economic loss caused by cysticercosis infection,but also can effectively guarantee food safety and human health.

整理并提交申报《犬细粒棘球绦虫抗原ELISA检测试剂盒》新兽药注册证书材料。通过盐析和亲和层析分别制备多克隆抗体、阴性粪抗原—多克隆抗体结合物和阳性粪抗原—多克隆抗体结合物。重组腺病毒AdC68-EgM123、AdHu5-EgM123接种小鼠未产生针对EgM123较好的免疫原性;EGM123+BCG分支杆菌激发小鼠产生血清抗体，但犬较差;EGM123+CTB/LTB肠毒素佐剂免疫犬实验未促进预期更好的保护效果。EgM9混合佐剂Quil A 或 ISCOMs免疫犬原头蚴口服感染45天后观察，成虫发育和产卵量与对照组相比较产生明显的抑制作用。牛棘球蚴(包虫)病EG95基因工程疫苗研制完成了所有实验室研究，中试产品的试制，组织整理了临床试验备案材料，预计2021年初提交农业部兽药评审中心。完成牛、羊包虫病 ELISA 诊断试剂盒新兽药证书的注册申报。完成羊、牛包虫病快速诊断试剂盒(金标试纸条)新兽药注册申报。完成对抗原的标记，优化控制供体受体荧光染料间间距和荧光共振能量转移距离；优化包虫等重要寄生虫相关诊断产品的生产工艺及产业化技术；构建微流控芯片，将本课题的FRET均相定量分析方法搭载微流控技术，实现高通量、快反应；开发配套的小型便携荧光检测仪，集信号采集，数据处理分析于一体。Sort out and submit the application materials for the new veterinary drug registration certificate of Echinococcus granulosus antigen ELISA kit.Polyclonal antibody,negative fecal antigen polyclonal antibody conjugate and positive fecal antigen polyclonal antibody conjugate were prepared by salting out and affinity chromatography,respectively.Mice inoculated with recombinant adenovirus adc68-egm123 and adhu5-egm123 did not produce good immunogenicity against egm123;Egm123+BCG Mycobacterium stimulated mice to produce serum antibodies,but dogs were poor;The experiment of immunizing dogs with egm123+CTB/LTB enterotoxin adjuvant did not promote the expected better protective effect.After 45 days of oral infection with EgM9 mixed adjuvant quil a or ISCOMs,adult development and oviposition were significantly inhibited compared with the control group.The Eg95 genetic engineering vaccine for bovine echinococcosis(echinococcosis)has completed all laboratory studies,trial production of pilot products,organized and sorted out the clinical trial filing materials,and is expected to be submitted to the veterinary drug evaluation center of the Ministry of agriculture in early 2021.Complete the registration and application of new veterinary drug certificate of bovine and sheep hydatidosis ELISA diagnostic kit.Complete the registration and application of new veterinary drugs for sheep and cattle hydatidosis rapid diagnosis kit(gold standard test strip).Complete the labeling of antigen,optimize the spacing between donor and recipient fluorescent dyes and the fluorescence resonance energy transfer distance;Optimize the production process and industrialization technology of diagnostic products related to important parasites such as hydatid;The microfluidic chip is constructed,and the FRET homogeneous quantitative analysis method of this subject is equipped with microfluidic technology to realize high flux and fast reaction;A small portable fluorescence detector is developed,which integrates signal acquisition,data processing and analysis.

本课题针对猪旋毛虫病早期活体诊断缺乏快速、灵敏、特异诊断技术以及尚无有效防治疫苗等问题，根据旋毛虫具有不同发育时期的特点，聚焦早期诊断标识与新型疫苗候选分子，利用最新的高灵敏显示技术，研发更为灵敏的、适用于现场的的诊断技术，研制新型候选分子基因工程疫苗。In view of the lack of rapid,sensitive and specific diagnostic technology for early diagnosis of swine trichinosis and the lack of effective vaccine and so on,according to the characteristics of different developmental stages of Trichinella spiralis,this project focuses on early diagnostic markers and new vaccine candidate molecules,and utilizes the latest highly sensitive display technology to develop more sensitive and suitable for field diagnosis technology,and to develop new candidate molecular genetic engineering vaccine.

通过本课题的实施，建立了家畜日本血吸虫病快速诊断技术3种，华支睾吸虫病早期检测诊断ELISA 1种，建立了淡水鱼华支睾吸虫感染胶体金免疫层析检测技术1种;《日本血吸虫抗体检测试纸条》完成了新兽药证书申报，正对照审核意见中提出的要求进行材料修改；牛、羊血吸虫病ELISA检测技术已完成新兽药注册申报的主要工作。筛选、鉴定家畜日本血吸虫病诊断标识10种。挖掘适于多种动物感染华支睾吸虫病活体诊断标识分子3-4种。优化了棘球蚴疫苗建立了20L发酵工艺和纯化工艺，研制了新型水溶性免疫佐剂棘球蚴疫苗，建立了血吸虫蛋白发酵工艺及中试纯化工艺。In these research,three rapid diagnosis techniques for domestic animal schistosomiasis,one early detection and diagnosis ELISA for clonorchiasis,and one colloidal gold immunochromatographic detection technique for freshwater fish Clonorchis sinensis infection were established;The"Schistosomiasis Antibody Test Strip"has completed the application of the new veterinary drug certificate,and the materials are revised in accordance with the requirements in the review opinions;the ELISA detection technology for schistosomiasis in cattle and sheep has completed the main work of the registration and application of new veterinary drugs.Screen and identify 10 types of diagnostic markers for schistosomiasis japonica in livestock.Discover 3-4 kinds of biodiagnostic marker molecules suitable for various animal infections with clonorchiasis.Optimized hydatid vaccine,established a 20L fermentation process and purification process,developed a new water-soluble immune adjuvant hydatid vaccine,established a schistosome protein fermentation process and a pilot-scale purification process.