【作者】 周丽；

【导师】 王君伟；

【作者基本信息】 东北农业大学 ， 预防兽医学， 2007， 硕士

【摘要】 鹅副粘病毒病又称鹅源新城疫，是由鹅副粘病毒(Goose Paramyxovirus，GPMV)引起的一种急性、烈性传染病。该病以鹅消化道和呼吸道病变为主要特征，具有高度的发病率和死亡率，给养鹅业带来了严重的危害。由于目前我国对鹅副粘病毒病的危害性缺乏足够重视，尚缺乏完善的疾病诊断、监测技术。面对该病在我国有逐步扩大的趋势，开展鹅副粘病毒病的诊断和检测技术的研究迫在眉睫。在血清学的临床检测方面，目前已经有多次关于鸡新城疫ELISA与HI检测比较的报道，然而对于鹅副粘病毒病却未见这方面的报导。本研究以NP蛋白为检测抗原对鹅副粘病毒病进行了ELISA与HI检测抗体相关性的比较。GPMV属于副粘病毒科，副粘病毒亚科，腮腺炎病毒属，禽副粘病毒(Avian Paramyxovirus APMV)Ⅰ型中的成员，具有基因Ⅶ型新城疫(NDV)的典型特征。NP蛋白是鹅副粘病毒核衣壳的主要结构蛋白，在自然感染或者疫苗免疫后的动物中都可以产生针对NP蛋白的抗体，具有重要的诊断价值。本研究通过原核表达载体pET-30a，成功构建了重组质粒pET-30a-NP，并通过大肠杆菌表达系统原核表达GPMV-NP蛋白。以纯化的NP蛋白为包被抗原建立了ELISA检测方法，通过对各个反应条件的优化，最终确定了最佳反应条件。本实验选取50只来源于肇东非疫区的鹅，随机分为A、B、C、D 4组。A组为非免疫对照组；B组为免疫组；C组为免疫攻毒组；D组为人工感染组。分别在第一次免疫前、免疫后及攻毒后第1、2、3、4、5、6、7、8、9、10、11、12、14、18周采血。对上述血清进行ELISA定量检测及HI抗体的检测。比较各组鹅群NP蛋白ELISA抗体和HI抗体的相关性，分别建立了相关的回归方程。免疫组：ELISA=0.1981 HI+0.0541(r=0.9134，P＜0.05)；免疫攻毒组：ELISA=0.1807 HI-0.0744 (r=0.8240，P＜0.05)；人工感染组：ELISA=0.2728 HI-0.4498(r=0.8795，P＜0.05)。对各组ELISA与HI的敏感性进行比较的结果显示，ELISA检测敏感性均高于HI方法。结果表明：GPMV两种检测方法所测得抗体之间呈显著相关，该结果为ELISA检测方法取代HI试验提供了理论依据。本研究对鹅副粘病毒两种检测方法作了初步实验室探索，而在实际临床检测中，能否采用NP蛋白为包被抗原的ELISA检测方法代替传统的HI试验还有待大量动物实验的验证。从而为深入研究GPMV的临床检测提供一定依据。更多还原

【Abstract】 Goose Paramyxovirus disease named Newcastle Disease viruses of goose origin again, whichis an acute contagious disease caused by Goose Paramyxovirus(GPMV), a member of Rubulavirusfamily. Which was characerized by the lesion of digestive tract with high mortality and occurrence,and has caused a large loss in economy. Goose Paramyxovirus disease infection has spreaded inmany province in China, but attention has not been directed towards Goose Paramyxovirusinfection, so far neither Goose Paramyxovirus disease vaccine applied nor adoption of measures tocontrol of Goose Paramyxovirus disease. It has shown that Goose Paramyxovirus disease infectionhas expand in China, so it is of great interest to study the pathogen from the molecular biology. Atpresent,there are many reports about comparison of ELBA and HI detection methods of NDV ondetection technology of serology. But no report on GPMV. This test first time explores the growthand decline rule of the antibody for neucleocapsid protein by ELBA whose coating antigen isneucleocapsid protein(NP) expressed by E.coli.Neucleocapsid protein (NP) is the main structural protein of GPMV, so it has importantdiagnostic significance. So gaining NP gene and its expressing product is key to the preparation ofthe specific diagnosis antigen. The NP gene of JS/1/97/GO strain was cloned into Prokarytoicexpression vector pET-30a to construct the positive recombinant plasmid pET-30a-NP sucessfully toexpress NP. The result of SDS-PAGE analysis showed that the NP was efficiently expressed and theimmunity reactivity of the recombinant protein was confirmed by Western-blot. The purified NPprotein could be used as antigen in the development of an indirect ELISA for the detection ofantibody against NP protein of GPMV, and the optimal reactive condition were determined.Healthy gooses were vaccinated with Oil-adjvant Inactive Vaccine of goose paramyxovirus. Animalswere randomly divided into four groups. A:non-vaccinated contronl; B:the vaccinated group C:theinfected group D:the post-vaccination infected group.Harvesting the blood of gooses in 1、2、3、4、5、6、7、8、9、10、11、12、14、18 weeks respectively after vaccination or infected. We detectgrowth and decline condition of the antibody for neucleocapsid protein by ELISA whose coatingantigen is neucleocapsid protein(NP) expressed by E.coli. Comparing the linear relationship betweenthe ELISA value and the HI value of each group,Construct the regression analysis of each group. (1)the equation of the vaccinated group was ELISA=0.1981 HI+0.0541(r=0.9134, P＜0.05); (2) theequation of the infected post-vaccination group was ELISA= 0.1807 HI-0.0744 (r=0.8240, P＜ 0.05); (3) the equation of the infected group was ELISA=0.2728 HI-0.4498 (r=0.8795, P＜0.05),the result demonstrates that antibody titer detected by two methods are correlated significantly.This study only explored the relationship of two antibodies in laboratory, however, in practicalclinical detection, a great quantity of animal experiments still need to do to check if the ELISAdetection method with NP as coating antigen can replace the HI test.更多还原