新城疫病毒蛋白特异性抗血清的研制

发布时间：2018-11-25 08:32

【摘要】：新城疫是由新城疫病毒引起的鸡和火鸡以及其他200多种鸟类的一种传染病。该病具有较高的发病率和死亡率,被世界动物卫生组织列为法定报告疫病。在我国新城疫的暴发和流行虽然得到了控制,但是新城疫隐性感染以及免疫鸡群流行新城疫等现象依然存在,生产性能和产品质量因此遭受严重影响。改进检测技术以及开发新的免疫评价方法成为当前新城疫防控工作的重点。新城疫病毒包括6种结构蛋白:大分子RNA依赖性RNA聚合酶蛋白(Large polymerase protein,L)、血凝素-神经氨酸酶(Hemagglutinin-neuraminidase,HN)、融合蛋白(Fusion protein,F)核衣壳蛋白(Nucleocapsid protein,NP)、磷蛋白(Phosphoprotein,P)和基质蛋白(Matrix protein,M)。本研究通过SDS-PAGE分离纯化了新城疫病毒的结构蛋白,并以其免疫豚鼠,成功制备了HN蛋白抗血清和NP蛋白抗血清,为新城疫的临床诊断和实验室研究奠定了基础。通过试验研究,主要得到了以下成果。1.病毒的纯化30%蔗糖密度梯度超速离心法,只需要一个蔗糖密度梯度,操作简单有效。经鉴定,采用此方法浓缩纯化的新城疫病毒血凝滴度达15log2,蛋白含量约为5.3 mg/mL,病毒纯度达99.4%。2.蛋白的分离制备利用SDS-PAGE对蛋白质进行电泳分离后,采用0.25 M KCl处理使蛋白条带呈现乳白色,并分别对蛋白HN、NP、M条带予以切开、分离,进一步采用以1%SDS为主要成分的回收液煮沸回收其中的蛋白。经验证,该方法蛋白回收效率理想,并且不会破坏蛋白的一级结构。3.动物试验采用分离制备的新城疫病毒蛋白免疫豚鼠,能够引起良好的免疫反应。并且成功制备了HN蛋白抗血清和NP蛋白抗血清,经鉴定,HN蛋白抗血清特异性良好,抗体效价约为1:20000,综合质量较高;NP蛋白抗血清效价约为1:60000,综合质量理想。
[Abstract]:Newcastle disease is an infectious disease caused by Newcastle disease virus in chickens, turkeys and more than 200 other birds. The disease has a high morbidity and mortality rate, and is listed as a reported disease by the World Organization of Animal Health (OIE). Although the outbreak and epidemic of Newcastle disease have been controlled in our country, the recessive infection of Newcastle disease and the epidemic of Newcastle disease in immunized chickens still exist, and the production performance and product quality are seriously affected. Improving detection technology and developing new immune evaluation methods have become the focus of Newcastle disease prevention and control. Newcastle disease virus (NDV) includes six structural proteins: macromolecule RNA dependent RNA polymerase (Large polymerase protein,L), hemagglutinin neuraminidase (Hemagglutinin-neuraminidase,HN), fusion protein (Fusion protein,F) nucleocapsid protein (Nucleocapsid protein,NP). Phosphorous protein (Phosphoprotein,P) and matrix protein (Matrix protein,M). In this study, the structural proteins of Newcastle disease virus (NDV) were isolated and purified by SDS-PAGE, and the antisera of HN and NP were successfully prepared by immunizing guinea pigs, which laid a foundation for the clinical diagnosis and laboratory study of Newcastle disease. Through experimental research, the following results are obtained. 1. The purification of virus by 30% sucrose density gradient ultracentrifugation requires only one sucrose density gradient and the operation is simple and effective. The results showed that the hemagglutination titer of Newcastle disease virus was 15log2, and the protein content was about 5.3 mg/mL, virus. The purity of the purified Newcastle disease virus was 99.40.2. The protein was separated by SDS-PAGE electrophoresis. The protein bands were treated with 0.25 M KCl to make the protein bands appear milky white, and the protein HN,NP,M bands were cut open and separated, respectively. The protein was further recovered by boiling the recovery solution with 1%SDS as the main component. It has been proved that this method has ideal recovery efficiency and does not destroy the primary structure of protein. 3. 3. The guinea pigs immunized with Newcastle disease virus (NDV) protein in animal experiment can induce a good immune response. HN antiserum and NP antiserum were successfully prepared. The specificity of HN antiserum was good, the antibody titer was about 1: 200000.The comprehensive quality of NP antiserum was about 1: 600000.The comprehensive quality was ideal.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S855.3

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