新城疫病毒蛋白特異性抗血清的研制
發(fā)布時間：2018-11-25 08:32
【摘要】：新城疫是由新城疫病毒引起的雞和火雞以及其他200多種鳥類的一種傳染病。該病具有較高的發(fā)病率和死亡率,被世界動物衛(wèi)生組織列為法定報告疫病。在我國新城疫的暴發(fā)和流行雖然得到了控制,但是新城疫隱性感染以及免疫雞群流行新城疫等現(xiàn)象依然存在,生產(chǎn)性能和產(chǎn)品質(zhì)量因此遭受嚴(yán)重影響。改進(jìn)檢測技術(shù)以及開發(fā)新的免疫評價方法成為當(dāng)前新城疫防控工作的重點。新城疫病毒包括6種結(jié)構(gòu)蛋白:大分子RNA依賴性RNA聚合酶蛋白(Large polymerase protein,L)、血凝素-神經(jīng)氨酸酶(Hemagglutinin-neuraminidase,HN)、融合蛋白(Fusion protein,F)核衣殼蛋白(Nucleocapsid protein,NP)、磷蛋白(Phosphoprotein,P)和基質(zhì)蛋白(Matrix protein,M)。本研究通過SDS-PAGE分離純化了新城疫病毒的結(jié)構(gòu)蛋白,并以其免疫豚鼠,成功制備了HN蛋白抗血清和NP蛋白抗血清,為新城疫的臨床診斷和實驗室研究奠定了基礎(chǔ)。通過試驗研究,主要得到了以下成果。1.病毒的純化30%蔗糖密度梯度超速離心法,只需要一個蔗糖密度梯度,操作簡單有效。經(jīng)鑒定,采用此方法濃縮純化的新城疫病毒血凝滴度達(dá)15log2,蛋白含量約為5.3 mg/mL,病毒純度達(dá)99.4%。2.蛋白的分離制備利用SDS-PAGE對蛋白質(zhì)進(jìn)行電泳分離后,采用0.25 M KCl處理使蛋白條帶呈現(xiàn)乳白色,并分別對蛋白HN、NP、M條帶予以切開、分離,進(jìn)一步采用以1%SDS為主要成分的回收液煮沸回收其中的蛋白。經(jīng)驗證,該方法蛋白回收效率理想,并且不會破壞蛋白的一級結(jié)構(gòu)。3.動物試驗采用分離制備的新城疫病毒蛋白免疫豚鼠,能夠引起良好的免疫反應(yīng)。并且成功制備了HN蛋白抗血清和NP蛋白抗血清,經(jīng)鑒定,HN蛋白抗血清特異性良好,抗體效價約為1:20000,綜合質(zhì)量較高;NP蛋白抗血清效價約為1:60000,綜合質(zhì)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.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S855.3
本文編號：2355457
[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.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S855.3
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