分类号：单位代码：10019密级：学号：66043学位论文猪O型口蹄疫高倍浓缩灭活疫苗的工艺研究 Technical Study on Highly Concentrated Inactivated Vaccine ofSwine FMDV Type O研究生：尹忠良指导教师：陈福勇教授合作指导教师：申请学位类别：兽医硕士专业领域名称：预防兽医研究方向：动物疫苗生产工艺所在学院：动物医学院2010年11月独创性声明本人声明所呈交的论文是我个人在导师指导下进行的研究工作及取得的研究成果。
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(保密的学位论文在解密后应遵守此协议)研究生签名：时间：年月日导师签名：时间： 年 月 日2中文摘要口蹄疫（Foot and Mouth Disease, FMD）是由口蹄疫病毒（FMDV）引起的主要危害牛、猪、羊等偶蹄动物的烈性传染病，具有发病率高，传染性强的特点，能形成国际大流行，国际兽疫局（OIE）将其排在A类家畜传染病的首位。
世界各国政府都高度重视本病的防治工作，近一个世纪以来，许多国家成立了专门的研究机构，对口蹄疫的预防和控制进行研究，在口蹄疫疫苗的研究方面一直也是研究热点。
在我国，预防控制口蹄疫疫病的重要措施是应用疫苗进行免疫，目前主要使用不经过浓缩的病毒培养液配制的普通灭活疫苗，每头剂量2ml仅含3个PD50。
因普通疫苗的抗原未经浓缩处理抗原含量偏低，同时存在一定量的细胞碎片等非抗原蛋白成份，所以保护力低、免疫期相对较短，并有一定的副反应。
本研究通过对猪O型口蹄疫病毒抗原进行高倍浓缩等一系列手段，制备出更高标准的新型疫苗，并探索出一套口蹄疫浓缩疫苗生产工艺。
通过对口蹄疫疫苗毒株的各种生物学特性尤其是病毒毒力和免疫原性、病毒液的前处理和高倍超滤浓缩工艺、浓缩后病毒含量检测方法和标准的建立（细胞TCID50与乳鼠LD50试验平行比较）、应用国际通行标准（OIE）对疫苗进行PD50测定并与血清中和抗体相比较等研究试验，最终研制成功的三批高倍浓缩疫苗，并对该三批疫苗的安全性、效力等指标进行检验，提高安全性,动物注射疫苗后安全而无副作用；提高免疫效力，每头剂达到6个PD50以上；均达到了预先设定的各项标准。
为将来研究猪口蹄疫多价疫苗提供基础数据。
关键词：猪口蹄疫，病毒浓缩，疫苗IIIAbstractFoot and Mouth Disease (FMD), a major threat to cloven-hoofed animals such as bovine, swine and sheep, is a highly contagious disease caused by Foot and Mouth Disease Virus (FMDV). Since it has a high incidence and can easily cause an epidemic outbreak, Office International des Epizooties (OIE) ranks it in the first in all contagious diseases for domestic animals. Governments all over the world have attached great importance to the prevention and treatment of this disease.For nearly a century, many countries have established special research institutions, which research focused on the prevention and control of the outbreak and prevalence of FMD. Vaccine research in the foot and mouth disease has also been a research hotspot since vaccination was one of the major means to prevent domestic animals from this disease.In China, the way widely adopted to prevent domestic animals from the infection of FMDV is vaccination, mainly through the use of non-concentrated liquid preparation of the common viral cultures inactivated vaccine; generally, the dosage per animal is 2ml and only contains 3 PD50. Since the concentration of viral antigens in the non-concentrated crude viral preparation (common vaccine) is relatively low and it contains a trace amount of non-antigenic protein components such as the host cell debris, these make the vaccines usually in a weak protection, a relatively short duration for immune response and having some side effects.In this study, we produced a higher standard new vaccine by through a series of means to highly concentrated swine type O FMDV antigen, and explored a set of production process for concentrated FMDV vaccine. Through examining the biological characteristics of the vaccine strain especially for its virulence and immunogenicity; evaluating the pre-process for the viral culture and the concentration technology; establishing the testing standard protocol for the viral antigen content after concentration (the results from the established new testing protocol obtained from ELISA and TCID50 were consistent with the results obtained from the mouse LD50 protocol); using OIE standard to determine the PD50 and comparing the efficacy by Neutralization test, we successfully produced three batches of high-concentrated vaccines. Improving the security,Animal safety without side effects after vaccination. Enhancing the immune effect,up to 6 PD50 dose or more per head. All three have been tested their safety, efficacy, and all indicators have met the pre-set standards. Providing valuable data for the research of swine FMD multivalent vaccines.To date, these vaccines have been passed the assessment and evaluation by the Ministry of Agriculture of the People’s Republic of China and will be certificated for the license soon.Key words: Swine Foot and mouth disease, virus concentration, vaccineIV目录中文摘要 (III)Abstract (IV)第一章文献综述：猪口蹄疫简述及疫苗的研究现状 (7)1 病原学 (7)2 流行病学 (8)3 症状 (8)4 防制措施 (9)第二章猪口蹄疫病毒O型(OR/80)毒株的鉴定试验 (11)前言 (11)1 材料与方法 (11)1.1 材料 (11)1.2 方法 (12)2 结果 (14)2.1 猪水泡皮毒F1～F3复壮与传代结果 (14)2.2 乳鼠毒适应与传代结果 (15)2.3 BHK21细胞毒适应培养结果 (15)2.4 毒力测定结果 (16)2.5 型特异性鉴定 (20)2.6 纯净检验 (20)2.7 免疫原性试验结果 (21)3 讨论与小结 (23)第三章猪口蹄疫高倍浓缩灭活疫苗制苗工艺研究 (24)前言 (24)1 材料与方法 (24)1.1 材料 (24)1.2 方法 (25)2 结果 (27)3 讨论 (31)4小结 (31)第四章猪口蹄疫高倍浓缩灭活疫苗的制备及安全、效力试验 (33)前言 (33)1 材料 (33)V1.1 毒种 (33)1.2 BHK21细胞 (33)1.3 试验动物 (34)1.4 其它试剂及设备 (34)2 方法 (35)2.1 病毒液的繁殖扩量 (35)2.2 病毒液的检验 (35)2.3 病毒抗原液的纯化与浓缩 (35)2.4 超滤浓缩后的病毒抗原液进行过滤 (36)2.5 病毒液纯化浓缩后的病毒含量测定 (36)2.6 灭活 (36)2.7 阻断 (36)2.8 半成品检验 (36)2.9 配苗 (37)3 成品检验 (37)3.1 性状 (37)3.2 无菌检验 (37)3.3 安全检验 (37)3.4 效力检验 (38)4 结果 (38)4.1 制苗病毒液的制备 (38)4.2 半成品检验 (39)4.3 配苗 (40)4.4 成品检验 (40)5 讨论 (43)6 小结 (44)第五章结论 (45)参考文献 (47)致谢 (51)附录 (53)作者简历 (57)VI第一章 文献综述：猪口蹄疫简述及疫苗的研究现状家畜口蹄疫（Foot and Mouth Disease，FMD）是一种主要感染偶蹄动物的急性、热性传染病，主要危害牛、猪、羊等偶蹄家畜。