猪O型口蹄疫高倍浓缩灭活疫苗工艺研究

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 of

Swine 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

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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。尽我所知，除了文中特别加以标注和致谢的地方外，论文中不包含其他人已经发表或撰写过的研究成果，也不包含为获得中国农业大学或其它教育机构的学位或证书而使用过的材料。与我一同工作的同志对本研究所做的任何贡献均已在论文中作了明确的说明并表示了谢意。

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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

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Abstract

Foot 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, vaccine

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