猪圆环病毒2型悬浮培养技术及冻干灭活疫苗研究

发布时间：2018-05-02 23:27

  本文选题：猪圆环病毒2型 + 微载体悬浮培养　； 参考：《中国农业大学》2016年博士论文

【摘要】：猪圆环病毒2型及其相关性疫病已成为危害养猪业的主要疫病之一,疫苗是防控PCV2的有效工具之一,但目前商品化疫苗存在诸如有效抗原含量低、免疫应激大等弱点,因此开发具有抗原含量高、纯度高、应激小、免疫剂量少等优势的PCV2疫苗,是今后的研究热点之一。本研究旨在利用细胞悬浮培养技术、抗原纯化技术和灭活冻干技术,研究PCV2-ZJ/C株的规模化培养工艺、抗原高效纯化工艺,并在此基础上,研制了PCV2冻干灭活疫苗,为PCV2冻干灭活疫苗的研究与临床应用提供参考。PCV2 ZJ/C株微载体悬浮培养工艺研究：采用有限稀释法对PK15细胞在微载体上进行驯化培养PK15细胞,获得了微载体培养适应性PK15细胞克隆株(命名为PK-15-S株),在此基础上,利用微载体生物反应器研究PK1 5细胞悬浮培养参数和PCV2-ZJ/C株的悬浮培养参数。结果表明,PCV2-ZJ/C株病毒滴度为108.3-108.5TCID50/mL,相当于传统转瓶培养工艺的6.3-10倍。PCV2 ZJ/C株悬浮培养病毒液的纯化工艺研究：采用澄清、浓缩、层析纯化等技术组合,对病毒滴度为108.3-108.5TCID50/mL的悬浮培养病毒液进行纯化,获得高纯度的病毒蛋白,并按照中华人民共和国农业部第1893号公告中的猪圆环病毒2型灭活疫苗(ZJ/C株)的质量标准进行免疫效力检验,结果表明,当纯化抗原的总蛋白浓度为20有效PCV2蛋白浓度0.34μg/mL时,免疫保护率在80%以上。PCV2冻干灭活疫苗的制备：采用冻干技术和抗原耐热保护技术,设计10种耐热保护剂(B1-10)和3种冻干曲线,优化冻干曲线和耐热保护剂,批量生产了3批疫苗。按照现有冻干疫苗的质量标准和农业部第1893号公告中的猪圆环病毒2型灭活疫苗(ZJ/C株)的质量标准进行耐热稳定性、免疫效力和保存期研究,结果表明,3批PCV2冻干灭活苗的各项指标均符合规程要求；37℃条件下保存14天,2-8℃保存期至少18个月,具备良好的临床应用前景。
[Abstract]:Porcine circovirus type 2 and its associated blight have become one of the major diseases that harm the pig industry. Vaccine is one of the effective tools to prevent and control PCV2. However, the commercialized vaccine has such weaknesses as low effective antigen content and high immune stress. Therefore, the development of PCV2 vaccine with high antigen content, high purity, low stress and low immune dose is one of the research hotspots in the future. The purpose of this study was to study the scale culture and high efficiency purification of PCV2-ZJ/C strain by cell suspension culture technique, antigen purification technique and inactivated freeze-drying technique. On this basis, a freeze-dried PCV2 vaccine was developed. To provide a reference for the research and clinical application of PCV2 freeze-dried inactivated vaccine. The suspension culture technology of PK15 cell line ZJ/C strain was studied. PK15 cells were domesticated and cultured on microcarrier by finite dilution method. An adaptive PK15 cell clone (named PK-15-S strain) was obtained, and the suspension culture parameters of PK1 5 cell and PCV2-ZJ/C strain were studied by microcarrier bioreactor. The results showed that the virus titer of PCV2-ZJ / C strain was 108.3-108.5 TCID _ (50) / mL, which was equivalent to 6.3-10 times of the traditional culture technology. The purification process of suspension culture of PCV2-ZJ / C strain ZJ/C strain: clarification, concentration, chromatographic purification, and so on. The suspension culture solution with 108.3-108.5TCID50/mL titer was purified to obtain high purity virus protein. In accordance with the quality standard of inactivated porcine circovirus type 2 vaccine ZJ / C strain, published by Ministry of Agriculture No. 1893 of the people's Republic of China, the results showed that when the total protein concentration of purified antigen was 0.34 渭 g/mL, the total protein concentration of purified antigen was 0.34 渭 g/mL. Preparation of freeze-dried vaccine with immune protection rate of more than 80%: 10 kinds of heat-resistant protective agents (B1-10) and 3 kinds of freeze-dried curves were designed by using freeze-drying technique and antigenic heat-resistant protection technique. The freeze-drying curve and heat-resistant protectant were optimized and three batches of vaccines were produced in batches. According to the quality standards of the existing freeze-dried vaccine and the quality standard of the inactivated porcine circovirus type 2 vaccine strain ZJ / C in announcement No. 1893 of the Ministry of Agriculture, the heat stability, immune efficacy and preservation life of the vaccine were studied. The results showed that all the indexes of three batches of PCV2 freeze-dried seedlings were in accordance with the requirements of regulations. The storage period of 14 days and 8 鈩，

本文编号：1835934