Vero细胞H5N1流感病毒的培养及其后处理工艺研究
发布时间：2019-01-03 09:48
【摘要】：近年来我国发生了多次禽流感疫情,造成了巨大的社会恐慌和经济损失。疫苗接种是目前预防和控制流感最经济有效的措施。在我国获得批准使用的流感疫苗均以鸡胚作为病毒生产基质。鉴于鸡胚的供应需要较长的时间,不能在短期内提供足够的疫苗以应对随时可能爆发的流感大流行。而Vero细胞能够利用细胞工厂和微载体-生物反应器系统进行大规模培养,生产过程易于控制及实现规模化生产,且培养出的流感病毒较鸡胚培养的病毒变异小,用其生产出的流感疫苗与当时流行株匹配程度高。因此,开发Vero细胞大流行流感疫苗显得尤为必要。在国内外,细胞工厂培养技术已经是非常成熟和普遍的体外大量培养细胞的技术,如培养KMB-17、2BS、MRC-5等细胞。其具有便捷灵活安全的操作方式,较少的占用空间,可控性好,既有优于转瓶的优势,又没有类似生物反应器的应用限制。本文主要考察了 Vero细胞在细胞工厂中的生长情况以及利用细胞工厂培养流感病毒条件的优化,筛选出了细胞工厂培养H5N1流感病毒较适培养条件为:胞龄为60～72 h、TPCK-胰酶含量为2μg/ml,MOI为0.1,收毒时间为接种后72 h,培养温度为33±0.5℃,维持液pH值约为7.6,病毒滴度达到1:256～1:1024。免疫荧光检测发现H5N1亚型流感病毒能够较好地感染Vero细胞;H5N1 Va流感病毒(A/Anhui/1/2005 va)在Vero细胞上扩增的过程中型别保持不变;基因序列比对结果证实H5N1Va两个表面抗原基因确实来源于A/Anhui/1/2005(H5N1)。由于Vero细胞为传代细胞系,生产时存在一些问题,如残余宿主蛋白可能引起过敏反应以及宿主细胞DNA有致瘤性风险。疫苗生产时需要控制Vero细胞的代次在150代以内,并进行后处理纯化以减少残余Vero细胞DNA和蛋白含量。本文对Vero细胞流感疫苗后处理纯化工艺进行了摸索,确定使用0.65、0.45、0.22 μm滤芯进行三级过滤澄清、300 KD超滤膜包浓缩、加Benzonase酶37 ℃处理病毒浓缩液4 h、1000 KD超滤膜包低于5 psi洗滤浓缩,然后再经Sepharose 4FF和DEAE Sepharose FF两步柱层析纯化。病毒回收率70%以上,残余DNA含量小于100 pg/剂,宿主蛋白含量小于2 μg/剂,BSA含量小于50 ng/剂,都达到了药典要求。该研究为以Vero细胞为基质的大流行流感疫苗的研发奠定了基础。
[Abstract]:In recent years, there have been many outbreaks of avian flu in China, resulting in huge social panic and economic losses. Vaccination is the most economical and effective measure to prevent and control influenza. Chicken embryo is used as the production medium of influenza vaccine approved in China. Given the long supply of chicken embryos, it is not possible to provide enough vaccines in the short term for an influenza pandemic at any time. However, Vero cells can be cultured on a large scale by cell factory and microcarrier bioreactor system. The production process is easy to control and realize large-scale production, and the influenza virus produced is less variant than that of chicken embryo. The flu vaccine produced by the vaccine matched well with the prevailing strain at the time. Therefore, the development of Vero cell pandemic influenza vaccine is particularly necessary. At home and abroad, cell factory culture technology has been a very mature and universal in vitro culture of cells, such as the culture of KMB-17,2BS,MRC-5 cells. The utility model has the advantages of convenient, flexible and safe operation, less space occupation, good controllability, which is superior to the flask rotation, and has no application limitations similar to that of bioreactor. In this paper, the growth of Vero cells in cell factories and the optimization of the conditions for the cultivation of influenza viruses in cell factories were investigated. The optimum conditions for the culture of H5N1 influenza viruses in cell factories were as follows: the cell age was 6072h. The TPCK- trypsin content was 2 渭 g / ml moi 0.1, the inoculation time was 72 h, the culture temperature was 33 卤0.5 鈩，
本文编号：2399212
[Abstract]:In recent years, there have been many outbreaks of avian flu in China, resulting in huge social panic and economic losses. Vaccination is the most economical and effective measure to prevent and control influenza. Chicken embryo is used as the production medium of influenza vaccine approved in China. Given the long supply of chicken embryos, it is not possible to provide enough vaccines in the short term for an influenza pandemic at any time. However, Vero cells can be cultured on a large scale by cell factory and microcarrier bioreactor system. The production process is easy to control and realize large-scale production, and the influenza virus produced is less variant than that of chicken embryo. The flu vaccine produced by the vaccine matched well with the prevailing strain at the time. Therefore, the development of Vero cell pandemic influenza vaccine is particularly necessary. At home and abroad, cell factory culture technology has been a very mature and universal in vitro culture of cells, such as the culture of KMB-17,2BS,MRC-5 cells. The utility model has the advantages of convenient, flexible and safe operation, less space occupation, good controllability, which is superior to the flask rotation, and has no application limitations similar to that of bioreactor. In this paper, the growth of Vero cells in cell factories and the optimization of the conditions for the cultivation of influenza viruses in cell factories were investigated. The optimum conditions for the culture of H5N1 influenza viruses in cell factories were as follows: the cell age was 6072h. The TPCK- trypsin content was 2 渭 g / ml moi 0.1, the inoculation time was 72 h, the culture temperature was 33 卤0.5 鈩，
本文编号：2399212
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