17D黄热病病毒在鸡胚细胞上的适应性传代及其相关特性的研究

发布时间：2018-06-04 03:53

本文选题：黄热病病毒 + 黄热疫苗　；参考：《北京生物制品研究所》2010年硕士论文

【摘要】： 黄热疫苗(YFV)自1936年开始在黄热病流行地区用于预防黄热病的感染,已经在世界范围内安全使用了60多年,有效控制了黄热病的大规模流行,是目前最安全、有效的虫媒病毒疫苗之一。但是至今尚未完全控制非洲和南美洲地区的黄热病爆发,除了疫苗的推广使用未能覆盖所有易感人群外,越来越多的黄热疫苗相关不良反应也引起了世界卫生组织和科学家的高度重视。黄热疫苗病毒在鸡胚卵黄囊中培养,因其特有的培养基质和病毒特性,在后续纯化工艺中,很难在保持有效免疫原性的同时获得良好的纯度。本实验通过改变其培养基质,以期在不降低免疫原性的同时,减少疫苗中鸡胚杂质残留。将17D黄热疫苗工作毒种在鸡胚成纤维细胞中连续传代,通过对传代病毒的生物学相关特性和基因遗传稳定性研究,探讨将传统用于黄热疫苗生产的全鸡胚改换为鸡胚成纤维细胞后,是否会对疫苗病毒的基因组和病毒的生物学特性产生影响,从而为使用鸡胚成纤维细胞作为黄热疫苗生产基质的可能性提供依据。本实验选择黄热疫苗国际标准品99/616-CE(天坛生物编号)和我国黄热疫苗工作毒种2008(54)01(54代)5#(天坛生物编号)作为实验用毒种,99/616为世界卫生组织参考品。两者都是17D疫苗株,均源于17D204株,并经特异性抗血清鉴别试验确认毒种。两株毒种分别在鸡胚成纤维细胞中进行适应性传代,各自盲传10代,记作“99/616-传代病毒代次”和“YFVBJ-传代病毒代次”。并分别对两株病毒的传代病毒的生物学相关特性和基因遗传稳定性进行了研究,内容包括传代病毒病毒滴度检测、LD50检测、小鼠平均生存时间检测、蚀斑大小检测、免疫原性检测、杂蛋白含量以及非结构基因NS1序列测定。实验结果的数据分析如下: 一.两株毒种的传代病毒滴度都存在一个下降趋势,但滴度均在5.0lgPFU/ml以上,各代次之间差异不显著。二.两株毒种的传代病毒经LD50检测都存在一个下降趋势,但除99/616-1外滴度都在5.0lgLD50以上,各代次之间差异不显著。三.小鼠平均生存时间、免疫原性等研究表明,传代病毒各代次之间未见显著性统计学差异。四.两株毒种的传代病毒在蚀斑大小试验中显示蚀斑大小存在变小的趋势,部分代次之间统计学差异显著。五.YFVBJ各传代病毒非结构基因NS1序列分析表明碱基序列未发生改变。这些结果提示虽然改变了培养基质,但我国的黄热疫苗工作毒种无论在生物学相关特性还是基因序列遗传稳定性上都表现出良好的稳定性。综上所述:本研究完成了黄热疫苗工作毒种和99/616黄热国际标准品在鸡胚成纤维细胞中的传代,并初步研究了培养基质改变对黄热病病毒的生物学相关特性以及基因遗传稳定性的影响,除了传代病毒的蚀斑大小有明显的差异之外,传代病毒表现出了良好的稳定性。这些工作为今后更深入的研究以鸡胚成纤维细胞为基质培养17D黄热病疫苗提供了基础数据。
[Abstract]:Yellow fever vaccine ( YFV ) has been used for the prevention of yellow fever in the epidemic area of yellow fever since 1936 . It has been safely used in the world for more than 60 years . It is one of the most secure and effective vaccine for the disease of yellow fever . However , it has not fully controlled the outbreak of yellow fever in Africa and South America . Besides , it has not completely controlled the outbreak of yellow fever in Africa and South America . In addition to the popularization and use of the vaccine , more and more adverse reactions related to yellow fever vaccine have also attracted much attention from the World Health Organization and scientists .

in ord to reduce immunogenicity , it is difficult to obtain good purity while maintaining effective immunogenicity .

In this experiment , the international standard of yellow fever vaccine 99 / 616 - CE ( Tiantan Biological No . ) and China Yellow Fever Vaccine Working Poison 2008 ( 54 ) 01 ( 54 generation ) 5 # ( Tiantan Biological No . ) were selected as the reference products for the experiment . Both of them were derived from 17D204 strains .

The data analysis of the experimental results is as follows :

One . There was a downward trend in the titer of the two strains of virus , but the titer was above 5.0lgPFL / ml , and there was no significant difference between successive generations .

2 . There was a downward trend in the LD50 test of the two strains of the virus , but the titer was more than 5.0g LD50 except for 99 / 616 - 1 , and there was no significant difference between the generations .

3 . The average survival time and immunogenicity of mice showed that there was no significant statistical difference between the generations of the passaging virus .

4 . There was a tendency for the size of plaque to be smaller in the test of plaque size . There was a significant difference between the partial generations .

5 . The analysis of the non - structural gene of the non - structural gene of YFVBJ showed that the nucleotide sequence had not changed . These results suggested that although the culture substrate was changed , the yellow fever vaccine work in China showed good stability both in the biology - related characteristic and the genetic stability of the gene sequence .

In conclusion , this study has completed the passage of yellow fever vaccine work toxin and 99 / 616 yellow fever international standard in chicken embryo fibroblasts , and preliminarily studied the effect of the change of culture matrix on the biological characteristics and genetic stability of yellow fever virus .
【学位授予单位】：北京生物制品研究所
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

【参考文献】