乙型脑炎SA14-14-2株E、NS1蛋白的原核表达及其免疫原性研究

发布时间：2018-01-04 03:31

本文关键词：乙型脑炎SA14-14-2株E、NS1蛋白的原核表达及其免疫原性研究　出处：《中国药品生物制品检定所》2006年硕士论文　论文类型：学位论文

【摘要】：流行性乙型脑炎病毒(Japanese encephalitis virus，JEV)简称乙脑病毒，又称日本脑炎病毒，是一种严重危害人畜(尤其是儿童)健康的虫媒病毒。目前没有治疗乙脑的有效药物。接种乙脑疫苗是控制乙脑的最重要措施。我国是乙型脑炎发病人数最多的国家。但近十几年来，乙脑的发病率明显下降，其中原因主要归功于疫苗的接种。目前国际普遍接种的疫苗是鼠脑来源的灭活疫苗。如日本、韩国、新加坡、印度以及美国等使用鼠脑来源的Nakayama株和／或Beijing-1株的灭活疫苗。由于灭活疫苗存在一些缺点，即(1)需要多次接种才能产生良好的免疫效果，(2)不良反应渐多，如局部反应率达20％左右，全身反应率达10％，(3)价格昂贵，生产工艺复杂等大大影响其进一步推广。因此需要研究生产更有效、安全、方便的疫苗。目前研究的新疫苗有两类，第一为减毒活疫苗，由SA14衍生出的SA14-14-2已经被多方面证明毒力低、安全性好。目前我国广泛使用的疫苗有SA14-14-2株原代地鼠肾细胞减毒活疫苗和北京P3株地鼠肾细胞灭活疫苗。SA14-14-2株原代地鼠肾细胞减毒活疫苗在国内已经成功接种逾2亿人，经证实安全、有效、便宜。第二类为基因重组疫苗。IC47为无毒重组的JEV，但由于没有解决稳定剂型、清除毒力逆转的问题，在作为正规疫苗应用之前，有很长的一段距离。因此，，减毒活疫苗SA14-14-2病毒株引起国内国际疫苗界人士的高度重视。本课题选用JEV SA14-14-2第一个被翻译的非结构基因ns1进行研究，采用PCR方法扩增NS1蛋白基因和E蛋白基因片段，将各基因片断克隆到原核表达载体pET系统上，并使其在大肠杆菌中诱导表达，筛选出表达菌株pET-32Et和pET—30NS1，通过亲和层析对两种蛋白进行纯化，得到纯度在96％以上的目的蛋白。
[Abstract]:Japanese encephalitis virus (je) is also known as Japanese encephalitis virus (JEV). Is a serious threat to people and animals (especially children). There is no effective drug to treat Japanese encephalitis. Vaccination with je vaccine is the most important measure to control Japanese encephalitis. China is the country with the highest incidence of encephalitis B, but in recent ten years. The incidence of encephalitis B has declined significantly, mainly due to vaccination. Currently, inactivated vaccines from the brain are widely used in the world, such as Japan, South Korea, and Singapore. Inactivated vaccines using brain-derived Nakayama and / or Beijing-1 strains in India and the United States. That is to say, many times of inoculation is needed to produce good immune effect. (such as local reaction rate of 20% or so, systemic reaction rate of 10 ~ 3%) is more and more expensive. The complexity of production technology has greatly affected its further promotion. Therefore, it is necessary to study and produce more effective, safe and convenient vaccines. There are two kinds of new vaccines, the first is live attenuated vaccine. SA14-14-2 derived from SA14 has been proved to be low virulence in many ways. At present, the widely used vaccines in China include SA14-14-2 strain primary hamster kidney cell attenuated vaccine and Beijing P3 strain hamster kidney cell inactivated vaccine .SA14-14-2 primary hamster kidney vaccine. Live attenuated cell vaccines have been successfully vaccinated against more than 200 million people in China. The second type is the gene recombinant vaccine. IC47 is the non-toxic recombinant JEV, but the problem of virulence reversal has not been solved because the stabilizer type has not been solved. Before being used as a regular vaccine, there is a long distance. Therefore, the attenuated live vaccine SA14-14-2 strain has attracted great attention from domestic and international vaccine researchers. In this study, the first translated non-structural gene ns1 of JEV SA14-14-2 was used to amplify NS1 gene and E protein gene by PCR. The gene fragments were cloned into the prokaryotic expression vector pET system and induced in Escherichia coli. The expression strains pET-32Et and pET-30NS1 were screened out. The two proteins were purified by affinity chromatography and the purity of the two proteins was over 96%.
【学位授予单位】：中国药品生物制品检定所
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392

【引证文献】