肠道病毒71型病毒样颗粒的制备、纯化与鉴定
发布时间：2018-11-07 20:10
【摘要】： 手足口病是婴幼儿的一种常见传染病。EV71是引起患者手足口病的主要病原体之一,同时还能引起无菌性脑膜炎、脑干脑炎和脊髓灰质炎样麻痹等多种与神经系统相关的疾病,严重威胁了儿童的生命健康,预防EV71的流行十分重要。据统计,2009年我国共报告手足口病病例约115.5万例(不含港、澳、台),死亡病例353例,占我国丙类传染病发病数第一位。至今尚无有效的针对性的疫苗预防EV71的感染,只能提高监测的强度和灵敏度,早发现,早治疗。因此研制针对EV71的预防性疫苗对控制婴幼儿手足口病的流行具有十分重要的意义。 本课题选择利用酿酒酵母表达系统发酵表达EV71 VLP。首先参照酿酒酵母基因中的优势密码子,运用分子生物学技术对EV71 P1和3CD基因进行设计和全基因合成。将合成的基因序列克隆到酿酒酵母表达载体上,转化酿酒酵母进行诱导表达。同时扩增EV71 VP1、VP2、VP3和VP4四个基因,克隆到酿酒酵母表达载体上,转化酿酒酵母进行诱导表达。其次,对诱导后的酵母细胞进行破碎,利用蛋白沉淀、氯化铯密度梯度离心等纯化方法初步纯化分离VLP。对纯化后样品进行Western Blot检测鉴定;并通过负染电镜观察VLP。 本课题利用两种方法在酿酒酵母中表达EV71 VLP:1.将VP1、VP2、VP3和VP4四个基因共同转化酵母细胞,可以在酿酒酵母细胞中表达,并自行组装成VLP。2. P1和3CD蛋白可以在酿酒酵母中成功表达,蛋白酶3CD将P1蛋白切割成VP1、VP2、VP3和VP4,后者在酿酒酵母中自行组装成VLP。实验中初步纯化的VLP可作为EV71预防性疫苗进行下一步研究。
[Abstract]:Hand, foot and mouth disease is a common infectious disease in infants and young children. EV71 is one of the main pathogens of hand, foot and mouth disease, and it can also cause aseptic meningitis, brainstem encephalitis, poliomyelitis and other diseases related to the nervous system. It is very important to prevent the prevalence of EV71 because it is a serious threat to the life and health of children. According to statistics, 1.155 million cases of HFMD (excluding Hong Kong, Macao and Taiwan) were reported in China in 2009, and 353 cases of death were reported, accounting for the first place in the number of Class C infectious diseases in China. So far, there is no effective vaccine to prevent EV71 infection, which can only improve the intensity and sensitivity of surveillance, early detection and early treatment. Therefore, the development of preventive vaccine against EV71 is of great significance in controlling the prevalence of HFMD in infants. In this paper, we choose to ferment EV71 VLP. by using yeast expression system of Saccharomyces cerevisiae. According to the dominant codon in Saccharomyces cerevisiae gene, the EV71 P1 and 3CD genes were designed and synthesized by molecular biology. The synthesized gene sequence was cloned into Saccharomyces cerevisiae expression vector and transformed into Saccharomyces cerevisiae for induction and expression. The four genes of EV71 VP1,VP2,VP3 and VP4 were amplified and cloned into the expression vector of Saccharomyces cerevisiae and transformed into Saccharomyces cerevisiae for induction expression. Secondly, the induced yeast cells were broken and purified by protein precipitation and cesium chloride density gradient centrifugation. The purified samples were identified by Western Blot, and VLP. was observed by negative staining electron microscope. Expression of EV71 VLP:1. in Saccharomyces cerevisiae by two methods The four genes of VP1,VP2,VP3 and VP4 were transformed into yeast cells, which could be expressed in Saccharomyces cerevisiae and assembled into VLP.2.. P1 and 3CD proteins can be successfully expressed in Saccharomyces cerevisiae, and protease 3CD dissects P1 protein into VP1,VP2,VP3 and VP4, which self-assemble into VLP. in Saccharomyces cerevisiae. The purified VLP can be used as a prophylactic EV71 vaccine for further study.
【学位授予单位】：北京工业大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392
本文编号：2317444
[Abstract]:Hand, foot and mouth disease is a common infectious disease in infants and young children. EV71 is one of the main pathogens of hand, foot and mouth disease, and it can also cause aseptic meningitis, brainstem encephalitis, poliomyelitis and other diseases related to the nervous system. It is very important to prevent the prevalence of EV71 because it is a serious threat to the life and health of children. According to statistics, 1.155 million cases of HFMD (excluding Hong Kong, Macao and Taiwan) were reported in China in 2009, and 353 cases of death were reported, accounting for the first place in the number of Class C infectious diseases in China. So far, there is no effective vaccine to prevent EV71 infection, which can only improve the intensity and sensitivity of surveillance, early detection and early treatment. Therefore, the development of preventive vaccine against EV71 is of great significance in controlling the prevalence of HFMD in infants. In this paper, we choose to ferment EV71 VLP. by using yeast expression system of Saccharomyces cerevisiae. According to the dominant codon in Saccharomyces cerevisiae gene, the EV71 P1 and 3CD genes were designed and synthesized by molecular biology. The synthesized gene sequence was cloned into Saccharomyces cerevisiae expression vector and transformed into Saccharomyces cerevisiae for induction and expression. The four genes of EV71 VP1,VP2,VP3 and VP4 were amplified and cloned into the expression vector of Saccharomyces cerevisiae and transformed into Saccharomyces cerevisiae for induction expression. Secondly, the induced yeast cells were broken and purified by protein precipitation and cesium chloride density gradient centrifugation. The purified samples were identified by Western Blot, and VLP. was observed by negative staining electron microscope. Expression of EV71 VLP:1. in Saccharomyces cerevisiae by two methods The four genes of VP1,VP2,VP3 and VP4 were transformed into yeast cells, which could be expressed in Saccharomyces cerevisiae and assembled into VLP.2.. P1 and 3CD proteins can be successfully expressed in Saccharomyces cerevisiae, and protease 3CD dissects P1 protein into VP1,VP2,VP3 and VP4, which self-assemble into VLP. in Saccharomyces cerevisiae. The purified VLP can be used as a prophylactic EV71 vaccine for further study.
【学位授予单位】：北京工业大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392
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相关期刊论文 前3条
1 周世力,李琳琳,何雅青,杨洪,喻子牛,金奇;我国分离的肠道病毒71型(SHZH03病毒株)全基因组核苷酸序列分析[J];病毒学报;2004年01期
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3 ;Expression,purification and characterization of enterovirus-71 virus-like particles[J];World Journal of Gastroenterology;2006年06期
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