發(fā)布時(shí)間：2018-11-07 20:10

【摘要】： 手足口病是嬰幼兒的一種常見(jiàn)傳染病。EV71是引起患者手足口病的主要病原體之一,同時(shí)還能引起無(wú)菌性腦膜炎、腦干腦炎和脊髓灰質(zhì)炎樣麻痹等多種與神經(jīng)系統(tǒng)相關(guān)的疾病,嚴(yán)重威脅了兒童的生命健康,預(yù)防EV71的流行十分重要。據(jù)統(tǒng)計(jì),2009年我國(guó)共報(bào)告手足口病病例約115.5萬(wàn)例(不含港、澳、臺(tái)),死亡病例353例,占我國(guó)丙類傳染病發(fā)病數(shù)第一位。至今尚無(wú)有效的針對(duì)性的疫苗預(yù)防EV71的感染,只能提高監(jiān)測(cè)的強(qiáng)度和靈敏度,早發(fā)現(xiàn),早治療。因此研制針對(duì)EV71的預(yù)防性疫苗對(duì)控制嬰幼兒手足口病的流行具有十分重要的意義。 本課題選擇利用釀酒酵母表達(dá)系統(tǒng)發(fā)酵表達(dá)EV71 VLP。首先參照釀酒酵母基因中的優(yōu)勢(shì)密碼子,運(yùn)用分子生物學(xué)技術(shù)對(duì)EV71 P1和3CD基因進(jìn)行設(shè)計(jì)和全基因合成。將合成的基因序列克隆到釀酒酵母表達(dá)載體上,轉(zhuǎn)化釀酒酵母進(jìn)行誘導(dǎo)表達(dá)。同時(shí)擴(kuò)增EV71 VP1、VP2、VP3和VP4四個(gè)基因,克隆到釀酒酵母表達(dá)載體上,轉(zhuǎn)化釀酒酵母進(jìn)行誘導(dǎo)表達(dá)。其次,對(duì)誘導(dǎo)后的酵母細(xì)胞進(jìn)行破碎,利用蛋白沉淀、氯化銫密度梯度離心等純化方法初步純化分離VLP。對(duì)純化后樣品進(jìn)行Western Blot檢測(cè)鑒定;并通過(guò)負(fù)染電鏡觀察VLP。 本課題利用兩種方法在釀酒酵母中表達(dá)EV71 VLP:1.將VP1、VP2、VP3和VP4四個(gè)基因共同轉(zhuǎn)化酵母細(xì)胞,可以在釀酒酵母細(xì)胞中表達(dá),并自行組裝成VLP。2. P1和3CD蛋白可以在釀酒酵母中成功表達(dá),蛋白酶3CD將P1蛋白切割成VP1、VP2、VP3和VP4,后者在釀酒酵母中自行組裝成VLP。實(shí)驗(yàn)中初步純化的VLP可作為EV71預(yù)防性疫苗進(jìn)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.
【學(xué)位授予單位】：北京工業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392

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本文編號(hào)：2317444