本文關(guān)鍵詞： 手足口病 EV71 類病毒顆粒 Bac-to-Bac桿狀病毒表達(dá)系統(tǒng) 免疫評(píng)價(jià)　出處：《北京協(xié)和醫(yī)學(xué)院》2013年碩士論文　論文類型：學(xué)位論文

【摘要】：手足口病(Hand, foot and mouth disease, HFMD)是由多種腸道病毒引起的常見傳染病。近幾年來,手足口病在亞太地區(qū)呈上升趨勢(shì),成為危害嬰幼兒健康的重要傳染病。腸道病毒71型(Enterovirus71,EV71)是HFMD最重要的病原體,柯薩奇A16型(Coxsackies A16, CA16)也在HFMD中占有一定的比例,目前EV71和CA16是我國近幾年流行的兩種重要病原體。由EV71感染引起的手足口病臨床表現(xiàn)為手足口部位出現(xiàn)皮疹或皰疹、潰瘍等,在嬰幼兒常會(huì)伴有嚴(yán)重的神經(jīng)性并發(fā)癥,包括病毒性腦膜炎、腦炎以及脊髓灰質(zhì)炎樣麻痹,引起的肺水腫、肺出血常導(dǎo)致嬰幼兒死亡。迄今為止尚無特效抗病毒藥物問世,疫苗將成為預(yù)防控制手足口病流行最有效的手段之一。疫苗的研制包括減毒活疫苗、滅活疫苗及基因工程疫苗,這些疫苗各有特點(diǎn),本文重點(diǎn)研究了桿狀病毒作為表達(dá)系統(tǒng)表達(dá)EV71主要抗原基因形成類病毒顆粒及其免疫效果。 在新型疫苗中,亞單位疫苗以類病毒顆粒(Virus-like Particles, VLPs)較為理想,因其由病毒外殼蛋白所組成,構(gòu)象上最接近天然病毒顆粒,具有較好的免疫原性,且不含病毒基因組成分,安全性較高,這些優(yōu)點(diǎn)使類病毒顆粒疫苗成為基因工程疫苗研究的熱點(diǎn)。桿狀病毒表達(dá)系統(tǒng)(baculovirus expression system, BES)是一個(gè)以昆蟲桿狀病毒為外源基因載體,以昆蟲細(xì)胞為受體的真核表達(dá)體系。因其可以容納大片段外源基因進(jìn)行表達(dá),使其在疫苗研究中更具有優(yōu)勢(shì),也由于是在真核系統(tǒng)中表達(dá),桿狀病毒表達(dá)系統(tǒng)被認(rèn)為是表達(dá)類病毒顆粒的最佳表達(dá)系統(tǒng)之一 目的：利用流行地區(qū)分離到的EV71毒株,應(yīng)用Bac-to-Bac桿狀病毒表達(dá)系統(tǒng)表達(dá)EV71類病毒顆粒,并對(duì)類病毒顆粒的免疫原性做了初步評(píng)價(jià)。 方法：首先對(duì)科室構(gòu)建保存的攜帶EV71結(jié)構(gòu)蛋白基因P1與非結(jié)構(gòu)蛋白基因3CD的供體質(zhì)粒pFastBac-Dual-Pl-3CD進(jìn)行雙酶切鑒定,然后將鑒定正確的供體質(zhì)粒轉(zhuǎn)入E. coli DH10中,與桿狀病毒質(zhì)粒Bacmid進(jìn)行同源重組,經(jīng)藍(lán)白斑篩選和PCR鑒定,構(gòu)建重組桿狀病毒質(zhì)粒Bacmid-Pl-3CD,將重組桿粒通過脂質(zhì)體轉(zhuǎn)染法轉(zhuǎn)染昆蟲細(xì)胞sf9細(xì)胞系,獲得攜帶EV71病毒結(jié)構(gòu)基因P1和3CD蛋白酶基因的重組桿狀病毒rBV-Pl-3CD,并通過蝕斑的方法對(duì)重組桿狀病毒的滴度進(jìn)行了測(cè)定。采用免疫熒光、Western Blot及電鏡觀察等方法檢測(cè)重組病毒表達(dá)產(chǎn)物特異性及形態(tài)學(xué)特征。表達(dá)產(chǎn)物經(jīng)蔗糖密度梯度離心純化,純化后的目的蛋白經(jīng)電鏡、Western Blot鑒定后免疫BALB/c小鼠,用ELISA、微量中和試驗(yàn)的方法對(duì)其免疫效果進(jìn)行初步評(píng)價(jià)。 結(jié)果：PCR鑒定結(jié)果顯示構(gòu)建的重組桿狀病毒rBV-Pl-3CD中可擴(kuò)增到特異性的P1和3CD基因,間接免疫熒光表明重組桿狀病毒rBV-Pl-3CD能特異性表達(dá)EV71結(jié)構(gòu)蛋白,Western Blot結(jié)果可見分子量約為39KD,條帶符合VPl分子量,證明P1基因可被表達(dá)的3CD酶正確切割。電鏡結(jié)果可觀察到大小約為24nm-30nm的顆粒,ELISA法檢測(cè)到實(shí)驗(yàn)組小鼠血清中抗EV71特異性IgG抗體,效價(jià)為1：776,微量中和實(shí)驗(yàn)顯示,血清能中和EV71病毒,中和效價(jià)為1：588。 結(jié)論：利用Bac-to-Bac桿狀病毒表達(dá)系統(tǒng)共表達(dá)EV71的結(jié)構(gòu)蛋白P1和非結(jié)構(gòu)蛋白3CD,經(jīng)3CD正確切割P1,在sf9細(xì)胞中組裝形成EV71類病毒顆粒。經(jīng)動(dòng)物實(shí)驗(yàn)初步評(píng)價(jià),該類病毒顆粒能夠誘導(dǎo)實(shí)驗(yàn)小鼠產(chǎn)生特異性抗EV71抗體,具有較好的免疫原性。這項(xiàng)研究獲得的結(jié)果為EV71新型疫苗的研發(fā)提供了依據(jù)和參考。
[Abstract]:Foot and mouth disease (Hand foot, and mouth disease, HFMD) is a common infectious disease caused by a variety of intestinal virus. In recent years, HFMD showed a rising trend in the Asia Pacific region has become an important infectious disease harm the health of infants. Enterovirus 71 (Enterovirus71, EV71) HFMD is the most important pathogens. Coxsackie A16 (Coxsackies A16 CA16) also occupy a certain proportion in the HFMD, the EV71 and CA16 are two important pathogens prevalent in our country in recent years. The clinical manifestations of HFMD caused by EV71 infection of the foot and mouth were rash or herpes and ulcer in children, often accompanied by nerve of the serious complications, including viral meningitis, encephalitis and poliomyelitis like paralysis, caused by pulmonary edema, pulmonary hemorrhage often leads to death in infants. So far there is no effective antiviral drugs available, vaccine will become the prevention and control of hand foot and mouth One of the most effective ways of disease epidemics. The development of vaccines includes live attenuated vaccine, inactivated vaccine and genetic engineering vaccine. These vaccines have their own characteristics. This paper focuses on the baculovirus expression system as the main antigen gene expressing EV71, forming virus like particles and their immune effects.
In the new type of vaccine, subunit vaccine with the virus like particles (Virus-like Particles, VLPs) is ideal, which is due to the virus coat protein conformation, the closest natural virus particles has good immunogenicity, and does not contain the viral genome composition, high safety, these advantages make the virus like particles vaccine research of genetic engineering vaccine. The baculovirus expression system (baculovirus expression system, BES) is a baculovirus gene vector in insect cells for eukaryotic expression system of receptors. Because it can accommodate large fragments of exogenous gene expression, which has more advantages in vaccine research, but also because of is in the eukaryotic expression systems, the baculovirus expression system is considered to be one of the best expression of virus like particles
Objective: to express EV71 virus particles by using Bac-to-Bac baculovirus expression system, and to preliminarily evaluate the immunogenicity of viroid particles by using EV71 isolates isolated from epidemic areas.
Methods: First Department building preservation with EV71 structural protein P1 gene and non structural protein gene 3CD donor plasmid pFastBac-Dual-Pl-3CD by double enzyme digestion, and then correct identification of donor plasmid into E. coli DH10, homologous recombination and baculovirus plasmid Bacmid by blue white screening and PCR identification, the recombinant baculovirus plasmid Bacmid-Pl-3CD, the recombinant bacmids transfected insect cells Sf9 cells by liposome transfection method, the recombinant baculovirus carrying the rBV-Pl-3CD structure of EV71 virus gene P1 and 3CD protease gene, and through the method of plaque titer of recombinant baculovirus was determined. Using immunofluorescence and morphological characteristics, product specific detection of recombinant Western virus Blot and electron microscopy expression. The expression products were purified by sucrose density gradient centrifugation, purified by protein The immune effect of BALB/c mice was evaluated by ELISA and microneutralization test by electron microscopy and Western Blot.
Results: PCR analysis showed that P1 can be amplified and 3CD specific gene in recombinant baculovirus constructed by rBV-Pl-3CD, indirect immunofluorescence showed that the recombinant baculovirus rBV-Pl-3CD can express EV71 protein structure specificity, Western Blot showed that molecular weight is about 39KD, with compliance with the molecular weight of VPl, that 3CD enzyme P1 gene the correct expression is cut. Electron microscopy results can be observed about the size of 24nm-30nm particles, the detection of anti EV71 specific IgG antibody in the serum of mice in experimental group ELISA, the titer of 1:776 display, micro neutralization test, serum neutralized EV71 virus neutralizing antibody titer was 1:588.
Conclusion: the Bac-to-Bac baculovirus expression system co expression of EV71 structural proteins and non structural proteins P1 3CD, by 3CD cut P1, EV71 assemble into virus like particles in Sf9 cells. The animal experiment preliminary evaluation, the virus particles can induce the mice to produce specific anti EV71 antibody, has good immunogenicity the results obtained in this research provide a reference basis for the research and development of new EV71 vaccine.

【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R392;R373.2

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相關(guān)期刊論文 前10條

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本文編號(hào)：1486580