EV71类病毒颗粒的表达及免疫原性的初步评价

发布时间：2018-02-03 05:40

本文关键词： 手足口病 EV71 类病毒颗粒 Bac-to-Bac杆状病毒表达系统免疫评价　出处：《北京协和医学院》2013年硕士论文　论文类型：学位论文

【摘要】：手足口病(Hand, foot and mouth disease, HFMD)是由多种肠道病毒引起的常见传染病。近几年来,手足口病在亚太地区呈上升趋势,成为危害婴幼儿健康的重要传染病。肠道病毒71型(Enterovirus71,EV71)是HFMD最重要的病原体,柯萨奇A16型(Coxsackies A16, CA16)也在HFMD中占有一定的比例,目前EV71和CA16是我国近几年流行的两种重要病原体。由EV71感染引起的手足口病临床表现为手足口部位出现皮疹或疱疹、溃疡等,在婴幼儿常会伴有严重的神经性并发症,包括病毒性脑膜炎、脑炎以及脊髓灰质炎样麻痹,引起的肺水肿、肺出血常导致婴幼儿死亡。迄今为止尚无特效抗病毒药物问世,疫苗将成为预防控制手足口病流行最有效的手段之一。疫苗的研制包括减毒活疫苗、灭活疫苗及基因工程疫苗,这些疫苗各有特点,本文重点研究了杆状病毒作为表达系统表达EV71主要抗原基因形成类病毒颗粒及其免疫效果。在新型疫苗中,亚单位疫苗以类病毒颗粒(Virus-like Particles, VLPs)较为理想,因其由病毒外壳蛋白所组成,构象上最接近天然病毒颗粒,具有较好的免疫原性,且不含病毒基因组成分,安全性较高,这些优点使类病毒颗粒疫苗成为基因工程疫苗研究的热点。杆状病毒表达系统(baculovirus expression system, BES)是一个以昆虫杆状病毒为外源基因载体,以昆虫细胞为受体的真核表达体系。因其可以容纳大片段外源基因进行表达,使其在疫苗研究中更具有优势,也由于是在真核系统中表达,杆状病毒表达系统被认为是表达类病毒颗粒的最佳表达系统之一目的：利用流行地区分离到的EV71毒株,应用Bac-to-Bac杆状病毒表达系统表达EV71类病毒颗粒,并对类病毒颗粒的免疫原性做了初步评价。方法：首先对科室构建保存的携带EV71结构蛋白基因P1与非结构蛋白基因3CD的供体质粒pFastBac-Dual-Pl-3CD进行双酶切鉴定,然后将鉴定正确的供体质粒转入E. coli DH10中,与杆状病毒质粒Bacmid进行同源重组,经蓝白斑筛选和PCR鉴定,构建重组杆状病毒质粒Bacmid-Pl-3CD,将重组杆粒通过脂质体转染法转染昆虫细胞sf9细胞系,获得携带EV71病毒结构基因P1和3CD蛋白酶基因的重组杆状病毒rBV-Pl-3CD,并通过蚀斑的方法对重组杆状病毒的滴度进行了测定。采用免疫荧光、Western Blot及电镜观察等方法检测重组病毒表达产物特异性及形态学特征。表达产物经蔗糖密度梯度离心纯化,纯化后的目的蛋白经电镜、Western Blot鉴定后免疫BALB/c小鼠,用ELISA、微量中和试验的方法对其免疫效果进行初步评价。结果：PCR鉴定结果显示构建的重组杆状病毒rBV-Pl-3CD中可扩增到特异性的P1和3CD基因,间接免疫荧光表明重组杆状病毒rBV-Pl-3CD能特异性表达EV71结构蛋白,Western Blot结果可见分子量约为39KD,条带符合VPl分子量,证明P1基因可被表达的3CD酶正确切割。电镜结果可观察到大小约为24nm-30nm的颗粒,ELISA法检测到实验组小鼠血清中抗EV71特异性IgG抗体,效价为1：776,微量中和实验显示,血清能中和EV71病毒,中和效价为1：588。结论：利用Bac-to-Bac杆状病毒表达系统共表达EV71的结构蛋白P1和非结构蛋白3CD,经3CD正确切割P1,在sf9细胞中组装形成EV71类病毒颗粒。经动物实验初步评价,该类病毒颗粒能够诱导实验小鼠产生特异性抗EV71抗体,具有较好的免疫原性。这项研究获得的结果为EV71新型疫苗的研发提供了依据和参考。
[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.

【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R392;R373.2

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