呼吸道合胞病毒表位重组蛋白的初步研究

发布时间：2018-01-30 10:42

本文关键词： 呼吸道合胞病毒表位预测 F蛋白 G蛋白中和表位　出处：《西北农林科技大学》2009年硕士论文　论文类型：学位论文

【摘要】： 呼吸道合胞病毒( Respiratory syncytial virus,RSV)是引起婴幼儿和易感成人呼吸道感染最主要的病毒性病原。它除了引起上呼吸道感染外,还是引起毛细支气管肺炎等下呼吸道感染的重要原因。自然感染率极高,对婴幼儿、老年人及免疫抑制患者能引起严重的下呼吸道炎症反应。世界卫生组织(WHO)十分重视该疫苗的开发,呼吁开发安全有效的RSV疫苗。但由于多种限制因素,例如:RSV A、B两亚型交错出现、选择合适的载体蛋白较困难等问题,迄今尚无成功的疫苗问世。为寻求可以用于RSV疫苗研制的表位,在病毒吸附蛋白(G蛋白)和融合蛋白(F蛋白)中寻找合适的中和表位,以Balb/c小鼠为动物模型,通过动物试验检测中和表位的效果。通过对吸附蛋白和融合蛋白的生物信息学预测和文献调研查找,从G蛋白中筛选出五个比较集中的表位,其位于G142-204中,最终选择了G142-204(命名为G)这段区间作为研究对象。从F蛋白筛选出两个表位F205-223和F255-278,使用刚性linker(GPG)来连接,最终选择F205-223-GPG-F255-278(命名为F)作为研究对象。采用重叠PCR技术,构建了编码G142-204和Balb/c小鼠血清白蛋白结构域Ⅰ的融合基因(命名为G-M)、F205-223-GPG-F255-278和Balb/c小鼠血清白蛋白结构域Ⅰ的融合基因(命名为F-M)。将融合基因成功构建在原核表达载体pET28a(+)上,并在大肠埃希菌BL21(DE3)表达。经检测目的蛋白是以包涵体形式表达,采用尿素变性,亲和层析法纯化,获得高纯度的融合蛋白,纯度达到90%。融合蛋白免疫9周龄的雌性Balb/c小鼠,采集小鼠血液,通过ELISA法检测血清中特异性抗体的抗体亚型,F-M蛋白免疫组IgG1/IgG2a比值为1.47,相比之下,较G-M更趋向于Th1/Th2之间的平衡。免疫融合蛋白后Balb/c小鼠肺部及血清中均能产生抗RSV抗体。免疫小鼠RSV攻毒试验,F-M免疫组与G-M免疫组都能够起到保护肺部、降低肺部病毒滴度的作用。本试验制备的融合蛋白具有一定的病毒中和能力,经过进一步的试验印证后,有望开发出一种更加安全有效的RSV疫苗。
[Abstract]:Respiratory syncytial virus (Respiratory syncytial, virus, RSV) is caused by susceptible infants and adult respiratory tract infection is the main pathogenic viruses. In addition it cause upper respiratory tract infection, or cause capillary bronchial pneumonia and other lower respiratory tract infections. The natural infection rate is very high, for infants and young children, the elderly and immunosuppressed patients the lower respiratory tract caused by severe inflammatory reaction. The WHO (WHO) attaches great importance to the development of the vaccine, called for the development of safe and effective RSV vaccine. But due to the limitation of various factors, such as: RSV A, B two subtypes staggered, the appropriate carrier protein is difficult, so far there is no successful advent of vaccines.
In order to find epitope suitable for the development of RSV vaccine, find suitable neutralizing epitope in virus adsorbed protein (G protein) and fusion protein (F protein), and Balb/c mice as animal model, and detect the effect of neutralization epitope by animal test.
The adsorption of protein and fusion protein bioinformatics prediction and literature search, from the G protein were screened five more concentrated epitopes, which is located in G142-204, finally chose G142-204 (named G) of this section as the research object. From the F protein and selected two epitopes of F205-223 and F255-278 linker (GPG), the use of rigid connection, the final choice of F205-223-GPG-F255-278 (named F) as the research object. By using the overlapping PCR technology, constructed the recombinant encoding G142-204 and Balb/c mice serum albumin domain I gene (named G-M), serum albumin fusion domain I F205-223-GPG-F255-278 and Balb/c mouse gene (named F-M). The fusion gene was successfully constructed in prokaryotic expression vector pET28a (+), and in Escherichia coli BL21 (DE3). The expression of the target proteins were expressed in the form of inclusion bodies, using urea denaturation, affinity Chromatography, high purity fusion protein was obtained, the purity of 90%. protein in 9 week old female Balb/c mice were collected by blood, specific antibodies in serum were measured by ELISA antibody subtype, F-M protein IgG1/IgG2a ratio was 1.47, compared with G-M tend to Th1/Th2 balance. The immune fusion protein in lungs and serum of Balb/c mice can produce anti RSV antibody in immunized mice. RSV challenge test, F-M group and G-M group are immune immunity can protect the lungs and reduce the virus titer of lung function. The experimental preparation of fusion protein had the virus neutralizing ability, after further tests confirmed later, is expected to develop a more safe and effective RSV vaccine.

【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

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