猪瘟病毒E2蛋白碳纳米管抗原的制备及其初步研究

发布时间：2018-03-09 01:22

本文选题：碳纳米管　切入点：猪瘟病毒　出处：《河南科技大学》2015年硕士论文　论文类型：学位论文

【摘要】：碳纳米管属于石墨烯家族,具有较强的跨细胞膜能力,经过功能化的碳纳米管能够携带多种有机或无机粒子,如蛋白质、核酸和化学药物等活性分子进入细胞内部,激活先天性免疫细胞,如单核细胞、巨噬细胞和树突状细胞等。目前国内外学者对碳纳米管的研究主要集中在针对于肿瘤的诊断和治疗方面,随着对碳纳米管的深入研究,越来越多的学者开始关注碳纳米管在疫苗载体方面的应用。猪瘟(classical swine fever,CSF)是由猪瘟病毒(classical swine fever virus,CSFV)感染猪所引起的一种高致病性接触性传染病,在养猪业中危害非常严重。E2蛋白是猪瘟病毒的主要保护性抗原,含有猪瘟病毒高度保守的抗原决定簇,能诱导机体产生猪瘟病毒的中和抗体。为探究碳纳米管作为疫苗载体对动物免疫的影响,本实验通过制备碳纳米管-猪瘟病毒E2蛋白抗原,免疫小鼠后,检测抗体效价,以评估碳纳米管颗粒疫苗对免疫的影响。本实验主要包括三个部分:1)用大肠杆菌进行诱导表达CSFV-E2蛋白,SDS-PAGE鉴定重组蛋白E2的表达形式,采用His标签镍柱亲和层析法进行纯化,并经Western Blot鉴定表达蛋白的抗原特异性。2)用EDC/NHS作偶联剂,将纯化后的重组蛋白E2与羧基化多壁碳纳米管偶联,制备成碳纳米管-E2蛋白抗原,并通过考马斯亮蓝法检测连接蛋白量。3)以碳纳米管-E2蛋白、E2蛋白、弗氏佐剂-E2蛋白免疫小鼠,两次免疫,间隔两周,于二免后第10天,使用间接ELISA检测抗体效价。结果:经考马斯亮蓝法检测后,测得1mg羧基化多壁碳纳米管连接蛋白量为480μg。E2蛋白组抗体效价最高达到1:3200,碳纳米管-E2蛋白组抗体效价最高达到1:12800,弗氏佐剂-E2蛋白组抗体效价最高达到1:51200。结论:本研究通过构建碳纳米管-E2蛋白抗原,并对小鼠进行免疫,通过ELISA检测,证明碳纳米管能够增强免疫反应,促进机体特异性抗体的产生。
[Abstract]:Carbon nanotubes (CNTs) belong to the graphene family and have strong transmembrane capability. Functionalized CNTs can carry a variety of organic or inorganic particles, such as proteins, nucleic acids and chemical drugs, into the cells. Activation of congenital immune cells, such as monocytes, macrophages and dendritic cells. More and more scholars are beginning to pay attention to the application of carbon nanotubes in vaccine carriers. Classical swine virus is a highly pathogenic contact infectious disease caused by classical swine fever virus (CSFV) infection in pigs. E2 protein is the main protective antigen of CSFV and contains highly conserved antigenic determinant of CSFV. In order to investigate the effect of carbon nanotubes (CNTs) as vaccine carrier on animal immunity, we prepared E2 protein antigen of CSFV and immunized mice with antibody titers. In order to evaluate the effect of CNT particle vaccine on immunity, this experiment mainly includes three parts: 1) expression of recombinant protein E2 was identified by SDS-PAGE of CSFV-E2 protein induced by Escherichia coli, and purified by His label nickel column affinity chromatography. The antigen specificity of the expressed protein was identified by Western Blot. 2) using EDC/NHS as coupling agent, the purified recombinant protein E2 was coupled with carboxylated multiwalled carbon nanotubes to prepare a carbon nanotube protein antigen. The mice were immunized with carbon nanotube-E2 protein E2 protein and Freund's adjuvant -E2 protein by Coomassie brilliant blue method. The mice were immunized twice for two weeks, 10 days after the second immunization. Indirect ELISA was used to detect antibody titer. Results: after Coomassie brilliant blue assay, The highest titer of antibody in carboxylated multiwalled carbon nanotubes (480ug / E2) protein group was 1: 3200, the highest antibody titer of carbon nanotube E2 protein group was 1: 12800, and that of Freund's adjuvant-E2 protein group was 1: 51200.Conclusion:. By constructing carbon nanotube-E2 protein antigens, The results of ELISA test showed that carbon nanotubes could enhance the immune response and promote the production of specific antibodies.
【学位授予单位】：河南科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S855.3

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