人星状病毒1型衣壳蛋白的序列分析、克隆表达及多克隆抗体制备

发布时间：2017-12-31 21:22

本文关键词：人星状病毒1型衣壳蛋白的序列分析、克隆表达及多克隆抗体制备　出处：《东华大学》2011年硕士论文　论文类型：学位论文

【摘要】：人星状病毒(Human astrovirus, HAstV)是引起急性病毒性胃肠炎的常见致病原因之一。其包括8个血清型即人星状病毒1型至8型,但以人星状病毒1型(Human astrovirus serotype 1, HAstV-1)在世界范围内最为流行。HAstV-1的基因组长度约6.8 kb,是无包膜的单股正链RNA病毒,包括三个开放读码框架ORF1a、ORF1b、ORF2。生物信息学分析预测显示,由ORF2编码的衣壳蛋白的主要结构蛋白之一-VP26片段,可能在病毒与细胞间相互作用过程中必不可少,而且该片段可能还包含中和及免疫反应表位。本研究首先对11株来自不同地区HAstV-1的衣壳蛋白前体基因核苷酸、氨基酸序列进行同源性比较并绘制进化树,发现按地区差异可分为两个基因组；再利用若干生物学分析软件分析VP26片段的理化性质、结构、生物学特性和抗原特征。然后利用原核表达系统在大肠杆菌中克隆、表达重组VP26蛋白并以NTA-Ni2+亲和层析法纯化重组蛋白,运用SDS-PAGE电泳、BCA试验、Western blot等方法对重组蛋白纯度、浓度、抗原性进行评价鉴定。以重组VP26蛋白作为抗原,免疫新西兰大耳兔获得多抗血清并对其效价、特异性用ELISA方法鉴定。结果表明：(1)原核表达载体pET30a(+)-VP26构建成功,重组VP26蛋白可在大肠杆菌Rosetta 2宿主菌中大量表达。(2)该重组VP26蛋白是研究VP26片段抗原性、生化特性、结构功能的理想工具,可用于评价该片段在免疫诊断和疫苗研制中的作用。(3)该原核表达、亲和层析纯化系统可用于满足今后大规模疫苗制备和检测试剂盒开发的需求,尤其是在应用于检测抗体的ELISA试剂盒包被抗原时,结果和成本要优于使用全病毒。(4)免疫兔所得多抗血清效价达到1：8000可以满足夹心法ELISA实验的需要。综上所述,HAstV-1衣壳蛋白原核表达系统建立和多克隆抗体制备可以作为今后相关研究的基础,有助于对HAstV-1感染致病机理、免疫诊断和疫苗研制的更深入研究。
[Abstract]:Human stellate virus astrovirus. HAstV is one of the common causes of acute viral gastroenteritis, including eight serotypes, human stellate virus type 1 to 8. However, human astrovirus serotype 1 was isolated from human stellate virus type 1. HAstV-1) the genome length of HAstV-1, which is the most popular in the world, is about 6.8 kb and is a single-stranded positive strand RNA virus without capsule. The bioinformatics prediction showed that one of the main structural proteins of capsid protein encoded by ORF2 was VP26 fragment. It may be necessary in the process of virus-cell interaction, and the fragment may also contain neutralized and immunoreactive epitopes. In this study, 11 strains of capsid protein precursor genes from different regions of HAstV-1 were compared in nucleotide and amino acid sequence homology and the phylogenetic tree was drawn. It was found that two genomes could be divided into two genomes according to regional differences. The physicochemical properties, structure, biological characteristics and antigen characteristics of VP26 fragments were analyzed by several biological analysis softwares, and then cloned in E. coli by prokaryotic expression system. The recombinant VP26 protein was expressed and purified by NTA-Ni2 affinity chromatography. The recombinant protein was purified by SDS-PAGE electrophoresis. Western blot was used to evaluate the purity, concentration and antigenicity of the recombinant protein. The recombinant VP26 protein was used as antigen to immunize New Zealand rabbits to obtain the polyclonal antiserum and its titer. The specificity was identified by ELISA. The results showed that the prokaryotic expression vector pET30a (pET-VP26) was successfully constructed. Recombinant VP26 protein can be expressed in Escherichia coli Rosetta 2 host strain. (2) the recombinant VP26 protein is used to study the antigenicity and biochemical characteristics of VP26 fragment. An ideal structural and functional tool for evaluating the role of the fragment in immunological diagnosis and vaccine development. Affinity chromatography purification system can be used to meet the needs of large-scale vaccine preparation and detection kit development in the future, especially when used in the detection of antibody ELISA kit coated antigen. The results and cost were better than that of the whole virus. 4) the titer of the polyantiserum was 1: 8000, which could meet the needs of the sandwich ELISA test. To sum up, the establishment of prokaryotic expression system of HAstV-1 capsid protein and the preparation of polyclonal antibody can be used as the basis of related research in the future, which is helpful to the pathogenesis of HAstV-1 infection. Further research on immune diagnosis and vaccine development.
【学位授予单位】：东华大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

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