克里米亚—刚果出血热病毒核衣壳蛋白和糖蛋白分段表达及抗原表位鉴定

发布时间：2018-01-11 14:18

本文关键词：克里米亚—刚果出血热病毒核衣壳蛋白和糖蛋白分段表达及抗原表位鉴定　出处：《新疆大学》2009年硕士论文　论文类型：学位论文

【摘要】： 克里米亚-刚果出血热(Crimean-Congo hemorrhagic fever, CCHF)是一种存在于中国新疆塔里木盆地和准噶尔盆地、俄罗斯北部、中东、南部欧亚大陆及非洲撒哈拉地区的烈性自然疫源性疾病,发病具有明显的季节性与地方性,其平均病死率在10%-50%。该病的病原是经蜱传播的克里米亚-刚果出血热病毒(CCHFV)。该病毒属于布尼亚病毒科内罗病毒属,是一种单股负链RNA病毒。目前,还没有有效的针对CCHF的预防和治疗手段。本研究利用DNA重组技术将克里米亚-刚果出血热病毒YL04057株S片段上的核衣壳蛋白基因(nucleocapsid protein, NP)编码区全序列和一系列分段片段克隆入原核表达载体,在大肠杆菌中进行表达,分离纯化表达产物,用抗克里米亚-刚果出血热病毒的多克隆抗体和2株单克隆抗体(14B7和43E5)对各表达产物进行抗原表位的检测和分析。结果显示,含有NP蛋白第235-305位氨基酸的三个表达蛋白片段可以被抗克里米亚-刚果出血热病毒的多克隆抗体检测出来,也可以与2株单克隆抗体反应。表明两株单抗14B7和43E5以及兔多抗所针对的抗原表位均定位在NP蛋白的第235-305位氨基酸区域,这个区域也是NP蛋白的一个高度保守的氨基酸区域。同时,我们对M片段上的糖蛋白(glycoprotein, GP)编码区分段片段克隆入原核表达载体,在大肠杆菌中表达,分离纯化表达产物,用抗克里米亚-刚果出血热病毒的多克隆抗兔抗体对各表达产物进行抗原表位的检测和分析。结果显示,重组分段糖蛋白均不与兔多抗血清反应。CCHFV糖蛋白结构复杂,而原核表达系统不具有完整的翻译后修饰功能。由于实验室制备的多抗血清多是针对核蛋白的,对糖蛋白的检测缺乏针对性。因此,有必要建立CCHFV糖蛋白的真核表达系统,获得糖蛋白及抗血清,以便于深入开展糖蛋白的研究。研究确定克里米亚-刚果出血热病毒结构蛋白的抗原特性,将有助于克里米亚-刚果出血热病毒感染与免疫机制的阐明,也将为其亚单位疫苗的研制提供理论基础。
[Abstract]:Crimean Congo hemorrhagic fever. CCHFs are a kind of severe natural epidemic diseases in Tarim Basin and Junggar Basin, northern Russia, Middle East, southern Eurasia and sub-Saharan Africa. The disease is seasonal and endemic. The average mortality is between 10 and 50. The pathogen of the disease is the Crimean Congo hemorrhagic Fever virus (CCHFV), which is transmitted by ticks. The virus belongs to the genus Conero virus of Bunia virus. It is a single-stranded negative-stranded RNA virus. At present, there is no effective prevention and treatment for CCHF. In this study, nucleocapsid protein, a nucleocapsid protein, was cloned from the S fragment of Crimean Congo hemorrhagic fever virus (YL04057) strain by DNA recombination technique. The whole sequence and a series of fragments of NPN coding region were cloned into prokaryotic expression vector and expressed in Escherichia coli. The expressed product was isolated and purified. The antigenic epitopes of the expressed products were detected and analyzed by polyclonal antibody against Crimean Congo hemorrhagic fever virus and two monoclonal antibodies (mAb 14B7 and 43E5). Three expressed protein fragments containing amino acids at position 235-305 of NP protein can be detected by polyclonal antibodies against Crimean Congo hemorrhagic fever virus. It also showed that the epitopes of the two McAbs 14B7 and 43E5 and rabbit polyantibodies were located in the amino acid region of the 235-305 amino acid of NP protein. This region is also a highly conserved amino acid region of NP protein. At the same time, we cloned the glycoprotein glycoprotein (GP) coding fragment of M fragment into prokaryotic expression vector, expressed in Escherichia coli, isolated and purified the expressed product. The antigenic epitopes of the expressed products were detected and analyzed with polyclonal anti-rabbit antibodies against Crimean Congo hemorrhagic fever virus. The recombinant segmental glycoprotein did not react with rabbit polyantiserum. The structure of CCHFV glycoprotein was complex. But the prokaryotic expression system does not have the complete post-translational modification function. Because the polyclonal antibody serum prepared in the laboratory is aimed at the nucleoprotein, the detection of the glycoprotein is not targeted. It is necessary to establish eukaryotic expression system of CCHFV glycoprotein to obtain glycoprotein and antiserum for further study of glycoprotein. The determination of the antigenic characteristics of Crimean-Congo hemorrhagic fever virus structural protein will be helpful to clarify the infection and immune mechanism of Crimean-Congo hemorrhagic fever virus. It will also provide a theoretical basis for the development of its subunit vaccine.
【学位授予单位】：新疆大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R373

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