戊型肝炎病毒ORF2和ORF3多肽的抗原性及ORF3多肽的免疫原性研究

发布时间：2018-06-07 02:05

本文选题：HEV + ORF2　；参考：《吉林大学》2009年博士论文

【摘要】： 戊型肝炎是全世界范围内的一个公共卫生问题,在发展中国家尤甚。本论文从3例戊型肝炎患者和1例感染戊型肝炎病毒的猪的粪便悬液中克隆4株HEV的基因组,并将其基因构建成cDNA克隆。序列分析显示该4株HEV中有1株为基因1型,3株为基因4型。在此基础上,本论文构建了18个分别表达1型和4型HEV ORF2和ORF3蛋白片段的克隆,进行原核表达和纯化。将纯化后的各多肽分别制备成HEV IgM和IgG抗体EIA检测试剂。使用制备的抗体检测试剂分别检测实验感染了不同基因型HEV的猴系列血清及临床HE患者的系列血清,比较不同基因型HEV对应位置多肽的抗原性差异、同一基因型HEV不同位置多肽之间的抗原性差异。结果显示,对于HEV IgM试剂,ORF2 N端和C端抗原组合使用可以取得最佳的检测效果;对于HEV IgG试剂,主要抗原位点集中在ORF2蛋白的两末端:N端多肽主要参与HE急性期抗体反应,且持续时间较短,C端多肽对应抗体出现也较早,但是持续时间相对较长,该多肽和恢复期血清有很强的结合能力;ORF2蛋白的抗原性在不同基因型之间无明显差异;ORF3蛋白的抗原位点主要集中在C端,且其抗原性在不同基因型之间存在差异。以上结论为优化HEV抗体检测试剂提供了数据支持。本论文还将原核表达的4型HEV ORF3抗原免疫恒河猴,并分别用1型和4型HEV毒株攻击免疫的恒河猴,以考察原核表达的HEV ORF3蛋白的免疫原性。结果显示,原核表达的ORF3蛋白可以部分保护试验动物免于HEV感染。这部分工作为HEV ORF3的功能研究及HE疫苗的开发提供了数据参考。
[Abstract]:Hepatitis E is a worldwide public health problem, especially in developing countries. In this paper, four strains of HEV were cloned from fecal suspension of 3 patients with hepatitis E and 1 pig infected with hepatitis E virus, and their genes were cloned into cDNA clone. Sequence analysis showed that one of the four HEV strains was gene type 1 and 3 were gene 4. On this basis, 18 HEV ORF2 and ORF3 protein fragments expressing type 1 and type 4 were constructed and expressed in prokaryotic expression and purification. The purified polypeptides were prepared into HEV IgM and IgG antibody EIA detection reagents respectively. The sera of monkeys infected with different genotypes of HEV and those of clinical patients with HE were detected using the prepared antibody detection reagents, and the antigenicity differences of peptides corresponding to different genotypes of HEV were compared. The antigenicity of polypeptides at different locations of the same genotype HEV was different. The results showed that the combination of HEV IgM reagent ORF2 N-terminal antigen and C-terminal antigen could obtain the best detection effect, and for HEV IgG reagent, the main antigen site was concentrated in the two ends of ORF2 protein, the N-terminal peptide was mainly involved in HE acute antibody reaction. The corresponding antibody of C-terminal peptide appeared earlier, but the duration was relatively long. The antigenicity of ORF2 protein was not significantly different from that of different genotypes, and the antigenicity of ORF3 protein was mainly at the C-terminal, and the antigenicity of ORF2 protein was different among different genotypes. These conclusions provide data support for optimizing HEV antibody detection reagent. The rhesus monkeys were immunized with prokaryotic HEV ORF3 antigen 4, and the rhesus monkeys were immunized with type 1 and type 4 HEV strains to investigate the immunogenicity of HEV ORF3 protein expressed in prokaryotic cells. The results showed that prokaryotic expression of ORF3 protein could partially protect experimental animals from HEV infection. This work provides a data reference for the functional study of HEV ORF3 and the development of HE vaccine.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R392.11

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