本文選題：HEV + ORF2��； 參考：《吉林大學》2009年博士論文

【摘要】： 戊型肝炎是全世界范圍內(nèi)的一個公共衛(wèi)生問題,在發(fā)展中國家尤甚。本論文從3例戊型肝炎患者和1例感染戊型肝炎病毒的豬的糞便懸液中克隆4株HEV的基因組,并將其基因構建成cDNA克隆。序列分析顯示該4株HEV中有1株為基因1型,3株為基因4型。 在此基礎上,本論文構建了18個分別表達1型和4型HEV ORF2和ORF3蛋白片段的克隆,進行原核表達和純化。將純化后的各多肽分別制備成HEV IgM和IgG抗體EIA檢測試劑。使用制備的抗體檢測試劑分別檢測實驗感染了不同基因型HEV的猴系列血清及臨床HE患者的系列血清,比較不同基因型HEV對應位置多肽的抗原性差異、同一基因型HEV不同位置多肽之間的抗原性差異。結果顯示,對于HEV IgM試劑,ORF2 N端和C端抗原組合使用可以取得最佳的檢測效果;對于HEV IgG試劑,主要抗原位點集中在ORF2蛋白的兩末端:N端多肽主要參與HE急性期抗體反應,且持續(xù)時間較短,C端多肽對應抗體出現(xiàn)也較早,但是持續(xù)時間相對較長,該多肽和恢復期血清有很強的結合能力;ORF2蛋白的抗原性在不同基因型之間無明顯差異;ORF3蛋白的抗原位點主要集中在C端,且其抗原性在不同基因型之間存在差異。以上結論為優(yōu)化HEV抗體檢測試劑提供了數(shù)據(jù)支持。 本論文還將原核表達的4型HEV ORF3抗原免疫恒河猴,并分別用1型和4型HEV毒株攻擊免疫的恒河猴,以考察原核表達的HEV ORF3蛋白的免疫原性。結果顯示,原核表達的ORF3蛋白可以部分保護試驗動物免于HEV感染。這部分工作為HEV ORF3的功能研究及HE疫苗的開發(fā)提供了數(shù)據(jù)參考。
[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.
【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2009
【分類號】：R392.11

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本文編號：1989209