本文關(guān)鍵詞：戊型肝炎病毒在家禽、家畜中的流行及其ORF3蛋白與人肝細(xì)胞蛋白相互作用的研究　出處：《北京協(xié)和醫(yī)學(xué)院》2011年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 戊型肝炎病毒 HEV-抗原 HEV-抗體 HEVORF3蛋白 酵母雙雜交實(shí)驗(yàn) 蛋白質(zhì)相互作用 免疫共沉淀實(shí)驗(yàn)

【摘要】：戊型肝炎病毒(Hepatitis E Virus, HEV)是戊型肝炎的病原體,是一種重要的傳染因子。逐漸積累起來的研究結(jié)果顯示戊型肝炎是一種人獸共患病,自然界存在HEV的動(dòng)物宿主。我國是戊型肝炎的流行區(qū),既有爆發(fā)流行也有散發(fā)病例,近年來散發(fā)病例報(bào)告數(shù)持續(xù)增加,可能與動(dòng)物源性HEV的傳播有關(guān)。了解HEV在我國動(dòng)物中的流行情況,對于預(yù)防和控制戊型肝炎的流行、減少發(fā)病具有重要意義。在本研究中,我們從不同省份采集我國主要飼養(yǎng)動(dòng)物的血清,對HEV在各種動(dòng)物中的感染率、感染水平進(jìn)行了調(diào)查分析,并對HEV基因型、基因亞型的分布進(jìn)行了分析研究。為了進(jìn)一步探索HEV感染、致病以及不同基因型別HEV宿主差異的分子機(jī)制,我們應(yīng)用酵母雙雜交系統(tǒng)對人肝cDNA文庫進(jìn)行篩選,篩選了與HEV ORF3蛋白有相互作用的蛋白分子,并對不同基因型HEV ORF3蛋白與肝細(xì)胞蛋白的相互作用進(jìn)行比較；通過對這些蛋白分子的生物學(xué)信息分析,對ORF3蛋白的功能及其對宿主細(xì)胞生物過程可能產(chǎn)生的影響進(jìn)行了分析、推測。 1.HEV在我國家畜、家禽中流行情況的調(diào)查 從我國的27個(gè)省(市)共采集了豬、牛、羊、家兔、雞和鴨血清6329份,應(yīng)用EIA方法測定血清中HEV抗體和抗原。結(jié)果顯示,豬、牛、羊和家兔的平均HEV抗體陽性率分別為82.2%、28.2%、10.4%和15.4%,豬HEV抗體陽性率明顯高于牛、羊和家兔。雞和鴨的抗體陽性率分別為3.58%和7.1%,二者都明顯低于豬、牛羊和家兔。來源于不同省份的動(dòng)物HEV抗體的陽性率有明顯差異,豬、牛、羊和家兔HEV抗體陽性率范圍分別為10.9%-100%、0～92.9%、0～48%和0-53.4%。大約1.9%的豬、0.8%的牛、1.6%的羊以及3.8%的兔血清樣本為HEV抗原陽性。雞和鴨的抗原陽性率分別為1.79%和1.43%。 應(yīng)用Nested RT-PCR在15份豬血清和22份不同品種的家兔的血清中檢測到了HEV RNA,而牛、羊、雞和鴨血清中未檢測到HEV RNA。通過對ORF2區(qū)域304bp長的核苷酸序列進(jìn)行比對和進(jìn)化樹分析,15株豬HEV均屬于基因4型。在進(jìn)化樹上,這15株4型HEV被分在不同的四組,分別為4a、4d及兩個(gè)新的亞型。綜合分析表明,我國豬群中流行的HEV主要為基因4型HEV,包括4a,4b,4d,4f,4g以及本研究新發(fā)現(xiàn)的兩個(gè)亞型。從家兔中分離到的22株HEV均屬于兔HEV,在進(jìn)化樹上,兔HEV又進(jìn)一步分成了3組,很可能代表三個(gè)基因亞型。 本研究表明HEV在我國家畜、家禽中普遍流行,豬HEV感染率最高,我國流行的豬HEV主要為基因4型,包括4a,4b,4d,4f,4g以及兩個(gè)新亞型。證實(shí)了兔HEV在我國不同地區(qū)不同品種的家兔中均有流行,支持兔HEV屬于一個(gè)新基因型的觀點(diǎn)。牛、羊、雞和鴨血清樣本中都檢測到了HEV抗原,但未能檢測出HEVRNA,仍需進(jìn)一步研究。 2. HEV ORF3蛋白與肝細(xì)胞蛋白相互作用的研究 本研究以基因1型和4型HEV ORF3蛋白為誘餌,通過酵母配對雜交實(shí)驗(yàn)對人肝cDNA文庫進(jìn)行篩選,篩選結(jié)果應(yīng)用酵母雜交共轉(zhuǎn)化實(shí)驗(yàn)進(jìn)行驗(yàn)證。經(jīng)過三次實(shí)驗(yàn),篩選出了32種與ORF3蛋白有相互作用的肝細(xì)胞蛋白。其中4種蛋白(4/32,12.5%)已經(jīng)有報(bào)告能夠與ORF3蛋白結(jié)合。進(jìn)一步應(yīng)用哺乳動(dòng)物細(xì)胞內(nèi)化學(xué)發(fā)光免疫共沉淀實(shí)驗(yàn)對酵母雜交實(shí)驗(yàn)的篩選結(jié)果的可靠性進(jìn)行評估,顯示篩選結(jié)果的可信度比較高。 生物信息學(xué)分析發(fā)現(xiàn)這32種相互作用蛋白在細(xì)胞核、細(xì)胞質(zhì)、細(xì)胞膜、細(xì)胞外基質(zhì)、以及線粒體、內(nèi)涵體膜中均有分布,大多數(shù)蛋白定位于細(xì)胞的多個(gè)部位。這些蛋白分別參與細(xì)胞代謝、細(xì)胞免疫、細(xì)胞黏附等多個(gè)生物學(xué)過程。因此推測ORF3蛋白可能通過與這些蛋白相互作用,發(fā)揮多重作用。通過對蛋白功能富集聚類分析,發(fā)現(xiàn)分別有顯著多數(shù)量的相互作用蛋白參與血液凝固、氧化應(yīng)激和鐵代謝過程。結(jié)合戊型肝炎臨床表現(xiàn)和肝組織的病理特征進(jìn)行分析,推測ORF3可能通過與宿主細(xì)胞蛋白的相互作用,影響凝血、脂質(zhì)代謝以及鐵代謝,而引發(fā)相應(yīng)的病理過程。 通過酵母雜交共轉(zhuǎn)化實(shí)驗(yàn)分別檢測基因1型HEV、4型HEV、兔HEV以及禽HEV ORF3蛋白與以基因4型和1型HEV ORF3為誘餌篩選到的這32種人肝細(xì)胞蛋白是否有相互作用。結(jié)果顯示,基因1型、4型和兔HEV的ORF3蛋白都能夠與這些蛋白結(jié)合,然而禽HEV的ORF3蛋白只與其中的13種蛋白有相互作用。因此,禽HEV對人肝細(xì)胞的影響作用可能不同于哺乳動(dòng)物HEV,這種蛋白相互作用的差別是否與禽HEV與哺乳動(dòng)物HEV宿主范圍等的差別有關(guān)聯(lián),還需要進(jìn)一步研究。 這些發(fā)現(xiàn)為我們進(jìn)一步研究ORF3蛋白的致病作用,以及研究HEV感染、復(fù)制和宿主限制性的分子機(jī)制提供了線索和方向。
[Abstract]:Hepatitis E Virus ( HEV ) is a pathogen of HEV , and it is an important infection factor . The research result shows that hepatitis E is a kind of animal host with HEV . In this study , we screened the infection rate and infection level of HEV in different provinces . In order to further explore the epidemic situation of HEV in our country , we screened the protein molecule interacting with HEV ORF3 protein and compared the interaction of HEV ORF3 protein with liver cell protein . 1 . Investigation of the prevalence of HEV in domestic animals and poultry in China The positive rates of anti HEV antibodies in pigs , cattle , sheep and rabbits were 82 . 2 % , 28 . 2 % , 10 . 4 % and 15.4 % , respectively . The positive rates of antibodies in pigs , cattle , sheep and rabbits were significantly lower than those in cattle , sheep and rabbits . The positive rates of antibodies in pigs , cattle , sheep and rabbits were respectively 10.9 % -100 % , 0 - 92.9 % , 0 - 48 % and 0 - 53.4 % . The positive rates of antibodies in pigs , cattle , sheep and rabbits were respectively 1 . 79 % and 1 . 43 % . HEV RNA was detected by Nested RT - PCR in serum of 15 pigs and 22 different breeds . HEV RNA was not detected in serum of cattle , sheep , chicken and duck . This study indicates that HEV is prevalent in domestic animals and poultry , and the prevalence of HEV infection is the highest . In our country , HEV is the most common type , including 4a , 4b , 4d , 4f , 4g and two new subtypes . It is confirmed that HEV is a new genotype in different regions of our country . HEV antigen is detected in the serum samples of cattle , sheep , chicken and duck . However , it is not possible to detect HEVRNA . 2 . Study on the Interaction of HEV ORF3 Protein with Hepatocytes Three experiments were carried out to select 32 liver cell proteins interacting with ORF3 protein . Four of these proteins ( 4 / 32 , 12.5 % ) had been reported to bind to ORF3 protein . Four of these proteins ( 4 / 32 , 12.5 % ) had been reported to bind to ORF3 protein . Bioinformatic analysis showed that the 32 interacting proteins were distributed in the nucleus , cytoplasm , cell membrane , extracellular matrix , and mitochondria and connotative body membranes . Most of the proteins were located in many parts of the cell . These proteins were involved in many biological processes such as cell metabolism , cellular immunity , cell adhesion and so on . The results showed that the ORF3 protein of HEV , 4 - type HEV , HEV and ORF3 protein could be combined with these proteins . These findings provide clues and directions for our further study of the pathogenic role of ORF3 protein , as well as to investigate the molecular mechanisms of HEV infection , replication and host restriction .

【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2011
【分類號】：R373.21;S852.65

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 ;The Leu477 and Leu613 of ORF2-Encoded Protein Are Critical in Forming Neutralization Antigenic Epitope of Hepatitis E Virus Genotype 4[J];Cellular & Molecular Immunology;2008年06期

2 ;Successful rescuing a pregnant woman with severe hepatitis E infection and postpartum massive hemorrhage[J];World Journal of Gastroenterology;2003年03期

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本文編號：1382202