本文選題：丙型肝炎病毒 + 高變區(qū)1��； 參考：《第二軍醫(yī)大學(xué)》2013年博士論文

【摘要】：丙型肝炎病毒(hepatitis C virus, HCV)屬于黃病毒科、丙型肝炎病毒屬,為有包膜的單正鏈RNA病毒。目前全球約有1.8億人感染HCV,大多數(shù)為慢性感染,部分感染者可發(fā)展為肝硬化和肝細(xì)胞癌。HCV的RNA聚合酶缺少校對(duì)功能,致使HCV整個(gè)基因組具有很高的變異性,但HCV基因的變異呈不平衡分布.位于包膜蛋白2(E2)氨基端的27個(gè)氨基酸殘基變異性最為突出,即HVRl。HIVR1高度暴露于包膜蛋白表面,其中含有中和抗體表位,可以誘導(dǎo)宿主產(chǎn)生針對(duì)該區(qū)域的抗體,產(chǎn)生的抗體可以高效中和HCV的感染,但HVR1的基因序列高度變異,先前由宿主免疫細(xì)胞產(chǎn)生的抗體不能識(shí)別變異后的HVRl,因此病毒得以逃避宿主細(xì)胞的免疫監(jiān)控并在病人體內(nèi)持續(xù)感染進(jìn)而使感染慢性化。眾多實(shí)驗(yàn)室都將HVR1作為HCV疫苗研發(fā)的重要靶標(biāo),但自1989年丙型肝炎病毒首次命名至今,仍沒有一個(gè)真正有效的保護(hù)性疫苗問世。 HCV感染會(huì)引起肝內(nèi)生化代謝紊亂,包括脂質(zhì)代謝障礙和載脂蛋白合成異常,與其他病毒性肝炎相比,HCV感染慢性化后,中重度肝細(xì)胞脂肪變性包括胞質(zhì)內(nèi)脂滴聚集是其典型組織病理學(xué)特征。HCV是有包膜RNA病毒,其表面有不同數(shù)量的脂蛋白包裹,所以病人血清中純化得到的HCV病毒顆粒呈現(xiàn)不同的密度分布,并且不同密度的病毒顆粒感染性也不同。有研究發(fā)現(xiàn)HVR1的刪除會(huì)導(dǎo)致低密度病毒顆粒數(shù)目下降,也就是減弱了HCV的脂蛋白結(jié)合能力,可見HVR1在病毒與脂蛋白結(jié)合的過程中亦發(fā)揮重要的作用。多項(xiàng)研究證明HVR1同時(shí)還在與宿主SR-B1受體結(jié)合以及介導(dǎo)病毒入侵的過程中起關(guān)鍵作用,HVR1具有如此重要的生物學(xué)功能,那么其結(jié)構(gòu)必然具有相對(duì)穩(wěn)定性和保守性,雖然HVR1是HCV基因組中變異頻率最高的部分之一,但對(duì)HCV一系列分離株的序列分析表明一些氨基酸殘基的位點(diǎn)是高度、或者相對(duì)保守的。盡管人們已經(jīng)發(fā)現(xiàn)HVR1同時(shí)行駛著誘導(dǎo)宿主免疫反應(yīng),脂蛋白結(jié)合以及介導(dǎo)病毒與受體結(jié)合的多重功能,但目前尚不清楚其結(jié)構(gòu)與生物學(xué)功能的對(duì)應(yīng)關(guān)系。 一、HCV高變區(qū)1中介導(dǎo)病毒細(xì)胞入侵關(guān)鍵氨基酸殘基的鑒定 首先我們以HCVpp作為研究模型,以HCVla亞型的H77株為模式株,用單一氨基酸殘基刪除掃描的方法分析HVR1中27個(gè)氨基酸殘基分別對(duì)HCV細(xì)胞入侵的影響,實(shí)驗(yàn)結(jié)果明確的鑒定出HVRl中介導(dǎo)HCV細(xì)胞入侵的關(guān)鍵氨基酸殘基(Ala-14,Gly-15, Lys-25, Gln-26以及Asn-27),其中任意一個(gè)殘基的刪除對(duì)HCVpp感染性的影響都是致命的,相比之下,其他22個(gè)氨基酸殘基的刪除對(duì)HCVpp感染性并未造成明顯影響。再用HCV入侵相關(guān)受體結(jié)合實(shí)驗(yàn)進(jìn)行機(jī)制分析,我們證明這5位氨基酸殘基介導(dǎo)了病毒包膜蛋白與宿主細(xì)胞SR-B1受體的結(jié)合。氨基酸殘基合并刪除實(shí)驗(yàn)進(jìn)一步的證明了我們的結(jié)論,HVRl氨基端13個(gè)殘基的刪除以及第16-24位9個(gè)氨基酸殘基的刪除不會(huì)顯著降低HCVpp的感染力。除了決定HCV細(xì)胞入侵的5個(gè)氨基酸殘基之外,HVR1氨基端的13個(gè)氨基酸殘基可以通過適當(dāng)?shù)耐蛔?增強(qiáng)病毒與受體的結(jié)合能力從而增強(qiáng)病毒的細(xì)胞入侵能力,并且這一區(qū)域適當(dāng)?shù)耐蛔冞�可以增強(qiáng)病毒的抗中和能力從而更利于病毒的免疫逃避和持續(xù)感染。 二、HCV高變區(qū)1中誘導(dǎo)中和抗體產(chǎn)生的抗原表位的鑒定 我們將HVR1分為氨基段,中段和羧基段三部分肽段分別與TRX融合表達(dá)后免疫家兔來檢測(cè)HVR1的27個(gè)氨基酸殘基在誘導(dǎo)宿主免疫反應(yīng)過程中是否存在差異,我們發(fā)現(xiàn)HVRl的氨基端13個(gè)氨基酸殘基不能誘導(dǎo)宿主產(chǎn)生抗體,而第16到24位的9個(gè)氨基酸殘基是HVRl中唯一的中和表位,這9個(gè)氨基酸殘基對(duì)于病毒細(xì)胞入侵無關(guān)緊要,但含有肝素結(jié)合位點(diǎn),有利于HCV的粘附,更為重要的是,高密度脂蛋白(HDL)介導(dǎo)的HCV感染增強(qiáng)作用依賴于該9個(gè)氨基酸殘基的存在,該九個(gè)殘基還可通過與HDL的作用顯著下調(diào)E2抗體的中和活性,小鼠DNA免疫試驗(yàn)也進(jìn)一步驗(yàn)證了我們的結(jié)論。 三、HCV高變區(qū)1中介導(dǎo)病毒顆粒與脂蛋白結(jié)合關(guān)鍵區(qū)域的鑒定 將濃縮的HCVcc進(jìn)行碘克沙醇密度梯度離心,發(fā)現(xiàn)密度偏低的病毒顆粒具有強(qiáng)感染性,而隨著HCVcc病毒顆粒密度的升高,其感染性也逐漸降低。此外,HDL對(duì)不同密度HCVcc感染的促進(jìn)作用差異顯著,其對(duì)低密度以及中等密度HCVcc感染的促進(jìn)作用不明顯,卻顯著增強(qiáng)高密度HCVcc的感染性。先前我們已證明HVRl中存在一個(gè)由9個(gè)氨基酸殘基組成的功能結(jié)構(gòu)域是誘導(dǎo)宿主產(chǎn)生抗體的主要抗原表位,并且HDL介導(dǎo)的HCV感染增強(qiáng)作用依賴于該9個(gè)氨基酸殘基的存在,本部分實(shí)驗(yàn)我們發(fā)現(xiàn)刪除了這9個(gè)氨基酸殘基后,HCVcc病毒顆粒的密度分布曲線發(fā)生了明顯改變,低密度的病毒顆粒數(shù)目明顯下降,并且感染性也顯著降低,而更多的病毒顆粒則集中在密度偏高幾層,這和刪除了整個(gè)HVRl的HCVcc所呈現(xiàn)出的病毒密度曲線趨勢(shì)幾乎是一致,這說明在HVRl中,決定HCV病毒顆粒與脂蛋白結(jié)合的關(guān)鍵部位恰恰就是這個(gè)由9個(gè)氨基酸殘基組成的抗原決定區(qū)。在HDL對(duì)不同密度病毒顆粒的感染增強(qiáng)試驗(yàn)中,刪除了9個(gè)氨基酸殘基的任何密度層病毒顆粒的感染性均不再被HDL所增強(qiáng)。 小結(jié) 我們鑒定出HVR1中至少存在三個(gè)功能結(jié)構(gòu)域,它們之間相互協(xié)調(diào)發(fā)揮著微妙的作用：HVR1的抗原決定區(qū)在病毒顆粒表面形成一個(gè)高度凸起,在脂蛋白結(jié)合過程中行駛一個(gè)非特異性“脂抓手”作用,結(jié)合在病毒顆粒表面的脂蛋白又通過與宿主SR-BI受體的結(jié)合拉近病毒顆粒與靶細(xì)胞的空間距離,使得抗原決定區(qū)兩側(cè)的SR-BI結(jié)合位點(diǎn)更易于與SR-BI受體結(jié)合并啟動(dòng)HCV細(xì)胞入侵的一系列步驟；更為有意思的是抗原決定區(qū)的空間凸起又是誘導(dǎo)宿主產(chǎn)生抗體的關(guān)鍵抗原表位,但其自身又呈現(xiàn)了高度的變異,使得宿主產(chǎn)生的抗體不能識(shí)別宿主體內(nèi)新釋放出的病毒顆粒,從而達(dá)到免疫逃避和持續(xù)感染的目的；而恰恰也是這樣一個(gè)抗原表位,因?yàn)槠渥陨淼目臻g高度凸起,使得HCV病毒顆粒表面包裹著大量的脂蛋白,脂蛋白覆蓋住了HCV病毒顆粒表面其他真正意義上的保守中和抗體表位,使得宿主細(xì)胞不能大量產(chǎn)生可以識(shí)別病毒顆粒表面保守位點(diǎn)的中和抗體,我們認(rèn)為這個(gè)“脂抓手”同時(shí)又起到了“免疫誘餌”的作用。人們往往將HVR1的27個(gè)氨基端殘基作為一個(gè)整體進(jìn)行研究,我們的實(shí)驗(yàn)對(duì)HVR1的功能意義進(jìn)行了深刻的剖析與闡釋,并精確地鑒定出HVR1結(jié)構(gòu)與功能之間的對(duì)應(yīng)關(guān)系,這為今后保護(hù)性疫苗以及抗病毒藥物的研發(fā)都提供了重要的啟示。
[Abstract]:Hepatitis C virus (hepatitis C virus, HCV) belongs to the family of the family yellirid family, hepatitis C virus and the envelope of single positive chain RNA virus. At present, about 180 million people are infected with HCV in the world, most of them are chronic infection. Some infected people can develop the RNA polymerase of liver cirrhosis and hepatocellular carcinoma.HCV without proofreading function, resulting in the whole genome of HCV. High variability, but the variation of the HCV gene is unevenly distributed. The 27 amino acid residues in the amino terminal of the envelope protein 2 (E2) are most prominent, that is, HVRl.HIVR1 is highly exposed to the surface of the envelope protein, which contains neutralizing antibody epitopes, which can induce the host to produce the antibody against the region, and the antibody produced can effectively neutralize the HCV. Infection, but the gene sequence of HVR1 is highly variable, and the antibodies previously produced by the host immune cells cannot identify the variant HVRl, so the virus can escape the monitoring of the host cells and keep the infection in the patient and cause the chronicity of the infection. Many laboratories have taken HVR1 as an important target for the development of the HCV vaccine, but since 1989 Since the first name of the hepatitis C virus, there is still no effective protective vaccine.
HCV infection causes biochemical metabolic disorders in the liver, including lipid metabolism disorders and apolipoprotein synthesis. Compared with other viral hepatitis, after chronic HCV infection, fatty degeneration of medium and severe hepatocytes, including intracellular lipid droplet aggregation, is a typical histopathological feature of.HCV, a enveloped RNA virus with different amounts of fat eggs on the surface. White parcels, so the HCV virus particles purified from the patient's serum show a different density distribution, and the virus particles of different densities are different. Studies have found that the deletion of HVR1 leads to a decrease in the number of low density virus particles, that is, the ability to reduce the lipoprotein binding capacity of HCV, and it can be seen that HVR1 is combined with the virus and lipoprotein. Many studies have shown that HVR1 also plays a key role in the combination of the host SR-B1 receptor and the invasion of the virus. HVR1 has such important biological functions that its structure is bound to be relatively stable and conserved, although HVR1 is the highest variation in the HCV genome. The sequence analysis of a series of isolates of HCV shows that some amino acid residues are highly or relatively conservative. Although it has been found that HVR1 is driving simultaneously the host immune response, lipoprotein binding, and the multiple functions of the combination of the virus and the receptor, it is not yet clear about its structure and biological function. The corresponding relationship.
Identification of key amino acid residues in HCV mediated hypervariable region 1 mediated virus cell invasion
First of all, we use HCVpp as the model, using the H77 strain of HCVla subtype as the model plant, and using the single amino acid residue deletion scanning method to analyze the effect of 27 amino acid residues in HVR1 on the invasion of HCV cells respectively. The experimental results clearly identify the key amino acid residues of the HVRl mediated HCV cell invasion (Ala-14, Gly-15, Lys-25, Gln-26). As well as Asn-27), the deletion of any one of the residues was fatal to the infection of HCVpp. In contrast, the deletion of the other 22 amino acid residues had no significant effect on HCVpp infection. The HCV invasion related receptor binding assay was used for mechanism analysis. We proved that the 5 amino acid residues mediate the envelope protein of the virus. The combination of the SR-B1 receptor with the host cell. The combined deletion experiment of amino acid residues has further demonstrated our conclusion that the deletion of 13 residues in the HVRl amino terminal and the deletion of the 16-24 site 9 amino acid residues will not significantly reduce the infection force of HCVpp. In addition to determining the 5 amino acid residues of the HCV cell invasion, 13 of the HVR1 amino terminus Amino acid residues can enhance the virus's cell invasiveness by increasing the binding ability of the virus to the receptor by appropriate mutations, and the appropriate mutation in this region can also enhance the anti neutralization ability of the virus and thus benefit the immune escape and persistent infection of the virus.
Two, identification of epitopes produced by neutralizing antibodies in HCV hypervariable region 1.
We divided HVR1 into amino group, the three part of the middle segment and the carboxyl segment were fused with TRX after the fusion expression of the three amino acid residues to detect the difference between the 27 amino acid residues of HVR1 in the induction of the host immune response. We found that the 13 amino acid residues of the amino terminal of HVRl did not induce the host to produce the antibody, and the sixteenth to 24 bits of the 9 ammonia. Base acid residues are the only neutralization epitopes in HVRl. These 9 amino acid residues are insignificant for virus cell invasion, but contain heparin binding sites, which are beneficial to HCV adhesion and, more importantly, the enhancement of HCV infection mediated by high density lipoprotein (HDL) depends on the existence of the 9 amino acid residues, and the nine residues can also be passed through HDL The neutralizing activity of E2 antibody was significantly reduced, and the DNA immunization test in mice further confirmed our conclusion.
Three, identification of 1 critical regions of HCV mediated hypervirion and lipoprotein binding.
The concentrated HCVcc was centrifuged with iodipol density gradient. It was found that the virus particles with low density were strongly infective, and with the increase of HCVcc virus particle density, its infectivity gradually decreased. In addition, the promoting effect of HDL on different density HCVcc infection was significantly different, and its promoting effect on low density and medium density HCVcc infection was promoted. It was not obvious, but significantly enhanced the infectivity of high density HCVcc. Previously, we have shown that a functional domain consisting of 9 amino acid residues in HVRl is the main epitope that induces the host to produce antibodies, and the enhancement of HDL mediated HCV infection depends on the presence of the 9 amino acid residues. In addition to the 9 amino acid residues, the density distribution curve of HCVcc virus particles changed significantly, the number of low density virus particles decreased significantly, and the infection was significantly reduced, while more virus particles were concentrated in a few layers of density, and this and the deletion of the virus density curve of the entire HVRl HCVcc was almost the same. It is consistent, which indicates that in HVRl, the key site to determine the binding of HCV virus particles to lipoprotein is precisely the antigen determining area consisting of 9 amino acid residues. In the HDL infection enhancement test for different density virus particles, any of the 9 amino acid residues of any dense lamina virus particles are no longer increased by HDL Strong.
Summary
We identified that there are at least three functional domains in HVR1 that play a subtle role in coordinating each other: the antigen determinant of HVR1 forms a high elevation on the surface of the virus particles, driving a nonspecific "fat gripper" in the process of lipoprotein binding, and by combining the lipoproteins on the surface of the virus particles. The combination of the host SR-BI receptor close to the space distance between the virus particles and the target cells makes the SR-BI binding site on both sides of the antigen determinant more easily combined with the SR-BI receptor and initiates a series of steps for the invasion of the HCV cells; more interesting is that the spatial protruding of the antigen determining region is the key epitope of the host to induce the host to produce antibodies. It has a high variation in itself, making the antibody produced by the host can not identify the newly released virus particles in the host, so as to achieve the purpose of immune escape and continuous infection. It is also an epitope of the antigen, because of its high elevation of its own space, so that a large amount of lipoprotein is wrapped on the surface of the HCV virus particles. Lipoproteins cover the other true conserved and antibody epitopes on the surface of the HCV virus particles, making the host cells unable to produce a large number of neutralizing antibodies that can identify the conservative sites on the surface of the virus particles. We think the "fat grab" also plays the role of "immune bait". People tend to take the 27 amino groups of HVR1. As a whole, the end residues are studied as a whole. Our experiments have deeply analyzed and explained the functional significance of HVR1, and accurately identified the corresponding relationship between the structure and function of HVR1, which provides important inspiration for the future protection of vaccine and the development of antiviral drugs.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：R512.63

【共引文獻(xiàn)】

相關(guān)期刊論文 前10條

1 李陽超;;簡(jiǎn)陽地區(qū)丙型肝炎病毒感染情況分析[J];國際檢驗(yàn)醫(yī)學(xué)雜志;2013年17期

2 郭江龍;李德周;王志斌;文金生;;重要病毒CTL表位肽疫苗的研究現(xiàn)狀[J];現(xiàn)代免疫學(xué);2013年04期

3 陶格斯;沈曉玲;博曉真;譚文杰;;慢性丙型肝炎患者中丙型肝炎病毒準(zhǔn)種變異研究進(jìn)展[J];生物技術(shù)通訊;2013年06期

4 王穎;葉波平;華子春;;補(bǔ)體系統(tǒng)及其在肝損傷再生中的作用[J];生命科學(xué);2013年12期

5 Mortada Hassan El-Shabrawi;Naglaa Mohamed Kamal Alanani;;Burden of pediatric hepatitis C[J];World Journal of Gastroenterology;2013年44期

6 Yue Wang;;Scotomas in molecular virology and epidemiology of hepatitis C virus[J];World Journal of Gastroenterology;2013年44期

7 金博;李楠;吳凱;張林;王艷梅;翟俊山;朱超慧;宋久剛;;HCV NS3誘導(dǎo)小鼠特異性T細(xì)胞反應(yīng)的抗原多肽序列篩選[J];中華臨床醫(yī)師雜志(電子版);2013年10期

8 Raquel Francine Liermann Garcia;Simone Moreira;Ana Lucia de Araújo Ramos;Leslie Ecker Ferreira;Angelo Alves de Mattos;Cristiane Valle Tovo;Lysandro Alsina Nader;Juliene Antonio Ramos;Edson Rondinelli;Arnaldo de Jesus Dominici;Christian Evangelista Garcia;Mauro de Souza Leite Pinho;Carlos Eduardo Brand嗾o-Mello;Cristiane Alves Villela-Nogueira;Paulo Henrique Condeixa de Fran，

本文編號(hào)：2050829