本文關(guān)鍵詞：高變區(qū)1抑制丙型肝炎病毒包膜蛋白E2誘生交叉中和抗體　出處：《第二軍醫(yī)大學(xué)》2011年博士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 丙型肝炎病毒 包膜蛋白E2 高變區(qū)1 體液免疫反應(yīng)

【摘要】：丙型肝炎病毒(hepatitis C virus,HCV)屬于黃病毒科,主要經(jīng)血液傳播,是引起人類(lèi)丙型肝炎的病原體。全球約有1.7億人感染HCV,每年新增感染者達(dá)300萬(wàn)-400萬(wàn)。我國(guó)HCV感染者估計(jì)近4000萬(wàn)。HCV感染易慢性化,其慢性化率可高達(dá)85%,部分慢性丙型肝炎患者可發(fā)展為肝硬化,甚至肝細(xì)胞癌。目前主要采用聚乙二醇干擾素α聯(lián)合利巴韋林治療丙型肝炎,其效果明顯受HCV基因型影響。2、3型HCV感染對(duì)該療法的持續(xù)病毒學(xué)應(yīng)答率(SVR)可達(dá)到75%,但1型HCV感染的SVR不到50%。目前全球范圍內(nèi)經(jīng)血液以及血制品途徑感染的丙型肝炎病例與上世紀(jì)九十年代以前相比有大幅下降,但在發(fā)展中國(guó)家血液和血制品仍然是傳播HCV的重要途徑。此外,每年散發(fā)性以及傳播途徑不明的丙型肝炎新發(fā)病例仍高居不下�？刂艸CV感染的關(guān)鍵措施還是依賴于開(kāi)發(fā)出有效的疫苗,從1989年HCV基因組被克隆以來(lái),丙型肝炎疫苗的研究在歐美和日本等發(fā)達(dá)國(guó)家一直是研究熱點(diǎn),但迄今未有有效的疫苗問(wèn)世。 HCV包膜蛋白E2位于HCV蛋白前體第384-746位氨基酸殘基,由一個(gè)大的N端膜外區(qū)和一個(gè)C端疏水錨定區(qū)組成,與HCV另一包膜蛋白E1通過(guò)非共價(jià)鍵形成異源性二聚體。E2蛋白是介導(dǎo)病毒吸附和進(jìn)入宿主細(xì)胞的關(guān)鍵蛋白,是誘導(dǎo)宿主產(chǎn)生HCV中和抗體的關(guān)鍵抗原,因此是疫苗研究的最主要靶標(biāo)。然而,包膜蛋白高度變異,尤其E2蛋白氨基末端的含有27個(gè)氨基酸殘基的高變區(qū)1(hypervariable region 1,HVR1)是HCV蛋白中變異頻率最高的區(qū)域,也是公認(rèn)的HCV中和抗體表位所在區(qū)域。HVR1是介導(dǎo)HCV與受體SR-BI結(jié)合的關(guān)鍵區(qū)域,HVR1與SR-BI的作用通過(guò)幾種不同的機(jī)制促進(jìn)HCV細(xì)胞侵入。HVR1抗體和SR-BI抗體均能阻斷HCV感染。HVR1的高度變異曾被認(rèn)為是遏制丙肝疫苗發(fā)展的瓶頸問(wèn)題,近幾年來(lái)基于HCV假病毒(HCVpp)以及細(xì)胞培養(yǎng)產(chǎn)生的HCV(HCVcc)的中和試驗(yàn)發(fā)現(xiàn),HVR1并不是唯一的中和抗體表位所在區(qū)域,在HCV包膜E2蛋白中還存在一系列構(gòu)象依賴的中和表位和線性中和表位,其中一些表位在不同基因型、亞型間高度保守。 本實(shí)驗(yàn)室前期分別用分泌表達(dá)E2的表達(dá)質(zhì)粒以及刪除了HVR1的E2表達(dá)質(zhì)粒免疫小鼠,然后檢測(cè)、分析小鼠血清中的抗體以及病毒中和活性,結(jié)果顯示:E2質(zhì)粒免疫的小鼠血清中,HVR1抗體在總E2抗體中占很大比重,針對(duì)HVR1以外區(qū)域的抗體在中E2抗體中只占較小比重;刪除HVR1可顯著增強(qiáng)E2質(zhì)粒免疫誘導(dǎo)的針對(duì)HVR1以外區(qū)域的抗體,小鼠免疫血清與其他基因型E2蛋白的交叉反應(yīng)以及對(duì)其他基因型HCVpp的交叉中和活性也隨之顯著增強(qiáng)。這和急性HCV感染者血清中可檢測(cè)到HVR1抗體而難以檢測(cè)到E2抗體相似。結(jié)果提示:HVR1有可能抑制E2蛋白中保守表位的免疫原性,從而從另一個(gè)方面導(dǎo)致HCV免疫逃避。目前基于HCV包膜蛋白為主要靶抗原的疫苗均含有HVR1,這類(lèi)疫苗免疫黑猩猩只能抵抗同株病毒攻擊,但不能抵抗異株的攻擊。HVR1的存在可能是這類(lèi)疫苗免疫保護(hù)效果不理想的重要原因,刪除HVR1也許能增強(qiáng)疫苗免疫對(duì)異株病毒攻擊的保護(hù)效果。 核酸疫苗在大動(dòng)物的免疫效果遠(yuǎn)遠(yuǎn)不及在小動(dòng)物的免疫效果,目前還沒(méi)有核酸疫苗用于人類(lèi)。為了進(jìn)一步探討用刪除了HVR1的包膜E2蛋白作為HCV疫苗的發(fā)展前景,本研究用CHO細(xì)胞表達(dá)了E2以及刪除了HVR1的E2蛋白,用親和層析純化出了兩種E2蛋白,分析兩種E2蛋白的構(gòu)象、受體結(jié)合功能和抗原性,通過(guò)小動(dòng)物免疫分析HVR1對(duì)E2蛋白免疫原性的影響,與我們用核酸免疫獲得的結(jié)果進(jìn)行對(duì)比和驗(yàn)證,探討缺失HVR1的E2蛋白在丙肝疫苗中的應(yīng)用前景。 一、穩(wěn)定表達(dá)HCV包膜E2-人IgG Fc融合蛋白的CHO細(xì)胞株的建立 我們首先分別拼接HCV包膜蛋白E2-Fc和E2Δ-Fc融合基因(E2Δ指缺失HVR1),使E2與人IgG Fc段作為融合蛋白表達(dá),方便目的蛋白的親和層析純化。將融合基因插入具有自主知識(shí)產(chǎn)權(quán)的CHO細(xì)胞表達(dá)質(zhì)粒載體pCIDA-GS-neo(專(zhuān)利號(hào):ZL 200610024202.5,發(fā)明名稱:一種用哺乳動(dòng)物細(xì)胞高效分泌表達(dá)丙型肝炎病毒包膜蛋白E2的方法),然后分別將構(gòu)建的表達(dá)質(zhì)粒轉(zhuǎn)染HEK 293T細(xì)胞,用ELISA及Western blotting方法檢測(cè)培養(yǎng)上清中E2-Fc以及E2Δ-Fc融合蛋白,證實(shí)兩者均可有效表達(dá)且表達(dá)水平相當(dāng)。爾后,我們分別將這兩種E2-Fc融合蛋白表達(dá)質(zhì)粒及EGFP表達(dá)質(zhì)粒(GS-EGFP)分別轉(zhuǎn)染CHO細(xì)胞,以蛋氨酸亞氨基代砜(methionine sulphoximine,MSX)在不含谷氨酰胺的培養(yǎng)基內(nèi)篩選表達(dá)目的蛋白的細(xì)胞克隆,用有限稀釋法對(duì)粗篩出的細(xì)胞克隆進(jìn)行進(jìn)一步篩選,從而建立和獲得了穩(wěn)定分泌表達(dá)E2-Fc和E2Δ-Fc融合蛋白的細(xì)胞株。緊接著,我們對(duì)篩選出的貼壁CHO細(xì)胞株進(jìn)行了無(wú)血清無(wú)蛋白培養(yǎng)的馴化,通過(guò)逐步下降培養(yǎng)基內(nèi)血清的濃度,使細(xì)胞由貼壁狀態(tài)轉(zhuǎn)為懸浮生長(zhǎng),隨后對(duì)懸浮細(xì)胞株的培養(yǎng)體系進(jìn)行了放大,從75T方瓶培養(yǎng)經(jīng)由250 ml三角燒瓶的小量放大,最后實(shí)現(xiàn)在Wave Bioreactor EH2/10細(xì)胞培養(yǎng)系統(tǒng)中2L細(xì)胞培養(yǎng)袋的灌流培養(yǎng)。細(xì)胞密度由最初的2×105cells/ml,達(dá)到最后的2×107cells/ml,細(xì)胞活率始終保持在80%以上,目的蛋白的分泌量也在灌流培養(yǎng)開(kāi)始后穩(wěn)定在3-4 mg/L左右。 二、E2-Fc、E2Δ-Fc融合蛋白純化及功能鑒定 以上述的細(xì)胞培養(yǎng)上清為原料,離心去除細(xì)胞,繼以GE公司超濾系統(tǒng)(QuixStand? Benchtop System)對(duì)上清液進(jìn)行濃縮和超濾,將其濃縮約10倍,并去除其中分子量低于30 KD的蛋白質(zhì)。隨后,以ProteinA親和層析柱HiTrap? Protein A HP Columns從超濾液中純化融合蛋白,通過(guò)對(duì)柱壓、洗脫條件、收集方式等參數(shù)的優(yōu)化,從每升培養(yǎng)上清中可純化出3-4 mg目的蛋白。然后用截留分子量為30 KD的離心型蛋白超濾柱以低溫低速離心的方式將純化出蛋白樣品的緩沖液置換為pH為7.0的磷酸鹽緩沖液。用構(gòu)象依賴性單抗對(duì)最終純化產(chǎn)物進(jìn)行的Western blotting分析表明E2蛋白保持了天然空間構(gòu)象。以糖苷酶EndoH、PNGase F分別對(duì)兩種蛋白進(jìn)行去糖基化處理,然后觀察其分子量的變化,結(jié)果提示這兩種蛋白的糖基化程度和形式相似。ELISA及Pull down實(shí)驗(yàn)顯示E2-Fc,E2Δ-Fc均能與人CD81大胞外環(huán)(hCD81-LEL)結(jié)合,E2Δ-Fc結(jié)合活性較E2-Fc為高。檢測(cè)兩種蛋白與細(xì)胞膜表面表達(dá)的人CD81和SR-BI分子的結(jié)合活性,結(jié)果顯示缺失HVR1后,E2蛋白與SR-BI的結(jié)合能力明顯降低,與CD81的結(jié)合略有增強(qiáng)。流式細(xì)胞術(shù)檢測(cè)發(fā)現(xiàn)兩種蛋白均能與HCV易感的Huh7.5細(xì)胞結(jié)合且效率相當(dāng)。將與E2蛋白結(jié)合后的Huh7.5細(xì)胞裂解,通過(guò)免疫共沉淀的方法檢測(cè)介導(dǎo)E2蛋白與細(xì)胞結(jié)合的分子,結(jié)果顯示,E2-Fc能將CD81及SR-BI共沉淀下來(lái),而HVR1缺失的E2Δ-Fc共沉淀較多的CD81及少量SR-BI,提示HVR1是介導(dǎo)E2蛋白與靶細(xì)胞表面SR-BI結(jié)合的重要區(qū)域。兩種蛋白均能抑制HCVpp和HCVcc的感染性,抑制率最高可達(dá)90%以上,且株特異性不強(qiáng)。 三、刪除HVR1增強(qiáng)E2-Fc融合蛋白的免疫原性 分別將上述兩種融合蛋白聯(lián)合弗氏佐劑免疫小鼠,于0、3和8周每只小鼠皮下注射10μg佐劑乳化的融合蛋白,初免第2、7和10周采血檢測(cè)小鼠血清中的E2ΔHVR1抗體及HVR1抗體,發(fā)現(xiàn)E2-Fc免疫組10只小鼠中,有5只E2ΔHVR1及HVR1抗體均為陽(yáng)性,4只E2ΔHVR1抗體單陽(yáng)性,1只兩種抗體一直是陰性。E2ΔHVR1及HVR1抗體滴度隨免疫次數(shù)增多逐步升高,E2Δ-Fc蛋白的確能誘導(dǎo)更高的E2ΔHVR1抗體水平,其滴度約為E2-Fc免疫組的5-10倍。證實(shí)HVR1的存在抑制了E2ΔHVR1抗體的產(chǎn)生。爾后,為進(jìn)一步檢測(cè)小鼠血清抗體中和活性,我們將各組小鼠血清混合后純化出IgG,行病毒感染中和實(shí)驗(yàn),結(jié)果顯示,兩組IgG對(duì)于與蛋白來(lái)源同株的H77株HCVpp感染中和效率沒(méi)有顯著性差異,中和率在80%以上。但對(duì)于異株HCV,E2-Fc組中和活性顯著低于E2Δ-Fc組。含有HVR1的E2-Fc融合蛋白誘導(dǎo)的抗體中,HVR1抗體陽(yáng)性則其交叉中和活性明顯低于HVR1抗體陰性者,也說(shuō)明HVR1抑制交叉中和抗體的產(chǎn)生。這些結(jié)果與我們DNA免疫的研究一致,高度提示缺失HVR1可有力促進(jìn)HCV包膜E2蛋白誘導(dǎo)交叉中和抗體。此外,考慮到Fc段本身有特殊的免疫學(xué)功能,可能有助于免疫應(yīng)答,尤其是細(xì)胞免疫應(yīng)答的誘導(dǎo)。本研究中,采用融合蛋白免疫小鼠,該融合蛋白是否能在動(dòng)物體內(nèi)誘生針對(duì)E2蛋白特異且高效的細(xì)胞免疫反應(yīng),是我們需要進(jìn)一步驗(yàn)證的內(nèi)容。 小結(jié) 本研究著眼于丙型肝炎病毒蛋白疫苗的開(kāi)發(fā)思路,在前期DNA免疫的基礎(chǔ)上,構(gòu)建了穩(wěn)定表達(dá)E2-Fc/E2Δ-Fc的無(wú)蛋白無(wú)血清懸浮CHO細(xì)胞培養(yǎng)體系并成功純化出相應(yīng)的目的蛋白。功能試驗(yàn)結(jié)果顯示,HVR1缺失并未明顯影響E2蛋白的天然構(gòu)象,HVR1刪除后,E2蛋白與其受體CD81的結(jié)合增強(qiáng),與SR-BI的結(jié)合能力減弱。動(dòng)物免疫結(jié)果顯示,HVR1的缺失顯著增強(qiáng)了E2蛋白誘導(dǎo)的交叉中和抗體。綜上所述,該研究首次從蛋白層面揭示了HVR1對(duì)E2蛋白結(jié)構(gòu)、功能及免疫原性的影響,為HCV蛋白疫苗的開(kāi)發(fā)提供了新思路。
[Abstract]:Hepatitis C virus (hepatitis C, virus, HCV) belongs to the family Flaviviridae, mainly through blood transmission, is caused by human hepatitis C virus pathogens. Approximately 170 million people worldwide are infected with HCV, new infections each year up to 3 million -400 million. China's HCV infected.HCV estimates that nearly 40 million of the infection is likely to slow, high rate of chronicity 85%, part of patients with chronic hepatitis C cirrhosis, and hepatocellular carcinoma. At present the main pegylated interferon alpha combined with Leigh Bhave Lin in the treatment of hepatitis C, the effect was significantly affected by the influence of HCV genotypes on the sustained virologic response to therapy of HCV infection rate of.2,3 (SVR) can reach 75%, but the 1 type of HCV infection SVR to 50%. worldwide through infected blood and blood products of hepatitis C patients and the last century before 90s compared to a sharp decline, but in the blood and blood products in developing countries is still. An important way to broadcast HCV. In addition, each of the sporadic and the transmission of unknown new cases of HCV infection still remains high. Key measures to control HCV infection is dependent on the development of an effective vaccine, since the 1989 HCV genome was cloned on hepatitis C vaccine in Europe and Japan and other developed countries have been studied hot, but so far no effective vaccine available.
HCV envelope protein E2 in HCV protein precursor in 384-746 amino acid residues, by a large N ectodominant terminal and a C terminal hydrophobic anchor region, and another HCV envelope protein E1 by non covalent bond to form heterologous dimers with two.E2 is the key protein mediated virus adsorption and entry the host cell is the key antigen induce neutralizing antibody to HCV, it is the main target of vaccine research. However, the envelope protein is highly variable and hypervariable region of 27 amino acid residues containing especially E2 protein amino terminal 1 (hypervariable region 1, HVR1) is a regional variation in the highest frequency of HCV protein HCV, is recognized as a neutralizing epitope region of.HVR1 is the key area of HCV mediated by receptor binding of SR-BI, HVR1 and SR-BI promote HCV cell invasion of.HVR1 antibody and SR-BI antibody can block the infection of HCV.HVR through several different mechanisms The height variation of 1 was considered to be a bottleneck in HCV vaccine development in recent years, based on the HCV virus (HCVpp) and cell culture produced HCV (HCVcc) found the neutralization test, HVR1 is not the only neutralizing epitope region, also depends on a series of conformational neutralization epitopes and linear neutralizing epitopes in HCV envelope protein E2, some epitopes in different genotypes and subtypes are highly conserved.
Ourprevious respectively with secretory expression plasmid E2 and the deletion of HVR1 expression plasmid of E2 immunized mice, then detection, analysis of antibody and virus neutralizing activity in serum of mice showed that the serum E2 of mice immunized, HVR1 antibody accounted for a large proportion in the total E2 antibody, antibody to outside the HVR1 area only a small proportion in E2 antibody; deletion of HVR1 significantly enhanced the antibody to HVR1 outside the E2 plasmid induced immune cross reaction, immune serum of mice with other genotypes of E2 protein and HCVpp of other genotypes cross neutralizing activity also increased significantly. And the acute HCV infection detected HVR1 antibody but it is difficult to detect the serum E2 antibody. The results suggest that HVR1 may inhibit the conservative form immunogenicity of the E2 protein, which can lead to HCV immune escape from another.. The HCV envelope protein based on the main target antigen vaccine containing HVR1, this kind of immune vaccine can resist chimpanzee monoecious virus attack, may exist but they cannot resist the different strains of the.HVR1 attack is an important reason for this type of vaccine immune protection effect is not ideal, perhaps can enhance the protective effect of deletion of HVR1 vaccine on different strains of virus attack.
The nucleic acid vaccine on the immune effect of large animal is far less than in the immune effect of small animal, there is no vaccine for human use. In order to further investigate the deletion of HVR1 envelope protein E2 as the development prospects of HCV vaccine, the expression of E2 and deletion of HVR1 E2 protein in CHO cells, purified by affinity chromatography two kinds of E2 protein, conformation analysis of two E2 protein, receptor binding function and antigenicity, through small animal immune analysis on the effect of HVR1 on E2 protein immunogenicity, were compared and validated with the results obtained by our DNA immunization, and to discuss the application of HVR1 deletion of E2 protein in hepatitis C vaccine.
Establishment of a CHO cell line stably expressing the HCV envelope E2- human IgG Fc fusion protein
We are first splicing HCV envelope protein E2-Fc and E2 -Fc fusion gene (E2 Delta HVR1, which refers to the lack of E2 and IgG) Fc as a fusion protein, convenient protein affinity chromatography. The fusion gene was inserted with independent intellectual property rights CHO expression plasmid vector pCIDA-GS-neo (Patent No.: ZL 200610024202.5. The invention: a method for mammalian cells secreting expression of hepatitis C virus envelope protein E2), then the plasmid was transfected into HEK cells by 293T, ELISA and Western blotting method to detect the culture supernatant of E2-Fc and E2 -Fc fusion protein, both of which can be confirmed and the expression level is quite effective. Then, we are the two kinds of E2-Fc fusion protein expression plasmid and expression plasmid EGFP (GS-EGFP) CHO cells were transfected with methionine imino sulfone (methionine sulphoximine, MSX generation) Clones were screened for the expression of target protein in the culture medium without glutamine, the cell clones were further screened by the screening method of limited dilution to establish and obtain a stable expression and secretion of E2-Fc and E2 a -Fc fusion protein in cell line. Then, we carried out domestication and serum-free culture of selected protein adherent CHO cells, through the medium of serum concentrations gradually decreased, the cell growth by adherence to the suspension, then the training system of suspension cell lines were amplified, amplified by 250 ml three 75T from the small angle flask flask culture, and finally realize the cultivation of 2L cell culture bag perfusion the culture system of Wave Bioreactor in EH2/10 cells. The cell density from 2 * 105cells/ml, reaching 2 * 107cells/ml, cell survival rate has remained above 80%, the secretion of protein in irrigation The flow culture began to stabilize at about 3-4 mg/L.
Purification and functional identification of two, E2-Fc, E2 Delta -Fc fusion protein
In the cell culture supernatant as raw materials and centrifuged to remove cells, followed by GE (QuixStand Benchtop System ultrafiltration system?) concentration and ultrafiltration of supernatant, which is concentrated about 10 times, and the removal of the molecular weight of less than 30 KD protein. Then, using ProteinA affinity chromatography column HiTrap Protein A HP Columns? The fusion protein was purified by ultrafiltration, the column pressure, elution conditions, optimization parameter collection methods, from per liter of culture supernatant can be purified 3-4 mg protein. Then with MWCO of 30 KD type centrifugal ultrafiltration column protein by low temperature and low speed centrifugal method and purified protein buffer replacement the samples of pH 7 phosphate buffer. With conformation dependent monoclonal antibody analysis of the purified products were Western blotting showed that E2 protein conformation. In order to maintain the natural glycosidase enzymes EndoH, PNGase and F respectively to two Protein deglycosylation treatment, then observe the changes of molecular weight, the results showed that two kinds of protein glycosylation and similar forms.ELISA and Pull down showed that E2-Fc, E2 -Fc and CD81 a large extracellular loop (hCD81-LEL) combined with E2, a -Fc binding activity was higher than E2-Fc binding activity. Detection of two kinds of protein expression and cell surface CD81 and SR-BI molecules, the results showed that HVR1 deletion, binding ability of E2 protein and SR-BI were significantly decreased, and the combination of CD81 is slightly enhanced. Flow cytometry assay showed that two proteins were HCV and susceptible Huh7.5 cell binding and efficiency Huh7.5. After cell lysis combined with E2 protein, molecular methods, by immunoprecipitation detection mediated by E2 protein binding to the cells showed that E2-Fc CD81 and SR-BI can be co precipitated, and deletion of the HVR1 E2 -Fc CD81 and a little more precipitation SR-BI indicates that HVR1 is an important region that mediates the binding of E2 protein to SR-BI on target cell surface. All two proteins can inhibit the infectivity of HCVpp and HCVcc, the inhibition rate is above 90%, and the plant specificity is not strong.
Three, deleting the immunogenicity of HVR1 enhanced E2-Fc fusion protein
The above two kinds of immunized mice with Freund's adjuvant in 0,3 and 8 weeks each mice were injected subcutaneously with 10 g adjuvant fusion protein, immunization of 2,7 and 10 weeks of blood testing E2 HVR1 antibody and HVR1 antibody in sera of mice immunized with E2-Fc, found in a group of 10 mice. 5 E2 HVR1 and HVR1 antibodies were positive, 4 E2 HVR1 1 single antibody positive, only two kinds of antibodies has been negative for.E2 and HVR1 HVR1 antibody titer with immunization times increased gradually increased, E2 Delta -Fc protein can indeed induce higher E2 HVR1 antibody level, its titer was about 5-10 times E2-Fc immune group. To confirm the presence of HVR1 inhibited E2 HVR1 antibodies. Then, for the further detection of serum antibody neutralizing activity, we will in serum were mixed and purified for IgG, virus neutralization test, results showed that the two groups for IgG and protein from strain H77 Strain HCVpp infection efficiency and no significant difference, and the rate is more than 80%. But for different strains of HCV, E2-Fc group of neutralizing activity was significantly lower than that in E2 group. A -Fc HVR1 containing E2-Fc fusion protein induced by HVR1 antibody, the antibody positive cross neutralizing activity was significantly lower than that of HVR1 antibody negative, indicating that HVR1 suppress the cross neutralizing antibodies. These results are consistent with our DNA immunity, highly suggestive of deletion of HVR1 can effectively promote the HCV envelope protein E2 induced cross neutralizing antibodies. In addition, taking into account the Fc itself has special immunological function, may contribute to the immune response, especially the induction of cellular immune response. In this study, the mice were immunized with fusion protein, whether the fusion protein in animal in vivo induced cellular immune responses against E2 protein specifically and efficiently, we need to further verify the contents.
Summary
This study focuses on the development of hepatitis C virus protein vaccine, based on the previous DNA immunity, constructed the stable expression of E2-Fc/E2 Delta -Fc protein free serum-free CHO cell culture system and purified protein. The corresponding objective function test results show that HVR1 deficiency did not affect E2 protein natural conformation HVR1 after deletion, enhanced binding protein E2 and its receptor CD81, and weaken the binding ability of SR-BI. Animal immunization results showed that loss of HVR1 significantly enhanced the cross neutralizing antibodies induced by E2 protein. In summary, this study first from the protein level revealed that HVR1 of E2 protein structure, function and influence of immunogenicity. Provides a new idea for the development of HCV protein vaccine.

【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】：2011
【分類(lèi)號(hào)】：R392

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