高变区1抑制丙型肝炎病毒包膜蛋白E2诱生交叉中和抗体
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摘要
丙型肝炎病毒(hepatitis C virus,HCV)属于黄病毒科,主要经血液传播,是引起人类丙型肝炎的病原体。全球约有1.7亿人感染HCV,每年新增感染者达300万-400万。我国HCV感染者估计近4000万。HCV感染易慢性化,其慢性化率可高达85%,部分慢性丙型肝炎患者可发展为肝硬化,甚至肝细胞癌。目前主要采用聚乙二醇干扰素α联合利巴韦林治疗丙型肝炎,其效果明显受HCV基因型影响。2、3型HCV感染对该疗法的持续病毒学应答率(SVR)可达到75%,但1型HCV感染的SVR不到50%。目前全球范围内经血液以及血制品途径感染的丙型肝炎病例与上世纪九十年代以前相比有大幅下降,但在发展中国家血液和血制品仍然是传播HCV的重要途径。此外,每年散发性以及传播途径不明的丙型肝炎新发病例仍高居不下。控制HCV感染的关键措施还是依赖于开发出有效的疫苗,从1989年HCV基因组被克隆以来,丙型肝炎疫苗的研究在欧美和日本等发达国家一直是研究热点,但迄今未有有效的疫苗问世。
HCV包膜蛋白E2位于HCV蛋白前体第384-746位氨基酸残基,由一个大的N端膜外区和一个C端疏水锚定区组成,与HCV另一包膜蛋白E1通过非共价键形成异源性二聚体。E2蛋白是介导病毒吸附和进入宿主细胞的关键蛋白,是诱导宿主产生HCV中和抗体的关键抗原,因此是疫苗研究的最主要靶标。然而,包膜蛋白高度变异,尤其E2蛋白氨基末端的含有27个氨基酸残基的高变区1(hypervariable region 1,HVR1)是HCV蛋白中变异频率最高的区域,也是公认的HCV中和抗体表位所在区域。HVR1是介导HCV与受体SR-BI结合的关键区域,HVR1与SR-BI的作用通过几种不同的机制促进HCV细胞侵入。HVR1抗体和SR-BI抗体均能阻断HCV感染。HVR1的高度变异曾被认为是遏制丙肝疫苗发展的瓶颈问题,近几年来基于HCV假病毒(HCVpp)以及细胞培养产生的HCV(HCVcc)的中和试验发现,HVR1并不是唯一的中和抗体表位所在区域,在HCV包膜E2蛋白中还存在一系列构象依赖的中和表位和线性中和表位,其中一些表位在不同基因型、亚型间高度保守。
本实验室前期分别用分泌表达E2的表达质粒以及删除了HVR1的E2表达质粒免疫小鼠,然后检测、分析小鼠血清中的抗体以及病毒中和活性,结果显示:E2质粒免疫的小鼠血清中,HVR1抗体在总E2抗体中占很大比重,针对HVR1以外区域的抗体在中E2抗体中只占较小比重;删除HVR1可显著增强E2质粒免疫诱导的针对HVR1以外区域的抗体,小鼠免疫血清与其他基因型E2蛋白的交叉反应以及对其他基因型HCVpp的交叉中和活性也随之显著增强。这和急性HCV感染者血清中可检测到HVR1抗体而难以检测到E2抗体相似。结果提示:HVR1有可能抑制E2蛋白中保守表位的免疫原性,从而从另一个方面导致HCV免疫逃避。目前基于HCV包膜蛋白为主要靶抗原的疫苗均含有HVR1,这类疫苗免疫黑猩猩只能抵抗同株病毒攻击,但不能抵抗异株的攻击。HVR1的存在可能是这类疫苗免疫保护效果不理想的重要原因,删除HVR1也许能增强疫苗免疫对异株病毒攻击的保护效果。
核酸疫苗在大动物的免疫效果远远不及在小动物的免疫效果,目前还没有核酸疫苗用于人类。为了进一步探讨用删除了HVR1的包膜E2蛋白作为HCV疫苗的发展前景,本研究用CHO细胞表达了E2以及删除了HVR1的E2蛋白,用亲和层析纯化出了两种E2蛋白,分析两种E2蛋白的构象、受体结合功能和抗原性,通过小动物免疫分析HVR1对E2蛋白免疫原性的影响,与我们用核酸免疫获得的结果进行对比和验证,探讨缺失HVR1的E2蛋白在丙肝疫苗中的应用前景。
一、稳定表达HCV包膜E2-人IgG Fc融合蛋白的CHO细胞株的建立
我们首先分别拼接HCV包膜蛋白E2-Fc和E2Δ-Fc融合基因(E2Δ指缺失HVR1),使E2与人IgG Fc段作为融合蛋白表达,方便目的蛋白的亲和层析纯化。将融合基因插入具有自主知识产权的CHO细胞表达质粒载体pCIDA-GS-neo(专利号:ZL 200610024202.5,发明名称:一种用哺乳动物细胞高效分泌表达丙型肝炎病毒包膜蛋白E2的方法),然后分别将构建的表达质粒转染HEK 293T细胞,用ELISA及Western blotting方法检测培养上清中E2-Fc以及E2Δ-Fc融合蛋白,证实两者均可有效表达且表达水平相当。尔后,我们分别将这两种E2-Fc融合蛋白表达质粒及EGFP表达质粒(GS-EGFP)分别转染CHO细胞,以蛋氨酸亚氨基代砜(methionine sulphoximine,MSX)在不含谷氨酰胺的培养基内筛选表达目的蛋白的细胞克隆,用有限稀释法对粗筛出的细胞克隆进行进一步筛选,从而建立和获得了稳定分泌表达E2-Fc和E2Δ-Fc融合蛋白的细胞株。紧接着,我们对筛选出的贴壁CHO细胞株进行了无血清无蛋白培养的驯化,通过逐步下降培养基内血清的浓度,使细胞由贴壁状态转为悬浮生长,随后对悬浮细胞株的培养体系进行了放大,从75T方瓶培养经由250 ml三角烧瓶的小量放大,最后实现在Wave Bioreactor EH2/10细胞培养系统中2L细胞培养袋的灌流培养。细胞密度由最初的2×105cells/ml,达到最后的2×107cells/ml,细胞活率始终保持在80%以上,目的蛋白的分泌量也在灌流培养开始后稳定在3-4 mg/L左右。
二、E2-Fc、E2Δ-Fc融合蛋白纯化及功能鉴定
以上述的细胞培养上清为原料,离心去除细胞,继以GE公司超滤系统(QuixStand? Benchtop System)对上清液进行浓缩和超滤,将其浓缩约10倍,并去除其中分子量低于30 KD的蛋白质。随后,以ProteinA亲和层析柱HiTrap? Protein A HP Columns从超滤液中纯化融合蛋白,通过对柱压、洗脱条件、收集方式等参数的优化,从每升培养上清中可纯化出3-4 mg目的蛋白。然后用截留分子量为30 KD的离心型蛋白超滤柱以低温低速离心的方式将纯化出蛋白样品的缓冲液置换为pH为7.0的磷酸盐缓冲液。用构象依赖性单抗对最终纯化产物进行的Western blotting分析表明E2蛋白保持了天然空间构象。以糖苷酶EndoH、PNGase F分别对两种蛋白进行去糖基化处理,然后观察其分子量的变化,结果提示这两种蛋白的糖基化程度和形式相似。ELISA及Pull down实验显示E2-Fc,E2Δ-Fc均能与人CD81大胞外环(hCD81-LEL)结合,E2Δ-Fc结合活性较E2-Fc为高。检测两种蛋白与细胞膜表面表达的人CD81和SR-BI分子的结合活性,结果显示缺失HVR1后,E2蛋白与SR-BI的结合能力明显降低,与CD81的结合略有增强。流式细胞术检测发现两种蛋白均能与HCV易感的Huh7.5细胞结合且效率相当。将与E2蛋白结合后的Huh7.5细胞裂解,通过免疫共沉淀的方法检测介导E2蛋白与细胞结合的分子,结果显示,E2-Fc能将CD81及SR-BI共沉淀下来,而HVR1缺失的E2Δ-Fc共沉淀较多的CD81及少量SR-BI,提示HVR1是介导E2蛋白与靶细胞表面SR-BI结合的重要区域。两种蛋白均能抑制HCVpp和HCVcc的感染性,抑制率最高可达90%以上,且株特异性不强。
三、删除HVR1增强E2-Fc融合蛋白的免疫原性
分别将上述两种融合蛋白联合弗氏佐剂免疫小鼠,于0、3和8周每只小鼠皮下注射10μg佐剂乳化的融合蛋白,初免第2、7和10周采血检测小鼠血清中的E2ΔHVR1抗体及HVR1抗体,发现E2-Fc免疫组10只小鼠中,有5只E2ΔHVR1及HVR1抗体均为阳性,4只E2ΔHVR1抗体单阳性,1只两种抗体一直是阴性。E2ΔHVR1及HVR1抗体滴度随免疫次数增多逐步升高,E2Δ-Fc蛋白的确能诱导更高的E2ΔHVR1抗体水平,其滴度约为E2-Fc免疫组的5-10倍。证实HVR1的存在抑制了E2ΔHVR1抗体的产生。尔后,为进一步检测小鼠血清抗体中和活性,我们将各组小鼠血清混合后纯化出IgG,行病毒感染中和实验,结果显示,两组IgG对于与蛋白来源同株的H77株HCVpp感染中和效率没有显著性差异,中和率在80%以上。但对于异株HCV,E2-Fc组中和活性显著低于E2Δ-Fc组。含有HVR1的E2-Fc融合蛋白诱导的抗体中,HVR1抗体阳性则其交叉中和活性明显低于HVR1抗体阴性者,也说明HVR1抑制交叉中和抗体的产生。这些结果与我们DNA免疫的研究一致,高度提示缺失HVR1可有力促进HCV包膜E2蛋白诱导交叉中和抗体。此外,考虑到Fc段本身有特殊的免疫学功能,可能有助于免疫应答,尤其是细胞免疫应答的诱导。本研究中,采用融合蛋白免疫小鼠,该融合蛋白是否能在动物体内诱生针对E2蛋白特异且高效的细胞免疫反应,是我们需要进一步验证的内容。
小结
本研究着眼于丙型肝炎病毒蛋白疫苗的开发思路,在前期DNA免疫的基础上,构建了稳定表达E2-Fc/E2Δ-Fc的无蛋白无血清悬浮CHO细胞培养体系并成功纯化出相应的目的蛋白。功能试验结果显示,HVR1缺失并未明显影响E2蛋白的天然构象,HVR1删除后,E2蛋白与其受体CD81的结合增强,与SR-BI的结合能力减弱。动物免疫结果显示,HVR1的缺失显著增强了E2蛋白诱导的交叉中和抗体。综上所述,该研究首次从蛋白层面揭示了HVR1对E2蛋白结构、功能及免疫原性的影响,为HCV蛋白疫苗的开发提供了新思路。
Hepatitis C virus (HCV), the only member of the hepacivirus genus, family Flaviviridae, is a major cause of posttransfusional non-A and non-B hepatitis. HCV infects more than 170 million people worldwide, among which 40 million are in China. About 85% patients fail to clear HCV and contract persistent infection which frequently leads to chronic liver disease, including cirrhosis and hepatocellular carcinoma. The current therapy for HCV infection is peg-IFNαin combination with ribavirin, but not all genotypies of HCV infected patients could receive the sustained viral response. Therefore, what needs dealing with urgently is developing vaccines to protect healthy persons as well as clear HCV from infected patients. Unfortunately, there is no effective vaccine available until now.
HCV E2 envelope protein extends from aa 384-746 of the polyprotein, containing a N-terminal ectodomain and a C-terminal hydrophobic anchor domain. E1 and E2, is thought to form a heterodimer stabilized by non-covalent interactions as envelope of the HCV particles. As an envelope protein, E2 is thought to play a major role in virus attachment to the target cell by binding to receptors on the cell plasma membrane followed by membrane fusion and entry. Meanwhile, as E2 may elicit production of neutralizing antibodies against the virus, it was considered a major candidate for anti-HCV vaccine development. However, the most variable region of HCV (HVR-1) is also located within the N-terminal 27 residues (aa 384-411) of E2, it is also widely recognized as an epitope to which the neutralizing antibody binds. The high variation of HVR1 was considered to be a bottleneck in HCV vaccine development. However, in recent years, virus neutralizing tests based on HCV pseudoparticles (HCVpp) and HCV cell culture model (HCVcc) found that HVR1 is not the only neutralizing epitope and parts of E2 other than HVR1 might be involved in the induction of virus neutralizing antibodies.
In our previous research, we constructed some eukaryotic plasmids to express the carboxyl-terminal truncated E2 protein with or without HVR1. Then these plasmids were used to immune mice and the relationship between HVR1 antibody and the total E2 antibody on amount and neutralizing activities were analyzed. What we found was 1. Secretionary expression of E2 is more effective to induce antibody response. 2. In addition to HVR1, there exist exactly other neutralizing epitopes in E2 protein. 3. anti-HVR1 is the main part of the neutralizing antibodies. 4. HVR1 attenuates the cross-reaction of antibody induced by E2. These results implies us whether we can enhance the immunogenicity of E2 by deleting HVR1 which might cover other conserved neutralizing epitopes existing in other regions of E2? If this alteration of E2 could induce effective cross-neutralizing humoral immune response, it could provide us a new strategy on developing HCV vaccine. However, DNA immunization might be interfered with confounding factors induced by other elements exist on the expression vector, which could mislead us. Protein vaccine using purified proteins as immunogens, can avoid these problems. Therefore, from this perspective, in this research, we constructed soluble E2 protein and soluble HVR1-deleting E2 expression plasmids, transfected into CHO cells and establish the E2 proteins’expression system. Based on affinity chromatography, E2 proteins were separated and purified from cell cultural supernatant, their conformation, function and immunogenicity was investigated and compared with each other. Our study was aimed to confirm the conclusion obtained from DNA immunization by E2 protein immunization and further explore the potential use of HVR1-deleting E2 protein in protective HCV vaccine.
1. Construction of HCV E2-human IgG Fc fusion protein eukaryotic expression system
In this part of our research, first, we constructed HCV E2-Fc/E2Δ-Fc fusion protein expression plasmids. This kind of fusion strategy was convenient for affinity chromatography of proteins. Next, we inserted glutamine synthetase gene into the downstream of target gene, by adding glutamine synthetase inhibitor (MSX) into the cell culture medium, recombinant CHO cell lines with high expression levels of fusion proteins could be selected. On the other hand, glutamine can be produced by cell metabolism of amines glutamine, exhibiting better growth and proliferation in the medium without glutamine; this might avoid cell injury induced by accumulation of ammonia, and greatly facilitate the follow-up of cell culture and protein purification. Then, these plasmids were transfected into HEK 293T cells to detect the expression levels of E2-Fc/E2Δ-Fc proteins in the supernatant based on ELISA and Western blotting. Results showed that these two proteins were expressed well and had got similar levels. After that, these two plasmids as well as another negative control plasmid- GS-EGFP were transfected into CHO cells respectively. Recombinant CHO cell clones were screened out under the pressure of MSX, these clones were then picked out and undertaken limited dilution to select clones with higher expression levels of target proteins. In order to improve space and medium utilization as well as reduce the mixed composition in FBS containing cell culture supernatant, serum-free protein-free medium adaptation were performed on these adherent cell lines, FBS concentration of cultural medium was decreased step by step, cells were transferred to suspension state instead. After adaptation, the suspension cell culture system was further enlarged, through 75T to 250ml flask with the small amount of amplification, and finally achieves the perfusion culture in 2L cellbags on Wave Bioreactor EH2/10 cell culture system. Cell density increased from 2×105cells/ml to the final 2×107cells/ml, cell viability was maintained above 80% and the secretion of protein was also stable at 3-4mg/L after perfusion. This part of our study has provided sufficient material for the following protein function analysis. Of course, the way to improve serum protein-free adaption efficiency and optimize the process of cell culture system enlargement still needs further exploration.
2. Purification and identification of E2-Fc fusion protein
As an intermediate steps between protein expression and function analysis, the separation and purification of target proteins play an important role. In this part of the experiment, we used the cultural supernatant collected from HCV E2-Fc fusion protein secreting suspension cell as raw materials, through the clarification step to remove most of the impurities, followed by concentration step based on ultrafiltration system, QuixStand? Benchtop System and hollow fiber ultrafiltration column, to reach a 10 times concentration and remove the molecules less than 30KD. Then, the ultrafiltrate was further purified with protein A affinity column HiTrap? Protein A HP Columns and purification system-?KTApurifier System (GE). During the purification process, through optimizing the column pressure, elution conditions collection, about 3-4mg of pure protein could be obtained from per liter culture supernatant, comparable with the reported E2-Fc fusion protein purification efficiency (2mg/L). Then, we exchanged the buffer of crude protein sample to PBS with ultrafiltration centrifuge column by way of low-temperature low-speed centrifugation, and further enrichment of the samples (3-5 times). Finally, we detected the concentration of protein samples by BCA protein quantitative method, and performed native-PAGE and Western blotting to confirm the conformation of protein samples maintain correct after ultrafiltration, purification, buffer exchange and other which can be used the following function analysis and animal experiments. At the same time, two crude purified proteins were undertaken glycosylation by glycosidase EndoH, PNGase F treatment, results suggest that glycosylation of both crude purified protein remained similar with their natural state. ELISA binding assay and pull down assays showed that E2-Fc, E2Δ-Fc could bind with human CD81 LEL, E2Δ-Fc exhibited higher binding activity than E2-Fc. Then, through flow cytometry, we investigated that both proteins can bind to HCV susceptible cell Huh7.5 cells well and had no much difference with each other. Further, we examined the binding activity between crude protein and CHO cells with expression of human CD81, SR-BI molecules respectively. Results showed that HVR1 deletion, binding efficiency between E2 protein and CHO-SR-BI was greatly decreased but slightly enhanced when binding with CHO-CD81. Next, we performed immunoprecipitation with lysis of Huh7.5 cells after E2 protein binding process to detect molecules that mediated E2 protein and cell binding. As a result, E2-Fc were able to precipitate CD81 and SR-BI, but E2Δ-Fc could co-precipitate little SR-BI and more CD81 when comparing with E2-Fc. This was consistent with report that HVR1 is important in mediating E2 and cell binding by affinity with SR-BI on target cells. Through infection assays of HCVcc and HCVpp, we observed concentration-dependent efficiency of crude pure fusion protein in inhibition of HCVcc and HCVpp infection. About 25-30μg of protein could be sufficient to completely suppress the infection of HCV infectious particles exhibiting no much strain specificity.
3. Characteraztion of immunogenecity enhacement by HCV HVR1 deletion in purified E2-Fc fusion protein
This section, we immunized BALB/c mice with crude purified proteins. Through the strategy of immunization for 3 times at week 0, 3 and 8 with a dose of 10μg once, serum was collected at week 2, 7 and 10 for anti-HVR1 and anti-E2ΔHVR1 antibody detection. In E2-Fc immunizing group, 5 in 10 were anti-HVR1 and anti-E2ΔHVR1 positive, another 4 were anti-E2ΔHVR1 positive only. It was supposed that Fc segment fused with the E2 protein affected the HVR1 epitopes exposure. Similar with results of DNA immunization, E2Δ-Fc protein induced higher anti-E2Δantibody levels. With the same quantity of total IgG, anti-HVR1 positive serum exhibted weaker neutralizing activitiy against the infection of J6/JFH1 HCVcc and Con1 HCVpp, but no much different from anti-HVR1 negative serum in inhibiting the infection of H77 HCVpp. These results confirmed the conclusions that anti-HVR1 had got strong neutralizing activity against the attack of HCV comes from the same strain but weak cross-neutralizing activity against the virus of other strains or genotypes.
In summary, we have established a eukaryotic cell culture system to produce secretory HCV E2-Fc and E2Δ-Fc fusion protein and purified the target proteins. Based on E2-Fc and E2Δ-Fc, we investigated the roles of HVR1 in structure, function and immunogenicity of E2 protein. No obvious conformational changes were found in HVR1-deleting E2 protein. Although maintaining major function of E2 protein, HVR1-deleting E2 does decrease affinity with SR-BI, suggesting HVR1 be important in E2 binding with SR-BI. In terms of the immunogenicity, HVR1-deleting E2 protein induced higher cross-neutralizing antibodies protecting Huh7.5 cells from HCVpp and HCVcc infection in vitro. These results indicated protective humoral immune response could be induced by HVR1-deleting E2 protein, which provide a promising approach for the development of prophylactic vaccines against HCV infection.
HCV包膜蛋白E2位于HCV蛋白前体第384-746位氨基酸残基,由一个大的N端膜外区和一个C端疏水锚定区组成,与HCV另一包膜蛋白E1通过非共价键形成异源性二聚体。E2蛋白是介导病毒吸附和进入宿主细胞的关键蛋白,是诱导宿主产生HCV中和抗体的关键抗原,因此是疫苗研究的最主要靶标。然而,包膜蛋白高度变异,尤其E2蛋白氨基末端的含有27个氨基酸残基的高变区1(hypervariable region 1,HVR1)是HCV蛋白中变异频率最高的区域,也是公认的HCV中和抗体表位所在区域。HVR1是介导HCV与受体SR-BI结合的关键区域,HVR1与SR-BI的作用通过几种不同的机制促进HCV细胞侵入。HVR1抗体和SR-BI抗体均能阻断HCV感染。HVR1的高度变异曾被认为是遏制丙肝疫苗发展的瓶颈问题,近几年来基于HCV假病毒(HCVpp)以及细胞培养产生的HCV(HCVcc)的中和试验发现,HVR1并不是唯一的中和抗体表位所在区域,在HCV包膜E2蛋白中还存在一系列构象依赖的中和表位和线性中和表位,其中一些表位在不同基因型、亚型间高度保守。
本实验室前期分别用分泌表达E2的表达质粒以及删除了HVR1的E2表达质粒免疫小鼠,然后检测、分析小鼠血清中的抗体以及病毒中和活性,结果显示:E2质粒免疫的小鼠血清中,HVR1抗体在总E2抗体中占很大比重,针对HVR1以外区域的抗体在中E2抗体中只占较小比重;删除HVR1可显著增强E2质粒免疫诱导的针对HVR1以外区域的抗体,小鼠免疫血清与其他基因型E2蛋白的交叉反应以及对其他基因型HCVpp的交叉中和活性也随之显著增强。这和急性HCV感染者血清中可检测到HVR1抗体而难以检测到E2抗体相似。结果提示:HVR1有可能抑制E2蛋白中保守表位的免疫原性,从而从另一个方面导致HCV免疫逃避。目前基于HCV包膜蛋白为主要靶抗原的疫苗均含有HVR1,这类疫苗免疫黑猩猩只能抵抗同株病毒攻击,但不能抵抗异株的攻击。HVR1的存在可能是这类疫苗免疫保护效果不理想的重要原因,删除HVR1也许能增强疫苗免疫对异株病毒攻击的保护效果。
核酸疫苗在大动物的免疫效果远远不及在小动物的免疫效果,目前还没有核酸疫苗用于人类。为了进一步探讨用删除了HVR1的包膜E2蛋白作为HCV疫苗的发展前景,本研究用CHO细胞表达了E2以及删除了HVR1的E2蛋白,用亲和层析纯化出了两种E2蛋白,分析两种E2蛋白的构象、受体结合功能和抗原性,通过小动物免疫分析HVR1对E2蛋白免疫原性的影响,与我们用核酸免疫获得的结果进行对比和验证,探讨缺失HVR1的E2蛋白在丙肝疫苗中的应用前景。
一、稳定表达HCV包膜E2-人IgG Fc融合蛋白的CHO细胞株的建立
我们首先分别拼接HCV包膜蛋白E2-Fc和E2Δ-Fc融合基因(E2Δ指缺失HVR1),使E2与人IgG Fc段作为融合蛋白表达,方便目的蛋白的亲和层析纯化。将融合基因插入具有自主知识产权的CHO细胞表达质粒载体pCIDA-GS-neo(专利号:ZL 200610024202.5,发明名称:一种用哺乳动物细胞高效分泌表达丙型肝炎病毒包膜蛋白E2的方法),然后分别将构建的表达质粒转染HEK 293T细胞,用ELISA及Western blotting方法检测培养上清中E2-Fc以及E2Δ-Fc融合蛋白,证实两者均可有效表达且表达水平相当。尔后,我们分别将这两种E2-Fc融合蛋白表达质粒及EGFP表达质粒(GS-EGFP)分别转染CHO细胞,以蛋氨酸亚氨基代砜(methionine sulphoximine,MSX)在不含谷氨酰胺的培养基内筛选表达目的蛋白的细胞克隆,用有限稀释法对粗筛出的细胞克隆进行进一步筛选,从而建立和获得了稳定分泌表达E2-Fc和E2Δ-Fc融合蛋白的细胞株。紧接着,我们对筛选出的贴壁CHO细胞株进行了无血清无蛋白培养的驯化,通过逐步下降培养基内血清的浓度,使细胞由贴壁状态转为悬浮生长,随后对悬浮细胞株的培养体系进行了放大,从75T方瓶培养经由250 ml三角烧瓶的小量放大,最后实现在Wave Bioreactor EH2/10细胞培养系统中2L细胞培养袋的灌流培养。细胞密度由最初的2×105cells/ml,达到最后的2×107cells/ml,细胞活率始终保持在80%以上,目的蛋白的分泌量也在灌流培养开始后稳定在3-4 mg/L左右。
二、E2-Fc、E2Δ-Fc融合蛋白纯化及功能鉴定
以上述的细胞培养上清为原料,离心去除细胞,继以GE公司超滤系统(QuixStand? Benchtop System)对上清液进行浓缩和超滤,将其浓缩约10倍,并去除其中分子量低于30 KD的蛋白质。随后,以ProteinA亲和层析柱HiTrap? Protein A HP Columns从超滤液中纯化融合蛋白,通过对柱压、洗脱条件、收集方式等参数的优化,从每升培养上清中可纯化出3-4 mg目的蛋白。然后用截留分子量为30 KD的离心型蛋白超滤柱以低温低速离心的方式将纯化出蛋白样品的缓冲液置换为pH为7.0的磷酸盐缓冲液。用构象依赖性单抗对最终纯化产物进行的Western blotting分析表明E2蛋白保持了天然空间构象。以糖苷酶EndoH、PNGase F分别对两种蛋白进行去糖基化处理,然后观察其分子量的变化,结果提示这两种蛋白的糖基化程度和形式相似。ELISA及Pull down实验显示E2-Fc,E2Δ-Fc均能与人CD81大胞外环(hCD81-LEL)结合,E2Δ-Fc结合活性较E2-Fc为高。检测两种蛋白与细胞膜表面表达的人CD81和SR-BI分子的结合活性,结果显示缺失HVR1后,E2蛋白与SR-BI的结合能力明显降低,与CD81的结合略有增强。流式细胞术检测发现两种蛋白均能与HCV易感的Huh7.5细胞结合且效率相当。将与E2蛋白结合后的Huh7.5细胞裂解,通过免疫共沉淀的方法检测介导E2蛋白与细胞结合的分子,结果显示,E2-Fc能将CD81及SR-BI共沉淀下来,而HVR1缺失的E2Δ-Fc共沉淀较多的CD81及少量SR-BI,提示HVR1是介导E2蛋白与靶细胞表面SR-BI结合的重要区域。两种蛋白均能抑制HCVpp和HCVcc的感染性,抑制率最高可达90%以上,且株特异性不强。
三、删除HVR1增强E2-Fc融合蛋白的免疫原性
分别将上述两种融合蛋白联合弗氏佐剂免疫小鼠,于0、3和8周每只小鼠皮下注射10μg佐剂乳化的融合蛋白,初免第2、7和10周采血检测小鼠血清中的E2ΔHVR1抗体及HVR1抗体,发现E2-Fc免疫组10只小鼠中,有5只E2ΔHVR1及HVR1抗体均为阳性,4只E2ΔHVR1抗体单阳性,1只两种抗体一直是阴性。E2ΔHVR1及HVR1抗体滴度随免疫次数增多逐步升高,E2Δ-Fc蛋白的确能诱导更高的E2ΔHVR1抗体水平,其滴度约为E2-Fc免疫组的5-10倍。证实HVR1的存在抑制了E2ΔHVR1抗体的产生。尔后,为进一步检测小鼠血清抗体中和活性,我们将各组小鼠血清混合后纯化出IgG,行病毒感染中和实验,结果显示,两组IgG对于与蛋白来源同株的H77株HCVpp感染中和效率没有显著性差异,中和率在80%以上。但对于异株HCV,E2-Fc组中和活性显著低于E2Δ-Fc组。含有HVR1的E2-Fc融合蛋白诱导的抗体中,HVR1抗体阳性则其交叉中和活性明显低于HVR1抗体阴性者,也说明HVR1抑制交叉中和抗体的产生。这些结果与我们DNA免疫的研究一致,高度提示缺失HVR1可有力促进HCV包膜E2蛋白诱导交叉中和抗体。此外,考虑到Fc段本身有特殊的免疫学功能,可能有助于免疫应答,尤其是细胞免疫应答的诱导。本研究中,采用融合蛋白免疫小鼠,该融合蛋白是否能在动物体内诱生针对E2蛋白特异且高效的细胞免疫反应,是我们需要进一步验证的内容。
小结
本研究着眼于丙型肝炎病毒蛋白疫苗的开发思路,在前期DNA免疫的基础上,构建了稳定表达E2-Fc/E2Δ-Fc的无蛋白无血清悬浮CHO细胞培养体系并成功纯化出相应的目的蛋白。功能试验结果显示,HVR1缺失并未明显影响E2蛋白的天然构象,HVR1删除后,E2蛋白与其受体CD81的结合增强,与SR-BI的结合能力减弱。动物免疫结果显示,HVR1的缺失显著增强了E2蛋白诱导的交叉中和抗体。综上所述,该研究首次从蛋白层面揭示了HVR1对E2蛋白结构、功能及免疫原性的影响,为HCV蛋白疫苗的开发提供了新思路。
Hepatitis C virus (HCV), the only member of the hepacivirus genus, family Flaviviridae, is a major cause of posttransfusional non-A and non-B hepatitis. HCV infects more than 170 million people worldwide, among which 40 million are in China. About 85% patients fail to clear HCV and contract persistent infection which frequently leads to chronic liver disease, including cirrhosis and hepatocellular carcinoma. The current therapy for HCV infection is peg-IFNαin combination with ribavirin, but not all genotypies of HCV infected patients could receive the sustained viral response. Therefore, what needs dealing with urgently is developing vaccines to protect healthy persons as well as clear HCV from infected patients. Unfortunately, there is no effective vaccine available until now.
HCV E2 envelope protein extends from aa 384-746 of the polyprotein, containing a N-terminal ectodomain and a C-terminal hydrophobic anchor domain. E1 and E2, is thought to form a heterodimer stabilized by non-covalent interactions as envelope of the HCV particles. As an envelope protein, E2 is thought to play a major role in virus attachment to the target cell by binding to receptors on the cell plasma membrane followed by membrane fusion and entry. Meanwhile, as E2 may elicit production of neutralizing antibodies against the virus, it was considered a major candidate for anti-HCV vaccine development. However, the most variable region of HCV (HVR-1) is also located within the N-terminal 27 residues (aa 384-411) of E2, it is also widely recognized as an epitope to which the neutralizing antibody binds. The high variation of HVR1 was considered to be a bottleneck in HCV vaccine development. However, in recent years, virus neutralizing tests based on HCV pseudoparticles (HCVpp) and HCV cell culture model (HCVcc) found that HVR1 is not the only neutralizing epitope and parts of E2 other than HVR1 might be involved in the induction of virus neutralizing antibodies.
In our previous research, we constructed some eukaryotic plasmids to express the carboxyl-terminal truncated E2 protein with or without HVR1. Then these plasmids were used to immune mice and the relationship between HVR1 antibody and the total E2 antibody on amount and neutralizing activities were analyzed. What we found was 1. Secretionary expression of E2 is more effective to induce antibody response. 2. In addition to HVR1, there exist exactly other neutralizing epitopes in E2 protein. 3. anti-HVR1 is the main part of the neutralizing antibodies. 4. HVR1 attenuates the cross-reaction of antibody induced by E2. These results implies us whether we can enhance the immunogenicity of E2 by deleting HVR1 which might cover other conserved neutralizing epitopes existing in other regions of E2? If this alteration of E2 could induce effective cross-neutralizing humoral immune response, it could provide us a new strategy on developing HCV vaccine. However, DNA immunization might be interfered with confounding factors induced by other elements exist on the expression vector, which could mislead us. Protein vaccine using purified proteins as immunogens, can avoid these problems. Therefore, from this perspective, in this research, we constructed soluble E2 protein and soluble HVR1-deleting E2 expression plasmids, transfected into CHO cells and establish the E2 proteins’expression system. Based on affinity chromatography, E2 proteins were separated and purified from cell cultural supernatant, their conformation, function and immunogenicity was investigated and compared with each other. Our study was aimed to confirm the conclusion obtained from DNA immunization by E2 protein immunization and further explore the potential use of HVR1-deleting E2 protein in protective HCV vaccine.
1. Construction of HCV E2-human IgG Fc fusion protein eukaryotic expression system
In this part of our research, first, we constructed HCV E2-Fc/E2Δ-Fc fusion protein expression plasmids. This kind of fusion strategy was convenient for affinity chromatography of proteins. Next, we inserted glutamine synthetase gene into the downstream of target gene, by adding glutamine synthetase inhibitor (MSX) into the cell culture medium, recombinant CHO cell lines with high expression levels of fusion proteins could be selected. On the other hand, glutamine can be produced by cell metabolism of amines glutamine, exhibiting better growth and proliferation in the medium without glutamine; this might avoid cell injury induced by accumulation of ammonia, and greatly facilitate the follow-up of cell culture and protein purification. Then, these plasmids were transfected into HEK 293T cells to detect the expression levels of E2-Fc/E2Δ-Fc proteins in the supernatant based on ELISA and Western blotting. Results showed that these two proteins were expressed well and had got similar levels. After that, these two plasmids as well as another negative control plasmid- GS-EGFP were transfected into CHO cells respectively. Recombinant CHO cell clones were screened out under the pressure of MSX, these clones were then picked out and undertaken limited dilution to select clones with higher expression levels of target proteins. In order to improve space and medium utilization as well as reduce the mixed composition in FBS containing cell culture supernatant, serum-free protein-free medium adaptation were performed on these adherent cell lines, FBS concentration of cultural medium was decreased step by step, cells were transferred to suspension state instead. After adaptation, the suspension cell culture system was further enlarged, through 75T to 250ml flask with the small amount of amplification, and finally achieves the perfusion culture in 2L cellbags on Wave Bioreactor EH2/10 cell culture system. Cell density increased from 2×105cells/ml to the final 2×107cells/ml, cell viability was maintained above 80% and the secretion of protein was also stable at 3-4mg/L after perfusion. This part of our study has provided sufficient material for the following protein function analysis. Of course, the way to improve serum protein-free adaption efficiency and optimize the process of cell culture system enlargement still needs further exploration.
2. Purification and identification of E2-Fc fusion protein
As an intermediate steps between protein expression and function analysis, the separation and purification of target proteins play an important role. In this part of the experiment, we used the cultural supernatant collected from HCV E2-Fc fusion protein secreting suspension cell as raw materials, through the clarification step to remove most of the impurities, followed by concentration step based on ultrafiltration system, QuixStand? Benchtop System and hollow fiber ultrafiltration column, to reach a 10 times concentration and remove the molecules less than 30KD. Then, the ultrafiltrate was further purified with protein A affinity column HiTrap? Protein A HP Columns and purification system-?KTApurifier System (GE). During the purification process, through optimizing the column pressure, elution conditions collection, about 3-4mg of pure protein could be obtained from per liter culture supernatant, comparable with the reported E2-Fc fusion protein purification efficiency (2mg/L). Then, we exchanged the buffer of crude protein sample to PBS with ultrafiltration centrifuge column by way of low-temperature low-speed centrifugation, and further enrichment of the samples (3-5 times). Finally, we detected the concentration of protein samples by BCA protein quantitative method, and performed native-PAGE and Western blotting to confirm the conformation of protein samples maintain correct after ultrafiltration, purification, buffer exchange and other which can be used the following function analysis and animal experiments. At the same time, two crude purified proteins were undertaken glycosylation by glycosidase EndoH, PNGase F treatment, results suggest that glycosylation of both crude purified protein remained similar with their natural state. ELISA binding assay and pull down assays showed that E2-Fc, E2Δ-Fc could bind with human CD81 LEL, E2Δ-Fc exhibited higher binding activity than E2-Fc. Then, through flow cytometry, we investigated that both proteins can bind to HCV susceptible cell Huh7.5 cells well and had no much difference with each other. Further, we examined the binding activity between crude protein and CHO cells with expression of human CD81, SR-BI molecules respectively. Results showed that HVR1 deletion, binding efficiency between E2 protein and CHO-SR-BI was greatly decreased but slightly enhanced when binding with CHO-CD81. Next, we performed immunoprecipitation with lysis of Huh7.5 cells after E2 protein binding process to detect molecules that mediated E2 protein and cell binding. As a result, E2-Fc were able to precipitate CD81 and SR-BI, but E2Δ-Fc could co-precipitate little SR-BI and more CD81 when comparing with E2-Fc. This was consistent with report that HVR1 is important in mediating E2 and cell binding by affinity with SR-BI on target cells. Through infection assays of HCVcc and HCVpp, we observed concentration-dependent efficiency of crude pure fusion protein in inhibition of HCVcc and HCVpp infection. About 25-30μg of protein could be sufficient to completely suppress the infection of HCV infectious particles exhibiting no much strain specificity.
3. Characteraztion of immunogenecity enhacement by HCV HVR1 deletion in purified E2-Fc fusion protein
This section, we immunized BALB/c mice with crude purified proteins. Through the strategy of immunization for 3 times at week 0, 3 and 8 with a dose of 10μg once, serum was collected at week 2, 7 and 10 for anti-HVR1 and anti-E2ΔHVR1 antibody detection. In E2-Fc immunizing group, 5 in 10 were anti-HVR1 and anti-E2ΔHVR1 positive, another 4 were anti-E2ΔHVR1 positive only. It was supposed that Fc segment fused with the E2 protein affected the HVR1 epitopes exposure. Similar with results of DNA immunization, E2Δ-Fc protein induced higher anti-E2Δantibody levels. With the same quantity of total IgG, anti-HVR1 positive serum exhibted weaker neutralizing activitiy against the infection of J6/JFH1 HCVcc and Con1 HCVpp, but no much different from anti-HVR1 negative serum in inhibiting the infection of H77 HCVpp. These results confirmed the conclusions that anti-HVR1 had got strong neutralizing activity against the attack of HCV comes from the same strain but weak cross-neutralizing activity against the virus of other strains or genotypes.
In summary, we have established a eukaryotic cell culture system to produce secretory HCV E2-Fc and E2Δ-Fc fusion protein and purified the target proteins. Based on E2-Fc and E2Δ-Fc, we investigated the roles of HVR1 in structure, function and immunogenicity of E2 protein. No obvious conformational changes were found in HVR1-deleting E2 protein. Although maintaining major function of E2 protein, HVR1-deleting E2 does decrease affinity with SR-BI, suggesting HVR1 be important in E2 binding with SR-BI. In terms of the immunogenicity, HVR1-deleting E2 protein induced higher cross-neutralizing antibodies protecting Huh7.5 cells from HCVpp and HCVcc infection in vitro. These results indicated protective humoral immune response could be induced by HVR1-deleting E2 protein, which provide a promising approach for the development of prophylactic vaccines against HCV infection.
引文
[1] Goldfield M. Some epidemiologic studies of transfusion-associated hepatitis. Infusionstherapie.1974; 1(8):645-649.
[2] Alter, H. J, and L. B. Seeff. Recovery, persistence, and sequelae in hepatitis C virus infection: a perspective on long-term outcome. Semin. Liver Dis 2000, 20:17-35.
[3] Lau GKK, Zhuang H. Top hepatology issue in China. J Gastroent Hepatol. 2003; 17(Suppl):A9.
[4] Takamizawa A, Mori C, Fuke I, et al. Structure and organization of the hepatitis C virus genome isolated from human carriers. J Virol,1991; 65(3):1105-1113.
[5] Kato N. Genome of human hepatitis C virus (HCV): gene organization, sequence diversity, and variation.Microb Comp Genomics. 2000, 5:129-51.
[6] Reed KE, Rice CM. Overview of hepatitis C virus genome structure, polyprotein processing, and protein properties.Curr Top Microbiol Immunol. 2000, 242:55-84.
[7] Penin F, Dubuisson J, Rey FA, Moradpour D, Pawlotsky JM. Structural biology of hepatitis C virus. Hepatology. 2004, 39: 5-19.
[8] Goffard A, Dubuisson J. Glycosylation of hepatitis C virus envelope proteins. Biochimie.. 2003;85:295-301
[9] Voisset C, Dubuisson J. Functional hepatitis C virus envelope glycoproteins.Biol Cell. 2004; 96: 413-20.
[10] Petit MA, Lievre M, Peyrol S, et al. Enveloped particles in the serum of chronic hepatitis C patients.Virology.2005, 336:144 -53.
[11] Pileri P, Ueatsu Y, Campagnoli S, et al. Binding of hepatitis C virus to CD81. Science, 1998, 282:938-41.
[12] Scarse lli E, Ansuini H, Cerino R, et a1.The human scavenger receptor class B type I is a novel candidate receptor for the hepatitis C virus. EMB0 J, 2002, 21: 5017-5025.
[13] Bartoschi B,Vitelli A, Granier C, et al. Cell entry of hepatitis C virus requires a set of co-receptors that include the CD81 tetraspanin and the SR-BI scavenger receptor. J Biol Chem. 2003; 278: 41624-30.
[14] Lozach PY, Amara A, Bartosch B, et al. C-type lectins L-SIGN and DC-SIGN capture and transmit infectious hepatitis C virus pseudotype particles. J Bio Chem, 2004, 279: 32035-45.
[15] Lee SH, Kim YK, Kim CS, et al. E2 of hepatitis C virus inhibits apoptosis. J Immunol. 2005; 175:8226-35.
[16] Von Hahn, T.& Rice, C .M. Hepatitis C virus entry. J. Biol. Chem. 2008; 283,3689-3693.
[17] Alexander Ploss, Matthew J, et al. Human occluding is a hepatitis C virus entry factor required for infection of mouse cells. Nature. 2009; 457: 882-886.
[18] Oren R, Takahashi S, Doss C, et al. TAPA-1, the target of an antiproliferative antibody, defines a new family of transmembrane proteins. Mol Cell Biol.1990; 10: 4007-15.
[19] Wright MD, Tomlinson MG. The ins and outs of the transmembrane 4 superfamily. Immunol Today. 1994; 15: 588-94.
[20] Cocquerel L, Meunier JC, Pillez A, et al. A retention signal necessary and sufficient for endoplasmic reticulum localization maps to transmembrane domain of hepatitis C virus glycoprotein E2. J Virol. 1998; 72: 2183-91.
[21] Kein F, Abraham JD, Schuster C, et al.Analysis of the subcellular localization of hepatitis C virus E2 glycoprotein in live cells using EGFP fusion proteins. J Gen Virol. 2003; 84: 561-6.
[22] Flint M, Dubuisson J, Maidens C, et al. Functional characterization of intracellular and secreted forms of a truncated hepatitis C virus E2 glycoprotein. J Virol. 2000; 74(: 702-9.
[23] Flint M, Dubuisson J, Maidens C, et al. Functional characterization of intracellular and secreted forms of a truncated hepatitis C virus E2 glycoprotein. J Virol. 2000, 74: 702-709.
[24] Koshy R, et al. Evaluation of hepatitis C virus protein epitopes for vaccine development.Trends Biotechnol,1996, 14(10):364-369.
[25] Tarr AW, Owsianka AM, Timms JM, et al. Characterization of the hepatitis C virus E2 epitope defined by the broadly neutralizing monoclonal antibody AP33. Hepatology, 2006, 43(3): 592-601.
[26] Meunier JC, Engle RE, Faulk K, et al. Evidence for cross-genotype neutralization of hepatitis C virus pseudo-particles and enhancement of infectivity by apolipoprotein C1. Proc Natl Acad Sci U S A. 2005;102:4560-4565.
[27] Esumi M, Rikihisa T, Nishimura S, et al. Experimental vaccine activities of recombinant E1 and E2 glycoproteins and hypervariable region 1 peptides of hepatitis C virus in chimpanzees. Arch Virol, 1999; 144: 973-80.
[28] Fenin Francois, Christophe Combet, Georgios Germanidis, et al. Conservation of the conformation and positive charges of hepatitis C virus E2 envelope glycoprotein hypervariable region 1 points to a role in cell attachment. J Virol, 2001; 75: 5703-10.
[29] Joachim Lupberger, Mirjam B. Zeisel, Fei Xiao, Christine Thumann, Isabel Fofana, Laetitia Zona, et al. EGFR and EphA2 are host factors for hepatitis C virus entry and possible targets for antiviral therapy. Nature Medicine. 2011.17, 589-595.
[30] Bartosch B, Dubuisson J, Cosset FL. Infectious hepatitis C virus pseudo-particles containing functional E1-E2 envelope protein complexes. J Exp Med. 2003;197:633-642.
[31] Lindenbach BD, Evans MJ, Syder AJ, et al. Complete replication of hepatitis C virus in cell culture. Science. 2005;309:623-626.
[32] Wakita T, Pietschmann T, Kato T, et al. Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nat Med.2005;11:791-796.
[33] Zhong J, Gastaminza P, Cheng G, et al. Robust hepatitis C virus infection in vitro. Proc Natl Acad Sci U S A.2005;102:9294-9299.
[34] Lambot M, Fretier S, Op De Beeck A, et al. Reconstitution of hepatitis C virus envelope glycoproteins into liposomes as a surrogate model to study virus attachment. J Biol Chem, 2002, 277: 20625-20630.
[35] Simonsen CC, McGrogan M. The molecular biology of production cell lines. Biologicals. 1994 Jun; 22(2):85-94. Review.
[36] Omasa T, Onitsuka M, Kim WD. Cell engineering and cultivation of Chinese hamster ovary (CHO) cells. Curr Pharm Biotechnol. 2010; 11(3):233-40. Review.
[37] Butler M. Optimization of the cellular metabolism of glycosylation for recombinant proteins produced by Mammalian cell systems. Cytotechnology. 2006; 50(1-3):57-76.
[38] Eisenberg D, Gill H S, Pfluegl GMU, et al. Structure function relationships of glutamine synthetases. Biochim Biophy Acta,2000,1477:122.
[39] Lee GM, Kim EJ, Kim NS, et al. Development of a serum-free medium for the production of erythropoietin by suspension culture of recombinant CHO cells using a statistical design. J Biotechnol,1999,69:85.
[40] Keen MJ, Rapson NT. Development of a serum-free culture media for t he large scale production of recombinant protein f rom a Chinese hamster ovary cell line. Cyrotechnology. 1995, l7:153-163.
[41] Glassy MC, Tharakan JP, Chao PC. Serum-free media inhybridoma culture and monoclonal antibody production. Biochemical and Biotechnology. 1988,32:1015-1028.
[42] Birch JR, Arathoon R. Suspension culture of mammalian cells. Bioprocess Technol. 1990;10:251-70. Review.
[43] Omasa T, Onitsuka M, Kim WD. Cell engineering and cultivation of Chinese hamster ovary (CHO) cells. Curr Pharm Biotechnol. 2010;11(3):233-40. Review.
[44] Singh V. Disposable bioreactor for cell culture using wave-induced agitation. Cytotechnology. 1999 Jul;30(1-3):149-58.
[45] Yang JD et al. Achievement of high cell density and high antibody productivity by a controlled-fed perfusion bioreactor process. Biotechnol Bioeng 2000;69:74-82.
[46] Mirabet M, Navarro A , Lopea A et al . Ammonium toxicity in different cell lines. Biotechnol Bioeng, 1997, 56(5):530-537.
[47] Dempsey J, Ruddock S, Osborne M et al. Improved fermentation processes for NS0 cell lines expressing human antibodies and glutamine synthetase. Biotechnol Prog, 2003, 19:175-178.
[1] Ordo?o D, Enjuanes L, Casasnovas JM. Methods for preparation of low abundance glycoproteins from mammalian cell supernatants. Int J Biol Macromol. 2006 Aug15; 39(1-3):151-6.
[2] K?lln J, Braren I, Bredehorst R, Spillner E. Purification of native and recombinant cobra venom factor using thiophilic adsorption chromatography. Protein Pept Lett. 2007; 14(5):475-80.
[3] D.R.Marshak, J.T.Kadonaga, R.R.Burgess, M.W.Knuth. Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Cold Spring Harbor Laboratory Press, 1996.
[4]应国清,王玉姣,易喻,等。免疫亲和层析技术及其医药应用进展。药物生物技术,2005,12(5):342-34。
[5] Takeshi I, Ken-ichi N, Ryuichi H, Akio K. Single-step af?nity puri?cation of recombinant proteins using the silica-binding Si-tag as a fusion partner. Protein Expression and Puri?cation.2010: 71, 91-95.
[6] Iwane M, Kitamura Y, Kaisho Y, Yoshimura K, Shintani A, Sasada R, Nakagawa S, Kawahara K, Nakahama K, Kakinuma A. Production, purification and characterization of biologically active recombinant human nerve growth factor. Biochem Biophys Res Commun. 1990, 31;171(1):116-22.
[7] Karlsson K, Nyberg F. Purification of substance P endopeptidase activity in the rat ventral tegemental area with the Akta-Purifier chromatographic system. J Chromatogr A. 2000. 29; 893(1):107-13.
[8] Whidby J, Mateu G, Scarborough H, Demeler B, Grakoui A, Marcotrigiano J.Blocking hepatitis C virus infection with recombinant form of envelope protein 2 ectodomain. J Virol.2009; 83(21):11078-89.
[9] Bankwitz D, Steinmann E, Bitzegeio J, Ciesek S, Friesland M, Herrmann E, Zeisel MB, Baumert TF, Keck ZY, Foung SK, Pécheur EI, Pietschmann T. Hepatitis C virus hypervariable region 1 modulates receptor interactions, conceals the CD81 binding site, and protects conserved neutralizing epitopes. J Virol. 2010; 84(11):5751-63.
[10] McCaffrey K, Gouklani H, Boo I, Poumbourios P, Drummer HE. The variable regions of hepatitis C virus glycoprotein E2 have an essential structural role in glycoprotein assembly and virion infectivity. J Gen Virol. 2011; 92:112-21.
[11] Callens N, Ciczora Y, Bartosch B, Vu-Dac N, Cosset FL, Pawlotsky JM, Penin F, Dubuisson J. Basic residues in hypervariable region 1 of hepatitis C virus envelope glycoprotein e2 contribute to virus entry. J Virol. 2005; 79(24):15331-41.
[12] Scarselli E, Ansuini H, Cerino R, Roccasecca RM, Acali S, Filocamo G, Traboni C, Nicosia A, Cortese R, Vitelli A. The human scavenger receptor class B type I is a novel candidate receptor for the hepatitis C virus. EMBO J. 2002; 21(19):5017-25.
[13] Dao Thi VL, Dreux M, Cosset FL. Scavenger receptor class B type I and the hypervariable region-1 of hepatitis C virus in cell entry and neutralisation. Expert Rev Mol Med. 2011,14 ;13:e13.
[14] Q. Yang, F. Faucher, M. Coseno, J. Heckman and S. Doublié. Purifcation, crystallization and preliminary X-ray diffraction of a disulfide cross-linked complex between bovine poly (A) polymerase and a chemically modified 15-mer oligo(A) RNA. Acta Cryst. 2011, F67, 241-244.
[15] Eric J, Enemark K, Grace Chen, et a1. Crystal structure of tDNA binding domain of the replication initiation protein E1 fro papillomavirus. Molecular Cell. 2000 (6): l, 149-158.
[1] Bukh J, Miller RH, Purcell RH. Genetic heterogeneity of hepatitis C virus: quasispecies and genotypes. Seminars in Liver Disease. 1995;15(1):41-63.
[2] Farci P, Shimoda A, Coiana A, et al. The outcome of acute hepatitis C predicted by the evolution of the viral quasispecies. Science. 2000;288(5464):339-344.
[3] Kato, N., Y. Ootsuyama, H. Sekiya, S. Ohkoshi, T. Nakazawa, M. Hijikata, and K. Shimotohno. Genetic drift in hypervariable region 1 of the viral genome in persistent hepatitis C virus infection. J. Virol. 1994.68:4776-4784.
[4] Bartosch B, Bukh J, Meunier JC, et al. In vitro assay for neutralizing antibody to hepatitis C virus: evidence for broadly conserved neutralization epitopes. Proceedings of the National Academy of Sciences of the United States of America. 2003;100(24):14199-14204.
[5] Farci P, Shimoda A, Wong D, et al. Prevention of hepatitis C virus infection in chimpanzees by hyperimmune serum against the hypervariable region 1 of the envelope 2 protein. PNAS USA. 1996;93(26):15394-15399.
[6] Farci P, Alter HJ, Wong DC, et al. Prevention of hepatitis C virus infection in chimpanzees after antibody-mediated in vitro neutralization. PNAS USA, 1994; 91: 7792-6.
[7] Major ME, Mihalik K, Puig M, et al. Previously infected and recovered chimpanzees exhibit rapid responses that control hepatitis C virus replication upon rechallenge. J Virol, 2002; 76(13): 6586-95.
[8] Keck, Z. Y., T. K. Li, J. Xia, M. Gal-Tanamy, O. Olson, S. H. Li, A. H. Patel, J. K. Ball, S. M. Lemon, and S. K. Foung. De?nition of a conserved immunodominant domain on hepatitis C virus E2 glycoprotein by neutralizing human monoclonal antibodies. J. Virol. 2008. 82:6061-6066.
[9] Owsianka, A. M., Timms, J. M., Tarr, A. W., Brown, R. J. P., Hickling, T. P., Szwejk, A., Bienkowska-Szewczyk, K., Thomson, B. J., Patel, A. H., Ball, J. K.. Identification of ConservedResidues in the E2 Envelope Glycoprotein of the Hepatitis C Virus That Are Critical for CD81 Binding. J. Virol. 2006.80: 8695-8704.
[10] Drummer HE, Boo I, Maerz AL, Poumbourios PA. Conserved Gly436-Trp-Leu-Ala-Gly-Leu-Phe-Tyr Motif in Hepatitis C Virus Glycoprotein E2 Is a Determinant of CD81 Binding and Viral Entry. J. Virol. 2006,80: 7844-7853
[11] Dubuisson J, Helle F, Cocquerel L. Early steps of the hepatitis C virus life cycle. Cell Microbiol. 2008 Apr;10(4):821-7. 2007,13. Review.
[12] Helle F, Dubuisson J. Hepatitis C virus entry into host cells. Cell Mol Life Sci. 2008 Jan;65(1):100-12. Review.
[13] Diedrich G. How does hepatitis C virus enter cells? FEBS J. 2006;273(17):3871-85. Review.
[14] Taniguchi, S., H. Okamoto, M. Sakamoto, M. Kojima, F. Tsuda, T. Tanaka, E. Munekata, E. E. Muchmore, D. A. Peterson, and S. Mishiro. A structurally ?exible and antigenically variable N-terminal domain of the hepatitis C virus E2/NS1 protein: implication for an escape from antibody. Virology. 1993.195:297-301.
[15] Mondelli M U, Cerino A, Meola A and Nicosia A 2003 Variability or conservation of hepatitis C virus hypervariable region 1? Implications for immune responses. J. Biosci. 28 305-310.
[16] Keck, Z. Y., J. Xia, Z. Cai, T. K. Li, A.M. Owsianka, A. H. Patel, G. Luo, and S. K. Foung. 2007. Immunogenic and functional organization of hepatitis C virus (HCV) glycoprotein E2 on infectious HCV virions. J. Virol. 81:1043-1047.
[17] Stamataki, Z., Coates, S., Evans, M.J., Wininger, M., Crawford, K., Dong, C., Fong, Y.L., Chien, D., Abrignani, S., Balfe, P., Rice, C.M., McKeating, J.A., Houghton, M.,. Hepatitis C virus envelope glycoprotein immunization of rodents elicits cross-reactive neutralizing antibodies. Vaccine. 2007,25, 7773-7784.
[1] Choo QL, Kuo G, Weiner AJ, Overby LR, Bradley DW, Houghton M. Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science 1989; 244: 359-362.
[2] Wyatt CA, Andrews L, Brotman B, Huang F, Lee D, Prince A. Immunity in chimpanzees chronically infected with hepatitis C virus:role of minor quasispecies in reinfection. J Virol 1998; 72: 1725-1730.
[3] Angnello V, Abel G, Knight GB, Muchmore E. Detection of wildspread hepatocyte infection in chronic hepatitis C. Hepatology 1998; 28: 573-584.
[4] Tanaka JK, K; Kumagai, J; Komiya, Y; Yugi, H; Kishimoto, S; Mizui, M; Tomoguri, T; Miyakawa, Y; Yoshizawa, H. Early dynamics of hepatitis C virus in the circulation of chimpanzees with experimental infection. Intervirology 2005; 48: 120-123.
[5] Shoukry NH, Sidney J, Sette A, Walker CM. Conserved hierarchy of helper T cell responses in a chimpanzee during primary and secondary hepatitis C virus infections. J Immunol 2004; 172: 483-492.
[6] Folgori A, Capone S, Ruggeri L, Meola A, Sporeno E, Ercole BB, Pezzanera M, Tafi R, Arcuri M, Fattori E, Lahm A, Luzzago A, Vitelli A, Colloca S, Cortese R, Nicosia A. A T-cell HCV vaccine eliciting effective immunity against heterologous virus challenge in chimpanzees. . Nat Med 2006; 12: 190-197.
[7] Major ME, Dahari H, Mihalik K, Puig M, Rice CM, Neumann AU, Feinstone SM. Hepatitis C virus kinetics and host responses associated with disease and outcome of infection in chimpanzees. Hepatology 2004; 39: 1709-1720.
[8] Martin A, Bodola F, Sangar DV, Goettge K, Popov V, Rijnbrand R, Lanford RE, Lemon SM. Chronic hepatitis associated with GB virus B persistence in a tamarin after intrahepatic inoculation of synthetic viral RNA. Proc Natl Acad Sci U S A 2003; 100: 9962-9967.
[9] Nam JH, Faulk K, Engle RE, Govindarajan S, St Claire M, Bukh J. In vivo analysis of the 3' untranslated region of GB virus B after in vitro mutagenesis of an infectious cDNA clone: persistent infection in a transfected tamarin. J Virol 2004; 78: 9389-9399.
[10] Garson JA, Whitby K, Watkins P, Morgan AJ. Lack of susceptibility of the cottontop tamarin to hepatitis C infection. J Med Virol 1997; 52: 286-288.
[11] Kock;, J M, Nassal;, S M, ;T F, Baumert;, H E, Blum; , F vW. Efficient infection of primary tupaia hepatocytes with purified human and woolly monkey hepatitis B virus. J Virol 2001; 7(75): 5084-5089.
[12] Xie Z, C; Riezu Boj, J,I; Lasarte, J.J; Guillen, J; Su, J.H; Civeira, M.P; Prieto, J. Transmission of hepatitis C virus infection to tree shrews. Virology 1998; 244: 513-520.
[13] Xu XC, H.; Cao, X.; Ben, K. Efficient infection of tree shrew (Tupaia belangeri) with hepatitis C virus grown in cell culture or from patient plasma. J Gen Virol 2007; 88: 2504-2512.
[14] Amako;Tsukiyama;Katsume;Hirata;Sekiguchi;Tobita;Hayashi;Hishima;Funata;Yonekawa;Kohara. Pathogenesis of hepatitis C virus infection in Tupaia belangeri . J Virol 2010; 84(1): 303-311.
[15] Yimin Tong, Yongzhe Zhu, Xueshan Xia, Yuan Liu, Yue Feng, Xian Hua, Zhihui Chen, Hui Ding, Li Gao, Yongzhi Wang, Mark A. Feitelson, Ping Zhao, and Zhongtian Qi. Tupaia CD81, SR-BI, claudin-1, and occludin support hepatitis C virus infection. J Virol. 2011 Mar;85(6):2793-802.
[16] Koike K, Moriya K, Ishibashi K, Matsuura Y, Suzuki T, Saito I, Iino S, Kurokawa K, Miyamura T. Expression of hepatitis C virus envelope proteins in transgenic mice. J Gen Virol 1995; 76(12): 3031-3038.
[17] Tanaka N, Moriya K, Kiyosawa K, Koike K, Gonzalez FJ, Aoyama T. PPARalpha activation is essential for HCV core protein-induced hepatic steatosis and hepatocellular carcinoma in mice. J Clin Invest 2008; 118: 683-694.
[18] Majumder M, Ghosh AK, Steele R, Zhou XY, Phillips NJ, Ray R, Ray RB. Hepatitis C virus NS5A protein impairs TNF-mediated hepatic apoptosis, but not by an anti-FAS antibody, in transgenic mice. Virology 2002; 294: 94-105.
[19] Wang AG, Lee DS, Moon HB, Kim JM, Cho KH, Choi SH, Ha HL, Han YH, Kim DG, Hwang SB, DY Y. Non-structural 5A protein of hepatitis C virus induces a range of liver pathology in transgenic mice. J Pathol 2009; 219(2): 253-262.
[20] Kanda T, Steele R, Ray R, RB R. Inhibition of intrahepatic gamma interferon production by hepatitis C virus nonstructural protein 5A in transgenic mice. J Virol 2009; 83(17): 8463-8469.
[21] Ilan EA, J.; Nussbaum, O.; Zauberman, A.; Eren, R.; Lubin, I.; Neville, L.; Ben-Moshe, O.; Kischitzky, A.; Litchi, A.; Margalit, I.; Gopher, J.; Mounir, S.; Cai, W.; Daudi, N.; Eid, A.; Jurim, O.; Czerniak, A.; Galun, E.; Dagan, S. The hepatitis C virus (HCV)-Trimera mouse: a model for evaluation of agents against HCV. J Infect Dis 2002; 182: 153-161.
[22] Eren RL, D.; Terkieltaub, D.; Nussbaum, O.; Zauberman, A.; Ben-Porath, J.; Gopher, J.; Buchnick, R.; Kovjazin, R.; Rosenthal-Galili, Z.; Aviel, S.; Ilan, E.; Shoshany, Y.; Neville, L.; Waisman, T.; Ben-Moshe, O.; Kischitsky, A.; Foung, S.K.; Keck, Z.Y.; Pappo, O.; Eid, A.; Jurim, O.; Zamir, G.; Galun, E.; Dagan, S. Preclinical evaluation of two neutralizing human monoclonal antibodies against hepatitis C virus (HCV): a potential treatment to prevent HCV reinfection in liver transplant patients. J Virol 2006; 80: 2654-2664.
[23] Ernst ES, K.; Bugert, J.J.; Blaker, H.; Pfaff, E.; Stremmel, W.; Encke, J. . Generation of inducible hepatitis C virus transgenic mouse lines. J Med Virol 2007; 79: 1103-1112.
[24] Kaul AW, I.; Meuleman, P.; Leroux-Roels, G.; Bartenschlager, R. Cell culture adaptation of hepatitis C virus and in vivo viability of an adapted variant. J Virol 2007; 81: 13168-13179.
[25] Kneteman NM, Weiner AJ, O'Connell J, Collett M, Gao T, Aukerman L, Kovelsky R, Ni ZJ, Zhu Q, Hashash A, Kline J, Hsi B, Schiller D, Douglas D, Tyrrell DL, Mercer DF. Anti-HCV therapies in chimeric scid-Alb/uPA mice parallel outcomes in human clinical application. Hepatology 2006; 43: 1346-1353.
[26] Vanwolleghem T, Meuleman P, Libbrecht L, Roskams T, De Vos R, Leroux-Roels G. Ultra-rapid cardiotoxicity of the hepatitis C virus protease inhibitor BILN 2061 in the urokinase-type plasminogen activator mouse. Gastroenterology 2007; 133: 1144-1155.
[27] Vanwolleghem T, Bukh J, Meuleman P, et al. Polyclonal immunoglobulins from a chronic hepatitis C virus patient protect human liver-chimeric mice from infection with a homologous hepatitis C virus strain. Hepatology, 2008, 47(6): 1846-1855.
[28] Law M, Maruyama T, Lewis J, et al. Broadly neutralizing antibodies protect against hepatitis C virus quasispecies challenge. Nat Med, 2008, 14(1): 25-27.
[29] Legrand N, Ploss A. Humanized mice for modeling human infectious disease: challenges, progress, and outlook. Cell Host Microbe, 2009, 6(1): 5-9.
[30] Strowig T, Gurer C, Ploss A. Priming of protective T cell responses against virus-induced tumors in mice with human immune system components. J Exp Med,2009, 206(6): 1423-1434.
[2] Alter, H. J, and L. B. Seeff. Recovery, persistence, and sequelae in hepatitis C virus infection: a perspective on long-term outcome. Semin. Liver Dis 2000, 20:17-35.
[3] Lau GKK, Zhuang H. Top hepatology issue in China. J Gastroent Hepatol. 2003; 17(Suppl):A9.
[4] Takamizawa A, Mori C, Fuke I, et al. Structure and organization of the hepatitis C virus genome isolated from human carriers. J Virol,1991; 65(3):1105-1113.
[5] Kato N. Genome of human hepatitis C virus (HCV): gene organization, sequence diversity, and variation.Microb Comp Genomics. 2000, 5:129-51.
[6] Reed KE, Rice CM. Overview of hepatitis C virus genome structure, polyprotein processing, and protein properties.Curr Top Microbiol Immunol. 2000, 242:55-84.
[7] Penin F, Dubuisson J, Rey FA, Moradpour D, Pawlotsky JM. Structural biology of hepatitis C virus. Hepatology. 2004, 39: 5-19.
[8] Goffard A, Dubuisson J. Glycosylation of hepatitis C virus envelope proteins. Biochimie.. 2003;85:295-301
[9] Voisset C, Dubuisson J. Functional hepatitis C virus envelope glycoproteins.Biol Cell. 2004; 96: 413-20.
[10] Petit MA, Lievre M, Peyrol S, et al. Enveloped particles in the serum of chronic hepatitis C patients.Virology.2005, 336:144 -53.
[11] Pileri P, Ueatsu Y, Campagnoli S, et al. Binding of hepatitis C virus to CD81. Science, 1998, 282:938-41.
[12] Scarse lli E, Ansuini H, Cerino R, et a1.The human scavenger receptor class B type I is a novel candidate receptor for the hepatitis C virus. EMB0 J, 2002, 21: 5017-5025.
[13] Bartoschi B,Vitelli A, Granier C, et al. Cell entry of hepatitis C virus requires a set of co-receptors that include the CD81 tetraspanin and the SR-BI scavenger receptor. J Biol Chem. 2003; 278: 41624-30.
[14] Lozach PY, Amara A, Bartosch B, et al. C-type lectins L-SIGN and DC-SIGN capture and transmit infectious hepatitis C virus pseudotype particles. J Bio Chem, 2004, 279: 32035-45.
[15] Lee SH, Kim YK, Kim CS, et al. E2 of hepatitis C virus inhibits apoptosis. J Immunol. 2005; 175:8226-35.
[16] Von Hahn, T.& Rice, C .M. Hepatitis C virus entry. J. Biol. Chem. 2008; 283,3689-3693.
[17] Alexander Ploss, Matthew J, et al. Human occluding is a hepatitis C virus entry factor required for infection of mouse cells. Nature. 2009; 457: 882-886.
[18] Oren R, Takahashi S, Doss C, et al. TAPA-1, the target of an antiproliferative antibody, defines a new family of transmembrane proteins. Mol Cell Biol.1990; 10: 4007-15.
[19] Wright MD, Tomlinson MG. The ins and outs of the transmembrane 4 superfamily. Immunol Today. 1994; 15: 588-94.
[20] Cocquerel L, Meunier JC, Pillez A, et al. A retention signal necessary and sufficient for endoplasmic reticulum localization maps to transmembrane domain of hepatitis C virus glycoprotein E2. J Virol. 1998; 72: 2183-91.
[21] Kein F, Abraham JD, Schuster C, et al.Analysis of the subcellular localization of hepatitis C virus E2 glycoprotein in live cells using EGFP fusion proteins. J Gen Virol. 2003; 84: 561-6.
[22] Flint M, Dubuisson J, Maidens C, et al. Functional characterization of intracellular and secreted forms of a truncated hepatitis C virus E2 glycoprotein. J Virol. 2000; 74(: 702-9.
[23] Flint M, Dubuisson J, Maidens C, et al. Functional characterization of intracellular and secreted forms of a truncated hepatitis C virus E2 glycoprotein. J Virol. 2000, 74: 702-709.
[24] Koshy R, et al. Evaluation of hepatitis C virus protein epitopes for vaccine development.Trends Biotechnol,1996, 14(10):364-369.
[25] Tarr AW, Owsianka AM, Timms JM, et al. Characterization of the hepatitis C virus E2 epitope defined by the broadly neutralizing monoclonal antibody AP33. Hepatology, 2006, 43(3): 592-601.
[26] Meunier JC, Engle RE, Faulk K, et al. Evidence for cross-genotype neutralization of hepatitis C virus pseudo-particles and enhancement of infectivity by apolipoprotein C1. Proc Natl Acad Sci U S A. 2005;102:4560-4565.
[27] Esumi M, Rikihisa T, Nishimura S, et al. Experimental vaccine activities of recombinant E1 and E2 glycoproteins and hypervariable region 1 peptides of hepatitis C virus in chimpanzees. Arch Virol, 1999; 144: 973-80.
[28] Fenin Francois, Christophe Combet, Georgios Germanidis, et al. Conservation of the conformation and positive charges of hepatitis C virus E2 envelope glycoprotein hypervariable region 1 points to a role in cell attachment. J Virol, 2001; 75: 5703-10.
[29] Joachim Lupberger, Mirjam B. Zeisel, Fei Xiao, Christine Thumann, Isabel Fofana, Laetitia Zona, et al. EGFR and EphA2 are host factors for hepatitis C virus entry and possible targets for antiviral therapy. Nature Medicine. 2011.17, 589-595.
[30] Bartosch B, Dubuisson J, Cosset FL. Infectious hepatitis C virus pseudo-particles containing functional E1-E2 envelope protein complexes. J Exp Med. 2003;197:633-642.
[31] Lindenbach BD, Evans MJ, Syder AJ, et al. Complete replication of hepatitis C virus in cell culture. Science. 2005;309:623-626.
[32] Wakita T, Pietschmann T, Kato T, et al. Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nat Med.2005;11:791-796.
[33] Zhong J, Gastaminza P, Cheng G, et al. Robust hepatitis C virus infection in vitro. Proc Natl Acad Sci U S A.2005;102:9294-9299.
[34] Lambot M, Fretier S, Op De Beeck A, et al. Reconstitution of hepatitis C virus envelope glycoproteins into liposomes as a surrogate model to study virus attachment. J Biol Chem, 2002, 277: 20625-20630.
[35] Simonsen CC, McGrogan M. The molecular biology of production cell lines. Biologicals. 1994 Jun; 22(2):85-94. Review.
[36] Omasa T, Onitsuka M, Kim WD. Cell engineering and cultivation of Chinese hamster ovary (CHO) cells. Curr Pharm Biotechnol. 2010; 11(3):233-40. Review.
[37] Butler M. Optimization of the cellular metabolism of glycosylation for recombinant proteins produced by Mammalian cell systems. Cytotechnology. 2006; 50(1-3):57-76.
[38] Eisenberg D, Gill H S, Pfluegl GMU, et al. Structure function relationships of glutamine synthetases. Biochim Biophy Acta,2000,1477:122.
[39] Lee GM, Kim EJ, Kim NS, et al. Development of a serum-free medium for the production of erythropoietin by suspension culture of recombinant CHO cells using a statistical design. J Biotechnol,1999,69:85.
[40] Keen MJ, Rapson NT. Development of a serum-free culture media for t he large scale production of recombinant protein f rom a Chinese hamster ovary cell line. Cyrotechnology. 1995, l7:153-163.
[41] Glassy MC, Tharakan JP, Chao PC. Serum-free media inhybridoma culture and monoclonal antibody production. Biochemical and Biotechnology. 1988,32:1015-1028.
[42] Birch JR, Arathoon R. Suspension culture of mammalian cells. Bioprocess Technol. 1990;10:251-70. Review.
[43] Omasa T, Onitsuka M, Kim WD. Cell engineering and cultivation of Chinese hamster ovary (CHO) cells. Curr Pharm Biotechnol. 2010;11(3):233-40. Review.
[44] Singh V. Disposable bioreactor for cell culture using wave-induced agitation. Cytotechnology. 1999 Jul;30(1-3):149-58.
[45] Yang JD et al. Achievement of high cell density and high antibody productivity by a controlled-fed perfusion bioreactor process. Biotechnol Bioeng 2000;69:74-82.
[46] Mirabet M, Navarro A , Lopea A et al . Ammonium toxicity in different cell lines. Biotechnol Bioeng, 1997, 56(5):530-537.
[47] Dempsey J, Ruddock S, Osborne M et al. Improved fermentation processes for NS0 cell lines expressing human antibodies and glutamine synthetase. Biotechnol Prog, 2003, 19:175-178.
[1] Ordo?o D, Enjuanes L, Casasnovas JM. Methods for preparation of low abundance glycoproteins from mammalian cell supernatants. Int J Biol Macromol. 2006 Aug15; 39(1-3):151-6.
[2] K?lln J, Braren I, Bredehorst R, Spillner E. Purification of native and recombinant cobra venom factor using thiophilic adsorption chromatography. Protein Pept Lett. 2007; 14(5):475-80.
[3] D.R.Marshak, J.T.Kadonaga, R.R.Burgess, M.W.Knuth. Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Cold Spring Harbor Laboratory Press, 1996.
[4]应国清,王玉姣,易喻,等。免疫亲和层析技术及其医药应用进展。药物生物技术,2005,12(5):342-34。
[5] Takeshi I, Ken-ichi N, Ryuichi H, Akio K. Single-step af?nity puri?cation of recombinant proteins using the silica-binding Si-tag as a fusion partner. Protein Expression and Puri?cation.2010: 71, 91-95.
[6] Iwane M, Kitamura Y, Kaisho Y, Yoshimura K, Shintani A, Sasada R, Nakagawa S, Kawahara K, Nakahama K, Kakinuma A. Production, purification and characterization of biologically active recombinant human nerve growth factor. Biochem Biophys Res Commun. 1990, 31;171(1):116-22.
[7] Karlsson K, Nyberg F. Purification of substance P endopeptidase activity in the rat ventral tegemental area with the Akta-Purifier chromatographic system. J Chromatogr A. 2000. 29; 893(1):107-13.
[8] Whidby J, Mateu G, Scarborough H, Demeler B, Grakoui A, Marcotrigiano J.Blocking hepatitis C virus infection with recombinant form of envelope protein 2 ectodomain. J Virol.2009; 83(21):11078-89.
[9] Bankwitz D, Steinmann E, Bitzegeio J, Ciesek S, Friesland M, Herrmann E, Zeisel MB, Baumert TF, Keck ZY, Foung SK, Pécheur EI, Pietschmann T. Hepatitis C virus hypervariable region 1 modulates receptor interactions, conceals the CD81 binding site, and protects conserved neutralizing epitopes. J Virol. 2010; 84(11):5751-63.
[10] McCaffrey K, Gouklani H, Boo I, Poumbourios P, Drummer HE. The variable regions of hepatitis C virus glycoprotein E2 have an essential structural role in glycoprotein assembly and virion infectivity. J Gen Virol. 2011; 92:112-21.
[11] Callens N, Ciczora Y, Bartosch B, Vu-Dac N, Cosset FL, Pawlotsky JM, Penin F, Dubuisson J. Basic residues in hypervariable region 1 of hepatitis C virus envelope glycoprotein e2 contribute to virus entry. J Virol. 2005; 79(24):15331-41.
[12] Scarselli E, Ansuini H, Cerino R, Roccasecca RM, Acali S, Filocamo G, Traboni C, Nicosia A, Cortese R, Vitelli A. The human scavenger receptor class B type I is a novel candidate receptor for the hepatitis C virus. EMBO J. 2002; 21(19):5017-25.
[13] Dao Thi VL, Dreux M, Cosset FL. Scavenger receptor class B type I and the hypervariable region-1 of hepatitis C virus in cell entry and neutralisation. Expert Rev Mol Med. 2011,14 ;13:e13.
[14] Q. Yang, F. Faucher, M. Coseno, J. Heckman and S. Doublié. Purifcation, crystallization and preliminary X-ray diffraction of a disulfide cross-linked complex between bovine poly (A) polymerase and a chemically modified 15-mer oligo(A) RNA. Acta Cryst. 2011, F67, 241-244.
[15] Eric J, Enemark K, Grace Chen, et a1. Crystal structure of tDNA binding domain of the replication initiation protein E1 fro papillomavirus. Molecular Cell. 2000 (6): l, 149-158.
[1] Bukh J, Miller RH, Purcell RH. Genetic heterogeneity of hepatitis C virus: quasispecies and genotypes. Seminars in Liver Disease. 1995;15(1):41-63.
[2] Farci P, Shimoda A, Coiana A, et al. The outcome of acute hepatitis C predicted by the evolution of the viral quasispecies. Science. 2000;288(5464):339-344.
[3] Kato, N., Y. Ootsuyama, H. Sekiya, S. Ohkoshi, T. Nakazawa, M. Hijikata, and K. Shimotohno. Genetic drift in hypervariable region 1 of the viral genome in persistent hepatitis C virus infection. J. Virol. 1994.68:4776-4784.
[4] Bartosch B, Bukh J, Meunier JC, et al. In vitro assay for neutralizing antibody to hepatitis C virus: evidence for broadly conserved neutralization epitopes. Proceedings of the National Academy of Sciences of the United States of America. 2003;100(24):14199-14204.
[5] Farci P, Shimoda A, Wong D, et al. Prevention of hepatitis C virus infection in chimpanzees by hyperimmune serum against the hypervariable region 1 of the envelope 2 protein. PNAS USA. 1996;93(26):15394-15399.
[6] Farci P, Alter HJ, Wong DC, et al. Prevention of hepatitis C virus infection in chimpanzees after antibody-mediated in vitro neutralization. PNAS USA, 1994; 91: 7792-6.
[7] Major ME, Mihalik K, Puig M, et al. Previously infected and recovered chimpanzees exhibit rapid responses that control hepatitis C virus replication upon rechallenge. J Virol, 2002; 76(13): 6586-95.
[8] Keck, Z. Y., T. K. Li, J. Xia, M. Gal-Tanamy, O. Olson, S. H. Li, A. H. Patel, J. K. Ball, S. M. Lemon, and S. K. Foung. De?nition of a conserved immunodominant domain on hepatitis C virus E2 glycoprotein by neutralizing human monoclonal antibodies. J. Virol. 2008. 82:6061-6066.
[9] Owsianka, A. M., Timms, J. M., Tarr, A. W., Brown, R. J. P., Hickling, T. P., Szwejk, A., Bienkowska-Szewczyk, K., Thomson, B. J., Patel, A. H., Ball, J. K.. Identification of ConservedResidues in the E2 Envelope Glycoprotein of the Hepatitis C Virus That Are Critical for CD81 Binding. J. Virol. 2006.80: 8695-8704.
[10] Drummer HE, Boo I, Maerz AL, Poumbourios PA. Conserved Gly436-Trp-Leu-Ala-Gly-Leu-Phe-Tyr Motif in Hepatitis C Virus Glycoprotein E2 Is a Determinant of CD81 Binding and Viral Entry. J. Virol. 2006,80: 7844-7853
[11] Dubuisson J, Helle F, Cocquerel L. Early steps of the hepatitis C virus life cycle. Cell Microbiol. 2008 Apr;10(4):821-7. 2007,13. Review.
[12] Helle F, Dubuisson J. Hepatitis C virus entry into host cells. Cell Mol Life Sci. 2008 Jan;65(1):100-12. Review.
[13] Diedrich G. How does hepatitis C virus enter cells? FEBS J. 2006;273(17):3871-85. Review.
[14] Taniguchi, S., H. Okamoto, M. Sakamoto, M. Kojima, F. Tsuda, T. Tanaka, E. Munekata, E. E. Muchmore, D. A. Peterson, and S. Mishiro. A structurally ?exible and antigenically variable N-terminal domain of the hepatitis C virus E2/NS1 protein: implication for an escape from antibody. Virology. 1993.195:297-301.
[15] Mondelli M U, Cerino A, Meola A and Nicosia A 2003 Variability or conservation of hepatitis C virus hypervariable region 1? Implications for immune responses. J. Biosci. 28 305-310.
[16] Keck, Z. Y., J. Xia, Z. Cai, T. K. Li, A.M. Owsianka, A. H. Patel, G. Luo, and S. K. Foung. 2007. Immunogenic and functional organization of hepatitis C virus (HCV) glycoprotein E2 on infectious HCV virions. J. Virol. 81:1043-1047.
[17] Stamataki, Z., Coates, S., Evans, M.J., Wininger, M., Crawford, K., Dong, C., Fong, Y.L., Chien, D., Abrignani, S., Balfe, P., Rice, C.M., McKeating, J.A., Houghton, M.,. Hepatitis C virus envelope glycoprotein immunization of rodents elicits cross-reactive neutralizing antibodies. Vaccine. 2007,25, 7773-7784.
[1] Choo QL, Kuo G, Weiner AJ, Overby LR, Bradley DW, Houghton M. Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science 1989; 244: 359-362.
[2] Wyatt CA, Andrews L, Brotman B, Huang F, Lee D, Prince A. Immunity in chimpanzees chronically infected with hepatitis C virus:role of minor quasispecies in reinfection. J Virol 1998; 72: 1725-1730.
[3] Angnello V, Abel G, Knight GB, Muchmore E. Detection of wildspread hepatocyte infection in chronic hepatitis C. Hepatology 1998; 28: 573-584.
[4] Tanaka JK, K; Kumagai, J; Komiya, Y; Yugi, H; Kishimoto, S; Mizui, M; Tomoguri, T; Miyakawa, Y; Yoshizawa, H. Early dynamics of hepatitis C virus in the circulation of chimpanzees with experimental infection. Intervirology 2005; 48: 120-123.
[5] Shoukry NH, Sidney J, Sette A, Walker CM. Conserved hierarchy of helper T cell responses in a chimpanzee during primary and secondary hepatitis C virus infections. J Immunol 2004; 172: 483-492.
[6] Folgori A, Capone S, Ruggeri L, Meola A, Sporeno E, Ercole BB, Pezzanera M, Tafi R, Arcuri M, Fattori E, Lahm A, Luzzago A, Vitelli A, Colloca S, Cortese R, Nicosia A. A T-cell HCV vaccine eliciting effective immunity against heterologous virus challenge in chimpanzees. . Nat Med 2006; 12: 190-197.
[7] Major ME, Dahari H, Mihalik K, Puig M, Rice CM, Neumann AU, Feinstone SM. Hepatitis C virus kinetics and host responses associated with disease and outcome of infection in chimpanzees. Hepatology 2004; 39: 1709-1720.
[8] Martin A, Bodola F, Sangar DV, Goettge K, Popov V, Rijnbrand R, Lanford RE, Lemon SM. Chronic hepatitis associated with GB virus B persistence in a tamarin after intrahepatic inoculation of synthetic viral RNA. Proc Natl Acad Sci U S A 2003; 100: 9962-9967.
[9] Nam JH, Faulk K, Engle RE, Govindarajan S, St Claire M, Bukh J. In vivo analysis of the 3' untranslated region of GB virus B after in vitro mutagenesis of an infectious cDNA clone: persistent infection in a transfected tamarin. J Virol 2004; 78: 9389-9399.
[10] Garson JA, Whitby K, Watkins P, Morgan AJ. Lack of susceptibility of the cottontop tamarin to hepatitis C infection. J Med Virol 1997; 52: 286-288.
[11] Kock;, J M, Nassal;, S M, ;T F, Baumert;, H E, Blum; , F vW. Efficient infection of primary tupaia hepatocytes with purified human and woolly monkey hepatitis B virus. J Virol 2001; 7(75): 5084-5089.
[12] Xie Z, C; Riezu Boj, J,I; Lasarte, J.J; Guillen, J; Su, J.H; Civeira, M.P; Prieto, J. Transmission of hepatitis C virus infection to tree shrews. Virology 1998; 244: 513-520.
[13] Xu XC, H.; Cao, X.; Ben, K. Efficient infection of tree shrew (Tupaia belangeri) with hepatitis C virus grown in cell culture or from patient plasma. J Gen Virol 2007; 88: 2504-2512.
[14] Amako;Tsukiyama;Katsume;Hirata;Sekiguchi;Tobita;Hayashi;Hishima;Funata;Yonekawa;Kohara. Pathogenesis of hepatitis C virus infection in Tupaia belangeri . J Virol 2010; 84(1): 303-311.
[15] Yimin Tong, Yongzhe Zhu, Xueshan Xia, Yuan Liu, Yue Feng, Xian Hua, Zhihui Chen, Hui Ding, Li Gao, Yongzhi Wang, Mark A. Feitelson, Ping Zhao, and Zhongtian Qi. Tupaia CD81, SR-BI, claudin-1, and occludin support hepatitis C virus infection. J Virol. 2011 Mar;85(6):2793-802.
[16] Koike K, Moriya K, Ishibashi K, Matsuura Y, Suzuki T, Saito I, Iino S, Kurokawa K, Miyamura T. Expression of hepatitis C virus envelope proteins in transgenic mice. J Gen Virol 1995; 76(12): 3031-3038.
[17] Tanaka N, Moriya K, Kiyosawa K, Koike K, Gonzalez FJ, Aoyama T. PPARalpha activation is essential for HCV core protein-induced hepatic steatosis and hepatocellular carcinoma in mice. J Clin Invest 2008; 118: 683-694.
[18] Majumder M, Ghosh AK, Steele R, Zhou XY, Phillips NJ, Ray R, Ray RB. Hepatitis C virus NS5A protein impairs TNF-mediated hepatic apoptosis, but not by an anti-FAS antibody, in transgenic mice. Virology 2002; 294: 94-105.
[19] Wang AG, Lee DS, Moon HB, Kim JM, Cho KH, Choi SH, Ha HL, Han YH, Kim DG, Hwang SB, DY Y. Non-structural 5A protein of hepatitis C virus induces a range of liver pathology in transgenic mice. J Pathol 2009; 219(2): 253-262.
[20] Kanda T, Steele R, Ray R, RB R. Inhibition of intrahepatic gamma interferon production by hepatitis C virus nonstructural protein 5A in transgenic mice. J Virol 2009; 83(17): 8463-8469.
[21] Ilan EA, J.; Nussbaum, O.; Zauberman, A.; Eren, R.; Lubin, I.; Neville, L.; Ben-Moshe, O.; Kischitzky, A.; Litchi, A.; Margalit, I.; Gopher, J.; Mounir, S.; Cai, W.; Daudi, N.; Eid, A.; Jurim, O.; Czerniak, A.; Galun, E.; Dagan, S. The hepatitis C virus (HCV)-Trimera mouse: a model for evaluation of agents against HCV. J Infect Dis 2002; 182: 153-161.
[22] Eren RL, D.; Terkieltaub, D.; Nussbaum, O.; Zauberman, A.; Ben-Porath, J.; Gopher, J.; Buchnick, R.; Kovjazin, R.; Rosenthal-Galili, Z.; Aviel, S.; Ilan, E.; Shoshany, Y.; Neville, L.; Waisman, T.; Ben-Moshe, O.; Kischitsky, A.; Foung, S.K.; Keck, Z.Y.; Pappo, O.; Eid, A.; Jurim, O.; Zamir, G.; Galun, E.; Dagan, S. Preclinical evaluation of two neutralizing human monoclonal antibodies against hepatitis C virus (HCV): a potential treatment to prevent HCV reinfection in liver transplant patients. J Virol 2006; 80: 2654-2664.
[23] Ernst ES, K.; Bugert, J.J.; Blaker, H.; Pfaff, E.; Stremmel, W.; Encke, J. . Generation of inducible hepatitis C virus transgenic mouse lines. J Med Virol 2007; 79: 1103-1112.
[24] Kaul AW, I.; Meuleman, P.; Leroux-Roels, G.; Bartenschlager, R. Cell culture adaptation of hepatitis C virus and in vivo viability of an adapted variant. J Virol 2007; 81: 13168-13179.
[25] Kneteman NM, Weiner AJ, O'Connell J, Collett M, Gao T, Aukerman L, Kovelsky R, Ni ZJ, Zhu Q, Hashash A, Kline J, Hsi B, Schiller D, Douglas D, Tyrrell DL, Mercer DF. Anti-HCV therapies in chimeric scid-Alb/uPA mice parallel outcomes in human clinical application. Hepatology 2006; 43: 1346-1353.
[26] Vanwolleghem T, Meuleman P, Libbrecht L, Roskams T, De Vos R, Leroux-Roels G. Ultra-rapid cardiotoxicity of the hepatitis C virus protease inhibitor BILN 2061 in the urokinase-type plasminogen activator mouse. Gastroenterology 2007; 133: 1144-1155.
[27] Vanwolleghem T, Bukh J, Meuleman P, et al. Polyclonal immunoglobulins from a chronic hepatitis C virus patient protect human liver-chimeric mice from infection with a homologous hepatitis C virus strain. Hepatology, 2008, 47(6): 1846-1855.
[28] Law M, Maruyama T, Lewis J, et al. Broadly neutralizing antibodies protect against hepatitis C virus quasispecies challenge. Nat Med, 2008, 14(1): 25-27.
[29] Legrand N, Ploss A. Humanized mice for modeling human infectious disease: challenges, progress, and outlook. Cell Host Microbe, 2009, 6(1): 5-9.
[30] Strowig T, Gurer C, Ploss A. Priming of protective T cell responses against virus-induced tumors in mice with human immune system components. J Exp Med,2009, 206(6): 1423-1434.