【作者】 任艳丽；

【导师】 戚中田； 赵平；

【作者基本信息】 第二军医大学 ， 微生物学， 2009， 博士

【摘要】 丙型肝炎病毒(hepatitis C virus,HCV)是带有包膜的正链RNA病毒,主要经血液传播,引起急、慢性丙型肝炎,并可导致肝硬化和肝细胞癌。HCV感染除引起肝脏病变外,还可引起多种肝脏外组织损害,与B细胞淋巴瘤、冷球蛋白血症等多种肝外疾病有关。目前全球HCV感染者约有1.7亿,HCV感染的慢性率高达80%。HCV的基因变异,对宿主细胞内免疫的拮抗作用以及相对低水平表达和复制等均与HCV慢性感染有关。最近的一些研究发现,HCV包膜E2蛋白与CD81分子结合所致免疫细胞的功能紊乱也可能是HCV慢性感染的重要原因,并且与B细胞淋巴瘤以及冷球蛋白血症具有重要相关性。HCV包膜E2蛋白的CD81结合活性还可能与HCV感染难以诱导中和抗体而导致慢性感染有关。一、HCV包膜蛋白E2促进B淋巴细胞活化和增殖的作用机制HCV包膜E2蛋白通过与B淋巴细胞表面上CD81分子的作用,改变了B细胞激活的正常信号转导通路,导致B淋巴细胞非特异性活化。我们构建了HCV包膜E2蛋白的表达质粒,转染细胞表达HCVE2蛋白。将表达的E2蛋白固定在事先包被了E2单抗的酶标微孔板上,然后加入Burkitt淋巴瘤Raji细胞,可观察到该细胞聚集贴附生长。然而用siRNA下调Raji细胞表面CD81的表达,则不能使Raji细胞聚集贴附生长,说明表达的E2蛋白能够与Raji细胞表面的CD81分子结合。进一步我们制备固定化的E2蛋白,并分别用固定化的E2和可溶性E2蛋白刺激Raji细胞,然后分析细胞内蛋白酪氨酸磷酸化的水平。结果表明,固相化的E2蛋白可有效上调Raji细胞内的蛋白酪氨酸磷酸化的水平,而可溶性E2对Raji细胞内的蛋白酪氨酸磷酸化水平则无明显影响。这可能是因为固相化的E2蛋白类似天然的病毒颗粒,在构象上要更接近于天然的病毒颗粒表面E2蛋白,而且固相化后的E2蛋白在单位体积的密度要高于可溶性的E2蛋白单位体积的密度,使其能够更充分的与细胞表面的CD81分子相互接触作用。构建CD81分子siRNA的慢病毒载体,转染293T细胞包装出病毒后感染Raji细胞,流式检测Raji细胞表面CD81分子表达被抑制,进一步用固相化的E2蛋白刺激该CD81表达下调的Raji细胞,则观察到Raji细胞内的蛋白酪氨酸磷酸化水平无明显的变化,这说明E2蛋白对Raji细胞的信号调节是经由CD81分子作用的。固相化的E2蛋白刺激Raji细胞后用western blotting技术检测细胞内凋亡相关蛋白Bcl-2和Bax蛋白的表达,可以观察到Bcl-2蛋白表达下降,而Bax蛋白表达增加。Bcl-2基因是人体最主要的抗凋亡基因,它编码的蛋白质产物定位于线粒体内膜、内质网膜及核膜上,其主要生物学功能是延长细胞的寿命,增加细胞对各种凋亡刺激因素的抵抗力.它可导致DNA受损的细胞持续生存,突变产物聚集,从而促进肿瘤的发生与发展。Bcl-2相关X蛋白(Bax)具有加速细胞凋亡的功能, Bax可与抗凋亡基因Bcl-2形成异源二聚体,具有抑制Bcl-2,促进细胞凋亡的作用。Bcl-2与Bax两者之间的比例决定了细胞的命运,若Bax占多数,则Bcl-2被抑制,凋亡被诱导,细胞死亡;反之则Bax受到抑制,细胞得以生存。E2蛋白刺激Raji细胞后促进Bcl-2的表达而抑制Bax的表达,这说明E2蛋白能够增强Raji细胞的抗凋亡能力。E2蛋白能够诱导B细胞蛋白酪氨发生酸磷酸化且促进B细胞的抗凋亡的能力,这些可能是E2促进B细胞增殖的重要机制。用流式细胞技术检测固相化HCV E2蛋白刺激的Raji细胞表面CD80、CD86和CD21分子的表达,结果可看到CD80和CD86分子的表达上调,而CD21分子的表达下降,CD80、CD86和CD21是与B淋巴细胞的活化紧密相关的膜表面分子,这说明E2蛋白能刺激Raji细胞的活化,从而也促进Raji细胞的增殖。二、HCV E2蛋白突变体免疫原性分析研究表明HCV包膜E2蛋白可诱导B淋巴细胞蛋白酪氨酸磷酸化,通过上调抗凋亡蛋白Bcl-2的表达,下调促凋亡蛋白Bax的表达而促进B淋巴瘤细胞的增殖,并且还能上调活化分子的表达,说明E2蛋白能通过激活胞内信号转导通路发挥一系列生物学效应。而E2对B细胞的激活可能导致HCV感染难以有效诱导中和抗体,因此,消除E2蛋白的CD81结合活性可能有利于中和抗体的诱导。我们分别将H77株的E2蛋白的第442位苯丙氨酸(F)和529位的色氨酸(W)突变为丙氨酸(A),构建质粒转染细胞,用E2多抗对细胞培养上清以及细胞裂解液中的E2蛋白表达进行检测,结果表明突变对E2的表达和分泌无明显影响。用ELISA分析两种突变的E2蛋白与大肠杆菌表达的Trx(硫氧还蛋白)-CD81 LEL(大胞外环)融合蛋白的结合情况,结果显示,该两个位点的突变体不再具有与CD81大胞外环(LEL)的结合活性;进一步用流式细胞术(FACS)分析两种E2突变体与表达CD81分子的CHO细胞结合情况表明,该两个位点突变的E2蛋白不再具有与膜表面的CD81分子结合的功能。用突变体质粒E2-W529/A和E2-W442/A转染细胞后,免疫荧光检测两株构象依赖性单抗与突变体E2蛋白的反应,结果表明,该两个位点的突变对E2蛋白空间构象无明显影响。将固相化的两种突变体E2蛋白分别刺激Raji细胞,没有检测到细胞内蛋白酪氨酸磷酸化水平发生明显变化,这表明,消除CD81结合活性可消除E2蛋白对Raji细胞胞内信号转导的激活作用。HCV E2蛋白第442和529位氨基酸分别位于两个线性B细胞表位中,其中,前者所在表位抗体具有中和活性,而后者病毒中和活性并不明显。因此,选择529位的突变体进行免疫可有利于保留更多的中和抗体表位。我们将野生E2与突变体E2-W529/A表达质粒分别免疫小鼠,眼眶取血收集免疫小鼠的血清,分别用野生和突变的E2蛋白为靶抗原检测小鼠的抗体动力学,结果表明,两种DNA免疫小鼠血清的E2抗体动力学水平相似,这说明W529/A突变对于E2蛋白的免疫原性无明显影响。524～531位氨基酸残基APTYSWGA为一线性B细胞表位,其抗体能抑制E2蛋白与CD81的结合。我们将该表位与红色荧光蛋白融合,构建表达质粒转染细胞,用免疫荧光分析该表位与小鼠血清的结合,结果表明,W529A的突变部分降低了该表位的免疫原性/抗原性。AP33表位为HCV E2蛋白第412～423位氨基酸残基QLINTNGSWHIN,也为一线性B细胞表位,其在HCV的六个基因型中高度保守,AP33能有效中和HCV的感染性。同样构建该表位与红色荧光蛋白RFP融合表达的质粒,转染细胞用免疫荧光分析其与小鼠血清的结合表明,W529/A突变不影响该表位的抗体应答,野生或突变E2均可诱导针对该表位的抗体。构建11株HCV(涵盖6个基因型)包膜E2蛋白的表达质粒,转染细胞用免疫荧光分析小鼠免疫血清与这些不同株不同基因型E2蛋白的反应,结果表明,E2-W529/A免疫血清可与该11株HCV包膜蛋白发生交叉反应。为了评价免疫血清的中和作用,我们构建了五株HCV包膜蛋白的HCVpp进行中和试验。从结果可见,野生型E2以及E2-W529/A突变体免疫血清均可有效中和这八株HCVpp对靶细胞Huh7.5的感染性,且对同株HCVpp的中和效率无明显差异。进一步我们用JHF-1株HCVcc评价小鼠血清的中和作用,结果表明两种质粒免疫血清均可抑制HCVcc的感染性,抑制效率相似。三、小结1.本研究中用固相化的HCV包膜蛋白E2刺激人B淋巴细胞Raji细胞,可诱导Raji细胞内的蛋白酪氨酸的磷酸化,而用siRNA干扰掉B细胞表面CD81的表达后用E2蛋白再刺激Raji细胞,细胞内酪氨酸磷酸化的水平未发生明显的变化。而且E2蛋白刺激Raji细胞可上调Raji细胞内凋亡相关蛋白Bcl-2蛋白的表达,下调Bax蛋白的表达,并促进膜表面CD80、CD86分子的表达,下调膜表面CD21分子的表达。这些结果表明HCV包膜蛋白E2可通过与CD81分子作用改变B淋巴细胞内信号的转导,从而引起的B细胞的增殖和非特异性激活。2.在此基础上,我们构建了一种E2蛋白突变体,将第529位色氨酸突变为丙氨酸,该W529/A突变体不影响E2蛋白的空间构象,但是消除了与CD81的结合活性。该突变体刺激B淋巴细胞后,对B细胞的胞内蛋白酪氨酸磷酸化水平变化影响不明显。以DNA免疫的方式进行小鼠免疫,一次免疫可使小鼠抗体阳转率达到100%,抗体应答(包括E2总抗体以及针对中和抗体表位的抗体)与天然E2免疫无明显差异,并且能有效诱导HCV交叉中和抗体。由于该突变体对B细胞无另外刺激,不导致B细胞的非特异激活,但可有效诱导中和抗体,因此可望作为HCV疫苗的有效研究方向,可代表一种HCV疫苗的发展策略。更多还原

【Abstract】 Hepatitis C virus is an enveloped virus with a positive-sense single-standed RNA genome. HCV is a major factor of post-transfusion and community-acquired hepatitis in the world and causes a variety of liver diseases, including acute and chronic hepatitis, cirrhosis, liver failure and hepatocellular carcinoma. Except causing liver pathological changes, HCV infection may induce extra-hepatic immune related manifestations in high percentage of infected patients.Immune system related manifestations include a myriad of conditions ranging from sub-clinical cryoglobulinemia to overt lymphoproliferative malignancy. Over 170 million people worldwide are infected with HCV, 80% of these develop chronic hepatitis C. HCV infection is associated with HCV gene variation, antagonistic reaction to immunity system of host cell and relative low levels of expression and replication of HCV. Recent studies show that the binding of HCV enveloped E2 protein to CD81 molecule leads to the lymphocyte functional disorders, which may play an important role in HCV chronic infection and have closed relation with B cell lymphadenoma and cryoglobulinemia. Associated with formation of HCV infection is hard to induce neutralizing antibody and thus leading to chronic infection. The interaction of HCV enveloped E2 protein with CD81 molecule has been considered to be related with this situation.1. The interaction of HCV envelope E2 protein with CD81 promotes activation and proliferation of B lymphocytesThe binding of HCV envelope E2 protein with CD81 molecule on the surface of B lymphocyte alters the normal signal transductional pathway of B lymphocyte activation, which may lead to nonspecific activation of B cell. We transfected 293T cell with plasmid expressing E2 protein to producing E2 protein. The expressed E2 protein was conjugated to ELISA microplate which was precoated with anti-E2 mAb H53. Then Raji cells were seeded onto the microplate and cultured . The condition of adherent growth of cells was observed. The recombinant lentivirus of human CD81 siRNA wan constructed and infected the Raji cells and down-regulated CD81 expression on the surface of Raji cells. The same experiment was repeated in the cell and the state of adherent growth wasn’t viewed. These results showed that the expressing E2 protein was be able to interact with CD81 molecule on the surface of Raji cells. We prepared immobilized HCV envelop E2 protein and then stimulated Raji cells with the immobilized E2 protein and soluble E2 protein,respectively. The level of phosphorate-tyrosine of the proteins in Raji cells was detected by western blot. The results indicated that immobilized E2 protein remarkably raises the level of phosphorate-tyrosine in Raji cells , but the soluble E2 protein hasn’t obvious impact to phosphorylation of tyrosine of Raji cell. It was presumed that immobilized E2 protein was resembled to natural viron enveloped E2 protein and may more efficiently interact with CD81 molecule. The Raji cells , CD81 expression silenced, was stimulated by immobilized E2 protein.The level of phosphorate-tyrosine of Raji cell didn’t change, which illustrates that E2 protein is via CD81 molecule to regulates the signal pathway of Raji cell. The expression of Bcl-2 protein and Bax protein in Raji cell, which were apoptosis-associated proteins, were analized by western blott when the Raji cells were stimulated by immobilized E2 protein. The results showed that expression level of Bcl-2 protein was decreased, thus Bax protein increased, which indicated that HCV E2 protein had important contribution to promote Raji cell proliferation. The CD80、CD86 and CD21 molecules on the surface of Raji cell, which were related proteins with the activation of B lymphocyte, were detected by Flow cytometric analysis. The results show that the expression of CD80 and CD86 molecules were elevated and the CD21 was decreased.These indicated that E2 protein probrably activated the Raji cell and promoted proliferation of the Raji cell.2. Immunogenic analysis of mutational HCV E2 proteinHCV E2 protein induces phosphorylation of tyrosine in B lymphocyte and promotes proliferation of B lymphocyte by up-regulating expression of anti-apoptosis protein Bcl-2 and down-regulating expression of enhancing apoptosis protein Bax. HCV E2 protein also increases the expression of activated molecule of B lymphocyte. These results indicate that E2 protein may activate signal transduction pathway and initiate a series of biological effect of B lymphocyte. The activation of B cell by E2 protein make it difficulty to induce the neutralizing antibody after HCV infection. So ,we presume that elimination of the linked activity of E2 protein with CD81 would be profited to producing of neutralizing antibody.Plasmids expressing mutant of HCV E2 protein of H77 strain were constructed, in which the codons encoding the aa442 Phe(F) and the aa529 Trp(W) of E2 protein were both changed into the codon encoding Ala(A). HEK293T cells were transfected with the two mutant plasmids E2-W529/A and E2-W442/A and then the culture supernatant and cell lysate were analyzed by E2 polyclonal antibody.The results showed that the mutation didn’t affect the expression and secretion of E2 proteins. We analyzed the binding by ELISA between the two mutans and the fusion protein Trx (thioredoxin)-CD81 (LEL, large extracellular loop)expressed by E. coli. The results showed that the mutants lost the binding function with CD81 LEL.The analysis of FACS showed that the two mutants were also no longer binding with the CD81 on the surface of cell. The two plasmids were transfected to HEK293T cells and then analysed the conformation of the two mutants with E2 conformation-dependent monoclonal antibody by immunofluorescent technique(IF). The results showed that the mutation of the two sits didn’t affect the conformation of E2 protein. The two mutants were immobilized and then to stimulate the Raji cell. There were no obvious changes about the the level of protein phosphorate-tyrosine in Raji cells, which indicated that elimination of binding activity of E2 protein with CD81 molecule deprivated the activation function of E2 proein to the signal transduction pathway in Raji cell.The aa442 and aa529 of HCV E2 protein located respectively in two linear epitopes of B cell. The epitope including aa442 has neutralizing activity and the other including aa529 has no obvious neutralizing activity. For keeping more epitopes of neutralizing antibody,we chose the plasmids of mutant E2-W529/A and wide-type E2 to immune BALB/c mice. Blood samples were collected regularly by retro-orbital puncture from immunized mice.The antibody kinetics of the mice serum were analysed by using wide E2 and mutational E2 proteins as antigen. The results showed that the level of serum antibody kinetics of these mice immuned with mutative E2 protein were similar to the wide E2 ,which indicated that the mutation of W529 didn’t affect the immunogenicity of E2 protein. The amino acid residue APTYSWGA(aa524-aa531) was a linear epitope of HCV. The antibody of the epitope may inhibit the binding of E2 protein with CD81.We constructed expression plasmid fusing the red fluorescent protein(RFP)into the C-termination of the epitope and then analyzed the reaction between this fused epitope and mouse serum by IF after transfecting the 293T cell. The results indicated that the mutation of W529A decreased the antigenicity of the epitope. The epitope AP33 , amino acid residue QLINTNGSWHIN (aa412-aa423) in HCV E2 protein, was also a linear epitope of B cell, which was high conservation in the six geneotypes of HCV and may effectively neutralize the infection of HCV. We fused the RFP with the epitope and analyzed the antibody response of mice serum by IF. The result showed the mutation of W529/A didn’t influence the epitope antigencity and both wide and mutant E2 may induce the antibody to the epitope. We transfected the 293T cell with plasmids of expressing 11 strains HCV E2 proteins including 6 genotypes and detected the cross-reaction of mice serum. Results: the mutant E2-W529/A immuned mice serum has cross-reaction with these 11 strains HCV E2 proteins. For analyzing the neutralizing effects of mouse serum, we constructed 8 strains HCVpp and the 8 strains HCV were from different countries and involved 4 genotypes. The data showed that the infection of the 8 strains HCVpp to Huh7.5 cell were effectively neutralized by the wide-type E2 and mutant E2-W529/A. The neutralizing effects of the both to the same strain HCVpp were parallel. Except using the HCVpp, We also evaluated the neutralizing effects of these mice serum with JFH-1 HCVcc . The infection of HCVcc could be inhibited by the both immuned mouse serum and the inhibition efficiency was similar.Summary:1. In this study ,we stimulated Raji cells with immobilized HCV envelope E2 protein, which induced the phosphorylation of the protein tyrosine in Raji cells ,up-regulated the expression of Bcl-2 protein and down-regulated the expresse of Bax protein. The expression of CD80 and CD86 molecules expressed on the surface of Raji cells was increased and the CD21 was decreased.These changes were inhibited in the Raji cells on which CD81 expression was silenced. The results indicated that HCV E2 protein may promote the proliferation and nonspecific activation of B lymphocyte via CD81 molecule.2. According to this study, we constructed an E2 protein mutant by changing the aa529 Trp(W) into Ala. The E2-W529/A mutant didn’t affect the conformation of E2 protein. For elimination of the binding activity with CD81, the mutant was no longer inducing the phosphorylation of the protein tyrosine and regulating the expression of apoptosis-associated proteins. We used the plasmids E2-W529/A to immunize the mice and detected the antibody positive rate up to 100% after the first immunization. The antigenicity of the E2 protein mutant was similar with wide-type E2 protein and may induce effective HCV cross-reaction neutralizing antibody. This mutant may represent a strategy for effective HCV vaccine development.更多还原