【作者】 徐进平；

【导师】 喻子牛； 叶林柏；

【作者基本信息】 华中农业大学 ， 微生物学， 2003， 博士

【摘要】 本文包括两个部分：（1）人丙型肝炎病毒（Hepatitis C virus，HCV）E1基因的表达及E1蛋白质免疫学性质研究。（2）人巨细胞病毒（Human Cytomegalovirus，HCMV）最早期蛋白与转录调控蛋白相互作用研究。 本文首次报道了HCV武汉株（WH strain）全长E1基因cDNA全序列，将该cDNA分别克隆于载体pQE30、pBlueBacHisB和pcDNA3.1（-）中，分别在大肠杆菌、真核细胞和实验动物肌肉组织中进行表达。全面研究了HCV E1 cDNA在大肠杆菌和真核细胞中表达的HCV E1蛋白的抗原性和免疫原性。该研究有助于进一步了解HCV包膜蛋白的功能和揭示HCV E1的变异规律及其特点，为将E1蛋白质应用于HCV的临床诊断和研制有效的、HCV疫苗，预防和治疗丙型肝炎提供了重要的参考和依据。 HCV全长E1 cDNA含576bp，编码含192个氨基酸的蛋白质。HCV武汉株和日本株（HCV-J型）E1基因核苷酸的同源性为74.48％，编码的氨基酸的同源性为78.13％。其免疫优势表位P1（210-223aa）和P2（315-327aa）氨基酸序列同源性分别为71.43％和100％。这证实了Ray以前所推测的P1可能是一个变异较大的区域，而P2较保守的观点。 大肠杆菌中表达的HCV E1为23kDa、非糖基化的融合蛋白，形成包涵体。以包涵体作为抗原，采用ELISA可检测出HCV阳性患者血清中的抗体，在确定的HCV阳性患者中阳性检出率为48/90。推测E1的抗原性可能与其B细胞表位，即RMAWDMMMNW（317-326aa）相关，而与E1蛋白是否糖基化无关。以包涵体作为免疫原，小鼠抗HCV E1 IgG抗体水平在第8周达到高峰，抗体滴度可达1：2400；家兔抗HCV E1IgG抗体水平在第8周达到高峰，抗体滴度可达1：5760，并能诱导小鼠粘膜产生免疫应答。E1蛋白能诱生小鼠较高水平的CTL应答、DTH应答，并引起CD₈^- T细胞数量在免疫后明显升高。推测E1蛋白表达所形成的包涵体结构直接作为免疫原，有利于HCV E1抗原的呈递。说明HCV E1包涵体蛋白具有良好的抗原性和免疫原性。 HCV E1基因cDNA在昆虫细胞中的表达产物主要为40kDa的融合蛋白，极弱地表达29-40kDa蛋白。推测该蛋白分子量差异是因表达产物经不同程度糖基化所致。以40kDa E1蛋白为抗原，采用ELISA方法检测HCV抗体，在确定的HCV阳性患者中阳性检出率48/90。该检测结果与包涵体HCV E1蛋白临床检测一致，但反应强弱有差异，糖基化蛋白反应弱。该蛋白可诱生免疫实验动物产生特异性体液免疫应答。小鼠抗HCV E1 IgG抗体水平在第6周达到高峰，抗体滴度可达1：2400；家兔抗HCV E1华中农业大学博士学位论文工gG抗体水平在第8周达到高峰，抗体滴度可达1:7680。HCvEI糖基化蛋白能诱生小鼠抗s工gG反应，但不能诱生小鼠slgA反应。通过LDH检测，发现该蛋白也能诱生小鼠CTL应答，并引起小鼠免疫后CD扩T细胞数量明显升高以及DTH应答，说明HCV El糖基化蛋白能诱生细胞免疫应答。 采用脂质体转染技术，用pcEI转染肝癌细胞HepGZ，HCvEI基因cDNA在HepGZ细胞中表达，表达产物为35kDa的单一糖基化成熟蛋白，且该蛋白能与临床HCV抗体阳性血清特异结合。采用亲和免疫细胞组织化学技术证实小鼠股四头肌细胞能摄取质粒pcEI DNA，并表达了HcvEI蛋白。通过肌肉注射途径，直接注射裸露质粒pcEI DNA免疫实验动物，发现pcEI DNA能引起7/8小鼠，5/5家兔抗HCVEI抗体阳转，诱导机体产生特异性抗HCv El抗体。通过LDH检测和比较CD;+/CD，‘比例，peEI引起小鼠免疫后CDs+T细胞数量明显升高以及DTH应答，说明pcEI能诱生小鼠特异性细胞免疫应答。 以人巨细胞病毒的最早期蛋白工E86为材料，研究工E86蛋白与细胞和其它病毒的转录调控因子和通用转录因子相互作用形成复合物的能力，建立基因表达调控的大分子体系研究模型。为在大分子体系水平上，进一步了解大分子体系中蛋白质的相互作用方式，以及这些相互作用对基因表达调控的具体意义奠定了基础。 将HCMV IE86 eDNA克隆入pGEX一 ZT表达载体，在大肠杆菌中表达出GST一IE86融合蛋白。SDS一PAGE和Western blot分析表明，GST一IE86融合蛋白存在于细胞裂解上清中，为可溶性蛋白，分子量为92kDa。通过亲和层析柱纯化，分别获得纯化的GST和GST一IE86融合蛋白。采用特异性亲和层析共分离技术研究HCMV IE86蛋白与转录调控因子和通用转录因子的相互作用。表明HCMV工E86蛋白能与直接结合启动子DNA的转录调控因子SPI、API、APZ及通用转录因子TFllB相互作用而形成异源蛋白复合物，该转录调控因子及通用转录因子与HCMV工E86蛋白同时被吸留在亲和层析柱中。但是，HCMV工E86蛋白不能与NF一KB相互作用。 HCMV IE86蛋白与转录调控因子及通用转录因子相互作用的活性与IE86蛋白的糖基化无关，而IE86蛋白的锌指结构可能是关键。IE86通过与SPI、API、APZ等转录调控因子、RNA多聚酶H所用的通用转录因子TFllD、TFllB等相互作用形成大分子体系，通过这种大分子体系的作用启动和增强转录。工E86蛋白在识别和结合启动子特异序列的转录调控因子与RNA多聚酶H的通用转录因子如TFllD、TFllB之间可能起到连系的桥梁作用，从而加速转录起始复合物的装配过程。我们推测工E86?更多还原

【Abstract】 This paper included two parts: (1) study on the expression of Hepatitis C Virus (HCV) El gene and the immunology character of El protein; (2) The Human Cytomegalovirus (HCMV) IE2 protein interacts with transcription activating protein.The full length of HCV El gene cDNA sequence of Wuhan strain was reported first time, and it was cloned respectively into pQE30, pBlueBacHisB and pcDNA3.1 (-). HCV El cDNA expression, antigenity and immunity of El protein from E. coli and eukaryote cells were overall analyzed. It is significant for further understanding the variation rule of El gene, the characteristics and functions of El protein. The studies on the expression of HCV El gene in E. coli and eukaryote cells, and the immunological properties of different forms of El protein and El DNA vaccine, will contribute greatly to the development of HCV diagnosis and vaccine.HCV El cDNA contained 576bp and encoded a protein of 192 amino acids. 74.48% nucleotide acids of El cDNA and 78. 13% amino acid of El protein are homologous between WH strain and Japan strain (HCV-J type). There were two preponderant epitopes, P1 (210-223aa) and P2 (315-327), which related to the immunity of El protein. 71.43% and 100% amino acids are homologous in P1 and in P2 respectively between the two strains. This indicated that P1 was a high variant, but P2 was high conservative.El fusion protein expressed in E. coli was non-glycosylated with the molecular weight of 23 kDa and formed inclusion body. HCV El inclusion body was used as antigen. El antibody could be detected in HCV positive human sera by ELISA. 48/90 of HCV positive sera could be detected by El antigen. The immunoreaction characteristic may correlate to a B-cell epitope in El protein at position 317-326aa, the RMAWDMMMNW amino acids domain. The reaction to the antibody was not related to protein glycosylation. The inclusion body of El protein was used as immunogen and could induce immune animals to produce IgG antibody specific to El. The mice IgG reached the highest value at the eighth week post immunization with the titer of 1 : 2400. The rabbits IgGreached the highest value at the eighth week post immunization with the titer of 1 : 5760. The antibody even could be detected in mucosa secretion. The protein induced mice CTL response at rather high level and DTH response. It also increase distinctly the ratio of CD8+T cells. It was presumed that the structure of the inclusion body was propitious to immunogen submitting. It showed that El protein had nice antigenity and immunogenicity.HCV El cDNA was expressed in sf9 cells by infection with Baculovirus. The products were mainly a protein with 40kDa and a series of protein expressed weakly with 29-40kDa. It was presumed that the difference in molecular weight resulted from glycosylation at different extents. El protein with 40kDa was used as antigen to detect HCV antibody in ELISA. 48/90 of HCV positive sera could be detected. The result was consistent with the inclusion body. But their reaction to antibody were different. The reaction produced by El glycoprotein was weaker than the El nonglycoprotein. HCV El glycoprotein with 40kDa as an immunogen could induce immune animals to produce specific immune response to HCV El. The mice IgG reached the highest titer at the sixth week post immunization. The titer was 1 I 2400. The rabbits IgG reached the highest value at the eighth week. The titer was 1 : 7680. HCV El glycoprotein induced mice slgG but not slgA response. The glycoprotein induced mice CTL response, distinct increase of CD8+T cells and DTH response. It indicated that HCV El glycoprotein could induce cell immunity response.HepG2 cells were transfected with pcE1 by lipofectamine. HCV El gene cDNA was experessed in HepG2 cells. The product was a single mature glycoprotein with 35kDa. The protein bound to HCV positive sera specifically. The mice thigh quadriceps cells incepted pcEl DNA and expressed as a glycoprotein that could be detected by affinity immunocytochemistry technology. Animals were immunized with bare pc更多还原