HCV E2蛋白诱导的体液免疫及CTL应答研究

应用PCR方法扩增出HCVE2基因编码417a.a～750a.a的DNA片段,克隆到原核表达载体pQE30 LacZ启动子下游,转化JM109菌株.在JM109菌株中诱导表达出N端含6个组氨酸的E2融合蛋白,用Ni-NTA-Superflow亲和层析柱纯化作为抗原免疫实验兔和BALB/c鼠.定期取免血,采用间接ELISA方法检测兔子体内针对E2的抗体水平和维持规律.结果显示,距初次免疫14d兔子体内已有抗体产生,直至免疫第55d抗体水平持续上升,之后抗体水平保持稳定,抗体滴度达到13200.六周后,取鼠脾脏制备淋巴细胞,定向刺激扩增后与经过重组真核表达质粒pCE2转染的P815细胞作用,利用LDH释放试验检测作用效果.在ET=2001的情况下,杀伤率超过30%.这些结果表明工程菌株表达的HCV E2蛋白具有良好的免疫原性,可以诱发免疫实验动物机体产生较高滴度的抗体及特异性CTL应答.由此我们认为E2蛋白是发展HCV预防工程蛋白疫苗的合适候选者.     

丙型肝炎病毒包膜蛋白E2可溶性单链可变区抗体在大肠杆菌中的表达

利用分子生物学技术,构建表达丙型肝炎病毒(HCV)包膜蛋白E2的人源单链可变区抗体(ScFv)的原核表达载体,并在大肠杆菌JM109中表达可溶性的HCV-E2-ScFv.以重组的HCVE2蛋白为包被抗原,利用噬菌体抗体库的表面展示技术,筛选到含有HCV-E2-ScFv基因的噬菌体克隆,从噬菌体抗体阳性克隆中提取质粒,经Ncol/NotI酶切鉴定后,该ScFv基因由750bp组成,将其亚克隆到pCANTAB5E载体中,转化大肠杆菌JM109,提取质粒进行DNA序列测定,符合ScFv的基因结构特点.IPTG诱导转化的大肠杆菌JM109,在其培养上清中获得了可溶性HCVE2单链可变区抗体的表达.酶联免疫吸附法(ELISA)证实表达的HCV-E2-ScFv具有与重组HCVE2蛋白的反应活性和特异性,对转化的JM109大肠杆菌上清中表述的HCV-E2-ScFv进行聚丙烯酰胺凝胶电泳(PAGE),证实表达的HCV-E2-ScFv的分子量为28kD.为应用HCV-E2-ScFv进行肝组织免疫组织化学和细胞内免疫基因治疗研究奠定了基础.     

hPLIC-2可溶性表达及多克隆抗体的制备

利用多聚酶联反应(PCR)扩增获得人hPLIC-2基因,并将该基因克隆到原核表达载体pGEX-2T上。转化大肠杆菌JM109,在27℃经IPTG诱导后,GST-hPLIC-2融合蛋白获得高效可溶性表达,并通过Glutathione Sephorase 4B获得纯化。使用结合有GST-hPLIC-2的Glutathione Sephorase 4B凝胶珠免疫大白兔制备了兔抗hPLIC-2多克隆抗体,抗体滴度大于1：16000。该抗体制备为研究hPLIC-2的功能提供了有用的工具。     

HCV包膜蛋白E2的克隆、表达与检测

构建丙型肝炎HCV包膜蛋白糖蛋白的E2基因原核表达载体,获得大量重组HCVE2蛋白,进行E2蛋白的抗原性及潜在保护作用研究。通过RT-PCR从HCVRNA阳性血清标本中扩增出975bp(383～708)E2基因片段,PCR产物经EcoR I和Sall I双酶切后连接到经同样酶切的PET-41a原核表达载体上,转化到大肠杆菌BL21(DE3)菌株,经Amp筛选,得到阳性重组质粒PET41a-HCVE2菌株,并以IPTG诱导蛋白表达,SDS-PAGE鉴定,表达产物经固定化金属配体亲和层析纯化,用ELLSA方法检测生物学活性。结果表明,构建的HCVE2包膜蛋白基因片段原核表达质粒所表达产物主要以包涵体形式存在,表达的融合蛋白与HCV阳性血清具有较好的反应原性。以HCVE2融合蛋白检测患者阳性血清具有良好的抗原性,有望能提高HCV抗体检测试剂盒的检出率。     

重组黄热病毒E蛋白结构域Ⅲ作为亚单位疫苗的抗原性和免疫原性

目的：表达纯化黄热病毒（YFV）囊膜蛋白（E蛋白）结构域Ⅲ,研究其作为亚单位疫苗预防YFV、日本脑炎病毒（JEV）感染的可能。方法：扩增YFVE蛋白结构域Ⅲ（YFDⅢ）的cDNA片段333bp,将其连接到原核表达载体pET-32a（＋）中,构建原核表达载体pET-YFDⅢ,转化感受态大肠杆菌Rosetta（DE3）,IPTG诱导表达重组YFDⅢ;用纯化的YFDⅢ免疫新西兰兔和BALB/c鼠,检测相关抗体滴度。结果：在大肠杆菌中可溶性表达了YFDⅢ融合蛋白,表达量约占菌体蛋白的50%;Western印迹及ELISA分析表明,纯化的YFDⅢ具有良好的抗原性和免疫原性;利用纯化的YFDⅢ免疫新西兰兔,获得了高达1∶4×105滴度的抗YFV抗体和1∶2×104滴度的抗JEV抗体;利用纯化的YFDⅢ免疫BALB/c鼠,获得了1∶7×104滴度的抗YFV抗体和1∶2×103滴度的抗JEV抗体。结论：重组YFDⅢ有较好的免疫原性,具有开发成亚单位疫苗的潜能。     

SARS冠状病毒S蛋白片段2的表达纯化与多克隆抗体的制备

采用RTPCR技术从SARS冠状病毒基因组扩增编码S蛋白的S2基因片段(第2170到2814位碱基),克隆到pMD18T载体并测序。用限制性内切酶消化后,S2基因亚克隆至表达载体pGEX4T2,转化大肠杆菌JM109,筛选鉴定阳性菌落。扩增培养含pGEXS2质粒的JM109大肠杆菌,经IPTG诱导,超声破菌,GSHSepharose亲和层析纯化目的蛋白,Westernblot检测SARS患者血清可以识别纯化的蛋白。用此蛋白免疫NIH小鼠,获得了高滴度抗GSTS2抗体的血清,为进一步研究SARS冠状病毒的亚单位疫苗奠定基础。     

SARS冠状病毒S蛋白片段2的表达纯化与多克隆抗体的制备

采用RT-PCR技术从SARS冠状病毒基因组扩增编码S蛋白的S2基因片段(第2170到2814位碱基),克隆到pMD18-T载体并测序.用限制性内切酶消化后,S2基因亚克隆至表达载体pGEX-4T-2,转化大肠杆菌JM109,筛选鉴定阳性菌落.扩增培养含pGEX-S2质粒的JM109大肠杆菌,经IPTG诱导,超声破菌,GSH-Sepharose亲和层析纯化目的蛋白,Western-blot检测SARS患者血清可以识别纯化的蛋白.用此蛋白免疫NIH小鼠,获得了高滴度抗GST-S2抗体的血清,为进一步研究SARS冠状病毒的亚单位疫苗奠定基础.     

丙型肝炎病毒E2基因DNA 免疫实验动物研究

将编码丙型肝炎病毒(HCV)E2蛋白417～750位氨基酸的DNA片段克隆到真核表达载体pcDNA 3.1(-)中的CMVIE启动子下游,构建成HCV E2重组真核表达质粒pcE2.ELISA法检测pcE2 DNA免疫兔血清中的E2抗体变化和维持规律,结果显示免疫20d已有抗体产生,30d后开始进入高峰,40d时达到最高值,至第90d抗体水平保持平稳,抗体滴度达到11600左右.流式细胞计数仪(FACS)检测pcE2 DNA免疫鼠CD4+、CD8+T淋巴细胞变化情况,与注射空载体pCDNA3.1(-)的阴性鼠相比,CD4+淋巴细胞水平略有上升,CD8+细胞水平有较大升高,增幅达35.46%.免疫组化检测结果显示注射pcE2的小鼠组织中有明显的阳性着色,而注射pcDNA3.1(-)的对照组小鼠免疫组化结果为阴性.以上结果表明pcE2在实验动物内表达出的HCV E2蛋白可以引起免疫动物的体液免疫应答和细胞免疫应答,尤其是MHC-1限制性杀伤性CD8+T淋巴细胞水平的提高对清除病毒是十分有利的,因此HCV E2 DNA免疫有可能成为预防和治疗HCV感染的一条新途径.     

表达重组猪瘟病毒E290多肽的干酪乳杆菌口服免疫特性及诱导特异性CTL反应的研究

经PCR扩增获得约60bp编码猪瘟病毒T细胞表位E290多肽基因片段,克隆至表达载体pPG-VP2中VP2基因5′端上游,命名为pPG-VP2-E290,电转化干酪乳杆菌,构建了表达猪瘟病毒E290多肽的重组乳酸菌系统。口服免疫BALB/c鼠和新西兰兔,检测诱导小鼠和兔体内产生特异性抗猪瘟病毒E290多肽IgG水平,并对E290多肽的CTL活性进行检测,同时对免疫兔进行猪瘟病毒攻毒实验,检测E290多肽抗体对免疫兔的保护作用。构建的重组猪瘟病毒T细胞表位的干酪乳杆菌具有良好的免疫性,口服免疫后的小鼠和兔血清中均检测到了较高水平的抗E290多肽抗体IgG,且能诱导小鼠机体产生抗猪瘟病毒的特异性CTL反应,亦证实猪瘟病毒E290免疫兔能够抵抗猪瘟病毒的攻击。     

糖基因Glt8d2重组蛋白的表达、纯化及多克隆抗体的制备

目的构建新糖基转移酶基因Glt8d2的原核表达载体,诱导融合蛋白的表达,并对其进行纯化;制备兔抗GltSd2蛋白多克隆抗体并进行鉴定。方法应用RT—PCR技术,以HepG2细胞mRNA为模板,扩增Glt8d2目的基因片段,构建原核表达载体pET-32a（＋）-Glt8d2。转化大肠埃希菌B121,异丙基-D-半乳糖苷诱导并通过SDS—PAGE凝胶电泳分析、Western印迹分析、生物质谱分析证实Gh8d2重组蛋白表达正确。大量表达后利用Ni’亲和柱对表达蛋白进行纯化及柱上复性。纯化蛋白免疫新西兰大白兔,获得抗Gh8d2蛋白的多克隆抗体。以纯化的Glt8d2重组蛋白为抗原,分别以免疫前后的新西兰兔血清作为第一抗体,利用Western印迹和酶联免疫吸附法对多克隆抗体进行特异性分析及效价检测。结果扩增获得Glt8d2基因片段,成功表达了Gh8d2重组蛋白,经SDS—PAGE凝胶电泳和Western印迹分析得到证实。成功获得重组蛋白及兔抗Gh8d2多克隆抗体。ELISA检测证实多克隆抗体效价〉1：320000。免疫组织化学分析显示,该基因编码的糖基转移酶在肝实质细胞内呈胞质模式表达分布。结论利用大肠埃希菌B121能够成功表达Gh8d2重组蛋白,获得高特异性、高效价兔抗Glt8d2重组蛋白的多克隆抗体,为今后研究Glt8d2基因的生物学功能研究奠定了基础。     

Induction of hepatic injury by hepatitis C virus-specific CD8+ murine cytotoxic T lymphocytes in transgenic mice expressing the viral structural genes

In the present study, we generated killer cells specific for hepatitis C virus (HCV) structural protein by re-stimulation of immune spleen cells from H-2(d) haplotype transgenic (Tg) mice, expressing the core, E1, E2, and NS2 genes of HCV regulated by the Cre/loxP switching system. The generated killer cells were conventional CD8(+)L(d) class-I MHC molecule-restricted cytotoxic T lymphocytes (CTLs) and specific for the HCV E1 structural protein. Because the CTLs could also kill hepatocytes from the Tg mice expressing HCV structural proteins in vitro, we attempted to transfer those CTLs intravenously into interferon regulatory factor-1 (IRF-1) negative, CD8-deficient Tg mice representing the HCV structural genes on hepatocytes to examine whether the inoculated CD8(+) CTLs can eliminate hepatocytes expressing the HCV genes in vivo. We observed an elevation of serum ALT level as well as damage of the liver tissue histologically. To our knowledge, this is the first demonstration to show that HCV-specific CD8(+) CTLs specifically attack hepatocytes expressing the HCV structural proteins both in vitro and in vivo.     

His-FemA融合蛋白表达载体的构建及其在原核细胞中的表达

目的 构建His标签的金黄色葡萄球菌甲氧西林耐药相关蛋白FemA的融合蛋白表达载体,并在大肠埃希菌中表达,为进一步研究femA基因的生物学功能和临床应用奠定基础.方法 根据GenBank中金黄色葡萄球菌femA基因序列,利用Primer Premier 5.0设计PCR引物,并在引物的5'加入BamHI及SalI酶切位点;以金黄色葡萄球菌基因组DNA为模版,PCR扩增出femA基因片段.将目的DNA片段及质粒pQE30分别进行双酶切、连接并转化大肠埃希菌DH5α;阳性克隆以PCR、双酶切及测序进行鉴定.将鉴定正确的pQE30-femA重组质粒转化大肠埃希菌JM109,异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达His-femA融合蛋白;采用SDS-PAGE及Western blot分析对表达蛋白进行验证.结果 经PCR、双酶切签定及序列测定,证实重组质粒pQE30-femA构建成功;重组质粒pQE30-femA转化大肠埃希菌JM109经IPTG诱导后,SDS-PAGE和Western blot分析显示表达出53 kD目的蛋白;经Bandscan软件分析,目的蛋白质在4h的表达量占细胞总蛋白的27.5％.结论 成功构建了His-FemA原核表达载体(pQE30-femA),并在大肠埃希菌中高效表达.     

Genetic Immunization of Wild-Type and Hepatitis C Virus Transgenic Mice Reveals a Hierarchy of Cellular Immune Response and Tolerance Induction against Hepatitis C Virus Structural Proteins

To study the effect of genetic immunization on transgenic expression of hepatitis C virus (HCV) proteins, we evaluated the immunological response of HCV transgenic mice to HCV expression plasmids. FVB/n transgenic mice expressing HCV structural proteins (core, E1, and E2) and wild-type (WT) FVB/n mice were immunized intramuscularly with plasmids expressing core (pHCVcore) or core/E1/E2 (pHCVSt). After immunization, HCV-specific humoral and cellular immune response was studied. Both WT and transgenic mice immunized with either HCV construct produced antibodies and exhibited T-cell proliferative responses against core or envelope. In WT mice immunized with pHCVSt, cytotoxic T-lymphocyte (CTL) activities were detected against E2 but not against core or E1, whereas strong CTL activities against core could be detected in WT mice immunized with pHCVcore. In pHCVSt-immunized, transgenic mice, CTL activities against the core or envelope were completely absent, but core-specific CTL activities could be detected in pHCVcore-immunized transgenic mice. A similar pattern of immune responses was also observed in other mouse strains, including a transgenic line expressing human HLA-A2.1 molecules (AAD mice). Despite the presence of a peripheral cellular immunity against HCV, no liver pathology or lymphocytic infiltrate was observed in these transgenic mice. Our study suggests a hierarchy of CTL response against the HCV structural proteins (E2 > core > E1) in vivo when the proteins are expressed as a polyprotein. The HCV transgenic mice can be induced by DNA immunization to generate anti-HCV antibodies and anticore CTLs. However, they are tolerant at the CTL level against the E2 protein despite DNA immunization.     

Structural elucidation of critical residues involved in binding of human monoclonal antibodies to hepatitis C virus E2 envelope glycoprotein

Human monoclonal antibodies derived from B cells of HCV-infected individuals provide information on the immune response to native HCV envelope proteins as they are recognized during infection. Monoclonal antibodies have been useful in the determination of the function and structure of specific immunogenic domains of proteins and should also be useful for the structure/function characterization of HCV E1 and E2 envelope glycoproteins. The HCV E2 envelope glycoprotein has at least three immunodistinctive conformation domains, designated A, B, and C. Conformational epitopes within domain B and C are neutralizing antibody targets on HCV pseudoparticles as well as from infectious cell culture virus. In this study, a combination of differential surface modification and mass spectrometric limited proteolysis followed by alanine mutagenesis was used to provide insight into potential conformational changes within the E2 protein upon antibody binding. The arginine guanidine groups in the E2 protein were modified with CHD in both the affinity bound and free states followed by mass spectrometric analysis, and the regions showing protection upon antibody binding were identified. This protection can arise by direct contact between the residues and the monoclonal antibody, or by antibody-induced conformational changes. Based on the mass spectrometric data, site-directed mutagenesis experiments were performed which clearly identified additional amino acid residues on E2 distant from the site of antibody interaction, whose change to alanine inhibited antibody recognition by inducing conformational changes within the E2 protein.     

HCV全长NS3基因表达及在抗体检测中的应用

丙型肝炎病毒 (ＨＣＶ)是引起非甲非乙型肝炎的主要病原因子。被ＨＣＶ感染的病例中 ,超过 5 0 %以上会引起持续性感染、慢性肝炎 ,最终可能引起肝硬化和肝细胞癌[1] 。ＨＣＶ严重威胁人类健康 ,但目前对丙肝患者尚缺乏有效的治疗手段 ,因此 ,严格把好血源关 ,提高对丙肝患者检出的灵敏度 ,是阻止丙肝血源传播的有效手段。丙型肝炎病毒基因组为单股正链ＲＮＡ ,核苷酸长约 9.5ｋｂ ,仅含一个开放阅读框 ,翻译成一个大的聚蛋白前体 ,由宿主细胞信号肽酶和病毒蛋白酶加工成多个成熟蛋白。其中非结构蛋白ＮＳ3分子量为 70ｋＤ ,有丝氨酸蛋白酶…     

Flt3配体增强HCV核心-包膜E2融合基因DNA疫苗诱导的细胞免疫应答

建立一种可高效诱导细胞免疫应答 ,对丙型肝炎病毒 (HCV)感染可能起预防和治疗作用的DNA疫苗。将小鼠Flt3配体 (FL)信号肽和胞外段cDNA插入结构优化的HCV核心 包膜E2融合抗原DNA疫苗pST CE2t,构建成pST CE2t FL。将pST CE2t FL转染COS7细胞 ,Westernblot和ELISA检测表明该重组质粒能表达HCV核心 包膜E2融合抗原和可溶性小鼠FL。分别将pST CE2t、pST CE2t FL和空载体pCI neo肌肉注射接种BALB c小鼠 ,检测小鼠的体液和细胞免疫应答。结果表明两种DNA结构均能在小鼠体内诱生细胞和体液免疫应答 ,但pST CE2t诱导的体液免疫应答强于pST CE2t FL ,而后者诱导的细胞免疫应答明显强于前者。FL能明显增强HCV核心 包膜E2融合抗原DNA疫苗诱导的细胞免疫应答 ,对于发展HCV预防和治疗性疫苗有潜在的应用价值。     

抗DAZAP2抗体的制备及在多发性骨髓瘤研究中的应用

为了进一步从蛋白水平上检测DAZAP2(deleted in azoospermia associated protein 2)在多发性骨髓瘤患者中的表达及研究DAZAP2的功能,以正常人的骨髓单个核细胞的总RNA为模板,RT-PCR扩增DAZAP2完整编码序列,构建原核表达重组质粒pQE30-DAZAP2,转化大肠杆菌JM109后,加IPTG诱导表达4h时,表达蛋白显著增加,Ni-NTA层析柱纯化蛋白。以该纯化蛋白免疫新西兰大白兔,制备抗DAZAP2抗体,ELISA检测抗体的效价在1：6400以上,Western blot检测抗体的特异性较好．用该抗体检测出DAZAP2雀6例正常人及4例多发性骨髓瘤患者中有表达,其他7例患者中没有表达,与RT-PCR结果一致,该抗体具有一定的临床应用前景,并能进一步用于功能研究．     

口服型HCV融合抗原DNA疫苗在小鼠诱导免疫应答

将编码一个外源信号肽、一个通用型辅助性T淋巴细胞抗原表位和HCV核心 包膜蛋白E2融合抗原基因的真核表达质粒pST CE2t(DNA疫苗 )转化到减毒鼠伤寒沙门菌SL72 0 7.将该重组菌口服接种BALB c小鼠 3次 .小鼠的抗HCV核心和E2抗体阳转率分别达 6 0 %和 70 % .体外以重组HCV核心或E2抗原刺激小鼠脾细胞 ,均使之发生明显的增殖反应 ,且小鼠脾细胞能有效杀伤表达HCV核心抗原的同系骨髓瘤细胞SP2 0 .这为研制高效免疫、成本低廉、接种方便的HCV疫苗提供了一个新的可行途径     

丙型肝炎病毒复合高变区1模拟表位蛋白的免疫原性分析

将丙型肝炎病毒高变区1(HVR1)模拟表位融合基因插入原核表达载体pGEX-4T-1,在大肠杆菌BL21(DE3)中进行表达,经亲和层析和凝胶过滤层析获得HCVHVR1模拟表位融合蛋白。用Westernblot和ELISA检测融合蛋白与HCV抗体阳性血清的结合情况。皮下注射免疫BALB/c小鼠,用ELISA检测小鼠血清中的抗HCV抗体水平及其与天然HCV高变区1合成肽的交叉反应。结果表明融合蛋白能与HCV抗体阳性血清特异结合,融合蛋白与HCV抗体阳性血清的结合频率为71.6%(25/35)。融合蛋白免疫小鼠后能有效诱导免疫应答,其诱生的特异性抗体最高滴度达104(免疫后第8周),且该抗体能同2条天然HCVHVR1合成肽发生交叉反应。本研究提示,HCV复合HVR1模拟表位融合蛋白在丙型肝炎疫苗的研发中可能具有潜在应用价值。     

Elicitation of immunity in mice after immunization with the S2 subunit of the severe acute respiratory syndrome coronavirus

The S2 domain of the severe acute respiratory syndrome coronavirus (SARS-CoV) spike (S) protein is responsible for fusion between virus and target cell membranes, and is expected to be immungenic. In this study, we investigated the immune responses against the S2 subunit in BALB/c mice, which were vaccinated either with plasmid DNA encoding the S2 domain (residues 681-1120), the recombinant S2 fragment (residues 681-980) in incomplete Freund's adjuvant, or with inactivated SARS-CoV. The increased number of specific cytotoxic cells (CTLs) and the high titer of specific antibody showed stimulation of both arms of the immune system in these groups. The shift in cytokines suggested that Th1-polarized immune response was induced by plasmid pCoVS2, meanwhile the Th2-dominant response was induced by recombinant S2 fragment and inactivated vaccine. However, the titer of neutralizing antibodies was only detectable in mice immunized with inactivated virus, but not with pCoVS2 plasmid. Taken together, the S2 domain could induce specific cellular immune response and a high level of total IgG but little neutralizing antibodies against infection by SARSCoV.