检测Epstein—Barr病毒特异性细胞毒性T淋巴细胞方？…

目的 建立一种非放射性、简便易行的可检测特异性细胞毒性Ｔ淋巴细胞的方法，并且初步应用于Ｅｐｓｔｅｉｎ－Ｂａｒｒ病毒的细胞免疫应答。方法 用重组的ＥＢＶ－ＬＭＰ１痘苗病毒、ＴＫ＾＋痘苗病毒和杆状病毒系统表达的ＥＢＶ－ＬＭＰ！蛋白分别免疫Ｂａｌｂ／Ｃ小鼠，用Ｐ８１５细胞和乳酸脱氢酶法检测ＥＢ病毒特异性细胞毒性Ｔ细胞的杀伤效应。结果 重组ＥＢＶ－ＬＭＰ１痘苗病毒免疫组原发ＣＴＬ水平和体外诱生的二次ＣＴＬ     

特异性细胞毒性T细胞检测方法的建立及其在重组痘苗病毒活疫苗细胞免疫研究中的应用

报道了用ＣＢＡ／Ｊ小鼠、Ｌ９２９细胞及ＭＴＴ比色法建立的特异性细胞毒性Ｔ细胞（ＣＴＬ）的检测方法及其在重组痘苗病毒活疫苗细胞免疫研究中的应用。通过检测痘菌病毒特异性初发ＣＴＬ的动态变化及其二次反应ＣＴＬ，表明，本法能较好地检测初发及二次反应ＣＴＬ水平的变化。用本法检测了不同启动子控制下的表达Ｅｐｓｔｅｉｎ－Ｂａｒｓ（ＥＢ）病毒膜抗原（ＥＢＶ－ＭＡ）的重组痘菌病毒ＥＢＶ－ＭＡ特异性ＣＴＬ、以及与表达人白细胞介素－２的重组痘苗病毒联合免疫时的ＥＢＶ－ＭＡ特异性ＣＴＬ。结果表明，Ｐ７５００启动子表达的ＥＢＶ－ＭＡ比Ｐ１１０００启动子表达的ＥＢＶ－ＭＡ诱导的ＥＢＶ－ＭＡ特异性ＣＴＬ高，但无显著性差异。当上述重组痘苗病毒与表达人白细胞介素－２的重组痘苗病毒联合免疫时，均能使二者的ＥＢＶ－ＭＡ特异性ＣＴＬ增高，但增高幅度本身及其二者之间均无显著性差异。     

含HPV16 E7的嵌合型VLPs引发细胞溶解反应

目的 探讨ＢＰＶ（牛乳头状瘤病毒）Ｌ１／ＨＰＶ（人乳头状瘤病毒）１６Ｅ７嵌合型乳头状瘤病毒样颗粒（ＶＩＰｓＸ）的免疫学特性。方法 将表达纯化的含ＢＰＶＬ１／ＨＰＶ１６Ｅ７的嵌合型ＶＬＰｓ作为抗原在体外刺激ＥＬ－４细胞并对其进行细胞毒性Ｔ淋巴细胞（ＣＴＬ）反应分析。结果 嵌合型ＶＬＰｓ可引发特异的ＣＴＬ反应。Ｌ１－ＶＬＰｓ抗体能阻断这种特异性细胞溶解反应。结论 含ＢＰＶＬ１／ＨＰＶ１６Ｅ７嵌合型的Ｖ     

Epstein—Barr病毒潜伏膜蛋白2A重组痘苗病毒转染DC及特异性CTL的体外诱导

通过ＥＢ病毒ＬＭＰ２Ａ重组痘苗病毒转染的ＤＣＳ体外诱导ＬＭＰ２Ａ特异性ＣＴＬ,并通过ＧＭ－ＣＳＦ、ＩＬ－４和ＴＮＦ－ａ培养体系,我们诱导出了人外周血单核细胞来源的ＤＣ。同时选用在鼻咽癌患者中表达的ＥＢ病毒潜伏蛋白之一ＬＭＰ２Ａ作为靶基因,利用重组痘苗病毒转染诱导的ＤＣＳ。ＤＣＳ与自体ＰＢＭＣＳ混合培养,在ＩＬ－２的刺激作用下获得特异性ＣＴＬ。结果如下：１．人外周血单核细胞经ＧＭ－ＣＳＦ、ＩＬ－４、ＴＮＦ－ａ的混合培养,１０天可获得成熟的功能性ＤＣＳ。ＦＡＣＳ检测显示ＤＣ表面相对特异性标志ＣＤ８３…     

中国流行株HIV—1gag和hIL—2／hIL—6核酸疫苗构建与实验免疫研究

目的 探讨中国流行株ＨＩＶ－１ｇａｇ与ｈＩＬ－２／ｈＩＬ－６共表达重组核酸疫苗闰的免疫效果。方法 以核酸疫苗质粒ｐＩＲＥＳ１ｎｅｏ为表达载体,构建重组核酸疫苗质料ｐＩＲＥＳ１－ｇａｇ、ｐＩＲＥＳ１－ｇａｇ－ｈＩＬ－２、ｐＩＲＥＳ１－ｇａｇ－ｈＩＬ－６,通过间接免疫荧光试验、Ｄｏｔ－ＥＬＩＳＡ检测ｇａｇ／ｈＩＬ－２／ｈＩＬ－６基因的表达产物。另将此重组核酸疫苗质粒免疫Ｂａｌｂ／ｃ小鼠,进行淋巴细胞转化试验、ＣＤ４＾＋、ＣＤ８＾＋Ｔ淋巴细胞数量测定、细胞毒性Ｔ淋巴细胞（ＣＴＬ）特异性杀伤作用检测及血清抗体检测,结果 构建的重组质粒转染ＢＨＫ细胞后可表达目的基因,免疫小鼠后可有效地刺激淋巴细胞增殖、诱导特异性ＣＴＬ反应,当和ｈＩＬ－２／ｈＩＬ－６共表达时免疫效果更加显著。讨论 与Ｇａｇ蛋白共表达的ｈＩＬ－２／ｈＩＬ－     

Epstein—Barr病毒膜抗原在昆虫细胞sf9中的表达,纯 …

将Ｅｐｓｔｅｉｎ－Ｂａｒｒ（ＥＢ）病毒膜抗原基因（ＥＢＶ－ＭＡ）称ＭＡ１和截去穿膜区的ＭＡ基因称ＭＡ２分别插入杆状病毒表达载体ｐＶＬ－９４１。将两种重组质粒分别与杆状病毒ＤＮＡ共转染ｓｆ９细胞后获得Ｂａｃｕｌｏ－ＭＡ１和Ｂａｃｕｌｏ－ＭＡ２两种重组病毒，表达产物分别位于重组病毒感染的细胞表面或释放到细胞培养液中。免疫荧光方法检查，在感染的细胞表面表达产物与抗ＭＡ的单克隆抗体特异性地结合，ＳＤＳ－Ｐ     

系统性红斑狼疮患者B淋巴细胞EBV-LMP1和ZEBRA的表达研究

目的：探讨ＥＢＶ－ＬＭＰ１和ＺＥＢＲＡ在系统性红斑狼疮患者（ＳＬＥ）的表达情况。方法：间接荧光免疫标记，流式细胞仪检测。结果：ＳＬＥ患者Ｂ淋巴细胞中ＥＢＶ－ＬＭＰ１和ＺＥＢＲＡ的表达显著高于正常对照组（Ｐ＜０．０１）。活动期患者ＣＤ２３＋细胞ＥＢＶ－ＬＭＰ１和ＺＥＢＲＡ的表达率均高于ＣＤ１９＋细胞（Ｐ＜０．０１）。非活动期患者ＣＤ２３＋细胞ＥＢＶ－ＬＭＰ１表达也高于ＣＤ１９＋细胞（Ｐ＜０．０１）。但ＥＢＶ－ＺＥＢＲＡ表达在两亚群间差异没有统计学意义（Ｐ＞０．０５）。结论：朋病毒参与了ＳＬＥ的发病机制，病毒主要以潜伏期状态存在于患者中，病毒复制促进病情发展，检测Ｂ淋巴细胞ＥＢＶ－ＬＭＰ１和ＺＥＢＲＡ的表达率，有助于病情活动指标的判断。     

重组rAAV-LMP诱导的特异性细胞毒T细胞对LMP阳性靶细胞的识别与杀伤

在正常个体中Ｅｐｓｔｅｉｎ－Ｂａｒ（ＥＢ）病毒是由病毒特异性细胞毒Ｔ淋巴细胞（ＣＴＬ）所控制，虽不能清除病毒，却对于控制细胞处于潜伏感染状态是必需的。抽取病人血分离淋巴细胞，在实验室制备ＥＢ病毒特异性ＣＴＬ，然后回输到病人体内，具有预防和治疗ＥＢ病毒相关疾病的意义。我们将ＥＢ病毒潜伏感染膜蛋白（ＬＭＰ）基因重组到腺病毒伴随病毒载体ｐＡＣＰ中去，与包装质粒Ａｄ８共转染已感染了Ⅱ型腺病毒的２９３细胞，获得重组病毒ｒＡＡＶ－ＬＭＰ，用此病毒感染淋巴细胞并表达ＬＭＰ，用高能Ｘ线照射灭活，与自体淋巴细胞共培养产生特异性ＣＴＬ。以ＥＢ病毒转化的类淋巴母细胞作靶细胞与ＣＴＬ反应，用ＢＬＴ活性法测定ＣＴＬ活性。结果表明，４株ＣＴＬ均能够识别和杀伤对应的靶细胞，并且随着ＣＴＬ数量的增加和反应时间的延长，上清中ＢＬＴ活性也增强。     

人巨细胞病毒基质蛋白PP150是CTL识别的提呈抗原之一

用痘苗病毒载体ＰＳＣ１１构建编码人巨细胞病毒（ＣＭＶ）基质蛋白ＰＰ１５０的重组痘苗病毒，刺激５例血清ＣＭＶ阳性供体全部诱导出ＰＰ１５０特异性细胞毒性Ｔ淋巴细胞（ＣＴＬ）反应。ＰＰ１５０特异性ＣＴＬ不仅能溶解Ｖａｃ．ＰＰ１５０感染细胞，也溶解ＣＭＶ感染细胞。在病毒感染早期（２小时）和用ＲＮＡ合成抑制剂ＡｃｔＤ阻止感染细胞病毒基因转录的条件下，ＰＰ１５０特异性ＣＴＬ能同样有效地溶解ＣＭＶ感染细胞。提示随病毒颗粒渗入的ＰＰ１５０在ＣＭＶＤＮＡ复制前即能有效地被提呈。上述结果表明，ＰＰ１５０是ＣＴＬ识别的抗原之一。     

人脐带血抗巨细胞病毒和EB病毒的特异性细胞毒？…

目的：探讨用人的脐带血单个核细胞体外同时扩增对抗ＥＢ病毒（ＥＢＶ）和巨细胞病毒（ＣＭＶ的特异性细胞毒性Ｔ淋巴细胞的可行性。方法：利用人的脐带血单个核细胞（ＣＢＭＣ）,通过ＥＢＶ感染转化成Ｂ成淋巴细胞细胞株（ＢＬＣＬ）,再通过逆转录病毒载体,将ＣＭＶ蛋白基因ｐｐ６５导入ＢＬＣＬ,用这种细胞体外刺激同一供者脐带血的ＣＢＭＣ产生细胞毒性Ｔ细胞（ＣＴＬ）,经「＾５１Ｃｒ」释放实验（ＣＲＡ）检测产生的ＣＴＬ     

Effective induction of type 1 cytotoxic T cell responses in mice with DNA vaccine encoding two hepatitis C virus cytotoxic T lymphocyte epitopes

The aims of this study were to explain whether a multiple cytotoxic T lymphocyte (CTL) epitope-based anti-hepatitis C virus (HCV) DNA vaccine can induce specific CTL responses to each HCV CTL epitope independently and long-term CD8(+) T cell memory responses, and to determine the cytokine secretion pattern and subtype of epitope-specific cytotoxic T cells. A multi-CTL epitope gene, which consists of two epitopes of HCV (H-2(d)-restricted HCV core(133142) and E1(315322)), was cloned into the eukaryotic expression vector pcDNA3.1. BALB/c mice (H-2(d) restricted) were vaccinated intramuscularly with this multi-CTL epitope-based DNA vaccine. The epitope-specific CTLs against target cells (P815,H-2(d) restricted) pulsed with various CTL epitope peptides were detected by lactate dehydrogenase release assay, and the precursor frequency of epitope-specific CTLs was determined by limiting dilution analysis. Cytokines (interleukin [IL]-2, IL-4, and interferon-) in culture supernatants were determined by enzyme-linked immunosorbent assay. The multi-CTL epitope-based DNA vaccine directed against two HCV CTL epitopes could induce specific CTL responses to each of the two CTL epitopes independently and long-term CD8(+) T cell memory responses. The epitope-specific cytotoxic T cells produced helper T cell type 1 cytokines. This work demonstrated that multiepitope DNA vaccination is a potential strategy to control HCV infection.     

The induction of murine cytotoxic T lymphocytes by bluetongue virus

Summary After inoculation with live bluetongue virus, mice produced cytotoxic T lymphocytes (CTL) which showed virus and H-2 restriction. Inactivated preparations failed to induce CTLs. On secondaryin vitro stimulation, specifically sensitised memory cells also produced high numbers of CTLs. The need for replicating virus to induce primary CTLs, evidence for partial type specificity and the role which cell-mediated immunity might play in the early stages of a bluetongue virus infection are discussed.With 1 Figure     

H-2 restriction and serotype crossreactivity of anti-reovirus cytotoxic T lymphocytes (CTL)

Murine anti-reovirus cytotoxic T lymphocytes (CTLs) were analyzed for H-2 restricted recognition of virus infected target cells and for potential cross-reactivity with cells infected by reovirus serotype 1 (T1; Lang strain) or by serotype 3 (T3; Dearing strain). Anti-reovirus CTL specifically lysed virus infected cells and lysis was shown to be H-2 restricted by the H-2Dd, H-2Ld, H-2Kd, H-2Kb, and H-2Kk antigens. No H-2 antigens were identified which failed to restrict virus recognition by anti-reovirus CTL. Anti-T1 and anti-T3 CTLs were also shown to crossreact completely with cells infected with the opposite virus serotype. Thus, anti-reovirus CTLs are restricted by a broad spectrum of H-2 antigens and they detect common rather than unique structural components of these two viral serotypes.     

PRRSV M 蛋白细胞系的培育及其在细胞毒性T淋巴细胞检测方法中的应用

目的 探讨PRRSV M基因在哺乳动物细胞NIH/3T3细胞中的表达,建立一种非放射性、简便易行的检测特异性细胞毒T淋巴细胞的方法.方法 应用RT-PCR扩增得到M基因并克隆入真核表达载体pcDNA3,构建成重组表达载体pcDNA3-M;将重组质粒转染至NIH/3T3细胞,G418筛选克隆;IFA检测M蛋白在转基因NIH/3T3细胞中的表达.用表达M蛋白的重组腺病毒rAd-M免疫小鼠,用表达M蛋白的NIH/3T3细胞和乳酸脱氢酶法检测PRRSV特异性细胞毒T淋巴细胞的杀伤效应. 结果 获得有G418抗性的NIH/3T3/M细胞克隆;IFA检测到有PRRSV M蛋白表达.重组腺病毒rAd-M免疫组可以诱发CTL应答,并明显高于wtAd和PBS对照组. 结论 培育成功一株能表达PRRSV M蛋白的细胞株NIH/3T3/M,可作为PRRSV CTL检测方法中的靶细胞,证明PRRSV M基因能够诱发特异性的细胞免疫.     

The reovirus-specific cytotoxic T cell response is not restricted to serotypically unique epitopes associated with the virus hemagglutinin

Reovirus, a virus that contains neither an envelope nor glycosylated polypeptides, has been found to induce virus-specific, major histocompatibility complex (MHC) class I antigen restricted, cytotoxic T lymphocyte (CTL) responses. The cytotoxic T cells require in vitro stimulation in the presence of virus to phenotypically express cytotoxic activity. Utilizing reovirus types 1 and 3, the CTLs derived from mice infected with one serotype can lyse target cells infected with a second serotype of reovirus. In addition, lymphocytes primed in vivo with one serotype develop into fully functional CTLs during in vitro stimulation with the other serotype of reovirus. Therefore, these results suggest that reovirus induced CTLs are virus, but not serotype specific. Common determinants shared by reovirus polypeptides from reovirus types 1 and 3 are most likely the stimuli for the majority of CTLs responses to reovirus.     

H-2-linked murine cytotoxic T cell responses specific for sendai virus-infected cells.

CBA (H-2k) mouse-derived lymphochoriomeningitis virus and herpes simplex virus-specific cytotoxic T lymphocytes lyse virus-infected target cells compatible on either the H-2k or H-2D region. In contrast, CBA, C3H and AKR (H-2k) mouse-derived sendai virus-specific cytotoxic T lymphocytes (CTL) fail to lyse H-2D-compatible virus-infected cells. A similar lack of H-2D region-associated lytic activity was found with C57BL/6 and C57BL/10 (H-2b) mice as well as with the recombinants B10.A (2R) [Kb-Db] and B10.A (4R) [Kk-Db]. On the other hand, BALB/c (H-2d) mice and A/J (H-2a) mice do generate H-2Dd-associated sendai virus-specific CTL. These results are in contrast to those obtained with (CBA X BALB/c)F1 and B10.HTT [Ks-Dd] mice, which failed to mount Dd region-associated CTL responses. It is concluded that D region-associated sendai virus-specific CTL responsiveness varies with the H-2 genotype of the responder cells.     

An influenza haemagglutinin-specific IgG enhances class I MHC-restricted CTL killing in vitro.

Lungs of H-2k mice co-inoculated with type A/WSN influenza virus+detective interfering WSN virus contain haemagglutinin (HA)-specific IgG which have three different activities. These have been purified by adsorbtion and elution using different forms of HA. The first IgG recognizes HA in a form present on H-2k cells infected with a vaccinia virus recombinant expressing the WSN HA gene (vaccinia-HA virus), but not on virus particles, and enhances class I major histocompatibility complex (MHC)-restricted killing of WSN-infected H-2k target cells by primary cytotoxic T lymphocytes (CTL) from the lungs of WSN-infected H-2k mice; it also confers on primary CTL from the lungs of WSN-infected H-2d mice the ability to lyse WSN-infected H-2k targets. This IgG is therefore analogous to the T-cell receptor in that it is antigen specific and MHC restricted. A second IgG recognizes HA in a form present on both H-2k and H-2d cells infected with the vaccinia-HA virus but not present on virus particles and inhibits CTL lysis of WSN-infected syngeneic target cells. Only the third binds to virus particles; this inhibits agglutination of red cells, but is non-neutralizing. It also inhibits CTL lysis of WSN-infected syngeneic targets. Thus we present evidence that HA-specific IgG may have a significant role in regulating CTL responses to influenza virus in vivo and that one of these IgG is MHC-restricted in its recognition of viral antigen. Finally, in vivo significance of these antibodies is indicated by the finding that adoptively transferred CTL-enhancing IgG protects mice from lethal WSN infection.     

Secondary cytotoxic allograft response in vitro. I. Antigenic requirements

The antigenic requirementsfor in vitro induction of secondary murine cytotoxic allograft responses were tested. The proliferative responses were assayed by the [³H]thymidine uptake technique; the generation of cytotoxic T lymphocytes (CTL) was tested in a ⁵¹Cr-cytotoxicity assay. Spleen cells from normal or alloantigen preimmunized CBA mice (H-2^k) were used as responder cells. Allogeneic x-irradiated splenic lymphocytes (normal stimulator cells) were UV light treated, heat treated or glutaradehyde fixed and subsequently tested for their capacity to induce CTL in a primary or secondary mixed lymphocyte culture (MLC). In addition allogeneic fibroblasts were tested as stimulator cells. The results obtained suggest that although they fail to trigger significant proliferative and cytotoxic T cell responses, in a primary MLC certain allogeneic stimulator cells, are able to induce strong cytotoxic T cell activity in a secondary MLC. The generation of these secondary CTL is preceded by only marginal cell proliferation.     

IL-12和IL-18基因免疫对HBcAg核酸疫苗诱导小鼠(H-2d)特异性免疫应答的影响

目的：观察IL-12和IL-18基因免疫对HBcAg核酸疫苗诱导小鼠（H-2d）特异性体液免疫和细胞免疫应答的影响.方法：用肌肉注射法将HBV核心区DNA疫苗、IL-12质粒和IL-18 质粒接种BALB/c小鼠;ELISA法检测小鼠血清抗-HBc（IgG）及IgG亚类（IgG1、IgG2a）;LDH释放法检测小鼠脾细胞HBcAg特异性CTL活性.结果：免疫6周后,HBcAg DNA疫苗联合IL-12质粒、IL-18质粒和IL-12＋IL-18质粒组小鼠的血清抗HBc终点滴度均明显高于单纯注射HBcAg DNA疫苗组小鼠（P＜0.05）,抗HBc IgG亚类以IgG2a占优.DNA疫苗免疫的各组小鼠,HBcAg特异性细胞毒性T淋巴细胞杀伤率均高于对照组（P组）,其中C＋IL-18组和C＋IL-12＋IL-18组中CTL值明显高于C组,尤以C＋IL-12＋IL-18组中的CTL杀伤率最高.结论：IL-12和IL-18基因与HBcAg DNA疫苗联合免疫,不仅能增强HBcAg特异性体液免疫应答,而且能增强HBcAg特异性CTL的杀伤活性.