EB病毒潜伏膜蛋白2A抗原表位的融合表达及其意义

为了探讨含有EB病毒潜伏膜蛋白2A(LMP2A)抗原表位片断表达的融合蛋白在鼻咽癌血清学检测中的应用意义,通过重叠延伸PCR方法,合成了3对相互重叠的寡核苷酸引物,涵盖LMP2A的主要的4个抗原表位,将它们拼接在一起构建一个多肽融合基因,克隆到PGEX-4T-2载体中表达融合蛋白,以GST亲和层析柱法纯化融合蛋白,鉴定后以此为抗原检测鼻咽癌患者的血清。结果表明,获得了含EB病毒LMP2A主要的4个抗原表位的融合蛋白(EC2A),蛋白纯度达90%以上,ELISA结果显示鼻咽癌患者血清的检出率为77.9%,正常人群血清为阴性,与常规的VCA-IgA法进行比较,有9份(13.3%)血清VCA-IgA为阴性而EC2A-IgG检出阳性,为鼻咽癌的临床检测提供了新思路,也为后续的单克隆抗体制备奠定了基础。     

复制缺陷型人IL-2重组腺病毒的制备与鉴定

目的将人ＩＬ－２的重组腺病毒载体与Ａｄ５腺病毒ＤＮＡ末端肽复合物同源重组，高效制备人ＩＬ－２的复制缺陷型重组腺病毒，并进行体外表达和活性检测。方法将含全部编码序列的人ＩＬ－２ｃＤＮＡ置于真核表达载体ｐＣＩｃｃ的ＣＭＶ启动子下游，切出含ＣＭＶ启动子、人ＩＬ－２ｃＤＮＡ和ＳＶ４０ｐｏｌｙＡ信号肽序列的ＣｌａＩ片段，插入Ｅ１区替代的腺病毒载体ｐＡｘｌｏｗ，选择左向转录的人ＩＬ－２重组腺病毒载体ｐＡｘｌｃｗ．ＣＩｈＩＬ－２与经ＥｃｏＴ２２Ⅰ酶切的Ａｄ５腺病毒ＤＮＡ－末端肽复合物共转染２９３细胞；通过同源重组获得人ＩＬ－２的复制缺陷型重组腺病毒；体外转染人宫颈癌ＨｅＬａ细胞、人Ｔ细胞淋巴瘤Ｈｕｔ７８细胞和原代人皮肤成纤维细胞。结果扩增到的病毒滴度达２．１×１０９ＰＦＵ／ｍｌ；体外转染的人肿瘤细胞均检测到ＩＬ－２的表达（４００～２６００Ｕ／１０６细胞／２４小时）。结论所制备的复制缺陷型重组腺病毒能有效介导人ＩＬ－２的基因转移，可望用于肿瘤基因治疗的临床研究。     

EBV-LMP2A重组腺病毒体外转染树突状细胞激发特异性CTL的研究

目的：用EBV潜伏膜蛋白2A(EBV-LMP2A)重组腺病毒转染树突状细胞(DC)激发特异性细胞毒性T细胞(CTL)，分析CTL的特性。方法：用AdS-LaMP2A重组腺病毒转染EBV健康携带者及鼻咽癌患者的DC，与自体来源的外周血单个核细胞(PBMC)混合培养，激发LMP2A特异性CIL。用LDH释放法检测CIL杀伤活性；流式细胞术(FACS)检测培养细胞群体中CD3^ 、CD4^ 、CD8^ 、CD56^ 细胞的组成；生物活性法检测细胞培养上清中IFN-γ含量；RT-PCR分析CTL的FasL mRNA表达。结果：EBV健康携带者及鼻咽癌患者的PBMC，经AdS-LMP2A转染的自体DC两次刺激后，都能诱导出显著的EBV-LMP2A特异的CIL。EBV健康携带者CTL的杀伤活性，随DC刺激次数的增加而逐渐增强，诱导的CTL细胞群体以CD^4 和CD8^ 细胞组成为主，且CD4^ 细胞比例高于CD8^ 细胞，另含少量CD56^ 细胞；在不同时段所诱导的CTL上清中，均含一定量的IFN-γ并随刺激次数和诱导时间的延长呈上升趋势；RT-PCR研究表明，所诱导的CTL有FasL mRNA的表达。结论：以腺病毒载体介导EBV-LMP2A基因转染的成熟DC，在体外能激发较强的LMP2A特异的功能性CTL，可用于EBV相关NPC的免疫治疗。     

EB病毒相关胃癌组织中病毒潜伏期基因表达的研究

Epstein Barrvirus(EBV)与多种人类恶性肿瘤如鼻咽癌、Burkitt淋巴瘤和免疫功能缺陷病人的淋巴组织过度增生性疾病等密切相关 ,近年来研究表明约 2 %～ 16 %的胃癌组织EBV阳性。EBV具有潜伏感染的特点 ,通常认为EBV潜伏期基因如核抗原家族基因 (EBNAs)和潜伏膜蛋白基因 (LMP) 1与病毒的细胞转化功能有关。明确EBV在肿瘤组织中的存在状态和表达活动 ,是研究其致癌机理的基础。本研究首先应用PCR Southern杂交检测 12 6例胃癌和相应癌旁组织中EBVDNA ,进一步采用原位杂交检测PCR阳性标本石蜡包埋组织中EBV编码小RNA(EBER1…     

EB病毒潜伏膜蛋白2A基因重组逆转录病毒载体和稳定表达细胞株的构建及鉴定

目的:克隆EB病毒(EBV)潜伏膜蛋白2A(LMP2A)全长基因, 并构建含有该目的基因的重组逆转录病毒载体pGEZ-LMP2A, 转染真核细胞后获得稳定表达LMP2A的细胞株.方法:采用RT-PCR技术从B95.8细胞中获得EB病毒LMP2A全长基因, 克隆到测序载体pMD18-T中, 经测序后再亚克隆到逆转录病毒载体pGEZ-Term中, 与两辅助病毒载体PHIT456、 PHIT60通过Lipofectamine2000共转染包装细胞293T, 测定产生的重组逆转录病毒滴度并感染小鼠成纤维细胞L929, 经Zeocine筛选后得到稳定的细胞克隆, 用RT-PCR和Western blot法检测细胞中LMP2A mRNA和蛋白表达情况.结果:重组逆转录病毒载体pGEZ-LMP2A经测序鉴定正确;转染后上清中病毒的滴度为5×108 cfu/L, 感染L929细胞后用Zeocine筛选出稳定的细胞克隆;RT-PCR和Western blot检测结果表明筛选出的细胞克隆能稳定表达EBV-LMP2A.结论:表达EBV-LMP2A细胞株的构建为蛋白制备与纯化奠定了物质基础, 也为后续的EBV相关性疾病疫苗的研究提供了新思路.     

LMP1基因重组慢病毒载体的构建及表达性能鉴定

为了进一步探讨EB（Epstein Barr）病毒潜伏膜蛋白1（LMP1）的作用机制及其功能，我们构建了EDL2-N-LMP-慢病毒载体，并对所构建的EDL2-N-LMP-慢病毒载体结构及表达进行鉴定。     

EB病毒LMP2A特异性CTL的体外诱导及分析

用EBV LMP2A重组痘苗病毒 (rVV LMP2A )转染人树突状细胞 (DC ) ,转染后的DC分别在第 1、 7、 14天刺激相同MHC背景的T细胞 ,在IL 2作用下诱导LMP2A特异性CTL。用LDH释放法检测CTL杀伤活性 ;流式细胞术 (FACS )检测CTL诱导分化过程中CD3+ 、CD4 + 、CD8+ 、CD5 6 + 等细胞的分群变化 ;RT PCR检测细胞分化过程中FasLmRNA表达 ;生物活性法检测功能性细胞因子IFN γ的分泌。结果显示本法诱导的CTL对靶细胞有特异性杀伤活性 ,第 2次和第 3次DC刺激后杀伤活性有所上升 ;在CTL诱导分化的第 7、 14、 2 1天细胞分群以CD4 + 、CD8+ 细胞为主 ;RT PCR证实所诱导的细胞内有FasLmRNA的表达 ;随细胞培养天数的增加IFN γ分泌增加 ,在第 14天达到较高水平。研究表明重组痘苗病毒载体rVV LMP2A转染的DC刺激T细胞可诱导出EBV LMP2A特异性CTL。     

EB病毒潜伏膜蛋白LMP1有关信号传导通路的研究进展

EB病毒的致癌性与细胞源性基因和潜伏感染基因有关，其中潜伏膜蛋白（1atent membrane protein，LMP）家族LMP1、LMP2A、LMP2B可干扰信号传导通路并诱导B细胞转化。     

EB病毒潜伏膜蛋白2的研究回顾

Epstein Barr病毒 (EBV)属于疱疹病毒科γ亚科 ,在人类中广泛传播。大多数EBV的初次感染是在患者幼儿时期 ,而且没有明显的临床症状 ,终身携带病毒。EBV与越来越多的人类肿瘤相关 ,包括由于免疫抑制引起的免疫增生性淋巴瘤、Burkitt淋巴瘤、NPC、HD和多种T细胞淋巴瘤等疾病。EBV潜伏感染时表达 8种蛋白 (6种核蛋白EBNA1、EBNA2、EBNA3A、EBNA3B、EBNA3C、EBNALP和 2种膜蛋白LMP1和LMP2 )。在这些与转化相关的病毒蛋白中 ,EBNA1、LMP1、LMP2是在NPC肿瘤标本和EBV相关肿瘤中可以检测到的蛋白。1　LMP2的功能198…     

EB病毒转化淋巴母细胞LMP-1,LMP-2A及LMP-2B的表达

目的了解EB病毒转化淋巴母细胞LMP-1,LMP-2A,LMP-2B基因的表达改变。方法采用实时定量PCR方法分别检测并比较同一献血员来源的正常人淋巴细胞与EBV转化淋巴母细胞前后LMP基因(LMP-1、LMP-2A、LMP-2B)的表达改变。采用Western bolt检测LMP-1蛋白表达。结果 LMP-1、LMP-2A、LMP-2B基因在转化淋巴母细胞比在正常人淋巴细胞的表达分别上调863倍、1 763倍、90 078倍。Western bolt检测LMP-1蛋白在转化淋巴母细胞中的表达比正常人淋巴细胞明显增强。结论在EBV转化淋巴母细胞过程中LMP-1、LMP-2A和LMP-2B表达均上调。     

Minimal protein domain requirements for the intracellular localization and self-aggregation of Epstein-Barr Virus Latent Membrane Protein 2

The EBV Latent Membrane Protein 2 (LMP2) may have a role in the establishment and maintenance of in vivo latency. The gene is transcribed into two mRNAs that produce two LMP2 protein isoforms. The LMP2a protein isoform has 12 transmembrane segments (TMs) and an amino terminal cytoplasmic signaling domain (CSD) while the LMP2b isoform is identical but lacks the CSD. There has not been a consensus on the cellular membrane localization being sometimes ascribed to either a plasma membrane or an intracellular location [M. Rovedo, R. Longnecker, J. Virol. 81:89–94, 2007; D. Lynch, J. Zimmerman, D.T. Rowe, J. Gen. Virol. 83:1025–1035, 2002; C. Dawson, J. George, S. Blake, R. Longnecker, L.S. Young, Virology 289:192–207, 2001]. Fluorescent marker and epitope tagged LMP2b truncation mutants progressively removing TMs from the N and C termini were used to assess the localization and aggregation properties of LMP2b. wtLMP2b had an exclusively intracellular perinuclear localization, while all truncations of the protein resulted in localization to the cell surface. By epitope loop-tagging, all the truncated LMP2b proteins were verified to be in the predicted membrane orientation. In co-transfection experiments, the C-terminal region was implicated in the self-aggregation properties of LMP2b. Thus, an intact 12 TM domain was required for intracellular localization and protein–protein interaction while a C-terminal region was responsible for auto-aggregative properties.     

Ad-LMP2重组腺病毒疫苗在恒河猴体内免疫效果的研究

目的观察带有EBV-LMP2的非复制型Ad-LMP2重组腺病毒疫苗免疫恒河猴诱导的针对EBV-LMP2的特异性细胞和体液免疫应答。方法分别使用高剂量(4·5×1011VP/kg)、中剂量(1·5×1011VP/kg)、低剂量(0·5×1011VP/kg)三个剂量的Ad-LMP2重组腺病毒,肌内注射免疫恒河猴,每5d免疫一次,共免疫6次,第7周时使用ELISPOT方法检测猴外周血细胞毒性T细胞应答,同时应用免疫酶方法检测血清中抗LMP2抗体。结果3个剂量免疫恒河猴均可以诱导出有效的细胞免疫应答及一定的抗体应答,免疫应答水平的高低与病毒剂量的高低有一定的关系,较高剂量产生的细胞及体液免疫应答水平比低剂量的要高。抗腺病毒中和抗体和抗LMP2抗体免疫2周后就可以检测到,其中抗LMP2抗体在免疫3~4周时滴度较高,7周时则与3~4周时接近或有所下降。结论非复制型Ad-LMP2重组腺病毒疫苗可以有效的诱导恒河猴产生EBV-LMP2特异性细胞和体液免疫反应。     

Cholesterol is critical for Epstein-Barr virus latent membrane protein 2A trafficking and protein stability

Latent membrane protein 2A (LMP2A) of Epstein-Barr virus (EBV) plays a key role in regulating viral latency and EBV pathogenesis by functionally mimicking signals induced by the B cell receptor (BCR) altering normal B cell development. LMP2A specifically associates with Nedd4 family ubiquitin-protein ligases which downmodulate LMP2A activity by ubiquitinating LMP2A and LMP2A-associated protein tyrosine kinases (PTKs). Since specific ubiquitin tags provide an endocytic sorting signal for plasma membrane proteins which traffic to membrane vesicles, we examined LMP2A localization and trafficking. We found that LMP2A is secreted through exosomes, small endocytic membrane vesicles, as previously demonstrated for LMP1. Interestingly, the treatment of cells with methyl-beta-cyclodextrin (MCD), which depletes cholesterol from plasma membrane, dramatically increased LMP2A abundance and LMP2A exosome secretion. Cholesterol depletion also blocked LMP2A endocytosis resulting in the accumulation of LMP2A on plasma membrane. LMP2A phosphorylation and ubiquitination were blocked by cholesterol depletion. LMP2A in the exosomal fraction was ubiquitinated but not phosphorylated. These results indicate that cholesterol-dependent LMP2A trafficking determines the fate of LMP2A degradation.     

Sequence Variations of Epstein-Barr Virus LMP2A Gene in Gastric Carcinoma in Japan

The latent EBV gene products expressed in Epstein-Barr virus (EBV)-associated gastric carcinoma (EBVaGC), are only LMP2A, EBNA-1, BARF-0 and EBERs. To examine the correlation between LMP2A sequence variation in EBVaGC and transformation of the cells, the complete sequence of the LMP2A gene was determined in three cases of Japanese EBVaGC and compared with the prototype B95-8 strain. In addition, the sequences of exons 2,6 and 7 of LMP2A were determined in four to six EBVaGC cases. The results of sequence analysis indicated that LMP2A of EBVaGC was structurally very similar to B95-8, but contained a significant nucleotide variation. Ten nucleotide substitutions were identified in almost all cases tested, and three of these caused amino acid changes. Of these three, two amino acid substitutions were not expected to change any known functions of LMP2A. The other amino acid substitution from serine to threonine was located at codon 348 within one of the target epitopes of EBV-specific cytotoxic T-lymphocytes. The LMP2A of EBV in peripheral blood lymphocytes from six healthy individuals showed serine (4/6 cases) or threonine (2/6 cases) substitution at codon 348, while LMP2A with the threonine substitution was the major form (5/6 cases) observed in EBVaGC, indicating that EBV with the threonine substitution may confer an advantage for viral persistence in tumor cells. However, our sequencing results suggested that the LMP2A protein in EBVaGC is functionally similar to that of the B95-8 strain and is not unique to gastric carcinoma, indicating the importance of LMP2A for EBV latency.     

表达轮状病毒VP7基因重组腺病毒载体的构建及免疫活性研究

目的 包装表达轮状病毒VP7基因的重组腺病毒,并检测其免疫活性.方法 RT-PCR扩增病毒结构蛋白VP7基因,定向克隆于腺病毒穿梭质粒pAdtrack-CMV中,在细菌中与缺陷型腺病毒基因组pAdeasy-1进行同源重组,并用RT-PCR和Western blot检测.将包装好的腺病毒免疫小鼠,应用间接ELISA检测小鼠血清中特异性轮状病毒IgG抗体.结果 酶切和测序鉴定证实成功构建携带VP7基因的重组腺病毒表达载体,并在293细胞中成功包装病毒;RT-PCR和Western blot 均能特异检测VP7基因的表达;重组腺病毒免疫小鼠后可诱导针对轮状病毒的特异性免疫.结论 重组腺病毒的成功包装,为新型轮状病毒基因疫苗的研制提供了一种可行的途径.     

Computational Prediction and Identification of Epstein-Barr Virus Latent Membrane Protein 2A Antigen-Specific CD8^＋ T-Cell Epitopes

Epstein-Barr virus （EBV） associated nasopharyngeal carcinoma （NPC） is a high incidence tumor in Southeast Asia. Among EBV encoded proteins, latent membrane protein 2A （LMP2A） is an important antigen for T cell therapy of EBV. In this study, we predicted six HLA-A2 restricted CTL candidate epitopes of LMP2A by SYFPEITHI, NetMHC and MHCPred methods combined with the polynomial method. Subsequently, biological functions of these peptides were tested by experiments in vitro. In ELISPOT assay, the positive response of the LMP2A specific CTL stimulated by three （LMP2A264.272, LMP2A426-434 and LMP2A3s6.364） of six peptides respectively showed that the numbers of spots forming cells （SFC） ranged from 55.7 to 80.6 SFC/5 x 104 CO8^＋ T cells and the responding index （RI） ranged from 5.4 to 7. These three epitope-specific CTLs could effectively kill specific HLA-A2- expressing target cells. As a result, LMP2A264.272 （QLSPLLGAV）, LMP2A426.434 （CLGGLLTMV） and LMP2A356.364 （FLYALALLL） were identified as LMP2A-specific CD8^＋ T-cell epitopes. It would be useful to clarify immune response toward EBV and to develop a vaccine against EBV-correlative NPC.     

含EBV-LMP2基因重组腺病毒疫苗的构建及其诱导CTL应答的初步探讨

目的　构建包含EBV LMP2基因的重组腺病毒疫苗并探讨它在体内外的免疫性质。方法　采用pAdeasy 1系统构建包含EBV LMP2基因的重组腺病毒疫苗 ,并用IFA、PCR和TCID50 等方法对其特异性进行鉴定。通过重组腺病毒感染人树突状细胞 (DC) ,在体外活化自体T细胞 ;以及通过重组腺病毒感染小鼠淋巴细胞 ,皮下免疫同种小鼠 ,体内活化CTL评价其免疫效果。结果　通过PCR以及间接免疫荧光试验分别证实了病毒目的基因LMP2的存在以及蛋白在 2 93细胞中的表达。采用TCID50 方法 ,测定第 6代的病毒滴度为 2× 10 8。体内外的免疫实验结果显示通过这两种方式均可以有效地引发针对EBV LMP2的CTL反应。结论　包含EBV LMP2基因的重组腺病毒疫苗可以在体内外有效地引发CTL应答 ,这些资料为下一步临床应用含LMP2的重组腺病毒作为疫苗治疗和预防EBV相关肿瘤奠定了基础。