EB病毒潜伏膜蛋白2A特异性CTL表位筛选及免疫功能研究
发布时间：2018-07-30 06:34
【摘要】：EB病毒(Epstein-Barr virus,EBV)属疱疹病毒科嗜B淋巴细胞病毒,已知它与多种人类恶性肿瘤相关。EBV相关鼻咽癌(nasopharyngeall carcinoma,NPC)是上皮来源的恶性肿瘤,高发于东南亚及我国华南地区。江苏目前发病率也在不断增高,平均每周新增病例10～20例,每年约960例新发病例。临床上NPC不能手术治疗,主要是通过放疗和化疗,但这种治疗方法并不能完全有效地清除肿瘤细胞,而且副反应较大,部分患者易复发和转移。因此在传统治疗方案的基础上加以生物治疗,发展以CTL为基础的免疫治疗已成为目前研究的方向。病毒蛋白在NPC肿瘤细胞中呈潜伏Ⅱ型表达,即仅表达EBV核抗原(EBNA1)和EBV潜伏膜蛋白(LMP1、LMP2A,2B)。其中LMP2A是一个疏水蛋白,本身不是转化基因,序列相对保守,且在肿瘤组织中能持续表达,并含有多种受HLA限制的CTL表位,被认为是EBV相关NPC免疫治疗的理想靶抗原。 我们以往的研究表明,树突状细胞转染LMP2A重组腺病毒载体或负载LMP2A蛋白后可激发特异性CTL反应。本研究进一步根据NPC患者常见的HLA型别(以HLA-A2为主),采用生物信息学方法,利用三种不同的计算机软件,SYFPEITHI(http: //www.uni-tuebingen.de/uni/kxi/),NetMHC(http: //www. jenner.ac.uk/MHCPred/)和MHCPred(http://www.cbs.dtu.dk/services/ NetMHC-2.0/)分析LMP2A蛋白全长序列,预测并合成了六条HLA-A2限制性的CTL候选表位肽。随后在EBV健康携带者和NPC患者中进行体外实验(包括酶联免疫斑点试验、胞内细胞因子检测和细胞杀伤试验),对候选肽段进行功能上的分析验证,最终确定了三条有效的LMP2A特异性的表位,分别是: LMP2A264 (QLSPLLGAV)、LMP2A426 (CLGGLLTMV)和LMP2A356 (FLYALALLL)。其中LMP2A264 (QLSPLLGAV)为本实验首次鉴定的新的LMP2A特异性表位。体外实验结果显示这三条肽段均可以有效的诱导CTL反应,杀伤表达LMP2A蛋白的特异性靶细胞。进一步采用有限稀释法建立了三株多肽特异性的T细胞克隆,经鉴定为两株多肽CLGGLLTMV特异性克隆和一株多肽QLSPLLGAV特异性克隆,并对克隆进行了表型和功能的初步分析。 综上所述,本研究采用生物信息学与体外实验相结合的方法,①鉴定了三条HLA-A2限制性的LMP2A特异性表位肽;②无论在EBV健康携带者还是在NPC患者中,这三条表位肽均可有效地诱导免疫应答;③通过T细胞克隆技术,得到了针对其中两条表位肽的三株CD8+T细胞克隆。所得结果将有助于阐明T淋巴细胞在控制EBV感染中的作用,对发展EBV相关肿瘤多肽疫苗,为NPC及其他病毒相关性肿瘤的免疫治疗提供理论和实验依据。
[Abstract]:Epstein-Barr virus (EBV) belongs to B lymphocytic virus of herpes viridae. It is known that EBV-associated nasopharyngeal carcinoma (nasopharyngeall carcinoma-NPC) is an epithelial-derived malignant tumor, which is highly prevalent in Southeast Asia and South China. The incidence rate in Jiangsu is increasing, with an average of 10 new cases per week, and about 960 new cases per year. Clinically, NPC can not be treated surgically, mainly through radiotherapy and chemotherapy, but this method can not completely and effectively remove tumor cells, and the side effects are larger, some patients are prone to recurrence and metastasis. Therefore, biotherapy based on traditional therapy and immunotherapy based on CTL has become the research direction. The viral protein was expressed as latent type 鈪，
本文编号：2154130
[Abstract]:Epstein-Barr virus (EBV) belongs to B lymphocytic virus of herpes viridae. It is known that EBV-associated nasopharyngeal carcinoma (nasopharyngeall carcinoma-NPC) is an epithelial-derived malignant tumor, which is highly prevalent in Southeast Asia and South China. The incidence rate in Jiangsu is increasing, with an average of 10 new cases per week, and about 960 new cases per year. Clinically, NPC can not be treated surgically, mainly through radiotherapy and chemotherapy, but this method can not completely and effectively remove tumor cells, and the side effects are larger, some patients are prone to recurrence and metastasis. Therefore, biotherapy based on traditional therapy and immunotherapy based on CTL has become the research direction. The viral protein was expressed as latent type 鈪，
本文编号：2154130
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