鼻咽癌组织中EBV潜伏膜蛋白2跨膜区CTL表位序列分析

背景与目的：Epstein-Barr病毒在华南地区鼻咽癌细胞中表达核抗原1（EBNA1）、潜伏膜蛋白1（LMP1）和潜伏膜蛋白2（LMP2）等病毒蛋白质。LMP2 mRNA不仅几乎100％表达于鼻咽肿瘤细胞,而且LMP2蛋白还具有较强的免疫原性,是一个较理想的免疫治疗靶点。本研究分析广州地区来源鼻咽癌组织的LMP2基因跨膜区的CTL表位序列,为设计以LMP2抗原为靶点的鼻咽癌免疫治疗提供依据。方法：收集广州地区鼻咽癌患者鼻咽活检组织20例和正常鼻咽粘膜活检组织3例,提取DNA,半巢式PCR扩增LMP2基因跨膜区．直接测序．分析CTL表位序列。结果：与标准株B95．8相比．鼻咽癌和正常鼻咽粘膜活检组织来源的LMP2基因跨膜区存在14处碱基替换,形成6处氨基酸替换．导致4处CTL表位序列变异（SSC、TYG、CLG和VMS）,其中VMS多态性为初次报道。由于从鼻咽癌组织扩增的LMP2序列与从正常鼻咽粘膜扩增的LMP2序列相同,表明这些变化是地域相关的病毒多态性而非鼻咽癌相关的病毒变异。结论：广州地区来源的Epstein-Barr病毒LMP2基因存在多态性．产生4处CTL表位序列变化。在设计以LMP2为靶点的免疫治疗时,应充分考虑病毒基因多态性的影响。

Sequence analysis of the CTL epitopes in transmembrane region of latent membrane protein 2 of Epstein-Barr virus derived from nasopharyngeal carcinoma cells

BACKGROUND & OBJECTIVE: Epstein-Barr virus (EBV) in nasopharyngeal carcinoma (NPC) cells expresses Epstein-Barr nuclear antigen 1 (EBNA1), latent membrane protein 1 (LMP1), and LMP2. LMP2 is an ideal target for immunotherapy because LMP2 mRNA is detected in 100% nasopharyngeal carcinoma cells and LMP2 protein shows stronger immunogenity than the rest 2 viral proteins. This study was to analyze the sequence of CTL epitopes in the transmembrane region of LMP2 to optimize LMP2-targeted immunotherapy. METHODS: Genomic DNA was extracted from 20 biopsies of NPC and 3 biopsies of normal nasopharynx from Cantonese. The transmembrane region of LMP2 gene was amplified with hemi-nest polymerase chain reaction (PCR), and then sequenced directly. RESULTS: As compared with prototype B95.8 cells, the transmembrane region of LMP2 gene, derived from Cantonese NPC and normal nasopharynx tissues, had 14 base pair substitutions, resulting in 6 amino acid substitutions. Among these substitutions, 3 changed amino acids were located in 4 HLA-restricted CTL epitopes (SSC, TYG, CLG, and VMS). Among these polymorphisms, the VMS variation was first identified. The sequence changes of the LMP2 derived from NPC was the same as those of the LMP2 from normal nasopharynx, indicating that those variations were due to geographic-associated polymorphisms rather than NPC-associated mutations. CONCLUSION: Polymorphisms of LMP2 exist in EBV derived from Cantonese, resulting in 4 CTL epitope variations, which implicates that the effect of LMP2 polymorphisms should be considered when LMP2-targeted vaccine is designed for immunotherapy.