禽流感病毒T细胞表位与体外重建鸡MHCⅠ类分子结合试验  

Study on Avian Influenza Virus T Cell Epitopes Binding with Reconstructed Chicken MHC Ⅰ Molecules in vitro

作　　者：李新生[1] 陈红英[1] 闫若潜 高凤山[3] 方勤美[4] 夏春[4] 

机构地区：[1]河南农业大学牧医工程学院,郑州450002 [2]河南省兽医防治站,郑州450002 [3]大连大学,大连116622 [4]中国农业大学动物医学院农业部预防兽医学重点实验室,北京100094

出　　处：《畜牧兽医学报》2007年第12期1335-1340,共6页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金资助项目(30371098)

摘　　要：为探讨体外重建的MHCⅠ复合体BF2-linker-β2 m鉴定病毒抗原肽系统能否在体外结合抗原多肽,以及不同类的MHCⅠ类分子结合相同和不同抗原多肽表位能力的差异,本研究采用人工合成的禽流感病毒的3条多肽,猪口蹄疫病毒的2条多肽,以及来源于草鱼呼肠孤病毒的2条多肽分别与4类体外重建的串联鸡MHCⅠ类分子复合体BF2-linker-β2 m进行了体外结合试验。BF2-linker-β2 m与不同的病毒抗原九肽按照1∶10的摩尔比进行混合,作用后取反应混合物离心除去未与MHCⅠ类分子结合的抗原多肽;然后取BF2-linker-β2 m分别与抗原多肽形成的复合体,利用酸洗法将结合的多肽自MHCⅠ类分子的抗原多肽结合槽中洗脱,洗脱后的蛋白和多肽混合物离心收集洗脱的多肽溶液,随后利用C18柱对洗脱的多肽进行去盐、脱酸处理;将多肽样品冻干,用基质溶解后直接点样进行一级质谱(MS)和二级质谱(MSMS)测定,结果表明,3类体外重建的BF2-linker-β2 m可与禽流感病毒多肽KILTIYSTV和LLLAIVSLV结合,而不与禽流感病毒多肽TIGECPKYV以及猪口蹄疫病毒和草鱼呼肠孤病毒的4条多肽结合。证实由于MHCⅠ类分子的多态性导致复等位基因的MHCⅠ类分子结合病毒抗原表位的能力存在差异;病毒的T细胞抗原表位是MHCⅠ类分子限制性的;KILTIYSTV和LLLAIVSLV是禽流感病毒的候选表位。In order to identify the peptides binding with different type of chicken MHC Ⅰ molecules, the constructed BF2 ＊ 04sh-linker-β2m, BF2 ＊ 09sh-linker-β2m, BF2 ＊ 12sh-linker-β2m, BF2 ＊ 05wj-linker-β2m proteins were mixed with seven designed and synthesized T cell epitopes （three derived from avian influenza virus, two derived from swine foot-and-mouth diseases virus and two derived from grass carp reovirus） at a respective mole ratio of 1 ： 10, incubated at 37℃ for 12 h. The mixtures were centrifuged in Amicon Ultra-15 protein Centrifugal Filter （3000） at 4 500 rpm for 45 min to remove unbound nonameric peptides. Then, the retained solution was mixed with 5 mL citrate-phosphate buffer, incubated at 37℃ for 5 min, then centrifuged as above to obtain the filtered solution. The peptides in acid elution solution were concentrated and desalted using Sep-Pak C18 cartridges. The sample plate which obtained the lyophilized peptides dissolved in 10 μL of matrix solution was put into a 4700 Proteomic Analyzer, and the primary mass spectra （MS） and secondary spectra （MS/MS） were conducted. The results revealed that peptides KILTIYSTV and LLLAIVSLV which derived from avian influenza viruses （AIV） could bind with three different types of chicken MHC Ⅰ. The results confirmed that the binding ablity of different MHC Ⅰ with peptides are different, the T cell epitopes derived from different virus are MHC Ⅰ restricted. KILTIYSTV and LLLAIVSLV are avian influenza＇s candidate T cell epitopes.

关 键 词：鸡 MHC Ⅰ类分子 T细胞表位 质谱 

分 类 号：S852.659.5[农业科学—基础兽医学]