【作者】 李新生； 陈红英； 闫若潜； 崔保安； 杜向党； 夏春；

【Author】 Li Xin-sheng~(1,2) Chen Hong-ying~1 Yan Ruo-qian~3 Gao Feng-shan~2 Cui Bao-an~1 Du Xiang-dang~1 Xia Chun~(2*) (1,The Key Laboratory for Preventive Veterinary Medicine,Ministry of Agriculture,College of Veterinary Medicine,China Agricultural University,beijing,100094,China;2,College of Husbandry and Veterinary,Henan Agricultural University,Zhengzhou,450002,China;3,Henan province veterinary preventive and treatment station,zhengzhou,450002,China.)

【机构】 河南农业大学牧医工程学院； 中国农业大学动物医学院,农业部预防兽医学重点实验室； 河南省兽医防治站； 中国农业大学动物医学院农业部预防兽医学重点实验室； 大连大学；

【摘要】 为探讨体外重建的MHC I复合体BF2-linker-β2m鉴定病毒抗原肽系统能否在体外结合抗原多肽,不同类的MHC I类分子结合同一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三条多肽,猪口蹄疫病毒的两条多肽,以及来源于草鱼呼肠孤病毒的两条多肽分别与四类体外重建的串联鸡MHC I类分子复合体BF2-linker-β2m进行了体外结合实验。首先BF2-linker-β2m与不同的病毒抗原九肽按照1:10的摩尔比进行混合,37℃作用12h后,取反应混合物经蛋白超滤浓缩管4500g离心45分钟以除去未与MHC I类分子结合的抗原多肽;然后将BF2-linkcr-β2m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MHC I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使用3K的超滤浓缩管4500g离心45分钟以收集洗脱的多肽溶液,随后利用C₁₈柱对洗脱的多肽去盐、脱酸处理;将多肽样品冻干,用基质溶解后直接点样上靶进行一级质谱（MS）和二级质谱（MSMS）测定,结果表明,三类体外重建的BF2-linker-β2m可与禽流感病毒多肽KILTIYSTV和LLLMVSLV发生结合,而不与禽流感多肽TIGECPKYV、及猪口蹄疫病毒和草鱼呼肠孤病毒的四条多肽结合。证实由于MHC I类分子的多态性导致复等位基因的MHC I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MHC I类分子限制性的:KILTIYSTV和LLLAIVSLV是禽流感病毒的候选表位。更多还原

【Abstract】 In order to identify the peptides binding with different type of chicken MHC I molecules,chicken four types of BF2-（G₄S）₃-β2m gene （BF2*04sh-linker-β2m,BF2*09sh-linker-β2m,BF2*12sh-linker-β2m） was constructed by splicing overlap extension PCR （SOE-PCR） method.BF2*04sh-linker-β2m,BF2*09sh-linker-β2m,BF2*12sh-linker-β2m,BF2*05wj-linker-β2m proteins were mixed with our designed and synthesized seven 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,mixing sufficiently and incubated at 37℃for 12h.The mixture was centrifuged in Amicon Ultra-15 protein Centrifugal Filter （3000） at 4500 rpm 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 10μL of matrix solution was put into a 4700 Proteomic Analyzer,and the primary mass spectra （MS） and secondary spectra （MS/MS） were analyzed.The result revealed peptides KILTIYSTV and LLLAIVSLV which derived from avian of influenza viruses （AIV） could bind with three different type of chicken MHC I.This confirmed 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.更多还原