CN114835778B - 一种h9n2亚型aiv mhc b2限制性表位肽及其应用 - Google Patents
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Abstract
本发明公开了一种H9N2亚型AIVMHCB2限制性表位肽及其应用,涉及基因工程技术领域。该表位肽的氨基酸序列如SEQIDNO.1、SEQIDNO.2、SEQIDNO.3或SEQIDNO.4所示。本发明通过建立H9N2亚型AIV感染B2单倍型鸡的动物模型,证明细胞免疫应答在B2单倍型鸡抵抗AIV感染中的重要作用,并利用B2单倍型MHCI类分子基序系统地筛选H9N2亚型AIV病毒蛋白中潜在的表位,最终通过功能实验确定具有免疫原性的多肽表位,为AIV表位疫苗的研发提供条件。
Description
技术领域
本发明涉及基因工程技术领域,特别是涉及一种H9N2亚型AIV MHC B2限制性表位肽及其应用。
背景技术
禽流感病毒(Avian influenza virus,AIV)是一种属于正粘病毒科,甲型流感病毒属的分节段病毒,宿主范围涉及各种禽类及包括人类在内的哺乳动物。根据血凝素(Hemagglutinin,HA)和神经氨酸酶(Neuraminidase,NA)的血清学差异,可以将AIV分为18种HA亚型和11种NA亚型,其中H9N2亚型AIV在中国家禽中普遍存在。尽管是低致病性禽流感,H9N2亚型AIV同样可以通过减少产蛋量或与家禽中的其他病原体共同感染鸡只造成巨大的经济损失,因此,加强对H9N2 AIV的防控和研究也是必不可少。
目前关于该病毒的防控主要以灭活疫苗为主,但是,长期免疫选择压力下导致病毒容易变异逃避抗体的识别,进而导致疫苗产生的特异性抗体在不同亚型间的保护力不足。因此,开发具有更广覆盖率和更持久保护力的疫苗对禽流感的防控十分重要。
大量研究表明,流感特异性CD8+ T细胞不仅参与病毒的清除,而且能提供针对其他亚型流感病毒的交叉保护,如Dai等人通过比较H9N2 AIV感染和疫苗免疫诱导无特定病原体鸡免疫应答产生的关键保护因子,发现CD8+ T细胞应答反应在对抗AIV感染中起重要作用;Seo等人发现鸡感染H9N2 AIV后,在H5N1 AIV的攻毒实验中具有更高的存活率,随后将活化的H9N2 AIV特异性CD8+ T细胞注射到雏鸡体内,能提高H5N1 AIV感染后鸡的存活率,因此,开发能诱导T细胞免疫应答的疫苗能弥补目前常用疫苗的不足,对于H9N2 AIV的防控有重要意义。
具有免疫原性的表位是诱导T细胞产生免疫效应的先决条件。截至2022年3月份,免疫表位数据库(IEDB)显示了一共34条AIV针对鸡的T细胞表位,其中24条已经功能验证具有免疫原性,包括22条CD8+ T细胞表位和2条CD4+ T细胞表位,这些表位位于核蛋白、聚合酶蛋白、基质蛋白1和血凝素上,涵盖了H5N1,H5N8和H7N1三种亚型,但未见关于H9N2亚型AIV的表位报道。因此针对目前长期流行的H9N2亚型AIV,系统开展筛选具有免疫原性的AIV表位的工作十分重要。
抗原表位通过与MHC I类分子结合从而被下游TCR识别,然而,MHC I类分子具有多态性,即使是针对同一病原,不同的MHC I类分子能结合的抗原表位也不同,所以,在筛选抗原表位的同时要明确MHC的限制性。目前,根据MHC B基因区的基因序列,可以将鸡分为B1到B29共29种单倍型,其中B2单倍型作为一种常见的倍型,已有大量报道其对某些疾病具有抗性,是进行AIV实验研究及疫苗开发的优良材料。
发明内容
本发明的目的是提供一种H9N2亚型AIV MHC B2限制性表位肽及其应用,以解决上述现有技术存在的问题,本发明通过建立H9N2亚型AIV感染B2单倍型鸡的动物模型,证明细胞免疫应答在B2单倍型鸡抵抗AIV感染中的重要作用,并利用B2单倍型MHC I类分子基序系统地筛选H9N2亚型AIV病毒蛋白中潜在的表位,最终通过功能实验确定具有免疫原性的多肽表位,为AIV表位疫苗的研发提供条件。
为实现上述目的,本发明提供了如下方案:
本发明提供一种H9N2亚型AIV MHC B2限制性表位肽,所述表位肽的氨基酸序列如SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3或SEQ ID NO.4所示。
本发明还提供上述的表位肽在制备H9N2亚型AIV疫苗中的应用。
本发明还提供一种H9N2亚型AIV疫苗,包含上述的表位肽。
本发明公开了以下技术效果:
本发明首先用H9N2亚型AIV(A/Chicken/Hunan/HN/2015)株感染B2单倍型(BW/G3)SPF鸡,通过检测鸡只的泄殖腔排毒情况,喉头排毒情况,PBMC中T细胞亚型的变化情况以及PBMC中免疫相关基因的变化等,确定感染模型的成功建立以充当后续的试验材料。然后,根据本实验室确定的B2单倍型鸡MHC I类分子结合多肽的基序(X-A/V/I/L/P/S/G-X-X-X-X-X-X-V/I/L),筛选出可能具有免疫原性的针对H9N2亚型AIV的候选多肽表位,最后经ELISpot试验验证上述多肽的免疫原性,确定有效的T细胞表位。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为B2单倍型鸡喉头病毒滴度;n=7;
图2为血清中抗体水平;n=4;
图3为攻毒后B2单倍型鸡PBMC中CD8α+ T细胞比例变化;n=4;
图4为攻毒后B2单倍型鸡PBMC中CD4+ T细胞比例变化;n=4;
图5为攻毒后B2单倍型鸡PBMC中CD4+和CD8α+ T细胞比例变化;n=4;
图6为攻毒后B2单倍型鸡PBMC中CD4+/CD8α+ T细胞的变化;n=4;
图7为攻毒后B2单倍型鸡PBMC中天然免疫相关基因表达情况;
图8为攻毒后B2单倍型鸡PBMC中CTLs相关基因表达情况;
图9为攻毒后B2单倍型鸡PBMC中Th2相关基因表达情况;
图10为肽池刺激淋巴细胞后IFN-γ的表达水平;其中a为pool_1~pool_28的ELISpot结果;b为pool_29~pool_56的ELISpot结果;c为pool_57~pool_85的ELISpot结果;除阳对照外,n=3;
图11为#1鸡ELISpot部分斑点展示;
图12为#2鸡ELISpot部分斑点展示;
图13为#3鸡ELISpot部分斑点展示;
图14为#1鸡脾脏淋巴细胞IFN-γ的分泌水平;除阳性对照外,每条肽三个技术重复;
图15为#2鸡脾脏淋巴细胞IFN-γ的分泌水平;除阳性对照外,每条肽三个技术重复;
图16为#3鸡脾脏淋巴细胞IFN-γ的分泌水平;除阳性对照外,每条肽三个技术重复。
具体实施方式
现详细说明本发明的多种示例性实施方式,该详细说明不应认为是对本发明的限制,而应理解为是对本发明的某些方面、特性和实施方案的更详细的描述。
应理解本发明中所述的术语仅仅是为描述特别的实施方式,并非用于限制本发明。另外,对于本发明中的数值范围,应理解为还具体公开了该范围的上限和下限之间的每个中间值。在任何陈述值或陈述范围内的中间值,以及任何其他陈述值或在所述范围内的中间值之间的每个较小的范围也包括在本发明内。这些较小范围的上限和下限可独立地包括或排除在范围内。
除非另有说明,否则本文使用的所有技术和科学术语具有本发明所述领域的常规技术人员通常理解的相同含义。虽然本发明仅描述了优选的方法和材料,但是在本发明的实施或测试中也可以使用与本文所述相似或等同的任何方法和材料。本说明书中提到的所有文献通过引用并入,用以公开和描述与所述文献相关的方法和/或材料。在与任何并入的文献冲突时,以本说明书的内容为准。
在不背离本发明的范围或精神的情况下,可对本发明说明书的具体实施方式做多种改进和变化,这对本领域技术人员而言是显而易见的。由本发明的说明书得到的其他实施方式对技术人员而言是显而易见得的。本发明说明书和实施例仅是示例性的。
关于本文中所使用的“包含”、“包括”、“具有”、“含有”等等,均为开放性的用语,即意指包含但不限于。
术语解释:
AIV:禽流感病毒;MHC I:I型主要组织相容性复合体;ELISpot:酶联免疫斑点试验;PBMC:外周血单个核细胞;SPF鸡:无特定病原体鸡;CTL:细胞毒性T细胞;IFN-γ:γ干扰素;DPI:攻毒后天数;EID50:鸡胚半数感染量;FBS:胎牛血清;PMA+Ionomycin:佛波酯和离子霉素。
实施例1
1试验材料
1.1试验动物和病毒
本试验所用的鸡为4周龄B2单倍型SPF鸡(BW/G3),购自国家禽类实验动物资源库;H9N2亚型AIV(A/Chicken/Hunan/HN/2015)株为华南农业大学人兽共患病防控制剂国家地方联合工程实验室保存。
1.2试验主要试剂
总RNA提取试剂盒购自简石生物公司;鸡外周血淋巴细胞分离液试剂盒、鸡脏器组织单个核细胞分离液试剂盒、红细胞裂解液购自天津灏洋生物公司;ChamQ SYRB qPCRMaster Mix购自南京诺唯赞生物科技有限公司;Chicken IFN-γELISpotBASIC kit购自Mabtech公司,流式抗体Anti-chicken CD3 antibody、Anti-chicken CD4 antibody、Anti-chicken CD8αantibody、Anti-chicken IFN-γ-FITC antibody购自SouthernBiotech公司;佛波酯和离子霉素(PMA+Ionomycin)、TMB ELISpot专用显色液购自中国达科为公司;RPMI-1640培养基、FBS澳洲胎牛血清购自美国GIBCO公司。
1.3主要溶液配制
(1)1640完全培养基:50mL离心管中加45mL RPMI-1640培养基,5mL的灭活FBS及500μL青链霉素(100×),混匀后4℃备用。
(2)流式Buffer:50mL离心管中加49mL RPMI-1640培养基,1mL的灭活FBS,混匀后4℃备用。
(3)细胞冻存液:50mL离心管中加45mL灭活FBS,5mL的DMSO(二甲基亚砜),混匀后4℃备用。
2试验方法
2.1病毒的扩繁
H9N2 AIV原毒融化后无菌PBS稀释1000倍。9~11日龄SPF鸡胚消毒后置于工作台中,每个鸡胚尿囊腔接种100μL稀释的病毒液。封口后鸡胚继续培养,并在接种24h后观察鸡胚死活,弃死胚,剩余活胚继续培养至72h即可收毒。用移液器吸取尿囊液至离心管中,液体4℃,2000rpm离心10min,取上清过0.22μm滤膜后分装,-80℃保存备用。
2.2血凝效价的测定
效价测定参照国家标准最新版(GB/T 18936-2020)。
2.3EID50的测定
鸡胚半数感染量(50%Embryo infective dose,EID50)的测定如下:扩繁的病毒液取出后冰上融化,并用PBS作10倍倍比稀释。取10-4~10-9稀释度的病毒液按2.1的方法接种鸡胚并培养,每个稀释度5个鸡胚。72h后,每个鸡胚收取25μL尿囊液,按2.2方法测定血凝效价,按Spearman-Karber法计算EID50。
2.4H9N2亚型AIV感染B2单倍型鸡动物模型的建立
无菌PBS稀释H9N2 AIV病毒液至107EID50/200μL。将实验动物分成2组,包括B2单倍型鸡实验组、B2单倍型鸡对照组,每组7只鸡。采用点眼滴鼻的方法攻毒,每只攻毒200μL,左右眼先各滴一滴,剩余病毒液全部经一侧鼻腔注入。对照组则用同样的方法接种等体积PBS。攻毒后3、5、7、9、11天采集动物的咽喉和泄殖腔拭子,外周抗凝血及非抗凝血进行检测。
2.4.1感染鸡排毒情况检测
采样日采集的拭子存放于-80℃保存,统一检测。拭子取出后冰上融化,充分涡旋,然后4℃,12000rpm、离心5min除杂。取上清过0.22μm滤头,按2.3测定EID50,一般做100~10-6稀释度,以此评估排毒情况。
2.4.2血清抗体水平的检测
采集的非抗凝血室温放置至血清析出后,1.5mL离心管收集血清。随后4℃、2000rpm离心10min去除红细胞,剩余血清用于检测血清抗体水平。
抗体水平的检测使用血凝抑制试验(Hemagglutination inhibition,HI),具体步骤参照国家标准最新版(GB/T 18936-2020)。
2.4.3检测鸡PBMC中T细胞亚型变化情况
按试剂盒说明书分离PBMC,取适量细胞作流式染色,以下按每管106个细胞说明:取细胞于流式管中,加入1mL流式Buffer,440g离心6min,期间按说明书推荐浓度避光稀释CD3、CD4及CD8抗体。离心后弃上清,每管加入100μL稀释好的抗体重悬,4℃避光孵育30min。随后加入1mL Buffer,440g离心6min,沉淀用250μL流式Buffer重悬,上流式细胞仪采集数据,数据用FlowJo软件分析。
2.4.4荧光定量PCR检测鸡PBMC免疫相关基因变化
按照简石生物总RNA提取试剂盒提取RNA。简单地说,取适量细胞,440g离心6min,弃上清,加入1mL TRIzol后涡旋。随后加入等体积无水乙醇,混匀,液体转移到2号柱内,离心1min,去除滤出液。往柱上加入400μL RNA洗涤液2,离心1min,弃滤液。往柱上加入80μLDNase I反应液,室温作用15min,然后添加400μLRNA洗涤液1,离心1min,弃滤液。然后加700μL RNA洗涤液2,离心1min,弃滤液。空转2min,2号柱转移到无RNA酶的离心管中,加入50μL事先预热至70℃的RNase-free水,放置2min后,离心1min洗脱RNA,超微量分光光度计检测浓度,-80℃保存。
RNA按照表1中的体系进行反转录,反转录程序如下:37℃反转15min,85℃灭活5s,4℃保存。
表1反转录体系
对反转录后的cDNA进行荧光定量PCR扩增,以检测PBMC中免疫相关基因的变化。检测的目的基因及引物如表2,体系如表3。反应程序:预变性95℃,30s;循环反应95℃,10s,60℃,30s,共40个循环;溶解曲线分析95℃,15s,60℃,60s,95℃,15s。以GAPDH基因为内参,ΔΔCt法分析结果。
表2免疫相关基因qPCR引物
表3荧光定量体系
2.5筛选可能具有免疫原性的多肽表位
根据本实验室确定的B2单倍型鸡MHC I类分子结合多肽的基序(X-A/V/I/L/P/S/G-X-X-X-X-X-X-V/I/L),筛选可能具有免疫原性的针对H9N2亚型AIV的候选多肽表位。筛选出的多肽由上海淘普生物科技有限公司合成,纯度为95%,每条肽合成5mg。
2.6候选多肽免疫原性检测
2.6.1ELISpot实验检测肽池免疫原性
合成的多肽用200μL的DMSO溶解,分装后置于-80℃备用。5条肽混合为一个池,分别命名为pool_1~pool_85,并用ELISpot实验检测免疫原性。实验操作参照Chicken IFN-γELISpotBASIC Kit说明书,具体如下:
第一天:
(1)排枪每孔加入15μL 35%乙醇活化细胞孔内的PVDF膜,活化时间不超过1min。然后每孔加入200μL无菌水洗涤,重复4遍;1:33稀释抗鸡IFN-γ单克隆抗体,每孔加入100μL,4℃包被过夜;
第二天:
(2)倒弃包被液,每孔用200μL无菌PBS洗涤4次;
(3)每孔加入200μL 1640完全培养基,室温封闭1~2h;
(3)倒弃封闭液,每孔加入100μL脾脏淋巴细胞悬液(含3.5×105个细胞),同时向试验组加入肽库(终浓度每条肽10μg/mL);阴性对照组加等体积DMSO;阳性对照组加入10μL达科为公司的PMA+Ionomycin混合物;
(4)所有样品加完后,将细胞板放入含5%CO2的37℃细胞培养箱中,培养至少18h;
第三天:
(5)培养结束后甩去培养基和细胞,每孔加入200μL无菌PBS洗涤5次,向每孔中加入100μL含有0.5%FBS和1μg/mL生物素标记的检测抗体的PBS,在室温下孵育2小时。甩弃液体,每孔加入200μL无菌PBS洗涤5次;
(6)向每孔中加入100μL稀释好的链霉亲和素标记的HRP,室温孵育1h。甩弃液体,每孔中加入200μL无菌PBS洗涤5次;
(7)每孔加入100μL TMB显色液,直至底部出现明显斑点,用超纯水冲洗细胞板子以终止反应,晾干后在自动读板仪中计数,并对斑点数进行统计学分析。
2.6.2ELISpot实验检测肽段免疫原性
将2.6.1中能显著刺激细胞产生斑点的肽库挑选出来,用ELISpot实验检测其中每一条多肽的免疫原性,具体步骤同2.6.1。
2.7数据分析
使用软件GraphPad Prism 8对所有实验数据进行统计学分析,其中ns表示P>0.05,差异不显著;*表示P<0.05,差异显著;**表示P<0.01,差异极显著;***表示P<0.001,差异极显著,****表示P<0.0001,差异极显著。
3结果
3.1感染鸡排毒情况检测
H9N2 AIV感染B2单倍型鸡后,根据实验安排采集拭子,按2.4.1检测排毒情况。如图1所示为喉头排毒情况,B2单倍型鸡的排毒高峰为3DPI,从5DPI开始喉头排毒量下降(P<0.001),到11DPI均检测不到排毒,表4所示为B2单倍型鸡泄殖腔排毒情况,在3DPI和5DPI,B2单倍型鸡攻毒组仅有2只鸡被检测呈阳性。7DPI B2单倍型鸡即检测不出泄殖腔排毒。对照组排毒检测均为阴性(数据未显示)。
表4 B2单倍型鸡泄殖腔排毒情况
注:括号外数字表示阳性鸡的泄殖腔排毒量,为log10EID50的值,括号内/前的数字表示检测到排毒的鸡只数量,/后的数字表示鸡只总数。
3.2感染鸡血清抗体水平检测
结果如图2所示,3DPI时抗体为阴性(均小于2孔),5DPI全部被检测鸡抗体水平转阳,且HI抗体水平至11DPI不断升高。结果表明,到5DPI时体液免疫应答已启动,从5DPI开始喉头拭子中排毒减少与抗体水平的升高相关。对照组抗体检测均为阴性(数据未显示)。
3.3感染后鸡PBMC中T细胞亚型的变化情况
为检测攻毒后B2单倍型鸡T细胞免疫应答情况,按实验安排颈静脉采集鸡外周血,分离PBMC,用流式抗体进行染色。如图3所示,在5DPI、7DPI、9DPI,攻毒组CD8+ T细胞比例相比对照组显著上升(P<0.001)。结果表明H9N2 AIV感染B2单倍型鸡后从第5天开始,在鸡PBMC中能检测到明显的CD8+ T细胞增殖,并持续到9DPI,提示从第5DPI开始病毒的清除不仅与抗体水平升高有关,CD8+ T细胞免疫应答也发挥了重要作用。
CD4+ T细胞亚型的变化如图4。H9N2 AIV感染B2单倍型鸡后,在5DPI、7DPI、9DPI会导致CD4+ T细胞比例显著下降(P<0.05),到11DPI即回复到正常水平。攻毒后B2单倍型鸡PBMC中CD4+CD8α+双阳性T细胞比例变化如图5。攻毒组与对照组相比CD4+CD8α+双阳性T细胞比例无统计学差异。此外,攻毒后B2单倍型鸡PBMC中CD4+/CD8α+T细胞的变化如图6。在5DPI、7DPI、9DPI攻毒组的CD4+/CD8α+T细胞比值显著低于对照组,说明在此阶段机体处于免疫抑制状态。
3.4感染后B2单倍型鸡PBMC免疫相关基因变化
为进一步验证宿主免疫应答在H9N2 AIV感染B2单倍型鸡过程中发挥的作用,荧光定量PCR检测体内感染5天后PBMC中重要免疫基因mRNA表达量的变化,其中检测主要包括三部分:天然免疫相关基因、CTLs基因和Th2基因。
天然免疫基因部分(图7),与对照组相比,5DPI感染组中抗病毒基因ISG12-2(Interferon-stimulated gene 12-2)、OASL(2’,5’-Oligoadenylate synthetase-like)、IFIT5(Interferon-inducedproteins with tetratricopeptide repeats 5)、USP18(Ubiquitin Specific Peptidase 18)、MX1(Myxovirus resistance 1)基因表达量显著上升(P<0.05);CTLs基因部分(图8),Granzyme k、IFN-γ(Interferon gamma)、lysin、PARP(Poly(ADP-ribose)polymerase)等基因表达量显著上升(P<0.05);检测的Th2基因(图9)表达量均没有显著性差异。结合3.3中CD8α+ T细胞比例增加,进一步说明AIV感染成功激活了B2单倍型鸡的细胞毒性T细胞免疫应答。综合上述结果,H9N2亚型AIV感染诱导B2单倍型SPF鸡(BW/G3)细胞免疫应答的模型建立成功。
3.5筛选可能具有免疫原性的多肽表位
根据B2单倍型鸡MHC I类分子结合多肽的基序(X-A/V/I/L/P/S/G-X-X-X-X-X-X-V/I/L),筛选可能具有免疫原性的针对H9N2亚型AIV的候选多肽表位,筛选出来的肽如表5所示。
表5根据基序筛选的可能具有免疫原性的多肽
3.6候选多肽免疫原性检测
3.6.1ELISpot实验检测肽池的免疫原性
将合成的多肽按5条肽为一个池的要求混合,分别刺激感染H9N2 AIV 28天后的B2单倍型鸡脾脏淋巴细胞。如图10,统计学分析发现pool_2、pool_3、pool_52、pool_75能显著刺激脾脏淋巴细胞产生IFN-γ斑点,说明组成上述肽池的肽段中存在具有免疫原性的表位。
3.6.2ELISpot实验检测多肽的免疫原性
如图11-13所示,具有免疫原性的单一多肽同样可以刺激脾脏淋巴细胞产生IFN-γ。根据参考文献(Identification ofnovel avian influenza virus derived CD8+ T-cell epitopes)提供的标准,和阴性对照组相比,能引起3只鸡中至少2只鸡显著产生IFN-γ的多肽可以认为具有免疫原性。如图14-16,P10、P11、P373及P257这四条肽段符合上面标准,可认为是B2单倍型限制性的H9N2AIV T细胞表位(见表6)。
表6四条针对B2单倍型鸡的H9N2亚型AIV T细胞表位信息
以上所述的实施例仅是对本发明的优选方式进行描述,并非对本发明的范围进行限定,在不脱离本发明设计精神的前提下,本领域普通技术人员对本发明的技术方案做出的各种变形和改进,均应落入本发明权利要求书确定的保护范围内。
序列表
<110> 华南农业大学
<120> 一种H9N2 亚型AIV MHC B2限制性表位肽及其应用
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 9
<212> PRT
<213> 人工序列(Artificial Sequence)
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Ala Val Lys Gly Ile Gly Thr Met Val
1 5
<210> 2
<211> 9
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Asp Val Ser Phe Gln Gly Arg Gly Val
1 5
<210> 3
<211> 9
<212> PRT
<213> 人工序列(Artificial Sequence)
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Met Ser Arg Asp Trp Leu Met Leu Ile
1 5
<210> 4
<211> 9
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Trp Ile Ile Arg Asn Trp Glu Thr Val
1 5
Claims (3)
1.一种H9N2亚型AIV MHC B2限制性表位肽,其特征在于,所述表位肽的氨基酸序列如SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3或SEQ ID NO.4所示。
2.一种根据权利要求1所述的表位肽在制备H9N2亚型AIV疫苗中的应用。
3.一种H9N2亚型AIV疫苗,其特征在于,包含权利要求1所述的表位肽。
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