CN112226408B - 一种猪病原或外源性蛋白特异性抗原肽筛选和鉴定的方法 - Google Patents
一种猪病原或外源性蛋白特异性抗原肽筛选和鉴定的方法 Download PDFInfo
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- CN112226408B CN112226408B CN202011057070.2A CN202011057070A CN112226408B CN 112226408 B CN112226408 B CN 112226408B CN 202011057070 A CN202011057070 A CN 202011057070A CN 112226408 B CN112226408 B CN 112226408B
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Abstract
本发明通过对免疫原(病原或蛋白抗原)上的有效抗原肽序列进行筛选,去除免疫原中对机体不具有免疫活性(即不能激活机体适应性免疫应答)的无用肽段,筛选出免疫活性较强,可高效刺激机体产生免疫应答的抗原肽。抗原肽相对于原始免疫原,因其序列较短,亲水性较强,因此易于在体外进行表达,并且可随意将不同抗原肽或不同免疫原来源的抗原肽串联制备复合抗原肽,或将同一抗原肽进行多次重复以增强T细胞识别,同时可与其他具有免疫增强蛋白等融合表达,进一步增强机体免疫系统对抗原肽所产生的免疫应答反应。
Description
技术领域:
本发明属于生物技术领域,具体涉及一种猪病原或外源性蛋白特异性抗原肽筛选和鉴定的方法,特别涉及一种分离和鉴定PRRSV抗原肽的方法及其应用。
背景技术:
宿主机体的免疫系统在对外源性的病原感染,抗原刺激或疫苗(以下统称为外源性免疫原)免疫的过程做出反应的过程中,主要通过宿主免疫系统中的抗原提呈细胞(APCs)对外源性的抗原进行捕获,并进行加工提呈,将完整的外源性免疫原蛋白降解切割为长度为12-24的氨基酸长度的抗原肽片段,以上的抗原肽片段可以装配与主要组织相容性复合物(MHC)II类分子的抗原肽结合槽中,形成“MHC-II-抗原肽”复合物。在猪体内,负责完成此过程的MHC-II类分子为猪白细胞抗原-II DR(Swine leukocyte antigen DR,SLA-DR)并表达于多种APCs表面,如猪肺泡巨噬细胞,或淋巴组织内的巨噬细胞和树突状细胞。猪APCs表面的SLA-DR分子所加工提呈的抗原肽,能够作为猪特异性CD4+T细胞表位,被猪体内相应的CD4+T细胞所识别,最终激活猪机体的适应性免疫应答,产生免疫记忆,保护机体免受病原的感染。
因此在猪免疫系统活化的过程中,最终引发免疫应答的有效抗原成分仅仅是外源性免疫原被APCs加工提呈后长度为12-24个氨基酸的抗原肽片段(即猪特异性CD4+ T表位),而并非完整的免疫原。因此,直接使用来源于免疫原的CD4+T表位片段刺激机体即可激活机体的免疫系统,产生与使用完整外源性蛋白抗原刺激机体免疫系统类似的免疫应答。此外,使用来源于完整外源性免疫原的一个或多个CD4+T表位片段通过全人工合成或是基因工程重组表达的方法可以表达为一种仅含有CD4+T表位的重组蛋白,所获得的重组蛋白可以作为一种基因工程疫苗用于猪传染病的预防。
猪繁殖与呼吸综合征(Porcine reproductive and respiratory syndrome,PRRS)是由猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndromevirus,PRRSV)引起的以母猪流产和仔猪呼吸障碍为主要特征的病毒性传染病,并能引起严重的免疫抑制。该病毒已在全球猪群中广泛传播,给世界养猪业造成了巨大的经济损失,已成为全球规模化猪场的主要疫病之一,也是全球猪病控制上的一大难题。现有的PRRSV的防控主要依靠减毒活疫苗和灭活疫苗,现有的证据显示减毒活疫苗的在生产实践中尽管可诱导机体产生对于疫苗毒株同源性较高的野毒感染产生免疫保护,但可导致被免疫动物排毒(疫苗毒株),疫苗毒在免疫猪群中长期存在,同时疫苗株在被免疫猪群中长期存在的过程中存在毒力返祖以及与野生流行毒株重组重新演化为质病毒株的缺点,因此长期被认为存在安全性隐患。另一方面,尽管灭活疫苗安全性较好,但由于灭活疫苗免疫动物后刺激适应性免疫应答较弱,因此难以形成有效保护,市场接受度不高。
本发明以猪及猪繁殖与呼吸综合征病毒(PRRSV)为例,以体外培养的猪骨髓诱导树突状细胞(Bone Marrow derived dendritic cells, BM-DCs)作为猪特异性抗原提呈细胞(APCs),使用PRRSV感染BM-DCs模拟PRRSV感染宿主或免疫宿主后其编码的病毒抗原在猪体内加工提呈的过程,通过SLA-DR富集BM-DCs上的“SLA-DR-抗原肽”复合物,洗脱抗原肽后通过质谱技术测定抗原肽氨基酸序列的方法,从而获得来源于PRRSV的抗原肽氨基酸序列。
发明内容:
本发明旨在提供一种利用猪源APCs在猪种属上对病原或外源性蛋白进行特异性抗原肽(即CD4+T表位)筛选鉴定的方法,并且以猪繁殖呼吸道综合征为例来进行说明。
本发明的第二个目的以第一个目的中所获得的具有活化T细胞能力激活机体适应性免疫应答的抗原肽,通过基因工程表达的方法,替代原始病原或抗原,设计产生新型基于抗原肽的基因工程重组疫苗。
一种利用猪源APCs在猪种属上对病原或外源性蛋白进行特异性抗原肽(即CD4+T表位)筛选鉴定的方法,其特征在于,包括如下步骤:
步骤一,猪源APCs细胞的制备;
步骤二,抗原肽的质谱鉴定;
步骤三,基于NanoLuc融合蛋白的抗原肽的ELISA验证。
优选的,本发明中所述猪源APCs细胞的制备采用如下方法:(1)猪骨髓源树突状细胞,通过分离仔猪的长腿骨,开孔后使用细胞培养液冲洗骨髓腔,获得的骨髓细胞经过猪粒细胞巨噬细胞-集落刺激因子(GM-CSF)的诱导后经体外培养7天后获得(如图1所示);(2)猪肺泡巨噬细胞,通过冲洗猪肺脏后,将肺泡灌洗液离心后获得(如图2所示);(3)猪外周血单核细胞源巨噬细胞,收集猪抗凝血,通过使用淋巴细胞分离液,从抗凝血中获得猪外周血单核细胞,加入猪粒细胞巨噬细胞-集落刺激因子(GM-CSF)和猪白介素4(IL-4)体外诱导培养7天后获得(如图3所示);或者(4)使用外来免疫原(包括并不限于病原,疫苗,蛋白抗原等)免疫猪个体,其后,分离猪的脾脏,淋巴结,经过体外在单细胞滤网上研磨获得包含有猪内源性巨噬细胞的免疫细胞群(如图4所示)。
优选的,本发明所述的抗原肽的质谱鉴定的方法包括如下步骤:(1)制备富集“SLA-DR-抗原肽”复合物;(2)获得抗原肽氨基酸序列;(3)使用质谱仪测定抗原肽的分子量,并通过质谱分析软件搜索病原来源蛋白氨基酸数据库的方式以确定抗原肽氨基酸序列。
所述制备富集“SLA-DR-抗原肽”复合物的方法为:使用APCs经过免疫原刺激(如病毒感染,加入抗原和APCs共培养等方式),再使用细胞裂解缓冲液裂解APCs制备细胞裂解液,使用识别SLA-DR的抗体从全细胞裂解液中富集“SLA-DR-抗原肽”复合物。
所述获得抗原肽氨基酸片段的方法为:将富集“SLA-DR-抗原肽”复合物使用三氟乙酸缓冲液处理,从SLA-DR上分离洗脱抗原肽,再使用截留分子量为5kD的超滤管,去除残留抗体及SLA-DR分子单体,使滤过液中仅含有抗原肽,再通过脱盐处理后,即得到所述抗原肽氨基酸片段。
优选的,本发明所述的基于NanoLuc融合蛋白的抗原肽的ELISA验证的方法包括如下步骤:(1)将所获的抗原肽依照其cDNA序列人工合成克隆进入pET28-NanoLucC1载体,表达为NanoLuc融合抗原肽,并使用NanoLuc抗体检测融合;(2),分别将NanoLuc融合抗原肽作为包板抗原,通过ELISA检测相应病原感染猪血清样本,ELISA检测为阳性率超过60%的肽段即为有效的抗原肽,否则不是有效的抗原肽。
本发明还请求保护所述抗原肽在制备疫苗中的应用,优选的,所述疫苗为基因工程重组疫苗,优选的,所述基因工程重组疫苗采用如下方法制备得到:将质谱技术所测定的来源于免疫原的“抗原肽”,依据抗原肽的数量,将其氨基酸编码序列进行串联或单独表达,通过体外重组蛋白表达系统(大肠杆菌,酵母,昆虫细胞,哺乳动物细胞等)进行重组表达,所表达获得的重组蛋白,即为相应免疫原的“抗原肽基因工程重组疫苗”。
一种分离和鉴定PRRSV抗原肽的方法,其特征在于,包括如下步骤:
步骤一,猪源APCs细胞的制备;
步骤二,抗原肽的质谱鉴定;
步骤三,基于NanoLuc融合蛋白的抗原肽的ELISA验证。
优选的,所述猪源APCs细胞的制备的方法包括如下步骤:(1)使用4-6周龄仔猪腿骨,从中段使用无菌锯条锯断后,在断骨的另一端钻孔,使用含有EDTA的无血清RPMI 1640培养基冲洗骨髓腔,以获得猪骨髓细胞悬液;(2)将所获得的猪骨髓细胞悬液以300g离心力离心10分钟后弃去上清,加入红细胞裂解液在37℃培养20分钟以充分裂解红细胞,再加入2倍体积的PBS以终止反应,再次以300g离心10分钟以获得纯化的骨髓细胞;(3)将骨髓细胞进行计数,以1×107个骨髓细胞置于10mL含有10%胎牛血清的RPMI 1640培养基中,按照40ng每毫升的浓度加入猪GM-CSF进行培养,每间隔2天更换一次培养液,连续培养7天后,收集悬浮细胞,此即为作为猪源APCs的骨髓树突状细胞。
优选的,所述抗原肽的质谱鉴定的方法包括如下步骤:(1)使用PRRSV-JXA1毒株以1MOI剂量感染1×108个APCs细胞,在病毒感染24小时后离心收集细胞,使用膜蛋白提取试剂盒提取APCs细胞膜蛋白以避免胞浆蛋白的污染,通过特异性识别SLA-DR的抗体(Purified Mouse Anti-Pig SLA-DR,BD Pharmingen™,Material Number: 553642)从APCs细胞膜蛋白中富集“SLA-DR抗原肽”复合物;(2)使用10%甘氨酸溶液从抗体上洗脱SLA-DR抗原肽复合物,再使用10%三氟乙酸(TFA)从SLA-DR抗原肽复合物上洗脱抗原肽;(3)将10%三氟乙酸(TFA)洗脱液通过截留分子量为5kD的超滤管进行过滤后去除SLA-DR分子,收集滤过液,此即为SLA-DR上洗脱的抗原肽;(4)将抗原肽滤过液脱盐冻干后,使用分子纯水重悬,在Orbitrap Fusion Lumos Tribrid质谱仪上机检测,获得抗原肽的质谱图谱,将PRRSV-JXA1所有的编码蛋白氨基酸序列设置为PRRSV蛋白序列库,使用ProteomeDiscoverer或PEAKS® Studio X软件对所获得的质谱图谱进行搜库,即可获得与PRRSV-JXA1编码蛋白匹配的抗原肽序列。
优选的,所述基于NanoLuc融合蛋白的抗原肽的ELISA验证的方法包括如下步骤:(1)将所获的PRRSV-JXA1免疫肽依照其cDNA序列人工合成克隆进入pET28-NanoLucC1载体,表达为NanoLuc融合抗原肽,并使用NanoLuc抗体检测融合;(2)分别将NanoLuc融合抗原肽作为包板抗原,通过ELISA检测PRRSV-JXA1感染猪血清样本。
本发明还请求保护根据以上方法获得的PRRSV抗原肽,其包括序列为SEQ ID NO:3-43的抗原肽或其组合。
本发明还请求保护包括PRRSV抗原肽的重组蛋白,分别是PRRSV-FAnsp,其氨基酸序列如SEQ ID NO:1所示,或者PRRSV-FBsp,其氨基酸序列如SEQ ID NO:2所示。
本发明还请求保护基于抗原肽的PRRSV-JXA1基因工程重组疫苗,所述疫苗的制备方法包括:(1)将所获的PRRSV-JXA1免疫肽依照其来源,划分为非结构蛋白NSPs来源及结构蛋白SPs来源,将NSPs来源的抗原肽串联为一个单一的重组蛋白,并人工合成其编码cDNA序列,命名为PRRSV-FAnsp,其氨基酸序列如SEQ ID NO:1所示;另一方面,将SPs来源的抗原肽串联为一个单一的重组蛋白,并人工合成其编码cDNA序列,命名为PRRSV-FBsp,其氨基酸序列如SEQ ID NO:2所示;(2)分别将两个cDNA片段克隆至pET28a载体,在大肠杆菌中诱导进行表达为包涵体,获得重组蛋白rPRRSV-FAnsp与rPRRSV-FBsp;(3)将重组蛋白rPRRSV-FAnsp与rPRRSV-FBsp按比例混合后,加入油佐剂混合均匀,即得到所述基因工程重组疫苗。
基于以上技术方案,本发明具有如下优点和有益效果:
第一,本发明通过对免疫原(病原或蛋白抗原)上的有效抗原肽序列进行筛选,去除免疫原中对机体不具有免疫活性(即不能激活机体适应性免疫应答)的无用肽段,筛选出免疫活性较强,可高效刺激机体产生免疫应答的抗原肽。抗原肽相对于原始免疫原,因其序列较短(12-24氨基酸残基),亲水性较强(需具备与SLA-DR结合的能力,疏水性肽段无法结合SLA-DR从而被提呈激活CD4+T细胞),因此易于在体外进行表达,并且可随意将不同抗原肽或不同免疫原来源的抗原肽串联制备复合抗原肽,或将同一抗原肽进行多次重复以增强T细胞识别,同时可与其他具有免疫增强蛋白(如FC标签,IL-2)等融合表达,进一步增强机体免疫系统对抗原肽所产生的的免疫应答反应。
第二,本发明所产生的抗原肽基因工程重组疫苗免疫动物后,不具有弱毒疫苗免疫动物后的持续排毒,疫苗毒力返祖,与野毒重组等缺点。与灭活疫苗相比,无需病毒培养及灭活,生产工艺简单。
第三,本发明所产生的抗原肽基因工程重组疫苗,可以通过去除病原/疫苗特定蛋白抗原肽的方法,制备为缺失特定蛋白抗原肽的鉴别疫苗,在免疫动物后,可与野毒感染动物区分,作为一种鉴别疫苗被使用。
附图说明:
图1:使用GM-CSF刺激诱导分化培养7天后骨髓树突状细胞形态:A:未诱导的骨髓细胞;B:GM-CSF骨髓树突状细胞BM-DCs。
图2:新鲜分离的猪肺泡巨噬细胞形态照片。
图3:猪外周血单个核细胞显微镜下形态。
图4:猪淋巴结及脾脏研磨去除红细胞后淋巴细胞。
图5:抗原肽序列质谱分析代表性图谱(NSP1β来源)。
图6:荧光素酶NanoLuc融合免疫抗原肽的大肠杆菌表达示例。
图7:抗原肽串联后分段表达结果。
图8:使用重组rPRRSV-FA与rPRRSV-FB蛋白免疫动物的免疫方案示意图。
图9:对重组rPRRSV-FA与rPRRSV-FB蛋白免疫动物血清样本使用爱德士公司PRRSV抗体ELISA检测试剂盒检测结果(S/P值大于0.4即为阳性)。
图10:使用重组rPRRSV-FA与rPRRSV-FB蛋白免疫动物血清样本进行中和抗体效价检测。
具体实施方式:
下面列出具体实施方案对本发明做进一步阐述,以使本领域技术人员可以更清楚得得知本发明的技术方案,并非对本发明的限制。
实施例1:猪源APCs细胞的制备(以猪骨髓树突状细胞为例)
1. 使用4-6周龄仔猪腿骨,从中段使用无菌锯条锯断后,在断骨的另一端钻孔,使用含有EDTA的无血清RPMI 1640培养基冲洗骨髓腔,以获得猪骨髓细胞悬液。
将所获得的猪骨髓细胞悬液以300g离心力离心10分钟后弃去上清,加入红细胞裂解液在37℃培养20分钟以充分裂解红细胞,再加入2倍体积的PBS以终止反应,再次以300g离心10分钟以获得纯化的骨髓细胞。
将骨髓细胞进行计数,以1×107个骨髓细胞置于10mL含有10%胎牛血清的RPMI1640培养基中,按照40ng每毫升的浓度加入猪GM-CSF进行培养,每间隔2天更换一次培养液,连续培养7天后,收集悬浮细胞,此即为作为猪源APCs的骨髓树突状细胞,其形态如图1所示,可进行下一步实验。
实施例2、抗原肽的质谱鉴定
1.使用PRRSV-JXA1毒株以1MOI剂量感染1×108个APCs细胞,在病毒感染24小时后离心收集细胞,使用膜蛋白提取试剂盒提取APCs细胞膜蛋白以避免胞浆蛋白的污染,通过特异性识别SLA-DR的抗体(Purified Mouse Anti-Pig SLA-DR,BD Pharmingen™,Material Number: 553642)从APCs细胞膜蛋白中富集“SLA-DR抗原肽”复合物。
使用10%甘氨酸溶液从抗体上洗脱SLA-DR抗原肽复合物,再使用10%三氟乙酸(TFA)从SLA-DR抗原肽复合物上洗脱抗原肽。
将10%三氟乙酸(TFA)洗脱液通过截留分子量为5kD的超滤管进行过滤后去除SLA-DR分子,收集滤过液,此即为SLA-DR上洗脱的抗原肽。
4.将抗原肽滤过液脱盐冻干后,使用分子纯水重悬,在Orbitrap Fusion LumosTribrid质谱仪上机检测,获得抗原肽的质谱图谱(代表性图谱如图5所示)。将PRRSV-JXA1所有的编码蛋白氨基酸序列设置为PRRSV蛋白序列库,使用Proteome Discoverer或PEAKS®Studio X软件对所获得的质谱图谱进行搜库,即可获得与PRRSV-JXA1编码蛋白匹配的抗原肽序列,其结果如表1所示。
表1通过分析质谱技术获得的PRRSV-JXA1病毒全基因组免疫肽序列
实施例3:基于NanoLuc融合蛋白的抗原肽的ELISA验证
1. 将所获的PRRSV-JXA1免疫肽依照其cDNA序列人工合成克隆进入pET28-NanoLucC1载体,表达为NanoLuc融合抗原肽,并使用NanoLuc抗体检测融合,如图6所示。
分别将NanoLuc融合抗原肽作为包板抗原,通过ELISA检测PRRSV-JXA1感染猪血清样本。其结果如表2所示。
表2 使用5份PRRSV抗体阳性血清与PRRSV抗体阴性血清对荧光素酶NanoLuc融合免疫抗原肽进行检测寻找阳性肽段
表2(续)使用5份PRRSV抗体阳性血清与PRRSV抗体阴性血清对荧光素酶NanoLuc融合免疫抗原肽进行检测寻找阳性肽段
实施例4:基于抗原肽的PRRSV-JXA1基因工程重组疫苗的表达
1.将所获的PRRSV-JXA1免疫肽依照其来源,划分为非结构蛋白(NSPs)来源及结构蛋白(SPs)来源。将NSPs来源的抗原肽串联为一个单一的重组蛋白,并人工合成其编码cDNA序列,命名为PRRSV-FAnsp,其氨基酸序列如SEQ ID NO:1所示。另一方面,将SPs来源的抗原肽串联为一个单一的重组蛋白,并人工合成其编码cDNA序列,命名为PRRSV-FBsp,其氨基酸序列如SEQ ID NO:2所示。
分别将两个cDNA片段克隆至pET28a载体,在大肠杆菌中诱导进行表达为包涵体,获得重组蛋白rPRRSV-FAnsp与rPRRSV-FBsp,如图7所示。
实施例5:PRRSV抗原肽基因工程重组疫苗的免疫原性鉴定
1. 选取四周龄仔猪,按照每头猪100μg重组rPRRSV-FAnsp蛋白(1mg/mL PBS)与100μg 重组rPRRSV-FBsp(1mg/mL PBS)蛋白混合后(终末体积为200μL)与油佐剂按照蛋白佐剂比值(46:56)的方式混匀乳化后注射仔猪,每间隔2周免疫一次,共免疫3次,采集每次免疫前的血清样本,免疫程序如图8所示。
在每次免疫前采集动物的血清样本,将不同时间点获得血清样本,使用爱德士公司PRRSV X3 Herd Check试剂盒进行检测,以确定血清中产生PRRSV特异性抗体。如图9所示, 除第一次免疫前(0天),血清样本S/P值低于阳性cut-off值(0.4)外,其他时间点样本均为阳性反应,提示被免疫动物体内产生PRRSV特异性抗体。
将不同时间点获得血清样本,PRRSV-JXA1毒株在MARC-145细胞上进行体外中和实验,以确定被免疫动物的血清中已产生PRRSV特异性中和抗体并且可提供免疫保护,如图10所示,除第一次免疫前血清样本无中和抗体产生外,其他时间点血清样本均可检测到PRRSV特异性中和血清,其中免疫两次后(4周),体外病毒中和实验的血清中和效价平均值为1:16倍稀(即24),达到对野毒感染提供免疫保护的水平。
序列表
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1 5 10 15
Thr Arg Arg Gly Leu
20
<210> 23
<211> 10
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 23
Ala Ile Thr Ile Asp Ser Ser Gln Gly Ala
1 5 10
<210> 24
<211> 23
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 24
Ala Arg His Ala Ile Phe Val Tyr Asp Pro His Arg Gln Leu Gln Ser
1 5 10 15
Met Phe Asp Leu Pro Ala Lys
20
<210> 25
<211> 19
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 25
Asp Leu Pro Ala Lys Gly Thr Pro Val Asn Leu Ala Val Pro Arg Glu
1 5 10 15
Glu Gln Leu
<210> 26
<211> 10
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 26
Leu Val Ala Ser Leu Arg Pro Ile His Lys
1 5 10
<210> 27
<211> 21
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 27
Gly Glu Ala Gln Met Leu Pro Glu Thr Val Phe Ser Thr Gly Arg Ile
1 5 10 15
Glu Val Asp Cys Arg
20
<210> 28
<211> 10
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 28
Leu Pro His Ala Phe Ile Gly Asp Val Lys
1 5 10
<210> 29
<211> 9
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 29
Asp Lys Thr Ala Tyr Phe Gln Leu Glu
1 5
<210> 30
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 30
Asn Phe Leu Trp Met Leu Ser Arg
1 5
<210> 31
<211> 11
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 31
Ile Met Glu Lys Ala Gly Gln Ala Ala Trp Lys
1 5 10
<210> 32
<211> 14
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 32
Ala Glu Thr Cys Lys Tyr Leu Ala Ser Arg Leu Pro Met Leu
1 5 10
<210> 33
<211> 13
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 33
Pro Thr Pro Gly Ser Arg Pro Lys Leu His Asp Phe Gln
1 5 10
<210> 34
<211> 14
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 34
Ala Leu Thr Thr Ser His Phe Leu Asp Thr Val Gly Leu Ala
1 5 10
<210> 35
<211> 13
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 35
Val Leu Asp Gly Ser Ala Ala Thr Pro Leu Thr Arg Val
1 5 10
<210> 36
<211> 16
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 36
Ala Pro Gln Lys Val Leu Leu Ala Phe Ser Ile Thr Tyr Thr Pro Val
1 5 10 15
<210> 37
<211> 37
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 37
Gly Arg Leu Leu Gly Leu Leu His Leu Leu Ile Phe Leu Asn Cys Ala
1 5 10 15
Phe Thr Phe Gly Tyr Met Thr Phe Val His Phe Glu Ser Thr Asn Arg
20 25 30
Val Ala Leu Thr Met
35
<210> 38
<211> 12
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 38
Lys Tyr Ile Leu Ala Pro Ala His His Val Glu Ser
1 5 10
<210> 39
<211> 24
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 39
Ala Gly Phe His Pro Ile Ala Ala Asn Asp Asn His Ala Phe Val Val
1 5 10 15
Arg Arg Pro Gly Ser Thr Thr Val
20
<210> 40
<211> 12
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 40
Lys Gln Gly Val Val Asn Leu Val Lys Tyr Ala Lys
1 5 10
<210> 41
<211> 10
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 41
Lys Gly Asn Gly Gln Pro Val Asn Gln Leu
1 5 10
<210> 42
<211> 13
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 42
Lys Asn Pro Glu Lys Pro His Phe Pro Leu Ala Thr Glu
1 5 10
<210> 43
<211> 23
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 43
Thr Val Glu Phe Ser Leu Pro Thr Gln His Thr Val Arg Leu Ile Arg
1 5 10 15
Ala Thr Ala Ser Pro Ser Ala
20
Claims (6)
1.一种利用猪源APCs在猪种属上对病原或外源性蛋白进行特异性抗原肽筛选鉴定的方法,其特征在于,包括如下步骤:
步骤一,猪源APCs细胞的制备;
步骤二,抗原肽的质谱鉴定;
步骤三,基于NanoLuc融合蛋白的抗原肽的ELISA验证;
所述步骤一,猪源APCs细胞的制备采用如下方法:(1)猪骨髓源树突状细胞,通过分离仔猪的长腿骨,开孔后使用细胞培养液冲洗骨髓腔,获得的骨髓细胞经过猪粒细胞巨噬细胞-集落刺激因子GM-CSF的诱导后经体外培养7天后获得;(2)猪肺泡巨噬细胞,通过冲洗猪肺脏后,将肺泡灌洗液离心后获得;(3)猪外周血单核细胞源巨噬细胞,收集猪抗凝血,通过使用淋巴细胞分离液,从抗凝血中获得猪外周血单核细胞,加入猪粒细胞巨噬细胞-集落刺激因子GM-CSF和猪白介素4体外诱导培养7天后获得;或者(4)使用外来免疫原免疫猪个体,其后,分离猪的脾脏,淋巴结,经过体外在单细胞滤网上研磨获得包含有猪内源性巨噬细胞的免疫细胞群;
所述步骤二,抗原肽的质谱鉴定的方法包括如下步骤:(1)制备富集“SLA-DR-抗原肽”复合物;(2)获得抗原肽氨基酸序列;(3)使用质谱仪测定抗原肽的分子量,并通过质谱分析软件搜索病原来源蛋白氨基酸数据库的方式以确定抗原肽氨基酸序列;
所述制备富集“SLA-DR-抗原肽”复合物的方法为:使用APCs经过免疫原刺激,再使用细胞裂解缓冲液裂解APCs制备细胞裂解液,使用识别SLA-DR的抗体从全细胞裂解液中富集“SLA-DR-抗原肽”复合物;
所述获得抗原肽氨基酸片段的方法为:将富集“SLA-DR-抗原肽”复合物使用三氟乙酸缓冲液处理,从SLA-DR上分离洗脱抗原肽,再使用截留分子量为5kD的超滤管,去除残留抗体及SLA-DR分子单体,使滤过液中仅含有抗原肽,再通过脱盐处理后,即得到所述抗原肽氨基酸片段;
所述步骤三,基于NanoLuc融合蛋白的抗原肽的ELISA验证的方法包括如下步骤:(1)将所获的抗原肽依照其cDNA序列人工合成克隆进入pET28-NanoLucC1载体,表达为NanoLuc融合抗原肽,并使用NanoLuc抗体检测融合;(2)分别将NanoLuc融合抗原肽作为包板抗原,通过ELISA检测相应病原感染猪血清样本,ELISA检测为阳性的肽段即为有效的抗原肽,否则不是有效的抗原肽。
2.权利要求1所获得的抗原肽在制备疫苗中的应用,其特征在于,所述疫苗为基因工程重组疫苗。
3.根据权利要求2所述的应用,其特征在于,所述基因工程重组疫苗采用如下方法制备得到:将质谱技术所测定的来源于免疫原的“抗原肽”,依据抗原肽的数量,将其氨基酸编码序列进行串联或单独表达,通过体外重组蛋白表达系统进行重组表达,所表达获得的重组蛋白,即为相应免疫原的“抗原肽基因工程重组疫苗”。
4.一种分离和鉴定PRRSV抗原肽的方法,其特征在于,包括如下步骤:
步骤一,猪源APCs细胞的制备;
步骤二,抗原肽的质谱鉴定;
步骤三,基于NanoLuc融合蛋白的抗原肽的ELISA验证;
所述步骤一,猪源APCs细胞的制备的方法包括如下步骤:(1)使用4-6周龄仔猪腿骨,从中段使用无菌锯条锯断后,在断骨的另一端钻孔,使用含有EDTA的无血清RPMI 1640培养基冲洗骨髓腔,以获得猪骨髓细胞悬液;(2)将所获得的猪骨髓细胞悬液以300g离心力离心10分钟后弃去上清,加入红细胞裂解液在37℃培养20分钟以充分裂解红细胞,再加入2倍体积的PBS以终止反应,再次以300g离心10分钟以获得纯化的骨髓细胞;(3)将骨髓细胞进行计数,以1×107个骨髓细胞置于10mL含有10%胎牛血清的RPMI 1640培养基中,按照40ng每毫升的浓度加入猪GM-CSF进行培养,每间隔2天更换一次培养液,连续培养7天后,收集悬浮细胞,此即为作为猪源APCs的骨髓树突状细胞;
所述步骤二,抗原肽的质谱鉴定的方法包括如下步骤:(1)使用PRRSV-JXA1毒株以1MOI剂量感染1×108个APCs细胞,在病毒感染24小时后离心收集细胞,使用膜蛋白提取试剂盒提取APCs细胞膜蛋白以避免胞浆蛋白的污染,通过特异性识别SLA-DR的抗体从APCs细胞膜蛋白中富集“SLA-DR抗原肽”复合物;(2)使用10%甘氨酸溶液从抗体上洗脱SLA-DR抗原肽复合物,再使用10%三氟乙酸从SLA-DR抗原肽复合物上洗脱抗原肽;(3)将10%三氟乙酸洗脱液通过截留分子量为5kD的超滤管进行过滤后去除SLA-DR分子,收集滤过液,此即为SLA-DR上洗脱的抗原肽;(4)将抗原肽滤过液脱盐冻干后,使用分子纯水重悬,在Orbitrap FusionLumos Tribrid质谱仪上机检测,获得抗原肽的质谱图谱,将PRRSV-JXA1所有的编码蛋白氨基酸序列设置为PRRSV蛋白序列库,使用Proteome Discoverer或PEAKS® Studio X软件对所获得的质谱图谱进行搜库,即可获得与PRRSV-JXA1编码蛋白匹配的抗原肽序列;
所述步骤三,基于NanoLuc融合蛋白的抗原肽的ELISA验证的方法包括如下步骤:(1)将所获的PRRSV-JXA1免疫肽依照其cDNA序列人工合成克隆进入pET28-NanoLucC1载体,表达为NanoLuc融合抗原肽,并使用NanoLuc抗体检测融合;(2)分别将NanoLuc融合抗原肽作为包板抗原,通过ELISA检测PRRSV-JXA1感染猪血清样本。
5.包括PRRSV抗原肽的重组蛋白,分别是PRRSV-FAnsp,其氨基酸序列如SEQ ID NO:1所示,或者PRRSV-FBsp,其氨基酸序列如SEQ ID NO:2所示。
6.基于抗原肽的PRRSV-JXA1基因工程重组疫苗,所述疫苗的制备方法包括:(1)将权利要求4所述方法所获的PRRSV-JXA1免疫肽依照其来源,划分为非结构蛋白NSPs来源及结构蛋白SPs来源,将NSPs来源的抗原肽串联为一个单一的重组蛋白,并人工合成其编码cDNA序列,命名为PRRSV-FAnsp,其氨基酸序列如SEQ ID NO:1所示;另一方面,将SPs来源的抗原肽串联为一个单一的重组蛋白,并人工合成其编码cDNA序列,命名为PRRSV-FBsp,其氨基酸序列如SEQ ID NO:2所示;(2)分别将两个cDNA片段克隆至pET28a载体,在大肠杆菌中诱导进行表达为包涵体,获得重组蛋白rPRRSV-FAnsp与rPRRSV-FBsp;(3)将重组蛋白rPRRSV-FAnsp与rPRRSV-FBsp按比例混合后,加入油佐剂混合均匀,即得到所述基因工程重组疫苗。
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