CN117143206A - Alv-j mhc-b21限制性表位肽及其筛选方法和应用 - Google Patents
Alv-j mhc-b21限制性表位肽及其筛选方法和应用 Download PDFInfo
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Abstract
本发明公开了ALV‑J MHC‑B21限制性表位肽及其筛选方法和应用,涉及生物技术领域。所述ALV‑J MHC‑B21限制性表位肽的氨基酸序列如SEQ ID NO.1或SEQ ID NO.2所示。本发明首先根据B21单倍型SPF鸡MHC I类分子结合多肽的基序合成多肽,然后体外培养ALV‑J特异性CD8+T细胞,用多肽刺激CD8+T细胞后通过ELISpot实验筛选出具有免疫原性的多肽表位,并利用ELISA和qPCR实验验证其免疫原性,从而确定有效的T细胞表位。经验证本发明成功筛选出了两条具有免疫原性的表位肽,为后续ALV‑J表位疫苗研发提供了基础。
Description
技术领域
本发明涉及生物技术领域,特别是涉及ALV-J MHC-B21限制性表位肽及其筛选方法和应用。
背景技术
禽白血病病毒(Avian Leukosis Virus)为单股、正链RNA病毒,属于反转录病毒科α反转录病毒属,其基因组分子有三个主要编码基因,即衣壳蛋白基因(gag)、聚合酶基因(pol)和囊膜糖蛋白基因(env),表达Gag、Pol、Gp85、Gp37四种蛋白。目前根据宿主范围、病毒囊膜蛋白的抗原差异性等可以分为11种亚型,其中J亚型禽白血病病毒(ALV-J)感染会诱发鸡产生肿瘤(如髓细胞瘤血管瘤等),对全球的养禽业危害严重。到目前为止,预防ALV-J的感染尚无有效的商品化疫苗,也没有切实有效的治疗措施。有研究表明,细胞免疫在抵抗ALV-J感染过程中发挥了重要作用,尤其是CD8+ T细胞应答。
由CD8+ T细胞介导的细胞免疫应答过程主要是通过T细胞受体(T cellreceptor,TCR)对I型主要组织相容性复合体(major histocompatibility complexclass,MHC I)与抗原肽组成的复合物进行特异性识别而启动。抗原肽是由抗原递呈细胞(Antigen presenting cell,APC)的酶系统对吞噬入胞内的抗原物质进行消化处理后形成的短肽,又称为表位,MHC I分子与特定的抗原多肽片段结合后被转移到细胞表面,通过淋巴细胞特异识别诱导免疫应答发生。所以,刺激机体产生细胞免疫应答,筛选出具有免疫原性的表位至关重要。
以往对于CD8+ T细胞表位的筛选主要是根据不同MHC单倍型SPF鸡的基序合成随机肽库,然后利用多肽刺激PBMC或脾脏细胞,通过ELISpot检测IFN-γ的产生来确定是否为CD8+ T细胞表位,但是这个方法敏感性不高,因此有必要开发一种新的多肽表位免疫原性筛选方法,以增强其灵敏性。
发明内容
本发明的目的是提供ALV-J MHC-B21限制性表位肽及其筛选方法和应用,以解决上述现有技术存在的问题,该ALV-J MHC-B21限制性表位肽具有良好的免疫原性,为ALV-J表位疫苗的研发提供了基础。
为实现上述目的,本发明提供了如下方案:
本发明提供ALV-J MHC-B21限制性表位肽,所述ALV-J MHC-B21限制性表位肽的氨基酸序列如SEQ ID NO.1或SEQ ID NO.2所示。
本发明还提供上述的ALV-J MHC-B21限制性表位肽在制备ALV表位疫苗中的应用。
本发明还提供一种ALV表位疫苗,包含上述的ALV-J MHC-B21限制性表位肽。
进一步地,所述ALV表位疫苗还包括药学上可接受的辅料。
本发明还提供一种筛选ALV-J MHC-B21限制性表位肽的方法,包括利用体外扩增ALV-J特异性CD8+ T细胞筛选ALV-J MHC-B21限制性潜在表位肽,再对所述ALV-J MHC-B21限制性潜在表位肽进行免疫原性验证,从而得到所述ALV-J MHC-B21限制性表位肽的步骤。
进一步地,所述免疫原性验证采用ELISA检测和/或荧光定量PCR检测。
本发明公开了以下技术效果:
本发明首先根据文献报道的B21单倍型SPF鸡MHC I类分子结合多肽的基序(X-H/K/R-X-X-X-X-X-(X)-E/D-X-A/V/L/I/F/M)合成多肽,然后体外培养ALV-J特异性CD8+ T细胞,用多肽刺激CD8+ T细胞后通过ELISpot实验筛选出具有免疫原性的多肽表位,并利用ELISA和qPCR实验验证其免疫原性,从而确定有效的T细胞表位。本发明的多肽表位免疫原性筛选方法灵敏性更高,从而找到更全面靶向ALV-J的B21 MHC限制性表位,经验证本发明成功筛选出了两条具有免疫原性的表位肽Gag343-352和Gag531-541,为后续ALV-J表位疫苗研发提供了基础。
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为鸡PBMC体外培养形态;图中标尺均为100μm;
图2为CD8+ T细胞增殖圈门策略及数量和比例变化;其中,A为ALV-J刺激后T细胞增殖的圈门策略,B和C分别为CD8+ T细胞增殖数量和比例的变化;
图3为ALV-J刺激CFSE标记增殖实验;其中,A为流式圈门策略;B为流式检测结果;B中红色表示CFSE标记PBMC后2天对照组样本,蓝色表示ALV-J刺激CFSE标记PBMC后2天的样本,橙色表示ALV-J刺激CFSE标记PBMC后4天的样本,绿色表示ALV-J刺激CFSE标记PBMC后6天的样本,从左到右样本编号依次为#23、#26、#17;
图4为ELISpot检测IFN-γ的结果;其中,A为肽池刺激ELISpot结果图;B为Pool 1和Pool 2中单个肽段刺激ELISpot结果图;
图5为荧光定量PCR检测相关细胞因子表达的结果;其中,A为Gag343-352刺激活化T细胞后免疫相关细胞因子的表达结果;B为Gag531-541刺激后免疫相关细胞因子的表达结果;
图6为ELISA检测相关细胞因子的表达结果;其中,A为Gag343-352刺激活化T细胞相关细胞因子分泌结果;B为Gag531-541刺激活化T细胞相关细胞因子分泌结果。
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:
ALV-J:J亚型禽白血病病毒;
DPI:攻毒后天数;
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;
IL-2:白介素2;
APC:抗原递呈细胞;
ELISpot:酶联免疫斑点实验;
PBS:磷酸盐平衡生理盐水;
ELISA:酶联免疫吸附实验。
本发明首先根据文献报道的B21单倍型SPF鸡MHC I类分子结合多肽的基序(X-H/K/R-X-X-X-X-X-(X)-E/D-X-A/V/L/I/F/M)合成多肽,然后体外培养ALV-J特异性CD8+T细胞,用多肽刺激CD8+ T细胞后通过ELISpot实验筛选出具有免疫原性的多肽表位,并利用ELISA和qPCR实验验证其免疫原性,从而确定有效的T细胞表位,具体详述如下:
实施例1
1.1试验原料和病毒
本试验所用的细胞为本课题组保存的ALV-J攻毒后28DPI B21单倍型SPF鸡PBMC,购自国家禽类实验动物资源库;所用多肽(表1所列多肽)由金斯瑞科技股份有限公司合成;ALV-J SCAU-HN06株由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,已在文献“Systematic Identificatin of Host Immune Key Factors InfluencingViral Infection in PBL of ALV-J Infected SPF Chicke”中公开。
1.2试验主要试剂
RPMI-1640培养基、FBS澳洲胎牛血清,PBS PH7.4 basic 1×,2-巯基乙醇(2-ME)、L-谷氨酰胺(100X)(L-Glutamine),二巯基乙醇(1000X)、HEPES(100X)购自美国GIBCO公司;二甲基亚砜(DMSO)、半刀豆球蛋白A(ConA)购自美国Sigma公司;Recombinant Human IL-2购自美国PeproTech公司。ChamQ SYRBR qPCR Master Mix购自南京诺唯赞生物科技有限公司;TMB ELISpot专用显色液购自中国达科为公司,Chicken IFN-γELISpot BASIC kit购自Mabtech公司;鸡肿瘤坏死因子(TNF-α)ELISA试剂盒和白介素2(IL-2)ELISA试剂盒购自Eβios公司。
抗体Anti-chicken CD3 antibody、Anti-chicken CD8αantibody、Anti-chickenIFN-γ-FITC antibody购自SouthernBiotech公司。
1.3主要溶液配制
流式Buffer:2% FBS+98% PBS,配置完成后放于4℃备用。
细胞冻存液:10% DMSO+90% FBS,配置完成后放于4℃备用。
T细胞培养基:10% FBS+1% L-Glutamine+1% MEM(100×)+1%青霉素-链霉素溶液+1%非必须氨基酸(100×)+0.1%2-ME,配置完成后放于4℃备用。
RP5:5% FBS+95%1640培养基,配制完成后置于4℃保存。
RP10:10% FBS+90%1640培养基,配制完成后置于4℃保存。
2试验方法
2.1 ALV-J特异性CD8+ T细胞体外扩增
2.1.1体外培养ALV-J特异性CD8+ T细胞
①外周血单个核细胞制备
复苏保存的ALV-J攻毒后28DPI B21单倍型SPF鸡PBMC,用台盼蓝计数同时观察存活率,每个样品取每孔3×106个细胞,用T细胞培养基重悬,每孔1mL细胞悬液铺于48孔不贴壁的细胞板中,并且按20U/mL加入IL-2。
②ALV-J感染抗原递呈细胞
另取每孔6×105个细胞(即ALV-J攻毒后28DPI B21单倍型SPF鸡PBMC)做APC,置于生化反应管中,440×g,5min离心后用1640培养基重悬,按103TCID50接种ALV-J,每孔150μL,病毒液与细胞悬液的比例为1:1,混合均匀后置于39℃培养箱中孵育5小时。将感染了ALV-J的PBMC作为实验中的抗原递呈细胞(Antigen presenting cell,APC)。
③APC刺激PBMC
APC孵育完成后,补加RP5到5mL,440×g离心五分钟后,用每孔100μL T细胞培养基重悬,加入到铺好的48孔板中,作为实验组;阳对照组按2.5ng/mL加入ConA(储存浓度为1ng/μL),阴对照不做任何处理。培养箱培养6天,其中每隔两天进行换液,具体方法弃掉350μL培养基并且补加500μL新的T细胞培养基;每天观察细胞形态变化以及培养基的颜色变化。
④流式细胞术检测CD8+ T细胞比例变化
每2,4,6天染色上机监测CD8+ T细胞的数量及比例变化。ALV-J刺激鸡记忆性PBMC增殖培养后不同时间后,显微镜观察细胞形态有明显变化时,实验组,阳对照组和阴对照组每组取1×106个细胞进行染色,流式抗体CD3用流式Buffer按1:50稀释,CD8用流式Buffer按1:25稀释,四度避光孵育30min后离心,200μL流式Buffer重悬,流式细胞仪检测CD8+ T细胞数量和比例变化。
2.1.2 CFSE检测ALV-J特异性CD8+ T细胞增殖
①CFSE标记PBMC
实验前先将灭菌PBS与RP-10培养基置于37℃水浴锅进行预热。用PBS清洗细胞,400g离心5min,将细胞沉淀以1×107/mL的浓度重悬于含有0.5μM CFSE的PBS中,于37℃水浴锅避光孵育10min。孵育完成后,400g离心5min,弃掉上清,加入预热的RP10培养基清洗细胞。离心弃上清后用T细胞培养基重悬。
②ALV-J感染CFSE标记后的PBMC
取2×106个CFSE标记后的PBMC于生化反应管中,按103TCID50接种ALV-J,每孔150μL病毒液,病毒液与细胞悬液的比例为1:1,混合均匀后置于39℃培养箱中孵育5小时。
③CFSE-APC刺激CFSE-PBMC
将CFSE标记后的PBMC按2.1.1方法进行分组和培养,每天观察细胞形态学变化,并取细胞进行流式检测,记录细胞增殖变化情况。
2.2 B21单倍型SPF鸡CD8+ T细胞抗原表位筛选及鉴定
2.2.1 ELISpot检测候选多肽免疫原性
按2.1.1方法体外刺激PBMC增殖,在增殖第四天用合成的候选表位(表1所示)进行ELISpot试验,检测多肽表位刺激PBMC活化后产生IFN-γ的情况,首先构建肽池,每个肽池中含有4条候选表位(见表1),然后挑选出有效肽池进行其中单个肽的筛选。具体步骤如下:
(1)活化PVDF 96孔板:每孔加入15μL 35%乙醇,反应至多1min,然后用无菌水洗涤5次;
(2)包被抗体:将抗鸡IFN-γ稀释至15μg/mL,每孔加入100μL,4℃作用过夜;
(3)封闭:倒掉包被液,再用PBS洗涤5次,随后在灭菌的吸水纸上扣干板子。按200μL/孔在PVDF 96孔板上加入含10% FBS的RMPI 1640培养基,室温封闭反应30min;
(4)刺激:倒掉封闭液,每孔加入200μL的体外扩增的T细胞,细胞浓度为1×106cells/mL的淋巴细胞悬液。同时向实验组加入50μL稀释后的抗原多肽,使其终浓度为10μg/mL,阳对照组加入终浓度为10μg/mL的PMA,阴对照组则不加入任何刺激物。所有的样品加完后,将PVDF 96孔板放入CO2培养箱,37℃作用48h;
(5)二抗孵育:培养结束后,甩去细胞,用PBS洗涤5次,每孔加入100μL浓度为1μg/mL的生物素标记二抗,室温孵育2h;
(6)HRP孵育:甩掉二抗液,用PBS洗涤5次,随后向孔内加入100μL链霉亲和素标记的HRP,室温孵育1h;
(7)显色:甩掉HRP孵育液,用PBS洗涤5次,随后每孔加入100μL TMB显色底物,室温(或37℃)作用30min,当阳对照组出现明显斑点即可甩掉显色液,用纯水终止显色,风干读板。
表1
2.2.2荧光定量PCR检测免疫相关细胞因子的分泌
按2.1.1方法体外刺激PBMC增殖,在增殖第四天按照10μg/mL加入多肽刺激24小时,弃掉培养基后加入裂解液,利用TRIZOL法提取细胞RNA,分光光度计检测RNA浓度后。利用TAKARA公司的PrimeScriptTM RT Master Mix(Perfect Real Time)试剂盒进行RNA反转录,体系如表2。反应程序:37℃反应15min,85℃灭活5s。
表2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中细胞免疫相关基因的变化。检测的目的基因及引物如表3,体系如表4。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。上述实验结果运用GraphPad Prism 8软件进行统计分析。
表3细胞免疫相关基因qPCR引物
表4荧光定量反应体系
2.2.3 ELISA检测免疫相关细胞因子的分泌
按2.1.1方法体外刺激T细胞增殖,在增殖第四天按照10μg/mL加入多肽刺激24小时,取上清液进行ELISA试验检测多肽刺激活化T细胞后产生相关细胞因子的情况,具体步骤如下:
(1)样本处理
无菌管收集细胞培养上清,离心20min,仔细收集上清,用PBS稀释细胞悬液,使细胞浓度达到100万/mL左右,然后反复冻融,使细胞破碎并放出细胞内成分,再次离心20分钟,收集上清用作检测。
(2)标准曲线建立
在酶标包被板上设标准品孔10孔,在第一第二孔中分别加入标准品100μL,然后在第一第二孔中加标准品稀释液50μL,混匀;然后从第一第二孔中分别各取100μL加入到第三第四孔中,再在第三第四孔中分别加标准品稀释液50μL,混匀;然后再第三第四孔中先各取50μL弃掉,然后各取50μL加到第五第六孔中,按上述操作一直稀释到第九第十孔,混合均匀后在第九第十孔中各取50μL弃掉。(稀释后各孔加样量都为50μL,浓度分别为90ng/L,60ng/L,30ng/L,15ng/L,7.5ng/L)
(3)加样
设空白孔(不加样品及酶标试剂,其他的操作相同),在酶标板上先加样品稀释液40μL,然后加待测样品10μL。
(4)孵育
用封板膜封板后置37℃温育30分钟。
(5)洗涤
揭开封板膜,弃液甩干每孔加满洗涤液,静止30秒后弃去,重复5次,拍干。
(6)加酶
每孔加入50μL酶标试剂,空白除外。
(7)温育:同(4)。
(8)洗涤:同(5)。
(9)显色:
每孔先加入显色剂A 50μL,再加显色剂B 50μL,轻轻震荡混匀,37℃避光显色15分钟。
(10)终止:
每孔加终止液50μL,终止反应。
(11)测定
以空白空调零,450nm波长依序测量各孔的吸光度。
2.3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体外培养ALV-J特异性CD8+ T细胞细胞形态学变化
基于鸭子T细胞的体外培养方法(专利公开号:CN113234676A),按照2.1.1所示的具体实验步骤,首先使用TCID50=3的ALV-J感染的记忆性PBMC作为APC,孵育5小时后加入到未刺激的PBMC中作为刺激组,阴对照组不做处理。培养后每天观察培养基颜色以及细胞形态变化,如图1可以看出,实验组细胞开始聚集,细胞变大变圆,在聚集细胞的周围开始出现个头较小的细胞;阴对照组没有明显的变化,呈单个生长状态,细胞没有出现聚集,随着培养天数的增加有部分细胞死亡。
3.2体外培养ALV-J特异性CD8+ T细胞数量及比例变化
为了确定ALV-J特异性CD8+ T细胞增殖,通过流式细胞仪检测CD8+ T细胞数量和比例的变化。首先按照2.1.1所示的方法进行体外培养CD8+ T细胞,分别在培养后的2,4,6天,取ALV-J刺激组和对照组细胞进行流式染色,具体染色方法如2.1④所示。流式染色圈门策略如图2中A,用FlowJo软件对流式结果进行分析,并进行统计学分析。结果如图2中B和C。ALV-J可刺激PBMC中的记忆性CD8+ T细胞增殖,结果表明,ALV-J刺激后CD8+ T细胞从第四天(P<0.01)开始增殖,数量和比例开始出现明显差异,到第六天仍有显著差异(P<0.001)。
3.3 CFSE验证ALV-J特异性CD8+ T细胞增殖
为了进一步确定ALV-J特异性CD8+ T细胞增殖,按照2.1.2中方法对鸡PBMC进行CFSE标记与流式检测,根据图3中A所示的流式圈门策略,用FlowJo软件对流式结果进行分析。流式结果如图3中B显示,ALV-J刺激组细胞均在培养后第4天出现增殖小峰,且第六天增殖最明显,表明ALV-J刺激鸡PBMC后可促进病毒记忆性CD8+ T细胞的增殖。
3.3 ELISpot检测候选表位的免疫原性
为了筛选出有免疫原性的多肽,首先按照2.1.1的方法体外培养ALV-J特异性CD8+T细胞,并按2.2.1的方法进行ELISpot实验。利用本实验室保存的表位库构建肽池,其中每四条肽构建一个肽池,分别命名为Pool 1-5,具体见表1。结果如图4中A所示,以PMA作为刺激物产生较多斑点证明实验成立,Pol270-280(由本实验室筛选得到,专利公开号:CN115991733A)作为阳性对照组(P<0.01),与DMSO对照组相比,Pool 1(P<0.01)和Pool 2(P<0.05)这两组刺激明显产生更多的斑点,表明Pool 1和Pool 2这两个肽池中含有具有免疫原性的多肽。接着,用Pool 1和Pool 2中的八条肽分别刺激活化的T细胞,用2.2.1中的方法进行检测,结果如图4中B所示,与DMSO对照组相比,Gag343-352和Gag531-541这两条肽可以刺激产生更多的斑点(P<0.05),表明Gag343-352和Gag531-541为筛选出具有免疫原性的两条肽(表5)。
表5两条针对B21单倍型鸡的ALV-J T细胞表位信息
3.4荧光定量PCR检测相关细胞因子的表达情况
为了验证所筛选出的多肽的抗原性,首先按照2.1.1的方法体外培养ALV-J特异性CD8+ T细胞,然后用筛选出的两条多肽刺激活化后的CD8+ T细胞,按照2.2.2的方法进行荧光定量PCR的检测。结果如图5所示,与CTLs相关的细胞因子和与天然免疫相关的细胞因子都有上调趋势,其中,与CTLs相关的基因中,Granzy-A,NK-lysin等上调显著(P<0.05),与天然免疫相关的基因中IFN-α上调显著(P<0.01)。
3.5ELISA检测相关细胞因子的表达情况
为了进一步验证筛选出多肽的功能,首先按照2.1.1的方法体外培养ALV-J特异性CD8+ T细胞,然后用筛选出的两条多肽刺激活化后的CD8+ T细胞,按照2.2.3的方法进行ELISA实验检测,结果如图6所示,其中多肽刺激组与不经多肽刺激组相比,与细胞毒性相关的细胞因子TNF-α,IL-2有显著性上调(P<0.01),说明Gag343-352和Gag531-541是具有免疫原性的两条肽,且为CD8 T细胞表位。
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。
Claims (6)
1.ALV-J MHC-B21限制性表位肽,其特征在于,所述ALV-J MHC-B21限制性表位肽的氨基酸序列如SEQ ID NO.1或SEQ ID NO.2所示。
2.如权利要求1所述的ALV-J MHC-B21限制性表位肽在制备ALV表位疫苗中的应用。
3.一种ALV表位疫苗,其特征在于,包含权利要求1所述的ALV-J MHC-B21限制性表位肽。
4.根据权利要求3所述的ALV表位疫苗,其特征在于,所述ALV表位疫苗还包括药学上可接受的辅料。
5.一种筛选ALV-J MHC-B21限制性表位肽的方法,其特征在于,包括利用体外扩增ALV-J特异性CD8+T细胞筛选ALV-J MHC-B21限制性潜在表位肽,再对所述ALV-JMHC-B21限制性潜在表位肽进行免疫原性验证,从而得到所述ALV-J MHC-B21限制性表位肽的步骤。
6.根据权利要求5所述的方法,其特征在于,所述免疫原性验证采用ELISA检测和/或荧光定量PCR检测。
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