CN117304277A - 一种o型口蹄疫病毒vp4蛋白t细胞表位多肽及其应用 - Google Patents
一种o型口蹄疫病毒vp4蛋白t细胞表位多肽及其应用 Download PDFInfo
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Abstract
本发明公开了一种O型口蹄疫病毒VP4蛋白T细胞表位多肽及其应用,为以下任一或多个表位多肽,所述表位多肽的氨基酸序列如SEQ ID NO.1‑2的所示;或将SEQ ID NO.1‑2的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加由SEQ ID NO.1‑2衍生的且保持SEQ ID NO.1‑2所示的蛋白功能的氨基酸序列。本发明通过设计O型口蹄疫病毒的重叠肽段,利用酶联免疫斑点技术检测,筛选出功能性T细胞抗原表位多肽,利用IFN‑γ胞内细胞因子染色,进一步筛选确认能够有效诱导T淋巴细胞分泌IFN‑γ的肽段,可用于制备O型口蹄疫病毒多表位疫苗。
Description
技术领域
本发明涉及抗原表位多肽技术领域,尤其涉及一种O型口蹄疫病毒VP4蛋白T细胞表位多肽。
背景技术
口蹄疫(FootandmouthDisease,FMD)是由口蹄疫病毒(FMDvirus,FMDV)引起的一种急性传染病。该病感染偶蹄目动物,包括猪、牛、羊等常见家畜,感染动物口腔粘膜、蹄部和乳房皮肤发生水疱,给畜牧业带来严重经济损失。FMDV分为七个血清型,分别为A、O、C、南非1、南非2、南非3和亚洲1型,其中O型FMDV仍在我国流行。FMDV属于小RNA病毒科(Picornaviridae)口疮病毒属(Aphthovirus),在病毒的中心为一条单链的正链RNA,由大约8000个碱基组成,编码四种结构蛋白(VP1、VP2、VP4和VP4),病毒外壳为对称的20面体。
目前,临床上使用的FMDV疫苗以灭活疫苗为主,该类疫苗主要诱导体液免疫,诱导细胞免疫的能力较差。T细胞表位能够被T细胞受体识别,从而刺激特异性T细胞的活化,产生抗病毒免疫。目前,O型FMDVT细胞的表位尚不完全清晰,为此,提出一种O型口蹄疫病毒VP4蛋白T细胞表位多肽及其应用。
发明内容
本发明的目的是为了解决背景技术提出的问题,而提出的一种O型口蹄疫病毒VP4蛋白T细胞表位多肽及其应用。
为了实现上述目的,本发明采用了如下技术方案:
一种O型口蹄疫病毒VP4蛋白T细胞表位多肽,其为以下任一或多个表位多肽,所述表位多肽的氨基酸序列如SEQ ID NO.1-2的所示;或
将SEQ ID NO.1-2的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加由SEQ ID NO.1-2衍生的且保持SEQ ID NO.1-2所示的蛋白功能的氨基酸序列。
在另一方面,一种O型口蹄疫病毒VP4蛋白T细胞表位多肽的编码基因。
在另一方面,一种O型口蹄疫病毒VP4蛋白T细胞表位多肽编码基因的生物材料,所述生物材料为重组表达载体、表达盒、重组菌或宿主细胞。
在另一方面,一种药物,含有一种O型口蹄疫病毒VP4蛋白T细胞表位多肽。
在另一方面,一种多肽疫苗,含有所述O型口蹄疫病毒结构蛋白VP4的T细胞的抗原表位多肽,所述多肽疫苗还含有佐剂,所述佐剂包括壳聚糖、载体蛋白。
在另一方面,一种O型口蹄疫病毒VP4蛋白T细胞表位多肽或其编码基因或权利要求3所述的生物材料在提高机体产生细胞数量或提高机体分泌IFN-γ细胞数量中的应用,以及在制备预防口蹄疫病毒感染的疫苗,药物,试剂或试剂盒中的应用。
与现有技术相比,本发明提供了一种O型口蹄疫病毒VP4蛋白T细胞表位多肽,具备以下有益效果:
本发明通过设计O型口蹄疫病毒结构蛋白VP4的重叠肽段,利用酶联免疫斑点技术检测,筛选出功能性T细胞抗原表位多肽,利用IFN-γ胞内细胞因子染色,进一步筛选确认能够有效诱导T淋巴细胞分泌IFN-γ的肽段,可用于制备O型口蹄疫病毒多表位疫苗。
附图说明
图1为ELISPOT筛选O型FMDV结构蛋白VP4功能性抗原表位肽结果图。横坐标为刺激物名称,其中PC为阳性对照,NC为阴性对照。
纵坐标为斑点数;
图2为ELISPOT筛选出的105号肽进行验证结果图;
图3为ELISPOT筛选出的106号肽进行验证结果图;
图4为105、106号肽段IFN-γ胞内细胞因子染色验证实验阴性对照结果图;
图5为105、106号肽段IFN-γ胞内细胞因子染色验证实验阳性对照结果图;
图6是对图2-图5的定量分析结果示意图;
图7为为ELISPOT对105和106号肽的重叠序列105-106进行验证的结果图;其中横坐标为刺激物名称,其中PC为阳性对照,NC为阴性对照,纵坐标为斑点数;
图8为105-106号肽段IFN-γ胞内细胞因子染色验证实验阳性对照结果图;
图9为105-106号肽段IFN-γ胞内细胞因子染色验证实验阴性对照结果图;
图10为对105和106号肽的重叠序列流式结果示意图;
图11是对图8-图10的定量分析结果示意图;
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。
步骤1.蛋白肽库的合成
参照GenBank发表的O/BY/CHA/2010株(JN998085)的结构蛋白VP4氨基酸序列,设计一系列覆盖VP4蛋白全长氨基酸序列的重叠肽段,每个肽段长度为18AA,相邻肽段间重叠12AA,一共12个肽段,由南京肽谷生物科技公司合成。合成多肽纯度≥95%,溶解在纯水中,-80℃保存。步骤2.小鼠攻毒与脾脏淋巴细胞的分离将5-8周龄的IFNAR-/-C57/BL6小鼠分为免疫组和对照组(n=5),免疫组小鼠以5000PFUs/0.1mL进行腹部皮下注射攻毒,对照组小鼠以相同方式注射PBS溶液。免疫7天后,将小鼠安乐死,无菌取脾脏,碾磨均匀后过70μm细胞滤网,置于装有完全培养基(rpmI1640+10%FBS+1%双抗)的50mL离心管中,1700rpm离心5分钟,弃上清。采用红细胞裂解液重悬上述细胞,常温静置5分钟后,加入3倍体积的完全培养基,于1700rpm离心5分钟,弃上清。最后用完全培养基重悬细胞并计数。步骤3.ELISPOT试验初步筛选功能性表位肽小鼠免疫后7天,分离脾脏淋巴细胞,分别用肽库的每一条多肽刺激淋巴细胞,通过ELISPOT试验检测O型FMDV特异性T细胞受多肽刺激后分泌IFN-γ的水平,从而初步筛选出功能性抗原表位多肽。
96孔ELISPOT板(达科为,2210003),每孔加入2*105脾脏淋巴细胞,再加入1中合成的单个蛋白肽刺激(终浓度为10μg/mL),总体积100μL/孔。同时设立阳性对照孔:加入PMA(500ng/mL)+Lonomycine(10μg/mL),设立阴性对照孔:加入纯水。每类设立3个重复孔。置于37℃,5%CO2中培养20h后进行显色操作。
(1)裂解细胞:倾倒孔内细胞及培养基,加入冰冷的去离子水,200μL/well,4℃冰箱放置10分钟低渗裂解细胞。
(2)洗板:甩出孔内液体,加入1×Washing Buffer工作液,260μL/well,停留1分钟后弃去孔内液体,重复六次,每一次在吸水纸上扣干。
(3)检测抗体孵育:将1×Biotinylated Antibody工作液加入各实验孔,100μL/well。37℃孵育1小时。
(4)洗板:加入1×Washing Buffer工作液,260μL/well,停留1分钟后弃去孔内液体,重复六次,每一次在吸水纸上扣干。
(5)酶联亲和素孵育:将1×Streptavidin-HRP工作液加入各实验孔,100μL/well37℃孵育1小时。
(6)洗板:甩出孔内液体,加入1×Washing Buffer工作液,260μL/well,停留1分钟后弃去孔内液体,重复五次,每一次在吸水纸上扣干,洗涤完成后揭开板底座,用去离子水/自来水洗涤膜底面及底座,用吸水纸小心吸干底座及膜底残留的水迹,合上底座,加入1×Washing Buffer工作液,260μL/well,停留1分钟后弃去孔内液体,彻底扣干孔内液体。
(7)显色:将现配的AEC显色液加入各实验孔,100μL/well。室温避光静置5-30分钟,根据斑点生成情况选择终止显色时间。若室温低于20℃,建议在37℃温箱做显色,每隔5-10分钟检查一次。
(8)终止显色:倾倒孔内液体,揭开板底座,用去离子水/自来水洗涤各实验孔正反面及底座3-5遍,终止显色。将板放置在室温阴凉处,待其自然晾干后合上底座。
(9)ELISPOT板斑点计数,并记录斑点的各种参数,做统计分析。结果显示,肽段105、106能有效刺激免疫组小鼠脾脏淋巴细胞分泌IFN-γ,显著高于不加刺激物的阴性对照孔细胞(图1)。同时,不能刺激对照组小鼠脾脏淋巴细胞分泌IFN-γ,初步表明肽段105、106诱导脾脏淋巴细胞分泌IFN-γ是O型FMDV特异性T细胞激活的结果;功能性抗原表位肽氨基酸序列如表1所示。
表1O型FMDV结构蛋白功能性抗原表位多肽氨基酸序列
多肽名称 | 多肽序列 | 多肽所属结构蛋白 |
105 | NQSGNTGSIINNYYMQQY | VP4 |
106 | GSIINNYYMQQYQNSMDT | VP4 |
步骤4.IFN-γ胞内细胞因子染色试验
为进一步验证功能性抗原表位多肽所对应的T细胞表型,本试验将ELISPOT筛选获得的2功能性抗原表位多肽(105、106、)分别刺激免疫小鼠脾脏淋巴细胞,进行IFN-γ胞内细胞因子染色试验。
96孔板中每孔加入1×106个脾脏淋巴细胞,再分别加入单个肽刺激(终浓度为10μg/mL),同时设立阳性对照:PMA(500ng/mL)+Lonomycine(10μg/mL),
阴性对照:加入纯水。置于37℃,5%CO2中培养1h后,每孔加入Brefeldin A(MCE,2030421),终浓度10μg/mL,37℃,5%CO2中继续培养5h。
孵育完成后,将上述细胞转入EP管中,1500rpm5min离心收集细胞。加入0.5mL流式缓冲液(Biosharp,BL1136A)清洗细胞一次,1500rpm5min离心收集细胞。随后进行细胞表明染色:
1.加入Fc抗体进行孵育
1)每管加入25μL Fc Block(1:50)重悬,置于冰上20min。
2.加入CD8a、CD4、CD3e进行染色
1)每管加入25μL Abs Mix(1:100),置于冰上30min。
2)将Zombie Aqua Dye用PBS以1:500稀释,每100ul染色106细胞(即加入100ul染色)。室温避光静置15-30min。加入0.5mL流式缓冲液清洗细胞一次,离心,弃上清。
3.利用Fixation/Permeabilization Concentrate(Thermo,GAS003)试剂对细胞进行固定和透膜。
1.加入100ulA液(固定剂),室温下避光孵育15分钟。
2.用0.5mL流式缓冲液洗涤,1500rpm5min。
3.加入100ulB液(破膜剂)和适量体积的胞内抗体或相应同型对照.(mouse IFNγAbs)1:50。
4.涡旋1-2秒,室温下避光孵育20分钟.
5.用0.5mL流式缓冲液洗涤,1500rpm5min,弃上清。
6.用200ul流式缓冲液重悬细胞并及时上机分析。
结果发现肽段105和106能有效诱导8.68%(图2)、9.43%(图3)的CD8+T淋巴细胞分泌IFN-γ,与阴性对照(0.16%)(图4)相比差异显著(p<0.05),同时阳性对照激活9.77%的CD8+T淋巴细胞分泌IFN-γ(图5)。统计结果如图6所示。说明105和106号肽段包含CD8+T细胞表位。
步骤5.蛋白肽合成
此步骤根据上述实验结果,将105-106,重叠序列合成新的肽段,用肽段重复上述实验。
多肽名称 | 多肽序列 | 多肽所属结构蛋白 |
105-106 | GSIINNYYMQQY | VP4 |
步骤6.ELISPOT
ELISPOT试验进一步确认功能性表位肽
将3只小鼠免疫后7天,分离脾脏淋巴细胞,用步骤5中合成的多肽刺激淋巴细胞,通过ELISPOT试验检测O型FMDV特异性T细胞受多肽刺激后分泌IFN-γ的水平,从而进一步确认功能性抗原表位多肽。
如图7所示,肽段105、106的重叠序列105-106能有效刺激免疫组小鼠脾脏淋巴细胞分泌IFN-γ,显著高于不加刺激物的阴性对照孔细胞(图7)。同时,不能刺激对照组小鼠脾脏淋巴细胞分泌IFN-γ,初步表明肽段105-106诱导脾脏淋巴细胞分泌IFN-γ是O型FMDV特异性T细胞激活的结果。
步骤7.ICS
ICS试验进一步确认功能性表位肽。
将3只小鼠免疫后7天,分离脾脏淋巴细胞,用步骤5中合成的多肽刺激淋巴细胞,通过ICS试验检测O型FMDV特异性T细胞受多肽刺激后分泌IFN-γ的水平,从而进一步确认功能性抗原表位多肽。
如图8-11所示,肽段105、106的重叠序列105-106能有效诱导4.13%的CD8+T淋巴细胞分泌IFN-γ(图10),与阴性对照(0%)(图9)相比差异显著(p<0.05),同时阳性对照激活9.20%的CD8+T淋巴细胞分泌IFN-γ(图8)。统计结果如图11所示。说明105-106肽段包含CD8+T细胞表位。
以上所述,仅为本发明较佳的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,根据本发明的技术方案及其发明构思加以等同替换或改变,都应涵盖在本发明的保护范围之内。
Claims (10)
1.一种O型口蹄疫病毒VP4蛋白T细胞表位多肽,其特征在于,其为以下任一或多个表位多肽,所述表位多肽的氨基酸序列如SEQ ID NO.1-2的所示;或
将SEQ ID NO.1-2的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加由SEQ ID NO.1-2衍生的且保持SEQ ID NO.1-2所示的蛋白功能的氨基酸序列。
2.根据权利要求1所述一种O型口蹄疫病毒VP4蛋白T细胞表位多肽的编码基因。
3.一种含有权利要求1所述一种O型口蹄疫病毒VP4蛋白T细胞表位多肽编码基因的生物材料,所述生物材料为重组表达载体、表达盒、重组菌或宿主细胞。
4.一种药物,其特征在于,含有权利要求1所述一种O型口蹄疫病毒VP4蛋白T细胞表位多肽。
5.一种多肽疫苗,其特征在于,含有权利要求1所述O型口蹄疫病毒结构蛋白VP4的T细胞的抗原表位多肽。
6.根据权利要求5所述的多肽疫苗,其特征在于,所述多肽疫苗还含有佐剂,所述佐剂包括壳聚糖、载体蛋白。
7.根据权利要求1所述一种O型口蹄疫病毒VP4蛋白T细胞表位多肽或其编码基因或权利要求3所述的生物材料在提高机体产生细胞数量或提高机体分泌IFN-γ细胞数量中的应用。
8.权利要求1所述O型口蹄疫病毒结构蛋白VP4的T细胞的抗原表位多肽或其编码基因或权利要求3所述的生物材料在制备预防口蹄疫病毒感染的疫苗中的应用。
9.权利要求1所述O型口蹄疫病毒结构蛋白VP4的T细胞的抗原表位多肽或其编码基因或权利要求3所述的生物材料在制备治疗口蹄疫病毒感染的药物中的应用。
10.权利要求1所述O型口蹄疫病毒结构蛋白VP4的T细胞的抗原表位多肽或其编码基因或权利要求3所述的生物材料在制备检测口蹄疫病毒的试剂或试剂盒中的应用。
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