CN108840911B - 新城疫病毒基质蛋白的抗原表位、抗体、鉴定方法和应用 - Google Patents
新城疫病毒基质蛋白的抗原表位、抗体、鉴定方法和应用 Download PDFInfo
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- CN108840911B CN108840911B CN201810590651.9A CN201810590651A CN108840911B CN 108840911 B CN108840911 B CN 108840911B CN 201810590651 A CN201810590651 A CN 201810590651A CN 108840911 B CN108840911 B CN 108840911B
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Abstract
本发明公开了一种新城疫病毒基质(Matrix,M)蛋白的抗原表位、抗体、鉴定方法和应用,涉及生物学技术领域。本发明提供的新城疫病毒基质蛋白的抗原表位的序列1为77MIDDKP82,序列2为354HTLAKYNPFK363。本发明的2个新城疫病毒M蛋白的B细胞优势线性表位可用于新城疫病毒血清学分析的检测抗原;可作为标记去构建新城疫病毒的标记疫苗;本发明提供的2个M蛋白的B细胞优势线性表位序列在新城疫病毒所有基因型的M蛋白中具有较高的保守性,其M蛋白的表位区域77‑82和354‑363可适用于所有的新城疫病毒毒株。
Description
技术领域
本发明涉及生物学技术领域,尤其涉及一种新城疫病毒基质蛋白的抗原表位、抗体、鉴定方法和应用。
背景技术
新城疫(Newcastle Disease,ND)是由新城疫病毒(Newcastle Disease Virus,NDV)引起的一种禽类呼吸道、消化道和神经系统损伤为主要特征的急性、高度致死性传染病。NDV为单股负链RNA病毒,属于副黏病毒科禽副黏病毒属。NDV基因组全长约为15.2kb,与其它副黏病毒相似,其基因编码包括核蛋白(nucleoprotein,NP),磷蛋白(phosphoprotein,P),基质蛋白(matrix protein,M),融合蛋白(fusion protein,F),血凝素神经氨酸酶(hemagglutinin-nueraminidase,HN)和大聚合酶蛋白(RNA-dependent RNApolymerase or large ploymerase,L)在内的6个蛋白,基因组以3’-NP-P-M-F-HN-L-5’顺序排列。
其中,M基因核苷酸长度为1241bp,开放性阅读框长度为1095bp,共编码364个氨基酸,蛋白分子量约为40kDa。它是NDV的非糖基化膜相关蛋白,在病毒的装配、出芽过程中发挥重要作用。其作为非保护性抗原在表达量上非常丰富且高度保守,能够诱导动物产生高滴度的抗体。NDV只有一个血清型,准确的M蛋白抗原表位可以作为生物学标记为新城疫相关的诊断及标记疫苗的研发奠定基础,但目前相关技术公开较少。
发明内容
有鉴于此,本发明实施例提供了一种新城疫病毒基质蛋白的抗原表位、抗体、鉴定方法和应用,主要目的是鉴定新城疫病毒M蛋白B细胞的抗原表位。
为达到上述目的,本发明主要提供了如下技术方案:
一方面,本发明实施例提供了一种新城疫病毒基质蛋白的抗原表位,所述基质蛋白的B细胞优势线性抗原表位的氨基酸序列为如SEQ ID NO.1(77MIDDKP82)和SEQ ID NO.2(354HTLAKYNPFK363)。
作为优选,所述新城疫病毒的毒株类型包括Class II系中II型(LaSota株)、VI型(SX10株)、VII型(JS株)、IX型(F48E9)以及Class I系(pigeon/QH株)。
另一方面,本发明实施例提供了上述新城疫病毒基质蛋白的抗原表位的鉴定方法,所述方法包括以下步骤:
步骤1:利用B细胞线性表位分析软件,结合蛋白质二级结构预测以及亲水性方案、突出表面及转角方案、表面可及性方案、灵活性方案、极性方案、抗原性方案分析新城疫疫苗株的M蛋白B细胞潜在的线性表位分布,为后续表位鉴定提供参考;
步骤2:在不破坏步骤1获得的M蛋白优势线性表位预测区域的前提下,将M蛋白分为M1和M2两段,通过Western blot和IFA检测M蛋白全长和分段区域蛋白抗原性情况;
步骤3:对疫苗株免疫的鸡抗血清靶向其M蛋白重叠肽段进行肽扫描分析,根据抗原-抗体结合强度信号初步确定M蛋白的线性表位区域;将肽扫描得到的表位序列融合红色荧光蛋白进行表达,通过Western blot和IFA进行检测,初步确定准确的抗原表位区域;根据鉴定结果,将反应阳性的表位序列进行人工合成,得到合成肽,对所述合成肽进行Dot-blot和ELISA检测;
步骤4:利用步骤3的所述合成肽免疫小鼠制备抗血清;利用Dot-blot检测所述合成肽和所述抗血清之间的反应以及交叉反应情况;利用制备的鼠抗合成肽血清和疫苗株全病毒进行反应检测筛选表位序列;
步骤5:对筛选出的所述表位序列进行连续定点突变,然后和步骤4制备的小鼠抗血清进行分析验证,确定出最短优势表位序列,所述最短优势表位序列为权利要求1所述的序列1和序列2;
步骤6:将鉴定出的疫苗株M蛋白B细胞线性抗原表位区域的氨基酸序列和新城疫病毒其他所有基因型代表毒株M蛋白对应区域进行比对分析,确定其保守性;将步骤4制备的鼠抗血清和其他病毒反应,以确定鉴定出的抗原表位区域是否适用于其他病毒毒株。
作为优选,所述步骤1中的新城疫疫苗株类型为Class II系中II型(LaSota株);所述步骤6中的新城疫病毒其他所有基因型代表毒株类型为VI型(SX10株)、VII型(JS株)、IX型(F48E9)以及Class I系(pigeon/QH株)。
又一方面,本发明实施例提供了上述新城疫病毒抗体,所述抗体是采用上述的抗原表位序列偶联惰性蛋白,按照免疫程序免疫动物获得的抗血清,所述抗性血清即为所述新城疫病毒的抗体。
再一方面,本发明实施例提供了上述新城疫病毒抗体和抗原的鉴别方法,所述鉴别方法包括采用上述抗原表位序列作为抗原去检测新城疫病毒抗体;采用上述制备的抗体检测新城疫病毒。
又一方面,新城疫病毒疫苗,所述疫苗包括上述新城疫病毒基质蛋白的抗原表位经过包括缺失或插入修饰构建的标记疫苗。
又一方面,本发明实施例提供了上述新城疫病毒基质蛋白的抗原表位在制备新城疫病毒抗体中的应用。
又一方面,本发明实施例提供了上述新城疫病毒基质蛋白的抗原表位在制备新城疫病毒疫苗中的应用。
又一方面,本发明实施例提供了上述新城疫病毒基质蛋白的抗原表位在鉴定新城疫病毒抗原和抗体中的应用。
与现有技术相比,本发明的有益效果是:
1.本发明提供了2个新城疫病毒M蛋白的B细胞优势线性表位,详细的序列为:77MIDDKP82和354HTLAKYNPFK363;
2.本发明提供的2个新城疫病毒M蛋白B细胞表位可用于新城疫病毒血清学分析的检测抗原;
3.本发明筛选出的2个M蛋白B细胞优势表位可以作为标记去构建新城疫病毒的标记疫苗;
4.本发明通过不同基因型序列比对分析,提供了2个M蛋白的B细胞优势线性表位序列在新城疫病毒所有基因型的M蛋白中具有较高的保守性,其M蛋白的表位区域77-82和354-363可适用于所有的新城疫病毒毒株;例如Class II系中II型(LaSota株)、VI型(SX10株)、VII型(JS株)、IX型(F48E9)以及Class I系(pigeon/QH株);
5.本发明综合利用信息生物学,肽扫描技术和定点突变技术对M蛋白的B细胞线性表位进行了精确定位;该鉴定系统简便,准确,可以为其它病毒蛋白的抗原表位鉴定提供参考。
附图说明
图1A是本发明实施例提供的M蛋白二级结构预测结果图;
图1B是本发明实施例提供的M蛋白抗原表位预测结果图;
图2是本发明实施例提供的M蛋白及其分段产物与LaSota疫苗毒株免疫鸡抗血清的反应结果图;
(A:M蛋白以及分段真核表达产物的Western blot检测;B:M蛋白以及分段真核表达产物的IFA检测);
图3是本发明实施例提供的M蛋白与鸡抗血清的肽扫描信号结果图;
图4是本发明实施例提供的优势抗原表位序列融合RFP表达的鉴定结果图;
(A:优势抗原表位RFP融合蛋白的IFA检测;B:优势抗原表位RFP融合蛋白的Western blot检测);
图5是本发明实施例提供的优势抗原表位合成肽与鸡抗血清的反应性结果图;
(A:利用dot-blot检测合成肽和LaSota鸡阳性血清反应情况;B:MIDE2和MIDE6合成肽与鸡抗血清反应敏感性试验;C:ELISA检测合成肽和鸡阳性抗血清反应情况);
图6是本发明实施例提供的优势表位合成肽与其小鼠抗血清的反应情况图;
图7是本发明实施例提供的优势抗原MIDE2和MIDE6最短有效表位的鉴定图;
(A:MIDE2最短基序的鉴定;B:MIDE6最短基序的鉴定);
图8是本发明实施例提供的M2和M6血清和不同新城疫病毒毒株的反应结果图;
(N:空白对照组;L:NDV Class II基因II型LaSota株;S:NDV Class II基因VI型SX10株;J:NDV Class II基因VII型JS株;F:NDV Class II基因IX型F48E9株;P:NDV ClassI Pigeon/QH株)。
具体实施方式
为更进一步阐述本发明为达成预定发明目的所采取的技术手段及功效,以下以较佳实施例,对依据本发明申请的具体实施方式、技术方案、特征及其功效,详细说明如后。下述说明中的多个实施例中的特定特征、结构、或特点可由任何合适形式组合。
表位预测:表位预测方法是通过计算机软件对抗原分子结构和理化性质进行分析从而对表位存在的部位进行预测,该方法表示直观,可以作为表位进一步验证的实验基础。表位预测主要有以下几种方案:亲水性方案(Hydrophilicity),分子可及性(Accessibility),可塑性(Flexibility),二级结构预测(Secondary structure)等。
肽扫描:该技术是指在芯片上每隔几个氨基酸合成重叠的具有一定长度的小肽段,然后利用酶联免疫分析这些肽段和相应抗体的互相作用,以其与特异性抗体结合的牢固程度来预测线性表位。肽扫描技术是目前常用的B细胞线性表位预测方法,构象表位无法检出。
定点突变:是研究蛋白质结构与其功能之间复杂关系的有效手段,指的是通过突变目的蛋白的某个或某几个特定氨基酸,进一步比较突变后和野生型的抗原与抗体结合识别情况,从而实现抗原表位的鉴定。
实施例
一种新城疫病毒LaSota株M蛋白B细胞优势抗原表位鉴定方法如下:
步骤一:用DNAStar Protean软件对NDV LaSota毒株M蛋白(氨基酸序列为SEQ IDNO.7))进行二级结构进行分析;使用在线数据库Immune Epitope Database(IEDB),ABCpred,Bcepred综合对LaSota株M蛋白B细胞表位进行预测;使用以下7种预测方案:亲水性方案、突出表面及转角方案、表面可及性方案、灵活性方案、极性方案、抗原性方案;如图1所示,M蛋白具有丰富的α螺旋和β片层以及转角结构,疏水性较强,表面可及性和抗原指数都较高;而且M蛋白中得分较高且稳定存在的线性表位大约有9个,其位置如下:28-39,48-56,77-85,118-126,157-169,223-233,244-253,296-311和345-361;
步骤二:为了确定M蛋白大致的抗原性分布,根据信息生物学分析结果,在不破坏潜在抗原表位的前提下,将M蛋白分段为M1和M2,然后利用Western blot和间接免疫荧光(IFA)检测M蛋白及分段区域和LaSota疫苗免疫的鸡抗血清反应情况;如图2所示,制备的LaSota株全病毒鸡抗血清能够和M蛋白表达产物产生较好的反应,该结果表明制备的抗血清能够作为肽扫描待检样品,同时分段表达产物M1和M2蛋白也和抗血清产生了较好反应,推测M蛋白抗原表位呈现均匀分布,和步骤一中M蛋白B细胞抗原表位预测结果相符;
步骤三:对LaSota疫苗株免疫的鸡抗血清靶向其M蛋白重叠肽6段进行肽扫描分析;合成含有M蛋白全部序列的重叠肽段,每15个氨基酸合成一段,相邻两段有两个氨基酸移位,构建多肽微阵列;将多肽微阵列与LaSota鸡阳性血清1:500稀释孵育,然后于山羊抗鸡的二抗进行孵育,同时用无关小鼠单克隆抗体HA作为对照;最后根据抗原-抗体反应荧光强度扫描判定优势表位区域;如图3所示,共有6段区域可以作为M蛋白潜在的优势抗原表位区域,分别命名为:MIDE1,MIDE2,MIDE3,MIDE4,MIDE5和MIDE6;将肽扫描得到的潜在的表位序列融合红色荧光蛋白(RFP)构建真核表达质粒,转染HEp-2细胞进行IFA检测;如图4-A所示,MIDE2和MIDE6融合蛋白和抗血清具有较好的反应效果,MIDE5融合蛋白具有微弱的荧光反应,MIDE1,MIDE3和MIDE4和抗血清无反应;根据IFA结果,我们选择MIDE1,MIDE2,MIDE5和MIDE6融合蛋白进行Western blot鉴定;如图4-B所示,相比于其他序列,MIDE2和MIDE6融合蛋白和抗血清产生了较强的反应,和IFA结果相符;根据Western blot结果,将MIDE1(氨基酸序列为SEQ ID NO.3),MIDE2(氨基酸序列为SEQ ID NO4),MIDE5(氨基酸序列为SEQ ID NO.5)和MIDE6(氨基酸序列为SEQ ID NO.6)对应序列偶联KLH进行人工合成,随后进行Dot-blot和ELISA检测,如图5所示,发现MIDE2(氨基酸序列为SEQ ID NO.4)和MIDE6(氨基酸序列为SEQ ID NO.6)能够和鸡抗血清产生较强免疫反应;
步骤四:将人工合成的相应表位的多肽免疫小鼠,制备抗表位的小鼠血清抗体,分别命名为:M1,M2,M5,M6。按照每只小鼠大约200μg合成肽进行免疫;取4mg/mL合成肽200μL加入380μL ddH2O混匀,然后与400μL完全弗氏佐剂于三通阀中乳化,每只小鼠免疫200Μl,14天,28天后,将乳化剂换做不完全弗氏佐剂按照同样的剂量进行免疫,第38天,采血于EP管中,将EP管斜放,4℃过夜,然后1500r/min离心5min,取上清,-20℃储存待用;利用Dot-blot检测合成肽和抗血清之间的反应以及交叉反应情况,如图6所示,MIDE2和MIDE6合成肽能够较好的刺激小鼠产生抗体,同时可以观察到MIDE6合成肽和M2血清之间,以及MIDE2,MIDE5和M6血清之间存在微弱的交叉反应;利用制备的合成肽小鼠血清抗体分别和LaSota全病毒进行反应,Western blot结果显示M2和M6血清能够和LaSota全病毒产生较好的反应,至此判定MIDE2和MIDE6为有效的M蛋白抗原表位序列;
步骤五:为了实现对鉴定出的2个优势表位序列(MIDE2和MIDE6)最短有效基序的确定,本发明分别从N端和C端分别进行连续氨基酸突变,根据肽扫描分析结果,靶向的突变序列为:MIDE2中的75VGMIDDKP82(氨基酸序列为SEQ ID NO.1),以及MIDE6中的352KGHTLAKYNPFK363(氨基酸序列为SEQ ID NO.2);将突变体表达产物和鼠抗血清进行反应,如图7所示,对于MIDE2序列,75VGM77连续的突变或者81KP82联合突变会引起优势抗原表位序列抗原性丧失,判定MIDE2的最短基序为77MIDDKP82(氨基酸序列为SEQ ID NO.1);同样的,MIDE6序列中352KGHT355或者是362FK363的联合突变会引起其抗原性丧失,判定其最短基序为354HTLAKYNPFK363(氨基酸序列为SEQ ID NO.2),另外单独的K363A突变会引起MIDE6抗原性显著降低,推测363K氨基酸对其抗原性具有十分重要的作用;
步骤六:为了确定鉴定出的LaSota株M蛋白IDE序列的保守性,将NDV不同基因型的对应氨基酸序列进行了比对分析;结果表明,鉴定出的2段优势抗原表位序列在不同基因型中具有较高的保守性;将步骤4制备的鼠抗血清和其他病毒进行反应,如Class II系中II型(LaSota株)、VI型(SX10)、VII型(JS株)、IX型(F48E9)以及Class I系(pigeon/QH株),如图8所示,Western blot结果发现M2和M6和以上病毒均产生了明显反应,推测鉴定出的优势抗原表位区域适用于新城疫病毒其他所有的毒株。
本发明综合运用生物信息学软件(如,IEDB)预测了NDV疫苗株LaSota的M蛋白线性B细胞表位,利用肽扫描技术对疫苗免疫的鸡抗血清和M多肽进行抗原-抗体扫描分析,将扫描出的表位序列和红色荧光蛋白(RFP)融合表达后进行免疫印迹(Western blot)和间接免疫荧光(IFA)检测,初步确定优势抗原表位序列;然后结合点杂交(Dot-blot),间接ELISA对表位肽进行了鉴定;同时,根据鉴定的表位序列人工合成对应的多肽,免疫小鼠后得到相应的抗表位的抗体,利用这些抗体进行深入的表位分析和确定;最后结合定点突变技术,确定了2个最短M蛋白B细胞线性表位序列:77MIDDKP82(氨基酸序列为SEQ ID NO.1)和354HTLAKYNPFK363(氨基酸序列为SEQ ID NO.2),为建立新型血清学诊断试剂和标记疫苗的研发奠定基础。
本发明实施例中未尽之处,本领域技术人员均可从现有技术中选用。
以上公开的仅为本发明的具体实施方式,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,可轻易想到变化或替换,都应涵盖在本发明的保护范围之内。因此,本发明的保护范围应以上述权利要求的保护范围为准。
序列表
<110> 西北农林科技大学
<120> 新城疫病毒基质蛋白的抗原表位、抗体、鉴定方法和应用
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Claims (5)
1.新城疫病毒基质蛋白的抗原表位肽,其特征在于,所述基质蛋白的抗原表位肽的氨基酸序列如SEQ ID NO.1和SEQ ID NO.2所示。
2.新城疫病毒抗体,其特征在于,所述抗体是采用权利要求1所述的抗原表位肽偶联惰性蛋白,按照免疫程序免疫动物获得的抗血清,所述抗性血清即为所述新城疫病毒的抗体。
3.新城疫病毒疫苗,其特征在于,所述疫苗包括经过修饰的新城疫病毒基质蛋白的抗原表位肽;所述经过修饰的新城疫病毒基质蛋白的抗原表位肽的氨基酸序列如SEQ IDNO.4或SEQ ID NO.6所示。
4.权利要求1所述的新城疫病毒基质蛋白的抗原表位肽在制备新城疫病毒抗体中的应用。
5.权利要求1所述的新城疫病毒基质蛋白的抗原表位肽在制备新城疫病毒疫苗中的应用。
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