CN114315984A - 一种用于制备prrsv基因ii型表位缺失疫苗毒株的n蛋白表位突变标记及其应用 - Google Patents
一种用于制备prrsv基因ii型表位缺失疫苗毒株的n蛋白表位突变标记及其应用 Download PDFInfo
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Abstract
本发明提供了一种用于制备PRRSV基因II型表位缺失疫苗毒株的N蛋白表位突变标记及其应用，属于生物制品技术领域。所述突变标记在PRRSV基因II型N蛋白C端92～103位表位序列的基础上突变一个或多个氨基酸，所述表位突变标记的氨基酸序列如SEQ ID NO:1所示，其中X1可以为T、P或A，X2为V或A。本发明通过鉴定PRRSV基因II型病毒N蛋白中优势抗原表位，证明将此表位缺失后可以有效区分自然感染动物与表位缺失全病毒灭活疫苗或弱毒疫苗免疫动物，为PRRSV免疫净化防控产品的制备提供了依据。
Description
技术领域
本发明属于生物制品技术领域，具体涉及一种用于制备PRRSV基因II型表位缺失疫苗毒株的N蛋白表位突变标记及其应用。
背景技术
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)引起的严重危害世界养猪业的高度接触性传染病。该病主要引起仔猪、育肥猪的呼吸道疾病和母猪的繁殖障碍，给全球养猪业造成了巨大的经济损失。PRRSV是一种单股正链RNA病毒，基因组全长约15kb，主要感染肺泡巨噬细胞(PAM)、成熟单核细胞、小胶质细胞等抗原递呈细胞。
疫苗免疫对于PRRS的防控具有一定的效果，疫苗包括弱毒疫苗与灭活疫苗，但均存在一定的缺点。弱毒疫苗存在毒力返强、田间流行毒与疫苗毒重组变异等现象，如果临床使用的弱毒疫苗株多，则可能进一步促进病毒重组变异，使疫病流行形势复杂化。灭活疫苗的问题是对同源毒株的保护效果较好，对异源流行病毒的交叉免疫保护不足。随着，重组变异毒株的不断出现，越来越多的专家认识到，应重视灭活疫苗的使用，以改变PRRS防控的复杂局面。
目前，不管是弱毒疫苗或灭活疫苗均不能有效区分自然感染与疫苗免疫动物，无法进行感染状况评估与免疫猪群内疫病的净化。因此，制备PRRS表位缺失负标记疫苗是目前PRRS防控产品研究的重点。现有技术中用于区别PRRSV自然感染和疫苗免疫的方法，是利用疫苗免疫动物后，不产生针对美洲株PRRS病毒M蛋白线性表位的抗体，通过检测所述抗体，实现区分自然感染与疫苗免疫动物(CN 105749267A，公开日2016年7月13)，再例如，区分牛布鲁氏菌疫苗免疫与自然感染状态的方法只需对一个样本同时检测其IgG和IgM就可对自然感染和疫苗免疫进行区分(CN108037277A，公开日为2018年5月15日)。还有通过同源重组技术敲除靶基因或多肽位点，通过检测目标基因或多肽区分疫苗免疫动物和野生菌感染动物(CN111057671A，2020年4月24日；CN104165998A，公开日为2016年8月)。虽然构建敲除抗原表位的疫苗便于后续区分鉴定，但对敲除的位点有严格限制，比如不恰当敲除抗原表位可能会降低疫苗抗原的免疫原性，降低疫苗的免疫效果。因此鉴定PRRSV的优势非保护性抗原表位，将此表位缺失后制备标记疫苗，才能有效区分自然感染与表位缺失全病毒疫苗免疫动物。
发明内容
有鉴于此，本发明的目的在于提供一种PRRSV的N蛋白线性非中和表位突变标记位点，可以根据该位点制备表位缺失灭活疫苗或弱毒疫苗，用于区分PRRSV自然感染动物和全病毒疫苗免疫动物。
本发明提供了一种用于制备PRRSV基因II型表位缺失标记疫苗毒株的N蛋白表位突变标记，在PRRSV基因II型N蛋白C端92～103位氨基酸序列的基础上突变一个或多个氨基酸；
所述N蛋白表位突变标记的氨基酸序列如SEQ ID NO:1所示，其中X1为T、P或A，X2为V或A。
优选的，当突变1个氨基酸位点时，突变表位中的任何一个位点氨基酸，优选为突变93位丝氨酸。
优选的，当突变多个氨基酸时，包括连续或非连续突变2～12个氨基酸。
优选的，所述突变包括突变修饰、缺失或插入异源序列。
优选的，所述突变修饰包括氨基酸替换。
本发明提供了一种表位缺失全病毒PRRSV疫苗，所述疫苗含有所述N蛋白表位突变标记的PRRSV。
优选的，所述PRRSV为PRRSV基因II型。
本发明提供了PRRSV N蛋白的猪源单个B细胞抗体C8在制备检测所述PRRSV表位缺失疫苗的试剂或试剂盒中的应用。
本发明提供了一种用于制备PRRSV基因II型表位缺失疫苗毒株的N蛋白表位突变标记，在PRRSV的N蛋白C端92～103位氨基酸序列的基础上进行一个或多个氨基酸位点突变；所述表位突变标记的氨基酸序列如SEQ ID NO:1所示，其中X1为T、P或A，X2为V或A。本发明鉴定了PRRSV基因II型病毒N蛋白C端的非保护性优势抗原表位，将此表位突变后可有效区分自然感染动物与表位缺失全病毒疫苗免疫动物，为PRRSV免疫净化防控策略的制与实施提供技术依据。
附图说明
图1为在293T细胞中转染pcDNA3.1-N和pcDNA3.1空载体，用C8和GAPDH抗体进行Westernblot结果；
图2为合成肽ELISA检测C8单抗与N蛋白的N19肽段反应结果；
图3为N蛋白83～112位多肽连续三点突变后与C8单抗的反应性检测结果；
图4为N蛋白92-103位单点突变后与C8单抗的反应性检测结果；
图5为表位缺失N蛋白与正常N蛋白转染293T细胞后与C8单抗的反应性检测；
图6为利用N蛋白抗体竞争ELISA方法检测原核表达的N蛋白及N92-94A突变蛋白、GST蛋白免疫小鼠血清结果；
图7为不同基因型PRRSV毒株和LDV的N蛋白氨基酸序列比对结果；
图8为rGSWW2015/N/S93A和rGSWW2015/N/S93A+D94A感染Marc-145细胞48小时后SR30A单抗和C8单抗间接免疫荧光结果。
具体实施方式
本发明提供了一种制备PRRSV基因II型表位缺失疫苗毒株的N蛋白表位突变标记位点，在PRRSV基因II型N蛋白C端92～103位氨基酸序列的基础上突变一个或多个氨基酸；所述N蛋白表位突变标记的氨基酸序列如SEQ ID NO:1所示，其中X1为T、P或A，X2为V或A。
在本发明中，所述表位的氨基酸序列如SEQ ID NO:1(LSDGRISYX1X2EF)所示。在不同PRRSV毒株中，N蛋白C端92～103位的多肽的序列不是完全保守的，在100位和101位点存在氨基酸变异，X1可以为T、P或A，X2可为V或A。所述表位的来源毒株优选包括GSWW/2015、MN184A、MN184B和JXWN06等基因II型毒株。
在本发明中，所述突变优选包括缺失或替换。所述替换优选包括将表位序列中任一位置的氨基酸替换为其他种类的氨基酸，例如替换为丙氨酸。所述突变的位点数量为1个氨基酸位点时，优选突变93位丝氨酸。实施例研究结果表明，93位丝基酸突变后与C8的反应性明显降低，证明93位的丝氨酸是C8抗体识别的最关键位点。当突变多个氨基酸时，优选包括连续或非连续突变2～12个氨基酸，更优选为3～9个氨基酸。所述突变连续3个氨基酸优选包括突变92～94位LSD三个氨基酸残基、突变95～97位GRI三个氨基酸残基、突变98～100位SYX1三个氨基酸残基、突变101～103位X2EF三个氨基酸残基。本发明实施例结果表明，92-103位表位氨基酸序列三点突变后的蛋白均不与C8抗体反应，证明92-103位是C8抗体识别的表位，改变其中一个或几个氨基酸位点能够消除C8抗体识别的表位；利用表位缺失N蛋白与自然N蛋白免疫动物后，通过检测C8表位抗体能够区分两种蛋白免疫动物，因此，如将此缺失标记位点引入全病毒疫苗，则可以精确区分标记疫苗免疫动物与自然感染动物，实现PRRSV感染与免疫动物的准确鉴别，将为PRRSV基因II感染状况监测与免疫净化提供技术与产品支持。
基于PRRSV基因II型N蛋白92-103位表位缺失改造后，能够实现自然N蛋白与表位缺失N蛋白免疫动物的准确鉴别，因此，本发明提供了一种PRRSV基因II型N蛋白表位缺失标记全病毒疫苗的制备方法。
在本发明中，所述PRRSV优选为美洲型PRRSV毒株，即PRRSV基因2型。所述疫苗的疫苗毒株优选包括GSWW/2015和GSWW/2018等毒株。
在本发明中，由于线性表位突变标记位点来源于N蛋白，N蛋白具有较高的保守性和免疫原性，感染后7天左右即可在血清中检测到N蛋白抗体，但是N蛋白抗体不具有中和活性，因此，将全病毒疫苗携带线性表位突变标记位点后，不会影响全病毒疫苗的原始免疫原性和免疫保护效果。
在本发明中，所述表位突变全病毒PRRSV疫苗的构建方法不做特殊限制，采用本领域所熟知的表位缺失的疫苗构建方法即可。在本发明实施例中，优选采用基因工程的手段对N蛋白编码基因进行突变，将突变的N基因序列克隆至PRRSV全长质粒中，经病毒拯救，得到表位突变全病毒PRRSV疫苗。
本发明提供了PRRSV核衣壳蛋白的猪源单个B细胞抗体C8在制备区分PRRSV自然感染和所述表位缺失全病毒PRRSV疫苗免疫动物的检测试剂盒中的应用。
在本发明中，所述PRRSV核衣壳蛋白的猪源单个B细胞抗体在申请号为202110598030.7的专利中公开。所述试剂盒优选为基于ELISA法制备。所述ELISA法优选为竞争性ELISA法。所述试剂盒优选还包括抗原反应板、100×浓缩生物素标记抗体、100×浓缩酶标亲和素、25倍浓缩洗涤液、血清稀释液、底物溶液、终止液、阳性对照血清和阴性对照血清，具体成分参见申请号202110598030.7的专利记载。
在本发明中，所述试剂盒的检测方法，优选如下：
PRRSV野生型毒株和所述表位突变全病毒PRRSV疫苗分别免疫动物，收集两组抗血清；
将试剂盒的抗原反应板中分别添加抗血清，经孵育后，洗涤，得到反应板；
向所述反应板中添加生物素标记的C8抗体，经孵育后，洗涤，得到竞争反应后的反应板；
向所述竞争反应后的反应板中添加HRP标记亲和素，经孵育后洗涤，添加底物，经反应后终止反应，测定吸光度值。
在本发明中，PRRSVN蛋白抗体竞争ELISA方法可以很好的区分野生型和表位缺失的N蛋白免疫血清，从而实现自然感染和N蛋白表位缺失全病毒疫苗免疫动物的区别。
下面结合实施例对本发明提供的一种用于制备PRRSV基因II型表位缺失疫苗毒株的N蛋白表位缺失标记位点进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。
实施例1
C8抗体与美洲型PRRSV毒株N蛋白特异性结合实验将GSWW/2015毒株N蛋白CDS区基因序列(SEQ ID NO:2，AAGCTTACCATGTACCCATACGACGTCCCAGACTACGCTCCAAATAACAACGGCAAGCAGCAAAAGAAAAAGAAGGGGAATGGCCAGCCAGTCAATCAGCTGTGCCAAATGCTGGGTAAGATCATCGCCCAACAAAATCAGTCCAGAGGCAAGGGACCGGGGAAGAAAAATAGGAAGAAAAACCCGGAGAAGCCCCATTTCCCTCTAGCGACTGAAGATGACGTCAGGCATCACTTTACCCCTAGTGAGCGGCAATTGTGTCTGTCGTCGATCCAGACTGCCTTCAACCAGGGCGCTGGAACTTGTGCCCTGTCAGATTCAGGGAGGATAAGTTACACTGTGGAGTTTAGTTTGCCGACGCAACATACTGTGCGTCTGATCCGCGCCACAGCATCACCCTCAGCAGACTACAAGGACGACGACGACAAGGGCGACTACAAAGATGACGATGATAAGATCGATTACAAAGACGATGACGATAAGTGAGAATTC)合成后插入pCDNA3.1载体HindⅢ和ECOR I中，分别用无内毒素质粒提取试剂盒抽提pcDNA3.1空载体和pcDNA3.1-N质粒，利用Lipo3000转染293T细胞，转染后24小时用RIPA(碧云天P0013)裂解液冰上裂解细胞30分钟，4℃，12,000rpm离心10分钟，提取总蛋白。加入上样缓冲液，95℃处理5分钟，蛋白变性后用4％～12％丙烯酰胺凝胶进行SDS-PAGE，然后将蛋白转印到PVDF膜上，用5％脱脂奶粉封闭1小时，加入C8抗体(1:1000)4℃孵育过夜。用TBST溶液洗三次后加入HRP标记的山羊抗猪二抗(sigma,1:10000)，室温孵育1小时后用TBST溶液洗三次，然后加入化学发光液(Thermo fisher,Pierce ECL Plus)，5分钟后在暗室用胶片曝光。
结果见图1所示。结果显示，C8抗体与PRRSV的N蛋白发生结合反应。
实施例2
合成肽ELISA初步鉴定C8识别N蛋白抗原表位的实验
将GSWW15的N蛋白分段合成多肽，各多肽名称与序列的对应关系见表1。以50μg/ml浓度溶解于碳酸盐溶液(Sigma)中，每孔100μl包被96孔板，4℃孵育过夜。加入生物素标记的0.25μg/ml C8抗体，37℃反应1小时，用PBST洗涤5次，最后一次拍干。加入HRP标记亲和素100μl，37℃反应1小时，用PBST洗涤5次，最后一次拍干。加入底物溶液[3,3’,5,5’-四甲基联苯胺(TMB)底物]37℃避光反应15分钟，加终止液(0.3mol/L H2SO4溶液)，在10分钟内检测450nM吸光值。
表1 GSWW15的N蛋白各肽段的信息
结果见图2。结果显示，C8单抗与N19肽段反应，说明N19肽段包含了C8单抗的抗原表位。
实施例3
GST融合蛋白鉴定N蛋白抗原表位关键位点实验
将GSWW/2018毒株的N蛋白83-112位进行三点突变(点突变由公司基因合成后通过BamHI和XhoI连接到载体中)为丙氨酸(丙氨酸扫描结构较为简单，识别容易，目的仅是破坏该表位)，合成基因后克隆到pGEX-6T-1载体中。将含有不同突变体的质粒转化BL-21感受态细胞，挑单克隆过夜培养。加入终浓度为1mmol/L的IPTG诱导6小时，收集上清进行Westernblot检测。
结果见图3。结果显示，92-103位三点突变后的蛋白均不与C8抗体反应，证明92-103位是C8抗体识别的表位。
实施例4
N蛋白92位-94位突变导致该表位缺失实验
将GSWW/2018毒株的N蛋白，及92-94位突变为丙氨酸的N蛋白基因(序列见SEQ IDNO:47，GGATCCATGCCAAATAACAACGGCAAGCAGCAAAAGAAAAAGAAGGGGAATGGCCAGCCAGTCAATCAGCTGTGCCAAATGCTGGGTAAGATCATCGCCCAACAAAATCAGTCCAGAGGCAAGGGACCGGGGAAGAAAAATAGGAAGAAAAACCCGGAGAAGCCCCATTTCCCTCTAGCGACTGAAGATGACGTCAGGCATCACTTTACCCCTAGTGAGCGGCAATTGTGTCTGTCGTCGATCCAGACTGCCTTCAACCAGGGCGCTGGAACTTGTGCCGCGGCTGCCTCAGGGAGGATAAGTTACACTGTGGAGTTTAGTTTGCCGACGCAACATACTGTGCGTCTGATCCGCGCCACAGCATCACCCTCAGCATGACTCGAG)合成后克隆到pCMV-HA载体中，用无内毒素质粒提取试剂盒提取质粒。将pCMV-GS18N-HA、pCMV-GS18N92-94AA和pCMV-HA分别转染293T细胞，转染24小时后用4％多聚甲醛室温固定10分钟，用预冷的PBS洗5次，然后用封闭液室温封闭1小时，分别加入猪源C8单抗(1:500)，商品化的N蛋白鼠源单抗SR30A1(1:50)及HA兔源单抗(1；500)4℃过夜孵育。用预冷PBS洗5次，每次5分钟，加入分别荧光标记二抗(1:50)：FITC标记的山羊抗猪抗体、FITC标记的山羊抗小鼠抗体和Cy3标记的山羊抗兔抗体，37℃避光反应1小时，PBS洗涤5次后在荧光显微镜下观察并拍照。
结果见图5。间接免疫荧光实验结果显示发现野生型N蛋白可以与两株单抗反应，而92-94突变的N蛋白不与C8单抗反应，但是可以与SR30A反应，说明92-94位突变可以消除C8识别的表位。
实施例5
N蛋白竞争ELISA方法可以区分GST-N蛋白及GST-N92-94A蛋白免疫小鼠血清，具体方法如下：
重组蛋白的表达：由公司基因合成野生型N蛋白和92-94位突变为丙氨酸的N92-94A核酸序列，利用BamHI和XhoI连接到pGEX-6T-1载体中。将阳性质粒转化BL-21感受态，挑单克隆过夜培养，扩大培养后取500mL新鲜菌液，活化3小时后，分别加入IPTG至终浓度为1mmol/L，37℃诱导8小时。将菌液8,000rpm离心5分钟收集菌体，弃掉上清，用50mL平衡液(0.15M NaCl、0.0027M KCl、0.01M Na2HPO4、0.0018MKH2PO4，调节pH 7.3)将菌体沉淀吹打混匀悬浮在冰上超声破碎，超声功率为250W、超声3秒、间歇3秒处理15分钟；10,000r离心10分钟，最后分别收集上清裂解液与离心沉淀，4℃保存，并用SDS-PAGE检测。
重组蛋白纯化：取GST-4FF树脂4mL装入柱子，使其自然沉降。采用5倍柱体积平衡液冲洗平衡树脂。将上述蛋白样品加入树脂柱，混匀，4℃结合过夜，控制流速，收集流穿液。采用平衡液洗涤柱子5～6次，控制流速，并收集部分样品待检。最后采用还原性谷胱甘肽洗脱液(0.05M Tris-HCl、0.01M GSH，pH 8.0)洗涤柱子5～6次，洗脱目的蛋白，收集样品。对收集到的样品进行SDS-PAGE与Westernblot分析。
小鼠免疫：1)抗原制备：测定重组蛋白的浓度，并适当调整蛋白浓度，与201佐剂按1:1体积混合，充分乳化，然后按照每只小鼠20μg剂量，免疫100μL进行免疫。
2)动物免疫：选取30只4～6周龄健康的雌性BALB/c小鼠，分为3组(表2)，每组10只。免疫方式均为小鼠背部皮下免疫，第三周进行加强免疫。第五周再加强免疫一次，第六周眼球采血并处死小鼠，分离血清，-20℃保存备用。用本实验室建立的PRRSV N蛋白抗体竞争ELISA方法检测(专利202110598030.7)。
表2 Balb/C小鼠的分组及免疫剂量
结果见图6。采用本实验室建立的PRRSVN蛋白抗体竞争ELISA方法可以很好的区分野生型和表位缺失的N蛋白免疫血清，为标记疫苗的制备奠定了基础。
将基因1型和基因2型的PRRSV毒株(见图7)以及乳酸脱氢酶增高症病毒(LDV)毒株的N蛋白序列进行比对，发现该线性表位在基因2型毒株中是保守的。这表明本发明提供的N蛋白线性表位标记位点适用于所有PRRSV基因2型候选表位缺失标记疫苗株的构建。利用点突变PCR技术以本实验室已有的PRRSV感染性克隆全长质粒pGS-AB为模板构建N蛋白C末端表位单个或多个氨基酸的缺失/突变质粒，体外转录获得病毒全长RNA，转染Marc-145细胞拯救病毒。拯救成功的重组毒株在Marc-145细胞上连续传代，制备弱毒疫苗或灭活疫苗；在仔猪出生后第14天用候选表位缺失疫苗进行免疫，免疫后每隔7天采血分离血清，中和试验检测中和抗体水平，分别用IDEXX PRRSV ELISA抗体检测试剂盒及本实验室建立的PRRSVN蛋白竞争ELISA抗体检测方法检测血清中N蛋白的抗体。
实施例6
N蛋白93-94位突变病毒的拯救方法
以本实验室保存的包含GSWW/2015毒株基因组半长(7606-15347位)质粒pGS-B为模板，利用点突变试剂盒进行突变，获得pGS-B/N93A和pGS-B/N93-94A质粒。将pGS-B/N93A和pGS-B/N93-94A分别与pGS-A(1-7606位)质粒利用SphⅠ和NheⅠ进行双酶切后连接，获得全长感染性克隆，命名为pGS-B/N93A和pGS/N93-94A。将全长质粒用AclⅠ线性化后用酚氯仿法纯化，然后利用Ambion T7体外转录试剂盒转录合成病毒全长RNA。利用电转法转染BHK21细胞，48小时后收集细胞及上清，反复冻融3次后接种Marc-145细胞，待70％以上细胞发生病变时收集细胞和上清。提取RNA，PCR扩增，测序鉴定突变位点。成功拯救获得突变毒株，分别命名为rGSWW2015/N/S93A和rGSWW2015/N/S93A+D94A。将突变病毒接种Marc-145细胞48小时后进行间接免疫荧光检测。
结果显示，rGSWW2015/N/S93A毒株可以与C8单抗和商品化单抗SR30A反应，而rGSWW2015/N/S93A+D94A毒株只能与单抗SR30A反应，不与C8反应，证明表位缺失毒株构建成功。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
序列表
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Arg Lys Lys Asn Pro Glu Lys Pro His Phe Pro Leu Ala Thr Glu
1 5 10 15
<210> 13
<211> 15
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 13
Glu Lys Pro His Phe Pro Leu Ala Thr Glu Asp Asp Val Arg His
1 5 10 15
<210> 14
<211> 15
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 14
Pro Leu Ala Thr Glu Asp Asp Val Arg His His Phe Thr Pro Ser
1 5 10 15
<210> 15
<211> 15
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 15
Asp Asp Val Arg His His Phe Thr Pro Ser Glu Arg Gln Leu Cys
1 5 10 15
<210> 16
<211> 15
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 16
His Phe Thr Pro Ser Glu Arg Gln Leu Cys Leu Ser Ser Ile Gln
1 5 10 15
<210> 17
<211> 15
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 17
Glu Arg Gln Leu Cys Leu Ser Ser Ile Gln Thr Ala Phe Asn Gln
1 5 10 15
<210> 18
<211> 15
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 18
Leu Ser Ser Ile Gln Thr Ala Phe Asn Gln Gly Ala Gly Thr Cys
1 5 10 15
<210> 19
<211> 15
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 19
Thr Ala Phe Asn Gln Gly Ala Gly Thr Cys Ala Leu Ser Asp Ser
1 5 10 15
<210> 20
<211> 15
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 20
Gly Ala Gly Thr Cys Ala Leu Ser Asp Ser Gly Arg Ile Ser Tyr
1 5 10 15
<210> 21
<211> 15
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 21
Ala Leu Ser Asp Ser Gly Arg Ile Ser Tyr Thr Val Glu Phe Ser
1 5 10 15
<210> 22
<211> 15
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 22
Gly Arg Ile Ser Tyr Thr Val Glu Phe Ser Leu Pro Thr Gln His
1 5 10 15
<210> 23
<211> 15
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 23
Thr Val Glu Phe Ser Leu Pro Thr Gln His Thr Val Arg Leu Ile
1 5 10 15
<210> 24
<211> 18
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 24
Leu Pro Thr Gln His Thr Val Arg Leu Ile Arg Ala Thr Ala Ser Pro
1 5 10 15
Ser Ala
<210> 25
<211> 15
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 25
Met Pro Asn Asn Asn Gly Arg Gln Gln Asn Lys Lys Lys Gly Asp
1 5 10 15
<210> 26
<211> 15
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 26
Gly Arg Gln Gln Asn Lys Lys Lys Gly Asp Gly Gln Pro Val Asn
1 5 10 15
<210> 27
<211> 15
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 27
Lys Lys Lys Gly Asp Gly Gln Pro Val Asn Gln Leu Cys Gln Met
1 5 10 15
<210> 28
<211> 16
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 28
Gly Gln Pro Val Asn Gln Leu Cys Gln Met Leu Gly Lys Ile Ile Ala
1 5 10 15
<210> 29
<211> 16
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 29
Gln Leu Cys Gln Met Leu Gly Lys Ile Ile Ala Gln Gln Arg Gln Ser
1 5 10 15
<210> 30
<211> 15
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 30
Leu Gly Lys Ile Ile Ala Gln Gln Arg Gln Ser Lys Gly Arg Gly
1 5 10 15
<210> 31
<211> 15
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 31
Ala Gln Gln Arg Gln Ser Lys Gly Arg Gly Pro Gly Lys Lys Asn
1 5 10 15
<210> 32
<211> 15
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 32
Ser Lys Gly Arg Gly Pro Gly Lys Lys Asn Lys Asn Lys Asn Leu
1 5 10 15
<210> 33
<211> 15
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 33
Pro Gly Lys Lys Asn Lys Asn Lys Asn Leu Glu Lys Pro His Phe
1 5 10 15
<210> 34
<211> 15
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 34
Lys Asn Lys Asn Leu Glu Lys Pro His Phe Pro Leu Ala Thr Glu
1 5 10 15
<210> 35
<211> 15
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 35
Glu Lys Pro His Phe Pro Leu Ala Thr Glu Asp Asp Val Arg His
1 5 10 15
<210> 36
<211> 15
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 36
Pro Leu Ala Thr Glu Asp Asp Val Arg His His Phe Thr Pro Ser
1 5 10 15
<210> 37
<211> 15
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 37
Asp Asp Val Arg His His Phe Thr Pro Ser Glu Arg Gln Leu Cys
1 5 10 15
<210> 38
<211> 15
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 38
His Phe Thr Pro Ser Glu Arg Gln Leu Cys Leu Ser Ser Ile Arg
1 5 10 15
<210> 39
<211> 15
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 39
Glu Arg Gln Leu Cys Leu Ser Ser Ile Arg Thr Ala Phe Asn Gln
1 5 10 15
<210> 40
<211> 15
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 40
Leu Ser Ser Ile Arg Thr Ala Phe Asn Gln Gly Ala Gly Thr Cys
1 5 10 15
<210> 41
<211> 15
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 41
Thr Ala Phe Asn Gln Gly Ala Gly Thr Cys Thr Leu Ser Asp Ser
1 5 10 15
<210> 42
<211> 15
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 42
Gly Ala Gly Thr Cys Thr Leu Ser Asp Ser Gly Arg Ile Ser Tyr
1 5 10 15
<210> 43
<211> 15
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 43
Thr Leu Ser Asp Ser Gly Arg Ile Ser Tyr Thr Val Glu Phe Ser
1 5 10 15
<210> 44
<211> 15
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 44
Gly Arg Ile Ser Tyr Thr Val Glu Phe Ser Leu Pro Thr His His
1 5 10 15
<210> 45
<211> 15
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 45
Thr Val Glu Phe Ser Leu Pro Thr His His Thr Val Arg Leu Ile
1 5 10 15
<210> 46
<211> 18
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 46
Leu Pro Thr His His Thr Val Arg Leu Ile Arg Val Thr Thr Ser Pro
1 5 10 15
Ser Ala
<210> 47
<211> 384
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 47
ggatccatgc caaataacaa cggcaagcag caaaagaaaa agaaggggaa tggccagcca 60
gtcaatcagc tgtgccaaat gctgggtaag atcatcgccc aacaaaatca gtccagaggc 120
aagggaccgg ggaagaaaaa taggaagaaa aacccggaga agccccattt ccctctagcg 180
actgaagatg acgtcaggca tcactttacc cctagtgagc ggcaattgtg tctgtcgtcg 240
atccagactg ccttcaacca gggcgctgga acttgtgccg cggctgcctc agggaggata 300
agttacactg tggagtttag tttgccgacg caacatactg tgcgtctgat ccgcgccaca 360
gcatcaccct cagcatgact cgag 384
Claims (8)
1.一种用于制备PRRSV基因II型表位缺失疫苗毒株的N蛋白表位突变标记，其特征在于，在PRRSV基因II型N蛋白C端92～103位氨基酸序列的基础上突变一个或多个氨基酸；
所述N蛋白表位突变标记的氨基酸序列如SEQ ID NO:1所示，其中X1为T、P或A，X2为V或A。
2.根据权利要求1所述用于制备PRRSV基因II型表位缺失标记疫苗毒株的N蛋白线性表位突变标记，其特征在于，当突变1个氨基酸位点时，突变表位中的任何一个位点氨基酸；
优选的，突变93位丝氨酸。
3.根据权利要求1所述用于制备PRRSV基因II型表位缺失标记疫苗毒株的N蛋白线性表位突变标记，其特征在于，当突变多个氨基酸时，包括连续或不连续突变2～12个氨基酸。
4.根据权利要求1～3任意一项所述用于制备PRRSV基因II型表位缺失标记疫苗毒株的N蛋白线性表位突变标记，其特征在于，所述突变包括突变修饰、缺失或插入异源序列。
5.根据权利要求4所述用于制备PRRSV基因II型表位消除标记疫苗毒株的N蛋白线性表位突变标记，其特征在于，所述突变修饰包括氨基酸替换。
6.一种表位缺失全病毒PRRSV疫苗，其特征在于，所述疫苗为含有权利要求1～5任意一项所述N蛋白表位突变标记的PRRSV。
7.根据权利要求6所述表位缺失全病毒PRRSV疫苗，其特征在于，所述PRRSV为PRRSV基因II型。
8.PRRSVN蛋白的猪源单个B细胞抗体C8在制备检测权利要求6或7所述PRRSV疫苗的试剂或试剂盒中的应用。
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| CN202111638189.3A CN114315984B (zh) | 2021-12-29 | 2021-12-29 | 一种用于制备prrsv基因ii型表位缺失疫苗毒株的n蛋白表位突变标记及其应用 |
| PCT/CN2022/081330 WO2023123696A1 (zh) | 2021-12-29 | 2022-03-17 | 一种用于制备prrsv基因ii型表位缺失疫苗毒株的n蛋白表位突变标记及其应用 |
| US17/997,749 US20240148850A1 (en) | 2021-12-29 | 2022-03-17 | N protein epitope mutation marker for preparing epitope deletion-marked vaccine strain of type ii porcine reproductive and respiratory syndrome virus (prrsv) and use thereof |
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| CN114836392A (zh) * | 2022-04-27 | 2022-08-02 | 佛山科学技术学院 | 一种prrsv弱病毒及其制备方法和应用 |
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| CN114315984B (zh) | 2023-02-03 |
| WO2023123696A1 (zh) | 2023-07-06 |
| US20240148850A1 (en) | 2024-05-09 |
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