CN109678952B - 一种基于小麦黄条纹病毒g蛋白的多克隆抗体、制备方法及其应用 - Google Patents
一种基于小麦黄条纹病毒g蛋白的多克隆抗体、制备方法及其应用 Download PDFInfo
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Abstract
本发明公开了一种基于小麦黄条纹病毒G蛋白的多克隆抗体、制备方法及其应用。多克隆抗体的制备包括以下步骤:根据WYSV的G基因的序列,首次优化密码子合成了该基因并亚克隆到用于大肠杆菌表达的靶载体pET‑30a中;重组蛋白经原核表达,免疫新西兰大白兔制备得到了WYSV G蛋白的多克隆抗体。Western blot检测表明制备的抗体能与G重组蛋白、感病小麦样品有特异性结合,说明获得的抗体特异性高。基于制备的多克隆抗体建立了检测WYSV的DIBA杂交方法,能够特异、灵敏并且快速地检测田间禾本科农作物是否感染WYSV病毒。
Description
技术领域
本发明属于农业生物学技术领域,具体地涉及一种WYSV多克隆抗体、制备方法和基于抗体建立的免疫检测试剂盒以及应用,从而达到快速、高效检测WYSV的目的。
背景技术
小麦是我国的主要粮食作物之一,其安全生产关乎民生。近年来,小麦上的病毒病发生较重,尤其是大麦黄矮病毒(Barley yellow dwarf virus,BYDV)引起的小麦黄矮病和小麦黄花叶病毒(Wheat yellow mosaic virus,WYMV)引起的小麦黄花叶病,造成小麦严重减产和巨大经济损失。此外,小麦矮缩病毒(Wheat dwarf virus,WDV)引起的小麦矮缩病毒病,是由介体异沙叶蝉(Psammotettix striatus L.)以持久非增殖方式传播,小麦矮缩病毒罹病植株典型症状表现为矮化、黄化和分蘖增多等,在我国西北部局部麦区造成严重危害。小麦黄条纹病毒(Wheat yellow striate virus,WYSV)是2016年陕西韩城病害田间调查时发现的一种新的细胞核弹状病毒,也是由异沙叶蝉传播到小麦、大麦等禾本科作物上。与小麦矮缩病毒病产生的症状不同,受WYSV侵染的小麦无明显矮化,表现为严重黄化、发病叶片沿叶脉失绿,逐渐发展为从叶尖开始干枯,最后枯死。WYSV为负义单链RNA病毒,整个WYSV基因组全长为14,486nt,含有7个开放阅读框(ORF),在反义链上以“N-P-P3-M-G-P6-L”顺序依次编码蛋白。为进一步了解这种病毒病的发生、流行规律、建立早期监测和预警体系,急需建立快速、高效的病毒检测方法。
植物病毒的检测方法通常有生物学、血清学和分子生物学等手段。但就WDV和与新病毒WYSV而言,由于两种病毒均由异沙叶蝉传播,症状上又有相似之处,生物学鉴定无法完成鉴别;以PCR为代表的分子生物学手段具有快速、高度灵敏等特点,但需要昂贵的仪器和分子生物学试剂,且不适合大批量样品的检测;血清学具有快速简便、高通量等优点,且适合田间大规模样品检测,因此被广泛应用于植物病毒的诊断、流行规律的分析、预测预警、科学防控和抗病育种等方面。
发明内容
本发明的目的是提供一种小麦黄条纹病毒WYSV-G的抗体,并以此抗体为基础建立了斑点免疫杂交快速检测方法(Dot Immunobinding Assay,DIBA),同时提供了一种快速、便捷、灵敏以及特异性好的相应试剂盒。
一种基于小麦黄条纹病毒G蛋白的多克隆抗体的制备方法,其特征在于:以小麦黄条纹病毒G蛋白为免疫原通过大肠杆菌原核系统表达重组蛋白,纯化后得到的G蛋白抗原通过皮下多点注射新西兰大白兔制备得到多克隆抗体。
所述重组蛋白表达是将含有经密码子优化的小麦黄条纹病毒G蛋白重组质粒转入到大肠杆菌DL21原核系统中进行表达。
所述重组质粒是将含小麦黄条纹病毒G蛋白中第115至1828nt的核苷酸经密码子优化并添加有酶切位点、His标签以及终止密码子,按NdeI--(WYSV-G)--His tag--Stopcodon—HindIII的顺序合成基因,利用无缝克隆技术将合成基因转入载体pET-30a中,合成基因的氨基酸的序列如SEQ ID No.1,所述合成基因的核苷酸的序列如SEQ ID No.2所示。
所述注射是将纯化后的可溶性G蛋白与完全弗氏佐剂乳化后,皮下多点免疫于新西兰雄兔,10天后进行第二次免疫,以后每隔一周加强免疫,加强免疫采用与不完全弗氏佐剂乳化,大腿肌肉注射方法。
所述免疫后5d取少量血清,用间接ELISA测定抗体的效价,当效价达到l:100 000以上时,取血并分离血清,采用protein A-Sepharose affinity column从抗血清中提纯获得多克隆抗体anti-WYSV-G-IgG。
上述方法制备得到的小麦黄条纹病毒G蛋白多克隆抗体。
所述多克隆抗体在WYSV病毒检测中的应用。
小麦黄条纹病毒的斑点免疫杂交快速检测试剂盒,包括上述小麦黄条纹病毒G蛋白多克隆抗体和酶标二抗。
所述的酶标二抗为商品化的过氧化物酶(HRP)标记抗体,为羊抗兔IgG-HRP,其工作浓度为1:2000。
所述的试剂盒还包括阳性质控对照、杂交膜、样品研磨液、洗膜液、封闭液以及显色液。
所述的阳性质控对照为小麦黄条纹病毒G基因的原核表达蛋白。
所述的杂交膜为尼龙膜或者硝酸纤维素膜。
所述样品研磨液为0.05M碳酸钠包被缓冲液,pH=9.6;所述洗膜液为1×PBS缓冲液,所述封闭液为含5%脱脂奶粉的PBS缓冲液,所述显色液为HRP-DAB底物显色液。
所述的小麦黄条纹病毒的毒源采自陕西韩城,经PCR鉴定为阳性的植株,经介体异沙叶蝉活体饲喂毒源再次保存在感病小麦品种扬麦12号。
所述异沙叶蝉种群采自陕西韩城,常年在小麦幼苗上饲养。
所述的介体饲养和作为饲料的寄主小麦材料均种植在22℃培养箱内,16h光照、8h黑暗,光照强度为20000Lx。每四周将叶蝉转移到新种的幼苗上,以此方法来保存介体。
本发明所述的小麦黄条纹病毒免疫试剂盒的实验方法,其包括如下步骤:
1)将适量大小的尼龙膜或硝酸纤维素膜用镊子划成均一的正方格,置于干净的培养皿中备用。将待测植物样品0.1g放入1.5mL离心管中,加入1000μL样品研磨液并用小研磨棒捣碎后,吸取1.2μL上清液于划好膜上的方格中。其中,加入原核表达的G蛋白样品以小麦健康叶片研磨液分别作为实验阳性质控和阴性对照。
2)待室温晾干后加入封闭液(5%脱脂奶粉的PBS buffer),37℃下封闭2h,并轻轻摇晃;用洗膜液洗膜3次,每次10min。
3)取0.6μL WYSV病毒抗体,按1:10000倍稀释溶解于6mL封闭液,将尼龙膜浸入其中,37℃轻摇孵育1h;用洗膜液(PBS buffer)洗膜3次,每次10min。
4)取3μL羊抗兔二抗(SeraCare Life Sciences公司),溶解于6mL TBST buffer中(1:2000倍稀释),37℃轻摇孵育1h,并轻轻摇晃。用洗膜液洗膜3次,每次10min。
5)使用HRP-DAB底物显色试剂盒(天根公司产品)进行显色反应。向一个新的离心管中加入1mL HRP反应液,然后依次加入试剂A、B和C各50μL,混匀后滴加至尼龙膜上。室温下反应1min,此过程闭光。
6)通过观察显色反应判断实验结果:待测的样品如显示为蓝黑色,则携带WYSV病毒;不显色或只有叶片本色的待测样品不携带WYSV病毒。
本发明利用原核表达经密码子优化的WYSV-G蛋白,制备了多克隆抗体(polyclonal antibodies,PAbs),并利用制备的抗体建立WYSV的斑点免疫杂交检测体系。
本发明具有以下优点:
1)利用原核表达的经密码子优化的WYSV-G蛋白作为免疫原,克服了全长序列难以正常表达的缺点。制备的多克隆抗体与其他病毒或健康样品均无交叉反应,能特异性地检测含有WYSV的植株样品,说明该抗体能很好地用于WYSV病毒的检测。
2)利用制备的WYSV病毒的特异性多克隆抗体建立了一种斑点免疫杂交检测方法,以该方法组装了小麦黄条纹病毒的检测试剂盒。该试剂盒具有快速灵敏、特异性强,无需PCR、酶联测定仪等任何昂贵的仪器,操作简单方便,对基层单位十分适用,兼顾实用性和快速性,具有良好的应用前景。
附图说明
图1.重组G蛋白的NdeI和HindШ双酶切验证:1)Marker DL10000;2)pET-30a空载体;3)pET-30a-G。
图2.SDS-PAGE电泳分析WYSV-G重组蛋白的表达:PC1)BSA(1μg);PC2)BSA(2μg);M1)蛋白marker;NC)未诱导全细胞裂解物;1)15℃诱导16小时的细胞裂解物;2)37℃诱导4小时的细胞裂解物;NC1)未诱导的细胞裂解物上清液;NC2)未诱导的细胞裂解物的碎片;3)在15℃诱导16小时的细胞裂解物上清液;4)在15℃诱导16小时的细胞裂解物的碎片;5)在37℃诱导4小时的细胞裂解物上清液;6)在37℃诱导4小时的细胞裂解物的碎片。
图3.Western-blot分析WYSV-G重组蛋白的表达:M2)蛋白marker;1)15℃诱导16小时的细胞裂解物;2)37℃诱导4小时的细胞裂解物;3)在15℃诱导16小时的细胞裂解物上清液;4)在15℃诱导16小时的细胞裂解物的碎片;5)在37℃诱导4小时的细胞裂解物上清液;6)在37℃诱导4小时的细胞裂解物的碎片。
图4.Western-blot分析WYSV-G多克隆抗体的特异性:1)蛋白Marker;2)健株样品;3)病株样品。
图5.DIBA免疫检测技术方法的建立:1)WYSV阳性样品;2)健康小麦样品;3)空白对照。
图6.DIBA免疫检测技术方法的特异性检测:1)WYSV阳性样品;2-6)BYSMV、WDV、BYDV-GAV、GPV和PAV;7)健康小麦样品;8)空白对照。
图7.DIBA免疫检测技术方法的灵敏度检测:第1行第1格点WYSV-G的原核表达蛋白,第2-6格分别为携带WYSV的小麦叶片样品,浓度梯度依次按照1:10;1:20;1:40;1:80;1:160;1:320;1:640;1:1280的比例稀释后取1.2μL点于方格中央,第2行为阴性对照样品,
图8.DIBA方法用于2017-2018年度田间采集的疑似样品的检测。
具体实施方式
下面结合具体实施例,进一步阐述本发明,这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法,通常按照常规条件。
需特别指出的是,尽管本发明阐述的是针对小麦上WYSV病毒的鉴定技术,但WYSV病毒也侵染大麦、燕麦等多种麦类种质资源,因此应用本发明对任何引起WYSV病毒的寄主如大麦、燕麦等麦类种质资源均也包括在本发明所要求的权利范围之内。
下述实验病毒,本申请人实验室均有保存,可以对外公开发放。
实施例1.小麦黄条纹病毒G蛋白的基因优化及合成、亚克隆
根据WYSV的全长基因序列(GenBank登录号MG604920),利用软件分析该病毒的ORF5编码的蛋白即糖蛋白Glycoprotein(G),发现G蛋白基因全长1986nt,编码661个氨基酸,预测的分子量和等电点分别为74.9KDa和5.30。
利用在线软件SignalP(http://www.cbs.dtu.dk/services/SignalP/)和TMHMMServer v.2.0(http://www.cbs.dtu.dk/services/TMHMM/)预测蛋白的信号肽和跨膜区,经分析发现WYSV的G蛋白没有潜在的信号肽剪切位点,但含有两个跨膜区,分别在基因5’端的第7至24位和第611至633位。运用Optimum GeneTM密码子优化技术(南京金斯瑞公司科技有限公司),最终选取578个氨基酸(第115至1828nt)优化密码子,并在3’端加上His标签,按“NdeI--(WYSV-G)--His tag--Stop codon--HindIII”基因合成策略进行合成(氨基酸序列见SEQ1和对应的核苷酸序列见SEQ2),再将合成的基因利用无缝克隆技术克隆至pET-30a(+)(Novagen公司)的载体中,经NdeI和HindIII双酶切验证准确无误后(酶切图1),用于下一步实验。
实施例2.重组蛋白表达及纯化
将重组表达质粒pET-30a-(WYSV-G)通过热击发转化至BL21(DE3)表达菌株,经测序验证后,将挑选阳性克隆即含有正确重组表达质粒的BL21菌液接种于含50ug/mL卡那霉素的4mL LB液体培养基中,37℃、200rpm培养至OD600为0.6~0.8,加入IPTG至终浓度为0.3mmol/L在15℃条件下诱导16h,然后37℃继续诱导培养4h后收集1mL菌液,离心收集菌体。经12%的SDS-PAGE凝胶电泳检测,分别检测全菌、上清和沉淀中蛋白表达情况。发现在诱导处理的样品中检测到目的蛋白特异表达,大小约为65kDa,与预计的融合蛋白大小一致,而在未诱导的样品中没有检测到目的条带(图2)。因表达的蛋白带有6×His标签靶序列,故利用6×His标签单克隆抗体(南京金斯瑞生物技术有限公司,货号A00186)进行Western blot分析,结果发现表达的重组蛋白与6×His单克隆抗体有特异性反应(图3),表明WYSV的G蛋白基因已经在大肠杆菌DL21中融合表达。应用Ni2+NTA亲和层析柱纯化回收蛋白。获得含有G蛋白的上清液,将该上清液通过Ni2+亲和柱,再用5倍柱体积的250mM咪唑浓度洗脱缓冲液洗脱,收集洗脱下来的蛋白并利用分光光度仪测定A280处读数,当有蛋白峰时开始收集样品,直至A280又恢复至基准线,得到纯化的G蛋白,储存于含0.5%SDS的1×PBS缓冲液中(pH 7.4)。
实施例3.抗体制备
纯化后的可溶性蛋白与完全弗氏佐剂乳化后,皮下多点免疫于新西兰雄兔,10天后进行第二次免疫,以后每隔一周加强免疫,加强免疫采用与不完全弗氏佐剂乳化,大腿肌肉注射方法。每次免疫后5d取少量血清,用间接ELISA测定抗体的效价,当效价达到l:100000以上时,取血并分离血清。采用protein A-Sepharose affinity column从抗血清中提纯获得anti-WYSV-G-IgG,按照500×21(1000)至500×210(512000)连续倍比稀释,实验分别以免疫前血清为阴性对照和PBS缓冲液为空白对照,结果表明,间接ELISA测定效价大于1:512000。然后,提取感病小麦及健康小麦的总蛋白,经SDS-PAGE凝胶电泳后,电转至硝酸纤维素膜上,用封闭缓冲液稀释的多抗血清作为一抗(1:200稀释),进行Western blot特异性分析,结果表明感染WYSV植株的提取液中有一条约75kDa大小的条带,而健康植株的提取液中没有,说明提纯的抗体有较好的特异性(图4)。
实施例4.基于多克隆抗体建立DIBA免疫检测技术
基于实施例3得到的多克隆抗体,本发明建立了斑点免疫结合检测技术(Dotimmunobinding assay,DIBA)用于疑似WYSV的植株样品检测,具体步骤如下:
1)将杂交膜(尼龙膜或硝酸纤维素膜)用镊子划成均一的正方格,置于干净的培养皿中备用。将待测植物样品0.1g放入1.5mL离心管中,加入1000μL样品研磨液(1×PBS)并用小研磨棒捣碎后,吸取1.2μL上清液于划好膜上的方格中。选取WYSV带病小麦叶片为待测样品,以小麦健康叶片作为实验阴性对照,样品研磨液为实验空白对照。
2)待室温晾干后加入封闭液(5%脱脂奶粉的PBS buffer),37℃下封闭2h,并轻轻摇晃;用洗膜液(PBS buffer)洗膜3次,每次10min。
3)取3μL WYSV病毒抗体,按1:10000倍稀释溶解于6mL封闭液,将尼龙膜浸入其中,37℃轻摇孵育1h;用洗膜液洗膜3次,每次10min。
4)取3μL羊抗兔二抗(Seracare公司),溶解于6mL TBST buffer中(1:2000倍稀释),37℃轻摇孵育1h。用洗膜液洗膜3次,每次10min。
5)使用HRP-DAB底物显色试剂盒(天根)进行显色反应。向一个新的离心管中加入1mL HRP反应液,然后依次加入试剂A、B和C各50μL,混匀后滴加至尼龙膜上。室温下反应1min,此过程闭光。
6)通过观察显色反应发现:已知的WYSV阳性样品显示蓝黑色,健康样品均无反应,仅显示一些叶片本色(绿色),空白对照(样品研磨液)不显色,说明该抗体能特异检测含有WYSV的植株样品,具体结果见图5。
实施例5.DIBA免疫检测技术的特异性和灵敏度试验
为对实施例4建立的DIBA免疫检测技术的特异性进行评价,分别取侵染小麦的另一种弹状病毒-大麦黄条点病毒(Barley Yellow striate mosaic virus,BYSMV)、异沙叶蝉传播的小麦矮缩病毒(WDV)以及由麦蚜传播的大麦黄矮病毒(BYDV-GAV/GPV/PAV)的小麦植株叶片进行试验。待测样品0.1g放入1.5mL离心管中,加入样品研磨液1000μL,小研磨棒捣碎后,取上清液体1.2μL小心滴于杂交膜的小方格中央,室温风干;其余实验过程同实施例4中第2-5个步骤。通过观察显色反应发现:已知的WYSV阳性样品显示蓝黑色,与BYSMV、WDV和BYDV-GAV带病小麦叶片样品以及健康样品均无反应,仅显示一些叶片本色(绿色),空白对照(样品研磨液)不显色,说明该抗体能特异检测含有WYSV的植株样品,具体结果见图6。
此外,为对实施例4建立的DIBA免疫检测技术的灵敏度进行评价,将携带WYSV的小麦叶片样品0.1g放入1.5mL离心管中,加入样品研磨液1mL,小研磨棒捣碎后将原液按1:10、1:20、1:40、1:80、1:160、1:320、1:640和1:1280稀释后进行系列检测,分别取1.2μL小心滴于杂交膜的小方格中央,室温风干;其余实验过程同实施例4中第2-5个步骤,灵敏度的试验结果表明特异性的:WYSV抗血清能灵敏地检测含有WYSV的植株样品,健康样品也仅显示一些叶片本色(绿色),空白对照(样品研磨液)不显色,原液按1:160比例稀释后还可以清晰看见蓝色斑点,表明该抗体灵敏度达到能检测约8ug植物叶片中的病毒,具体结果见图7。
实施例6.DIBA免疫检测方法应用于田间样品的检测
实施例4和5的实验结果表明利用制备的WYSV多克隆抗体建立的针对WYSV的DIBA免疫检测方法可用于检测小麦样品中的WYSV的存在。2017-2018年小麦生长季在我国陕西、山西、山东、云南、贵州和湖北等6个省份采集到症状疑似为WYSV病毒的标样85份。利用实施例4和5建立的方法对采自田间的样品进行检测。每份标样称取0.1g叶片,研磨后取1.2μL点于杂交膜上,部分样品的检测结果图8。检测结果表明WYSV在陕西韩城和湖北武汉地区点片发生发现。
序列表
<110> 中国农业科学院植物保护研究所
<120> 一种基于小麦黄条纹病毒G蛋白的多克隆抗体、制备方法及其应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 578
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Met Asp Ser Gln Glu Asn Arg Pro Phe Thr Leu His Pro Ala His Gly
1 5 10 15
Ala Ile Pro Val Leu Pro Ser Ser Gln Pro Ser Gln Lys Val Ser Pro
20 25 30
Asp Asp Thr Asn Ser Leu Ile Arg Leu Thr Thr Ser Glu Gln Leu Trp
35 40 45
His Asn His Pro Gln Asp Ile Gly Pro Lys Asp Val Pro Ala Asp Met
50 55 60
Tyr Pro Ile Tyr Ser Cys Pro Asn Leu Ser Asn Ala Tyr Leu Leu Pro
65 70 75 80
Ile Trp Tyr Gly Ser Cys Leu Asp Ala Cys Gln Ile Thr Thr Pro Lys
85 90 95
His Thr Val Asn Val Lys Leu Trp Thr Ile Asn Ser Ser Val Thr Asp
100 105 110
Val Asp Gly Tyr Gln Ile Asp Val Tyr Tyr Asp Thr Lys Phe Ser His
115 120 125
Val Gly Pro Phe Gly Gly Cys Ser Val Ser Leu Ser Glu Ser Val Thr
130 135 140
Lys Glu Pro Lys Gln Glu Asp Ile Met Ile Trp Lys Ser Arg Leu Val
145 150 155 160
Ser Lys Pro Val Asn Asp Val Glu Ser Trp Ile Met Tyr Asp Glu Pro
165 170 175
Ser Cys Asn Tyr Phe Ser Asp Glu Tyr Ser Ser Gly Phe Arg Leu Val
180 185 190
Ile Thr Arg Thr Lys Leu Lys Leu Met Ile Asp Ser Val Gly Asn Leu
195 200 205
Tyr Ile Ala Asp Leu Arg Pro Gly Ser Tyr Asp Thr Tyr Lys Thr Gly
210 215 220
Tyr Ala Ile His Gly Ser Thr Ala Trp Ile Trp Asp Thr Asp Asp Ser
225 230 235 240
Met Asn His Gly Met Cys Tyr Phe Lys Gln Thr Asp Asp Thr Tyr Cys
245 250 255
Asp Tyr Asp Asn Asn Thr Lys Tyr Met Phe Cys Lys Met Ser Gly Val
260 265 270
Ser Phe Asp Thr Thr Val Gln Gln Arg Ile Thr Ser Ser Cys Ala Gly
275 280 285
Asp Leu Asn Ile Ser Thr Asp Gly Val Ile Tyr Gln Ile Gly Asp Ser
290 295 300
Gly Asp Thr Ala Ser Thr Gln Gln Arg Leu Ser Asp Ile Leu His Gln
305 310 315 320
Asn Val Glu Leu Gly Met Gln Ser Leu Val Ser Leu Ile Asn Asp Val
325 330 335
Phe Ile Asn Ile Glu Ser Ser Tyr Cys Thr Gly Val Cys Asp Ile Met
340 345 350
Glu Val Ile Val Ser Asn Tyr Pro Thr Ala Thr Thr Val Leu Glu Thr
355 360 365
Pro Ile Gly Pro Trp Leu Pro Ile Thr Ser Asp Gly His Thr Ile Met
370 375 380
Thr Pro Cys Met Ala Asp Val Asn Trp Ile Ile Gln Thr Pro Ile Val
385 390 395 400
Tyr Cys Phe Ser Lys Glu Met Ile Lys Val Ile Asn Lys Asp Thr Arg
405 410 415
Lys Glu Ala Trp Trp Arg Ile Val Asn Ser Tyr Ile Ile Leu Asn Glu
420 425 430
Thr Cys Ser Asp Thr Asn Ser Thr Ala Leu Glu Ile Leu Arg Asp Arg
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Met Ser Lys Arg Arg Asp Ile Val Tyr Ser Phe Trp Arg Gly Asp Leu
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Lys Tyr Lys His Pro Ile Thr Leu Asp Asn Ile Thr Ser Gln Leu Val
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Asn His Thr Ala Asp Leu Tyr Glu Trp His Met Gly Asp Lys Asn Gly
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Thr Ala Gly Gln Thr Thr Phe Ser Asp Leu Leu Gly Arg Val Glu Lys
530 535 540
Ala Gly Thr Asn Val Ile Lys Gly Cys Val Lys Met Thr Gly Asn Leu
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Leu Ile Trp Ile Thr Ser His Ile Glu Met Ile Gly His His His His
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His His
<210> 2
<211> 1749
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
catatggaca gccaagagaa ccgtccgttt accctgcatc cggcgcatgg tgcgatcccg 60
gtgctgccga gcagccaacc gagccagaaa gtgagcccgg acgataccaa cagcctgatc 120
cgtctgacca ccagcgagca gctgtggcac aaccacccgc aagacatcgg tccgaaggac 180
gttccggcgg atatgtaccc gatttatagc tgcccgaacc tgagcaacgc gtacctgctg 240
ccgatctggt atggcagctg cctggatgcg tgccagatta ccaccccgaa gcacaccgtg 300
aacgttaaac tgtggaccat caacagcagc gtgaccgacg ttgatggtta ccaaattgac 360
gtgtactatg ataccaaatt cagccacgtt ggcccgtttg gtggctgcag cgtgagcctg 420
agcgagagcg ttaccaagga gccgaaacag gaagacatca tgatttggaa gagccgtctg 480
gtgagcaaac cggtgaacga cgttgagagc tggatcatgt atgatgaacc gagctgcaac 540
tacttcagcg acgaatatag cagcggtttt cgtctggtga tcacccgtac caagctgaaa 600
ctgatgatcg atagcgttgg taacctgtac attgcggacc tgcgtccggg cagctacgat 660
acctataaga ccggttatgc gatccacggc agcaccgcgt ggatttggga caccgacgat 720
agcatgaacc acggcatgtg ctacttcaaa cagaccgacg atacctactg cgactatgat 780
aacaacacca agtatatgtt ctgcaaaatg agcggcgtga gctttgatac caccgttcag 840
caacgtatca ccagcagctg cgcgggtgac ctgaacatta gcaccgatgg cgtgatctac 900
caaattggtg acagcggcga taccgcgagc acccagcaac gtctgagcga catcctgcac 960
cagaacgttg agctgggtat gcaaagcctg gtgagcctga ttaacgacgt tttcatcaac 1020
attgagagca gctactgcac cggcgtgtgc gatatcatgg aagtgattgt tagcaactat 1080
ccgaccgcga ccaccgttct ggaaaccccg atcggtccgt ggctgccgat taccagcgac 1140
ggccacacca tcatgacccc gtgcatggcg gatgtgaact ggatcattca gaccccgatt 1200
gtttactgct ttagcaagga gatgatcaaa gtgattaaca aggacacccg taaagaagcg 1260
tggtggcgta tcgttaacag ctatatcatt ctgaacgaaa cctgcagcga taccaacagc 1320
accgcgctgg aaattctgcg tgaccgtatg agcaagcgtc gtgatatcgt gtacagcttc 1380
tggcgtggtg acctgattgt tagctacccg tataacaaaa gccgttggat cacctacaag 1440
gatgaaaaaa ttcaacgtag cagcaagtgg tttgacaaac tggtggatct gaagtataaa 1500
cacccgatca ccctggacaa cattaccagc cagctggtta accacaccgc ggacctgtac 1560
gagtggcaca tgggcgataa gaacggtacc gcgggccaaa ccacctttag cgatctgctg 1620
ggtcgtgttg aaaaagcggg caccaacgtg atcaagggtt gcgttaaaat gaccggcaac 1680
ctgctgattt ggattaccag ccacattgag atgatcggtc accaccacca ccaccactaa 1740
tgaaagctt 1749
Claims (8)
1.一种基于小麦黄条纹病毒G蛋白的多克隆抗体的制备方法,其特征在于:以小麦黄条纹病毒G蛋白为免疫原通过大肠杆菌原核系统表达重组蛋白,纯化后得到的G蛋白抗原通过皮下多点注射新西兰大白兔制备得到多克隆抗体;
所述重组蛋白表达是将含有经密码子优化的小麦黄条纹病毒G蛋白重组质粒转入到大肠杆菌DL21原核系统中进行表达;
所述重组质粒是将含小麦黄条纹病毒G蛋白中第115至1828nt的核苷酸经密码子优化并添加有酶切位点、His标签以及终止密码子,按NdeI--(WYSV-G)--His tag--Stopcodon—HindIII的顺序合成基因,利用无缝克隆技术将合成基因转入载体pET-30a中,合成基因的氨基酸的序列如SEQ ID No.1,所述的核苷酸的序列如SEQ ID No.2所示。
2.根据权利要求1所述的方法,所述注射是将纯化后的可溶性G蛋白与完全弗氏佐剂乳化后,皮下多点免疫于新西兰雄兔,10天后进行第二次免疫,以后每隔一周加强免疫,加强免疫采用与不完全弗氏佐剂乳化,大腿肌肉注射方法;所述免疫后5d取少量血清,用间接ELISA测定抗体的效价,当效价达到l:100 000以上时,取血并分离血清,采用protein A-Sepharose affinity column从抗血清中提纯获得多克隆抗体anti-WYSV-G-IgG。
3.权利要求1-2任一所述的方法制备得到的小麦黄条纹病毒G蛋白多克隆抗体。
4.权利要求3所述所述多克隆抗体在WYSV病毒检测中的应用。
5.根据权利要求4所述的应用,所述检测的方法为斑点免疫杂交检测。
6.小麦黄条纹病毒的斑点免疫杂交快速检测试剂盒,包括上述小麦黄条纹病毒G蛋白多克隆抗体和酶标二抗。
7.根据权利要求6所述的试剂盒,所述的酶标二抗为商品化的过氧化物酶HRP标记抗体,为羊抗兔IgG-HRP,其工作浓度为1:2000。
8.根据权利要求6所述的试剂盒,还包括阳性质控对照、杂交膜、样品研磨液、洗膜液、封闭液以及显色液;所述的阳性质控对照为小麦黄条纹病毒G基因的原核表达蛋白;所述的杂交膜为尼龙膜或者硝酸纤维素膜;所述样品研磨液为0.05M碳酸钠包被缓冲液,pH=9.6;所述洗膜液为1×PBS缓冲液,所述封闭液为含5%脱脂奶粉的PBS缓冲液,所述显色液为HRP-DAB底物显色液。
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