CN107664694A - 一种基于e2蛋白检测猪非典型瘟病毒抗体的elisa试剂盒 - Google Patents
一种基于e2蛋白检测猪非典型瘟病毒抗体的elisa试剂盒 Download PDFInfo
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- CN107664694A CN107664694A CN201710698052.4A CN201710698052A CN107664694A CN 107664694 A CN107664694 A CN 107664694A CN 201710698052 A CN201710698052 A CN 201710698052A CN 107664694 A CN107664694 A CN 107664694A
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- pig atypia
- elisa kit
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Abstract
本发明公开了一种基于E2蛋白检测猪非典型瘟病毒抗体的ELISA试剂盒,包括SEQ ID NO.1所示序列的猪非典型瘟病毒E2蛋白包被的反应板、酶标抗体、显色液、终止液、阳性血清和阴性血清。本发明首次创建了检测猪非典型瘟病毒抗体的ELISA试剂盒,而且该试剂盒可快速、特异、灵敏地检测血清中猪非典型瘟病毒的抗体,其检测血清抗体灵敏度为1:1000。本发明试剂盒使用方法简单、成本低廉,反应结果易于观察,特异性好,适用于猪非典型瘟病毒感染的监测、流行病学调查以及兽医临床样本的检测,适合大范围推广应用。
Description
技术领域
本发明涉及兽医免疫检测领域,更具体地,涉及一种基于E2蛋白检测猪非典型瘟病毒抗体的ELISA试剂盒。
背景技术
仔猪先天性震颤发生于新生仔猪,特征是仔猪在出生后观察到骨骼肌阵挛收缩,出现震颤现象,症状严重的会因饥饿而导致死亡。2015年,Hause研究小组证实了猪非典型瘟病毒(Atypical porcine pestivirus, APPV)是仔猪先天性震颤的病原。随后,德国、荷兰、奥地利等多个国家先后发现猪群存在APPV流行。华南农业大学宁章勇课题组首次发现了我国猪群APPV感染的发生,并分离了APPV GD1和GD2毒株,但是到目前为止我国猪群APPV流行的情况仍然不清楚。目前对于猪非典型瘟病毒感染尚无疫苗可以应用,所以目前缺乏检测猪非典型瘟病毒抗体的相关试剂盒。
发明内容
本发明的目的是为了克服现有技术的上述不足,提供一种基于E2蛋白检测猪非典型瘟病毒抗体的ELISA试剂盒。
为了实现上述目的,本发明是通过以下技术方案予以实现的:
一种检测猪非典型瘟病毒抗体的ELISA试剂盒,包括SEQ ID NO.1所示序列的猪非典型瘟病毒E2蛋白包被的反应板、酶标抗体、显色液、终止液、阳性血清和阴性血清。
优选地,所述猪非典型瘟病毒E2蛋白的制备方法为:将SEQ ID NO.2所示序列的猪非典型瘟病毒E2基因克隆至原核表达载体上,将得到的重组原核表达载体转化感受态细胞,筛选阳性重组菌株,诱导阳性重组菌株获得E2蛋白。
优选地,所述原核表达载体为PGEX-6P-1。
优选地,所述感受态细胞为BL21大肠杆菌。
优选地,使用IPTG诱导阳性重组菌株表达目的蛋白,IPTG终浓度为1.0 mmol/L、温度为37 ℃条件下诱导表达8小时。
优选地,所述包被方法为:10 μg/mL的E2蛋白按每孔100 μL加入反应板中,4 ℃过夜包被,次日PBST洗板3次,拍干。
优选地,所述显色液为TMB,所述终止液为浓度2 mol/L的H2SO4。
优选地,所述酶标抗体为HRP标记兔抗猪IgG。
优选地,所述阳性血清为通过猪非典型瘟病毒攻毒后的猪血清;阴性血清为健康猪血清。
优选地,诱导阳性重组菌株后,收集菌液,将菌液在12000 r/min条件下离心3min,菌体用PBS按1:10(V/V)重悬。将悬浮菌液在冰浴条件下超声破碎60次菌体破碎条件为,冰浴条件下超声破碎60次,3 sec/次,每次间隔5 sec。
与现有技术相比,本发明具有以下有益效果:
本发明首次创建了检测猪非典型瘟病毒抗体的ELISA试剂盒,且该试剂盒可快速、特异、灵敏地检测血清中猪非典型瘟病毒的抗体。具体体现在,该猪非典型瘟病毒抗体的ELISA试剂盒特异性强:对已知其他病毒的阳性血清进行交叉反应的检验,所有待检血清与APPV阴性血清的比值(P/N值)均小于2.1;重现性好:批内和批间的重复性检验,变异系数均在5 %以内;敏感度高:其检测血清抗体灵敏度为1:1000;准确性高:阳性检出率与采用荧光定量PCR方法检测的结果完全吻合。本发明试剂盒使用方法简单、成本低廉,反应结果易于观察,特异性好,适用于猪非典型瘟病毒感染的监测、流行病学调查以及兽医临床样本的检测,适合大范围推广应用。
目前对于猪非典型瘟病毒感染尚无疫苗可以应用,所以检测猪非典型瘟病毒血清抗体的存在对判定猪只的感染及采取相应的处理措施具有重要意义。由于猪非典型瘟病毒在我国是新发现的病原,目前还没有血清抗体的检测方法。本发明首次将E2重组表达蛋白(大肠杆菌原核表达蛋白)运用到APPV检测中。虽然利用真核表达系统可以将猪非典型瘟病毒E2蛋白表达的更加接近真实的天然蛋白的空间构象和功能,但是本发明还是选择了大肠杆菌表达系统,原因在于,大肠杆菌表达的重组蛋白抗原易于制备和纯化,而且表达重组蛋白抗原的宿主细胞大肠杆菌比制备APPV抗原的宿主细胞(如PK15等猪源细胞)与APPV的宿主(猪)亲缘关系更远,检测时产生非特异性的可能性也减少。
附图说明
图1为APPV GD1株的E2基因PCR扩增产物电泳结果(M:DL2000 DNA Marker,1:E2基因PCR产物)。
图2为APPV GD1株的E2基因质粒构建过程中双酶切鉴定结果(M:DL5000 DNAMarker,1:EcoR I/Xho I双酶切条带)。
图3为E2蛋白的诱导表达结果(M:蛋白质Marker,1:PGEX-6P-1空载体不诱导表达产物,2:PGEX-6P-1空载体诱导表达产物,3:PGEX-6P-1/APPV-E2重组质粒转化BL21(DE3)感受态细胞不诱导表达产物,4:PGEX-6P-1/APPV-E2重组质粒转化BL21(DE3)感受态细胞诱导表达产物)。
图4为E2蛋白Western-blot检测及纯化蛋白电泳结果(A为E2蛋白的免疫印迹检测结果,M:蛋白质Marker,1:E2蛋白免疫印迹;B为SDS-PAGE检测纯化的E2蛋白,M:蛋白质Marker,1:纯化的E2蛋白)。
具体实施方式
下面结合说明书附图和具体实施例对本发明作出进一步地详细阐述,所述实施例只用于解释本发明,并非用于限定本发明的范围。下述实施例中所使用的试验方法如无特殊说明,均为常规方法;所使用的材料、试剂等,如无特殊说明,为可从商业途径得到的试剂和材料。
实施例1
一种检测猪非典型瘟病毒抗体的ELISA试剂盒,所述试剂盒包括猪非典型瘟病毒E2蛋白包被的反应板、酶标抗体、显色液、终止液、阳性血清和阴性血清。
其中,猪非典型瘟病毒E2蛋白包被的反应板的制备方法如下:
一、猪非典型瘟病毒E2蛋白的获得:
1、克隆猪非典型瘟病毒E2基因:
根据APPV GD1株(GenBank:KX950761)的基因序列设计扩增E2基因的引物,具体引物如下:
F1:5′-GCCGTAGAATTCATGTCATGCCACAGAAGACAAG-3′(SEQ ID NO:3),下划线表示EcoRI酶切位点;
R1:5′-GCGAACCTCGAGTTAACTAGCTTCCACTTGTAG-3′(SEQ ID NO:4),下划线表示XhoI酶切位点。引物由英潍捷基(上海)贸易有限公司合成。
血清样本按照TRIzol试剂盒的说明进行总RNA提取。参照PrimeScript™ II 1stStrand cDNA Synthesis Kit说明进行反转录cDNA,后冻存于-80 ℃超低温冰箱备用。
cDNA的合成:按照PrimeScriptTM II 1st Strand cDNA Synthesis Kit的使用说明书,在0.2 mL PCR管中加入下列试剂:
在PCR仪上进行变性反应:65 ℃,5 min,冰上急冷。
在上述PCR管中加入下列反转录反应液:
PCR反应程序:42 ℃ 60 min;70 ℃ 15 min;50 ℃ 1 min。
PCR扩增:以上述得到的cDNA为模板进行扩增,PCR反应体系如下:
PCR程序为:94 ℃预变性5 min;94 ℃变性30 sec,56 ℃退火40 sec,72 ℃延伸30sec,30个循环,最后72 ℃延伸10 min。将PCR扩增产物进行琼脂糖凝胶电泳,结果(图1)显示,扩增获得753 bp的片段(不包括酶切位点、起始密码子、终止密码子和保护性碱基则为723 bp),与预期片段大小相符。根据宝生物工程(大连)有限公司的DNA凝胶回收试剂盒说明书回收目的条带,回收获得的片段命名为APPV-E2。
2、构建APPV GD1株的E2蛋白的表达载体
将回收的APPV-E2与PGEX-6P-1载体用EcoRI和XhoI进行双酶切,酶切条件为37 ℃作用1.5 h,酶切体系如下:
根据宝生物工程(大连)有限公司的DNA凝胶回收试剂盒说明书回收酶切产物,再将回收产物进行连接反应,反应条件为16 ℃连接3 h,连接体系如下:
将重组质粒转化至JM109感受态细胞,在含有Amp的LB固体培养基上培养12 h,挑选阳性菌落接种于含有Amp的LB液体培养基中,37 ℃摇床180 r/min培养12~14 h后,提取质粒,双酶切鉴定(图2),送英潍捷基(上海)贸易有限公司测序。测序正确的重组质粒命名为PGEX-6P-1/APPV-E2。测序后发现猪非典型瘟病毒E2蛋白的氨基酸序列如SEQ ID NO:1所示,猪非典型瘟病毒E2基因的核苷酸序列如SEQ ID NO:2所示。
3、APPV GD1株的E2蛋白的表达
BL21(DE3)感受态细胞购自北京鼎国生物技术有限公司;含APPV GD1株E2蛋白的表达质粒PGEX-6P-1/APPV-E2由步骤2中构建;各种分子生物学试剂均购自宝生物工程(大连)有限公司。
将步骤2获得的PGEX-6P-1/APPV-E2质粒转化BL21(DE3)感受态细胞。转化菌于Amp抗性的LB液体培养基中37 ℃、180 r/min条件下过夜培养,次日将过夜培养物与新制Amp抗性的LB液体培养基按体积比为1:100扩大培养约3小时,菌液OD600达0.6时加入IPTG至终浓度为1.0 mmol/L,并在37 ℃下诱导8 h,然后收集菌液,将菌液在12000 r/min条件下离心3min,菌体用PBS按1:10(V/V)重悬。同时以PGEX-6P-1载体转化相应表达菌作为阴性对照。
将上述制得的悬浮菌液在冰浴条件下超声破碎60次,3 sec/次,每次间隔5 sec,然后将破碎后的菌液在4 ℃、12000 r/min条件下离心10 min,弃上清,收集沉淀,沉淀用PBS按1:10(V/V)重悬。吸取80 μL上清,加入20 μL 5×SDS上样buffer,煮沸10 min后,在3000 r/min条件下常温离心30 sec,然后进行SDS-PAGE电泳检查,结果(图3)显示,通过将重组质粒PGEX-6P-1/APPV-E2转化到BL21(DE3)后,在约53 kD处出现了目的条带,而阴性对照不含有目的条带。
APPV GD1株E2蛋白Western-blot检测:按上述表达条件表达E2蛋白,然后进行SDS-PAGE,随后将电泳后的凝胶转印至PVDF膜上,80 V转印60 min,转印完毕后将PVDF膜取出,以10%脱脂奶粉于室温摇动封闭2 h,以TBST洗涤3次,每次5 min,然后以1:1000稀释的兔抗GST多克隆抗体在4 ℃过夜孵育后取出,以TBST洗涤3次,每次5 min,随后以1:5000稀释的辣根酶标记鼠抗兔IgG抗体于室温摇动孵育2 h,以TBS洗涤3次,每次5 min,洗涤后显影(图4A)。
APPV GD1株E2蛋白的纯化:将含重组质粒PGEX-6P-1/APPV-E2的BL21(DE3)感受态细胞在最适条件下表达,然后收集菌体,用PBS重悬后在冰浴条件下超声破碎60次,3 sec/次,每次间隔5 sec,然后将破碎后的菌液在4 ℃、12000 r/min条件下离心10 min,收集沉淀;然后将收集的沉淀用PBS重悬后,按1:4比例加入5×SDS上样buffer煮沸5 min,然后离心进行SDS-PAGE。电泳后,用蒸馏水反复清洗凝胶3遍,再用PBS清洗3遍。用4 ℃预冷的0.25mol/L KCl溶液避光染色10 min后,准确切下白色的含有目的蛋白的凝胶条带。研磨至碎,并于液氮快速冻融3次,期间不断研磨至粉末状,用适量PBS彻底溶解,4 ℃静置过夜。次日离心取上清即为纯化后的蛋白(图4B),将回收的上清液移到Millipore超滤管中,4000 g离心,20倍体积浓缩,调节蛋白浓度到1 mg/mL。
二:包被抗原包被反应板:用包被液将蛋白样品稀释至10 μg/mL,每孔100 μL,4℃过夜。次日PBST洗板3次,每次5 min,拍干;(2)封闭:每孔加100 μL 5%脱脂奶粉,于37 ℃保湿箱封闭2 h,同上洗板;
所述试剂盒的检测方法为:
(1)向猪非典型瘟病毒E2蛋白包被的反应板中加入待检猪血清100 μL,在37 ℃孵育2h,PBST洗板3次,每次5 min,拍干;
(2)加入工作浓度的兔抗猪酶标抗体100 μL,在37 ℃条件下孵育1 h,同上洗板;
(3)加入100 μL TMB显色液,室温避光反应10 min;
(4)加入终止液(2 mol/L H2SO4),50 μL/孔;
(5)酶标仪以双波长形式读取OD450-OD630值。
实施例2
检测猪非典型瘟病毒抗体的ELISA试剂盒的特异性检验
利用实施例1描述的方法对已知的各种病毒的阳性血清进行交叉反应的检验,以确定该方法的特异性。结果表明,利用该方法对猪瘟病毒(CSFV)、猪伪狂犬病毒(PRV)、猪繁殖与呼吸障碍综合征病毒(PRRSV)、猪流感病毒(SIV)及圆环病毒(PCV)标准阳性血清进行检验时,所有待检血清与APPV阴性血清的比值(P/N值)均小于2.1(表1),表明该方法具有很好的特异性。
表1特异性检验
实施例3
检测猪非典型瘟病毒抗体的ELISA试剂盒的重复性检验
利用实施例1描述的方法进行重复性检验,结果表明,利用该实验方法对同一样本进行批内和批间的重复性检验,变异系数均在5 %以内,说明该方法具有很高的重复性(表2)。
表2重复性检验
实施例4
检测猪非典型瘟病毒抗体的ELISA试剂盒的敏感性检验
利用实施例1描述的方法进行敏感性检验,对已知的血清进行10倍的倍比稀释,结果表明待检血清进行1:10、1:100和1:1000倍稀释时,结果判断为阳性;进行1:10000及1:100000倍稀释时,结果为阴性(表3)。
表3敏感性检验
实施例5
检测猪非典型瘟病毒抗体的ELISA试剂盒的适用性检验
利用实施例1描述的方法对目前已经发现的感染中国猪群的猪非典型瘟病毒的五个毒株(APPV GD1、GD2、GD3、GD和CH-GX毒株)感染的猪血清进行检测。结果表明,五种毒株感染猪血清均为阳性,说明本发明对于不同毒株均有很好的检测效果。
实施例6
检测猪非典型瘟病毒抗体的ELISA试剂盒的临床样品的检验
利用实施例1描述的方法对从广东省的收集的100份猪血清进行APPV阳性血清的检测,并采用荧光定量PCR方法进行比对。
结果表明,检测100份猪血清,APPV阳性血清的比例为7%,ELISA试剂盒和荧光定量PCR方法均完全检测了阳性血清,这两种方法之间的比对的检测重合率为100%。
综上可见,利用本发明制得的包被E2蛋白的ELISA试剂盒检测APPV血清抗体具有特异性强、重现性好、敏感度高和准确性高的优点,可用于兽医临床样品的检测。
SEQUENCE LISTING
<110> 华南农业大学
<120> 一种基于E2蛋白检测猪非典型瘟病毒抗体的ELISA试剂盒
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Claims (7)
1.一种检测猪非典型瘟病毒抗体的ELISA试剂盒,其特征在于,包括SEQ ID NO.1所示序列的猪非典型瘟病毒E2蛋白包被的反应板、酶标抗体、显色液、终止液、阳性血清和阴性血清。
2.根据权利要求1所述的检测猪非典型瘟病毒抗体的ELISA试剂盒,其特征在于,所述猪非典型瘟病毒E2蛋白的制备方法为:将SEQ ID NO.2所示序列的猪非典型瘟病毒E2基因克隆至原核表达载体上,将得到的重组原核表达载体转化感受态细胞,筛选阳性重组菌株,诱导阳性重组菌株获得E2蛋白。
3.根据权利要求2所述的猪非典型瘟病毒抗体的ELISA试剂盒,其特征在于,所述原核表达载体为PGEX-6P-1。
4.根据权利要求2所述的猪非典型瘟病毒抗体的ELISA试剂盒,其特征在于,所述感受态细胞为BL21大肠杆菌。
5.根据权利要求2所述的猪非典型瘟病毒抗体的ELISA试剂盒,其特征在于,使用IPTG诱导阳性重组菌株表达目的蛋白,IPTG终浓度为1.0 mmol/L、温度为37 ℃条件下诱导表达8小时。
6.根据权利要求1所述的猪非典型瘟病毒抗体的ELISA试剂盒,其特征在于,所述包被方法为:10 μg/mL的E2蛋白按每孔100 μL加入反应板中,4 ℃过夜包被,次日PBST洗板3次,拍干。
7.根据权利要求1所述的猪非典型瘟病毒抗体的ELISA试剂盒,其特征在于,所述显色液为TMB,所述终止液为浓度2 mol/L的H2SO4。
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