CN116754766A - 检测猫杯状病毒的试剂盒 - Google Patents
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- Peptides Or Proteins (AREA)
Abstract
本发明涉及生物技术检测领域,具体涉及基于检测猫杯状病毒的试剂盒。本发明提供了双抗体夹心酶联免疫法的检测猫杯状病毒的试剂。本发明通过噬菌体展示技术,筛选获得基于VP1蛋白作为猫杯状病毒重要的免疫原性蛋白的重组抗体,并将其用于FCV的ELISA检测试剂盒,具有特异性高、灵敏度高和稳定性好等优势。
Description
技术领域
本发明涉及生物技术检测领域,具体涉及基于检测猫杯状病毒的试剂盒。
背景技术
猫杯状病毒感染是猫病毒性呼吸道传染病,主要表现为上呼吸道症状,即精神沉郁、浆液性和粘液性鼻漏、结膜炎、口腔炎、气管炎、支气管炎,伴有双相热。该病自然条件下,仅猫科动物对此病毒易感,常发生于6~8日龄猫。主要传染源为病猫和带毒猫。患猫在急性期可随分泌物和***物排出大量病毒,直接传染易感猫。带毒猫经治疗,症状可消失,但长期排毒,是最危险的传染源。并且,FCV的强度变异株FCV-VSD导致动物全身性感染、死亡率增高,
目前,猫杯状病毒感染的诊断是通过根据病史、临床症状和流行特点进行诊断。临床确诊困难时可进行实验室诊断;刮取病理组织物荧光抗体染色,监测抗原的存在。为了提高猫杯状病毒的检测效果,降低对野生动物和宠物行业带来重大的经济损失,进一步研发检测猫杯状病毒的试剂盒仍是本领域的研究热点。
发明内容
有鉴于此,本发明要解决的技术问题在于提供猫杯状病毒的双抗体夹心酶联免疫法检测试剂盒,该具有良好的灵敏性、特异性和稳定性。
本发明提供的检测猫杯状病毒的试剂,包括包被于固相介质的捕获抗体和标记有生物标记或化学标记的检测抗体。
其中,捕获抗体或检测抗体可以相同也可以不同。
所述捕获抗体或检测抗体中:
重链可变区的CDR1的氨基酸序列为GXH1XH2LSXH3YXH4,其中XH1选自I或F;XH2选自D或S,XH3选自T或S,XH4选自A或V或Y或T;
重链可变区的CDR2的氨基酸序列为IXH5XH6XH7XH8XH9XH10,其中,XH5选自N或Y;XH6选自T或G;XH7选自D或S或T或G;XH8选自G或A;XH9选自S或N或Y或I;XH10选自A或T或R;
重链可变区的CDR3的氨基酸序列为XH11XH12XH13XH14XH15XH16XH17XH18XH19GXH20XH21XH2 2XH23XH24XH25,其中,XH11选自A或无;XH12选自R或无;XH13选自A或无;XH14选自G或无;XH15选自A或P或无;XH16选自G或R或无;XH17选自R或S或E或G或无;XH18选自G或Y;XH19选自R或V或I或无;XH20选自F或L或V或Y或I;XH21选自P或T或无;XH22选自D或A或N;XH23选自L或Y或F;XH24选自D或无;XH25选自P或S或无;
轻链可变区的CDR1的氨基酸序列为XL1SXL2XL3XL4XL5XL6XL7,其中,XL1选自Q或E;XL2选自V或I;XL3选自Y或无;XL4选自S或D或G或N;XL5选自D或S或N;XL6选自W或N;XL7选自Y或E或无;
轻链可变区的CDR2的氨基酸序列为XL8XL9S,其中,XL8选自D或Y或A;XL9选自V或A;
轻链可变区的CDR3的氨基酸序列为XL10XL11XL12XL13XL14YXL15XL16XL17XL18XL19XL20XL21,其中,XL10选自C或无;XL11选自C或无;XL12选自A或S或L;XL13选自G或N;XL14选自A或S或G;XL15选自S或D;XL16选自G或D或无;XL17选自N或D;XL18选自I或V或A;XL19选自Y或D;XL20选自T或G或A或S或N;XL16选自L或A或无。
一些实施例中,
所述捕获抗体中:
重链可变区的CDR1的氨基酸序列如SEQ ID NO:29、SEQ ID NO:35、SEQ ID NO:41、SEQ ID NO:47和/或SEQ ID NO:53所示中的至少一种;
重链可变区的CDR2的氨基酸序列如SEQ ID NO:30、SEQ ID NO:36、SEQ ID NO:42、SEQ ID NO:48和/或SEQ ID NO:54所示中的至少一种;
重链可变区的CDR3的氨基酸序列如SEQ ID NO:31、SEQ ID NO:37、SEQ ID NO:43、SEQ ID NO:49和/或SEQ ID NO:55所示中的至少一种;
轻链可变区的CDR1的氨基酸序列如SEQ ID NO:32、SEQ ID NO:38、SEQ ID NO:44、SEQ ID NO:50和/或SEQ ID NO:56所示中的至少一种;
轻链可变区的CDR2的氨基酸序列如SEQ ID NO:33、SEQ ID NO:39、SEQ ID NO:45、SEQ ID NO:51和/或SEQ ID NO:57所示中的至少一种;
轻链可变区的CDR3的氨基酸序列如SEQ ID NO:34、SEQ ID NO:40、SEQ ID NO:46、SEQ ID NO:52和/或SEQ ID NO:58所示中的至少一种;
所述检测抗体中:
重链可变区的CDR1的氨基酸序列如SEQ ID NO:29、SEQ ID NO:35、SEQ ID NO:41、SEQ ID NO:47和/或SEQ ID NO:53所示中的至少一种;
重链可变区的CDR2的氨基酸序列如SEQ ID NO:30、SEQ ID NO:36、SEQ ID NO:42、SEQ ID NO:48和/或SEQ ID NO:54所示中的至少一种;
重链可变区的CDR3的氨基酸序列如SEQ ID NO:31、SEQ ID NO:37、SEQ ID NO:43、SEQ ID NO:49和/或SEQ ID NO:55所示中的至少一种;
轻链可变区的CDR1的氨基酸序列如SEQ ID NO:32、SEQ ID NO:38、SEQ ID NO:44、SEQ ID NO:50和/或SEQ ID NO:56所示中的至少一种;
轻链可变区的CDR2的氨基酸序列如SEQ ID NO:33、SEQ ID NO:39、SEQ ID NO:45、SEQ ID NO:51和/或SEQ ID NO:57所示中的至少一种;
轻链可变区的CDR3的氨基酸序列如SEQ ID NO:34、SEQ ID NO:40、SEQ ID NO:46、SEQ ID NO:52和/或SEQ ID NO:58所示中的至少一种;
更进一步的,
所述检测抗体中:
轻链三个CDR区的氨基酸序列分别如SEQ ID NO:32~34所示;重链三个CDR区的氨基酸序列分别如SEQ ID NO:29~31中任一项所示;
所述捕获抗体中:
轻链三个CDR区的氨基酸序列分别如SEQ ID NO:50~52所示;重链三个CDR区的氨基酸序列分别如SEQ ID NO:47~49中任一项所示。
更具体的,
所述检测抗体重链可变区如SEQ ID NO:1所示;轻链可变区如SEQ ID NO:2所示;重链与轻链之间以linker连接,linker序列如SEQ ID NO.11所示。
所述捕获抗体重链可变区如SEQ ID NO:7所示;轻链可变区如SEQ ID NO:8所示;重链与轻链之间以linker连接,linker序列如SEQ ID NO.11所示。
本发明中,所述化学标记为同位素、免疫毒素和/或化学药物;所述生物标记为生物素、亲和素或酶标记。所述酶标记优选为辣根过氧化物酶或碱性磷酸酶。所述免疫毒素优选为黄曲霉毒素、白喉毒素、绿脓杆菌外毒素、蓖麻毒蛋白、相思子毒蛋白、槲寄生凝集素、蒴莲根毒素、PAP、造草素、白树毒素或丝瓜毒素。
本发明中,所述固相介质或半固体介质是指本发明所述的重组抗体、经标记的重组抗体能够附着至其上的任何支持物,包括但不限于硝酸纤维素膜、聚偏二氟乙烯(PVDF)膜、iPDMS芯片、微孔板、聚苯乙烯平板、微粒、微载体、凝胶等。
一些实施例中,所述捕获抗体包被于酶标板,所述检测抗体标记有HRP。
一些实施例中,所述捕获抗体与检测抗体的质量比为10:1。
本发明所述的检测试剂中,还包括包被缓冲液、洗涤液、封闭液、TMB显色液和终止液;其中:
所述包被缓冲液为pH7.2~7.4的PBS缓冲液,
所述洗涤液为含有质量分数0.05%Tween-20的PBST溶液。
所述封闭液为质量分数为2%的脱脂奶粉溶液;
所述终止液为2mol/L H2SO4溶液。
进一步的,本发明还提供了猫杯状病毒的检测方法,其包括以如前所述的检测试剂对样品进行检测。
本发明所述的检测方法可以是诊断目的的,也可以是非诊断目的的,本发明对此不做限定。其中,以诊断为目的的检测方法中,样品来自于动物体,非诊断目的的检测方法中,样品来自于环境,例如食物、水、实验室培养物或者器物表面的拭子等。
本发明中,所述检测方法具体包括:以捕获抗体对样品进行捕获后,加入检测抗体进行孵育;所述捕获的条件为37℃,1h;所述孵育的条件为37℃,1h;
本发明中,所述孵育后,还包括TMB显色、终止显色和读取450nm吸光度的步骤。
本发明所述的检测方法可以为定性检测的方法。
所述定性检测根据是否产生荧光判断结果,产生荧光的样品为阳性样品。
本发明提供了双抗体夹心酶联免疫法的检测猫杯状病毒的试剂。本发明通过噬菌体展示技术,筛选获得基于VP1蛋白作为猫杯状病毒重要的免疫原性蛋白的重组抗体,并将其用于FCV的ELISA检测试剂盒,具有特异性高、灵敏度高和稳定性好等优势。
附图说明
图1示本发明实施例提供的FCV VP1-scFv噬菌体文库的构建和免疫文库的多样性分析,其中,A为新西兰兔免疫4次的抗体效价;B为扩增条带为VL基因文库和VH基因文库,M:Marker;1:VH,2:VL,3:VL-linker-VH目的条带;
图2示本发明实施例提供的高亲和力FCV VP1-scFv的筛选,其中,A为电转后PCR鉴定结果;B为第一轮噬菌体展示筛选后的抗体文库多样性;C为第二轮噬菌体展示筛选后的抗体文库多样性;D为第二轮噬菌体ELISA确定筛选抗原FCV VP1高特异性结合的scFv抗体;
图3示本发明实施例提供的Ab-FCV VP1的表达与亲和力验证,其中,A为Ab-FCVVP1的SDS-PAGE结果,M:Marker,1:Ab-FCV VP1-1,2:Ab-FCV VP1-2,3:Ab-FCV VP1-3,4:Ab-FCV VP1-4,5:Ab-FCV VP1-5;B为Ab-FCV VP1-1抗体与抗原FCV VP1结合的EC50;C为Ab-FCVVP1-2抗体与抗原FCV VP1结合的EC50;D为Ab-FCV VP1-3抗体与抗原FCV VP1结合的EC50;E为Ab-FCV VP1-4抗体与抗原FCV VP1结合的EC50;F为Ab-FCV VP1-5抗体与抗原FCV VP1结合的EC50;
图4示本发明实施例提供的Ab-FCV VP1-4重组抗体与与FCV病毒的间接免疫荧光实验结果。
图5示本发明实施例提供的Ab-FCV VP1-4重组抗体与FCV、FPV、FHV病毒结合的特异性验证。
图6示本发明实施例提到的含有兔抗体骨架的载体pTT5-V5与pTT5-V6质粒图。
具体实施方式
本发明提供了基于VP1蛋白的检测猫杯状病毒的抗体及其应用,本领域技术人员可以借鉴本文内容,适当改进工艺参数实现。特别需要指出的是,所有类似的替换和改动对本领域技术人员来说是显而易见的,它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述,相关人员明显能在不脱离本发明内容、精神和范围内对本文的方法和应用进行改动或适当变更与组合,来实现和应用本发明技术。
单链抗体FCV VP1-scFv-1和重组抗体Ab-FCV VP1-1的重链可变区氨基酸序列:QSVKESGGRLVTPGTPLTITCTVSGIDLSTYAMSWVRQDPGKGLEYIGFINTDGSAYYAS WAKGRFTISKTSSTTVDLKMTSLTTEDTATYFCGRGFDLWGQGTLVTVSS(SEQ ID NO:1);
H1-CDR1:GIDLSTYA(SEQ ID NO:29);
H1-CDR2:INTDGSA(SEQ ID NO:30);
H1-CDR3:GRGFDL(SEQ ID NO:31);
单链抗体FCV VP1-scFv-1和重组抗体Ab-FCV VP1-1的轻链可变区氨基酸序列:DGMMTQTPSPVSAAVGGTVTIKCQSSQSVYSDWLSWFQQKPGQPPKRLIYDASTLASG VPSRFKGSGSGTQFTLTISDLECDDAATYYCAGAYSGNIYTFGGGTEVVVK(SEQ ID NO:2);
L1-CDR1:QSVYSDW(SEQ ID NO:32);
L1-CDR2:DAS(SEQ ID NO:33);
L1-CDR3:AGAYSGNIYT(SEQ ID NO:34);
单链抗体FCV VP1-scFv-2和重组抗体Ab-FCV VP1-2的重链可变区氨基酸序列:QSLEESGGRMVTPGTPLTLTCTVSGFSLSSYVVSWVRQAPGKGLEWIGIIYTSGNTYYAN WAKGRFTISKTSTTVDLKITSPTTEDTATYFCGRGGLPALWGQGTLVTVSS(SEQ ID NO:3);
H2-CDR1:GFSLSSYV(SEQ ID NO:35);
H2-CDR2:IYTSGNT(SEQ ID NO:36);
H2-CDR3:GRGGLPAL(SEQ ID NO:37);
单链抗体FCV VP1-scFv-2和重组抗体Ab-FCV VP1-2的轻链可变区氨基酸序列:DGVLTQTPSSTSAAVGGTVTISCQSSESVYDNNYLSWYQQKPGQPPKVLIYYVSTLASGV PSRFKGSGSGTQFTLTISDLECDDAATYYCAGAYSGNIYGFGGGTEVVVK(SEQ ID NO:4);
L2-CDR1:ESVYDNNY(SEQ ID NO:38);
L2-CDR2:YVS(SEQ ID NO:39);
L2-CDR3:AGAYSGNIYG(SEQ ID NO:40);
单链抗体FCV VP1-scFv-3和重组抗体Ab-FCV VP1-3的重链可变区氨基酸序列:QSVEESGGRLVTPGTPLTLTCTVSGFSLSTYAMIWVRQAPGKGLEWIGIIYGTAYRYYASW AKGRFTISKTSTTVDLKITSPTTEDTATYFCARSYVGVTAYDPVGQGTLVTVSS(SEQ ID NO:5);
H3-CDR1:GFSLSTYA(SEQ ID NO:41);
H3-CDR2:IYGTAYR(SEQ ID NO:42);
H3-CDR3:ARSYVGVTAYDP(SEQ ID NO:43);
单链抗体FCV VP1-scFv-3和重组抗体Ab-FCV VP1-3的轻链可变区氨基酸序列:DGVPTQTPSSVSAAVGGTVTISCQSSQSVYSNWLSWFQQKPGQPPKRLIYDASTLTSGVS SRFKGSGSGTQFILTISDLECDDAATYYCAGAYSGNIYAFGGGTEVVVK(SEQ ID NO:6);L3-CDR1:QSVYSNW(SEQ ID NO:44);
L3-CDR2:DAS(SEQ ID NO:45);
L3-CDR3:AGAYSGNIYA(SEQ ID NO:46);
单链抗体FCV VP1-scFv-4和重组抗体Ab-FCV VP1-4的重链可变区氨基酸序列:QSVEESGGRLVTPGTPLTLTCTVSGFSLSSYTMSWVRQAPGKGLEWIGIIYTGGITYYASW AKGRFTISRTSTTVDLKITSPTTEDTATYFCAREGVGYAFDSWGPGTLVTVSS(SEQ ID NO:7);
H4-CDR1:GFSLSSYT(SEQ ID NO:47);
H4-CDR2:IYTGGIT(SEQ ID NO:48);
H4-CDR3:AREGVGYAFDS(SEQ ID NO:49);
单链抗体FCV VP1-scFv-4和重组抗体Ab-FCV VP1-4的轻链可变区氨基酸序列:DGMMTQTPSPVSATVGGTVTIKCQASESIGSWLAWYQQKPGQRPKLLIYAASTLASGVP SRFSGSGSGTEFTLAISGVECADAATYYCQQSNSYSDVDSLFGGGTEVVVK(SEQ ID NO:8);
L4-CDR1:ESIGSW(SEQ ID NO:50);
L4-CDR2:AAS(SEQ ID NO:51);
L4-CDR3:QQSNSYSDVDSL(SEQ ID NO:52);
单链抗体FCV VP1-scFv-5和重组抗体Ab-FCV VP1-5的重链可变区氨基酸序列:QSLEESGGRLVTPGTPLTLTCTASGFSLSSYYMTWVRQAPGKGLEWIGIIYGTGYTYYAS WAKGRFTISKTSTTVDLKMTSLTTEDTATYFCARAGPGGGIGINLWGQGTLVTVSS(SEQ ID NO:9);
H5-CDR1:GFSLSSYY(SEQ ID NO:53);
H5-CDR2:IYGTGYT(SEQ ID NO:54);
H5-CDR3:ARAGPGGGIGINL(SEQ ID NO:55);
单链抗体FCV VP1-scFv-5和重组抗体Ab-FCV VP1-5的轻链可变区氨基酸序列:DGVMTQTESPVSAAVGSTVTINCQSSQSVYNNNELSWYQQKPGQPPKLLIYYASTLASG VSSRFKGSGSGTQFTLTISGVQCDDAATYYCLGGYDDDADNAFGGGTEVVVK(SEQ ID NO:10);
L5-CDR1:QSVYNNNE(SEQ ID NO:56);
L5-CDR2:YAS(SEQ ID NO:57);
L5-CDR3:LGGYDDDADNA(SEQ ID NO:58);
连接肽linker的氨基酸序列:GGGGSGGGGGGSSRSS(SEQ ID NO:11);
VH-scFv-F1:5’-ggtgggggtggttcctctagatcttcccagtcgktggaggagtcc-3’(SEQ IDNO:12);
VH-scFv-F2:5’-ggtgggggtggttcctctagatcttcccagwcagtgaaggagtcc-3’(SEQ IDNO:13);
VH-scFv-F3:5’-ggtgggggtggttcctctagatcttcccagtcgctggrggagtcc-3’(SEQ IDNO:14);
VH-scFv-F4:5’-ggtgggggtggttcctctagatcttcccagtcggtggaggagtcc-3’(SEQ IDNO:15);
VH-scFv-R1:5’-aggaggctattggccggcctggcctgargagayggtgaccagggtgcc-3’(SEQID NO:16);
VH-scFv-R2:5’-aggaggctattggccggcctggccgcagcagggggccagtgggaagactgac-3’(SEQ ID NO:17);
VL-scFv-F1:5’-aaaagaggcccaggcggccgagctcgtgmtgacccagactcca-3’(SEQ IDNO:18;
VL-scFv-F2:5’-aaaagaggcccaggcggccgagctcgatmtgacccagactcca-3’(SEQ IDNO:19);
VL-scFv-F3:5’-aaaagaggcccaggcggccgcycaagtgctgacccag-3’(SEQ ID NO:20);
VL-scFv-F4:5’-aaaagaggcccaggcggccgcccwagtgatgacccag-3’(SEQ ID NO:21);
VL-scFv-R1:5’-ggaagatctagaggaaccacccccaccaccgcccgagccaccgccacctttgatttccacattggtgcc-3’(SEQ ID NO:22);
VL-scFv-R2:5’-ggaagatctagaggaaccacccccaccaccgcccgagccaccgccacctaggatctccagctcggtccc-3’(SEQ ID NO:23);
重叠PCR-FP:5’-gaggaggaaaaagaggcccaggcggcc-3’(SEQ ID NO:24);
重叠PCR-RP:5’-agaggaggctattggccggcctggcc-3’(SEQ ID NO:25);
pComb3XSS-FP:5’-gctggtttcgctaccgtggcccaggcggcc-3’(SEQ ID NO:26);
pComb3XSS-RP:5’-gtgatggtgatggtgctggccggcctggcc-3’(SEQ ID NO:27);
FCV VP1蛋白的氨基酸序列:
MCSTCANVLKYYDWDPHFRLVVNPNKFLSVGFCDNPLLCCYPELLPEFGTVWDCNQSPLQIYLESILGDDDWSSTHEAIDPVVPPMHWDEAGKIFQPHPGVLMHYIVGEVAKAWDPNLPLFRLEADDGSITTPEQGTVVGGVIAEPSAQMAVAADTATGKSVDSEWEAFFSFHTSVNWGTSETQGKILFKQSLGPLLNPYLEHLAKLYVAWSGSIEVRFSISGSGVFGGKLAAIVVPPGVDPVQSTSMLQYPHVLFDARQVEPVIFTIPDLRSTLYHLMSDTDTTSLVIMVYNDLINPYARDSNSSGCIVTVETKPGPDFRFHLLKPPGSMLTHGSVPSDLIPKNSSLWIGNRHWTDITDFIIRPFVFQANRHFDFNQETAGWSTPRFRPITVTISQKDGAKLGIGIATDCIVPGIPDGWPDTTIPSRLTPAGDYAITSGDNSDIATKQQYETADEIKNNTNFKSMYICGALQRAWGDKKVSNTGFITTATISDNQLVPSNVIDQTKIAVFQDNRVSKEVQTSDVTLAILGYTGIGEEAVGADRDKVVRISVLPETGARGGNHPIFYKNSMKLGYVVGSIDVFNSQILHTSRQLSLNNYLLAPDSLAVYRIIDANGSWFDVGIDSDGFSFVGVSHIGKLEFPLSASYMGIQLAKIRLASNIRSNMVKL(SEQ IDNO:28)。
菌株、质粒、试验动物与试剂:成年新西兰兔购自安徽定远县永康清源养殖场;FCVVP1蛋白为本实验室保存(氨基酸序列为SEQ ID NO.28);FCV病毒由本实验室保存(GenBank:KT000003.1);含有兔抗体骨架的载体pTT5-V5与pTT5-V6质粒为本实验室保存(图5);HRP标记的重组蛋白A购自生工生物工程(上海)股份有限公司;HRP标记的鼠抗M13K07噬菌体购自成都阿帕克生物科技有限公司;HRP标记的羊抗兔IgG购自北京梅科万德生物科技有限公司;pComb3XSS质粒、大肠杆菌DH5α感受态细胞购自山东赫兹生物科技技术有限公司;M13K07辅助噬菌体、大肠杆菌ER2738感受态细胞购自南京禾鸣英固生物科技有限公司;SDS-PAGE蛋白胶试剂盒购自雅酶生物科技技术有限公司;青霉素-链霉素溶液购自GE公司(用于细胞培养);弗氏佐剂购自Sigma公司;ClonExpress II One Step CloningKit购自南京诺唯赞生物科技股份有限公司;Premix taq、Plasmid mini kit、GelExtraction及转染试剂均购自TaKaRa公司。
本发明采用的试材皆为普通市售品,皆可于市场购得。
下面结合实施例,进一步阐述本发明:
实施例1动物免疫
实验选用健康成年新西兰兔,免疫前静脉采血,分离血清作为阴性对照。取1×105TCID50/ml的FCV病毒在新西兰兔的后颈部注射,每周免疫一次,共免疫4次,在第0、7、14、21天的每次免疫前静脉采血0.5mL分离血清保存。第4次免疫后7天(即第28天),静脉采血0.5mL分离血清保存。ELISA结果显示第一次免疫血清抗体滴度显著上升,二免抗体滴度大于5×104,三免后达105,四免血清提滴度达1.5×105,与免疫前的血清抗体效价相比,均有显著提高(图1中A)。第28天处死新西兰兔,收集血液0.5mL分离血清保存,解剖并取出脾脏,用于淋巴细胞分离。
实施例2淋巴细胞总RNA的抽提及抗体轻重链基因的扩增
用Trizol试剂提取淋巴细胞总RNA,以其为模板用Oligo(dT)为引物反转录合成cDNA。根据兔抗序列的轻重链框架区设计引物序列组合(引物序列为SEQ ID NO.12~SEQID NO.23),进行PCR扩增抗体VH和VL的编码区。PCR反应条件为:95℃预变性体5min,95℃变性30s,55℃退火30s,72℃延伸60s,35个循环后,72℃再延伸5min。PCR产物经1%琼脂糖凝胶电泳验证在350bp左右有目的条带,纯化回收备用(图1中B)。
实施例3FCV VP1-scFv抗体库的构建
以纯化的VH和VL为模板,经重叠PCR后在VH和VL之间***linker(序列为SEQ IDNO.11),合成scFv片段VL-linker-VH,序列两端含有sfiI限制性酶切位点。
重叠延伸第一轮PCR:不加引物,98℃变性1min,45℃退火30s,72℃延伸1min,重复10个循环,最后72℃作用5min。加入引物重叠PCR-FP、重叠PCR-RP(SEQ ID NO.24~SEQ IDNO.25),98℃变性30s,然后58℃复性30s,72℃延伸1min,30个循环后72℃再延伸5min。PCR产物经1%琼脂糖凝胶电泳以胶回收的方式回收扩增产物,条带约800bp(图1中B),保存于-20℃。重叠PCR引物序列为SEQ ID NO.24~SEQ ID NO.25。
将胶回收后的PCR产物和pComb3XSS载体分别使用限制性内切酶sfiI在50℃条件下酶切30min,酶切产物经1%琼脂糖凝胶电泳验证后柱回收纯化并用T4 DNA连接酶连接得到FCV VP1-scFv-pComb3XSS文库。连接产物转化至ER2738感受态细胞中,用四环素和氨苄青霉素抗性2YT(2×Yeast extract and Tryptone)固体培养基筛选阳性克隆。随机挑选文库中的20个单克隆进行PCR鉴定(引物序列为SEQ ID NO.26~SEQ ID NO.27),计算得抗体库重组率为60%(图2中A)。将固体培养基上的所有克隆用2YT培养基洗下,4℃保存。
实施例4噬菌体的扩增
从上述用2YT培养基洗下的所有克隆的文库菌液中取2mL,接种于50mL 2YT液体培养基中,37℃200rpm活化至OD600达到0.8~1.0,加入终浓度为1×1012pfu/mL的M13K07辅助噬菌体进行侵染,静置30min后加入卡那霉素(终浓度为50μg/mL),在30℃200rpm培养过夜。次日10000rpm4℃离心20min,保留上清液并向其中加入1/2体积的PEG8000,在冰上静置2h使噬菌体沉淀。10000rpm 4℃离心20min,弃上清。用5mL PBS使沉淀重悬,并加入3mLPEG8000复沉,重复离心操作后,用4mL PBS重悬沉淀并过滤除菌后,保存于4℃备用。
实施例5FCV VP1-scFv筛选
提前将FCV VP1蛋白生物素化。扩增的FCV VP1-scFv文库噬菌体、标记了FCV VP1蛋白的Dynabeads M-280磁珠和2%脱脂奶粉封闭液混合后,25℃±5℃孵育1h,磁珠用PBST洗涤5~10次。用0.1M Glycine-HCl(pH=2.0)洗脱磁珠表面噬菌体,洗脱后用1MTris中和至pH为7.0。用洗脱后中和的噬菌体感染对数生长期的ER2738感受态细胞,在2YT固体培养基上培养富集,收获菌落进行下一轮筛选。
重复上述实验两次。从两轮富集亲和筛选文库中分别随机挑选20个单克隆进行PCR验证,结果显示所建FCV VP1-scFv文库第一轮淘选测得阳性率达70%,第二轮淘选测的阳性率达95%(图2中B和C)。
实施例6Phage ELISA
将FCV VP1抗原按每孔50μg/mL的浓度包被酶标板,4℃过夜。用含有0.05%Tween-20的PBST溶液洗涤5次,弃PBST,加入2%脱脂奶粉封闭液,37℃封闭1h。用PBST溶液洗涤5次,拍干孔中残留液体,将待筛阳性克隆抗体上清和2%脱脂奶粉封闭液按照3:2比例混合,25℃±5℃静置10min去干扰化处理后,加入酶标板中,37℃孵育1h。用PBST溶液洗涤5次,拍干孔中残留液体,加入标记HRP的鼠抗M13K07抗体(用封闭液1:3000稀释),37℃孵育1h。用PBST溶液洗涤5次,拍干孔中残留液体,加入TMB显色液,避光静置15min后,加入2mol/LH2SO4终止显色,读取450nm处吸光度(表1)。如图2中D显示,选择OD450值高的12株抗体测序,最终获得5株序列不同的抗体用于后续实验。
表1. 96个含Ab-FCV VP1-1抗体的噬菌体与抗原FCV VP1的亲和力检测
0.7499 | 0.9347 | 1.384 | 1.5467 | 1.4948 | 0.4876 | 1.3691 | 0.8919 | 1.3492 | 0.7921 | 1.8346 | 0.1093 |
1.2354 | 0.8379 | 1.0397 | 1.9346 | 1.2378 | 1.6392 | 1.7381 | 0.6732 | 1.8279 | 0.329 | 0.9218 | 0.5369 |
1.8796 | 1.2938 | 1.1289 | 1.0729 | 1.5386 | 1.5936 | 1.3918 | 2.6924 | 1.3926 | 0.6827 | 0.612 | 0.7361 |
0.5389 | 0.5379 | 0.6948 | 0.5379 | 1.5097 | 1.0527 | 1.3094 | 1.261 | 0.7912 | 1.0724 | 0.3719 | 1.7362 |
1.9346 | 1.3897 | 0.7124 | 0.839 | 0.973 | 0.7943 | 0.9682 | 1.0739 | 0.9368 | 1.2681 | 0.2768 | 0.9824 |
2.3469 | 0.8967 | 1.5432 | 1.549 | 1.3032 | 0.5392 | 1.3768 | 1.5927 | 1.0329 | 0.2917 | 1.0258 | 0.6381 |
1.3672 | 1.8631 | 1.7362 | 1.4739 | 0.5495 | 1.7935 | 2.0361 | 0.6719 | 0.5928 | 0.1368 | 1.2773 | 0.0431 |
0.9256 | 1.2395 | 0.3718 | 1.7935 | 1.0748 | 1.6927 | 1.8531 | 0.8529 | 0.3191 | 0.7183 | 0.6263 | 0.0482 |
实施例7FCV VP1重组抗体Ab-FCV VP1的重组、表达及纯化
扩增并纯化5株单链抗体FCV VP1-scFv的VH和VL序列,用EcoRI和HindIII限制性内切酶对含有兔抗体骨架的pTT5-V5与pTT5-V6载体进行双酶切,经同源重组得到抗体表达质粒VH-pTT5-V5-1~VH-pTT5-V5-5和VL-pTT5-V6-1~VL-pTT5-V6-5。将重组质粒VH-pTT5-V5-1~VH-pTT5-V5-5和VL-pTT5-V6-1~VL-pTT5-V6-5一一对应,通过PEI转染至HEK293f细胞中进行真核表达。48h后10000rpm 4℃离心20min收集培养液上清,利用Protein A亲和层析柱纯化抗体,最终用PBS溶液保存融合蛋白,分别命名为Ab-FCV VP1-1、Ab-FCV VP1-2、Ab-FCV VP1-3、Ab-FCV VP1-4、Ab-FCV VP1-5。
将纯化后的样品加入还原型蛋白上样缓冲液,变性后进行SDS-PAGE电泳检测,结果如图3中A所示,抗体的重链和轻链之间的二硫键被还原,在50kDa和25kDa左右处检测到目的条带,与预期结果一致。
实施例8间接ELISA法检测重组抗体Ab-FCV VP1与FCV VP1抗原的结合
采用间接ELISA方法测定Ab-FCV VP1与抗原FCV VP1的亲和力。酶标板中加入抗原孵育,4℃过夜,用2%脱脂奶粉封闭液37℃封闭2h。用2%脱脂奶粉将抗体Ab-FCV VP1稀释12个梯度加入孔中,37℃孵育1h。加入HRP标记羊抗兔IgG二抗,用TMB进行显色反应,并在酶标仪中检测450nm波长处吸光度(表2~表6)。
表2Ab-FCV VP1-1与抗原FCV VP1结合的EC50
表3Ab-FCV VP1-2与抗原FCV VP1结合的EC50
表4 Ab-FCV VP1-3与抗原FCV VP1结合的EC50
表5 Ab-FCV VP1-4与抗原FCV VP1结合的EC50
表6 Ab-FCV VP1-5与抗原FCV VP1结合的EC50
检测上述抗体Ab-FCV VP1结合效果如图3B~3F所示,量效曲线与回归模型成功拟合,Ab-FCV VP1的5株抗体都能够有效结合FCV VP1蛋白,抗体Ab-FCV VP1-4的亲和力最高,与重组表达FCV VP1蛋白的结合的EC50为1.243×10-1μg/mL,表明该重组抗体具有活性且亲和力良好,将Ab-FCV VP1-4用于后续实验。
实施例9间接免疫荧光检测重组抗体
使用含有10% FBS的EMEM复苏CRFK贴壁细胞,待细胞融合度达到80~90%时,接种MOI为0.1的FCV毒液,于37℃5% CO2培养箱共感染培养,同时设置病毒阴性组作为对照(即不加入病毒)。补加入等体积2% FBS EMEM,于细胞培养箱中培养,24h后用PBS洗涤细胞3次。加入500μL 4%多聚甲醛固定细胞,30min后用PBS洗涤细胞3次。加入500μL 0.1%TritonX100,15min后用PBS洗涤细胞3次。用含3%酪蛋白钠的PBS于37℃封闭1h,用PBS洗涤3次。将重组抗体Ab-FCV VP1作为间接免疫荧光的一抗,浓度为0.5μg/mL,37℃孵育1h,二抗为羊抗兔FITC,37℃孵育1h,洗板3次,加入500ul DAPI染色20min,PBS洗板3次使用倒置荧光显微镜观察。如图4所示,重组抗体Ab-FCV VP1-4与FCV病毒阳性样品出现明显的特异性绿色荧光信号,而病毒阴性对照组未见荧光信号。证明重组抗体Ab-FCV VP1-4可以与FCV病毒特异性结合,可以应用于猫杯状病毒FCV的特异性检测。
实施例10抗体特异性验证
分别用1×104TCID50/ml的FPV、FCV、FHV细胞毒液上清包被的浓度包被酶标板,4℃过夜。用含有0.05%Tween-20的PBST溶液洗涤5次,弃PBST,加入2%脱脂奶粉封闭液,37℃封闭1h。用PBST溶液洗涤5次,拍干孔中残留液体,将重组抗体Ab-FCV VP1-4和2%脱脂奶粉封闭液按照3:2比例混合,25℃±5℃静置10min去干扰化处理后,加入酶标板中,37℃孵育1h。用PBST溶液洗涤5次,拍干孔中残留液体,加入HRP标记羊抗兔IgG二抗,37℃孵育1h。用PBST溶液洗涤5次,拍干孔中残留液体,加入TMB显色液,避光静置15min后,加入2mol/LH2SO4终止显色,读取450nm处吸光度(表7)。如图6所示,重组抗体Ab-FCV VP1-4与FCV病毒显示出良好的亲和,与FPV和FHV病毒的结合水平差,表现出FCV病毒特异性,证明重组抗体Ab-FCV VP1-4可以与FCV病毒特异性结合,可以应用于猫杯状病毒FCV的特异性检测,且不与猫细小病毒和猫疱疹病毒的特异性检测混淆,是优秀的猫杯状病毒检测抗体。
表7Ab-FCV VP1-4与FCV、FPV、FHV特异性结合验证
1 | 2 | 3 | |
FCV | 2.0273 | 2.1523 | 2.0741 |
FPV | 0.0613 | 0.0801 | 0.0609 |
FHV | 0.0536 | 0.0512 | 0.0688 |
实施例11基于抗猫杯状病毒兔单链重组抗体建立双抗夹心法酶联免疫检测方法
辣根过氧化物酶(HRP)标记处理方法:按照HRP标记试剂盒(6012-1)说明书操作。
双抗夹心法酶联免疫检测方法的具体步骤:(1)将抗猫杯状病毒兔单链重组抗体Ab-FCV VP1-1、Ab-FCV VP1-2、Ab-FCV VP1-3、Ab-FCV VP1-4、Ab-FCV VP1-5使用PBS缓冲液(pH=7.2~7.4)稀释至5μg/mL进行包被,4℃孵育过夜,洗涤液洗板;(2)用2%脱脂奶粉封闭液37℃封闭2h;(3)加入50ng/mL的标准样品(FCV VP1蛋白),每孔100μL,37℃孵育1h,洗涤液洗板;(3)分别加入稀释2000倍的Ab-FCV VP1-1-HRP、Ab-FCV VP1-2-HRP、Ab-FCV VP1-3-HRP、Ab-FCV VP1-4-HRP和Ab-FCV VP1-5-HRP检测抗体,每孔100μL,37℃孵育1h,洗涤液洗板;(4)加入TMB显色液,常温避光显色10分钟,加入2mol/L H2SO4终止显色,读取450nm处吸光度,确定最佳配对抗体。
表8抗体配对实验结果
如表8数据可知,将5株抗体进行两两配对组合后,当捕获抗体为Ab-FCV VP1-4,检测抗体为Ab-FCV VP1-1时,同等浓度的样品测得OD450值最高,因此选择兔单链重组抗体Ab-FCV VP1-4作为捕获抗体,经辣根过氧化物酶标记的兔单链重组抗体Ab-FCV VP1-1作为检测抗体,二者组合使用用于双抗夹心法酶联免疫检测时,具有特异性高、灵敏度高和稳定性好等优势,这对于在临床诊断和科研应用上具有重要的意义。
以上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。
Claims (10)
1.检测猫杯状病毒的试剂,其特征在于,包括包被于固相介质的捕获抗体和标记有生物标记或化学标记的检测抗体;
所述捕获抗体中:
重链可变区的CDR1的氨基酸序列如SEQ ID NO:29、SEQ ID NO:35、SEQ ID NO:41、SEQID NO:47和/或SEQ ID NO:53所示中的至少一种;
重链可变区的CDR2的氨基酸序列如SEQ ID NO:30、SEQ ID NO:36、SEQ ID NO:42、SEQID NO:48和/或SEQ ID NO:54所示中的至少一种;
重链可变区的CDR3的氨基酸序列如SEQ ID NO:31、SEQ ID NO:37、SEQ ID NO:43、SEQID NO:49和/或SEQ ID NO:55所示中的至少一种;
轻链可变区的CDR1的氨基酸序列如SEQ ID NO:32、SEQ ID NO:38、SEQ ID NO:44、SEQID NO:50和/或SEQ ID NO:56所示中的至少一种;
轻链可变区的CDR2的氨基酸序列如SEQ ID NO:33、SEQ ID NO:39、SEQ ID NO:45、SEQID NO:51和/或SEQ ID NO:57所示中的至少一种;
轻链可变区的CDR3的氨基酸序列如SEQ ID NO:34、SEQ ID NO:40、SEQ ID NO:46、SEQID NO:52和/或SEQ ID NO:58所示中的至少一种;
所述检测抗体中:
重链可变区的CDR1的氨基酸序列如SEQ ID NO:29、SEQ ID NO:35、SEQ ID NO:41、SEQID NO:47和/或SEQ ID NO:53所示中的至少一种;
重链可变区的CDR2的氨基酸序列如SEQ ID NO:30、SEQ ID NO:36、SEQ ID NO:42、SEQID NO:48和/或SEQ ID NO:54所示中的至少一种;
重链可变区的CDR3的氨基酸序列如SEQ ID NO:31、SEQ ID NO:37、SEQ ID NO:43、SEQID NO:49和/或SEQ ID NO:55所示中的至少一种;
轻链可变区的CDR1的氨基酸序列如SEQ ID NO:32、SEQ ID NO:38、SEQ ID NO:44、SEQID NO:50和/或SEQ ID NO:56所示中的至少一种;
轻链可变区的CDR2的氨基酸序列如SEQ ID NO:33、SEQ ID NO:39、SEQ ID NO:45、SEQID NO:51和/或SEQ ID NO:57所示中的至少一种;
轻链可变区的CDR3的氨基酸序列如SEQ ID NO:34、SEQ ID NO:40、SEQ ID NO:46、SEQID NO:52和/或SEQ ID NO:58所示中的至少一种。
2.根据权利要求1所述的检测试剂,其特征在于,
所述检测抗体中:
轻链三个CDR区的氨基酸序列分别如SEQ ID NO:32~34所示;重链三个CDR区的氨基酸序列分别如SEQ ID NO:29~31中任一项所示;
所述捕获抗体中:
轻链三个CDR区的氨基酸序列分别如SEQ ID NO:50~52所示;重链三个CDR区的氨基酸序列分别如SEQ ID NO:47~49中任一项所示。
3.根据权利要求2所述的检测试剂,其特征在于,
所述检测抗体重链可变区如SEQ ID NO:1所示;轻链可变区如SEQ ID NO:2所示;
所述捕获抗体重链可变区如SEQ ID NO:7所示;轻链可变区如SEQ ID NO:8所示。
4.根据权利要求3所述的检测试剂,其特征在于,
所述检测抗体中,重链与轻链之间以linker连接,linker序列如SEQ ID NO.11所示;
所述捕获抗体中,重链与轻链之间以linker连接,linker序列如SEQ ID NO.11所示。
5.根据权利要求1~4任一项所述的检测试剂,其特征在于,所述固相介质为酶标板;所述检测抗体标记有HRP。
6.根据权利要求1~4任一项所述的检测试剂,其特征在于,所述捕获抗体与检测抗体的质量比为10:1。
7.根据权利要求1~4任一项所述的检测试剂,其特征在于,其还包括包被缓冲液、洗涤液、封闭液、TMB显色液和终止液;其中:
所述包被缓冲液为pH7.2~7.4的PBS缓冲液,
所述洗涤液为
所述封闭液为2%的脱脂奶粉溶液;
所述终止液为2mol/L H2SO4溶液。
8.非诊断目的的猫杯状病毒的检测方法,其包括以权利要求1~7任一项所述的检测试剂对样品进行检测。
9.根据权利要求8所述的检测方法,其特征在于,以捕获抗体对样品进行捕获后,加入检测抗体进行孵育;所述捕获的条件为37℃,1h;所述孵育的条件为37℃,1h。
10.根据权利要求9所述的检测方法,其特征在于,所述孵育后,还包括TMB显色、终止显色和读取450nm吸光度的步骤。
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