CN116836267A - 检测猫泛白细胞减少症病毒的抗体及其应用 - Google Patents
检测猫泛白细胞减少症病毒的抗体及其应用 Download PDFInfo
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Abstract
本发明涉及生物检测技术领域,具体涉及检测猫泛白细胞减少症病毒的抗体及其应用。本发明提供了检测猫泛白细胞减少症病毒的抗体及其应用。本发明通过构建噬菌体展示文库筛选出猫泛白细胞减少症病毒的特异性抗体。与现有技术相比,本发明提供的猫泛白细胞减少症病毒的抗体特异性强、亲和力高、可与病毒结合,可用于FPV的特异性检测,具有很好的市场应用前景。
Description
技术领域
本发明涉及生物检测技术领域,具体涉及检测猫泛白细胞减少症病毒的抗体及其应用。
背景技术
猫泛白细胞减少症病毒(Felinepanleukopeniavirus,FPV)又称猫细小病毒、猫瘟病毒。猫传染性肠炎病毒,属于细小病毒科,细小病毒属,是一种单链线状DNA病毒。FPV感染在临床上以白细胞急剧减少、腹泻、出血性肠炎、突发双相型高热、脱水及呕吐为特征,是目前细小病毒中致病性最强、感染范围最宽的一种。病毒感染发病率高,死亡率可达90%,对全球宠物行业造成重大的危害和经济损失。
FPV基因组由非结构蛋白NS1、NS2和结构蛋白VP1、VP2构成,VP2蛋白是FPV中相对保守的抗原特异性衣壳蛋白,具有刺激病毒产生中和抗体的重要表位,并且VP2蛋白氨基酸关键位点的突变在病毒感染发病机制中起关键性作用,可以有效刺激机体产生中和抗体并影响其致病性、宿主范围及血凝性。
目前,本领域内检测感染FPV血清抗体的诊断抗体灵敏度及特异性尚不能满足市场检测需求。
发明内容
有鉴于此,本发明要解决的技术问题在于提供检测猫泛白细胞减少症病毒的抗体及其应用,本发明提供了蛋白活性高且更加敏感特异的检测猫泛白细胞减少症病毒的抗体,可用于间接免疫荧光和间接ELISA等特异性检测。
本发明提供的猫泛白细胞减少症病毒的抗体,包含如下氨基酸序列:
(1)其重链三个CDR区分别具有如SEQ ID NO:1~18中任一项所示的氨基酸序列;其轻链三个CDR区分别具有如SEQ ID NO:19~36中任一项所示的氨基酸序列;
(2)与(1)所示的氨基酸序列中经过氨基酸的取代、添加或缺失一个或多个,但与(1)所示氨基酸功能相同或相似的氨基酸序列;或
(3)与(1)或(2)所示的氨基酸序列具备至少80%同源性的氨基酸序列。
一些实施例中,所述猫泛白细胞减少症病毒的抗体重链的三个CDR区分别具有如SEQ IDNO:1、2和3所示的氨基酸序列;
一些实施例中,所述猫泛白细胞减少症病毒的抗体轻链的三个CDR区分别具有如SEQ ID NO:19、20和21所示的氨基酸序列。
本发明中,所述猫泛白细胞减少症病毒的抗体中:
Ⅰ、其重链的四个FR区分别具有如SEQ ID NO:37~60中任一项所示的氨基酸序列;其轻链的四个FR分别具有如SEQ ID NO:61~84中任一项所示的氨基酸序列;
Ⅱ、与Ⅰ所示的氨基酸序列中经过氨基酸的取代、添加或缺失一个或多个,但与Ⅰ所示氨基酸功能相同或相似的氨基酸序列;或
Ⅲ、与Ⅰ或Ⅱ所示的氨基酸序列具备至少80%同源性的氨基酸序列。
本发明中,所述具有至少80%同源性的序列为在原序列的基础上,经取代、添加或缺失一个或多个氨基酸获得的氨基酸序列中,所述多个为2个、3个、4个、5个、6个、7个、8个、9个、10个、11个、12个、13个、14个、15个、16个、17个、18个、19个、20个、21个、22个、23个、24个、25个、26个、27个、28个、29个、30个、31个、32个、33个、34个、35个或36个。
一些实施例中,所述猫泛白细胞减少症病毒的抗体重链的四个FR区分别具有如SEQ ID NO:37、38、39和40所示的氨基酸序列;
一些实施例中,所述猫泛白细胞减少症病毒的抗体轻链的四个FR分别具有如SEQID NO:61、62、63和64所示的氨基酸序列。
一些具体实施例中,所述猫泛白细胞减少症病毒的抗体重链可变区包含SEQ IDNO:1、2和3所示的CDR区和SEQ ID NO:37、38、39和40所示的FR区。
一些具体实施例中,所述猫泛白细胞减少症病毒的抗体轻链可变区包含SEQ IDNO:19、20和21所示的CDR区和SEQ ID NO:61、62、63和64所示的FR区。
一些实施例中,所述猫泛白细胞减少症病毒的抗体重链可变区具有如SEQ ID NO:85~90任一项所示的氨基酸序列;
所述猫泛白细胞减少症病毒的抗体轻链可变区具有如SEQ ID NO:91~96任一项所示的氨基酸序列。
一些具体实施例中,所述猫泛白细胞减少症病毒的抗体重链可变区具有如SEQ IDNO:85所示的氨基酸序列;所述猫泛白细胞减少症病毒的抗体轻链可变区具有如SEQ IDNO:91所示的氨基酸序列。
一些实施例中,所述重链和轻链的连接肽linker具有如SEQ ID NO:97所示的氨基酸序列。
一些实施例中,所述猫泛白细胞减少症病毒的抗体还包括恒定区,所述重链的恒定区为IgGH,所述轻链的恒定区为κ链。
生物材料,其特征在于,包括以下Ⅰ~Ⅵ中至少一项:
Ⅰ、编码任一项所述猫泛白细胞减少症病毒的抗体的核酸;
Ⅱ、包含所述核酸的表达载体;
Ⅲ、转化或转染所述表达载体的宿主细胞;
Ⅳ、所述的猫泛白细胞减少症病毒的抗体与固体介质或半固体介质偶联制得的偶联物;
Ⅴ、经化学标记或生物标记的所述猫泛白细胞减少症病毒的抗体;
Ⅵ、经化学标记或生物的所述的猫泛白细胞减少症病毒的抗体与固体介质或半固体介质偶联制得的偶联物。
本发明所述的表达载体能够在宿主细胞体内表达产生所述猫泛白细胞减少症病毒的抗体,所述的宿主细胞选自大肠杆菌、酵母菌、昆虫细胞或哺乳动物细胞。
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。
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。
本发明提供了所述的抗体的制备方法,包括:所述生物材料中的宿主细胞,诱导所述猫泛白细胞减少症病毒的抗体的表达。
本发明提供了如下ⅰ~ⅲ所示中至少一项在制备猫泛白细胞减少症病毒的检测试剂或检测试剂盒中的应用:
ⅰ、所述的猫泛白细胞减少症病毒的抗体;
ⅱ、所述的生物材料;
ⅲ、经所述的制备方法制得猫泛白细胞减少症病毒的抗体。
本发明提供了检测猫泛白细胞减少症病毒的抗体及其应用。本发明通过构建噬菌体展示文库筛选出猫泛白细胞减少症病毒的特异性抗体。与现有技术相比,本发明提供的猫泛白细胞减少症病毒的抗体特异性强、亲和力高、可与病毒结合,可用于FPV的特异性检测,具有很好的市场应用前景。通过EC50验证抗体的亲和力,结果表明,本发明提供的猫泛白细胞减少症病毒的抗体与重组表达FPVVP2蛋白的结合的EC50为1.897×10-1μg/mL,表明该猫泛白细胞减少症病毒的抗体具有高活性且亲和力良好。
附图说明
图1为本发明实施例提供的FPVVP2-scFv噬菌体文库的构建和免疫文库的多样性分析,其中(A)新西兰兔免疫4次的抗体效价检测;(B)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为本发明实施例提供的高亲和力FPVVP2-scFv的筛选,其中:(A)电转后PCR鉴定结果;(B)第一轮噬菌体展示筛选后的抗体文库多样性;(C)第二轮噬菌体展示筛选后的抗体文库多样性;(D)第二轮噬菌体ELISA确定筛选抗原FPVVP2高特异性结合的scFv抗体;
图3为本发明实施例提供的Ab-FPVVP2的表达与亲和力验证,其中:(A)Ab-FPVVP2的SDS-PAGE结果,M:Marker,1:Ab-FPVVP2-1,2:Ab-FPVVP2-2,3:Ab-FPVVP2-3,4:Ab-FPVVP2-4,5:Ab-FPVVP2-5,6:Ab-FPVVP2-6;(B)Ab-FPVVP2-1抗体与抗原FPVVP2结合的EC50;(C)Ab-FPVVP2-2抗体与抗原FPVVP2结合的EC50;(D)Ab-FPVVP2-3抗体与抗原FPVVP2结合的EC50;(E)Ab-FPVVP2-4抗体与抗原FPVVP2结合的EC50;(F)Ab-FPVVP2-5抗体与抗原FPVVP2结合的EC50;(G)Ab-FPVVP2-6抗体与抗原FPVVP2结合的EC50;
图4为本发明实施例提供的Ab-FPVVP2-1重组抗体与FPV病毒的间接免疫荧光实验结果;
图5为pTT5-V5与pTT5-V6质粒图谱。
具体实施方式
本发明提供了检测猫泛白细胞减少症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。
本发明采用的试材皆为普通市售品,皆可于市场购得。其中:
成年新西兰兔购自安徽定远县永康清源养殖场;FPVVP2蛋白由本实验室制备和保存(GenBank:M38246.1);FPV病毒由本实验室保存(GenBank:M38246.1);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;ClonExpressIIOneStepCloningKit购自南京诺唯赞生物科技股份有限公司;Premixtaq、Plasmidminikit、GelExtraction及转染试剂均购自TaKaRa公司。
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FPV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结果显示接种7d后血清抗体滴度显著上升,14d后抗体滴度大于104,21d后达5×104,28d后达1.28×105(图1A)。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1B)。
引物序列组合如下所示:
VH-scFv-F1:
5’-GGTGGGGGTGGTTCCTCTAGATCTTCCCAGTCGKTGGAGGAGTCC-3’
VH-scFv-F2:
5’-GGTGGGGGTGGTTCCTCTAGATCTTCCCAGWCAGTGAAGGAGTCC-3’
VH-scFv-F3:
5’-GGTGGGGGTGGTTCCTCTAGATCTTCCCAGTCGCTGGRGGAGTCC-3’
VH-scFv-F4:
5’-GGTGGGGGTGGTTCCTCTAGATCTTCCCAGTCGGTGGAGGAGTCC-3’
VH-scFv-R1:
5’-AGGAGGCTATTGGCCGGCCTGGCCTGARGAGAYGGTGACCAGGGTGCC-3’
VH-scFv-R2:
5’-AGGAGGCTATTGGCCGGCCTGGCCGCAGCAGGGGGCCAGTGGGAAGACTGAC-3’
VL-scFv-F1:
5’-AAAAGAGGCCCAGGCGGCCGAGCTCGTGMTGACCCAGACTCCA-3’
VL-scFv-F2:
5’-AAAAGAGGCCCAGGCGGCCGAGCTCGATMTGACCCAGACTCCA-3’
VL-scFv-F3:
5’-AAAAGAGGCCCAGGCGGCCGCYCAAGTGCTGACCCAG-3’
VL-scFv-F4:
5’-AAAAGAGGCCCAGGCGGCCGCCCWAGTGATGACCCAG-3’
VL-scFv-R1:
5’-GGAAGATCTAGAGGAACCACCCCCACCACCGCCCGAGCCACCGCCACCTTTGATTTCCACATTGGTGCC-3’
VL-scFv-R2:
5’-GGAAGATCTAGAGGAACCACCCCCACCACCGCCCGAGCCACCGCCACCTAGGATCTCCAGCTCGGTCCC-3’
实施例3FPVVP2-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之间插入linker,连接肽linker的氨基酸序列为GGGGSGGGGGGSSRSS(SEQ ID NO:97),合成scFv片段VH-linker-VL,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,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,约750bp(图1B),保存于-20℃。重叠PCR引物序列如下所示:
重叠PCR-FP:5’-GAGGAGGAAAAAGAGGCCCAGGCGGCC-3’
重叠PCR-RP:5’-AGAGGAGGCTATTGGCCGGCCTGGCC-3’
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T4DNA连接酶连接得到FPVVP2-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×YeastextractandTryptone)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,计算得抗体库重组率为45%(图2A)。将固体培养基上的所有克隆用2YT培养基洗下,4℃保存。鉴定引物序列如下所示:
pComb3XSS-FP:5’-GCTGGTTTCGCTACCGTGGCCCAGGCGGCC-3’
pComb3XSS-FP:5’-GTGATGGTGATGGTGCTGGCCGGCCTGGCC-3’
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50mL2YT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。10000rpm4℃离心20min,弃上清。用5mLPBS使沉淀重悬,并加入3mLPEG8000复沉,重复离心操作后,用4mLPBS重悬沉淀并过滤除菌后,保存于4℃备用。
实施例5 FPVVP2-scFv筛选
提前将FPVVP2蛋白生物素化。扩增的scFv文库噬菌体、标记了FPVVP2蛋白的DynabeadsM-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0.1MGlycine-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随机挑选20个单克隆进行PCR验证,结果显示所建FPVVP2-scFv文库第一轮淘选测得阳性率达65%,第二轮淘选测的阳性率达85%(图2B和C)。
实施例6 PhageELISA(单链抗体亲和力检测)
将FPVVP2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(检测结果见表1)。
表1 FPVVP2待筛抗体与抗原FPVVP2的亲和力检测
0.0491 | 0.2119 | 0.3729 | 0.1299 | 0.4749 | 0.9438 | 0.4844 | 0.2792 | 0.3718 | 0.2723 | 0.1755 | 0.1739 |
0.2612 | 0.9128 | 0.1838 | 0.2363 | 0.7658 | 0.1856 | 0.1295 | 0.9749 | 0.0372 | 0.0847 | 0.1739 | 0.2842 |
0.1849 | 0.1744 | 0.2364 | 0.3947 | 0.8648 | 0.2847 | 0.8758 | 0.0283 | 0.084 | 0.0932 | 0.3629 | 0.2745 |
0.8733 | 0.1493 | 0.2848 | 0.0572 | 0.2849 | 0.1947 | 0.3849 | 0.1857 | 0.1085 | 0.0937 | 0.2469 | 0.047 |
0.1837 | 0.1395 | 0.0375 | 0.3837 | 0.8274 | 0.2746 | 0.3742 | 0.4848 | 0.3759 | 0.3856 | 0.2845 | 0.0285 |
0.2048 | 0.0385 | 0.2947 | 0.3859 | 0.0639 | 0.2855 | 0.7642 | 0.3038 | 0.0285 | 0.2848 | 0.4274 | 0.1945 |
0.2938 | 0.3927 | 0.1937 | 0.1834 | 0.3287 | 0.2039 | 0.0274 | 0.0948 | 0.1976 | 0.2849 | 0.1639 | 0.2857 |
0.1474 | 0.2353 | 0.2847 | 0.0382 | 0.2842 | 0.0376 | 0.0937 | 0.9547 | 0.0284 | 0.3937 | 0.1739 | 0.896 |
如图2D显示,选择OD450值高的11株抗体测序,最终获得6株序列不同的抗体用于后续实验。
实施例7 FPVVP2重组抗体Ab-FPVVP2的重组、表达及纯化
扩增并纯化单链抗体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-V5-6和VL-pTT5-V6-1~VL-pTT5-V6-6。将重组质粒VH-pTT5-V5-1~VH-pTT5-V5-6和VL-pTT5-V6-1~VL-pTT5-V6-6一一对应,通过PEI转染至HEK293f细胞中进行真核表达。48h后10000rpm4℃离心20min收集培养液上清,利用ProteinA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-FPV VP2-1、Ab-FPVVP2-2、Ab-FPVVP2-3、Ab-FPVVP2-4、Ab-FPVVP2-5、Ab-FPVVP2-6。
其中6株抗体的序列如下:
单链抗体FPVVP2-scFv-1和重组抗体Ab-FPVVP2-1的重链氨基酸序列:
QSLEESGGRLVTPGTPLTLTCTVSGFSLSSYSMSWVRQAPGKGLEWIGFINTEGSAYYASWAKGRFTISRTSTTVDLKIPSPTTEDTATYFCVRVNIANSGDFWGQGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGKAPSTCSKPTCPPGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGKGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK
单链抗体FPVVP2-scFv-1和重组抗体Ab-FPVVP2-1的轻链氨基酸序列:
DGVMTQTPASVSAAVGGTVTIKCQASQSIGSRLAWYQQKAGQRPKLLIYGASTLASGVPSRFRGSGSGTEFTLTISDLECADAATYYCQCNHYGSSYGAAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC*
单链抗体FPVVP2-scFv-2和重组抗体Ab-FPVVP2-2的重链氨基酸序列:
QSLEESGGRLVTPGTPLTLTCTVSGFSLGGYAMNWVRQAPGKGLEWIGLINTEGNTYYANWAKGRFTISKISTTVHLKITSPTTEDTATYFCARINAGNAADLWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGKAPSTCSKPTCPPGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGKGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK
单链抗体FPVVP2-scFv-2和重组抗体Ab-FPVVP2-2的轻链氨基酸序列:
DGDMTQTPASVSEPVGGTVTIKCQASQYISGRLAWYQQKPGQPPKLLIYSASTLASGVPSRFSGSGSGTEFTLTISDLECADAATYYCQCTYYGSTYGGAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC*
单链抗体FPVVP2-scFv-3和重组抗体Ab-FPVVP2-3的重链氨基酸序列:
QSVEESGGRLVTPGGSLTLTCTVSGIDLSIYEMNWVRQAPGKGLEWIGIIYRRGDTDYASWAKGRFTISKTSTTVDLKFTSPTTEDTATYFCAAFDPDTYTYPAFTLWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGKAPSTCSKPTCPPGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGKGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK
单链抗体FPVVP2-scFv-3和重组抗体Ab-FPVVP2-3的轻链氨基酸序列:
DGMMTQTPSSKSVPVGDTVTINCQASESVYSNNRLAWYQQKPGQPPKLLIYSASTLASGVPSRFKGSGFGTQFTLTISGVQCDDAATYFCAGYKSSSTAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC*
单链抗体FPVVP2-scFv-4和重组抗体Ab-FPVVP2-4的重链氨基酸序列:
QQQLEESGGRLVTPRTPLTLTCTASGFSLSRYEMSWVRQAPGKGLEWIGIIYASGDTDYASWAKGRFTISKTSTTVDLKMTSPTTEDTATYFCANFDPYDYTYPAFGLWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGKAPSTCSKPTCPPGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGKGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK
单链抗体FPVVP2-scFv-4和重组抗体Ab-FPVVP2-4的轻链氨基酸序列:
DGVMTQTPSSKSVPVGDTVTINCQASESVYSNNRLAWFQQKPGQPPKLLIYSASTLASGVPSRFKGSGSGTQFTLTISGVQCDDAATYYCAGYKSSSVAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC*
单链抗体FPVVP2-scFv-5和重组抗体Ab-FPVVP2-5的重链氨基酸序列:
QSVKESGGRLVTPGTPLTLTCTVSGFSLSSYSMSWVRQAPGKGLDYIGYINTDGTTYFATWAKGRFTISKTSTTVDLKITRPTTEDTATYFCARIGVANSDDYWGQGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGKAPSTCSKPTCPPGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGKGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK
单链抗体FPVVP2-scFv-5和重组抗体Ab-FPVVP2-5的轻链氨基酸序列:
DGVMTQTPASVSEPVGGTVTIKCQASQYISSRLAWYQQKPGQPPKLLIYSASTLASGVSSRFKGSGSGTQFTLTISDLECADAATYYCQSTYYTSNVAFGGGTEVVVRGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC*
单链抗体FPVVP2-scFv-6和重组抗体Ab-FPVVP2-6的重链氨基酸序列:
QSVEESGGRLVTPGTPLTLTCTVSGFSLSSYAVSWVRQAPGKGLEYIGIINTGGTTSYASWAKGRFTISRTSTTVDLKIPSLTTEDTATYFCARVQYDVYGYTDTTHAFDPWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGKAPSTCSKPTCPPGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGKGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK
单链抗体FPVVP2-scFv-6和重组抗体Ab-FPVVP2-6的轻链氨基酸序列:
DGVMTQTPSSVSAAVGGTVTIKCQASQSIGSSLAWYQQKPGQPPKLLIYRASTLASGVPSRFKGSGSGTQFTLTISDLECADAATYYCQSSYYSGTSSAYGYAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC*
6株抗体的重链可变区、轻链可变区及其相应的CDR与FR序列如下表2所示。
表2 6株抗体的重链可变区、轻链可变区及其相应的CDR与FR氨基酸序列
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单链抗体FPVVP2-scFv-1和重组抗体Ab-FPVVP2-1的重链可变区核苷酸序列
5’-CAGTCGCTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGA CACTCACCTGCACCGTCTCTGGATTCTCCCTCAGTAGCTATTCAATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGATTCATTAATACTGAGGGTAGCGCATACTACGCGAGCTGGGCAAAAGGCCGATTCACCATCTCCAGAACTTCGACCACGGTGGATCTGAAAATTCCCAGTCCGACAACCGAGGACACGGCCACCTATTTCTGTGTCAGAGTTAATATTGCTAATTCCGGTGACTTCTGGGGCCAAGGCACCCTGGTCACCGTCTCCTCA-3’
单链抗体FPVVP2-scFv-1和重组抗体Ab-FPVVP2-1的轻链可变区核苷酸序列
5’-GATGGCGTGATGACCCAGACTCCAGCCTCCGTGTCTGCAGCTGTGGGAGGCA CAGTCACCATCAAGTGCCAGGCCAGTCAGAGCATTGGTAGTAGGTTAGCCTGGTATCAGCAGAAAGCAGGGCAGCGTCCCAAGCTCCTGATCTATGGTGCATCCACTCTGGCATCTGGGGTCCCATCGCGGTTCAGGGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCGACCTGGAGTGTGCCGATGCTGCCACTTACTACTGTCAATGTAATCATTATGGTAGTAGTTATGGTGCTGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAAA-3’
单链抗体FPVVP2-scFv-2和重组抗体Ab-FPVVP2-2的重链可变区核苷酸序列
5’-CAGTCGCTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGA CACTCACCTGCACAGTCTCTGGATTCTCCCTCGGTGGCTATGCAATGAATTGGGTCCGCCAGGCTCCAGGGAAGGGACTGGAATGGATCGGATTGATTAATACTGAAGGTAACACATACTACGCGAACTGGGCGAAAGGCCGATTCACCATCTCCAAAATCTCGACCACGGTGCATCTGAAAATCACCAGTCCGACAACCGAGGACACGGCCACCTATTTCTGTGCCAGAATAAATGCTGGTAATGCGGCTGACTTGTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCA-3’
单链抗体FPVVP2-scFv-2和重组抗体Ab-FPVVP2-2的轻链可变区核苷酸序列
5’-GATGGCGATATGACCCAGACTCCAGCCTCTGTGTCTGAACCTGTGGGAGGCA CAGTCACCATCAAGTGCCAGGCCAGTCAATACATTAGTGGTAGATTAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTATTCTGCGTCCACTCTGGCCTCTGGGGTCCCATCGCGGTTCAGCGGCAGTGGATCTGGGACAGAGTTCACTCTCACCATCAGCGACCTGGAGTGTGCCGATGCTGCCACTTACTACTGTCAATGTACTTATTATGGTAGTACTTATGGGGGTGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAAA-3’
单链抗体FPVVP2-scFv-3和重组抗体Ab-FPVVP2-3的重链可变区核苷酸序列
5’-CAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTAACGCCTGGAGGATCCCTGA CACTCACCTGCACAGTCTCTGGAATCGACCTCAGTATCTATGAAATGAACTGGGTCCGCCAGGCCCCAGGGAAGGGGCTGGAATGGATCGGAATCATTTATCGTAGGGGTGACACAGACTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCGACCACGGTGGATCTGAAGTTCACCAGTCCGACAACCGAGGACACGGCCACCTATTTCTGTGCCGCGTTCGATCCTGATACTTATACTTATCCTGCCTTTACTTTGTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCA-3’
单链抗体FPVVP2-scFv-3和重组抗体Ab-FPVVP2-3的轻链可变区核苷酸序列
5’-GATGGCATGATGACCCAGACACCATCTTCCAAGTCTGTCCCTGTGGGAGACAC AGTCACCATCAATTGCCAGGCCAGTGAGAGTGTTTATAGTAACAACCGCTTAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTATTCTGCATCCACTCTGGCATCTGGGGTCCCATCGCGGTTCAAAGGCAGTGGATTTGGGACACAGTTCACTCTCACCATCAGCGGCGTGCAGTGTGACGATGCTGCCACTTATTTCTGTGCAGGATATAAAAGTTCTAGTACTGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAAA-3’
单链抗体FPVVP2-scFv-4和重组抗体Ab-FPVVP2-4的重链可变区核苷酸序列
5’-CAGCAGCAGCTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTAGGACACCC CTGACACTCACCTGCACAGCCTCTGGATTCTCCCTCAGTAGGTATGAAATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGAATCATTTATGCTAGTGGTGACACAGACTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCGACCACGGTGGATCTGAAAATGACCAGTCCGACAACCGAGGACACGGCCACCTATTTCTGTGCCAATTTCGATCCTTATGATTATACTTATCCTGCCTTTGGTTTGTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCA-3’
单链抗体FPVVP2-scFv-4和重组抗体Ab-FPVVP2-4的轻链可变区核苷酸序列
5’-GATGGCGTGATGACCCAGACTCCATCTTCCAAGTCTGTCCCTGTGGGAGACAC AGTCACCATCAATTGCCAGGCCAGTGAGAGTGTTTATAGTAACAACCGCTTAGCCTGGTTTCAACAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTACTCTGCATCCACTCTGGCATCTGGGGTCCCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGGCGTGCAGTGTGACGATGCTGCCACTTACTACTGTGCAGGATATAAAAGTAGTAGTGTTGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAAA-3’
单链抗体FPVVP2-scFv-5和重组抗体Ab-FPVVP2-5的重链可变区核苷酸序列
5’-CAGTCGGTGAAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGA CACTCACCTGCACAGTCTCTGGATTCTCCCTCAGTAGCTATTCAATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGATTACATCGGATACATTAATACTGATGGTACCACATACTTCGCGACCTGGGCAAAAGGCCGATTCACCATCTCCAAAACCTCGACCACGGTGGATCTGAAAATCACCCGTCCGACAACCGAGGACACGGCCACCTATTTCTGTGCCAGAATTGGAGTTGCTAATAGTGATGATTATTGGGGCCAAGGCACCCTGGTCACCGTCTCCTCA-3’
单链抗体FPVVP2-scFv-5和重组抗体Ab-FPVVP2-5的轻链可变区核苷酸序列
5’-GATGGCGTGATGACCCAGACTCCAGCCTCCGTGTCTGAACCTGTGGGAGGCA CAGTCACCATCAAGTGCCAGGCCAGTCAGTACATTAGTAGTAGATTAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTATTCTGCATCCACTCTGGCATCTGGGGTCTCATCGCGGTTCAAAGGCAGTGGATCTGGGACGCAGTTCACTCTCACCATCAGCGACCTGGAGTGTGCCGATGCTGCCACTTACTACTGTCAATCTACTTATTATACTAGTAATGTTGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAGA-3’
单链抗体FPVVP2-scFv-6和重组抗体Ab-FPVVP2-6的重链可变区核苷酸序列
5’-CAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGA CACTCACCTGCACGGTCTCTGGCTTCTCCCTCAGTAGCTATGCAGTGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATACATCGGAATCATTAATACTGGTGGTACCACATCCTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAGAACCTCGACCACGGTGGATCTGAAAATCCCCAGTCTGACAACCGAGGACACGGCCACCTATTTCTGTGCCAGAGTCCAGTACGATGTTTATGGTTATACTGATACTACCCATGCTTTTGATCCCTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCA-3’
单链抗体FPVVP2-scFv-6和重组抗体Ab-FPVVP2-6的轻链可变区核苷酸序列
5’-GATGGCGTGATGACCCAGACTCCATCCTCTGTGTCTGCAGCTGTGGGAGGCAC AGTCACCATCAAGTGCCAGGCCAGTCAGAGCATTGGTAGTAGCTTAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTACAGGGCATCCACTCTGGCATCTGGGGTCCCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGACCTGGAGTGTGCCGATGCTGCCACTTACTACTGTCAAAGCAGTTATTATAGTGGTACTAGTAGTGCTTATGGTTATGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAAA-3’
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3A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-FPVVP2与FPVVP2抗原的结合
采用间接ELISA方法测定Ab-FPVVP2与抗原FPVVP2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3~表8)。
检测上述抗体Ab-FPVVP2结合效果如图3B~3C所示,量效曲线与回归模型成功拟合,Ab-FPVVP2的6株抗体都能够有效结合FPVVP2蛋白,抗体Ab-FPVVP2-1的亲和力最高,与重组表达FPVVP2蛋白的结合的EC50为1.897×10-1μg/mL,表明该重组抗体具有活性且亲和力良好,将Ab-FPVVP2-1用于后续实验。
表3Ab-FPVVP2-1与抗原FPVVP2结合的EC50
表4Ab-FPVVP2-2与抗原FPVVP2结合的EC50
表5Ab-FPVVP2-3与抗原FPVVP2结合的EC50
表6Ab-FPVVP2-4与抗原FPVVP2结合的EC50
表7Ab-FPVVP2-5与抗原FPVVP2结合的EC50
表8Ab-FPVVP2-6与抗原FPVVP2结合的EC50
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FPV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。补加入等体积2%FBSEMEM,于细胞培养箱中培养,24h后用PBS洗涤细胞3次。加入500μL4%多聚甲醛固定细胞,30min后用PBS洗涤细胞3次。加入500μL0.1%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-FPVVP2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500ulDAPI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-FPVVP2-1与FPV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-FPVVP2可以与FPV病毒特异性结合,可以应用于猫泛白细胞减少症病毒FPV的特异性检测。
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.猫泛白细胞减少症病毒的抗体,其特征在于,
(1)其重链三个CDR区分别具有如SEQ ID NO:1~18中任一项所示的氨基酸序列;其轻链三个CDR区分别具有如SEQ ID NO:19~36中任一项所示的氨基酸序列;
(2)与(1)所示的氨基酸序列中经过氨基酸的取代、添加或缺失一个或多个,但与(1)所示氨基酸功能相同或相似的氨基酸序列;或
(3)与(1)或(2)所示的氨基酸序列具备至少80%同源性的氨基酸序列。
2.根据权利要求1所述的抗体,其特征在于,
其重链的三个CDR区分别具有如SEQ ID NO:1、2和3所示的氨基酸序列;
其轻链的三个CDR区分别具有如SEQ ID NO:19、20和21所示的氨基酸序列。
3.根据权利要求1或2所述的抗体,其特征在于,
其重链的四个FR区分别具有如SEQ ID NO:37~60中任一项所示的氨基酸序列;
其轻链的四个FR分别具有如SEQ ID NO:61~84中任一项所示的氨基酸序列。
4.根据权利要求1~3任一项所述的抗体,其特征在于,
其重链的四个FR区分别具有如SEQ ID NO:37、38、39和40所示的氨基酸序列;
其轻链的四个FR分别具有如SEQ ID NO:61、62、63和64所示的氨基酸序列。
5.根据权利要求1~4任一项所述的抗体,其特征在于,
其重链可变区具有如SEQ ID NO:85~90任一项所示的氨基酸序列;
其轻链可变区具有如SEQ ID NO:91~96任一项所示的氨基酸序列。
6.根据权利要求1~5任一项所述的抗体,其特征在于,
所述重链和轻链的连接肽linker具有如SEQ ID NO:97所示的氨基酸序列。
7.根据权利要求1~6任一项所述的抗体,其特征在于,还包括恒定区,所述重链的恒定区为IgGH,所述轻链的恒定区为κ链。
8.生物材料,其特征在于,包括以下Ⅰ~Ⅵ中至少一项:
Ⅰ、编码权利要求1~7任一项所述猫泛白细胞减少症病毒的抗体的核酸;
Ⅱ、包含所述核酸的表达载体;
Ⅲ、转化或转染所述表达载体的宿主细胞;
Ⅳ、权利要求1~7任一项所述的猫泛白细胞减少症病毒的抗体与固体介质或半固体介质偶联制得的偶联物;
Ⅴ、经化学标记或生物标记的权利要求1~7任一项所述猫泛白细胞减少症病毒的抗体;
Ⅵ、经化学标记或生物的权利要求1~7任一项所述的猫泛白细胞减少症病毒的抗体与固体介质或半固体介质偶联制得的偶联物。
9.权利要求1~7任一项所述的抗体的制备方法,其特征在于,包括:培养如权利要求8所述生物材料中的宿主细胞,诱导所述猫泛白细胞减少症病毒的抗体的表达。
10.如下ⅰ~ⅲ所示中至少一项在制备猫泛白细胞减少症病毒的检测试剂或检测试剂盒中的应用:
ⅰ、权利要求1~7任一项所述的猫泛白细胞减少症病毒的抗体;
ⅱ、权利要求8所述的生物材料;
ⅲ、经权利要求9所述的制备方法制得猫泛白细胞减少症病毒的抗体。
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CN117700535B (zh) * | 2024-02-05 | 2024-04-26 | 北京纳百生物科技有限公司 | 一种猫泛白细胞减少症病毒vp2蛋白单克隆抗体及其应用 |
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