CN116589565A - 一株非洲猪瘟p72单克隆抗体及应用 - Google Patents
一株非洲猪瘟p72单克隆抗体及应用 Download PDFInfo
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Abstract
本发明提供一株非洲猪瘟病毒p72单克隆抗体的制备及其在检测p72蛋白和建立非洲猪瘟病毒乳胶层析试纸条检测方法中的应用。本发明制备了一株ASFV p72蛋白单克隆抗体杂交瘤细胞株R3E9,确定了该株单抗的可变区序列。该株单抗,可用于IFA,识别杆状病毒表达的p72蛋白和ASFV感染细胞中的p72蛋白;也可用于western blotting,识别变性的p72蛋白。以该株单抗制备的非洲猪瘟病毒乳胶层析试纸条,具有良好的特异性和灵敏性,无需辅助设备即可实现ASFV现场快速检测,为我国猪场快速诊断ASFV提供重要技术支撑,将增强我国防控ASFV能力。
Description
技术领域
本发明涉及生物工程技术领域,尤其涉及一株非洲猪瘟病毒p72单克隆抗体的制备及其在检测p72蛋白和建立非洲猪瘟病毒层析试纸条检测方法中的应用。
背景技术
非洲猪瘟是由非洲猪瘟病毒(African swine fever virus,ASFV)感染猪引起的一种烈性、高度接触性传染病。家猪和野猪均对ASFV易感,临床症状随感染毒株毒力的不同而异:强毒株感染引致急性病例,主要表现为高热和内脏广泛性出血,病程快,4-10天出现死亡,死亡率达100%;中等毒力毒株则导致亚急性型病例,潜伏期延长,临床症状减轻,但体内出血和水肿明显,死亡率为30-70%;弱毒株感染引起慢性型病例,可持续5至12个月,不引起体内出血,主要表现为皮肤坏死溃疡、关节发炎肿胀、淋巴节增生和肺出现坏死矿化,生长迟缓甚至消瘦,种猪感染则出现繁殖障碍。ASF对养猪业危害巨大,世界动物卫生组织(OIE)将其列为法定报告的动物疫病,养猪业遭受了巨大冲击。ASFV已被我国列为一类传染病,是各猪场重点防控的头号猪病。
ASFV是一种线性双链DNA病毒,为非洲猪瘟病毒科非洲猪瘟病毒属的唯一成员。病毒基因组全长为170-194kbp,编码151-167个开放阅读框(Open reading frame,ORF)。ASFV形态为正二十面体,粒子直径为175-215nm,结构较为复杂,由内至外分五层组成:含病毒基因组的类核(nucleoid)、内核心壳(core shell)、内膜(inner envelope)、衣壳(capsid)和外囊膜(outer envelope)。ASFV在感染细胞的胞浆内复制,能通过出芽形式释放具有外囊膜的成熟病毒粒子。但除了成熟的病毒粒子有感染性外,细胞内已形成衣壳层的病毒粒子也具有感染性,并能在感染晚期通过裂解细胞得到释放。病毒外膜中含有病毒蛋白p12和CD2v,其中CD2v具有红细胞凝集活性,在病毒致病中发挥重要作用。病毒二十面体衣壳含有五种蛋白:一种主要衣壳蛋白p72,四种次要衣壳蛋白pH240R、p17、p49及pM1249L。p72是病毒粒子的最主要结构成分,占病毒粒子总质量近三分之一(Adv.Virus Res.100,41-74(2018))。同时,p72也是病毒的重要抗原蛋白,是病毒基因分型的主要依据,也是ASFV诊断和亚单位疫苗开发的重要靶标。
目前,ASFV尚无有效疫苗和药物可供使用,检测技术在ASFV防控中发挥着重要作用。本发明通过大肠杆菌表达系统制备p72蛋白,以p72蛋白免疫小鼠制备单克隆抗体。通过筛选及亚克隆纯化,获得了一株分泌p72单克隆抗体的杂交瘤细胞株。该杂交瘤细胞株分泌的单抗,可用于western blotting中识别p72蛋白,也能在间接免疫荧光实验中特异性识别杆状病中表达的p72蛋白和ASFV感染细胞中的p72蛋白。以乳胶微球标记该单克隆抗体,制备了ASFV抗原检测试纸条。该试纸条具有良好的特异性和灵敏性,无需辅助设备即可实现ASFV现场快速检测,为我国猪场快速诊断ASFV提供重要技术支撑,增强我国防控ASFV能力。
发明内容
本发明的目的提供一株ASFV p72单克隆抗体细胞株及其应用。所述的单克隆细胞株分泌的抗体,能用于western blotting检测p72蛋白,也能识别昆虫细胞中表达的p72蛋白和ASFV感染细胞中的p72蛋白。以乳胶微球标记该单克隆抗体,能用于建立检测ASFV的乳胶微球层析试纸条。
此外,本发明还提供了一种p72蛋白制备方法及应用。
此外,本发明还提供了一种ASFV抗原乳胶层析检测试纸条。
为了解决上述技术问题,本发明通过如下技术方案实现:
本发明通过将p72基因片段克隆进原核表达载体pCold I中,经16℃诱导表达,表达截短的p72蛋白包涵体经变性-柱纯化-复性,获得了纯化的p72蛋白。以p72蛋白免疫小鼠,二免后将小鼠的脾细胞与SP2/0细胞融合,通过p72抗体ELISA筛选阳性克隆杂交瘤,并通过稀释法获得一株纯化的杂交瘤细胞克隆。通过间接免疫荧光实验,证实该杂交瘤抗体能结合杆状病毒表达的p72蛋白和ASFV感染猪原代单核细胞表达的p72蛋白。以乳胶微球偶联该单克隆抗体作为免疫探针,建立ASFV抗原检测免疫层析方法。
本发明中使用的p72蛋白,是p72基因克隆于原核表单载体pCold I,经低温(16℃)诱导、包涵体变性、His柱纯化以及透析复性而制备的。
本发明中建立的p72乳胶微球层析检测试纸条为:以300nm的红色乳胶微球标记p72单克隆抗体作为检测探针,将宽度为4mm的玻璃纤维长条在探针溶液中浸润并自然干燥后作为结合垫;将1.25mg/mL的兔抗p72多抗喷涂于NC膜上作为检测线(T线),再将0.5mg/mL的SPA蛋白喷涂于同一NC膜上作为质控性(C线);玻璃纤维长条经样品垫处理液浸润并在37℃烘箱干燥后作为样品垫。将结合垫、吸水垫依次粘贴在NC膜两侧,再将样品垫粘贴在结合垫的另一端,各个垫彼此重叠2mm组装入塑料卡槽中,形成检测试纸条。将样品用样本稀释液进行1:2稀释后,滴3滴加于试纸条加样窗口,室温静置10min左右,观察试纸条显色结果。若T线和C线均为红色,判定为阳性;若T线不显色,C线为红色,判定为阴性;若T线和C线均不显色,检测结果无效。
附图说明
图1PCR扩增的ASFV p72基因片段,M:DNA Marker DL5000;1:p72基因片段;
图2ASFV p72截短蛋白的诱导表达,其中,M:蛋白分子质量标准;1:pColdⅠ空载体;2:pCold-p72未诱导;3:pCold-p72诱导菌体;4:pCold-p72诱导菌超声上清;5:pCold-p72诱导菌超声沉淀;
图3ASFV p72截短蛋白的纯化,M:蛋白分子质量标准;1:纯化的p72截短蛋白;
图4p72单克隆抗体的Western blotting,M:蛋白分子质量标准;1:p54蛋白;2:p72截短蛋白;
图5FA分析杆状病毒表达的p72蛋白;
图6IFA分析ASFV感染细胞,左:对照细胞;右:ASFV感染的细胞;
图7R3E9单抗的SDS-PAGE图,M:蛋白Marker;2:R3E9单抗;
图8不同直径乳胶微球的显色结果,(a):p72蛋白多抗喷膜检测线;(b)葡萄球菌A蛋白喷膜检测线质控线,1:200nm乳胶微球;2:300nm乳胶微球;3:400nm乳胶微球;
图9不同SPA蛋白浓度的C线显色结果,1:0.125mg/mL;2:0.25mg/mL;3:0.5mg/mL;
图10不同p72多抗浓度的T线显色结果,1:0.5mg/mL;2:0.75mg/mL;3:1.0mg/mL;4:1.25mg/mL;
图11乳胶微球层析试纸条组装示意图;
图12试纸条的特异性检测;
图13试纸条的灵敏性检测,其中,1:512ng/mL;2:256ng/mL;3:128ng/mL;4:64ng/mL;5:32ng/mL;6:16ng/mL;7:8ng/mL;8:4ng/mL;9:2ng/mL;10:1ng/mL;11:0.5ng/mL;12:0.25ng/mL;0.125ng/mL。
具体实施方式
下列实施例中,未注明具体条件的实验方法,通常按常规条件,如《精编分子生物学实验指南》(F.M.奥斯伯,R.E.金斯顿,J.G.塞德曼等主编,马学军,舒跃龙的译.北京:科学出版社,2004)中所述的方法进行。
ASFV在我国流行,给我国养猪业造成了惨重的经济损失,已成为威胁我国养猪业健康发展的主要传染病之一。为了提供一种现场快速检测ASFV的诊断方法,本发明通过原核表达系统制备一种截短的p72蛋白,以p72蛋白免疫小鼠制备单克隆抗体。通过ELISA筛选及亚克隆纯化,获得了一株分泌72单克隆抗体的杂交瘤细胞株。该杂交瘤细胞株分泌的单抗,能用于western blotting中识别p72蛋白,也能结合杆状病毒表达的p72蛋白和ASFV感染表达的p72蛋白。以红色乳胶微球标记该单克隆抗体作为探针,建立的层析试纸条,可特异性检测ASFV。
下面结合附图和具体实施方式对本发明作进一步详细的说明。
下面通过实施例,具体阐述本发明。
在本发明的下述实施例中,使用的实验材料如下:
BL21感受态细胞,购自Takara公司;p72基因序列,委托通用生物系统(安徽)有限公司合成,并分别克隆入pCold I载体中;His标签蛋白纯化试剂盒,购自Bio-Works公司。
其它试剂:质粒提取试剂盒,购自OMEGA公司;IPTG、BSA和TMB,购自碧云天生物科技公司;脱脂乳,购自美国BD公司;考马斯亮蓝R-250,购自Biosharp公司;乳胶微球,苏州为度生物技术有限公司;葡萄球菌A蛋白(SPA),北京沃凯生物科技有限公司。
实施案例1.截短p72蛋白的表达与纯化
1.1p72蛋白重组原核表达质粒的构建
通过DNA STAR中的Protean软件以及TMHMM对p72的亲水性和抗原性分析,结合p72蛋白的结构,截取p72蛋白421-647位氨基酸进行表达。以本实验室优化合成的p72基因序列为模板,设计合成引物P72O3F(5-ctccatatgCCCGAGATCCACAACCTGTTC-3)和P72O3R(5-gctctagattaGGTGCTGTACCTCAGCACGG-3),通过PCR扩增出681bp的p72基因片段(图1)。经NdeI/XbaI双酶切p72片段和pColdI,胶回收酶切的p72片段和pColdI,并以T4 DNA连接酶连接,转化TOP10感受态,经NdeI/XbaI双酶切筛选和测序确认,获得阳性重组质粒pCold-p72。
1.2p72蛋白的制备
将重组质粒pCold-p72转化BL21感受态。在重组菌转化平板上挑取单菌落,小量培养至OD600为0.6,加入诱导剂IPTG(终浓度1mM)在16℃培养16-20小时诱导蛋白表达,未诱导组不加IPTG,在相同条件下培养。以6000rpm离心5min收集诱导培养后菌体,并以1mL PBS重悬沉淀菌体。将装有菌液的离心管置于冰上,在超声破碎仪。超声好的菌液在4℃下6000rpm离心5min,分别收集上清和沉淀,沉淀以1mL PBS重悬。上清和沉淀分别取40μl于1.5mL EP管中,加入10μl 5×Loading Buffer,沸水煮样10min,冷却后10000rpm离心2min,取10μL样品进行SDS-PAGE,随后进行考马斯亮蓝染色。SDS-PAGE结果显示(图2),转化菌经诱导后,表达出约26kd的截短p72蛋白,且主要存在于沉淀中,以包涵体形式表达。
大量培养转化菌并诱导p72蛋白表达,超声破碎菌体后,离心获得包涵体沉淀。每200mL菌液的包涵体沉淀,以20mL包涵体变性缓冲液(8M尿素,100mM Tris-HCl,10mMβ-巯基乙醇,2mM EDTA,2mM脱氧胆酸钠)重悬,4℃、1500g离心30min,保留上清。根据His标签蛋白纯化试剂盒说明书,纯化变性的p72截短蛋白,并将纯化蛋白放入透析袋中依次经过含6M、4M、2M尿素的复性缓冲液和PBS逐步透析复性。以10ku超滤管浓缩离心浓缩复性的蛋白液,SDS-PAGE检测浓缩蛋白样品。SDS-PAGE电泳结果(图3)显示,纯化后的p72截短蛋白呈单一条带,在25-30kD间。同时以BCA蛋白测定试剂盒测定蛋白浓度为1.8mg/mL,将p72截短蛋白分装后,-80℃保存备用。
实施案例2.p72蛋白单克隆抗体杂交瘤细胞株的制备与鉴定
2.1小鼠免疫
5只6-8周龄的Balb/c小鼠,初免以弗氏完全佐剂乳化的p72截短蛋白皮下免疫接种。初免后21天,以弗氏不完全佐剂乳化的p72截短蛋白皮下接种再次免疫。二免10天后,通过尾静脉采血,制备血清,以本实验室建立的p72抗体间接ELISA进行抗体水平检测。ELISA结果显示,5只免疫小鼠血清中的抗体达到1:64000稀释倍以上。二免后21天,选择2只p72抗体水平高的小鼠,通过尾静脉注射纯化的p72截短蛋白加强免疫。
2.2杂交瘤细胞融合与筛选
第三次加强免疫后3天,取小鼠脾脏,制备小鼠脾细胞。按照常规程序,将脾细胞与骨髓瘤细胞SP2/0融合。融合后的细胞,分种含有饲养细胞的96孔板。融合后15天,取生长有杂交瘤细胞的孔的上清进行ELISA检测,阳性孔杂交瘤细胞以有限稀释法进行亚隆纯化。经过3轮单克隆纯化,同一杂交瘤细胞株在96孔板上检测孔阳性率达到100%,将杂交瘤细胞扩大培养保存,共保存27株杂交瘤细胞株。
2.3杂交瘤抗体生物学特性分析
将制备的p72截短蛋白进行SDS-PAGE,随后转膜,以实施实例2.2中27株单抗作为一抗,进行western blotting分析。如图4所示,所有27株单抗均识别变性电泳后的p72截短蛋白。
以本实验室构建的表达ASFV p72和B602L的重组家蚕杆状病毒rBm-p72感染BmN细胞,48h后固定细胞,以杂交瘤细胞培养上清中的单克隆抗体为一抗,进行间接免疫荧光(IFA)实验。结果如图5所示,6株杂交瘤细胞(2E5,5F3,H3E4,R1G4,R3E9,R3F7)的单抗能识别杆状病毒表达的p72蛋白。
为了进一步筛选可识别ASFV感染细胞中p72的单克隆抗体,在P3实验室中,以ASFV感染猪原代单核细胞,固定感染细胞后,以2E5、5F3、H3E4、R1G4、R3E9和R3F7六株细胞杂交瘤上清为一抗,进行间接免疫荧光分析。如图6所示,经R3E9单抗孵育,ASFV感染细胞呈现特异性荧光。这表明,R3E9结合ASFV感染细胞中的p72蛋白。
2.4R3E9株单克隆抗体亚型鉴定
应用proteintech公司的小鼠单克隆抗体亚型鉴定试剂盒分析R3E9株单克隆抗体的亚型。按照试剂盒说明书,将R3E9株培养上清进行鉴定。试剂盒测定的值(表1)显示,该株单抗重链为IgG1亚型,轻链为Kappa亚型。
表1.R3E9株单抗亚型鉴定
2.5R3E9株单克隆抗体可变区基因序列
培养于6孔板中的R3E9株杂交瘤细胞,移出上清后,每孔加入1mL Trizol裂解细胞,将裂解样品收集于1.5mL离心管,暂存于-80℃,将样品提交生物技术公司进行小鼠杂交瘤细胞单克隆抗体可变区测序。测序结果如下:
重链可变区(VH)核苷酸序列如下:
5-CAGGTTCAGCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATGTCCTGCAAGGCTTCGGGATA
CACATTCACTGACTATGTTATCACCTGGGTGAAGCAGAGAAATGGACAGGGCCTTGAATGGATTGGAGAGATTTATCCTG
GAATTGGTAGTACTTATTACAATGACAAGTTCAAGGGCAAGGCCACACTGACTGCAGACAAATCCTCCAACACAGCCTAC
ATGCAGCTCAGTAGCCTGACATCTGAGGACTCTGCGGTCTATTTCTGTGCAACAGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA-3(SEQ ID NO.1)
其中,
骨架区1(FR1):5-CAGGTTCAGCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATGTCCTGCAAGGCTTCG-3(SEQ ID NO.2)
互补决定区1(CDR1):5-GGATACACATTCACTGACTATGTT-3(SEQ ID NO.3)
FR2:5-ATCACCTGGGTGAAGCAGAGAAATGGACAGGGCCTTGAATGGATTGGAGAG-3(SEQ IDNO.4)
CDR2:5-ATTTATCCTGGAATTGGTAGTACT-3(SEQ ID NO.5)
FR3:5-TATTACAATGACAAGTTCAAGGGCAAGGCCACACTGACTGCAGACAAATCCTCCAACACAGCCTACATGCAGCTCAGTAG CCTGACATCTGAGGACTCTGCGGTCTATTTCTGT-3(SEQ ID NO.6)
CDR3:5-GCAACAGACTAC-3(SEQ ID NO.7)
FR4:5-TGGGGCCAAGGCACCACTCTCACAGTCTCCTCA-3(SEQ ID NO.8)
对应的氨基酸序列如下:
VH:
QVQLQQSGPELVKPGASVKMSCKASGYTFTDYVITWVKQRNGQGLEWIGEIYPGIGSTYYNDKFKGKATLTADKSSNTAY MQLSSLTSEDSAVYFCATDYWGQGTTLTVSS(SEQ ID NO.9)
FR1:QVQLQQSGPELVKPGASVKMSCKAS(SEQ ID NO.10)
CDR1:GYTFTDYV(SEQ ID NO.11)
FR2:ITWVKQRNGQGLEWIGE(SEQ ID NO.12)
CDR2:IYPGIGST(SEQ ID NO.13)
FR3:YYNDKFKGKATLTADKSSNTAYMQLSSLTSEDSAVYFC(SEQ ID NO.14)
CDR3:ATDY(SEQ ID NO.15)
FR4:WGQGTTLTVSS(SEQ ID NO.16)
轻链可变区(VL)核苷酸序列如下:
5-GATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATC TAGTCAGAGCATTGCACATAGTAATGGAAACACCTATTTACAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTATCAAGGTTCACATGTTCCTCCGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA-3(SEQ ID NO.17)
其中,
FR1:
5-GATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGT-3(SEQ ID NO.18)
CDR1:5-CAGAGCATTGCACATAGTAATGGAAACACCTAT-3(SEQ ID NO.19)
FR2:5-TTACAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTAC-3(SEQ IDNO.20)
CDR2:5-AAAGTTTCC-3(SEQ ID NO.21)
FR3:
5-AACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTG GAGGCTGAGGATCTGGGAGTTTATTACTGC-3(SEQ ID NO.22)
CDR3:5-TATCAAGGTTCACATGTTCCTCCGACG-3(SEQ ID NO.23)
FR4:5-TTCGGTGGAGGCACCAAGCTGGAAATCAAA-3(SEQ ID NO.24)
对应的氨基酸序列如下:
VL:
DVLMTQTPLSLPVSLGDQASISCRSSQSIAHSNGNTYLQWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKI SRVEAEDLGVYYCYQGSHVPPTFGGGTKLEIK(SEQ ID NO.25)
FR1:DVLMTQTPLSLPVSLGDQASISCRSS(SEQ ID NO.26)
CDR1:QSIAHSNGNTY(SEQ ID NO.27)
FR2:LQWYLQKPGQSPKLLIY(SEQ ID NO.28)
CDR2:KVS(SEQ ID NO.29)
FR3:NRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYC(SEQ ID NO.30)
CDR3:YQGSHVPPT(SEQ ID NO.31)
FR4:FGGGTKLEIK(SEQ ID NO.32)
实施实例3单克隆抗体R3E9在免疫层析试纸条中的应用
3.1R3E9腹水制备与纯化
5只8周龄BALB/c雌鼠,以腔注射降植烷,每只0.5mL。一周后,向小鼠腹部注射R3E9杂交瘤细胞0.5mL(1×106个细胞)。杂交瘤细胞接种后约7d,小鼠腹部明显膨大、被毛错乱、精神萎靡。处死小鼠,70%酒精浸泡10min后,剪开小鼠腹部皮肤,用枪头吸出腹水,2000rpm离心10min,取上清分装于1.5mL EP管,于-20℃保存。将收集的腹水用5%脱脂乳从1:100开始倍比稀释,用间接ELISA法检测抗体效价为1:51200。
以Protein G抗体纯化试剂盒纯化R3E9腹水中的抗体,取少量纯化后的抗体进行SDS-PAGE电泳鉴定,结果如图7所示,纯化后的抗体在55KDa和24KDa处各有一条特异性条带,符合抗体重链和轻链的特点。利用BCA法测定纯化抗体的浓度,R3E9抗体浓度为0.83mg/mL。
3.2乳胶微球标记R3E9单抗
以红色乳胶微球对p72单抗进行标记,标记步骤如下:(1)向2.0mL EP管加入1mL偶联缓冲液(50mM MES,pH=6.0),再向其中加入0.125mL微球(4%固含量)悬浮液。超声混匀,15℃,20000g离心10min,弃上清;(2)加入1mL偶联缓冲液,超声混匀后,向EP管中加入3.5μLEDC溶液(10mg/mL EDC),涡旋混匀,再加入33μL NHS溶液(10mg/mL NHS),超声混匀;(3)将EP管放于旋混合仪上,37℃,40r/min,活化20min,15℃,20000g离心10min,去除上清;(4)活化乳胶微球经偶联缓冲液洗涤一次后,并以偶联缓冲液重悬,并加入R3E9单抗(50μg),混匀后放于旋混合仪上,37℃,40r/min,偶联2h;(5)向EP管中加入0.5mL微球封闭液,涡旋混匀,将EP管放于旋混合仪上,37℃,40r/min,封闭1h;(6)封闭后的乳胶微球,以微球洗涤液洗涤两次,最后向EP管中加入0.5mL微球保存液(微球终浓度1mg/mL),混匀,4℃保存备用。
3.3层析试纸条的优化与组装
将玻璃纤维裁剪成宽度为2.2cm的长条,然后用样品垫处理液浸润,在37℃烘箱干燥5h,即为抗原检测试纸条的样品垫。将未处理的玻璃纤维裁剪成宽度为4mm的长条,放入实施实例3.2制备的乳胶微球标记物溶液中充分浸润,然后将润湿的玻璃纤维在室温环境中自然干燥,即为抗原检测试纸条的结合垫。
分别选取200nm、300nm、400nm粒径的乳胶微球对p72单抗进行标记,在NC膜上对检测线(p72蛋白多抗)和质控线(葡萄球菌A蛋白)单独喷膜后,初步组装试纸条。在试纸条上滴加杆状病毒表达的p72蛋白,结果如图所示,C线(图8a)和T线(图8b)均出现条带,但300nm直径的乳胶标记物显色效果最好并且条带清晰。因此,乳胶微球最佳直径为300nm。
以葡萄球菌A蛋白(SPA)作为纸条的质控线(C线)。将SPA蛋白浓度分别稀释为0.125mg/mL、0.25mg/mL、0.5mg/mL,将各个浓度的SPA蛋白分别喷涂在NC膜上,在室温下自然晾干,初步组装试纸条。在样品垫上滴加稀释后的p72抗原,如图9所示,不同浓度SPA的C线均显示清晰条带,但以0.5mg/mL的条带颜色最深。因此,选择C线喷膜浓度为0.5mg/mL的SPA。
以实验室制备的p72蛋白多抗为检测线(T线),将多抗分别稀释为0.5mg/mL、0.75mg/mL、1mg/mL、1.25mg/mL,将各个浓度的多抗分别喷涂在NC膜上,并在室温下自然晾干,初步组装试纸条。在样品垫上滴加利用杆状病毒表达制备的p72抗原,结果如图10所示,不同浓度p72多抗的T线均出现条带,但以多抗浓度为1.25mg/mL时,试纸条显色效果最佳。因此,确定p72多抗的喷膜浓度为1.25mg/mL。
将喷涂好检测线和质控线的NC膜粘贴在PVC底板上,将吸水垫裁剪成宽度为1.7cm的长条,然后将结合垫、吸水垫依次粘贴在NC膜两侧,再将样品垫粘贴在结合垫上方,各个垫彼此重叠2mm(图11)。将组装好的试纸长条用切割机裁剪成宽度为4mm的试纸条,装入塑料外壳中,在真空包装袋中密封保存备用。
3.4ASFV抗原检测试纸条检测结果判定标准
将ASFV灭活病毒用滴加在加样口,室温静置5-10min,观察试纸条显色情况。当试纸条T线和C线均显示为红色时,判定为阳性;当试纸条T线不显色,C线为红色时,判定为阴性;当试纸条T线和C线均不显色,检测结果无效。
3.5ASFV抗原检测试纸条特异性与灵敏性
将ASFV灭活病毒以及PRV、PRRSV、PEDV、PCV、PDCoV的细胞培养上清分别经样品稀释液稀释后滴加于试纸条加样口,室温静置5-10min,结果如图12所示,只有滴加了ASFV灭活病毒的试纸条呈现阳性结果,滴加了PRV、PRRSV、PCV、PEDV的细胞培养上清的试纸条显色结果为阴性,表明抗原检测试纸条具有良好的特异性。
将杆状病毒表达的p72蛋白分别稀释至浓度为512ng/mL、256ng/mL、128ng/mL、64ng/mL、32ng/mL、16ng/mL、8ng/mL、4ng/mL、2ng/mL、1ng/mL、0.5ng/mL、0.25ng/mL、0.125ng/mL,滴加于抗原检测试纸条的加样口,室温静置5-10min,结果如图13所示,当样品中p72抗原浓度≥1ng/mL时,试纸条T线显色,当检测样品中的p72抗原浓度为0.5ng/mL时,试纸条T线不显色,表明抗原检测试纸条的检出限为1ng/mL。
通过上述实施实例,本发明制备了一株ASFV p72蛋白单克隆抗体杂交瘤细胞株R3E9,确定了该株单抗的可变区序列。该株单抗,可用于IFA,识别杆状病毒表达的p72蛋白和ASFV感染细胞中的p72蛋白;也可用于western blotting,识别变性的p72蛋白。特别的是,以乳胶微球标记该株单克隆抗体,可用于制备检测ASFV抗原的乳胶层析试纸条。
Claims (10)
1.一株非洲猪瘟病毒p72单克隆抗体,其特征在于,所述抗体或其功能性片段具有重链可变区和轻链可变区,其中,所述重链可变区的互补决定区HCDR1、HCDR2和HCDR3的氨基酸序列和所述轻链可变区的互补决定区LCDR1、LCDR2和LCDR3的氨基酸序列为:HCDR1如SEQID NO.11所示,HCDR2如SEQ ID NO.13所示,HCDR3如SEQ ID NO.15所示;
LCDR1如SEQ ID NO.27所示,LCDR2如SEQ ID NO.29所示,LCDR3如SEQ ID NO.31所示。
2.根据权利要求1所述的抗体或其功能性片段,其特征在于,所述重链可变区和所述轻链可变区的氨基酸序列为:所述重链可变区如SEQ ID NO.9所示,所述轻链可变区如SEQ IDNO.25所示。
3.根据权利要求2所述的抗体或其功能性片段,其特征在于,所述抗体的轻链恒定区为κ型轻链恒定区或λ型轻链恒定区。
4.根据权利要求2所述的抗体或其功能性片段,其特征在于,所述抗体的重链恒定区为IgA、IgD、IgE、IgG或IgM抗体的重链恒定区。
5.根据权利要求1-4任一项所述的抗体或其功能性片段,其特征在于,所述功能性片段为Fab、Fab’、F(ab’)2、Fv或ScFv片段。
6.一种分离的核酸分子,其特征在于,其编码如权利要求1-5中任一项所述的抗体或其功能性片段。
7.含有权利要求6所述的核酸分子的重组载体。
8.含有权利要求7所述的重组载体的重组细胞。
9.如权利要求1-5中任一项所述的抗体或其功能性片段、如权利要求6所述的核酸分子、如权利要求7所述的重组载体或权利要求8所述的重组细胞在制备竞争ELISA检测非洲猪瘟病毒药物中的应用,所述应用为非疾病的诊断和治疗用途。
10.一种ASFV抗原乳胶层析检测试纸条,其特征在于所述乳胶层析检测试纸条中包被权利要求1所述的非洲猪瘟病毒p72单克隆抗体。
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