CN116444653A - 一株阻断性非洲猪瘟病毒单克隆抗体杂交瘤细胞株的制备与应用 - Google Patents
一株阻断性非洲猪瘟病毒单克隆抗体杂交瘤细胞株的制备与应用 Download PDFInfo
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Abstract
本发明提供了一株阻断性非洲猪瘟病毒单克隆抗体杂交瘤细胞株的制备与应用,确定了该株单抗的可变区序列。该株单抗,可用于IFA,识别真核细胞中表达的p30蛋白;也可用于western blotting,识别变性的p30蛋白。特别的是,ASFV阳性猪血清能抑制该株单抗与p30蛋白结合,显示该株单抗可用于建立p30抗体竞争ELISA检测方法。
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p30单克隆抗体的制备及其在检测p30蛋白和建立p30抗体竞争ELISA检测方法中的应用。
背景技术
非洲猪瘟(African swine fever,ASF)是由非洲猪瘟病毒(African swine fevervirus,ASFV)感染猪引起的一种烈性、高度接触性传染病,病死率可高达100%,是危害世界养猪业的一种重要传染病,世界动物卫生组织(OIE)将其列为法定报告动物疫病。自2018年8月我国辽宁沈阳猪场出现首例ASF疫情以来,一年时间内,我国32个省、市、自治区均爆发ASF疫情,给我国养猪业造成了迄今最为惨重的经济损失。随着ASFV流行时间延长,自2020年下半年以来,我国非洲猪瘟流行出现新情况,即出现非洲猪瘟病毒低毒力流行株。ASFV低毒力株感染严重迟缓育肥猪生长,引致种猪繁殖障碍,且感染潜伏期长,临床症状早期不易觉察,可在猪群中悄无声息传播,晚期可出现关节炎、皮肤坏死等典型症状,危害严重,防控难度大。ASF已成为我国各猪场全力防控的首要疫病,我国已将其列为一类动物疫病,亟需有效的技术方法和防控措施来控制ASFV在我国流行。
ASFV是一种有囊膜、线性双链DNA病毒,是非洲猪瘟病毒科非洲猪瘟病毒属的唯一成员。其病毒粒子结构较为复杂,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(outerenvelope)。ASFV基因组全长为170-194kbp,由中央保守区和两端可变区组成,编码151-167个开放阅读框(Open reading frame,ORF)。通过质谱分析,在体外培养的细胞中,可以检测到94种病毒蛋白的表达。而成熟的病毒粒子,则发现多达68种病毒蛋白,其中未知功能蛋白达20多种。在众多ASFV蛋白中,p30是ASFV编码的一种早期蛋白。在ASFV感染猪只中,p30抗体生成时间早,形成水平高,是检测ASFV感染的重要靶标。
ASFV感染猪的临床症状与感染毒株的毒力密切相关:强毒株引致急性型ASF,猪感染后4-10d几乎100%死亡,以高烧、皮肤出血和内脏广泛出血与水肿为主要特征;中等毒力毒株引致亚急性型,潜伏期延长,7-20d出现死亡,死亡率为30%-70%,除血管出现明显的出血和水肿外,临床症状与急性病例相似,但程度较轻;低毒力毒株则引致慢性型,可持续5至12个月,不引起体内出血,生长迟缓甚至消瘦,晚期出现临床症状,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,也能引致母猪流产[5]。慢性型患猪在持续感染的过程中,具有传染性,能使接触的易感动物感染[6]。亚急型和慢性型感染可出现一些康复猪,但ASF康复猪长期带毒,并能传播病毒[4,7]。
2018年ASFV传入我国,感染猪均呈现急性型症状,病程快,死亡率高。随着ASFV流行时间延长,自2020年下半年以来,我国非洲猪瘟流行出现新情况,即出现非洲猪瘟病毒低毒力流行株。ASFV低毒力株感染严重迟缓育肥猪生长,引致种猪繁殖障碍,且感染潜伏期长,临床症状早期不易觉察,可在猪群中悄无声息传播,晚期可出现关节炎、皮肤坏死等典型症状,危害严重,防控难度大。
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。在慢性型ASF中,抗体检测显现出明显优势。低毒力ASFV感染,虽然临床症状不易觉察,但能诱导猪只产生特异性的病毒抗体。特别是p30抗体,在感染猪中7-10天就可检测出,是早期检测低毒力ASFV感染的重要靶标。
本发明通过大肠杆菌表达***制备p30蛋白,以p30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p30单克隆抗体的杂交瘤细胞株。该杂交瘤细胞株分泌的单抗,可识别真核细胞中表达的p30蛋白,也能用于western blotting中识别p30蛋白,且在竞争ELISA检测中,阳性猪血清能高效阻断该单克隆抗体与p30蛋白结合,显示该单克隆抗体可应用于建立p30抗体阻断ELISA,为我国ASFV血清学检测技术开发提供坚实基础。
发明内容
本发明的目的提供一株ASFV p30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,能识别真核细胞中的p30蛋白和用于western blotting检测p30蛋白,ASFV阳性猪血清对该单克隆抗体与p30抗原结合具有抑制作用,能用于建立ASFV抗体竞争ELISA检测方法。
此外,本发明还提供了一种p30的制备方法及应用。
此外,本发明还提供了一种p30抗体竞争ELISA方法。
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p30基因克隆进原核表达载体pCold I中,经16℃诱导表达,表达的p30蛋白包涵体经变性-柱纯化-复性,获得了纯化的p30蛋白。以p30蛋白免疫小鼠,取产生高水平抗体小鼠的脾细胞与SP2/0细胞融合,通过p30抗体ELISA筛选阳性克隆杂交瘤,并通过稀释法获得纯化的杂交瘤细胞克隆。通过ASFV阳性猪血清的竞争ELISA检测,获得了一株能分泌与ASFV阳性血清竞争性抗体的杂交瘤细胞株。
本发明中使用的p30蛋白,是p30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。
本发明中使用的包被抗原p30蛋白,经复性缓冲液(50mM Tris-HCl,100mM NaCl,1%甘氨酸,5%甘油,0.2% PEG 3350,1mM氧化型谷胱甘肽,1mM还原型谷胱甘肽)透析复性后,具备良好的生物活性,与不同ASFV阳性猪血清均能高水平亲和作用,是一种优异的ELISA诊断抗原。
本发明中使用的p30抗体竞争ELISA方法为:每孔p30蛋白包被量为20ng;0.05M碳酸盐缓冲液为包被液,4℃包被过夜;5%脱脂乳为封闭液,37℃封闭时间1h;ASFV阳性血清作1:20倍稀释,37℃孵育1h;杂交瘤细胞培养上清37℃孵育1h;酶标羊抗小鼠抗体稀释度为1:1×104,37℃孵育1h;TMD室温下显色10min,建立了p30抗体间接ELISA方法。
附图说明
图1p30 PCR扩增产物电泳图;M:Marker;1.ASFV p30基因片段;
图2pCold-p30的XhoI-XbaI酶切鉴定M:Marker;1.pCold-p30;
图3p30蛋白SDS-PAGE电泳结果;
图4ASFV阳性猪血清对2-3株单抗的抑制作用;
图5 2-3株单抗识别WB中的p30蛋白;
图6间接免疫荧光(IFA)分析2-3株单抗识别真核表达的p30蛋白。
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。而ASFV低毒力株在我国流行,则需要大量准确可靠的血清学诊断试剂盒可供使用。本发明通过原核表达***制备p30蛋白,以p30蛋白免疫小鼠制备单克隆抗体。通过ELISA和竞争ELISA筛选及亚克隆纯化,获得了一株分泌p30单克隆抗体的杂交瘤细胞株。该杂交瘤细胞株分泌的单抗,可识别真核细胞中表达的p30蛋白,也能用于western blotting中识别p30蛋白,且与ASFV阳性猪血清中抗体具有高度竞争性,能用于建立p30抗体竞争ELISA检测方法。本发明涉及的p30单克隆抗体细胞株及应用,为我国ASFV检测技术开发提供坚实的物质和技术支撑。
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;ASFV基因序列,委托通用生物***(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;HRP标记羊抗猪,sigma公司;ELISA酶标板,购自深圳市金灿华实业有限公司。
实施案例1.p30蛋白的表达与纯化
1.1p30蛋白原核表达载体的构建
根据ASFV基因II型SY18株(GenBank:MH766894)p30蛋白的基因序列,设计合成为引物ColdI-p30F/ColdI-p30R(ColdI-p30F:5-CCctcgagATGGATTTTATTTTAAATATATCCATG-3(SEQ ID NO:1);ColdI-p30R:5-GCtctagaTTATTTTTTTTTTAAAAGTTTAATAACCATG(SEQ ID NO:2))。以本实验室保存p30质粒为模板,进行PCR扩增。50μl的PCR体系所含组分如下:2ⅹGCBuffer,25μl;dNTP,4μl;ColdI-p30F、ColdI-p30R(10μM)各1μl(10μM);Primestar高保真DNA聚合酶,0.5μl;质粒模板,1μl;ddH2O,17.5μl。PCR反应参数为:95℃预变性3min;95℃变性30s,58℃退火30s,72℃延伸40s,30个循环;72℃延伸7min。PCR扩增产物经核酸电泳显示(图1),获得一超过500bp的条带。以胶回收试剂盒回收纯化该条带,以XhoI和XbaI双酶切该回收片段和pColdI载体。酶切产物回收后,以T4 DNA连接酶将片段与载体进行连接,连接产物转化Top10感受态细胞,涂布含有氨苄青霉素筛选抗性平板,挑取单克隆菌落,至氨苄青霉素液体LB培养基摇菌过夜。用质粒提取试剂盒提取质粒,以XhoI和XbaI双酶切鉴定重组质粒(图2),并将酶切阳性克隆送擎科生物公司测序。鉴定正确的阳性质粒命名为pCold-p30。
1.2p30蛋白的制备
将pCold-p30转化BL21感受态,取100μL涂布到LB固体培养基上(氨苄抗性),37℃恒温培养箱中倒置培养12-16h。
在重组菌转化平板上挑取单菌落,小量培养,并加入诱导剂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1mLPBS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。蛋白电泳结果显示,p30蛋白在细菌中主要以包涵体形式表达。
将平板上的重组菌单菌落,挑取至装有5mL LB液体(含氨苄100μg/mL)的试管中,在37℃恒温摇床37℃恒温摇床培养过夜。将过夜培养的菌液按1:100的比例转接至一个装有200mL LB液体(含氨苄100μg/mL)的烧瓶中,进行细菌培养和蛋白诱导表达。离心收集菌体,并以PBS重悬菌体洗涤一次,再以30ml PBS重悬,并置于冰上超声破碎,300W,超3s、停3s,工作20min。将超声的样品在4℃以1500g离心20min,弃上清,保留沉淀。以30mL包涵体洗涤缓冲液(50mM Tris-HCl,2mM EDTA,100mM NaCl,0.5% TritonX-100(v/v),2M尿素,pH7.5)重悬,150W,超3s、停3s,超声3min,4℃、1500g离心10min,弃上清。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)重悬沉淀,室温放置30min,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p30蛋白,分管收集,1mL/管。以SDS-PAGE分析蛋白纯度,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(50mM Tris-HCl,100mM NaCl,1%甘氨酸,5%甘油,0.2% PEG 3350,1mM氧化型谷胱甘肽,1mM还原型谷胱甘肽)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2.1mg/mL,将p30蛋白分装后,-80℃保存备用。
实施案例2.p30蛋白单克隆抗体杂交瘤细胞株的制备
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p30蛋白皮下免疫接种。初免后21天,以弗氏不完全佐剂乳化的p30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p30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1:32000稀释倍以上。二免后21天,选择2只p30抗体水平高的小鼠,通过尾静脉注射p30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,阳性孔杂交瘤细胞进行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。
2.3具有竞争性单克隆抗体的杂交瘤细胞株筛选
在本实验室建立的p30抗体间接ELISA基础上稍加修改,建立p30抗体竞争ELISA,即:p30蛋白20ng/孔包被的酶标板,1:20稀释的灭活ASFV阳性猪血清和阴性猪血清以100μL/孔分别加入不同孔中,一列阳性血清间隔一列阴性血清,37℃孵育1h。经PBST洗涤后,加入杂交瘤细胞上清,37℃孵育1h。经PBST洗涤后,加入hrp标记的羊抗鼠抗体,37℃孵育1h。PBST洗涤后,加入TMD室温作用15min,2M的浓硫酸终止反应,酶标仪测定OD450值。竞争ELISA检测结果显示:一株杂交瘤细胞2-3的阴性猪血清/阳性猪血清(N/P)的值为3.12,显示出该株杂交瘤细胞分泌单抗与猪血清中的ASFV抗体有较好的竞争抑制作用。实施案例3.2-3株单克隆抗体的生物学特性
3.1不同猪血清对2-3株单克隆抗体抑制效果检测
将8份灭活ASFV阳性猪血清和8份阴性猪血清均作1:20稀释,应用实施实例2.3中的竞争ELISA方法进行检测,结果如图4所示,阳性样品与阴性样品OD值存在极显著差异,平均OD值N/P为4.25。这表明所测不同阳性血清对2-3株单抗均具有良好的抑制作用。
3.2 2-3株单克隆抗体对p30蛋白的识别
将制备的p30蛋白进行SDS-PAGE,随后转膜,以2-3株单抗作为一抗,进行westernblotting分析。如图5所示,2-3株单抗识别变性电泳后的p30蛋白。
将p30蛋白基因序列克隆入真核表达载体p3×Flag-CMV-7.1中,构建了重组表达质粒pFlag-P30。将pFlag-P30及p3×Flag-CMV-7.1转染293T细胞。转染后24h,固定细胞,以2-3株单抗作为一抗进行间接免疫荧光(IFA)分析。结果如图6所示,2-3株单抗特异性识别真核细胞中表达的p30蛋白。
3.3 2-3株单克隆杂交瘤的稳定性
将2-3株单克隆杂交瘤细胞株连续传代10代,计数第10代细胞数量,取150个细胞分种至96孔板。培养10天后,随机取22孔细胞上清,进行间接ELISA检测。各孔上清OD450值如表,所有孔均为阳性。这表明,2-3株杂交瘤细胞能稳定分泌p30抗体。
表1.二十二孔杂交瘤细胞的OD450值
3.4 2-3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2-3株单克隆抗体的亚型。按照试剂盒说明书,将2-3株培养上清进行鉴定。试剂盒测定的值(表2)显示,该株单抗重链为IgG2b亚型,轻链为Kappa亚型。
表2.2-3株单抗亚型鉴定
3.5 2-3株单克隆抗体亚型鉴定
培养于6孔板中的2-3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-GAGGTGCAACTGGTGGAGTCTGGGGGAGACTTAGTGAATCCTGGAGGGTCCCTGAAA CTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGTTATGGCATGTCTTGGTTTCGCCAGACTCCAGACAAGAGGCTGGAGTGGGTCGCAACCATTAGTAGTGGTGATTACACCTACTATCCAGACAGTGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACACCCTGTATCTGCAAATGAGCAGTCTGAGGTCTGAGGACACAGCCATGTATTACTGTGCAAGATTCTATGGTAACGACTTTGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA-3(SEQ ID NO:3)。其中,
骨架区(FR1):5-GAGGTGCAACTGGTGGAGTCTGGGGGAGACTTAGTGAATCCTGGAGGGTCCCTGAAA CTCTCCTGTGCAGCCTCT-3(SEQ ID NO:4);
互补决定区1(CDR1):5-GGATTCACTTTCAGTAGTTATGGC-3(SEQ ID NO:5);
FR2:
5-ATGTCTTGGTTTCGCCAGACTCCAGACAAGAGGCTGGAGTGGGTCGCAACC-3(SEQ IDNO:6);
CDR2:5-ATTAGTAGTGGTGATTACACC-3(SEQ ID NO:7);
FR3:
5-TACTATCCAGACAGTGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACA CCCTGTATCTGCAAATGAGCAGTCTGAGGTCTGAGGACACAGCCATGTATTACTGT-3(SE Q ID NO:8);
CDR3:5-GCAAGATTCTATGGTAACGACTTTGCTATGGACTAC-3(SEQ ID NO:9);
FR4:5-TGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA-3(SEQ ID NO:10)。
对应的氨基酸序列如下:
VH:
EVQLVESGGDLVNPGGSLKLSCAASGFTFSSYGMSWFRQTPDKRLEWVATISSGDYTYYPD SVKGRFTISRDNAKNTLYLQMSSLRSEDTAMYYCARFYGNDFAMDYWGQGTSVTVSS(SE Q ID NO:11);
FR1:EVQLVESGGDLVNPGGSLKLSCAAS(SEQ ID NO:12);
CDR1:GFTFSSYG(SEQ ID NO:13);
FR2:MSWFRQTPDKRLEWVAT(SEQ ID NO:14);
CDR2:ISSGDYT(SEQ ID NO:15);
FR3:YYPDSVKGRFTISRDNAKNTLYLQMSSLRSEDTAMYYC(SEQ ID NO:16);
CDR3:ARFYGNDFAMDY(SEQ ID NO:17);
FR4:WGQGTSVTVSS(SEQ ID NO:18)。
轻链可变区(VL)核苷酸序列如下:
5-GATGTTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGTCTTGTACACAGTAATGGAGACACCTACTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAACTCCTGATCTACAGAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAGTTCACATGTTCCGTGGACGTTCGGTGGAGGCACCAGGCTGGAAATCAAA-3(SEQ ID NO:19)。其中:FR1:5-GATGTTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCC ATCTCTTGCAGATCTAGT-3(SEQ ID NO:20);
CDR1:5-CAGAGTCTTGTACACAGTAATGGAGACACCTAC-3(SEQ ID NO:21)
FR2:5-TTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAACTCCTGATCTAC-3(SEQ IDNO:22)
CDR2:5-AGAGTTTCC-3(SEQ ID NO:23)
FR3:5-AACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCAC ACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGC-3(SEQ IDNO:24)
CDR3:5-TCTCAAAGTTCACATGTTCCGTGGACG-3(SEQ ID NO:25)
FR4:5-TTCGGTGGAGGCACCAGGCTGGAAATCAAA-3(SEQ ID NO:26)。
对应的氨基酸序列如下:
VL:DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGDTYLHWYLQKPGQSPKLLIYRVSNRFSG VPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSSHVPWTFGGGTRLEIK(SEQ ID NO:27)FR1:DVVMTQTPLSLPVSLGDQASISCRSS(SEQ ID NO:28)
CDR1:QSLVHSNGDTY(SEQ ID NO:29)
FR2:LHWYLQKPGQSPKLLIY(SEQ ID NO:30)
CDR2:RVS(SEQ ID NO:31)
FR3:NRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFC(SEQ ID NO:32)
CDR3:SQSSHVPWT(SEQ ID NO:33)
FR4:FGGGTRLEIK(SEQ ID NO:34)
通过上述实施实例,本发明制备了一株ASFV p30蛋白单克隆抗体杂交瘤细胞株2-3,确定了该株单抗的可变区序列。该株单抗,可用于IFA,识别真核细胞中表达的p30蛋白;也可用于western blotting,识别变性的p30蛋白。特别的是,ASFV阳性猪血清能抑制该株单抗与p30蛋白结合,显示该株单抗可用于建立p30抗体竞争ELISA检测方法。
尽管已用具体实施例来说明和描述了本发明,然而应意识到,在不背离本发明的精神和范围的情况下可以作出许多其它的更改和修改。因此,这意味着在所附权利要求中包括属于本发明范围内的所有这些变化和修改。
Claims (10)
1.一株阻断性非洲猪瘟病毒单克隆抗体或其功能性片段,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SEQ ID NO.13所示,HCDR2如SEQ ID NO.15所示,HCDR3如SEQ ID NO.17所示;
LCDR1如SEQ ID NO.29所示,LCDR2如SEQ ID NO.31所示,LCDR3如SEQ ID NO.33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.11所示,所述轻链可变区如SEQID NO.27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性或阻断性非洲猪瘟病毒检测药物中的应用,所述应用为非疾病的诊断和治疗用途。
10.如权利要求10所述的应用,所述应用具体为竞争性阻断ELISA。
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