CN116621976A - 抗PDCoV核衣壳蛋白的单克隆抗体2H7及其应用方法 - Google Patents
抗PDCoV核衣壳蛋白的单克隆抗体2H7及其应用方法 Download PDFInfo
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- CN116621976A CN116621976A CN202310806431.6A CN202310806431A CN116621976A CN 116621976 A CN116621976 A CN 116621976A CN 202310806431 A CN202310806431 A CN 202310806431A CN 116621976 A CN116621976 A CN 116621976A
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Abstract
本发明涉及生物技术领域,旨在提供一种抗PDCoV核衣壳蛋白的单克隆抗体2H7及其应用方法。该单克隆抗体包含抗体重链的Ig结构域VH CDR1、VH CDR2和VH CDR3,以及抗体轻链的Ig结构域VL CDR1、VL CDR2和VL CDR3;其中,前三者的氨基酸序列分别如SEQ ID NO:1~3所示,后三者的氨基酸序列分别如SEQ ID NO:4、SEQ ID NO:13、SEQ ID NO:5所示。该单克隆抗体为IgG1亚型,抗体的亲和力高,免疫能力强,具有稳定性好,活性高、亲和力强、灵敏度高、特异性强等优点;与PEDV和TGEV等其他猪肠道病毒无交叉反应,可用于PDCoV诊断试剂盒开发,能够有效进行PDCoV鉴别诊断的推广与应用。
Description
技术领域
本发明涉及生物技术领域,特别是涉及一种抗PDCoV核衣壳蛋白的单克隆抗体2H7及其应用。
背景技术
目前已发现的猪消化道冠状病毒传染性肠胃炎病毒(TGEV)、猪流行性腹泻病毒(PEDV)和猪代尔塔冠状病毒(PDCoV),常引起猪肠道疾病,导致呕吐、腹泻、脱水,发病率及死亡率较高。其中,PDCoV属于代尔塔冠状病毒属,为有囊膜的、不分节段的、单股正链RNA病毒,其基因组全长约25kb,包括5’端非编码区,3’端非编码区以及至少7个开放阅读框。其中开放阅读框编码主要的病毒蛋白,包括ORF1a/1b,纤突(Spike,S)蛋白、小膜(Envelope,E)蛋白、膜(Membrane,M)蛋白、非结构蛋白6(NS6)、核衣壳(Nucleocapsid,N)蛋白、非结构蛋白7(NS7)。
据研究,PDCoV与天然感染的临床病例相关,这些病例主要表现为仔猪严重腹泻,呕吐和脱水,以及典型的萎缩性肠炎的组织病理学损伤,发病率和死亡率高达50~100%,给养猪业造成严重的经济损失。该病毒感染在我国猪群中已经流行,其感染所导致的临床症状与PEDV和TGEV等其他猪肠道病毒感染症状非常相近,在临床上很难通过肉眼判断对其进行鉴别诊断,目前尚无可用于PDCoV治疗的有效药物或疫苗,也没有用于鉴别诊断PDCoV病毒感染的特异性抗体。
现有报道中,针对PDCoV的单克隆抗体主要是抗S蛋白单抗及抗N蛋白单抗。制备PDCoV单抗的抗原蛋白主要通过原核表达体系获得,但S蛋白其分子量较大,很难在体外表达体系中获得大量全长、保留原始构象、高纯度的蛋白,在其基础上制备的单克隆抗体存在与PDCoV毒株亲和力低、反应性差等问题。由原核表达体系获得的N蛋白活性、免疫原性和纯度较低也导致了其制备的单克隆抗体出现存在与PEDV和TGEV等其他猪肠道病毒交叉反应等特异性差、灵敏性低的问题。
发明内容
本发明要解决的技术问题是,克服现有技术中的不足,提供一种抗PDCoV核衣壳蛋白的单克隆抗体2H7及其应用。
为解决技术问题,本发明的解决方案是:
提供一种抗PDCoV核衣壳蛋白的单克隆抗体,所述单克隆抗体包含抗体重链的Ig结构域VH CDR1、VH CDR2和VH CDR3,以及抗体轻链的Ig结构域VL CDR1、VL CDR2和VL CDR3;其中,所述VH CDR1、VH CDR2和VH CDR3的氨基酸序列分别如SEQ ID NO:1~3所示;所述VLCDR1、VL CDR2和VL CDR3的氨基酸序列分别如SEQ ID NO:4、SEQ ID NO:13、SEQ ID NO:5所示。
作为本发明的优选方案,所述单克隆抗体为全长抗体、Fab抗体或F(ab’)2抗体。
作为本发明的优选方案,轻链可变区的氨基酸序列如SEQ ID NO.6所示,重链可变区的氨基酸序列如SEQ ID NO.7所示。
本发明进一步提供了编码前述单克隆抗体的基因,编码轻链可变区的基因序列如SEQ ID NO.8所示,编码重链可变区的基因序列如SEQ ID NO.9所示。
本发明还提供了前述任意一种单克隆抗体的应用方法,是以所述单克隆抗体制备用于检测PDCoV的试剂盒。
作为本发明的优选方案,是将所述试剂盒以血清学检测的方式加以使用。
本发明还提供了一种用于PDCoV检测的试剂盒,该试剂盒包括权利要求1或2所述的任意一种单克隆抗体。
作为本发明的优选方案,该试剂盒是ELISA检测试剂盒、使用免疫层析试纸条的免疫层析诊断试剂盒、免疫组化试剂盒中的任意一种,使用试剂盒时的检测方式为血清学检测。所取待检测样本为静脉血。
与现有技术相比,本发明的技术效果是:
1、本发明提供的抗PDCoV核衣壳蛋白的单克隆抗体为IgG1亚型,抗体的亲和力高,免疫能力强,具有稳定性好,活性高、亲和力强等优点。
2、本发明提供的单克隆抗体灵敏度高、特异性强,与PEDV和TGEV等其他猪肠道病毒无交叉反应;可以用于PDCoV诊断试剂盒开发,能够有效进行PDCoV鉴别诊断的推广与应用。
附图说明
图1为实施例1中表达及纯化的PDCoV N蛋白的SDS-PAGE及Western blot分析图。
图2为实施例2中PDCoV N蛋白免疫小鼠后血清抗体效价分析。1-5分别为5只不同小鼠的血清样本。
图3为实施例2中单克隆抗体的效价测定分析。
图4为实施例3中单克隆抗体的Western分析及特异性检测。
图5为实施例2中单克隆抗体在间接免疫荧光方法检测PDCoV中的应用。
图6为实施例2中单克隆抗体在间接免疫荧光方法中的特异性分析,该单抗不与PEDV病毒反应。
图7为实施例2中单克隆抗体在间接免疫荧光方法中的特异性分析,该单抗不与TGEV反应。
图8为实施例5中单克隆抗体在Western blot方法检测PDCoV中的应用。
具体实施方式
本发明通过真核表达体系获得纯度高、活性好、接近天然蛋白构象的PDCoV核衣壳蛋白,并免疫小鼠获得分泌特异性抗PDCoV核衣壳蛋白的单克隆细胞株。
“CDR”(complementarity-determining regions)在免疫学中叫做互补决定区或互补决定簇,它是Ig和TCR蛋白质的短片段,含有不同Ig或TCR间的大多数序列差异并能与抗原相接触。在本发明所述抗PDCoV核衣壳蛋白的单克隆抗体中,在每一条抗原受体多肽链上有3个CDR,每个完整Ig或TCR分子上有6个CDR,分别为VH CDR1、VH CDR2和VH CDR3以及VLCDR1、VL CDR2和VL CDR3,其中VH和VL分别指抗体重链和抗体轻链的Ig结构域。这些“超变”片段表现为环状结构,一起形成能与抗原的三维结构互补的表面。
实施例1:PDCoV N蛋白的制备
实验方法:
重组表达质粒构建:N蛋白基因序列如SEQ ID NO:10所示,使用特异性引物(F:GGGTTCCGGCAGCGGTTCTGCTGCACCAGTAGTCCCTACTACTGACG,序列SEQ ID NO.11;R:CGCTGCTGATTCCTGCTTTATCTC,序列SEQ ID NO.12),以病毒基因组cDNA为模板进行PCR,用同源重组方法将PCR纯化产物,插入到pFASTBac真核表达载体,获得有N蛋白基因序列的重组质粒pFASTBac-PDCoV-N。将测序验证正确的pFASTBac-PDCoV-N表达载体质粒转化至大肠杆菌DH10Bac中,涂布于含100μLX-gal和5μL IPTG的LB平板上,37℃过夜培养,进行蓝白斑筛选,挑选白色菌落于5mL含有三抗(卡那霉素、四环素和庆大霉素)的LB液体培养基中,振荡培养14h,取菌液做划线培养分离单菌落并保存。
真核表达:利用试剂盒抽提PDCoV N蛋白重组杆状病毒质粒,将1μL杆粒(质量小于5μg)加入200μL SF900Π培养基,将16μL转染试剂加入184μL SF900Π培养基,各放置5min后混合,孵育25min。期间,用无三抗的SF900Π培养基清洗SF9细胞两次。待孵育完成后,往细胞培养瓶中缓慢加入共孵育混合物,再补充无三抗的SF900Π培养基600μL。转染5h后换液,先用含有三抗的SF900Π培养基清洗细胞两次,再往培养瓶中加入5.5ml含有三抗的SF900Π培养基,每天观察细胞病变情况。5天后收获细胞冻融后离心取上清,得到P1代病毒储液,分装于1.5mL EP管中,每管500μL,置于-80℃保存。取1瓶75cm2方瓶的SF9细胞,中加入500μL P1代病毒储液,置于27℃烘箱中孵育,每天观察有无病变,3~5天后收获细胞,冻融后离心取上清,得到P2代病毒储液,分装于1.5mL EP管中,每管500μL,置于-80℃保存。采用同样的方法扩增P3代病毒储液。取2瓶75cm2方瓶的SF9细胞,将细胞转入含100mL SF 900ⅡSFM培养基的锥形瓶中培养,细胞计数板记下初始细胞密度,置于27℃培养箱中振荡培养,每12h进行细胞计数。待细胞密度达到1.2×106~2.0×106个细胞/mL时,向细胞中加入6mL P3代病毒储液,轻轻混匀,继续于27℃培养箱中振荡培养。72h左右收获细胞,冻融后离心取上清。
蛋白纯化:取2mL镍琼脂糖凝胶,加到12mL亲和层析柱空柱中,混匀镍柱填料。待镍柱内乙醇流尽后,向镍柱中加满50mM PBS洗涤缓冲液,平衡镍柱。将收集到的细胞上清小心的加入镍柱内,控制流速在5s一滴,收集流穿液。向镍柱中加三倍体积的5mM咪唑洗脱杂蛋白,收集洗脱液。再加入3mL 400mM咪唑,将镍柱内的琼脂糖凝胶吹匀,待镍琼脂糖沉淀后,收集洗脱下来的目的蛋白,分装,置于-20℃保存。
SDS-PAGE分析:配制SDS-PAGE胶;分别取40μLSF9细胞上清、过柱流穿液、5mM咪唑洗脱液、目的蛋白,加入10μL 5×Loading Buffer吹打混匀,在沸水锅内加热10min;组装好电泳槽,倒满电泳液,上样,按照先80V 30min后120V 90min的程序进行电泳;电泳结束后,将胶置于考马斯亮蓝染液中,37℃摇床上摇2h进行染色,后置于脱色液中脱色,每隔1h换一次脱色液,待条带清晰可见后置于凝胶成像仪上拍照观察结果。
Western blot分析:将SF9细胞上清、过柱流穿液、5mM咪唑洗脱液、目的蛋白进行SDS-PAGE电泳。转膜:剪去与需要转移的聚丙烯酰胺凝胶相同大小的PVDF膜,转膜前将PVDF膜在甲醇中激活1min后转入湿转缓冲液中浸泡,滤纸、海绵直接放入湿转缓冲液中浸泡。按照(+)厚海绵-薄滤纸-PVDF膜-胶-薄滤纸-厚海绵(-)顺序,200mA湿转1h。转膜结束后,将PVDF膜放入新配制的5%的脱脂奶粉封闭液中,37℃摇床封闭1h。封闭结束后,用TBST洗涤PVDF膜3次,每次5min。一抗孵育:用0.5%脱脂奶粉1∶2000稀释鼠源His单抗,4℃摇床过夜孵育。用TBST洗涤PVDF膜3次,每次5min。二抗孵育:用0.5%的脱脂奶粉1∶5000稀释HRP标记的羊抗鼠二抗,37℃摇床孵育1h。用TBST洗涤PVDF膜3次,每次5min。加200μL显色液,置于凝胶成像仪进行拍照记录实验结果。
实验结果:
成功获得PDCoV N基因片段(1029bp),并用融合PCR将蜂素信号肽及His标签融合后连接至pFASTBac载体上,经PCR及测序验证,获得重组pFASTBac-PDCoV N质粒。成功将质粒导入大肠杆菌DH10Bac中,经蓝白斑筛选后及PCR测序,获得重组大肠杆菌DH10Bac-pFASTBac-PDCoV N。抽取杆粒导入SF9细胞,成功获得P1、P2、P3代次的杆状病毒,并获得了大量表达PDCoV N蛋白的SF9细胞上清。将获得的细胞上清进行镍柱纯化,获得纯度较好的PDCoV N蛋白,大小约为42kDa,蛋白浓度为126μg/mL。蛋白SDS-PAGE考马斯亮蓝染色及Western Blot结果见附图1。获得的PDCoV N蛋白可用于后续单克隆抗体制备。
实施例2:PDCoV N单克隆抗体的制备
实验方法:
动物免疫:将纯化得到的重组PDCoV N蛋白与FCA等体积乳化后,背部皮下多点免疫小鼠。两周后换用FIA佐剂等体积乳化后免疫小鼠,间隔两周后进行第三次免疫,同时测定小鼠血清抗体效价。细胞融合前3天,腹腔注射重组PDCoV N蛋白用以冲击免疫。
细胞融合:获取免疫小鼠的新鲜血液和脾脏。将血液分离得到血清,用作单抗筛选时的阳性对照。将脾脏放入六孔板的一个孔中,清洗并去除脾脏外的组织、筋膜和脂肪。将洗净的脾脏放于铜网上。用灭菌过的试管碾压脾脏,直至脾脏被碾碎,滤过铜网。将过滤后的细胞转移至离心管中,1200rpm,28℃离心10min。将四个75cm2方瓶的SP2/0细胞用1640培养基重悬,转移至离心瓶中,1200rpm,28℃离心10min。用培养基将上述两个步骤获得的细胞重悬后合为一瓶,1200rpm,28℃离心10min。再用新的培养基重悬离心重复清洗一次后弃去上清。将离心管悬置在37℃水浴中,1min内滴入37℃预热的PEG1450,边滴边摇动离心管,静置30s后,1min内滴入1mL 1640培养基,静置30s,之后在1min内分别滴入2mL、3mL、4mL、5mL、6mL 1640培养基,分别静置30s以终止融合。1200rpm,28℃离心10min,弃去上清,加入1640筛选培养基(含有三抗+20%血清+50×HAT)重悬,分入10个96孔板中,37℃培养。
阳性杂交瘤细胞筛选及单克隆化:用建立的ELISA检测方法筛选阳性杂交瘤细胞。用2.5μg/mL的重组PDCoV N蛋白4℃包被过夜。次日弃去包被液,用PBST洗涤3次,每次3min。每孔加入200μL封闭液,37℃封闭2h后弃去。用PBST洗涤3次,每次3min。将杂交瘤培养上清分别加入检测酶标板对应孔,l00μL/孔,37℃孵育1h后弃去液体,用PBST洗涤3次,每次3min。用阳性和阴性小鼠血清4000倍稀释分别作为阳性和阴性对照。用二抗稀释液1∶5000稀释羊抗小鼠酶标二抗,以100μL/孔加入酶标板,37℃反应1h,用PBST洗涤3次,每次3min。每孔加入l00μL TMB显色液37℃避光反应10min。每孔加入50μL 2M H2SO4终止显色,测定450nm处吸光度值,筛选阳性孔细胞。
用含HAT的完全培养基将阳性孔细胞重悬,进行细胞计数,按单克隆化稀释后加入96孔板,37℃培养10天。观察细胞生长情况,每3-4天时加培养基50μL,在第10天时吸取100μL细胞上清进行ELISA检测,初步确定阳性单克隆细胞株,进行保存。
单抗腹水制备及效价测定:选取4周龄的雌性BALB/c小鼠,腹腔注射1mL灭菌的液体石蜡。约一周后取长满两个75cm2方瓶的单克隆细胞株,重悬进行细胞计数。用1640培养基配成约2×106个/mL的细胞悬液。给注射了液体石蜡的小鼠腹腔注射杂交瘤细胞悬液,每只小鼠腹腔注射1mL。接种后7天左右可见小鼠腹部明显膨大,此时可由腹腔抽取腹水。抽取的腹水13000rpm/min离心10min,收集上清,-20℃保存备用。将小鼠腹水用一抗稀释液从1∶2000倍比稀释到1∶2048000。按所建立的ELISA方法进行腹水效价的测定。
实验结果:
PDCoV N免疫小鼠后,小鼠血清抗体水平显著上升,检测结果如附图2所示。取免疫小鼠的脾细胞与瘤细胞进行细胞融合后,成功筛选得到PDCoV N单克隆抗体,命名为2H7。获得小鼠腹水10mL,经ELISA检测腹水效价达到1∶1024000,结果如附图3所示。
实施例3:单克隆抗体的特异性分析
实验方法:
取PDCoV病毒液20μL,分别加入5μL 5×Loading buffer,于水浴锅中煮样10min。将蛋白样品进行SDS-PAGE电泳;以阳性单克隆化杂交瘤细胞上清作为一抗,羊抗鼠HRP标记抗体作为二抗,进行Western blot及分析。以PEDV及TGEV病毒液作为对照,检验杂交瘤细胞分泌抗体的特异性。
实验结果:
如附图4所示,该单抗可与PDCoV结合产生特异性反应,且不与PEDV和TGEV反应。
实施例4:单克隆抗体在间接免疫荧光方法检测PDCoV中的应用
实验方法:
96孔细胞汇合度达到80%时,弃去旧培养基,用缓冲液洗涤两次,加入病毒液,每孔100μL,37℃培养箱培养22h后弃去旧培养基。每孔加入50μL 4%多聚甲醛,在室温固定30min。弃去固定液,用PBS轻轻洗涤两遍,每孔加入50μL 0.2%Triton X-100,室温透化15min。弃去透化液,用PBS轻轻洗涤两遍,每孔加入200μL 5%脱脂奶粉,37℃封闭2h。弃去封闭液,每孔加入1:500稀释的单克隆抗体腹水作为一抗,37℃孵育1h,以小鼠PDCoV多抗血清作一抗对照。弃去上清,用PBS轻轻洗涤两遍,加入1∶1000稀释的FITC标记的荧光二抗,每孔50μL,于37℃避光孵育1h。加入1∶200稀释的DAPI,每孔50μL,室温染核2min,用PBS轻轻洗涤两遍,进行荧光显微镜观察。
实验结果:
如附图5所示,单克隆抗体2H7可与病毒结合,显示病毒在细胞内的分布情况,且用小鼠PDCoV多抗血清作为一抗进行IFA检测背景较多噪点,且非特异性荧光较多,以单抗作为一抗进行IFA检测时结果更特异、清晰。附图6、附图7为PEDV及TGEV感染细胞对照试验,可见PEDV及TGEV的细胞样本中无荧光信号产生,说明该单克隆抗体特异性较好,可用于PDCoV等猪消化道病毒的鉴别诊断。
实施例5:单克隆抗体在Western blot方法检测PDCoV中的应用
实验方法:
取PDCoV病毒液、PDCoV N蛋白各20μL,分别加入5μL 5×Loading buffer,于水浴锅中煮样10min。将蛋白样品进行SDS-PAGE电泳,以带有his标签的无关蛋白作为对照;以1:500稀释的单抗腹水作为一抗,以小鼠PDCoV多抗血清作一抗对照。羊抗鼠HRP标记抗体作为二抗,进行Western blot分析。
实验结果:
如附图8所示,单克隆抗体2H7可与全病毒及N蛋白结合反应,显示病毒N蛋白在细胞内的表达情况,且不与无关蛋白反应。以单抗作为一抗时的Western结果比用小鼠PDCoV多抗血清作为一抗时条带更特异、清晰。
实施例6:单克隆抗体编码基因序列分析
实验方法:
用试剂盒提取抗PDCoV N单克隆抗体2H7的杂交瘤细胞的总RNA,逆转录成cDNA。根据抗体重链和轻链的恒定区设计2对简并引物,分别进行PCR扩增,目的片段回收并克隆至T载体测序。
实验结果:
解析得到单克隆抗体2H7包含VH CDR1、VH CDR2和VH CDR3以及VL CDR1、VL CDR2和VL CDR3,其中所述VH CDR1、VH CDR2和VH CDR3的氨基酸序列分别如SEQ ID NO:1~3所示,所述VL CDR1、VL CDR2和VL CDR3的氨基酸序列分别如SEQ ID NO:4、SEQ ID NO:13、SEQ IDNO:5所示。轻链可变区氨基酸序列如SEQ ID NO.6所示,重链可变区的氨基酸序列如SEQ IDNO.7所示;编码轻链可变区的基因序列如SEQ ID NO.8所示;编码重链可变区的基因序列如SEQ ID NO.9所示。
实施例7:单克隆抗体的具体应用示例
按下述配方制备ELISA试剂盒:
1)10mM pH 7.4PBS缓冲液
NaCl 8g,KCl 0.2g,Na2HPO4·12H2O 3.58g,KH2PO4 0.27g,加去离子水至800mL,调pH至7.4,定容至1L。
2)10mM pH7.4 PBST缓冲液
10mM pH7.4 PBS缓冲液,每升加入tween-20 500μL。
3)封闭液
取灭菌的10mM pH7.4 PBS缓冲液,加入各组分终浓度为蔗糖0.05g/mL、proclin-300 0.1%、BSA0.02 g/mL。过滤除菌,无菌分装。
4)一抗稀释液
取10mM pH7.4 PBS缓冲液,加入终浓度为0.05g/mL甘露醇、0.05g/mL蔗糖、0.1%proclin-300、0.01g/mL BSA。
5)酶标二抗稀释液
取10mM pH7.4 PBS缓冲液,加入终浓度为0.05%吐温-20(Tween-20)、0.1%proclin-300、0.01g/mL BSA。
6)CBS包被液
取一粒CBS胶囊剂,溶于100mL去离子水中,震荡混匀。
7)终止液
8%浓硫酸111mL,去离子水899mL。
8)0.1%酪蛋白盐
取灭菌的10mM pH7.4 PBS缓冲液,加入0.1%的酪蛋白盐粉末,沸水浴至粉末溶解。
将该试剂盒以下述方式进行使用:
1)用pH 9.6的碳酸盐缓冲液(CBS)稀释的PDCoV多克隆抗体,100μL/孔包被酶标板,4℃包被14h;
2)弃去包被液,用PBST洗板3次,每次3min,最后一次拍干;
3)加入封闭液,200μL/孔,置于湿盒中37℃封闭2h;
4)弃去封闭液,拍干;加入用一抗稀释液稀释的待检样品,100μL/孔,同时设2孔阳性对照和阴性对照。置于湿盒中37℃孵育60min;
5)弃去孔中液体,用洗涤液洗板3次,每次3min,最后一次拍干;
6)加入用一抗稀释液稀释的PDCoV单克隆抗体,100μL/孔,置于湿盒中37℃孵育30min;弃去孔中液体,洗涤液洗板3次,最后一次拍干;
7)加入用酶标二抗稀释液稀释的HRP标记抗猪IgG抗体,100μL/孔,置于湿盒中37℃孵育30min;弃去孔中液体,洗涤液洗板3次,最后一次拍干;
8)加入TMB显色液(A液、B液各50μL),100μL/孔,37℃避光显色10min;
9)加入反应终止液,50μL/孔,混匀后立即测定OD450nm值。
实施例8:单克隆抗体的具体应用示例
按下述配方制备免疫层析胶体金快速检测试剂盒:
1)包被液
取50mM pH 9.6的CBS缓冲液,加入3%甲醇。
2)NC膜封闭液
取10mM pH 7.4PBS缓冲液,加入0.5%Tween 20、0.5%PEG2000、2%BSA和0.02%NaN3。
3)金标抗体稀释液
取10mM硼酸盐缓冲液(BB,pH 8.2),加入8%蔗糖、2%BSA和0.05%NaN3的。
4)免疫层析胶体金胶体金免疫层析试纸条
检测线(T线)包被PDCoV多克隆抗体,质控线C线包被羊抗鼠IgG,NC膜上C线、T线从上往下依次排列,金标记的PDCoV单抗2H7固定于金标垫。
将该试剂盒以下述方式进行使用:
检测时取适量待测样本滴于加样孔,若样本中含有PDCoV病毒,会与包被于T线的多抗结合,并与相应的金标2H7单克隆抗体结合,从而显色。
实施例9:单克隆抗体的具体应用示例
按下述配方制备免疫组化试剂盒:
1)10mM pH 7.4PBS缓冲液
NaCl 8g,KCl 0.2g,Na2HPO4·12H2O 3.58g,KH2PO4 0.27g,加去离子水至800mL,调pH至7.4,定容至1L。
2)10mM pH7.4 PBST缓冲液
10mM pH7.4 PBS缓冲液,每升加入tween-20 500μL。
3)固定液
4%多聚甲醛。
4)透化液
0.5%Triton-X100。
5)一抗稀释液
取10mM pH7.4 PBS缓冲液,加入终浓度为0.05g/mL甘露醇、0.05g/mL蔗糖、0.1%proclin-300、0.01g/mL BSA。
6)酶标二抗稀释液
取10mM pH7.4 PBS缓冲液,加入终浓度为0.05%吐温-20(Tween-20)、0.1%proclin-300、0.01g/mL BSA。
将该试剂盒以下述方式进行使用:
1)取待测样本加入固定液,4℃过夜。
2)弃去固定液,用PBS洗3次,每次3min。
3)加入透化液,室温作用15min。
4)弃去透化液,用PBS洗3次,每次3min。
5)加入用一抗稀释液稀释的PDCoV单克隆抗体,置于湿盒中37℃孵育1h。
6)弃去一抗,用PBS洗3次,每次3min。
7)加入用酶标二抗稀释液稀释的HRP标记抗猪IgG抗体,置于湿盒中37℃孵育1h;弃去二抗,用PBS洗3次,每次3min。
8)用显微镜观察成像。
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Claims (8)
1.一种抗PDCoV核衣壳蛋白的单克隆抗体2H7,其特征在于,所述单克隆抗体包含抗体重链的Ig结构域VH CDR1、VH CDR2和VH CDR3,以及抗体轻链的Ig结构域VL CDR1、VL CDR2和VL CDR3;其中,所述VH CDR1、VH CDR2和VH CDR3的氨基酸序列分别如SEQ ID NO:1~3所示;所述VL CDR1、VL CDR2和VL CDR3的氨基酸序列分别如SEQ ID NO:4、SEQ ID NO:13、SEQ IDNO:5所示。
2.根据权利要求1所述的单克隆抗体,其特征在于,所述单克隆抗体为全长抗体、Fab抗体或F(ab’)2抗体。
3.根据权利要求1或2所述的单克隆抗体,其特征在于,轻链可变区的氨基酸序列如SEQID NO.6所示,重链可变区的氨基酸序列如SEQ ID NO.7所示。
4.编码权利要求3所述单克隆抗体的基因,其特征在于,编码轻链可变区的基因序列如SEQ ID NO.8所示,编码重链可变区的基因序列如SEQ ID NO.9所示。
5.权利要求1或2所述任意一种单克隆抗体的应用方法,其特征在于,是以所述单克隆抗体制备用于检测PDCoV的试剂盒。
6.权利要求5所述应用方法制备得到的试剂盒的使用方法,其特征在于,是将所述试剂盒以血清学检测的方式加以使用。
7.一种用于PDCoV检测的试剂盒,其特征在于,该试剂盒包括权利要求1或2所述的任意一种单克隆抗体。
8.根据权利要求7所述的试剂盒,其特征在于,该试剂盒是ELISA检测试剂盒、使用免疫层析试纸条的免疫层析诊断试剂盒、免疫组化试剂盒中的任意一种,使用试剂盒时的检测方式为血清学检测。
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