CN101413949A - 猪圆环病毒pcv2亚型鉴定试纸卡 - Google Patents
猪圆环病毒pcv2亚型鉴定试纸卡 Download PDFInfo
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Abstract
PCV2亚型鉴定检测试纸卡是适用于猪PCV2亚型鉴定的试纸卡。其反应试剂载体吸附层固定在支撑层上,吸附层包括纤维层,金标纤维层,纤维素膜层和吸水材料层;金标纤维层为吸附胶体金标记的识别PCV1和PCV2共同抗原表位的抗衣壳蛋白单克隆抗体的金标玻璃棉,纤维素膜层为印制检测印迹T1、T2和对照印迹C的硝酸纤维素膜;T1为识别1767PCV2亚型特异性抗原表位的抗衣壳蛋白单克隆抗体溶液印制的1767亚型检测印迹,T2为识别1766PCV2和1767PCV2共同抗原表位的抗衣壳蛋白单克隆抗体溶液印制的1766和1767亚型检测印迹,对照印迹C为抗小鼠IgG多克隆抗体溶液印制的印迹。试纸卡特异性强,敏感性高,检测结果直观,易于推广应用。
Description
技术领域
本发明属于生物技术领域,涉及一种猪圆环病毒PCV1和PCV2鉴别检测和PCV2亚型鉴定检测试纸卡。
背景技术
猪圆环病毒(porcine circovirus,PCV)是目前发现的最小的动物病毒,病毒粒子直径约17nm,为共价闭合,环状,单股DNA病毒,具有两种基因型:猪圆环病毒1型(PCV1)和猪圆环病毒2型(PCV2)。PCV1无致病性,但广泛存在于猪体内和猪源传代细胞系,其基因组全长1759nt或1758nt,PCV2对猪有致病性,是引发一系列猪圆环病毒病(porcine circovirusdiseases,PCVD),特别是断奶仔猪多系统衰竭综合征(post-weaning multisystemic wastingsyndrome,PMWS)的主要病原,导致猪群免疫抑制,给养猪业造成了巨大的经济损失。PCV2基因组全长1768nt或1767nt,目前已知PCV2 ORF1编码与病毒复制相关的Rep蛋白,ORF2编码PCV2免疫相关的衣壳蛋白,ORF3编码与PCV2复制无关、与致病性有关的蛋白。最近,我们从疑似猪“高热症”患猪中分离鉴定一种基因组全长1766nt的新的PCV2毒株(1766PCV2)。1766PCV2在1059位缺失一个核苷酸G,引起ORF2C末端密码子移位,使得1766PCV2缺失1767PCV2的特异性抗原表位。该特征性抗原表位可作为血清学标志,用于1766PCV2、1767PCV2和1768PCV2的抗原分型鉴定。
目前,国内外采用的PCV2抗原和抗体检测技术主要有:病毒分离培养,间接免疫荧光(IFA),免疫酶单层试验(IMPA),酶联免疫吸附试验(ELISA),原位杂交(ISH),聚合酶链式反应(PCR)等等。上述技术和方法虽然可以检测PCV2病毒,在实践中也取得一定的效果,但均无法实现以PCV2抗原亚型的鉴别诊断,并且存在试验操作复杂,耗时长,需要特定的专业技能和仪器设备等,常限于实验室内进行,很难在基层普及和推广。因此,在分析鉴定PCV2亚型抗原表位特征的基础上,研制开发适用于养殖基层的快捷、简便的PCV1和PCV2鉴别诊断和抗原分型检测器具,对诊断、监控猪群PCV2感染,有效控制PCVD的发生和危害有重要意义。
发明内容
本发明的目的是:提供一种适用于猪PCV1和PCV2感染快速鉴别检测和PCV2抗原亚型鉴定的试纸卡,在生产实践中易于推广应用。
本发明的PCV1和PCV2鉴别检测试纸卡,包括用不吸水薄片条制成的支撑层1,反应试剂载体吸附层固定在支撑层1上,从样品端11到手柄端12的反应试剂载体吸附层依次为:纤维层2,金标纤维层3,纤维素膜层4和吸水材料层5;金标纤维层3为吸附胶体金标记的识别PCV1和PCV2共同抗原表位的抗衣壳蛋白单克隆抗体的金标玻璃棉,纤维素膜层4为从样品端11至手柄端12依次印制检测印迹T1 6、检测印迹T2 7和对照印迹C 8的硝酸纤维素膜,试纸卡固定在塑料卡10内,在样品端11设有加样孔9;检测印迹T1 6为以识别PCV1型特异性抗原表位的单克隆抗体溶液在纤维素膜上印制的条状PCV1检测印迹;检测印迹T2 7为以识别PCV1和PCV2共同抗原表位的抗衣壳蛋白单克隆抗体溶液印制的条状PCV检测印迹,对照印迹C8为以抗小鼠IgG多克隆抗体溶液印制的条状对照印迹,上述3条印迹按序平行排列。
本发明的PCV2亚型鉴定检测试纸卡,包括用不吸水薄片条制成的支撑层1,反应试剂载体吸附层固定在支撑层1上,从样品端11到手柄端12的反应试剂载体吸附层依次为:纤维层2,金标纤维层3,纤维素膜层4和吸水材料层5,金标纤维层3为吸附胶体金标记的识别PCV1和PCV2共同抗原表位的抗衣壳蛋白单克隆抗体的金标玻璃棉,纤维素膜层4为从样品端11至手柄端12依次印制检测印迹T1 6、检测印迹T2 7和对照印迹C 8的硝酸纤维素膜,试纸卡固定在塑料卡10内,在样品端11设有加样孔9;检测印迹T1 6为以识别1767PCV2亚型特异性抗原表位的抗衣壳蛋白单克隆抗体溶液在纤维素膜上印制的1767亚型条状检测印迹,检测印迹T2 7为以识别1766PCV2和1767PCV2共同抗原表位的抗衣壳蛋白单克隆抗体溶液印制的1766和1767亚型条状检测印迹,对照印迹C 8为以抗小鼠IgG多克隆抗体溶液印制的条状对照印迹,上述3条印迹按序平行排列。
以上方案详述如下:
PCV1和PCV2鉴别检测试纸卡含有支撑层和反应试剂载体吸附层,支撑层为不吸水薄片条,反应试剂载体吸附层粘贴于支撑层上,由样品端到手柄端依次为纤维层、金标纤维层、纤维素膜层和吸水材料层,纤维素膜层为从样品端至手柄端依次印制检测印迹T1,检测印迹T2和对照印迹C的硝酸纤维素膜,试纸卡固定在塑料卡内,在样品端设有加样孔。
支撑层的不吸水薄片条可用硬质塑胶片条,或不吸水的硬纸条,纤维层可用玻璃棉,吸水材料层可用吸水纸,纤维素膜层可用硝酸纤维素膜,金标纤维层可用吸附金标蛋白玻璃棉,简称金标玻璃棉,金标蛋白为胶体金标记的识别PCV1和PCV2共同抗原表位的抗衣壳蛋白单克隆抗体mAb1,PCV1和PCV2鉴别检测试纸卡的检测印迹T1为以识别PCV1型特异性抗原表位的抗衣壳蛋白单克隆抗体mAb2溶液在纤维素膜上印制的PCV1检测印迹“|”,检测印迹T2为以识别PCV1和PCV2共同抗原表位的抗衣壳蛋白单克隆抗体mAb3溶液印制的PCV检测印迹“|”,对照印迹C为以抗小鼠IgG多克隆抗体pAb溶液印制的对照印迹C“|”,其组合排列为“|||”;PCV2亚型鉴定检测试纸卡的检测印迹T1为以识别1767PCV2亚型特异性抗原表位的抗衣壳蛋白单克隆抗体mAb4溶液在纤维素膜上印制的1767亚型检测印迹“|”,检测印迹T2为以识别1766PCV2和1767PCV2共同抗原表位的抗衣壳蛋白单克隆抗体mAb5溶液印制的1766和1767亚型检测印迹“|”,对照印迹C为以抗小鼠IgG多克隆抗体pAb溶液印制的对照印迹“|”,其组合排列为“|||”。
试纸卡具有下列各项优点:
(1)特异性强,敏感性高。PCV1和PCV2鉴别检测和PCV2亚型鉴定检测试纸卡以高亲和力单克隆抗体为基础制备而成,抗衣壳蛋白单克隆抗体分别特异性识别PCV1和PCV2共同抗原表位,PCV1型特异性抗原表位,以及PCV2亚型特异性抗原表位,有效区分PCV不同抗原型和抗原亚型,有高度的特异性。
(2)操作简便快速。使用PCV1和PCV2鉴别检测和PCV2亚型鉴定检测试纸卡检测PCV,能有效鉴别检测PCV1和PCV2感染,并进行PCV2抗原亚型鉴定,在5-10分钟内即可判定检测结果。
(3)显示检测结果形象、直观准确。PCV1和PCV2鉴别检测和PCV2亚型鉴定检测试纸卡以红棕色“|”,“||”或“|||”作为阴性,抗原型和抗原亚型检测标记,通过PCV1和PCV2鉴别检测试纸卡可鉴别检测PCV1和PCV2感染,即在PCV1和PCV2鉴别检测试纸卡上显示一条棕红色“|”印迹表示在被检测样品中未检出PCV病毒,两条棕红色“||”印迹表示被检样品PCV2病毒阳性,三条棕红色“|||”印迹表示被检样品PCV1病毒阳性;与PCV2亚型鉴定试纸卡的组合检测,可进一步鉴定PCV2的抗原亚型,即在PCV2亚型鉴定试纸卡上显示一条棕红色“|”印迹表示被检测样品中的PCV2病毒为1768抗原亚型,两条棕红色“||”印迹表示被检样品中的PCV2病毒为1766抗原亚型,三条棕红色“|||”印迹表示被检样品中的PCV2病毒为1767抗原亚型,试纸卡无显色印迹则表示检测失败或试纸卡失效,结果判定形象、直观、准确,简单明了,不易出现假阴性和假阳性误判。
(4)成本低,投资少。PCV1和PCV2鉴别检测和PCV2亚型鉴定检测试纸卡可在检测现场完成操作,检测一步到位,成本低廉,投资少,见效快。
本发明有益的积极效果是:操作简单,普通技术人员可以操作,能满足各种需要,如疫病诊断、疫病监测、口岸检疫、卫生防疫、集约化养殖到个体养殖等,易于大范围推广,具有广阔的市场前景。
附图说明
图1是PCV1和PCV2鉴别检测和PCV2亚型鉴定检测试纸卡结构示意图。图中,1.支撑层,2.样品端的纤维层,3.金标纤维层(金标玻璃棉),4.纤维素膜层,5.手柄端的吸水材料层,6.检测印迹T1,7.检测印迹T2,8.对照印迹C,9.加样孔,10.塑料卡,11.样品端,12.手柄端。
图2是PCV1和PCV2鉴别检测结果图。图中,1为PCV阴性,2为PCV2阳性,3为PCV1阳性,4为检测失败或试纸卡失效。
图3是PCV2抗原亚型鉴定检测结果图。图中,1为1768亚型PCV2,2为1766亚型PCV2,3为1767亚型PCV2,4为检测失败或试纸卡失效。
具体实施方式
以下结合附图对本发明进一步描述。
PCV1和PCV2鉴别检测和PCV2亚型鉴定检测试纸卡可应用于猪PCV1和PCV2的鉴别检测以及PCV2抗原亚型鉴定。制备PCV2鉴别检测和抗原亚型鉴定检测试纸卡,首先需制备衣壳蛋白重组蛋白,进而制备抗衣壳蛋白单克隆抗体,筛选获得识别PCV1和PCV2共同抗原表位,PCV1型特异性抗原表位,1767亚型特异性抗原表位,以及1766和1767共同抗原表位的单克隆抗体,其次需制备羊抗小鼠IgG多克隆抗体,分别用于制备PCV1和PCV2鉴别检测和PCV2亚型鉴定检测试纸卡的金标玻璃棉,PCV1和PCV2鉴别检测试纸卡的PCV检测印迹T2,PCV1和PCV2鉴别检测试纸卡的PCV1检测印迹T1,PCV2亚型鉴定检测试纸卡的1767亚型检测印迹T1,PCV2亚型鉴定检测试纸卡的1766和1767亚型检测印迹T2,以及两个试纸卡的对照印迹C。
实施例1 衣壳蛋白重组蛋白的制备
用基因工程技术高效表达PCV1和PCV2衣壳蛋白,制备衣壳蛋白重组蛋白。根据PCV1分离株(GenBank No.AY193712)和1767PCV2分离株HZ0201(GenBank No.AY188355)全基因序列,分别设计PCV1和PCV2衣壳蛋白特异性引物,PCV1上游引物为PV1up:5’-GCGGATCCACGGGTATCTTCAATTC-3’,含BamHI酶切位点,PCV1下游引物为PV1down:5’-CCGCTCGAGTTATTTATTTAGAGGGTC-3’,含XhoI酶切位点,PCV2上游引物为pG1:5’-GCGGATCCAATGGCATCTTCAACAC-3’,含BamHI酶切位点;PCV2下游引物pG2:5’-CCGCTCGAGTTAAGGGTTAAGTGGG-3’,含XhoI酶切位点。分别以PCV1和PCV2病毒DNA为模板,扩增573bp和579bp的去核定位信号衣壳蛋白基因,反应条件为:95℃预变性5min后,按95℃ 30s,58或61℃ 30s,72℃ 45s进行30个循环,最后72℃再延伸10min。PCR产物经BamHI和XhoI双酶切,分别连接到原核表达载体pET-28a和pGEX-4T-1的谷胱甘肽-S-转移酶基因(GST)下游BamHI和XhoI位点间,转化E.coli Top10感受态细胞,涂布于含100g/mL卡那霉素(Kan)或氨苄青霉素(Amp)的LB平板,37℃培养16h,挑取单克隆菌落,碱裂解法抽提重组质粒,以PCR和双酶切鉴定阳性克隆,经序列测定验证鉴定。经PCR和酶切鉴定,构建重组表达质粒pET-PCV1-dCap和pGEX-PCV2-dCap,其中PCV2衣壳蛋白在N端与GST融合。分别将重组表达质粒pET-PCV1-dCap和pGEX-PCV2-dCap转化表达菌E.coli BL21(DE3)和E.coli BL21,挑选单克隆接入5mL含100g/mL Kan或Amp)的LB培养基中,37℃、250r/min振荡培养10h,然后按1:100转接500mL LB(100g/mL Kan)或2×YTA(100g/mLAmp)的培养基,37℃、250r/min振荡培养3h至A600为0.6-0.8时,加入终浓度为0.1mmol/L的IPTG在37℃诱导表达4h,4℃ 4000r/min离心10min收集诱导表达菌体。
利用QIAGEN公司的Ni-NTA亲和纯化柱,在变性条件下采用pH值梯度洗脱法纯化PCV1重组His-dCap蛋白,主要步骤如下:将收集的菌体沉淀称量,按8mL/g菌体沉淀的比例重悬于缓冲液B(100mM NaH2PO4,10mMTris·Cl,8M urea,pH8.0)中,在室温下轻微振荡45~60min(避免气泡产生),400W超声波击打30次,12000rpm,4℃离心30min,取上清加入到含2mLNi-NTA填料的经缓冲液B平衡的纯化柱中,封闭纯化柱,200r/min,37℃振荡30min,使重组蛋白和Ni2+-NTA固相充分结合后,之后让纯化柱垂直放置,待Ni-NTA填料沉积后,将纯化柱用10mL缓冲液C(100mM NaH2PO4,10mM Tris-Cl,8M urea,20mMimidazol,pH6.3)洗脱2次后,再用10mL buffer D(100mM NaH2PO4,10mM Tris-Cl,8M urea,pH5.9)洗脱2次,最后用10mL缓冲液E(100mM NaH2PO4,10mM Tris-Cl,8M urea,pH4.5)洗脱目的蛋白,收集洗脱液进行SDS-PAGE鉴定。采用BCA法测定纯化His-dCap蛋白含量,计算表达量。结果显示,含pET-PCV1-dCap重组质粒的表达菌成功表达分子量为28kDa的PCV1 His-dCap蛋白,其表达量为36mg/L培养物,可用于制备抗衣壳蛋白单克隆抗体。
采用GSTrapFF亲和层析柱(Amersham公司)在天然条件下纯化PCV2 GST-dCap蛋白,主要步骤为:按每毫升培养菌液加入50μL 4℃预冷的Binding buffer(140mmol/L NaCl,2.7mmol/L KCl,10mmol/L Na2HPO4,1.8mmol/L KH2PO4,pH7.3)将收集的细菌沉淀重新悬浮,超声波裂解破碎(0.2-0.3kw,120次)直至菌液呈均一、半透明状或云雾状消失;加入终浓度为1%的Triton X-100,冰上振荡混匀30min,4℃、12000rpm离心30min,取上清液进一步用0.45μm滤器过滤。取上清过用Binding buffer预平衡的GSTrap FF纯化柱,流速为0.5mL/min(中间不能引入气泡)。用20-30倍柱床体积Binding buffer洗涤柱子(流速为1mL/min),用10倍柱床体积的Elution buffer(50mmol/L Tris-HCl,10mmol/L reducedglutathione,pH 8.0)洗脱(流速为1mL/min),或者用80U凝血酶(Thrombin)柱上酶解PCV2GST-dCap融合蛋白,纯化重组的dCap蛋白,用eppendorf管收集洗脱液,每管1.0mL,取样作SDS-PAGE分析,并用Bradford法测蛋白含量,计算表达量。结果显示,含pGEX-PCV2-dCap重组质粒的表达菌成功表达分子量为48kDa的GST-dCap融合蛋白,其表达量为6.14mg/L培养物,可用于制备抗衣壳蛋白单克隆抗体。
实施例2 抗衣壳蛋白单克隆抗体的制备与筛选
分别将重组PCV1和PCV2衣壳蛋白与弗氏佐剂等量混合,充分乳化,以50g-100g/只免疫BALB/c系小鼠3次,每次间隔15-30天;第3次加强免疫后3-4天,将免疫小鼠眼球放血,拉颈致死,于75%酒精浸泡5-10min,无菌取其脾细胞;剪碎并经100目尼龙网过滤,1000r/min离心10min,收集脾细胞;将1×108的脾细胞与2-5×107的SP2/0骨髓瘤细胞混合,1000r/min离心10min,弃上清,在37℃的水浴中将0.7-1ml的40%-50%PEG 4000(pH8.5-9.0)缓缓加入细胞,温育1min后,缓慢加入无血清1640培养基15ml,以终止PEG的作用,37℃水浴5-10min,1000r/min离心10min,弃上清,将细胞重悬于HAT选择培养基中,并加入96孔培养板(100 1-200 l/孔),置37℃ 5% CO2培养箱中培养。培养7-10天后,分别用5 g-10 g/ml的PCV1和PCV2衣壳蛋白重组蛋白包被96孔酶标板,以ELISA检测杂交瘤的培养上清,挑取强阳性细胞克隆(OD492=0.8以上),经连续3次的有限稀释法克隆化,建立杂交瘤细胞株,将杂交瘤细胞腹腔注射经降植烷致敏一周的BALB/c系小鼠,每只小鼠注射5×105个细胞,诱生小鼠腹水,制备抗衣壳蛋白单克隆抗体。所建立的25株杂交瘤细胞株染色体数为92-98,其分泌的单克隆抗体特异识别PCV1和/或PCV2衣壳蛋白,与GST及其它菌体蛋白不发生交叉反应,亲和力常数达10-9,轻链亚型为κ或λ,重链亚型为IgG1,IgG2a,IgG2b或IgG3。
实施例3 抗衣壳蛋白单克隆抗体的筛选
以间接免疫荧光(IFA)对所制备的25株抗衣壳蛋白单克隆抗体进行筛选和鉴定。分别将PCV1分离株,1768PCV2分离株,1767PCV2分离株和1766PCV2分离株按1:10接种胰酶消化的无PCV污染的PK-15细胞,将病毒细胞混合液加入96孔细胞培养板,每孔100μL,37℃ 5%CO2培养96h,加入1:1混合的甲醇丙酮固定液100μL,-20℃固定20min。将固定液弃去,自然干燥。用5%的脱脂奶粉封闭1h,每孔加入待检杂交瘤细胞上清100μL,37℃孵育1h,PBST洗涤5次,加入1:400稀释的FITC标记的羊抗鼠IgG 50μL,37℃孵育40min,PBST洗涤5次。加入50μL PBS,用倒置荧光显微镜观察阳性细胞,筛选识别不同PCV基因型和抗原亚型的单克隆抗体。经IFA检测和筛选,单克隆抗体3F6和4C3均能识别感染PK-15细胞的PCV1,1768PCV2,1767PCV2和1766PCV2病毒粒子,为识别PCV1和PCV2共同抗原表位的单克隆抗体mAb1和mAb3,单克隆抗体3F11特异性识别感染PK-15细胞的PCV1病毒粒子,而不与1768PCV2,1767PCV2和1766PCV2感染细胞反应,为识别PCV1型特异性抗原表位的单克隆抗体mAb2,单克隆抗体8A12仅特异性识别感染PK-15细胞的1767PCV2病毒粒子,而不与其他三株病毒感染细胞反应,为特异识别1767PCV2亚型单克隆抗体mAb4,单克隆抗体6H9识别感染PK-15细胞的1767PCV2和1766PCV2病毒粒子,但不与1768PCV2和PCV1感染细胞反应,为识别1766PCV2和1767PCV2共同抗原表位的单克隆抗体mAb5,上述识别PCV1和PCV2共同抗原表位,PCV1型特异性,1767PCV2亚型特异性,以及1766PCV2和1767PCV2共同抗衣壳蛋白单克隆抗体分别用于制备PCV1和PCV2鉴别检测和PCV2亚型鉴定检测的金标玻璃棉,PCV1和PCV2鉴别检测试纸卡的检测印迹T2,PCV1和PCV2鉴别检测试纸卡的检测印迹T1,以及PCV2亚型鉴定检测试纸卡的1767亚型检测印迹T1,PCV2亚型鉴定检测试纸卡的1767亚型和1766亚型检测印迹T2的印制。
实施例4 抗衣壳蛋白单克隆抗体识别抗原表位的鉴定
以合成多肽分析鉴定抗衣壳蛋白单克隆抗体识别的抗原表位。根据PCV2 HZ0201衣壳蛋白的氨基酸序列,设计由18肽组成的氨基酸重叠多肽片段,每条肽之间重叠8个氨基酸,移位10个氨基酸(aa),跨越PCV2衣壳蛋白氨基酸位25-233(未包含核定位信号NLS中的前24个氨基酸),共计20条肽,于每条肽N端加一半胱氨酸Cys,用于载体蛋白偶联,所设计多肽由上海波泰生物技术有限公司合成,每条合成多肽10mg,纯度大于90%,冻干保存。
用无菌的蒸馏水或去离子水、无氧水溶解冻干多肽,浓度为1mg/ml,应用异型双功能试剂Sulfo-SMCC(分子量436.37,美国Pierce公司产品)通过多肽N端Cys上的-SH基团与载体蛋白牛血清白蛋白BSA偶联,步骤为:1)称量4mg BSA,溶于500μl偶联缓冲液(0.1Mphosphate,0.15M NaCl,1mM EDTA,pH7.2);2)加入1mg Sulfo-SMCC于载体蛋白溶液;3)RT孵育60min或37℃孵育30min,并不时混匀;4)对偶联缓冲液充分透析,以除去多余的偶联剂,用偶联缓冲液调整蛋白浓度为5mg/ml;5)取20μl(100μg)Sulfo-SMCC活化的载体蛋白,加入N末端含Cys的溶解多肽100μg,充分混匀,于4℃孵育4h或过夜,完成偶联反应,4℃保存备用。
采用Peptide-ELISA分析抗衣壳蛋白单克隆抗体识别的抗原表位。以50mM Tris-HCl缓冲液(pH8.6)作包被液稀释BSA偶联多肽至1μg/ml,包被96孔酶标板,每孔100μl,4℃孵育过夜,PBST洗涤3次;用5%的脱脂奶PBS封闭酶标板,200μl/孔,37℃封闭3h,PBST洗涤3次;加入用5%的脱脂奶1:500-1000稀释的抗衣壳蛋白单克隆抗体,100μl/孔,重复3个孔,37℃孵育1.5h,PBST洗涤6次;加入1:10000稀释的HRP羊抗鼠IgG,100μl/孔,37℃孵育1h,PBST洗涤6次;加入TMB显色溶液100μl,室温孵育10min,观察显色结果,每孔加入50μl2M硫酸溶液终止反应,在酶标仪上读取450nm吸收值。对于Peptide-ELISA阳性多肽,进一步设计合成系列截短多肽,偶联于载体蛋白BSA,同上以Peptide-ELISA分析截短多肽与相应单克隆抗体的反应性,精确定位所获得抗衣壳蛋白单克隆抗体识别的抗原表位。合成多肽的抗原表位分析结果表明,单克隆抗体mAb1识别的PCV1和PCV2共同抗原表位为PCV1和PCV2衣壳蛋白的156YHSRYFT162,单克隆抗体mAb2识别的PCV1型特异性抗原表位为PCV1衣壳蛋白的92LPFQYYRIRKVK103,单克隆抗体mAb3识别的PCV1和PCV2共同抗原表位为PCV1和PCV2衣壳蛋白的175QPNNKRNQLWLRLQTAGN192,单克隆抗体mAb4识别的1767PCV2亚型特异性抗原表位为PCV2衣壳蛋白C末端226LKDPPLNP233,是1767PCV2新的血清学标志,单克隆抗体mAb5不能识别PCV2衣壳蛋白的任何一条合成多肽,表明该单克隆抗体识别的1766PCV2和1767PCV2共同抗原表位为构象型抗原表位。
实施例5 抗小鼠IgG多克隆抗体的制备
采取小鼠血液,分离血清,以辛酸-硫酸铵法粗提小鼠血清IgG,并用Superdex 200凝胶层析进行纯化。首先用4倍血清体积的0.06M pH4.8醋酸缓冲液稀释血清,以1M NaOH调pH至4.5;在室温下边搅拌边逐滴加入辛酸,每毫升血清加入25μL辛酸,4℃静置2h,12000r/min离心30min,取上清;在溶液中加10倍体积磷酸盐缓冲液PBS,用5M NaOH调pH至7.4,冰浴至4℃;按每毫升混合液加0.277g硫酸铵并搅拌30min,4℃静置4h,12000r/min离心30min,弃上清;沉淀以0.05M PBS pH7.4重悬于,透析除盐,直至用BaCl2检测无白色沉淀为止,用PEG8000进行浓缩。利用GE公司AKAT purifier蛋白纯化系统以Hiload16/60 Superdex 200prep pg凝胶过滤预装柱纯化粗提的小鼠IgG。用0.22μm滤器过滤粗提的小鼠血清IgG溶液,并经超声波除气;将Hiload 16/60 Superdex 200prep pg凝胶过滤预装柱与AKAT purifier蛋白纯化系统相连接,先用ddH2O清洗柱子,接着用含0.15M NaCl的0.05M磷酸盐缓冲液(PBS,pH8.0)平衡柱子,流速为2mL/min,直至UV280的吸光值和电导值基线稳定,平衡柱子时要清洗各阀位。将过滤的猪血清IgG样品用注射器(上样前注意排空注射器里的气泡,以免损害柱子)注入样品环中,设置洗脱速度和报警压,进行上样和洗脱,流速为1mL/min。观察UV280值基线的变化,收集紫外吸收峰的洗脱峰,每管收集2mL,SDS-PAGE电泳检测脱蛋白,合并收集液,装入透析袋,放入PEG8000中浓缩,用Nanodrop 1000分光光度仪测OD280和OD260吸光值,并根据公式计算IgG含量:蛋白含量(mg/mL)=(1.45×OD280-0.74×OD260)×稀释倍数,测得小鼠IgG的蛋白含量为12.0mg/mL,用于羊抗小鼠IgG多克隆抗体的制备。
以50~100μg/kg体重的小鼠IgG蛋白加弗氏佐剂充分乳化,经皮下和肌肉注射免疫健康山羊3~4次,末次免疫10天后,静脉采血,以ELISA测定其血清抗体效价在1:2000以上时,心脏采血或颈动脉放血,收集高免血清;以辛酸-硫酸铵法粗提免疫山羊血清IgG,并用Superdex 200凝胶层析进行纯化,不重述。所制备的抗小鼠IgG多克隆抗体用于PCV1和PCV2鉴别检测和PCV2亚型鉴定检测试纸卡对照印迹的印制。
实施例6 金标玻璃棉的制备
以柠檬酸钠还原法制备金溶胶,即在沸腾的50-100ml 0.01-0.05%氯金酸水溶液中加入2-4ml的0.5-2%柠檬酸三钠溶液,获得直径15nm左右的胶体金。以0.1mol/L K2CO3调胶体金pH至8.5-9.5,以1:1000-1:1300的标记比将待标记的单克隆抗体mAb1加入pH8.5-9.5金溶胶中,标记10min后,加20% PEG 10000至终浓度0.05%,4℃ 1500-3000g离心20min,除去未结合的金颗粒,4℃ 15000g离心1h,弃上清,获初步纯化金标蛋白混合物后,用丙烯葡聚糖S-400柱层析,分离纯化金标蛋白,获得胶体金标记的抗PCV2衣壳蛋白单克隆抗体。将1:100-1:1500稀释的胶体金标记蛋白吸附于精制玻璃棉中,4℃低温真空干燥,制备PCV1和PCV2鉴别检测和PCV2亚型鉴定检测试纸卡的金标玻璃棉。
实施例7 PCV1和PCV2鉴别检测和PCV2亚型鉴定检测试纸卡的实施结构
PCV1和PCV2鉴别检测和PCV2亚型鉴定检测试纸卡的实施结构参见图1,图中,1为支撑层用不吸水薄片条,实施中可采用塑胶薄片条或采用不吸水的硬质纸片材,反应试剂载体吸附层由2,3,4,5,组合而成,从样品端2开始,到手柄端5依次粘贴在支撑层1上面;其中2为样品端纤维层,实施中可使用玻璃纤维棉简称玻璃棉,3为吸附金标蛋白的纤维层,金标蛋白为胶体金标记的识别PCV1和PCV2共同抗原表位的抗衣壳蛋白单克隆抗体mAb1,可采用精制玻璃纤维棉,简称为金标玻璃棉,4为纤维素膜层,实施中可采用硝酸纤维素膜,5为手柄端吸水材料层,采用吸水纸,如滤纸或其它吸水纸均可,2,3,4,5各层彼此之间交界处纤维互相交叉渗透;PCV1和PCV2鉴别检测试纸卡的6为用识别PCV1型特异性抗原表位的抗衣壳蛋白单克隆抗体mAb2溶液在纤维素膜上印制的PCV1检测印迹T1“|”,7为用识别PCV1和PCV2共同抗原表位的抗衣壳蛋白单克隆抗体mAb3溶液印制的PCV检测印迹T2“|”,PCV2亚型鉴定检测试纸卡的6为识别1767PCV2亚型特异性抗原表位的抗衣壳蛋白单克隆抗体mAb4溶液在纤维素膜上印制的1767亚型检测印迹T1“|”,7为用识别1766PCV2和1767PCV2共同抗原表位的抗衣壳蛋白单克隆抗体mAb5溶液印制的1766和1767亚型检测印迹T2“|”,8为用抗小鼠IgG多克隆抗体pAb溶液印制的对照印迹C“|”,在纤维素膜上的检测印迹T1,检测印迹T2和对照印迹C组合排列为“|||”,9为样品溶液的加样孔,10为塑料卡,11为样品端,12为手柄端。
实施例8 PCV1和PCV2鉴别检测和PCV2亚型鉴定检测试纸卡实施检测的反应原理
PCV1和PCV2鉴别检测试纸卡的反应原理为:待检测样品溶液加入样品孔后,待检溶液通过虹吸带动待检抗原与金标蛋白一起向硝酸纤维素膜扩散,并最终渗透到滤纸层中,在扩散过程中PCV1或PCV2病毒抗原与胶体金标记的识别PCV1和PCV2共同抗原表位的抗衣壳蛋白单克隆抗体mAb1相结合,形成金标蛋白-抗原复合物,该复合物可与检测印迹T1中的识别PCV1型特异性抗原表位的抗衣壳蛋白单克隆抗体mAb2相结合,生成红棕色“|”标记,同时也可与检测印迹T2中的识别PCV1和PCV2共同抗原表位的抗衣壳蛋白单克隆抗体mAb3结合,生成另一条红棕色“|”标记,部分未与抗原结合的金标蛋白不能与检测印迹结合而继续扩散,在纤维膜上与对照印迹C中的抗小鼠IgG多克隆抗体pAb结合,生成红棕色标记“|”,三种标记组合叠加,形成三条红棕色阳性标记“|||”,代表样品为PCV1病毒阳性;同理,样品中的PCV2病毒抗原与胶体金标记的识别PCV1和PCV2共同抗原表位的抗衣壳蛋白单克隆抗体mAb1相结合形成金标蛋白-抗原复合物,但该复合物不能与检测印迹T1中的识别PCV1型特异性抗原表位的抗衣壳蛋白单克隆抗体mAb2结合,不能形成红棕色标记,复合物继续扩散与检测印迹T2中的识别PCV1和PCV2共同抗原表位的抗衣壳蛋白单克隆抗体mAb3相结合,生成红棕色“|”标记,部分未与抗原结合的金标蛋白不能与检测印迹结合而继续扩散,在纤维膜上与对照印迹C中的抗小鼠IgG多克隆抗体pAb结合,生成红棕色标记“|”,从而形成两条红棕色阳性标记“||”,代表样品为PCV2病毒阳性,而PCV1病毒阴性;如果样品溶液中既不含PCV1病毒,又不含PCV2病毒,则没有金标蛋白-抗原复合物形成,不能与检测印迹T1或T2结合,只有金标蛋白与对照印迹C相结合,生成一条红棕色标记“|”,代表样品为PCV1和PCV2病毒阴性。如果纤维素膜上没有红棕色标记显现,则表明检测失败或PCV1和PCV2鉴别检测试纸卡失效。
PCV2亚型鉴定检测试纸卡检测的反应原理为:待检测样品溶液加入样品孔后,待检溶液通过虹吸带动待检抗原与金标蛋白一起向硝酸纤维素膜扩散,并最终渗透到滤纸层中,在扩散过程中PCV2病毒抗原与胶体金标记的识别PCV1和PCV2共同抗原表位的抗衣壳蛋白单克隆抗体mAb1相结合,形成金标蛋白-抗原复合物,1767PCV2形成的复合物与检测印迹T1中的识别1767PCV2亚型特异性抗原表位的抗衣壳蛋白单克隆抗体mAb4相结合,生成红棕色“|”标记,同时也与检测印迹T2中的识别1766PCV2和1767PCV2共同抗原表位的抗衣壳蛋白单克隆抗体mAb5结合,生成另一条红棕色“|”标记,部分未与抗原结合的金标蛋白不能与检测印迹结合而继续扩散,在纤维膜上与对照印迹C中的抗小鼠IgG多克隆抗体pAb相结合,生成红棕色标记“|”,三种标记组合叠加,形成三条红棕色阳性标记“|||”,代表样品中PCV2病毒的抗原亚型为1767亚型;同理,1766PCV2形成的金标蛋白-抗原复合物不能与检测印迹T1中的识别1767PCV2亚型特异性抗原表位的抗衣壳蛋白单克隆抗体mAb4识别,不能形成红棕色标记,复合物继续扩散与检测印迹T2中的识别1766PCV2和1767PCV2共同抗原表位的抗衣壳蛋白单克隆抗体mAb5相结合,生成红棕色“|”标记,部分未与抗原结合的金标蛋白不能与检测印迹结合而继续扩散,在纤维膜上与对照印迹C中的抗小鼠IgG多克隆抗体pAb结合,生成红棕色标记“|”,从而形成两条红棕色阳性标记“||”,代表样品中PCV2病毒的抗原亚型1766亚型;而1768PCV2形成的金标蛋白-抗原复合物既不能与检测印迹T1中的识别1767PCV2亚型特异性抗原表位的抗衣壳蛋白单克隆抗体mAb4结合,也不能与检测印迹T2中的识别1766PCV2和1767PCV2共同抗原表位的抗衣壳蛋白单克隆抗体mAb5识别,因此两条检测印迹均不能形成红棕色检测标记,只能形成一条对照印迹C标记“|”,代表样品中PCV2病毒的抗原亚型为1768亚型。如果纤维素膜上没有红棕色标记显现,则表明检测失败或PCV2分型试纸卡失效。
实施例9 PCV1和PCV2鉴别检测和PCV2亚型鉴定检测试纸卡检测的实例操作方法
采集待检猪淋巴结或脾脏,以样品稀释液或生理盐水1:5倍稀释,充分研磨,制备样品溶液。取50μl样品溶液分别加入PCV1和PCV2鉴别检测和PCV2亚型鉴定检测试纸卡,水平放置约2-5分钟,检测结果见图2和图3。PCV1和PCV2鉴别检测试纸卡的显现一条红棕色标记“|”为PCV阴性,显现两条红棕色标记“||”为PCV2阳性而PCV1阴性,显现三条红棕色标记“|||”为PCV1阳性,如果PCV1和PCV2鉴别检测试纸卡没有标记显示,则表明检测失败或试纸卡失效;当PCV1和PCV2鉴别检测试纸卡显现两条红棕色标记“||”时,PCV2亚型鉴定检测试纸卡显现一条红棕色标记“|”表示PCV2的抗原亚型为1768亚型,显现两条红棕色标记“||”表示PCV2的抗原亚型为1766亚型,显现三条红棕色标记“|||”表示PCV2的抗原亚型为1767亚型,如果PCV2分型试纸卡没有标记显示,则表明检测失败或试纸卡失效(图3)。
序列表:
<110>浙江大学
<120>猪圆环病毒PCV2亚型鉴定试纸卡
<160>4
<210>1
<211>26
<212>DNA
<213>人工序列
<223>PCV1上游引物PV1up
<400>1
<210>2
<211>27
<212>DNA
<213>人工序列
<223>PCV1下游引物PV1down
<400>2
<210>3
<211>25
<212>DNA
<213>人工序列
<223>PCV2上游引物pG1
<400>3
<210>4
<211>25
<212>DNA
<213>人工序列
<223>PCV2下游引物pG2
<400>4
Claims (1)
1.PCV2亚型鉴定检测试纸卡,包括用不吸水薄片条制成的支撑层(1),反应试剂载体吸附层固定在支撑层(1)上,从样品端(11)到手柄端(12)的反应试剂载体吸附层依次为:纤维层(2),金标纤维层(3),纤维素膜层(4)和吸水材料层(5),其特征在于:金标纤维层(3)为吸附胶体金标记的识别PCV1和PCV2共同抗原表位的抗衣壳蛋白单克隆抗体的金标玻璃棉,纤维素膜层(4)为从样品端(11)至手柄端(12)依次印制检测印迹T1(6)、检测印迹T2(7)和对照印迹C(8)的硝酸纤维素膜,试纸卡固定在塑料卡(10)内,在样品端(11)设有加样孔(9);检测印迹T1(6)为以识别1767PCV2亚型特异性抗原表位的抗衣壳蛋白单克隆抗体溶液在纤维素膜上印制的1767亚型条状检测印迹,检测印迹T2(7)为以识别1766PCV2和1767PCV2共同抗原表位的抗衣壳蛋白单克隆抗体溶液印制的1766和1767亚型条状检测印迹,对照印迹C(8)为以抗小鼠IgG多克隆抗体溶液印制的条状对照印迹,上述3条印迹按序平行排列。
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CN102735680A (zh) * | 2012-07-05 | 2012-10-17 | 北京大北农科技集团股份有限公司 | 一种猪圆环病毒2型抗体胶体金快速检测试纸条 |
CN110903356A (zh) * | 2019-12-16 | 2020-03-24 | 中国农业大学 | 一种猪圆环病毒ii型抗原以及检测猪圆环病毒ii型抗体的胶体金免疫层析试纸条 |
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Cited By (4)
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CN102735680A (zh) * | 2012-07-05 | 2012-10-17 | 北京大北农科技集团股份有限公司 | 一种猪圆环病毒2型抗体胶体金快速检测试纸条 |
CN102735680B (zh) * | 2012-07-05 | 2017-05-10 | 北京大北农科技集团股份有限公司 | 一种猪圆环病毒2型抗体胶体金快速检测试纸条 |
CN110903356A (zh) * | 2019-12-16 | 2020-03-24 | 中国农业大学 | 一种猪圆环病毒ii型抗原以及检测猪圆环病毒ii型抗体的胶体金免疫层析试纸条 |
CN110903356B (zh) * | 2019-12-16 | 2021-07-13 | 中国农业大学 | 一种猪圆环病毒ii型抗原以及检测猪圆环病毒ii型抗体的胶体金免疫层析试纸条 |
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