CN110776564B - 两株抗新城疫病毒纳米抗体及其表达制备方法和应用 - Google Patents
两株抗新城疫病毒纳米抗体及其表达制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种抗新城疫病毒的纳米抗体NDV‑Nb4和NDV‑Nb49,氨基酸序列分别如SEQ ID NO:1和SEQ ID NO:2所示。还公开了由纳米抗体NDV‑Nb4与铁蛋白融合构建融合蛋白的方法,通过原核表达系统可溶性表达纳米抗体与铁蛋白的融合蛋白;以及由纳米抗体NDV‑Nb49与辣根过氧化物酶融合构建融合蛋白的方法,通过真核表达系统表达NDV‑Nb49与辣根过氧化物酶融合蛋白。还公开了一种检测新城疫病毒的方法,是以NDV‑Fe‑Nb4融合蛋白为捕获NDV的蛋白、NDV‑Nb49‑HRP融合蛋白为检测NDV的蛋白,配合用于检测NDV,该方法简便易操作,且特异性好,准确率高。
Description
技术领域
本发明涉及生物技术领域,特别是涉及一种两株抗新城疫病毒纳米抗体及其表达制备方法和应用。
背景技术
纳米抗体是1989年Hamers-Casterman等在骆驼血液里发现的一种天然缺失轻链的重链抗体,其分子量为15kDa,直径2.2nm,长4.8nm。与传统抗体相比,纳米抗体具有更高亲和力和水溶性,构象稳定,易于基因工程改造以及穿过血脑屏障等优势。近年来,随着对纳米抗体研究的不断深入,该抗体已在蛋白可视化示踪、结构解析以及人类和动物疫病的诊治领域等得到广泛应用。在现有病原的免疫学诊断技术中,抗体发挥着举足轻重的作用。例如,在病原抗体的ELISA检测方法中,二抗或抗病原抗体的生产决定着检测方法的生产成本和市场推广应用价值。然而,传统抗体的生产需要动物或发酵细胞培养,导致其生产成本高,生产工艺复杂。纳米抗体具有易基因工程改造,生产成本低和生产工艺简单的优点。因此,其可以替代传统抗体,应用于病原的免疫学诊断技术的研发中,具有广阔的市场应用前景。
新城疫(Newcastle disease,ND)是由新城疫病毒(Newcastle disease virus,NDV)引起的以感染禽类为主的一种急性、高度接触性传染病。该病以消化道和呼吸道病变为主要特征,发病率和死亡率高,给养禽业带来了严重的危害。在我国,NDV灭活和弱毒疫苗的广泛使用,使得ND的暴发和流行已得到较好的控制。但NDV不同毒株间毒力有较大差异,且在高免疫压力下,随着时间推移病毒也随之发生着一定的变异,给NDV检测工作带来了困难。
目前,NDV检测方法主要有病毒的分离与鉴定、电镜检测技术、血清学诊断方法以及分子生物学诊断方法等。ELISA方法由于其操作简单,通量高,是动物疫病诊断和病原检测的主要方法。ELISA方法很多,包括直接ELISA,间接ELISA,双抗体夹心ELISA等。其中双抗体夹心ELISA方法被广泛应用于病原的检测中,同时由于其通量高,敏感性高和特异性强的优点,已有很多商品化试剂盒的生产。一种病原双抗体夹心ELISA检测方法的建立,首先需要筛选和制备配对的两个抗体(捕获和检测抗体),捕获抗体需要很强的捕获抗原能力,检测抗体需要进行HRP或其它酶的标记。因此,在商品化试剂盒的制备过程中,需要大量的制备抗体并进行标记。但由于酶标记效率问题和抗体生产工艺问题,导致该类试剂盒生产成本居高不下,很难在临床或基层进行大规模的推广使用。而纳米抗体具有分子量小,易基因工程改造的特点。因此,利用纳米抗体替代传统抗体建立夹心ELISA方法,通过对纳米抗体进行改造,实现简化生产工艺,降低生产成本具有重要的意义。
发明内容
本发明的目的是提供一种抗新城疫病毒的纳米抗体NDV-Nb4和NDV-Nb49。同时还提供了由抗新城疫病毒的纳米抗体NDV-Nb4与铁蛋白融合构建融合蛋白的方法,通过构建原核表达系统,可溶性表达将纳米抗体与铁蛋白的融合蛋白;还提供一种由抗新城疫病毒的纳米抗体NDV-Nb49与辣根过氧化物酶融合构建融合蛋白的方法,利用真核表达系统HEK293T细胞,表达NDV-Nb49与辣根过氧化物酶融合蛋白。
本发明还有一个目的是提供一种检测新城疫病毒的方法,具体以所述NDV-Fe-Nb4融合蛋白作为捕获NDV的蛋白,NDV-Nb49-HRP融合蛋白作为检测NDV的蛋白,配合用于检测NDV。
为了实现本发明的这些目的和其它优点,提供了一种抗新城疫病毒的纳米抗体NDV-Nb4,氨基酸序列如SEQ ID NO:1所示。
本发明还提供一种抗新城疫病毒的纳米抗体NDV-Nb49,氨基酸序列如SEQ ID NO:2所示。
本发明还提供一种由所述的抗新城疫病毒的纳米抗体NDV-Nb4与铁蛋白融合构建融合蛋白的方法,包括以下步骤:
步骤1:根据编码铁蛋白的核苷酸序列设计引物NDV-4-FeNb-Nde I-F和NDV-4-Ferritin-R,并以编码铁蛋白重组质粒pUC-57-Fe为模板,通过PCR扩增得到铁蛋白的核苷酸序列;
步骤2:根据编码抗新城疫病毒纳米抗体NVD-Nb4的核苷酸序列设计引物NDV-4-Nb-F和NDV-4-FeNb-BamH I-R,对步骤1获取的含有NDV-Nb4基因的重组质粒pMERC-NDV-Nb4进行PCR扩增,得到纳米抗体序列;
步骤3:以步骤1和步骤2得到的两个目的基因片段为模板,利用扩增铁蛋白基因的上游引物NDV-4-FeNb-Nde I-F和扩增纳米抗体NDV-Nb4基因的下游引物NDV-4-FeNb-BamHI-R,经PCR扩增,获得NDV-Fe-Nb4融合基因;
步骤4:将所得的NDV-Fe-Nb4融合基因和原核表达载体pET-28a分别双酶切后,连接,然后转入感受态大肠杆菌,获取阳性重组质粒pET-28a-NDV-Fe-Nb4,并将所得阳性质粒pET-28a-NDV-Fe-Nb4转化感受态大肠杆菌,培养后分离纯化,获取NDV-Fe-Nb4融合蛋白。
优选的是,步骤1中引物NDV-4-FeNb-Nde I-F如SEQ ID NO:3所示;
引物NDV-4-Ferritin-R如SEQ ID NO:4所示。
优选的是,步骤2中引物NDV-4-Nb-F如SEQ ID NO:5所示;
引物NDV-4-FeNb-BamH I-R如SEQ ID NO:6所示。
本发明还提供一种由所述的抗新城疫病毒的纳米抗体NDV-Nb49与辣根过氧化物酶融合构建融合蛋白的方法,包括以下步骤:
步骤A:将NDV-Nb49片段和pCMV-HRP载体分别双酶切后,用连接酶连接,然后将连接产物转入感受态大肠杆菌中,涂布LB平板,获取阳性质粒;
步骤B:将所得阳性质粒转入HET293T细胞中,收集细胞分泌表达上清液,经纯化,获取NDV-Nb49-HRP融合蛋白。
优选的是,步骤B中将上清液用SDS-PAGE纯化后,电转化至硝酸纤维素膜上后,用Western blotting分析鉴定,得到所述NDV-Nb49-HRP融合蛋白。
本发明还提供一种利用所述的构建融合蛋白的方法获取的NDV-Fe-Nb4融合蛋白。
本发明还提供一种利用所述的构建融合蛋白的方法获取的NDV-Nb49-HRP融合蛋白。
本发明还提供一种检测新城疫病毒的方法,以所述NDV-Fe-Nb4融合蛋白作为捕获NDV的蛋白,NDV-Nb49-HRP融合蛋白作为检测NDV的蛋白,配合用于检测NDV。
本发明公开了以下技术效果:
本发明提供一株抗新城疫病毒纳米抗体NDV-Nb4,并通过构建NDV-Nb4纳米抗体与铁蛋白融合表达载体,利用原核表达系统表达该融合蛋白,该融合蛋白作为捕获抗体,可极大的提高捕获新城疫病毒的能力。同时构建了另一株NDV-Nb49纳米抗体与HRP融合表达载体,并利用真核表达系统表达了该融合蛋白,该蛋白作为检测抗体,可以很好的检测被捕获新城疫病毒。两株纳米抗体融合蛋白的配对使用,极大地提高了抗原捕获能力,同时无需对抗体进行纯化和标记,可直接应用于ELISA方法的建立,大大简化了生产工艺,降低了生产成本。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为本发明NDV-Nb-4基因与铁蛋白融合重组表达载体构建的电泳图;其中,a:PCR分别扩增铁蛋白的基因序列和NDV-Nb-4的基因序列,M:核酸Maker;1:扩增铁蛋白的基因;2:扩增NDV-Nb-4的基因;b:PCR扩增NDV-Fe-Nb4和铁蛋白的融合基因片段;c:双酶切重组载体和融合基因片段,M:核酸Maker;1:pET28a重组载体酶切后片段;2:融合基因片段酶切后片段;d:重构载体NDV-捕获抗体的菌液PCR电泳图,M:核酸Maker;NC:阴性对照;
图2为本发明NDV纳米抗体与铁蛋白融合的捕获抗体原核表达与纯化结果;其中,a:SDS-PAGE分析NDV纳米抗体与铁蛋白融合的捕获抗体的原核表达,M:蛋白Maker;Lane 1:诱导表达菌体裂解液;Lane 2:超声后上清;Lane 3:包涵体;b:纯化NDV纳米抗体与铁蛋白融合的捕获抗体,M:蛋白Maker;Lane 1:超声后上清;Lane 2:流出液;Lane 3-5:10mM咪唑洗脱杂蛋白;Lane 6-14:洗脱目的蛋白;c:Western blot分析NDV纳米抗体与铁蛋白融合的捕获抗体的原核表达;d:电镜观察NDV纳米抗体与铁蛋白融合的捕获抗体自组装;
图3为本发明ELISA验证NDV-Fe-Nb4特异性结合和捕获NDV病毒颗粒的结果;其中,a:NDV-Fe-Nb4与NDV病毒颗粒的特异性结合;b:NDV-Fe-Nb4与普通纳米抗体比较,捕获NDV病毒颗粒能力更强;
图4为本发明NDV纳米抗体融合HRP的检测抗体的真核表达与纯化结果;其中,a:双酶切重组载体和融合基因片段;b:重构载体NDV-检测抗体的菌液PCR电泳图;Lanes 1-5:不同的细菌克隆;c:IFA分析NDV纳米抗体融合HRP的检测抗体的真核表达;d:Western blot分析NDV纳米抗体与HRP融合的检测抗体的真核表达;
图5为本发明ELISA验证NDV-Nb49-HRP细胞分泌上清特异性结合NDV病毒颗粒的结果;其中,a:NDV-Nb49-HRP与NDV病毒颗粒的特异性结合;b:不同稀释度的NDV-Nb49-HRP细胞分泌上清与NDV病毒颗粒结合;
图6为本发明检测不同稀释度的NDV病毒颗粒的变化情况;其中,a:检测不同鸡胚尿囊液NDV病毒颗粒稀释梯度;b:检测不同质量的纯化的NDV病毒颗粒;c:不同血凝效价鸡胚尿囊液NDV病毒颗粒的线性回归方程;d:不同质量的纯化的NDV病毒颗粒的线性回归方程;
图7为本发明双抗体夹心ELISA的特异性分析结果。
具体实施方式
现详细说明本发明的多种示例性实施方式,该详细说明不应认为是对本发明的限制,而应理解为是对本发明的某些方面、特性和实施方案的更详细的描述。
应理解本发明中所述的术语仅仅是为描述特别的实施方式,并非用于限制本发明。另外,对于本发明中的数值范围,应理解为还具体公开了该范围的上限和下限之间的每个中间值。在任何陈述值或陈述范围内的中间值以及任何其他陈述值或在所述范围内的中间值之间的每个较小的范围也包括在本发明内。这些较小范围的上限和下限可独立地包括或排除在范围内。
除非另有说明,否则本文使用的所有技术和科学术语具有本发明所述领域的常规技术人员通常理解的相同含义。虽然本发明仅描述了优选的方法和材料,但是在本发明的实施或测试中也可以使用与本文所述相似或等同的任何方法和材料。本说明书中提到的所有文献通过引用并入,用以公开和描述与所述文献相关的方法和/或材料。在与任何并入的文献冲突时,以本说明书的内容为准。
在不背离本发明的范围或精神的情况下,可对本发明说明书的具体实施方式做多种改进和变化,这对本领域技术人员而言是显而易见的。由本发明的说明书得到的其他实施方式对技术人员而言是显而易见得的。本申请说明书和实施例仅是示例性的。
关于本文中所使用的“包含”、“包括”、“具有”、“含有”等等,均为开放性的用语,即意指包含但不限于。
实施例1
纳米抗体NDV-Nb4与铁蛋白融合蛋白的原核表达
1、纳米抗体NDV-Nb4与铁蛋白融合表达的原核表达载体的构建
(1)获取编码NDV-Nb4的基因与编码铁蛋白的基因
利用Primer 5.0软件进行设计引物,由西安擎科生物工程股份有限公司合成,具体如下:
上游引物NDV-4-FeNb-Nde I-F:
5’-CCGCATATGATGCTGAGCGAACGCATGCTGA-3’;
下游引物NDV-4-Ferritin-R:5’-ACTGCCTCCACCGCCACTGCCTCCACCGCCACTGCCTCCACCGCCGTCCTTGGCAAATTTCAGTTT;
以编码铁蛋白重组质粒(pUC-57-Fe,金唯智生物科技有限公司合成)为模板,通过PCR特异性扩增编码铁蛋白的核酸序列,扩增采用大连宝生物的GxL Polymerase,按照其操作说明书进行操作。扩增条件为:98℃预热3min,98℃10s、55℃45s、68℃1min,35个循环,68℃延伸5min,4℃保持。扩增完成后,琼脂糖凝胶电泳成功扩增获得了大小为522bp的目的基因(见图1a)。
同样利用Primer5.0软件,依据编码抗NDV纳米抗体NDV-Nb4的核苷酸序列,设计引物,由西安擎科生物工程股份有限公司合成,具体如下:
上游引物NDV-4-Nb-F:5’-AAACTGAAATTTGCCAAGGACGGCGGTGGAGGCAGTGGCGGTGGAGGCAGTGGCGGTGGAGGCAGTCTGCAGGAGTCTGGGGGAGGCT-3’;
下游引物NDV-4-FeNb-BamH I-R:5’-GACGGATCCTTAGGCGGCCGCTGAGGAGACGGTGACC;
以含有NDV-Nb4基因的重组质粒(pMERC-NDV-Nb4,由金唯智生物技术有限公司合成),PCR扩增,在用的聚合酶与扩增铁蛋白一样,扩增条件为:98℃预热1min,98℃10s、55℃15s、68℃30s,32个循环,68℃延伸2min,4℃保持。扩增完成后,核酸电泳分析得到大小为320bp的纳米抗体序列(见图1a)。
(2)编码NDV-Nb4与铁蛋白融合蛋白基因
胶回收上述PCR扩增获得的两个目的片段,然后以回收的目的片段为模板,利用扩增铁蛋白基因的上游引物NDV-4-FeNb-Nde I-F和扩增纳米抗体NDV-Nb4基因的下游引物NDV-4-FeNb-BamH I-R,PCR扩增铁蛋白和NDV-Nb4的融合片段,扩增条件为98℃预热3min,98℃10s、50℃55s、68℃1min,37个循环,68℃延伸5min,4℃保持。对融合扩增产物进行琼脂糖凝胶电泳分析得到大小为820bp的NDV-Fe-Nb4序列(见图1b),并进行胶回收。
将扩增获得NDV-Fe-Nb4融合基因的PCR产物用Nde I和BamH I进行双酶切处理,胶回收试剂盒回收获得NDV-Fe-Nb4基因。然后将原核表达载体pET-28a(商品化载体,Novagen公司)同样用Nde I和BamHI进行双酶切处理(见图1c),与回收获得的NDV-Fe-Nb4基因进行连接后,转入感受态大肠杆菌DH-5α并均匀涂在含卡那霉素抗性的LB平板上,获取重组质粒pET-28a-NDV-Fe-Nb4,然后挑取单克隆菌落并PCR鉴定后(见图1d)、测序鉴定正确后转入感受态Transetta(DE3)用于表达。
2、纳米抗体NDV-Nb4与铁蛋白融合蛋白的原核表达、纯化与鉴定
将所获取的阳性质粒pET-28a-NDV-Fe-Nb4转化的大肠杆菌Transetta(DE3),37℃震荡培养至菌液OD600值达0.6时,用终浓度为1.0mM/L,IPTG16℃150rpm,诱导20h。取15mL菌液,6000g离心10min,弃上清重悬沉淀。经超声处理后,6000g离心分离上清与沉淀样品,然后配制12%的分离胶,4%的浓缩胶,上清和沉淀样品上样后,SDS-PAGE电泳,液相脱色后,确定重组蛋白为可溶性表达(见图2a)。
利用Ni-NTA-6FF Beads按照常规方法(分子克隆指南)进行蛋白纯化,首先利用平衡液平衡柱子,然后将获得的上清直接上柱,流速控制为0.5ml/min,收集流出液;然后依次洗脱杂蛋白和目的蛋白,纯化样品进行SDS-PAGE检测,结果显示蛋白得到很好的纯化(见图2b)。
电泳后,小心取下凝胶放于转膜液中,取大小合适的PVDF膜,置于甲醇中15-20s活化。按湿转顺序依次放置厚滤纸、凝胶、PVDF膜、厚滤纸,注意叠放过程中无气泡产生;安装转膜仪,加入足量转膜液,并放置冰袋确保转膜在低温下进行;100V转膜30min,转膜结束后,用5%脱脂奶粉封闭PVDF膜,室温孵育1h;然后孵育一抗(鼠抗His标签,1:5000)1h,洗膜后;孵育HRP标记的羊抗鼠二抗(1:5000)1h;发光液ECL发光,结果发现表达的重组蛋白能够与标签抗体发生免疫反应(见图2c),证明铁蛋白与纳米抗体NDV-Nb4融合蛋白得到正确的表达。
随后将表达纯化的重组蛋白,滴在干燥的载玻片上,再把带有支持膜的铜网放在悬液的液珠上漂浮以沾取样品,然后,用滤纸吸干铜网上的多余悬液,再将铜网在染色液珠(醋酸铀)上悬浮,时间1-2min,最后再用滤纸将染液吸干即可观察,透射电镜观察其发生自组装(见图2d)。
3、原核表达的NDV-Fe-Nb4融合蛋白与NDV的免疫反应检测
将纯化的NDV颗粒包被ELISA板,H9N2型禽流感为阴性对照,包被量为400ng/孔,4℃孵育过夜后,200μL封闭液(2.5%脱脂奶粉的PBST(0.01M PBS,0.05%Tween 20),37℃封闭1小时后,洗涤液PBST洗板3次,加入400ng/孔的原核表达的NDV-Fe-Nb4蛋白,37℃孵育1h后,洗涤液PBST洗板3次,按效价1:2000加入鼠源抗His标签单抗,100μL/孔,37℃孵育1小时后,洗涤液PBST洗板3次,按效价1:5000加入羊抗鼠单抗,100μL/孔,37℃孵育1小时后,洗涤液PBST洗板3次,加入TMB(3,3’,5,5’-四甲基联苯胺)显色,ELISA自动酶标仪OD45nm读数。结果如图3a所示,构建的NDV-Fe-Nb4纳米抗体仍保持纳米抗体Nb4与NDV颗粒抗原结合的能力。
将原核表达的NDV-Fe-Nb4和NDV-Nb4包被ELISA板,800ng/孔,4℃孵育过夜后,200μL封闭液(2.5%脱脂奶粉的PBST),37℃封闭1h后,洗涤液PBST洗板3次,加入1μg/孔的纯化的NDV病毒颗粒,H9N2型禽流感作为阴性对照,37℃孵育1小时后,洗涤液PBST洗板3次,按效价1:5000加入鼠源抗NDV单抗(购自千寻生物)和阳性H9N2鸡血清,100μl/孔,37℃孵育1h后,洗涤液PBST洗板3次,1:5000,分别对应加入羊抗鼠和兔抗鸡单抗,100μl/孔,37℃孵育1h后洗涤液PBST洗板3次,TMB显色,ELISA自动酶标仪OD45nm读数。结果如图3b所示,构建的NDV-Fe-Nb4纳米抗体与纳米抗体Nb4相比,具有更强捕获NDV颗粒的能力。
实施例2
真核分泌表达NDV-Nb49与辣根过氧化物酶融合蛋白
1、纳米抗体NDV-Nb49与辣根过氧化物酶融合蛋白(NDV-Nb49-HRP)真核表达载体的构建与表达
将NDV-Nb49片段(pMERC-NDV-Nb49,由金唯智生物技术有限公司合成)和pCMV-HRP载体(载体构建方法参照文献Nanobody-horseradish peroxidase fusion protein as anultrasensitive probe to detect antibodies against Newcastle disease virus inthe immunoassay.J Nanobiotechnol,2019,17:35.)用限制性内切酶Not I和Pst I同时进行双酶切处理后(见图4a),胶回收并用T4连接酶连接,将连接产物转入感受态大肠杆菌DH-5α中,并均匀涂在含氨苄青霉素抗性的LB平板上,挑取单克隆,然后进行菌液PCR鉴定(见图4b)和测序鉴定,提取阳性质粒,并将阳性质粒转染至HEK293T细胞,收集细胞分泌表达上清。
将收集的上清SDS-PAGE后,电转化至硝酸纤维素膜上后,Western blotting分析(一抗鼠抗His,效价1:5000;羊抗鼠融合HRP,效价1:5000)鉴定上清中含有NDV-Nb49-HRP重组融合蛋白(图4d)。
此外,IFA检测(间接免疫荧光检测),将质粒转染的HEK293T细胞用70%乙醇固定,4℃条件下固定30min,弃掉固定液在生物安全柜中吹干;1%BSA,37℃封闭1h;PBS洗三次,每次10min;37℃孵育一抗鼠抗His,效价1:200,孵育1h;PBS洗三次,每次10min;孵育羊抗鼠FITC,效价1:400,37℃条件下孵育1h;DAPI室温孵育20min;PBS洗三次,每次10min。荧光显微镜观察,结果同样显示构建的纳米抗体NDV-Nb49-HRP成功表达(见图4c)。
2、真核分泌表达的NDV-Nb49-HRP与NDV颗粒的免疫反应性检测
将纯化的NDV颗粒包被ELISA板,H9N2型禽流感病毒为阴性对照,包被量为400ng/孔,4℃孵育过夜后,200μL封闭液(2.5%脱脂奶粉的PBST),37℃封闭1h后,洗涤液PBST洗板3次,直接加入不同稀释度(1:1-1:10000)的细胞培养上清(包含NDV-Nb49-HRP),37℃孵育1h后,直接加入TMB显色,ELISA自动酶标仪OD45nm读数。
结果如图5所示,构建的NDV-Nb49-HRP仍保持纳米抗体Nb49与NDV颗粒抗原结合的能力。
实施例3
纳米抗体NDV-Fe-Nb4与NDV-Nb49-HRP配对在NDV检测中的应用
1、NDV-Fe-Nb4作为捕获抗体、NDV-Nb49-HRP作为检测抗体,检测NDV的敏感性分析
将实施例1表达的纳米抗体与铁蛋白的融合蛋白NDV-Fe-Nb4作为包被抗体;将实施例2表达的纳米抗体与辣根过氧化物酶的融合蛋白NDV-Nb49-HRP作为检测抗体;然后将纯化的NDV(LaSota株)颗粒利用PBS缓冲液按照10倍梯度稀释,以确定两株纳米抗体配对检测NDV的敏感性。
首先,将原核表达的融合蛋白NDV-Fe-Nb4包被ELISA板,800ng/孔,4℃孵育过夜后,200μL封闭液(2.5%脱脂奶粉的PBST),37℃封闭1h后,洗涤液PBST洗板3次,平行加入不同稀释倍数的纯化的NDV颗粒和不同稀释梯度的NDV尿囊液毒(不同的血凝效价),H9N2型禽流感病毒作为阴性对照,37℃孵育1h后,洗涤液PBST洗板3次,直接按体积比1:10加入细胞培养上清(包含NDV-Nb49-HRP),37℃孵育1h后,直接加入TMB显色,ELISA自动酶标仪OD45nm读数。
结果如图6所示,NDV-Fe-Nb4与NDV-Nb49-HRP配对的纳米抗体检测NDV,可以最低检到血凝效价为22的NDV悬液以及10ng纯化的NDV。
2、NDV-Fe-Nb4作为捕获抗体、NDV-Nb49-HRP作为检测抗体,检测NDV的特异性分析
利用建立的ELISA分析两株纳米抗体配对检测NDV的特异性分析,首先将原核表达的融合蛋白NDV-Fe-Nb4包被ELISA板,800ng/孔,4℃孵育过夜后,200μL封闭液(2.5%脱脂奶粉的PBST),37℃封闭1h后,洗涤液PBST洗板3次;然后将其它感染禽类的主要病毒包括H9N2,H5N1,H7N9,IBV,IBDV,FADV和ALV-J以及阳性对照NDV分别加入每孔中(100μL/孔);37℃孵育1h后,洗涤液PBST洗板3次,直接按体积比1:10加入细胞培养上清(包含NDV-Nb49-HRP),37℃孵育1h后,直接加入TMB显色,ELISA自动酶标仪OD45nm读数。
结果如图7所示,NDV-Fe-Nb4与NDV-Nb49-HRP配对的纳米抗体仅能特异性检测NDV,不能检测其它感染禽类的主要病毒。
以上所述的实施例仅是对本发明的优选方式进行描述,并非对本发明的范围进行限定,在不脱离本发明设计精神的前提下,本领域普通技术人员对本发明的技术方案做出的各种变形和改进,均应落入本发明权利要求书确定的保护范围内。
序列表
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<120> 两株抗新城疫病毒纳米抗体及其表达制备方法和应用
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Claims (2)
1.一种抗新城疫病毒的纳米抗体NDV-Nb4,其特征在于,氨基酸序列如SEQ ID NO:1所示。
2.一种抗新城疫病毒的纳米抗体NDV-Nb49,其特征在于,氨基酸序列如SEQ ID NO:2所示。
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