CN108892723A - 用于检测猪流行性腹泻病毒的单域重链抗体、制备方法及应用 - Google Patents
用于检测猪流行性腹泻病毒的单域重链抗体、制备方法及应用 Download PDFInfo
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- CN108892723A CN108892723A CN201810761629.6A CN201810761629A CN108892723A CN 108892723 A CN108892723 A CN 108892723A CN 201810761629 A CN201810761629 A CN 201810761629A CN 108892723 A CN108892723 A CN 108892723A
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Abstract
本发明公开了一种用于检测猪流行性腹泻病毒的单域重链抗体、制备方法及应用。本发明采用猪流行性腹泻病毒免疫阿拉善双峰驼,利用其外周血淋巴细胞建立针对猪流行性腹泻病毒的单域重链抗体库,将表达纯化的猪流行性腹泻病毒S蛋白偶联在酶标板上作为抗原,利用噬菌体展示技术对单域重链抗体库进行免疫性筛选,获得针对猪流行性腹泻病毒S蛋白的单域重链抗体基因,将单域重链抗体基因转入大肠杆菌中,建立在大肠杆菌中高效表达的单域重链抗体株,分析每个单域重链抗体株的基因序列,获得针对猪流行性腹泻病毒S蛋白的单域重链抗体VHH链的氨基酸序列SEQ ID NO.8。本发明的抗体稳定性和特异性良好,可用于检测和治疗PEDV。
Description
技术领域
本发明涉及基因工程技术领域,尤其涉及一种用于检测猪流行性腹泻病毒的单域重链抗体、制备方法及应用。
背景技术
猪流行性腹泻是由猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)引起的,以腹泻、呕吐、脱水和对幼龄仔猪高致死率为主要特征的一种高度接触性肠道传染病。感染PEDV的猪病理变化主要表现在猪的空肠和回肠部分的肠绒毛的萎缩和脱落,对猪的危害很大,给养猪业带来严重的影响和经济损失。S蛋白是PEDV一个重要的结构蛋白,它是位于病毒粒子表面的纤突糖蛋白,在病毒感染宿主机体后介导中和抗体产生的过程中发挥重要生物学作用,S蛋白是PEDV基因工程疫苗和诊断技术等方面研究中最重要的一个靶蛋白。
常用防治PED发生的方法是将PEDV感染猪的粪便或发病仔猪的肠内容物,进行病毒的人工接种使其感染妊娠孕母猪,以刺激其产生乳源抗体,从而大幅度降低新生仔猪的死亡率和缩短PED在猪肠中的存在时间。
目前,对PEDV S蛋白抗原表位的研究较少,而抗原表位能够刺激机体产生抗体进而保护机体免受病原的侵害。
发明内容
有鉴于此,本发明实施例提供了一种用于检测猪流行性腹泻病毒的单域重链抗体、制备方法及应用,主要目的开发一种稳定且特异性良的抗体以用于检测或预防猪流行性腹泻病毒。
为达到上述目的,本发明主要提供了如下技术方案:
一方面,本发明实施例提供了一种用于检测猪流行性腹泻病毒的单域重链抗体,所述单域重链抗体的氨基酸序列为SEQ ID NO.8。
另一方面,本发明实施例提供了上述用于检测猪流行性腹泻病毒的单域重链抗体的制备方法,所述方法包括以下步骤:采用猪流行性腹泻病毒免疫阿拉善双峰驼,利用所述双峰驼外周血淋巴细胞建立针对猪流行性腹泻病毒的单域重链抗体库,将表达并纯化的猪流行性腹泻病毒S蛋白偶联在酶标板上作为抗原,利用噬菌体展示技术对所述单域重链抗体库进行免疫性筛选,获得针对猪流行性腹泻病毒S蛋白的单域重链抗体基因,通过测序获得其氨基酸序列。
作为优选,所述通过测序获得其氨基酸序列具体为:将所述单域重链抗体基因转入大肠杆菌中,建立在大肠杆菌中高效表达的单域重链抗体株,利用序列比对软件分析每个所述单域重链抗体株的基因序列,最后获得针对猪流行性腹泻病毒S蛋白的单域重链抗体VHH链的氨基酸序列。
再一方面,本发明实施例提供了上述用于检测猪流行性腹泻病毒的单域重链抗体在制备用于检测猪流行性腹泻病毒的检测试剂中的应用。
再一方面,本发明实施例提供了上述用于检测猪流行性腹泻病毒的单域重链抗体在制备用于治疗猪流行性腹泻病毒的药物中的应用。
又一方面,本发明实施例提供了上述用于检测猪流行性腹泻病毒和/或其含量的ELISA试剂盒,所述ELISA试剂盒中的检测抗体为上述用于检测猪流行性腹泻病毒的单域重链抗体。
又一方面,本发明实施例提供了用于预防和/或治疗猪流行性腹泻病毒的药物,所述药物包含上述用于检测猪流行性腹泻病毒的单域重链抗体。
又一方面,本发明实施例提供了用于预防和/或治疗猪流行性腹泻病毒的疫苗,所述疫苗是上述用于检测猪流行性腹泻病毒的单域重链抗体经过浓缩和冻干获得。
与现有技术相比,本发明的有益效果是:
本发明采用猪流行性腹泻病毒免疫阿拉善双峰驼,利用所述双峰驼外周血淋巴细胞建立针对猪流行性腹泻病毒的单域重链抗体库,将表达并纯化的猪流行性腹泻病毒S蛋白偶联在酶标板上作为抗原,利用噬菌体展示技术对所述单域重链抗体库进行免疫性筛选,获得针对猪流行性腹泻病毒S蛋白的单域重链抗体基因,将所述单域重链抗体基因转入大肠杆菌中,建立在大肠杆菌中高效表达的单域重链抗体株,利用序列比对软件分析每个所述单域重链抗体株的基因序列,最后获得针对猪流行性腹泻病毒S蛋白的单域重链抗体VHH链的氨基酸序列,实验证明本发明研发的上述抗体稳定性和特异性均良好;并也证明了该单域重链抗体可以同PEDV S蛋白和PEDV病毒特异性结合,从而可应用于猪流行性腹泻病毒检测试剂和猪流行性腹泻治疗药物的开发。
附图说明
图1是本发明实施例提供的PCR扩增得到的VHH基因片段检测电泳图;
图2是本发明实施例提供的针对PEDV单域重链抗体,经镍柱亲和层析纯化前后的SDS-PAGE的电泳图;
图3是本发明实施例提供的以PEDV单域重链抗体为核心材料制作的基于ELISA技术的PEDV检测试剂盒原理示意图。
具体实施方式
为更进一步阐述本发明为达成预定发明目的所采取的技术手段及功效,以下以较佳实施例,对依据本发明申请的具体实施方式、技术方案、特征及其功效,详细说明如后。下述说明中的多个实施例中的特定特征、结构、或特点可由任何合适形式组合。
噬菌体展示技术:M13单链丝状噬菌体展示系统是目前应用最广泛的文库展示系统,通过将蛋白、抗体或多肽与噬菌体pIII蛋白融合表达,使之参与噬菌体组装,并展示在噬菌体表面,形成噬菌体展示文库。使用噬菌体展示文库,针对特定靶分子进行3-5轮亲和筛选,可以富集能特异性结合靶分子的噬菌体,得到相应的DNA序列信息。
猪流行性腹泻病毒:本发明研究以下实验选用的猪流行性腹泻病毒购自中国科学院武汉病毒所。
实施例1(构建PEDV特异性的单域重链抗体库)
(1)由PEDV病毒与弗氏佐剂(Sigma)混合,免疫一峰阿拉善双峰驼,每10天免疫一次,共免疫3次,第一次使用弗氏完全佐剂,剩余两次使用弗式不完全佐剂,第一次免疫前采集基础血清,最后一次免疫后一周采集血清,测定抗体效价;
(2)3次免疫结束后,提取骆驼外周血淋巴细胞150毫升并提取总RNA;
(3)将总RNA RT-PCR反转录成cDNA;
(4)利用巢式PCR扩增VHH链,片段的大小约为400bp;图1是PCR扩增得到的VHH基因片段检测电泳图;
(5)利用限制性内切酶Sfi I及Not l酶切pCANTAB5E噬菌体展示载体及VHH基因片段的PCR纯化产物,并连接两个片段;
(6)连接产物电转化至感受态E.coli TG1中,构建PEDV特异性单域重链抗体噬菌体展示文库,并测定抗体库库容,库容的大小为5.5×106。
实施例2(筛选PEDV单域重链抗体)
(1)取2毫升转化TG1细胞加入到100毫升2×TY-AG培养基中,37℃培养2小时;
(2)加入辅助噬菌体M13K07进行感染,感染比例为M13K07:TG1=20:1,室温静置30分钟;
(3)离心10分钟,将离心沉淀下来的细胞重悬接入100毫升2×YT-AK培养基,37℃培养过夜;
(4)将培养液2200g离心30分钟,取上淸液,用PEG/NaCl沉淀扩增后的噬菌体,并将沉淀后的噬菌体溶解在PBS溶液中,0.22微米滤器过滤,4℃保存备用;
(5)用包被缓冲液稀释PEDV S蛋白并偶联在酶标板上,4℃放置过夜;
(6)第二天每孔中分别加入300微升封闭液,室温封闭2小时;
(7)将上述步骤4所述方法收集的噬菌体与等体积的封闭液混匀在室温孵育20分钟,然后将封闭后的噬菌体加入酶标板,每孔100微升,在室温下作用1小时;
(8)用PBST(PBS中含有0.05%吐温20)洗5遍,洗去不结合的噬菌体;
(9)以新鲜制备的100毫摩尔三乙胺将与PEDV S蛋白特异性结合的噬菌体洗脱,并感染处于对数生长期的E.coli TG1,再产生并纯化噬菌体用于下一轮的筛选,相同筛选过程重复3轮,使PEDV S蛋白特异性噬菌体不断富集。
实施例3(用phage-ELISA法筛选PEDV S蛋白特异性克隆)
(1)从上述3轮筛选后含有噬菌体的细胞培养皿中随机挑取96个单克隆接种于1毫升2×TY-AG培养基中,37℃培养过夜;
(2)将过夜培养的菌液转接入1.3毫升2×YT-AG培养基和109M13K07的4个24孔板中,37℃150rpm培养2h,1000g离心20min,沉淀用1毫升2×YT-AK培养基重悬沉淀,30℃250rpm过夜培养,次日离心收集噬菌体上清;
(3)将上述重组噬菌体与封闭液1:1体积混合,加入包被有10微克每毫升的PEDV S蛋白的96孔酶标板中,37℃孵育1h,阴性对照孔加100微升109M13K07;
(4)用PBST洗去未结合的噬菌体,100微升1:5000稀释的HRP/anti-M13单抗。37℃孵育1h;
(5)每孔加入100微升TMB,室温避光反应15-30min,每孔加入50微升2M H2SO4终止反应,酶标仪测波长450nm;
(6)当样品孔OD值大于对照孔OD值2倍以上时,判为阳性克隆孔;
(7)将阳性克隆接种至5毫升含100微克氨苄青霉素每毫升的LB液体中以便提取质粒并进行测序;
根据序列比对软件DNAStar分析各个克隆株的基因序列,把基因序列相同的株视为同一克隆株,而其序列不同的株视为不同克隆株,最终共有1株抗体;其抗体为FR1(SEQID No.1)-CDR1(SEQ ID No.5)-FR2(SEQ ID No.2)-CDR2(SEQ ID No.6)-FR3(SEQ IDNo.3)-CDR3(SEQ ID No.7)-FR4(SEQ ID No.4)区,构成整个VHH(其氨基酸序列为SEQ IDNo.8,其核苷酸序列与氨基酸混合序列为SEQ ID No.9)。
实施例4(抗PEDV单域重链抗体在宿主菌大肠杆菌中表达、纯化)
(1)将Phage-ELISA阳性并经测序正确克隆株的质粒酶切,将酶切得到的VHH基因连接pET-28a载体电转化到E.coli Transetta(DE3)中,并将其涂布在含有100微克每毫升氨苯青霉素LB固体培养基的板上,37℃培养过夜;
(2)挑选单个菌落接种在5毫升含有氨苄青霉素的LB培养液中,37℃培养过夜;
(3)接种1毫升的过夜菌种至100毫升LB培养基中,37℃摇床培养,培养到OD值达到0.4-0.6时,加入终浓度1毫摩尔IPTG,30℃培养过夜;
(4)第二天,离心收菌,将菌体利用超声破碎仪破碎,收集破碎上清和沉淀;
(5)以镍柱亲和层析纯化抗体蛋白,制备高纯度的纯化蛋白;
图2是表达和纯化的针对PEDV单域重链抗体SDS-PAGE的电泳结果;M:蛋白分子标记,单位为KDa;1:未诱导前细菌破碎粗提液;2:诱导后细菌破碎粗提物;3-5:共3步洗脱液洗脱的样品。
应用例1(纯化后的PEDV单域重链抗体在检测样品中PEDV含量的试剂盒中的应用)
如图3所示,为了便于制备PEDV检测试剂盒,本发明以PEDV免疫兔子制备了多克隆抗体,抗体效价为1:16000,将PEDV兔多克隆抗体包被在ELISA酶标板中,加入倍比稀释的抗原PEDV病毒,平行操作加入待检测的样品,在室温下轻摇1小时;用PBST洗去未结合的抗原后,再加入HRP标记的PEDV单域重链抗体,在室温下放置1小时,用PBST洗去未结合的抗体后,加入TMB显色液,于酶标仪上,在450nm波长读取吸收值。以将各浓度梯度的PEDV标准品稀释倍数为横坐标,PEDV标准品的吸收值为纵坐标作出标准曲线,然后将待检测样品的吸收值读数带入浓度标准曲线,即可判断出样品中的PEDV含量。
本发明实施例中未尽之处,本领域技术人员均可从现有技术中选用。
以上公开的仅为本发明的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,可轻易想到变化或替换,都应涵盖在本发明的保护范围之内。因此,本发明的保护范围应以上述权利要求的保护范围为准。
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Claims (8)
1.用于检测猪流行性腹泻病毒的单域重链抗体,其特征在于,所述单域重链抗体的氨基酸序列为SEQ ID NO.8。
2.权利要求1所述的用于检测猪流行性腹泻病毒的单域重链抗体的制备方法,其特征在于,所述方法包括以下步骤:采用猪流行性腹泻病毒免疫阿拉善双峰驼,利用所述双峰驼外周血淋巴细胞建立针对猪流行性腹泻病毒的单域重链抗体库,将表达并纯化的猪流行性腹泻病毒S蛋白偶联在酶标板上作为抗原,利用噬菌体展示技术对所述单域重链抗体库进行免疫性筛选,获得针对猪流行性腹泻病毒S蛋白的单域重链抗体基因,通过测序获得其氨基酸序列。
3.如权利要求2所述的用于检测猪流行性腹泻病毒的单域重链抗体的制备方法,其特征在于,所述通过测序获得其氨基酸序列具体为:将所述单域重链抗体基因转入大肠杆菌中,建立在大肠杆菌中高效表达的单域重链抗体株,利用序列比对软件分析每个所述单域重链抗体株的基因序列,最后获得针对猪流行性腹泻病毒S蛋白的单域重链抗体VHH链的氨基酸序列。
4.权利要求1所述的用于检测猪流行性腹泻病毒的单域重链抗体在制备用于检测猪流行性腹泻病毒的检测试剂中的应用。
5.权利要求1所述的用于检测猪流行性腹泻病毒的单域重链抗体在制备用于治疗猪流行性腹泻病毒的药物中的应用。
6.用于检测猪流行性腹泻病毒和/或其含量的ELISA试剂盒,其特征在于,所述ELISA试剂盒中的检测抗体为权利要求1-3任一项所述的用于检测猪流行性腹泻病毒的单域重链抗体。
7.用于预防和/或治疗猪流行性腹泻病毒的药物,其特征在于,所述药物包含权利要求1-3任一项所述的用于检测猪流行性腹泻病毒的单域重链抗体。
8.用于预防和/或治疗猪流行性腹泻病毒的疫苗,其特征在于,所述疫苗是所述权利要求1-3任一项所述的用于检测猪流行性腹泻病毒的单域重链抗体经过浓缩和冻干获得。
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