CN106008709B - 一种特异性结合PRRS病毒非结构蛋白Nsp4纳米抗体及其应用 - Google Patents
一种特异性结合PRRS病毒非结构蛋白Nsp4纳米抗体及其应用 Download PDFInfo
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Abstract
本发明涉及一种PRRS病毒非结构蛋白Nsp4的纳米抗体，同时公布了该纳米抗体的氨基酸序列以及编码该纳米抗体的DNA序列。本发明还提供了一种慢病毒载体，其可以将上述纳米抗体基因导入宿主细胞使其发挥抗病毒功能。本发明的Nsp4纳米抗体可以特异性结合PRRS病毒非结构蛋白Nsp4，并且具有抑制PRRS病毒在细胞中增殖的功能。
Description
技术领域
本发明属于生物技术领域，具体涉及一种PRRS病毒非结构蛋白Nsp4纳米抗体及其在抗病毒中的应用。
背景技术
猪繁殖与呼吸综合征(Porcine reproductive and respiratory syndrome，PRRS)又名蓝耳病，是由PRRS病毒感染所致的一种猪的急性传染病。该病使得我国养猪业遭受的巨大的经济损失，但是由于PRRS病毒具有抗原变异性、嗜巨噬细胞性、抗体依赖性增强作用(ADE)和持续性感染等特征(Chand R J：Pathogenesis of porcine reproductiveand respiratory syndrome virus.Curr Opin Virol，2012，2(3)：256-263)，现有疫苗对该病的保护作用非常有限，而且目前还没有针对该病的特效抗病毒药物。
PRRS病毒为单股正链RNA病毒，其基因组大小约为15kb，包括至少11个开放阅读框(ORF)。其中ORF1(包括ORF1a和ORF1b)是最大的一个阅读框约占病毒基因组的80％。ORF1a和ORF1b编码的多聚蛋白pp1a和pp1ab随后被病毒编码的蛋白酶酶解为成熟的非结构蛋白，这些非结构蛋白共同组成病毒的复制转录复合体来完成病毒的复制和增殖。Nsp4具有3C样丝氨酸蛋白酶活性，是PRRS病毒的主要蛋白酶，负责Nsp3-12的加工与成熟。最新的研究还表明Nsp4还与病毒的毒力以及宿主对病毒的固有免疫有关。因此Nsp4非常合适作为抗病毒药物靶点。
1993年Hamers等人发现骆驼体内存在一种仅具有重链的抗体，将其称为重链抗体(heavy-chain antibodies，HCAbs)(Hamers-Casterman C：Naturally occurringantibodies devoid of light chains.Nature，1993，6428(363)：446-448)。这类抗体的抗原结合位点仅由重链的可变区VHH(variabledomain of the heavy chain of HCAbs，VHH)单结构域形成，尽管天然缺失轻链可变区，却仍具有良好和广泛的抗原结合力。VHH抗体是目前所发现的具有完整功能的最小分子抗体片段，分子量为15kDa，仅为常规抗体的1/10，其分子高度4.8nm，直径2.2nm，故被称为纳米抗体(nanobody)。VHH抗体重要的特征之一是其具有更长的抗原互补结合区(CDR)，可结合一些常规抗体无法接近的抗原表位，如位于酶蛋白裂隙中的活性中心结构(De Genst E：Molecular basis for the preferentialcleft recognition by dromedary heavy-chain antibodies.Proc Natl Acad Sci USA，2006，103(12)：4586-4591)。另外，它还具有易表达，水溶性好，稳定性强，免疫原性弱，组织穿透性好等优点，使得该抗体作为一种小型化的基因工程抗体在基础研究、药物开发等领域有广阔的应用前景。
发明内容
发明目的
本发明所要解决的技术问题是提供一种特异性针对PRRS病毒非结构蛋白Nsp4并可以抑制病毒增殖的纳米抗体，同时提供这些纳米抗体的编码序和一种慢病毒载体。
技术方案
本发明的目的是通过如下措施来达到：
一种PRRS病毒非结构蛋白Nsp4的纳米抗体，包括框架区FR和互补决定区CDR，其特征在于，所述框架区FR选自下组的FR的氨基酸序列：SEQ ID NO：1所示的FR1，SEQ ID NO：2所示的FR2，SEQ ID NO：3所示的FR3，SEQ ID NO：4所示的FR4；或SEQ ID NO：5所示的FR1，SEQ ID NO：6所示的FR2，SEQ ID NO：7所示的FR3，SEQ ID NO：8所示的FR4；所述互补决定区CDR选自下组的CDR氨基酸序列：SEQ ID NO：9所示的CDR1，SEQ ID NO：10所示的CDR2，SEQID NO：11所示的CDR3；或SEQ ID NO：12所示的CDR1，SEQ ID NO：13所示的CDR2，SEQ ID NO：14所示的CDR3。优选地，所述的PRRS病毒非结构蛋白Nsp4的纳米抗体，其特征在于，具有SEQID NO：15或SEQ ID NO：16所示的氨基酸序列。
一种DNA分子，其特征在于，它编码本发明所述的Nsp4纳米抗体。优选地，所述DNA分子，其特征在于，具有SEQ ID NO：17所示的核苷酸序列。
一种慢病毒载体，其特征在于，它含SEQ ID NO：17或SEQ ID NO：18所示的核苷酸序列。优选地，所述慢病毒载体，其特征在于，其可以将PRRS病毒非结构蛋白Nsp4的纳米抗体基因导入宿主细胞使其发挥抗病毒功能。
有益效果
本发明的PRRS病毒非结构蛋白Nsp4特异性纳米抗体可以特异性结合Nsp4。利用慢病毒载体，其可以将上述纳米抗体基因导入宿主细胞，并在细胞中表达纳米抗体，从而发挥抗病毒功能。
附图说明
图1是ELISA实验鉴定Nsp4纳米抗体结合力及特异性的结果。
图2是用Western Blot检测经慢病毒转导后Marc-145细胞中Nanobody-eGFP融合蛋白表达的结果。泳道1是未经转导的Marc-145细胞，泳道2是Nb-NC慢病毒转导的Marc-145细胞，泳道3是Nb4-1慢病毒转导的Marc-145细胞，泳道4是Nb4-2慢病毒转导的Marc-145细胞。
图3 PRRS病毒接种细胞24和48小时后培养上清中子代病毒滴度(TCID50)。
具体实施方式
本发明首先将Nsp4重组蛋白免疫一只阿拉善双峰驼，经过5次免疫之后分离该双峰驼外周血淋巴细胞并构建了Nsp4特异的单域重链抗体文库。将可溶性Nsp4重组蛋白包被在酶标板上，利用噬菌体展示技术筛选Nsp4免疫性的纳米抗体基因库(骆驼重链抗体噬菌体展示基因库)，获得了Nsp4特异性纳米抗体。构建携带纳米抗体基因的慢病毒载体，将其导入Marc-145细胞，胞质内表达的Nsp4纳米抗体可以高效地抑制PRRS病毒在Marc-145细胞中的增殖。
下面结合具体实施例，进一步阐述本发明。
实施例1：针对PRRS病毒非结构蛋白Nsp4纳米抗体文库的构建：
(1)将5ml Nsp4重组蛋白(1mg/m1)与弗氏佐剂等体积混合并乳化均匀，免疫一只阿拉善双峰驼，每两周1次，共免疫5次，除第一次使用弗氏完全佐剂，剩余几次全部使用弗氏不全佐剂。(2)最后一次免疫后3天，提取骆驼外周血淋巴细胞并提取总RNA，参照QIAGENRNA提取试剂盒说明书操作。(3)按照InvitrogenIII第一链合成系统试剂盒说明书，将提取的RNA反转录成cDNA并利用套式PCR扩增VHH链，第一轮PCR：
上游引物：GTCCTGGCTGCTCTTCTACAAGG
下游引物：GGTACGTGCTGTTGAACTGTTCC
扩增重链抗体信号肽肽和抗体CH2之间的片段，55C退火，28个循环；回收700bp附近目的条带。
第二轮PCR：
以第一轮PCR回收产物作模板，
上游引物：CAGGTGCAGCTGCAGGAGTCTGGGGGAGR
下游引物：CTAGTGCGGCCGCTGAGGAGACGGTGACCTGGGT
扩增重链抗体可变区(VHH)基因，55℃退火，18个循环，回收目的片段。(4)使用限制性的内切酶(购自NEB)PstI及NotI酶切20μg pCANTAB 5E噬菌体展示载体，及7μg VHH，并用T4 DNA连接酶(NEB)连接两个片段。(5)将连接产物电转化至电转感受态细胞TG1中，活化后涂布于LB-AMP琼脂平板，37℃过夜培养，收集菌苔-80℃保存。(6)取50μl-80℃保存的甘油菌，接种于100ml LB-AMP培养基，待细菌生长至对数期，用20MOI M13KO7辅助噬菌体感染TG1细胞，过夜培养后纯化噬菌体，得到免疫骆驼纳米抗体噬菌展示基因库。
实施例2：针对Nsp4的纳米抗体筛选过程：
(1)将溶解在0.01M pH 7.4PBS中的Nsp4重组蛋白(10μg/孔)偶联在NUNC酶标板上，4℃放置过夜，同时设立阴性对照。(2)第二天加入200微升2.5％脱脂奶粉，室温封闭2小时。(3)2小时后，每孔加入100μl噬菌体(1×1011pfu免疫骆驼纳米抗体噬菌展示基因库)，在室温下作用1小时。(4)用PBST(PBS中含有0.05％吐温20)洗15遍，洗去不结合的噬菌体。(5)用三乙基胺(100mM)洗脱与Nsp4特异性结合的噬菌体，并感染处于对数期生长的大肠杆菌TG1，产生并纯化噬菌体用于下一轮的筛选。经过3轮筛选，富集阳性克隆。
实施例3：用酶联免疫方法(ELISA)鉴定单个阳性克隆：
(1)经过3轮筛选后，将感染噬菌体的TG1细胞按一定稀释比例涂布于LB-AMP琼脂平板，随机挑取96个单克隆接种于TB-AMP培养基中生长至对数期后，加入终浓度1mM的IPTG，37℃培养过夜。(2)收集菌体，-20℃冻融一次，上清中应含有纳米抗体片段。(3)取100μl上清加入经Nsp4包被的ELISA孔中，同时分别取100μl上清加入经对照蛋白Nsp9包被的ELISA孔中，室温放置1小时。(4)用PBST洗5次，加入Rabbit anti-E tag antibody(兔源多克隆抗体，购自南京金斯瑞公司)，室温放置1小时。(5)用PBST洗5次，加入HRP G antiRabbit antibody(山羊抗兔辣根过氧化物标记抗体，购自Jackson公司)，在室温下放置1小时。(6)用PBST洗5次，加入TMB显色底物，在405nm波长，读取吸收值。(7)当样品孔OD值大于对照孔OD值3倍以上时，判为阳性克隆孔。(8)对阳性克隆经行测序分析，最终得到两株Nsp4特异性纳米抗体，命名为Nb4-1和Nb4-2。ELISA结果如图1所示，核苷酸序列如SEQ ID NO：17和SEQ ID NO：18所示。
实施例4：慢病毒包装及转导
(1)通过overlap PCR将纳米抗体基因与增强型绿色荧光蛋白(EGFP)基因通过linker融合，并通过Xba I和BamH I酶切位点克隆入pTRIP-CMV-Puro载体，得到pTRIP-CMV-Nbx-Puro载体。
(2)将pTRIP-CMV-Nbx-Puro与载体psPAX2和pMD2.G共转染Marc-145细胞，参照罗氏X-tremeGENE-HP-DNA转染试剂说明书操作。(3)转染后16小时换液，转染后60小时收集含有慢病毒的培养上清。(4)用上述慢病毒转导Marc-145细胞，转导后36小时换用嘌呤霉素选择性培养基加压筛选。(5)Wessern-blot进一步验证纳米抗体EGFP融合蛋白的表达，结果如图2显示，在40kDa附近出现特异性的目的条带，说明融合蛋白表达。
实施例5：病毒感染实验
将Marc-145细胞和上述经慢病毒转导的Marc-145细胞铺在24孔细胞培养板，至细胞达到80％汇合度，接种0.1MOI的PRRS病毒SD-16毒株。感染后24和48小时收集细胞上清，检测病毒滴度(TCID50)，结果如图3显示，Nb4-1和Nb4-2慢病毒转导的Marc-145细胞产生的子代病毒滴度显著低于对照组(Nb-NC)。
Claims (6)
1.一种PRRS病毒非结构蛋白Nsp4的纳米抗体，其特征在于，具有SEQ ID NO：15或SEQID NO：16所示的氨基酸序列。
2.一种DNA分子，其特征在于，它编码权利要求1所述的Nsp4纳米抗体。
3.根据权利要求2所述的DNA分子，其特征在于，具有SEQ ID NO：17或SEQ ID NO：18所示的核苷酸序列。
4.一种慢病毒载体，其特征在于，它含SEQ ID NO：17或SEQ ID NO：18所示的核苷酸序列。
5.根据权利要求1所述的Nsp4纳米抗体在制备用于治疗和防治PRRS药物中的应用。
6.根据权利要求2所述的DNA分子在开发抗PRRS转基因猪中的应用。
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