CN114773458A - 猪流行性腹泻病毒Nsp15蛋白的多克隆抗体及其制备方法 - Google Patents
猪流行性腹泻病毒Nsp15蛋白的多克隆抗体及其制备方法 Download PDFInfo
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- G01N2469/10—Detection of antigens from microorganism in sample from host
Abstract
本发明涉及一种猪流行性腹泻病毒Nsp15蛋白的多克隆抗体及其制备方法。本发明成功构建了猪流行性腹泻病毒Nsp15蛋白的原核表达质粒，并摸索出37℃为最佳诱导温度，通过镍柱纯化得到高纯度Nsp15蛋白，纯度高达90％，蛋白浓度为0.69mg/mL，进而制备得到了效价较高的Nsp15蛋白多克隆抗体，抗体效价为2×105，WesternBlot试验验证Nsp15阳性血清特异性较好，可用于检测猪流行性腹泻病毒与Nsp15的功能机制研究。
Description
技术领域
本发明涉及医药技术领域，特别是涉及一种猪流行性腹泻病毒Nsp15蛋白的多克隆抗体及其制备方法。
背景技术
猪流行性腹泻(Porcine epidemic diarrhea,PED)于1971年发现于英国，是由猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)感染引起的一种猪的严重腹泻性传染病。PEDV一般通过直接或间接的粪口途径传播。在某些情况下，空气传播也可能在PEDV传播中发挥作用。PEDV主要通过腹泻粪便或呕吐物传播，也可通过临床或亚临床感染的猪、运输猪粪便或食物的工具、主人、访客等传播。其他污染源，如环境中的微生物、食品、饲料、母猪奶等都可能是病毒的潜在传播途径。新生仔猪由于母源抗体低下导致自身免疫力低下，对PEDV极为易感，并呈现出极高的病死率。目前，猪流行性腹泻成为世界上最具毁灭性的猪病毒性疾病之一，造成全球猪肉行业严重的经济损失。
PEDV可感染所有年龄阶段的猪，其临床症状为水样腹泻、呕吐、厌食和抑郁。新生仔猪发病率接近100％，但母猪的发病率不尽相同。根据发病猪场调查，PEDV通常的潜伏期约1-8天。猪场感染PEDV的猪粪便可在48小时内检测到病毒核酸，4周内都可能检测得到病毒核酸。1周龄仔猪感染PEDV后出现严重的水样腹泻和呕吐症状，症状维持3-4天，随后出现严重脱水和电解质失衡，最终导致死亡。1-3日龄仔猪死亡率接近100％。主要的症状出现在胃肠道，其特征是胃膨胀，内有未消化的乳凝块，肠壁薄而透明的，并伴有黄色液体。PEDV组织学特征主要是严重的弥漫性萎缩性肠炎、浅表绒毛细胞肿胀，伴有轻度细胞质空泡化，以及含有凋亡细胞的绒毛固有层皱缩。这过程阻碍了猪体对营养物质和电解质的消化和吸收，导致产生消化不良和腹泻等症状，最后仔猪出现严重和致命的脱水死亡。
PEDV蛋白中，发现有11种抑制干扰素的产生，包括Nsp1、Nsp3、Nsp7、Nsp14、Nsp15、Nsp16、E、M、N、ORF3。其中Nsp1是最有效的IFN拮抗剂，PEDVNsp1可以阻断IRF1的核易位并减少过氧化物酶体的数量，减少的过氧化物酶体和抑制IRF1介导的IFN-λ依赖于PEDV Nsp1的保守氨基酸。PEDVNsp1不干扰IRF3磷酸化和核易位，但是通过降解CREB结合蛋白(CBP)，中断了IRF3和CBP的增强体组装，PEDVNsp1通过降解CBP和抑制ISGs来调节宿主的先天免疫。PEDVNsp1也阻碍NF-κB核易位，这不仅影响干扰素的产生也会影响炎症因子的产生，如：TNF-α，IL-1β，IL-6，IL-15，IL-17。
PEDV Nsp3的PLP2结构也通过阻止RIG-I和STING介导的I型IFN活化而抑制固有免疫。PEDV PLpro域中的PLP2结构也通过阻止RIG-I和STING介导的I型IFN活化，这一机制主要是阻止了RIG-I 63位赖氨酸泛素化修饰，而63位赖氨酸链泛素化修饰对RIG-I介导的信号转导至关重要。PEDVNsp5通过裂解NF-κB必不可少的适配器NEMO(或IKKγ)从而抑制NF-κB IRF3核易位，最终抑制IFN-β生产。PEDV NSP9的晶体结构解析是发现，Nsp9二聚化可以增强核酸结合力，有助于与核酸结合，利于其复制。PEDV Nsp16下调RIG-1和MDA5从而抑制IFN-β和ISRE活性，可促进病毒增殖。此外，Nsp10可以增强IFN-βNsp16的抑制效果。PEDV编码的非结构蛋白Nsp12是一种RNA依赖的RNA聚合酶(RNA-dependent RNApolymerase，RdRp)，是病毒复制、增殖的关键复制酶。PEDVNsp12蛋白能够与宿主细胞蛋白RNF7相互作用，其具体作用机制不明。
针对PEDV非结构蛋白的研究还有待进一步开展，PEDV非结构蛋白是否具有有效中和表位，其与复制转录复合体(RTC)结合过程，是否存在保守的结合位点，及其如何拮抗或促进干扰素的作用机制有待进一步探究。PEDV的Nsp15蛋白具有核酸内切酶活性，属于NEndoU家族，其C端较为保守，冠状病毒家族Nsp15氨基酸序列同源性高，N端差异显著。NEndoU能够特异性识别尿嘧啶(U)，特定识别并且切割RNA底物3′端的嘧啶核苷酸残基所产生的2′-3′环磷酸和5′-OH末端，并参与加工小核仁RNA，组成复制酶转录酶复合体的一部分，在复制和转录中至关重要。但是，目前缺乏针对猪流行性腹泻病毒Nsp15蛋白的多克隆抗体，阻碍了后续对Nsp15拮抗干扰素的作用机制的研究。因此，本发明提供的Nsp15多克隆抗体对Nsp15的检测及相关研究工作具有重要意义。
发明内容
本发明的目的在于提供一种猪流行性腹泻病毒Nsp15蛋白的高效价多克隆抗体及其制备方法。
本发明提供了一种猪流行性腹泻病毒Nsp15蛋白的多克隆抗体，来源于Nsp15重组蛋白免疫小鼠后产生的血清。
在一些实施例中，所述Nsp15重组蛋白由转化重组表达载体的大肠杆菌诱导表达后经过纯化获得。
在一些实施例中，所述Nsp15重组蛋白的基因序列如SEQ ID NO:1所示。
本发明提供了一种Nsp15重组蛋白，所述Nsp15重组蛋白的基因序列如SEQ ID NO:1所示。
本发明提供了一种重组表达载体，其含有如SEQ ID NO:1所示的核苷酸序列。
在一些实施例中，所述重组表达载体基于pET30a或pcDNA3.1重组得到。
本发明提供了一种宿主细胞，所述宿主细胞被转化或转染如上所述的重组表达载体。
本发明提供了如上所述的多克隆抗体、Nsp15重组蛋白、重组表达载体或宿主细胞在制备用于检测猪流行性腹泻病毒的产品中的应用。
本发明提供了一种如上所述的多克隆抗体的制备方法，包括以下步骤：通过原核表达制备如上所述的Nsp15重组蛋白，纯化后对小鼠进行免疫接种，然后采集小鼠血清制成多克隆抗体。
本发明成功构建了猪流行性腹泻病毒Nsp15蛋白的原核表达质粒，并摸索出37℃为最佳诱导温度，通过镍柱纯化得到高纯度Nsp15蛋白，纯度高达90％，蛋白浓度为0.69mg/mL，进而制备得到了高效价的Nsp15蛋白多克隆抗体，抗体效价为2×105，Western Blot试验验证Nsp15阳性血清特异性较好，可用于检测猪流行性腹泻病毒，也可以用于Nsp15的功能机制研究。
附图说明
图1为本发明一实施例的pET-30a-Nsp15重组表达载体的结构示意图；
图2为本发明一实施例的pET-30a-Nsp15重组表达载体双酶切后核酸琼脂糖电泳结果；
图3为本发明一实施例的Nsp15蛋白表达、鉴定及纯化SDS-PAGE分析；其中，A为Western Blot诱导温度的摸索(His标签)，B为诱导温度摸索，C为纯化后SD-PAGE鉴定蛋白纯度；
图4为本发明一实施例的血清效价检测结果；
图5为本发明一实施例的鼠源抗Nsp15血清的Western Blot检测结果。
具体实施方式
为了更加简洁明了的展示本发明的技术方案、目的和优点，下面结合具体实施例及其附图对本发明做进一步的详细描述。可以理解，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
一、实验步骤
1.1材料
1.1.1细胞、质粒、动物
细胞：人胚胎肾细胞HEK-293T由本实验室保存，毒株：PEDV-19-P4毒株由本实验室分离并保存。
大肠杆菌Trans1-T1感受态细胞、pEASY Bluent Simple Cloning Kit表达载体购自北京全式金生物技术有限公司，原核表达载体pET30a(+)由本实验室保存，6-8周龄SPF级雌性BALB/c小鼠购自斯贝福生物技术有限公司，饲养于在军事医学研究院军事兽医研究所动物实验中心。
1.1.2主要试剂
胶回收试剂盒BioSpin Gel Extraction Kit购自杭州博日科技。IPTG、BL21(DE3)购自索莱宝，限制性内切酶NdeI和HindIII购自Thermo Scientific公司。弗氏完全佐剂及弗氏不完全佐剂均购自美国Sigma公司。HRP标记的羊抗鼠IgG购自碧云天生物。
1.2 PEDV Nsp15基因及其引物的合成
参考GenBank序列(GenBank:KJ184549.1)，对其氨基酸依据大肠杆菌表达系统密码子偏爱进行优化，扩增片段全长为1017bp，送至金斯瑞公司进行基因优化。利用PrimerPremier 5.0设计上述合成的Nsp15基因引物，引物序列为Nsp15-F：5'-CATATGCACCATCACCATCATCAGGTCTGGAAAATATTGCGTTTAATG-3'和Nsp15-R 5'-AAGCTTTCATTATTGCAGTTGCGGGTAGAAGG-3'，引物由库美生物工程股份有限公司合成。
1.3原核载体的构建
(1)目的片段的扩增
PCR反应体系如下：
| 试剂 | 体积 |
| cDNA | 2μL(根据cDNA浓℃) |
| 上游引物 | 1μL |
| 下游引物 | 1μL |
| dNTP | 4μL |
| 5×buffer | 10μL |
| Pfastfly | 1μL |
| H<sub>2</sub>O | 31μL |
| Total | 50μL |
试剂加入后，离心机混匀后离心，设置PCR程序如下图所示：
本实验中涉及到的cDNA来自于PEDV。
(2)胶回收
核酸凝胶电泳结束后切取1000bp左右(Nsp15)的条带，使用胶回收试剂盒回收琼脂糖凝胶中的DNA片段。加入500μL Binding buffer(2×)，50～60℃水浴至胶块完全融化至水状。加至离心柱中(离心柱置于2mL收集管内)，室温10000rpm离心1min，弃收集管中滤液。加300μL的固定剂Binding buffer，室温静置2min，10000rpm室温离心1min，弃去收集管中滤液。加500μL Wash Buffer，10000rpm室温离心1min，弃去收集管中滤液，步骤(3)重复一次。将离心柱置于收集管中，室温晾干5min，10000rpm空离2min。离心柱置于1.5mL EP管中，室温静置2min，晾干酒精以免影响后续连接，加50℃加入50μL ddH2O，室温静置1min，10000rpm室温离心1min。Nanodrop分光光度计测定浓度。
(3)连接
将上述双酶切回收产物连接pET-30a，连接体系如下：
| 试剂 | 浓度 |
| 片段 | X |
| 载体 | Y |
| LigationhighVer.2连接酶 | 2μL |
| Total | 20μL |
根据pET-30a和PCR产物的浓度进行连接，连接体系的计算公式如下：
(A×x/M)/(B×y/N)≈1:(3～10)
A：代表载体浓度，B：代表片段浓度，M：代表载体核酸碱基数个数，N：代表片段碱基个数。
(4)转化连接
负80℃冰箱中取出Trans1-T1感受态细胞，迅速置于解冻5-10min。将上述连接产物加至100μL Trans1-T1感受态细胞中，冰上孵育30min。准备42℃水浴锅，将样品放入42℃水浴锅热激60s，次迅速置于冰上2min。加入1mL无抗性的LB培养基，37℃摇床震然荡培养1h。取100mL菌液用涂布棒涂于含有Amp的LB固体培养基平板上。平板倒置37℃培养过夜。
(5)质粒提取
取5～8mL LB菌液于1.5mL EP管中(培养时间约12～16h)，10000rpm室温离心1min，收集沉淀，弃上清。加250μL P1(已加入RNaseA于4℃保存)，移液枪反复吹打至细胞完全悬浮无沉淀。加250μL P2，上下轻轻颠倒4～5次，充分混匀，至菌液澄清透明。加350μLP3，上下轻轻颠倒EP管4～5次充分混匀至形成白色絮状沉淀，12000rpn室温离心10min。取上清加至离心柱，10000rpm室温离心1min，收集管液体弃去。加500μL Buffer4，10000rpm室温离心1min，弃去收集管中液体。加700μL Wash Buffer，10000rpm室温离心1min，弃去收集管中液体，步骤重复一次。晾干5min，室温10000rpm空离2min。将离心柱置于干净1.5mL EP管中，室温静置2min待酒精挥发后，加50μL ddH2O，室温静置1min，10000rpm室温离心1min。Nanodrop分光光度计测定浓度。
1.4酶联免疫吸附测定(ELISA)
1)包被：选择ELISA级别的微孔板，包被液稀释Nsp15蛋白，4℃过夜，包被浓度的摸索：一般包被浓度为10μg/mL-20μg/mL。
2)封闭：弃去包被液，TBST清洗3次，拍干ELISA板，5％BSA 37℃封闭1h。
3)加样及孵育：弃去封闭液，TBST清洗3次，拍干ELISA板，加入小鼠免疫血清，加样时应将所加物加在ELISA板孔的底部，避免加在孔壁上部，不可产生气泡。37℃孵育的1h。
4)添加二抗：移液枪弃去血清，TBST清洗3次，拍干ELISA板，加入HRP的羊抗鼠二抗，37℃孵育的1h。
5)显色：移液枪弃去二抗，TBST清洗3次，拍干ELISA板，加入显色液。
6)终止：每孔添加终止液，立刻用酶标仪检测。
1.5重组Nsp15蛋白的纯化
重组质粒pET-30a-Nsp15诱导表达，对诱导条件及诱导时间的摸索，选取未诱导、100mM和200mM IPTG诱导剂诱导，选取15℃、37℃两个温度条件进行最适诱导温度的摸索，诱导培养4h后，菌体超声破碎，4℃12000rpm离心15min，取菌液沉淀重悬于pH值为7.8的8mol/L尿素溶液中，在变性条件下纯化重组蛋白，充分透析后与镍柱结合，不同浓度咪唑梯度洗脱。收取蛋白流穿液与洗脱液后续进行SDS-PAGE实验分析。
1.6鼠源多克隆抗体的制备
6-8周龄SPF级雌性BALB/c小鼠免疫之前尾静脉采血作为阴性对照，将纯化后重组蛋白与弗氏佐剂1:1混合乳化，每只小鼠50μg重组蛋白进行背部皮下免疫，首次免疫使用弗氏不完全佐剂与重组蛋白混合，后于21天和42天重组蛋白与弗氏完全佐剂乳化免疫，免疫剂量和免疫方法同首次免疫，于14天、21天、28天、35天、42天、49天、56天分别采血，进行效价测定。
二、结果与分析
2.1pET-30a-Nsp15原核表达质粒的构建与鉴定
Nsp15蛋白的DNA序列经PCR扩增、纯化后，连接构建至空载体pET-30(+)上，重组质粒经酶切及鉴定、测序成功，转化至BL21表达菌株中，为后续表达纯化打下基础，质粒结构如图1所示。如图2，核酸琼脂糖电泳结果显示，pET-30a-Nsp15重组表达载体双酶切后核酸琼脂糖显示在1000bp处有条带，其大小与预期相符。
2.2Nsp15蛋白表达、鉴定及纯化
重组质粒pET-30a-Nsp15构建鉴定完毕后，对诱导条件及诱导时间的摸索，选取未诱导、100mM和200mM IPTG诱导剂诱导，选取15℃、37℃两个温度条件进行最适诱导温度的摸索，诱导培养4h后，菌体超声破碎，4℃12000rpm离心15min，取菌液沉淀重悬于pH值为7.8的8mol/L尿素溶液中，在变性条件下纯化重组蛋白，充分透析后与镍柱结合，不同浓度咪唑梯度洗脱。收取蛋白流穿液与洗脱液后续进行SDS-PAGE实验分析对诱导条件及诱导时间的摸索试验结果显示，在37℃条件下诱导4h的沉淀中蛋白纯度更高。使用镍柱对Nsp15蛋白进行纯化，如图3，有SDS-PAGE图分析显示重组Nsp15蛋白，在35kDa处可见清晰条带。且纯度达90％以上，蛋白浓度为0.69mg/mL。
2.3Nsp15多克隆抗制备与检测
纯化后的Nsp15蛋白使用ELISA包被液稀释至以20μg/mL，每孔100μL4℃冰箱包被过夜，TBST洗涤三次，每孔加入50μLPBS稀释的鼠源血清，稀释度为1：200、1:400、1:800、1:1600、1：3200、1：16400、1：12800、1：25600、1：51200、1：102400、1：204800，同时设置稀释的鼠阴性血清作为对照，37℃孵育1h，TBST洗涤4次后，每孔加入100μL HRP标记的羊抗鼠IgG二抗，37℃孵育1h，TBST洗涤4次后，每孔加入100μL的底物，室温避光显示10min，2mol/LH2SO4终止显色，酶标仪检测OD490nm/OD650nm数值，P/N≥2.0判定为阳性。如图4结果显示，2号小鼠血清中特异性Nsp15蛋白抗体效价高达1:25600，说明血清特异性抗体水平较高，可用于后续实验。
2.4鼠源抗Nsp15血清的WesternBlot检测
构建pcDNA3.1-Nsp15-Flag真核表达载体，按照lipo3000操作方法转染至293T细胞，37℃恒温培养48h，裂解细胞，进行Western Blot实验分析，如图5所示，Western Blot结果显示在35kD出有明显条带，与预期相符。
三、讨论
蛋白的原核表达应用十分广泛，尤其是在基因工程疫苗上，以其特有的表达量大，价格相对便宜，操作技术简单且规范，易上手等优点，备受市场青睐，但原核表达的缺点也很明显，表达蛋白受菌体产生的内毒素的影响，蛋白毒性较大，原核细胞由于没有蛋白翻译后折叠修饰等功能，没有或是甚少存在高级结构。作为制备单克隆抗体来说，原核蛋白的高表达量的优势显得尤为突出，所以本实验选择原核表达系统表达Nsp15蛋白，用于制备鼠源多克隆抗体。
Nsp15具有核酸内切酶活性，其C端结构域较为保守，参与细胞内小核仁RNA的加工，是病毒复制酶转录酶复合体必不可少的，在病毒复制和转录中发挥关键作用。Nsp15鼠多抗的制备将为后续研究Nsp15拮抗干扰素的作用机制提供必要的研究工具。
四、小结
1、本实施例成功构建pET-30a-Nsp15原核表达质粒，并摸索出37℃为最佳诱导温度，通过镍柱纯化得到高纯度Nsp15蛋白，纯度高达90％，蛋白浓度为0.69mg/mL。
2、制备了Nsp15蛋白多克隆抗体，抗体效价为2×105，Western Blot试验验证Nsp15阳性血清特异性较好。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。
Claims (10)
1.一种猪流行性腹泻病毒Nsp15蛋白的多克隆抗体，其特征在于，来源于Nsp15重组蛋白免疫小鼠后产生的血清。
2.根据权利要求1所述的多克隆抗体，其特征在于，所述Nsp15重组蛋白由转化重组表达载体的大肠杆菌诱导表达后经过纯化获得。
3.根据权利要求1所述的多克隆抗体，其特征在于，所述Nsp15重组蛋白的基因序列如SEQ ID NO:1所示。
4.一种Nsp15重组蛋白，其特征在于，所述Nsp15重组蛋白的基因序列如SEQ ID NO:1所示。
5.一种重组表达载体，其特征在于，其含有如SEQ ID NO:1所示的核苷酸序列。
6.根据权利要求5所述的重组表达载体，其特征在于，所述重组表达载体基于pET30a或pcDNA3.1重组得到。
7.一种宿主细胞，其特征在于，所述宿主细胞被转化或转染权利要求5或6所述的重组表达载体。
8.权利要求1～3任一项所述的多克隆抗体、权利要求4所述的Nsp15重组蛋白、权利要求5或6所述的重组表达载体或权利要求7所述的宿主细胞在制备用于检测猪流行性腹泻病毒的产品中的应用。
9.一种用于猪流行性腹泻病毒的检测试剂盒，其特征在于，包括权利要求1～2任一项所述的多克隆抗体和缓冲液。
10.一种权利要求1～3任一项所述的多克隆抗体的制备方法，其特征在于，包括以下步骤：通过原核表达制备权利要求4所述的Nsp15重组蛋白，纯化后对小鼠进行免疫接种，然后采集小鼠血清制成多克隆抗体。
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| WO2006076007A2 (en) * | 2004-04-22 | 2006-07-20 | Vanderbilt University | Methods of detecting coronavirus infections |
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