CN109651488A - 猪丁型冠状病毒重组n蛋白及其多克隆抗体的制备方法 - Google Patents
猪丁型冠状病毒重组n蛋白及其多克隆抗体的制备方法 Download PDFInfo
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Abstract
本发明公开了一种猪丁型冠状病毒(PDCoV)重组N蛋白的制备方法,该法对PDCoV的N基因片段进行扩增后经过原核表达、纯化后获得重组N蛋白。据此,发明人还建立了重组N蛋白多克隆抗体的制备方法,即应用PDCoV重组N蛋白与弗氏佐剂乳化后免疫动物获得多克隆抗体血清。实验表明,本发明成功扩增了PDCoV的N基因并进行原核表达及纯化,初步研究其免疫原性,为研制诊断试剂盒或制备多克隆抗体奠定了基础。
Description
技术领域
本发明属于猪丁型冠状病毒技术领域,尤其涉及一种猪丁型冠状病毒重组N蛋白及其多克隆抗体的制备方法。
背景技术
猪丁型冠状病毒(porcine deltacoronavirus,PDCoV)最早发现于2012年,香港大学的Woo等从健康的商品猪中检测到猪丁型冠状病毒。冠状病毒分为甲、乙、丙、丁四个亚群,感染猪的丁型冠状病毒(PDCoV)是唯一一种感染非禽类的丁型冠状病毒。此后,在美国、中国、韩国等许多国家相继报道了PDCoV的流行。与PEDV和TGEV类似,PDCoV主要引起猪的肠道疾病,导致感染猪腹泻、呕吐和脱水,发病率和死亡率可高达50%-100%,尤以哺乳阶段仔猪发病最为严重,严重威胁着猪群健康,给养猪业带来了巨大的经济损失。
PDCoV全长25.4kb,其基因组结构与一般冠状病毒(Coronavirus,CoV)相似,基因组结构从5’-3’末端依次为复制酶开放阅读框lab(replicase ORFlab),S糖蛋白基因(spike,S)、小膜蛋白基因(envelope,E)、膜蛋白基因(membrane,M)以及核衣壳蛋白基因(nucleocapsid,N),5’端和3’端都有较短的非翻译区。其中,N蛋白是病毒组成成分中最为丰富和多功能的蛋白。N蛋白是与基因组RNA形成复合物的结构蛋白,在病毒体装配期间与病毒膜蛋白相互作用,并且在提高病毒转录和装配效率中,及在致病机理形成过程中起关键作用。
目前针对PDCoV的诊断方法主要为RT-PCR方法,针对PDCoV抗体的检测方法的研究较少。
发明内容
本发明要解决的技术问题是提供一种猪丁型冠状病毒重组N蛋白及其多克隆抗体的制备方法。
为解决上述技术问题,本发明采用以下技术方案:
猪丁型冠状病毒重组N蛋白的制备方法,包括以下步骤:
<1>PDCoV N基因片段的扩增
对PDCoV的RNA进行反转录获得cDNA,然后采用特异性引物N-P1和N-P2对PDCoV N基因进行PCR扩增,特异性引物N-P1和N-P2分别具有序列表SEQ.ID.No.1和SEQ.ID.No.2的碱基序列;
<2>原核表达质粒pET32a-N的构建
将PCR扩增得到的PDCoV N基因片段连接pMD18-T克隆载体,转化DH5α感受态细胞获得pMD18-N克隆质粒,将pMD18-N克隆质粒和pET-32a(+)双酶切胶回收,利用T4连接酶将pET-32a(+)和N连接过夜后转化到DH5α感受态细胞得到原核表达质粒pET32a-N;
<3>重组N蛋白的获取
将原核表达质粒pET32a-N转化BL21感受态细胞,加入IPTG诱导,菌液超声波裂解,离心,收集上清和沉淀,SDS-PAGE电泳确定重组N蛋白主要在上清中表达,按照康为世纪公司His标签蛋白纯化试剂盒的纯化方法获得重组N蛋白。
步骤<1>中:PCR扩增的反应体系和反应条件为:反应体系:Taq Mix12.5μL,灭菌ddH2O 9μL,上下游引物N-P1、N-P2各0.5μL,cDNA 2.5μL;反应条件:95℃预变性5min;95℃变性45s,56℃退火45s,72℃延伸90s,32个循环;最后72℃延伸5min。
步骤<2>按以下操作进行:将PCR产物电泳后进行胶回收纯化,得到PDCoV N基因片段,连接pMD18-T克隆载体,转化DH5α感受态细胞,涂板于含有Amp的LB平板上培养过夜,挑取单个菌落接种于含有Amp的LB液体培养基中震荡10-12小时,抽提获得pMD18-N克隆质粒。
上述猪丁型冠状病毒重组N蛋白的制备方法,按以下操作进行:将pET-32a(+)空载体和克隆质粒pMD18-N分别用BamHⅠ和HindⅢ进行双酶切,分别胶回收pET-32a(+)空载体和目的基因N;利用T4连接酶将pET-32a(+)和N以1:3的比例连接过夜,转化DH5α感受态细胞进行克隆;抽提质粒并用BamHⅠ和HindⅢ进行双酶切鉴定,获得正确的原核表达质粒pET32a-N。
步骤<3>按以下操作进行:将原核表达质粒pET32a-N转化BL21感受态细胞;将重组菌培养至OD600nm 0.6-0.8时,加入IPTG诱导;将经SDS-PAGE电泳确定重组蛋白得到表达的菌液用超声波裂解,裂解后12000rpm离心10分钟,分别取上清和沉淀进行SDS-PAGE电泳。SDS-PAGE电泳确定重组N蛋白主要在上清中表达,按照康为世纪公司His标签蛋白纯化试剂盒的纯化方法获得重组N蛋白。
使用上述猪丁型冠状病毒重组N蛋白制备多克隆抗体的方法,应用猪丁型冠状病毒重组N蛋白与弗氏佐剂乳化后免疫动物获得多克隆抗体血清。
猪丁型冠状病毒重组N蛋白与弗氏佐剂乳化按以下操作进行:将纯化得到的重组PDCoVN蛋白加入透析袋中透析除去咪唑,再用PEG-20000进行包埋,将蛋白适当浓缩后测定蛋白浓度;用乳化器将浓缩后的蛋白溶液与等体积弗氏完全佐剂进行混合乳化,至不分层时即为一免用的蛋白抗原;二免、三免时取浓缩蛋白溶液与等体积弗氏不完全佐剂混合乳化作为二免、三免用蛋白抗原。
免疫动物按以下操作进行:
1)首免:将蛋白溶液与等体积弗氏完全佐剂混匀乳化,静置30分钟不分层时用于小鼠免疫;实验组免疫重组蛋白剂量为200μg/只,对照组免疫等体积的弗氏完全佐剂与PBS的乳化液;
2)二免:两周后进行二免,二免时用蛋白溶液与等体积弗氏不完全佐剂的混匀乳化,免疫用抗原的剂量同首免;三免方案同二免;
3)三免后两周断尾采血,分离血清。
针对目前猪丁型冠状病毒缺乏有关研究方法的问题,考虑到N蛋白的多功能性和高表达量,且不同PDCoV株之间N蛋白高度保守,发明人建立了一种猪丁型冠状病毒重组N蛋白的制备方法,该法对PDCoV N基因片段进行扩增后经过原核表达、纯化后获得重组N蛋白。据此,发明人还建立了重组N蛋白多克隆抗体的制备方法,即应用猪丁型冠状病毒重组N蛋白与弗氏佐剂乳化后免疫动物获得多克隆抗体血清。实验表明,本发明成功扩增了PDCoV的N基因并进行原核表达及纯化,初步研究其免疫原性,为研制诊断试剂盒或制备单克隆抗体奠定了基础。
附图说明
图1是目的基因N的RT-PCR扩增结果图,图中:M:DNA分子质量标准;1-2:目的基因N扩增产物。
图2是重组质粒pET32a-N双酶切鉴定结果图,图中:M:DNA分子质量标准;1-2:重组质粒pET32a-N。
图3是最佳IPTG诱导浓度优化结果图,图中:M:预染蛋白Marker(SM0441);1-8:0mM、0.05mM、0.1mM、0.2mM、0.5mM、1.0mM、2.0mM、4.0mM IPTG诱导5h。
图4是最佳诱导时间优化结果图,图中:M:预染蛋白Marker(SM0441);1-2:未诱导对照;3-8:1.0mM IPTG分别诱导7h、6h、5h、4h、3h和2h。
图5是重组蛋白N表达形式的鉴定结果图,图中:M:预染蛋白Marker;1:诱导后重组菌全菌;2:诱导后重组菌裂解上清;3:诱导后裂解沉淀。
图6是纯化的重组N蛋白SDS-PAGE电泳分析结果图,图中:M:预染蛋白Marker(CW0986);1-2:纯化后的重组蛋白N。
图7是Western-blot验证纯化后重组蛋白的抗原性结果图,图中:M:预染蛋白Marker(CW0986);1:纯化的重组蛋白N。
图8是IFA分析鉴定小鼠抗PDCoV N蛋白多克隆抗体的结果图,图中:A:小鼠抗PDCoVN蛋白多克隆抗体血清组;B:阴性对照组。
具体实施方式
1材料与方法
1.1主要试验材料
猪丁型冠状病毒(CH/GX/1468B/2017)、猪丁型冠状病毒阴性血清、猪丁型冠状病毒人工感染猪的阳性血清由申请人实验室分离、制备并保存。病毒基因组DNA/RNA快速抽提试剂盒为Axygen生物科技有限公司产品;Taq Mix、DNA凝胶回收试剂盒、质粒小量抽提试剂盒购自北京天根生化科技有限公司;pMD18-T Vector、BamHⅠ、HindⅢ、DL2000DNAMarker均为大连宝生物工程有限公司产品;预染蛋白Marker(CW0986)、预染蛋白Marker(CW2841),His标签蛋白纯化试剂盒(CW0894S)为康为世纪公司产品;HRP标记的山羊抗猪IgG抗体为美国Earthox公司产品;E.coli DH5α、E.coli BL21菌种,原核表达载体pET-32a(+)由申请人实验室保存。
1.2引物的设计与合成
根据申请人实验室分离的PDCoV流行毒株CH/GX/1468B/2017的全基因组序列,利用Meg Align(DNA Star),Primer Premier 7.0软件设计一对特异性引物N-P1和N-P2,特异性的扩增PDCoV N基因,PCR扩增目的片段大小为1029bp。引物由大连宝生物工程有限公司合成。
引物序列如下:
N-P1:5’-CGCGGATCCATGGCTGCACCAGTAGTCC-3’(SEQ.ID.No.1),
N-P2:5’-CCAAGCTTCTACGCTGCTGATTCCTGC-3’(SEQ.ID.No.2),下划线部分分别为BamHⅠ和HindⅢ酶切位点。
1.3 RNA的提取和N基因的扩增
按照AxyPrep Body Fluid Viral DNA/RNA Miniprep Kit使用说明书进行病毒RNA的提取,反转录获得cDNA进行PDCoV N基因的PCR扩增。
PCR反应体系为:Taq Mix12.5μL,灭菌ddH2O 9μL,上下游引物N-P1、N-P2各0.5μL,cDNA 2.5μL。
PCR反应条件为95℃预变性5min;95℃变性45s,56℃退火45s,72℃延伸90s,32个循环;最后72℃延伸5min。
1.4 N基因的克隆
PCR产物电泳后进行胶回收纯化,连接pMD18-T克隆载体,转化DH5α感受态细胞,涂板于含有Amp的LB平板上培养过夜,挑取单个菌落接种于含有Amp的LB液体培养基中震荡10-12小时,抽提质粒进行酶切鉴定,将酶切鉴定正确的质粒送往生物公司进行测序,并命名为pMD18-N。
1.5原核表达质粒pET32a-N的构建
将pET-32a(+)空载体和克隆质粒pMD18-N分别用BamHⅠ和HindⅢ进行双酶切,分别胶回收pET-32a(+)空载体和目的基因N。利用T4连接酶将pET-32a(+)和N以1:3的比例连接过夜,转化DH5α感受态细胞进行克隆。抽提质粒用BamHⅠ和HindⅢ进行双酶切鉴定,对酶切鉴定正确的送往生物公司进行测序,鉴定是否成功构建原核表达载体,将构建正确的原核表达载体命名为pET32a-N。
1.6重组PDCoV N蛋白的诱导表达及表达形式的鉴定
将正确构建的原核表达质粒pET32a-N转化BL21感受态细胞。将重组菌培养至OD600nm0.6-0.8时,加入IPTG诱导,优化最佳IPTG浓度和诱导时间等表达条件。将经SDS-PAGE电泳确定重组蛋白得到表达的菌液用超声波裂解,裂解后12000rpm离心10分钟,分别取上清和沉淀进行SDS-PAGE电泳,确定重组蛋白的表达形式。
1.7重组蛋白的纯化
按照康为世纪公司His标签蛋白纯化试剂盒中的方法,纯化重组PDCoV N蛋白,方法步骤略。
1.8纯化重组蛋白的抗原性分析
以申请人保存的PDCoV人工感染猪的阳性血清为一抗,HRP标记的山羊抗猪IgG为二抗,Western-blot检测纯化重组蛋白的抗原性。同时以PDCoV阴性猪血清为一抗进行Western-blot做阴性对照。
1.9重组蛋白多克隆抗体的制备
1.9.1制备免疫用蛋白抗原
将纯化得到的重组PDCoV N蛋白加入透析袋中置于大体积的PBS中透析12小时除去咪唑,再用PEG-20000(聚乙二醇,分子量为20000)进行包埋,将蛋白适当浓缩后测定蛋白浓度。
用乳化器将浓缩后的蛋白溶液与等体积弗氏完全佐剂进行混合乳化,至不分层时即为一免用的蛋白抗原;二免、三免时取浓缩蛋白溶液与等体积弗氏不完全佐剂混合乳化作为二免、三免用蛋白抗原。
1.9.2小鼠免疫方法
SPF级昆明小白鼠(10g/只)12只,分成2组,1个实验组,8只/组;1个对照组,4只/组。
1)首免:将蛋白溶液与等体积弗氏完全佐剂混匀乳化,静置30分钟不分层时,即可用于小鼠免疫;实验组免疫重组蛋白剂量为200μg/只,对照组免疫等体积的弗氏完全佐剂与PBS的乳化液;
2)二免:两周后进行二免,二免时用蛋白溶液与等体积弗氏不完全佐剂混匀乳化,免疫用抗原的剂量同首免(表1);三免方案同二免
3)三免后两周断尾采血,分离血清。
表1小鼠免疫方案(注:蛋白的浓度为0.605mg/mL。)
1.9.3鼠抗PDCoV N蛋白多克隆抗体效价的测定
用纯化的PDCoV N蛋白为包被抗原包被ELISA板,4℃包被过夜,每孔包被1μg;PBST洗涤3次(5min/次,下同)后用5%的脱脂奶粉37℃封闭2小时,PBST洗涤3次,以PDCoVN蛋白免疫小鼠的血清为一抗(1:1000-1:128000),37℃孵育1h,PBST洗涤3次,加入HRP标记羊抗小鼠IgG抗体(1:4000),37℃孵育45min,PBST洗涤3次后加入TMB显色底物,显色10min后加入终比液,酶标仪检测OD450值。以免疫血清OD450值与阴性对照血清OD450值的比值≥2.1为阳性判定标准,判定制备的鼠抗PDCoV N蛋白多克隆抗体的最低检出效价。
1.9.4鼠抗PDCoV N蛋白多克隆抗体的鉴定
利用间接免疫荧光实验(IFA)验证制备的鼠抗PDCoV N蛋白多克隆抗体的特异性。将LLC-PK细胞接种至96孔板,每孔1×105个细胞,至于5%CO2培养箱,37℃培养至形成100%的单层细胞;用DMEM将病毒液稀释1000倍,每孔加入100μL的病毒液,37℃培养24-48小时。吸取上清,加入150μL的固定液(-20℃预冷的无水乙醇)室温固定15分钟。固定后用PBS洗涤3次,加入以PBS 1:100倍稀释的PDCoV N蛋白免疫小鼠血清,每孔加入50μL,37℃孵育1小时。PBS洗涤三次后加入1:3000稀释的FITC标记的山羊抗小鼠IgG二抗,37℃孵育1小时。PBS洗涤5次后吸干孔内的液体,置于倒置的荧光显微镜下观察特异性的黄绿色荧光。
2结果
2.1 PDCoV N基因的扩增及原核表达载体的构建
按照1.3中的方法,RT-PCR扩增得到大小约1029bp(SEQ.ID.No.3)的片段,均与预期目的片段大小相符(见图1)。对获得的重组原核表达质粒pET32a-N用BamHⅠ和HindⅢ进行双酶切鉴定,分别得到大小约1029bp的目的片段和5.9kp的pET32a(+)载体片段,与预期相符(见图2)。经测序构建的原核表达质粒pET32a-N已正确构建。
2.2重组蛋白N的诱导表达条件的优化及表达形式的鉴定
将含正确构建的pET32a-N重组表达质粒的BL21大肠杆菌,用IPTG诱导表达,并对诱导条件进行优化。IPTG浓度为1.0mM和2.0mM时拥有较高的表达量,表达的目的蛋白大小约为58kDa,与预期大小一致,因此将IPTG最佳诱导浓度定为1.0mM(见图3)。随着诱导时间的增加,蛋白表达量逐渐增大,但诱导6h后蛋白表达量无明显增加,故将最佳诱导时间定为6h(见图4)。
将诱导表达的菌液超声波裂解后离心,分别取上期和沉淀进行SDS-PAGE电泳。结果表明,重组蛋白N以可溶性蛋白和包涵体两种形式表达(见图5),其中以可溶性蛋白形式表达为主,且可溶性表达的重组蛋白具有更好的生物学活性,故下一步将纯化上清中的重组蛋白N。
2.3重组蛋白N的纯化
将上清中的带His标签的重组N蛋白按照康为世纪公司His标签蛋白纯化试剂盒中的方法进行纯化,纯化产物经SDS-PAGE电泳分析。结果表明纯化后获得了较纯的重组N蛋白(SEQ.ID.No.4)(见图6)。
2.4重组蛋白N的抗原性分析
将纯化的N蛋白与申请人保存的PDCoV人工感染猪的阳性血清为一抗进行Western-blot鉴定(图7),结果表明纯化后的重组蛋白N能够与PDCoV阳性血清发生特异性的结合,在相应位置出现目的条带,表明纯化的N蛋白具有较好的抗原性。
2.5多克隆抗体的制备及鉴定
2.5.1多克隆抗体效价的测定
用鼠抗PDCoV N蛋白多克隆抗体血清进行间接ELISA检测时,免疫血清OD450值与阴性对照血清OD450值的比值≥2.1为阳性判定标准。结果显示,此研究制备的鼠抗PDCoV N蛋白多克隆抗体血清平均效价为1:64000(表2)。
表2 ELISA方法测定多克隆抗体效价结果
2.5.2多克隆抗体的IFA鉴定
用制备的小鼠抗PDCoV N蛋白多克隆抗体血清和未免疫蛋白阴性对照组小鼠血清进行IFA试验,检测多克隆抗体的特异性。结果显示,制备的多克隆抗体血清组在荧光显微镜下能出现特异性的黄绿色荧光,而对照组则没有可见荧光(图8)。
序列表
<110> 广西壮族自治区兽医研究所
<120> 猪丁型冠状病毒重组N蛋白及其多克隆抗体的制备方法
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
cgcggatcca tggctgcacc agtagtcc 28
<210> 2
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
ccaagcttct acgctgctga ttcctgc 27
<210> 3
<211> 1029
<212> DNA
<213> 猪丁型冠状病毒(porcine deltacoro-navirus)
<400> 3
atggctgcac cagtagtccc tactactgac gcgtcttggt ttcaggtgct caaagctcaa 60
aacaaaaagg ctactcatcc tcagtttcgt ggcaatggag ttccgcttaa ctccgccatc 120
aaacccgttg aaaaccatgg ttactggctt cgttacacca gacaaaagcc gggtggtact 180
ccgattcctc catcctatgc cttttattat actggcacag gtcctagagg aaatcttaag 240
tatggtgaac tccctcctaa tgatacccca gcaaccactc gtgttacttg ggttaagggt 300
tcgggagctg acacttctat taaacctcat gttgccaaac gcaaccccaa caatcctaaa 360
catcagctgc tacctctccg attcccaacc ggagatggcc cagcccaagg tttcagagtt 420
gaccccttca acgctagagg aagacctcag gagcgtggaa gtggcccaag atctcaatct 480
gttaactcca gaggcacagg caatcagccc aggaaacgcg accaatctgc acccgctgcg 540
gtacgtcgta agacccaaca tcaagctccc aagcggactt tacccaaggg taaaaccatt 600
tctcaggtat ttggcaaccg gtctcgcact ggtgccaatg tcggctctgc agacactgag 660
aagacgggta tggctgatcc tcgcatcatg gctttagcca gacatgtgcc tggtgttcag 720
gaaatgcttt tcgctggcca ccttgagagc aactttcagg cgggggcaat tacccttacc 780
ttctcttact caatcacagt gaaggagggt tctcctgact atgagagact taaggatgcg 840
ctcaatacgg tcgttaacca gacctatgag ccacccacca aactaactaa ggacaagaag 900
cctgacaaac aagaccagtc tgctaaaccc aaacagcaga agaaacctaa aaaggtaact 960
ctgccagcag acaaacagga ttgggagtgg gatgatgctt ttgagataaa gcaggaatca 1020
gcagcgtag 1029
<210> 4
<211> 342
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Met Ala Ala Pro Val Val Pro Thr Thr Asp Ala Ser Trp Phe Gln Val
1 5 10 15
Leu Lys Ala Gln Asn Lys Lys Ala Thr His Pro Gln Phe Arg Gly Asn
20 25 30
Gly Val Pro Leu Asn Ser Ala Ile Lys Pro Val Glu Asn His Gly Tyr
35 40 45
Trp Leu Arg Tyr Thr Arg Gln Lys Pro Gly Gly Thr Pro Ile Pro Pro
50 55 60
Ser Tyr Ala Phe Tyr Tyr Thr Gly Thr Gly Pro Arg Gly Asn Leu Lys
65 70 75 80
Tyr Gly Glu Leu Pro Pro Asn Asp Thr Pro Ala Thr Thr Arg Val Thr
85 90 95
Trp Val Lys Gly Ser Gly Ala Asp Thr Ser Ile Lys Pro His Val Ala
100 105 110
Lys Arg Asn Pro Asn Asn Pro Lys His Gln Leu Leu Pro Leu Arg Phe
115 120 125
Pro Thr Gly Asp Gly Pro Ala Gln Gly Phe Arg Val Asp Pro Phe Asn
130 135 140
Ala Arg Gly Arg Pro Gln Glu Arg Gly Ser Gly Pro Arg Ser Gln Ser
145 150 155 160
Val Asn Ser Arg Gly Thr Gly Asn Gln Pro Arg Lys Arg Asp Gln Ser
165 170 175
Ala Pro Ala Ala Val Arg Arg Lys Thr Gln His Gln Ala Pro Lys Arg
180 185 190
Thr Leu Pro Lys Gly Lys Thr Ile Ser Gln Val Phe Gly Asn Arg Ser
195 200 205
Arg Thr Gly Ala Asn Val Gly Ser Ala Asp Thr Glu Lys Thr Gly Met
210 215 220
Ala Asp Pro Arg Ile Met Ala Leu Ala Arg His Val Pro Gly Val Gln
225 230 235 240
Glu Met Leu Phe Ala Gly His Leu Glu Ser Asn Phe Gln Ala Gly Ala
245 250 255
Ile Thr Leu Thr Phe Ser Tyr Ser Ile Thr Val Lys Glu Gly Ser Pro
260 265 270
Asp Tyr Glu Arg Leu Lys Asp Ala Leu Asn Thr Val Val Asn Gln Thr
275 280 285
Tyr Glu Pro Pro Thr Lys Leu Thr Lys Asp Lys Lys Pro Asp Lys Gln
290 295 300
Asp Gln Ser Ala Lys Pro Lys Gln Gln Lys Lys Pro Lys Lys Val Thr
305 310 315 320
Leu Pro Ala Asp Lys Gln Asp Trp Glu Trp Asp Asp Ala Phe Glu Ile
325 330 335
Lys Gln Glu Ser Ala Ala
340
Claims (8)
1.一种猪丁型冠状病毒重组N蛋白的制备方法,其特征在于包括以下步骤:
<1>PDCoV N基因片段的扩增
对PDCoV的RNA进行反转录获得cDNA,然后采用特异性引物N-P1和N-P2对PDCoV N基因进行PCR扩增,所述特异性引物N-P1和N-P2分别具有序列表SEQ.ID.No.1和SEQ.ID.No.2的碱基序列;
<2>原核表达质粒pET32a-N的构建
将PCR扩增得到的PDCoV N基因片段连接pMD18-T克隆载体,转化DH5α感受态细胞获得pMD18-N克隆质粒;将pMD18-N克隆质粒和pET-32a(+)分别进行双酶切,回收N基因和pET-32a(+)载体,利用T4连接酶将pET-32a(+)和N基因连接过夜后转化到DH5α感受态细胞后提取质粒得到原核表达质粒pET32a-N;
<3>重组N蛋白的获取
将原核表达质粒pET32a-N转化BL21感受态细胞,加入IPTG诱导,菌液超声波裂解,离心,收集上清和沉淀,SDS-PAGE电泳确定重组N蛋白主要在上清中表达,按照康为世纪公司His标签蛋白纯化试剂盒的纯化方法获得重组N蛋白。
2.根据权利要求1所述的猪丁型冠状病毒重组N蛋白的制备方法,其特征在于步骤<1>中:PCR扩增的反应体系和反应条件为:反应体系:Taq Mix12.5μL,灭菌ddH2O 9μL,上下游引物N-P1、N-P2各0.5μL,cDNA 2.5μL;反应条件:95℃预变性5min;95℃变性45s,56℃退火45s,72℃延伸90s,32个循环;最后72℃延伸5min。
3.根据权利要求1所述的猪丁型冠状病毒重组N蛋白的制备方法,其特征在于步骤<2>按以下操作进行:将PCR产物电泳后进行胶回收纯化,得到PDCoV N基因片段,连接pMD18-T克隆载体,转化DH5α感受态细胞,涂板于含有Amp的LB平板上培养过夜,挑取单个菌落接种于含有Amp的LB液体培养基中震荡10-12小时,抽提获得pMD18-N克隆质粒。
4.根据权利要求1所述的猪丁型冠状病毒重组N蛋白的制备方法,其特征在于步骤<2>按以下操作进行:将pET-32a(+)空载体和克隆质粒pMD18-N分别用BamHⅠ和HindⅢ进行双酶切,分别胶回收pET-32a(+)空载体和目的基因N;利用T4连接酶将pET-32a(+)和N以1:3的比例连接过夜,转化DH5α感受态细胞进行克隆;抽提质粒并用BamHⅠ和HindⅢ进行双酶切鉴定,获得正确的原核表达质粒pET32a-N。
5.根据权利要求1所述的猪丁型冠状病毒重组N蛋白的制备方法,其特征在于步骤<3>按以下操作进行:将原核表达质粒pET32a-N转化BL21感受态细胞;将重组菌培养至OD600nm0.6-0.8时,加入IPTG诱导;将经SDS-PAGE电泳确定重组蛋白得到表达的菌液用超声波裂解,裂解后12000rpm离心10分钟,分别取上清和沉淀进行SDS-PAGE电泳,SDS-PAGE电泳确定重组N蛋白主要在上清中表达,按照康为世纪公司His标签蛋白纯化试剂盒的纯化方法获得重组N蛋白。
6.使用权利要求1所述猪丁型冠状病毒重组N蛋白制备多克隆抗体的方法,其特征在于使用猪丁型冠状病毒重组N蛋白与弗氏佐剂乳化后免疫动物获得多克隆抗体血清。
7.根据权利要求6所述的制备多克隆抗体的方法,其特征在于所述猪丁型冠状病毒重组N蛋白与弗氏佐剂乳化按以下操作进行:将纯化得到的重组PDCoV N蛋白加入透析袋中透析除去咪唑,再用PEG-20000进行包埋,将蛋白适当浓缩并测定蛋白浓度;用乳化器将浓缩后的蛋白溶液与等体积弗氏完全佐剂进行混合乳化,至不分层时即为一免用的蛋白抗原;二免、三免时取浓缩蛋白溶液与等体积弗氏不完全佐剂混合乳化作为二免、三免用蛋白抗原。
8.根据权利要求6所述的制备多克隆抗体的方法,其特征在于所述免疫动物按以下操作进行:
1)首免:用乳化器将蛋白溶液与等体积弗氏完全佐剂混匀乳化,静置30分钟不分层时用于小鼠免疫;实验组免疫重组蛋白剂量为200μg/只,对照组免疫等体积的弗氏完全佐剂与PBS的乳化液;
2)两周后进行二免,二免时用蛋白溶液和等体积弗氏不完全佐剂的混匀乳化,免疫用抗原的剂量同首免;三免方案同二免;
3)三免后两周断尾采血,分离血清。
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