CN109824767A - 猪流行性腹泻病毒重组s2蛋白及其多克隆抗体的制备方法 - Google Patents
猪流行性腹泻病毒重组s2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流行性腹泻病毒重组S2蛋白的制备方法,该法对PEDV S2A基因片段进行扩增后经过原核表达、纯化后获得重组S2蛋白。据此,发明人还建立了重组S2蛋白多克隆抗体的制备方法,即应用猪流行性腹泻病毒重组S2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PEDV流行变异毒株CH/GX/2015/750A S2基因部分抗原性强的区域,并进行原核表达,初步研究其反应原性和免疫原性,为下一步开展S2蛋白的功能研究以及为诊断试剂盒或亚单位疫苗的研发奠定了基础。
Description
技术领域
本发明属于猪流行性腹泻病毒技术领域,尤其涉及一种猪流行性腹泻病毒重组S2蛋白及其多克隆抗体的制备方法。
背景技术
猪流行性腹泻(porcine epidemic diarrhea,PED)是由猪流行性腹泻病毒(porcine epidemic diarrhea Virus,PEDV)引起的一种高度接触性的急性传染病,以粪口途径传播为主,感染猪会出现呕吐、水样腹泻、脱水等症状,乳猪会出现脱水、酸中毒死亡,猪只年龄越小死亡率越高。猪流行性腹泻病最初于20世纪70年代报道于英国和比利时,随后包括中国、菲律宾、韩国等在内的多个亚洲国家也先后报道本病的发生。2010年底开始,我国多个省市或地区陆续报道PED大规模暴发,且免疫过流行性腹泻疫苗的规模化猪场也爆发了PED,新生仔猪大量死亡,损失惨重。美洲于2013年也首次暴发PED,后证实是病毒的变异导致PEDV在美洲国家和地区的大流行,给养猪业造成了巨大的经济损失。
猪流行性腹泻病毒(PEDV)是单链正股RNA病毒,在遗传分类上属于套式病毒目冠状病毒科冠状病毒属。PEDV基因组共编码4种主要结构蛋白,即核衣壳蛋白(N)、包膜蛋白(E)、膜蛋白(M)和纤突蛋白(S)。S蛋白位于病毒粒子表面,由1383个氨基酸组成,分为两个功能区即S1(1~789aa)和S2(790~1 383aa),S蛋白在介导病毒粒子进入细胞和诱导机体产生中和抗体的过程中起到关键作用。我国2010年底以后,从发病猪肠道或粪便中分离到的PEDV流行毒株主要为变异毒株,其与经典疫苗株CV777相比,在S基因上有很多的变异,这可能使病毒毒力增强或者改变了病毒的免疫逃避机制,促使PED在中国的大暴发。Sun等报道,S1蛋白636~789aa是一段高度保守的区域,能够诱导机体产生中和抗体。孙东波使用丝状噬菌体展示系统鉴定出S蛋白的S1区域具有5个线性抗原表位区域和1个中和表位。桂锐等原核表达了PEDV部分S1蛋白(60-845bp),重组蛋白得到了表达且具有良好的反应原性,能够被兔PEDV多抗血清识别。前述研究主要针对PEDV S蛋白S1区域功能,而对PEDV S蛋白S2区域的抗原性等功能研究较少。
发明内容
本发明要解决的技术问题是提供一种猪流行性腹泻病毒重组S2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:
猪流行性腹泻病毒重组S2蛋白的制备方法,包括以下步骤:
<1>PEDV S2A基因片段的扩增
对PEDV的RNA进行反转录获得cDNA,然后采用特异性引物S2A-P1和S2A-P2对PEDVS2A基因进行PCR扩增,特异性引物S2A-P1和S2A-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-S2A的构建
将PCR扩增得到的PEDV S2A基因片段连接pMD18-T克隆载体,转化DH5α感受态细胞获得pMD18-S2A克隆质粒;将pMD18-S2A克隆质粒和pET-32a(+)分别进行双酶切,回收S2A基因和pET-32a(+)载体,利用T4连接酶将pET-32a(+)和S2A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-S2A;
<3>重组S2蛋白的获取
将原核表达质粒pET32a-S2A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S2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S2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S2A-P1、S2A-P2各0.5μL,cDNA 2.5μL;反应条件:95℃预变性5min;95℃变性45s,55℃退火45s,72℃延伸90s,34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PEDV S2A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-S2A克隆质粒。
步骤<2>按以下操作进行:将pET-32a(+)空载体和克隆质粒pMD18-S2A分别用BamHⅠ和XholⅠ进行双酶切,分别胶回收pET-32a(+)空载体和目的基因S2A;利用T4连接酶将pET-32a(+)和S2A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XholⅠ进行双酶切鉴定,获得正确的原核表达质粒pET32a-S2A。
步骤<3>按以下操作进行:将原核表达质粒pET32a-S2A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,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PEDV S2蛋白。
使用上述猪流行性腹泻病毒重组S2蛋白制备多克隆抗体的方法,使用猪流行性腹泻病毒重组S2蛋白与弗氏佐剂乳化后免疫动物获得多克隆抗体血清。
猪流行性腹泻病毒重组S2蛋白与弗氏佐剂乳化按以下操作进行:将纯化得到的重组PEDV S2蛋白加入透析袋中透析,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。
上述制备多克隆抗体的方法,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的混合液;
2)二免: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。
针对目前猪流行性腹泻病毒缺乏有关研究方法的问题,发明人建立了一种猪流行性腹泻病毒重组S2蛋白的制备方法,该法对PEDV S2A基因片段进行扩增后经过原核表达、纯化后获得重组S2蛋白。据此,发明人还建立了重组S2蛋白多克隆抗体的制备方法,即应用猪流行性腹泻病毒重组S2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PEDV流行变异毒株CH/GX/2015/750A S2基因部分抗原性强的区域,并进行原核表达,初步研究其反应原性和免疫原性,为下一步开展S2蛋白的功能研究以及为诊断试剂盒或亚单位疫苗的研发奠定了基础。
附图说明
图1是目的基因S2A的RT-PCR扩增结果图,图中:M:DNA分子质量标准;1:目的基因S2A。
图2是pET32a-S2A的双酶切鉴定结果图,图中:1重组质粒pET-S2A;M:DNA分子质量标准。
图3是最佳IPTG诱导浓度优化结果图,图中:M:预染蛋白Marker(SM0441);1-7:0mM、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:未诱导对照;2-7:0.2mM IPTG分别诱导1h、2h、3h、4h、5h和6h。
图5是最佳诱导温度的优化结果图,图中:M:预染蛋白Marker(CW0986);1-4:40℃、37℃、34℃和30℃诱导3h。
图6是重组蛋白S2A表达形式的鉴定结果图,图中: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;M:预染蛋白Marker。
图7是S2A纯化蛋白SDS-PAGE电泳分析结果图,图中:M:预染蛋白Marker(CW0986);1-3:纯化后的重组蛋白S2A。
图8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:S2A纯化蛋白。:
具体实施方式
1材料与方法
1.1主要试验材料
PEDV分离毒株CH/GX/2015/750A(NCBI登录号KY793536)、PEDV阴性血清、PEDV分离毒株CH/GX/2015/750A人工感染猪的阳性血清由申请人研究室分离、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、XholⅠ、DL2000DNA Marker均为大连宝生物工程有限公司产品;预染蛋白Marker(CW0986),His标签蛋白纯化试剂盒为康为世纪公司产品;预染蛋白Marker(SM0441)为索莱宝公司产品;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PEDV分离毒株CH/GX/2015/750A的S基因组序列,利用Meg Align(DNA Star),Primer Premier 7.0软件针对S2基因抗原性较好的区域设计一对特异性引物S2A-P1和S2A-P2,特异性的扩增PEDV部分S2基因(位于S基因2422nt-3945nt),命名为S2A。PCR扩增目的片段大小为1524bp。引物由大连宝生物工程有限公司合成。
引物序列如下:
S2A-P1:5’-CGCGGATCCTGTGCCACATATGTTTG-3’(SEQ.ID.No.1),
S2A-P2:5’-CCGCTCGAGCTCAAGGTCAACTAGTG-3’(SEQ.ID.No.2),下划线部分为BamHⅠ和XholⅠ酶切位点。
1.3 RNA的提取和S2A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S2A的PCR扩增。
PCR反应体系为:Taq Mix12.5μL,灭菌ddH2O 9μL,上下游引物S2A-P1、S2A-P2各0.5μL,cDNA 2.5μL。
PCR反应条件为95℃预变性5min;95℃变性45s,55℃退火45s,72℃延伸90s,34个循环;最后72℃延伸5min。
1.4 S2A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液体培养基中震荡培养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-S2A。
1.5原核表达质粒pET32a-S2A的构建
将pET-32a(+)空载体和克隆质粒pMD-S2A分别用BamHⅠ和XholⅠ进行双酶切,分别胶回收pET-32a(+)空载体和目的基因S2A。利用T4连接酶将pET-32a(+)和S2A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XholⅠ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-S2A。
1.6重组PEDV S2A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-S2A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,优化包括IPTG浓度、诱导温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重组蛋白的纯化与复性
1.7.1重组S2A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PEDV S2A蛋白。
1.7.1重组S2A蛋白的复性
重组蛋白S2A采用透析袋梯度透析的方法除去尿素及重新折叠复性。
1.8纯化重组蛋白的抗原性分析
以申请人实验室制备保存的PEDV分离毒株CH/GX/2015/750A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PEDV阴性猪血清为一抗进行Western-blot做阴性对照。
1.9小鼠抗S2A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、复性得到的重组S2A蛋白加入透析袋中用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。
用乳化器将等量浓缩后的蛋白与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、三免时取浓缩蛋白与等量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,对照组注射与实验组等量的弗氏完全佐剂与PBS的混合液;
2)二免:两周后进行二免,二免时用蛋白液和等量的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0.802mg/mL。)
1.10小鼠抗S2A蛋白多克隆抗体效价的测定
以纯化的S2A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,以免疫小鼠对的血清为一抗(1:1 000~1:128 000),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,判定制备的小鼠抗S2A蛋白多克隆抗体的最低检出效价。
2结果
2.1 PEDV S2A基因的扩增及原核表达载体的构建
按照1.3中的方法,以PEDV分离毒株CH/GX/2015/750A的RNA为模板通过RT-PCR扩增得到片段大小约1 524bp(SEQ.ID.No.3),与预期目的片段大小相符(见图1)。对获得的原核表达质粒pET32a-S2A用BamHⅠ和XholⅠ进行双酶切鉴定,分别得到大小约1524bp的目的片段和5.9kb的pET32a(+)载体片段,与预期相符(见图2)。测序证实目的片段与750A株的S2基因同源性为100%,说明重组原核表达质粒pET32a-S2A构建正确。
2.2重组蛋白S2A诱导表达条件的优化及表达形式的鉴定
将含pET32a-S2A重组表达质粒的BL21重组菌,用IPTG诱导表达,并对诱导条件进行优化。IPTG浓度为0.2mM时拥有较高的表达量,将IPTG最佳诱导浓度定为0.2mM(见图3)。随着诱导时间的增加,蛋白表达量逐渐增大,但诱导3h后蛋白表达量无明显增加,故将最佳诱导时间定为3h(见图4)。对最佳诱导温度进行优化,重组蛋白S2A在37℃时的表达量最高,故将最佳诱导温度定为37℃(见图5)。
将诱导表达的菌液超声波裂解后离心,分别取上清和沉淀进行SDS-PAGE电泳。结果表明,重组蛋白S2A主要在以包涵体的形式表达(见图6)。
2.3重组蛋白S2A的纯化
将以包涵体形式表达的带His标签的重组S2A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S2A蛋白(SEQ.ID.No.4)(见图7)。
2.4重组蛋白S2A的抗原性分析
将纯化的S2A蛋白与申请人实验室保存的PEDV分离毒株CH/GX/2015/750A人工感染猪的阳性血清为进行Western-blot鉴定,结果表明纯化后的重组蛋白S2A能够与PEDV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S2A蛋白具有较好的抗原性(见图8)。
2.5小鼠多克隆抗体效价的测定
用鼠抗S2A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。经计算,本研究制备的鼠抗S2A蛋白多克隆抗体血清平均效价为1:32000(表2)。
表2 ELISA方法测定多克隆抗体效价结果
序列表
<110> 广西壮族自治区兽医研究所
<120> 猪流行性腹泻病毒重组S2蛋白及其多克隆抗体的制备方法
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
cgcggatcct gtgccacata tgtttg 26
<210> 2
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
ccgctcgagc tcaaggtcaa ctagtg 26
<210> 3
<211> 1524
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
tgtgccacat atgtttgtaa tggtaactct cgttgtaaac aattactcac ccagtacact 60
gcagcatgta agaccataga gtcagcatta caactcagcg ctaggcttga gtctgttgaa 120
gttaactcta tgcttactat ttctgaagag gctctacagt tagccaccat tagttcgttt 180
aatggtgatg gatataattt tactaatgtg ctgggtgttt ctgtgtatga tcctgcaagt 240
ggcagggtgg tacaaaaaag gtcttttatt gaagacctgc tttttaataa agtggttact 300
aatggccttg gtactgttga tgaagactat aagcgctgtt ctaatggtcg ctctgtggca 360
gatctagtct gtgcacagta ttactctggt gtcatggtac tacctggtgt tgttgacgct 420
gagaagcttc acatgtatag tgcgtctctc atcggtggta tggtgctagg aggttttact 480
tctgcagcgg cattgccttt tagctatgct gttcaagcta gactcaatta tcttgctcta 540
cagacggatg ttctacagcg gaaccaacaa ttgcttgctg agtcttttaa ctctgctatt 600
ggtaatataa cttcagcctt tgagagtgtt aaagaggcta ttagtcaaac ttccaagggt 660
ttgaacactg tggctcatgc gcttactaag gttcaagagg ttgttaactc gcagggtgca 720
gctttgactc aacttaccgt acagctgcaa cacaatttcc aagccatttc tagttctatt 780
gatgacattt actctcgact ggacattctt tcagccgatg ttcaggttga ccgtctcatc 840
accggcagat tatcagcact taatgctttt gttgctcaaa ccctcactaa gtatactgag 900
gttcaggcta gcaggaagct agcacagcaa aaggttaatg agtgcgttaa atcgcaatct 960
cagcgttatg gtttttgtgg tggtgatggc gagcacattt tctctctggt acaggcagca 1020
cctcagggcc tgctgttttt acatacagta cttgtaccgg gtgactttgt agatgttatt 1080
gccatcgctg gcttatgcgt taacgatgaa attgccttga ctctacgtga gcctggctta 1140
gtcttgttta cgcatgaact tcaaaatcat actgcgacgg aatattttgt ttcatcgcga 1200
cgtatgtttg aacctagaaa acctaccgtt agtgattttg ttcaaattga gagttgtgtg 1260
gtcacctatg tcaatttgac tagagaccaa ctaccagatg taatcccaga ttacatcgat 1320
gttaacaaaa cacttgatga gattttagct tctctgccca atagaactgg tccaagtctt 1380
cctttagatg tttttaatgc cacttatctt aatctcactg gtgaaattgc agatttagag 1440
cagcgttcag agtctctccg taatactaca gaggagctcc aaagtcttat atataatatc 1500
aacaacacac tagttgacct tgag 1524
<210> 4
<211> 508
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Cys Ala Thr Tyr Val Cys Asn Gly Asn Ser Arg Cys Lys Gln Leu Leu
1 5 10 15
Thr Gln Tyr Thr Ala Ala Cys Lys Thr Ile Glu Ser Ala Leu Gln Leu
20 25 30
Ser Ala Arg Leu Glu Ser Val Glu Val Asn Ser Met Leu Thr Ile Ser
35 40 45
Glu Glu Ala Leu Gln Leu Ala Thr Ile Ser Ser Phe Asn Gly Asp Gly
50 55 60
Tyr Asn Phe Thr Asn Val Leu Gly Val Ser Val Tyr Asp Pro Ala Ser
65 70 75 80
Gly Arg Val Val Gln Lys Arg Ser Phe Ile Glu Asp Leu Leu Phe Asn
85 90 95
Lys Val Val Thr Asn Gly Leu Gly Thr Val Asp Glu Asp Tyr Lys Arg
100 105 110
Cys Ser Asn Gly Arg Ser Val Ala Asp Leu Val Cys Ala Gln Tyr Tyr
115 120 125
Ser Gly Val Met Val Leu Pro Gly Val Val Asp Ala Glu Lys Leu His
130 135 140
Met Tyr Ser Ala Ser Leu Ile Gly Gly Met Val Leu Gly Gly Phe Thr
145 150 155 160
Ser Ala Ala Ala Leu Pro Phe Ser Tyr Ala Val Gln Ala Arg Leu Asn
165 170 175
Tyr Leu Ala Leu Gln Thr Asp Val Leu Gln Arg Asn Gln Gln Leu Leu
180 185 190
Ala Glu Ser Phe Asn Ser Ala Ile Gly Asn Ile Thr Ser Ala Phe Glu
195 200 205
Ser Val Lys Glu Ala Ile Ser Gln Thr Ser Lys Gly Leu Asn Thr Val
210 215 220
Ala His Ala Leu Thr Lys Val Gln Glu Val Val Asn Ser Gln Gly Ala
225 230 235 240
Ala Leu Thr Gln Leu Thr Val Gln Leu Gln His Asn Phe Gln Ala Ile
245 250 255
Ser Ser Ser Ile Asp Asp Ile Tyr Ser Arg Leu Asp Ile Leu Ser Ala
260 265 270
Asp Val Gln Val Asp Arg Leu Ile Thr Gly Arg Leu Ser Ala Leu Asn
275 280 285
Ala Phe Val Ala Gln Thr Leu Thr Lys Tyr Thr Glu Val Gln Ala Ser
290 295 300
Arg Lys Leu Ala Gln Gln Lys Val Asn Glu Cys Val Lys Ser Gln Ser
305 310 315 320
Gln Arg Tyr Gly Phe Cys Gly Gly Asp Gly Glu His Ile Phe Ser Leu
325 330 335
Val Gln Ala Ala Pro Gln Gly Leu Leu Phe Leu His Thr Val Leu Val
340 345 350
Pro Gly Asp Phe Val Asp Val Ile Ala Ile Ala Gly Leu Cys Val Asn
355 360 365
Asp Glu Ile Ala Leu Thr Leu Arg Glu Pro Gly Leu Val Leu Phe Thr
370 375 380
His Glu Leu Gln Asn His Thr Ala Thr Glu Tyr Phe Val Ser Ser Arg
385 390 395 400
Arg Met Phe Glu Pro Arg Lys Pro Thr Val Ser Asp Phe Val Gln Ile
405 410 415
Glu Ser Cys Val Val Thr Tyr Val Asn Leu Thr Arg Asp Gln Leu Pro
420 425 430
Asp Val Ile Pro Asp Tyr Ile Asp Val Asn Lys Thr Leu Asp Glu Ile
435 440 445
Leu Ala Ser Leu Pro Asn Arg Thr Gly Pro Ser Leu Pro Leu Asp Val
450 455 460
Phe Asn Ala Thr Tyr Leu Asn Leu Thr Gly Glu Ile Ala Asp Leu Glu
465 470 475 480
Gln Arg Ser Glu Ser Leu Arg Asn Thr Thr Glu Glu Leu Gln Ser Leu
485 490 495
Ile Tyr Asn Ile Asn Asn Thr Leu Val Asp Leu Glu
500 505
Claims (8)
1.一种猪流行性腹泻病毒重组S2蛋白的制备方法,其特征在于包括以下步骤:
<1>PEDV S2A基因片段的扩增
对PEDV的RNA进行反转录获得cDNA,然后采用特异性引物S2A-P1和S2A-P2对PEDV S2A基因进行PCR扩增,所述特异性引物S2A-P1和S2A-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-S2A的构建
将PCR扩增得到的PEDV S2A基因片段连接pMD18-T克隆载体,转化DH5α感受态细胞获得pMD18-S2A克隆质粒;将pMD18-S2A克隆质粒和pET-32a(+)分别进行双酶切,回收S2A基因和pET-32a(+)载体,利用T4连接酶将pET-32a(+)和S2A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-S2A;
<3>重组S2蛋白的获取
将原核表达质粒pET32a-S2A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S2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S2蛋白。
2.根据权利要求1所述的猪流行性腹泻病毒重组S2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S2A-P1、S2A-P2各0.5μL,cDNA 2.5μL;反应条件:95℃预变性5min;95℃变性45s,55℃退火45s,72℃延伸90s,34个循环;最后72℃延伸5min。
3.根据权利要求1所述的猪流行性腹泻病毒重组S2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PEDV S2A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-S2A克隆质粒。
4.根据权利要求1所述的猪流行性腹泻病毒重组S2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-S2A分别用BamHⅠ和XholⅠ进行双酶切,分别胶回收pET-32a(+)空载体和目的基因S2A;利用T4连接酶将pET-32a(+)和S2A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XholⅠ进行双酶切鉴定,获得正确的原核表达质粒pET32a-S2A。
5.根据权利要求1所述的猪流行性腹泻病毒重组S2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-S2A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,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PEDV S2蛋白。
6.使用权利要求1所述猪流行性腹泻病毒重组S2蛋白制备多克隆抗体的方法,其特征在于使用猪流行性腹泻病毒重组S2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,其特征在于所述猪流行性腹泻病毒重组S2蛋白与弗氏佐剂乳化按以下操作进行:将纯化得到的重组PEDV S2蛋白加入透析袋中透析,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: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的混合液;
2)二免: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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