CN111471701B - 高效表达鹅星状病毒可溶性衣壳蛋白的orf2基因的方法及其应用 - Google Patents
高效表达鹅星状病毒可溶性衣壳蛋白的orf2基因的方法及其应用 Download PDFInfo
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- CN111471701B CN111471701B CN202010344174.5A CN202010344174A CN111471701B CN 111471701 B CN111471701 B CN 111471701B CN 202010344174 A CN202010344174 A CN 202010344174A CN 111471701 B CN111471701 B CN 111471701B
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Abstract
本发明涉及一种高效表达鹅星状病毒可溶性衣壳蛋白的ORF2基因的方法及其应用，属于兽用生物制品领域。本发明将鹅星状病毒的ORF2基因根据基因序列功能的不同分为两段表达，表达产物分别为衣壳蛋白capsid precursor(简称cap)和刺突蛋白capsid spike(简称spike)。根据功能区进行分割的两段蛋白可用大肠杆菌、杆状病毒和CHO表达系统进行表达。通过纯化方法获得鹅星状病毒的capsid precursor表达蛋白和capsid spike表达蛋白。表达的衣壳蛋白(capsid precursor)和刺突蛋白(capsid spike)蛋白可用于制备鹅星状病毒亚单位疫苗和卵黄抗体。本发明制备的亚单位疫苗和卵黄抗体具有很好的免疫效果和治疗效果，可应用于预防鹅星状病毒引起的疾病，并具有生产成本低、操作简单、生物安全性好的优点。
Description
技术领域
本发明涉及兽用生物制品技术领域，具体涉及一种高效表达鹅星状病毒可溶性衣壳蛋白的ORF2基因的方法及其应用。
背景技术
鹅星状病毒(Goose Astrovirus，GAstV)是引起三周龄以内的雏鹅，发生高致死性内脏痛风病的一种星状病毒。其发病特点是：鹅群感染GAstV后，以组织器官及腿部肌肉、关节覆盖大量尿酸盐沉淀物为主要特征，发病率达50％以上，致死率可达30％以上，对三周龄以内的雏鹅具有较强感染性。感染后康复鹅易产生免疫抑制。其主要临床症状包括精神沉郁，食欲不振，腹泻，生长不良，还可导致肝炎、肾炎等疾病。死亡雏鹅剖检可发现肾小管上皮细胞损伤，心脏、肝脏、肺脏、腿肌、胸肌等部位出现严重的尿酸盐沉积，非化脓性脑炎等特征。雏鹅早期感染GAstV出现活动减少、四肢瘫痪、精神沉郁的症状。感染GAstV的雏鹅很快即可出现死亡。感染后的雏鹅通过排粪等途径持续排毒，经粪口传播，难以控制传染源。GAstV给养禽业带来了巨大的经济损失。
现有技术防控GAstV的主要方法仍然是延长空舍时间、消毒及隔离等手段，没有相关预防和治疗性产品。因此，亟需开发研制新型有效的疫苗和卵黄抗体以达到有效防治该类传染病之目的。
发明内容
为解决现有技术中存在的问题，本发明专利设计了一种能高效表达鹅星状病毒ORF2基因的方法，并利用该方法制备了一种能够有效预防鹅星状病毒感染的疫苗和卵黄抗体，为防治鹅星状病毒提供保障。首先，本发明设计了一种能将鹅星状病毒衣壳蛋白高效表达的方法。为能将ORF2基因表达出正确的结构，根据序列结构的功能分为两段表达，表达产物分别为astrovirus capsid precursor(简称cap)和capsid spike(简称spike)。表达后的蛋白分别形成具有衣壳蛋白和刺突蛋白的表面免疫原性结构域。包括以下步骤，
(1)ORF2基因的合成，参照鹅星状病毒株ORF2基因序列，对序列进行大肠杆菌的密码子偏嗜性优化，人工合成ORF2基因如SEQ ID NO.1所示，连入载体得pUC57-ORF2；
(2)克隆基因的设计及PCR扩增，将ORF2基因根据编码的蛋白的功能不同将其分为衣壳蛋白39nt～406nt和刺突蛋白394nt～665nt两段分别进行载体构建和表达；所述衣壳蛋白的氨基酸序列如SEQ ID NO.3所示，核酸序列如SEQ ID NO.2所示，所述刺突蛋白的氨基酸序列如SEQ ID NO.5所示，核酸序列如SEQ ID NO.4所示；
(3)扩增片段分别连接pET28a载体，将衣壳蛋白DNA片段和刺突蛋白DNA片段切胶后用DNA纯化回收试剂盒回收，从重组菌株DH5α-pET28a中提取质粒pET28a，将提取的pET28a载体质粒与纯化回收的衣壳蛋白DNA片段和刺突蛋白DNA片段均分别用BamHI和NotI进行双酶切；将回收后的pET28a质粒、衣壳蛋白DNA片段(cap)和刺突蛋白DNA片段(spike)用T4 DNA Ligase连接质粒和片段，构建重组质粒pET28a-cap和pET28a-spike；
(4)重组质粒的转化，cap片段和spike片段分别与pET28a的连接产物转化至大肠杆菌JM109感受态细胞，将转化菌液涂布于固体LB琼脂平板，再将LB琼脂平板放于37℃温箱中过夜培养；待LB平板上长出单克隆菌体后，挑出单菌落活化于LB液体，取微量菌液进行PCR鉴定，余下部分菌液用于提取重组质粒进行双酶切鉴定；
(5)重组菌株的构建与表达，将鉴定正确的重组质粒pET28a-cap和pET28a-spike分别转化至大肠杆菌BL21-DE3-pLysS，构建重组表达菌株BL21-DE3-pLysS-pET28a-cap和BL21-DE3-pLysS-pET28a-spike，将BL21-DE3-pLysS-pET28a-cap和BL21-DE3-pLysS-pET28a-spike分别活化后，按1：100的比例接种至液体TB培养基中，置于37℃摇床培养约3h，加入IPTG至终浓度0.5mM，过夜诱导后将菌液离心，去掉TB上清，余下菌体用PBS重悬超声破碎，将超声菌体离心分离上清和沉淀，利用SDS-PAGE和WesternBlot进行可溶性分析。
该序列构建方法可分别应用原核表达载体、杆状病毒表达载体和CHO细胞表达载体。
进一步的，所述步骤(2)中克隆基因的设计及PCR扩增的方法为，根据核苷酸序列分别设计引物进行基因扩增，引物序列为Seg1-FCGCGGATCCCAGAAACTGCCGATGAAAGCTGAAC；Seg1-RAAAAGGAAAAGCGGCCGCGCCCTGACCAGTGGTGTTAACGTTC用于扩增368个氨基酸的衣壳蛋白；Seg2-FCGCGGATCCATGCAGGTGACCCCGAGCCT；Seg2-RAAAAGGAAAAGCGGCCGCGCTGGTTTTCAGGCTCACCGCCA用于扩增272个氨基酸的刺突蛋白；在上游引物Seg1-F和Seg2-F的5’端引入限制性酶切位点BamHI，在下游引物Seg1-R和Seg2-R的5’端引入限制性酶切位点NotI；分别以Seg1-F，Seg1-R为引物，以pUC57-ORF2为模板对衣壳蛋白的基因片段进行扩增；并以Seg2-F，Seg2-R为引物，以pUC57-ORF2为模板对刺突蛋白的基因片段进行扩增。
进一步的，所述方法还包括通过Ni-NTA层析柱对蛋白进行纯化，操作步骤如下：(1)填装镍料：取10mL镍填料装填柱子中，用ddH2O缓慢流过镍柱，将20％乙醇溶液流出，反复灌柱清洗；(2)平衡镍柱：20mL Binding Buffer平衡柱子；(3)样品上样：向镍柱中分别加入10mLcap蛋白或spike蛋白，重复上样3次；(4)洗涤蛋白：50mL Washing Buffer持续洗涤杂蛋白；(5)洗脱蛋白：10mL Elution Buffer洗脱目的蛋白。
重组菌株BL21-DE3-pLysS-pET28a-cap和BL21-DE3-pLysS-pET28a-spike分别能够可溶性表达cap蛋白和spike蛋白，二者分别形成鹅星状病毒的衣壳蛋白结构域和刺突蛋白结构域。
同时本发明还设计了一种预防鹅星状病毒的亚单位疫苗，所述亚单位疫苗利用纯化后的cap蛋白和spike蛋白与免疫佐剂进行配比后进行乳化制得。
进一步的，所述免疫佐剂为油佐剂、铝佐剂、蜂胶佐剂、油水双相佐剂中的一种。
本发明表达的cap和spike目的蛋白可用于鹅星状病毒抗体检测试剂盒研制、亚单位疫苗和卵黄抗体的研制，更加简易、高效和经济，具有巨大的生产应用价值。其中亚单位疫苗由表达的鹅星状病毒衣壳蛋白和刺突蛋白按照一定比例混合，经纯化后和矿物油免疫佐剂混合制得。
现对于现有技术，本发明的进步之处在于，提供的衣壳蛋白和刺突蛋白是GAstV的主要结构蛋白之一，含有能诱导中和抗体的构象依赖性抗原决定簇，是GAstV的保护性抗原，其诱导的中和抗体能保护宿主不受GAstV的感染。可见，本发明提供的衣壳蛋白和衣壳刺突蛋白，具有更好的保护作用。
本发明利用大肠杆菌表达对密码子的偏嗜性合成的序列，衣壳蛋白核酸序列如SEQ ID NO.2所示，所述衣壳刺突蛋白的核酸序列如SEQ ID NO.4所示，合成的序列和病毒本身的ORF2基因序列表达相比，表达产量更高，蛋白活性更高。
因大肠表达时，常形成无活性的包涵体，需要通过复性等复杂步骤才能恢复部分活性。因大肠表达全长ORF2蛋白出现不可逆性沉淀，本发明将鹅星状髌骨的ORF2基因分为衣壳蛋白(cap)和衣壳刺突蛋白(spike)两部分进行表达。分别表达两种目标蛋白减小了目的蛋白的分子量，能够避免产生包涵体和自发沉淀，有利于表达蛋白的纯化。更重要的是蛋白在表达的过程中能获得正确折叠和二硫键，从而获得有活性的蛋白质。
本发明提供的将鹅星状病毒的ORF2基因分为衣壳蛋白(cap)和衣壳刺突蛋白(spike)两部分进行表达，与用全长ORF2表达相比，很大程度上提升了蛋白的产量，蛋白产量能够达到5g/L以上，蛋白表达产量增加，质量稳定，成本显著降低。
本发明的技术方案制备的鹅星状病毒亚单位疫苗免疫产蛋鸡获得的卵黄抗体效价高于用其他疫苗免疫后获得的卵黄抗体效价，获得的卵黄抗体质量稳定，抗体水平均一，能够有效治疗由鹅星状病毒感染引起的疾病。
本发明的技术方案制备的鹅星状病毒亚单位疫苗抗原纯度高、安全性好，免疫原性强，免疫后抗体效价显著增高。而且本发明的亚单位疫苗通过大肠杆菌表达系统表达，制备过程相对简单，成本低，有利于工业放大和生产。
附图说明
图1是表达capsidprecursor蛋白的cap基因和表达capsidspike蛋白的spike基因克隆到pET28a载体得到的重组质粒酶切鉴定图，其中M：DNAMarker8000，1～2：分别为重组质粒pET28a+GAstV-cap和pET28a+GAstV-spike，重组质粒用BamHI和Notl的酶切片段。
图2是表达cap蛋白和spike蛋白的SDS-PAGE检测图。1和2分别为cap蛋白和spike蛋白纯化产物，泳道3、4分别为cap蛋白超声破碎后上清和沉淀，5、6泳道分别为spike蛋白超声破碎后上清和沉淀。
图3是表达cap蛋白和spike蛋白的Western Blotting检测图。1和2分别为cap蛋白和spike蛋白纯化产物，泳道3和4分别为cap蛋白超声破碎后上清和沉淀，5和6泳道分别为spike蛋白超声破碎后上清和沉淀。
具体实施方式
下面结合附图和具体实施例对本发明做进一步的说明。但以下实施例只是用来详细说明本发明，并不以任何方式限制本发明的范围。
在以下实施例中所涉及的仪器设备如无特别说明，均为常规仪器设备；所涉及的试剂如无特别说明，均为市售常规试剂；所涉及的试验方法，如无特别说明，均为常规方法。
实施例1
鹅星状病毒ORF2基因在大肠杆菌原核表达载体中的表达
1.ORF2基因的合成
参照鹅星状病毒株ORF2基因序列，综合因素对序列进行优化，例如大肠杆菌的密码子偏嗜性。人工合成ORF2基因，如SEQ ID NO.1所示，连入载体得pUC57-ORF2。
2.克隆基因的设计及PCR扩增
将ORF2基因根据编码的蛋白的不同将其分为衣壳蛋白39nt～406nt和刺突蛋白394nt～665nt两段分别进行载体构建和表达。
根据核苷酸序列分别设计引物进行基因扩增，引物序列为Seg1-FCGCGGATCCCAGAAACTGCCGATGAAAGCTGAAC；Seg1-RAAAAGGAAAAGCGGCCGCGCCCTGACCAGTGGTGTTAACGTTC用于扩增368个氨基酸的衣壳蛋白；Seg2-FCGCGGATCCATGCAGGTGACCCCGAGCCT；Seg2-RAAAAGGAAAAGCGGCCGCGCTGGTTTTCAGGCTCACCGCCA用于扩增272个氨基酸的刺突蛋白；在上游引物Seg1-F和Seg2-F的5’端引入限制性酶切位点BamHI，在下游引物Seg1-R和Seg2-R和的5’端引入限制性酶切位点Notl；引物由上海生工生物工程有限公司合成。
分别以Seg1-F，Seg1-R为引物，以pUC57-ORF2为模板对衣壳蛋白的基因片段进行扩增；并以Seg2-F，Seg2-R为引物，以pUC57-ORF2为模板对刺突蛋白的基因片段进行扩增。反应体系及扩增条件如下：
表1 PCR反应体系
PCR反应条件：95℃预变性3min，95℃变性30s，60℃退火30s，72℃延伸1min，30个循环；最后72℃延伸7min。PCR产物用1％琼脂糖凝胶电泳进行鉴定，以Seg1-F，Seg1-R为引物扩增目的片段为1104bp，以Seg2-F，Seg2-R为引物扩增目的片段为816bp，与预期一致。
3.扩增片段连接pET28a载体
(1)将衣壳蛋白DNA片段和刺突蛋白DNA片段切胶后用DNA纯化回收试剂盒回收。
(2)从重组菌株DH5α-pET28a中提取质粒pET28a。
(3)将提取的pET28a载体质粒与纯化回收的衣壳蛋白DNA片段和刺突蛋白DNA片段均分别用BamHI和NotI进行双酶切，酶切反应条件为37℃，3h。
(4)将回收后的pET28a质粒、衣壳蛋白DNA片段(cap)和刺突蛋白DNA片段(spike)检测浓度，用T4 DNA Ligase连接质粒和片段。
4.重组质粒的转化与鉴定
(1)cap片段和spike片段分别与pET28a的连接产物转化至大肠杆菌JM109感受态细胞将转化菌液涂布于固体LB琼脂平板(Kan，50μg/ml)，再将LB琼脂平板(Kan，50μg/ml)放于37℃温箱中过夜培养。
(2)待LB平板上长出单克隆菌体后，挑出单菌落活化于LB液体(Kan，50μg/ml)。
(3)取微量菌液进行PCR鉴定，余下部分菌液用于提取重组质粒进行双酶切鉴定，如图1所示。
(4)鉴定的阳性质粒送上海生工生物工程有限公司测序。结果表明构建重组载体pET28a-cap和pET28a-spike测序正确。
实施例2
构建Astrovirus重组大肠杆菌表达cap蛋白和spike蛋白
1.重组菌株的构建与表达
(1)将鉴定正确的重组质粒pET28a-cap和pET28a-spike分别转化至大肠杆菌BL21-DE3-pLysS，构建重组表达菌株BL21-DE3-pLysS-pET28a-cap和BL21-DE3-pLysS-pET28a-spike。
(2)将BL21-DE3-pLysS-pET28a-cap和BL21-DE3-pLysS-pET28a-spike分别活化后，按1：100的比例接种至液体TB(Kan，50μg/ml)培养基中，置于37℃摇床培养约3h，加入IPTG至终浓度0.5mM，过夜诱导后将菌液离心，去掉TB上清，余下菌体用PBS重悬超声破碎。
(3)将超声菌体离心分离上清和沉淀，利用SDS-PAGE和WesternBlot进行可溶性分析。
2.重组菌株表达条件的优化
在确认重组菌株BL21-DE3-pLysS-pET28a-cap和BL21-DE3-pLysS-pET28a-spike能够可溶性表达cap蛋白和spike蛋白后，优化cap蛋白和spike蛋白的最佳表达条件。分别对诱导剂浓度、诱导温度及诱导时长进行摸索，将诱导菌液离心处理，重悬菌体用超声仪超声破碎，保留蛋白上清，弃去破碎沉淀。表达产物用SDS-PAGE和WesternBlot进行分析。
SDS-PAGE结果如图2所示：泳道1、3、4在45KD附近出现明显的蛋白条带，泳道2、5、6在35KD附近出现明显的蛋白条带。泳道1、2分别为纯化后的cap蛋白和spike蛋白，显示蛋白的纯化效果良好。
Western Blot结果如图3所示：泳道1、3、4在45KD左右出现明显条带，泳道2、5、6在35KD左右出现明显条带，表明重组蛋白与HIS单抗反应良好。泳道1、2分别为纯化后的cap蛋白和spike蛋白，泳道3、5分别为cap蛋白和spike蛋白上清液，泳道4、6分别为cap蛋白和spike蛋白超声破碎后的沉淀重悬。
重组菌株BL21-DE3-pLysS-pET28a-cap，重新命名为BL21-GAstV-Cap菌株，建议分类命名为大肠埃希氏菌(Escherichiacoli)，保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏日期为2020年3月12日，保藏号为CGMCC No.19465。
重组菌株BL21BL21-DE3-pLysS-pET28a-spike，重新命名为BL21-GAstV-Spike菌株，建议分类命名为大肠埃希氏菌(Escherichia coli)，保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏日期为2020年3月12日，保藏号为CGMCC No.19464。
实施例3
cap蛋白和spike蛋白的亲和层析纯化
将大肠杆菌周质破碎上清通过Ni-NTA层析柱进行纯化操作步骤如下：(1)填装镍料：取10mL镍填料装填柱子中，用ddH2O缓慢流过镍柱，将20％乙醇溶液流出，反复灌柱清洗；(2)平衡镍柱：20mL Binding Buffer平衡柱子；(3)样品上样：向镍柱中分别加入10mLcap蛋白或spike蛋白，重复上样3次；(4)洗涤蛋白：50mL Washing Buffer持续洗涤杂蛋白；(5)洗脱蛋白：10mL Elution Buffer洗脱目的蛋白；将纯化的样品进行SDS-PAGE和Western Blot鉴定，cap蛋白和spike蛋白经过亲和层析纯化后纯度可达90％以上。Westernblot鉴定结果如图3的泳道1和泳道2所示：纯化后的cap和spike蛋白用his抗体进行Western blot，显示分别在45KD和35KD附近出现明显且纯净的蛋白条带。
2.ORF2纯化蛋白浓度测定
用BCA蛋白含量测定试剂盒(Pierce Biotechnology)测定纯化的cap蛋白和spike蛋白的浓度，具体步骤参考说明书。所测得的cap蛋白的浓度为6.9g/L，所测得的spike蛋白的浓度为5.2g/L表达量较高，能够满足大规模生产的要求。
实施例4
含有鹅星状病毒cap蛋白疫苗的制备及其安全性和免疫原性检测
1.疫苗的制备
将大肠杆菌表达和纯化后的cap蛋白和spike蛋白调整好浓度，与商业化的免疫佐剂ISA71VG按说明书的配比进行乳化，乳化后制备出三批鹅星状病毒大肠表达cap蛋白和spike蛋白的亚单位疫苗。
2.疫苗性状检验
外观：乳白色乳剂
剂型：油包水型乳剂。取一清洁吸管，吸取少量疫苗滴入冷水中，除第1滴外，均不扩散。
稳定性：吸取疫苗10ml加入离心管中，以3000r/min离心15分钟，管底析出的水相不超过0.5ml。
黏度：按现行《中国兽药典》附录进行，均符合规定。
疫苗装量检查：按现行《中国兽药典》附录进行检查，均符合规定。
疫苗无菌检验：按现行《中国兽药典》附录进行检验，均无菌生长。
3.疫苗免疫原性试验
选取40只14日龄雏鹅，公母混搭，被随机分为4组，每组10只，第1～3组每只颈部皮下分别注射三批大肠杆菌表达cap和spike蛋白亚单位疫苗0.5ml；对照组10只，不做任何免疫。各组的免疫程序和饲养环境均相同。免疫后28日，免疫鹅和对照鹅分别采血，分离血清，用琼脂免疫扩散试验法测定血清抗体效价，记录抗体结果。结果表明，3批疫苗免疫28天，琼扩抗体效价均能达到10/10阳性，琼扩抗体效价高于1：128。而对照未免疫鹅，琼扩抗体均低于1：4。表明用大肠杆菌表达系统表达的鹅星状病毒cap和spike蛋白均具有很好的免疫效果。
4.安全检验
用7～14日龄雏鹅40只，分为4组(10只/组)，第1～3组每只颈部皮下分别注射三批大肠杆菌表达cap和spike蛋白亚单位疫苗1ml，对照组10只，不做任何免疫，观察14日，并记录试验鹅的反应情况，包括精神状态、采食量、生长情况、有否发病和死亡，比较免疫组与对照组有否差异。二周后每组免疫雏鹅进行剖检，仔细观察各脏器有否病变，各注射部位油苗吸收状况，有否局部炎症，包括变色、肿胀、溃烂等，免疫组与对照组有否差异。
结果显示：鹅注射亚单位疫苗后，鹅群无应激反应，精神、食欲、死淘率、每周的饲料消耗均正常，精神状况良好，排泄正常。分别在免后第二周和第四周进行观察触摸，检查肌肉注射部位，未发现有炎症反应，与对照组相比无明显差异。二周后从每小组中各取出2只扑杀检查病变，可见各脏器均无病变，注射部位均无变色、肿胀、溃烂等炎症反应，但有个别鹅油苗尚未完全吸收。由此表明，制备的亚单位疫苗具有良好的安全性，没有毒性反应，不影响鹅群的正常生长发育。
实施例5
鹅星状病毒卵黄抗体的制备和抗体治疗
1.免疫接种
(1)基础接种：选取健康产蛋鸡50只，肌肉/皮下接种鹅星状病毒疫苗，剂量为0.5ml/只。
(2)第2次接种：基础免疫接种后间隔15天，肌肉/皮下注射鹅星状病毒疫苗1.0ml/只，多点注射。
(3)第3次接种：第2次接种后间隔15天，肌肉/皮下注射鹅星状病毒疫苗2.0ml/只，多点注射。
(4)以后每隔8-10周，肌肉注射接种1次。
(5)第3次接种后第7天开始，收集高免鸡蛋，用于制备卵黄抗体。高免蛋于15℃-25℃储存，时间不超过3天，2-8℃储存时间不超过7天。
2.高免蛋的卵黄抗体检测
(1)选取40枚免疫蛋和8枚不免疫蛋按照蛋黄与生理盐水1：3(V/V)稀释，加入与蛋黄等体积的三氯甲烷，充分振摇，萃取，3500r/min离心10分钟分离上清液，即为待测样品。
(2)抗原包被将星状病毒抗原，用包被液做200倍稀释包被酶标板，每孔100μl，37℃温箱孵育2h。
(3)洗涤弃去包被液，每孔大约300μl洗涤液，洗涤2min，弃去洗液，重复3次，最后一次在吸水纸上拍干孔内洗液。
(4)封闭每孔加封闭液150μl，37℃封闭30min，拍干。
(5)加一抗将已知鸡抗星状病毒卵黄抗体用稀释液做200倍稀释，每孔100μl，37℃温箱孵育1h，洗涤，方法同上。
(6)加二抗将酶标二抗1：6000倍稀释，每孔加入100μl，37℃温箱孵育45min，洗涤，每次3min。
(7)显色加底物液，每孔100μl，避光显色20min(室温)。
(8)终止每孔加入50μl终止液，用酶标仪测定波长450nm的OD值。
检测结果表明选取的40枚鸡蛋的卵黄抗体质量高、抗体水平稳定、均一。结果如表2所示。
表2卵黄抗体水平检测结果
3.卵黄抗体的田间应用
选择发病鹅群两个，每个发病鹅群随机选择500只鹅，做抗体注射，其他按照养殖户以前方法常规处理(目前没有有效方法)作为对照。每日记录死亡情况。10日后统计实验组和对照组死亡率。
对死亡率做统计分析结果表明，在两个养殖场注射卵黄抗体组鹅的死亡率分别为5.4％和6.4％；而不注射卵黄抗体组鹅的死亡率分别为40.7％和36.9％。表明该卵黄抗体能够有效治疗由鹅星状病毒感染引起的疾病。
本发明采用分子生物学技术、分子免疫学技术及基因工程技术研制的鹅星状病毒病亚单位疫苗，试验证明具有良好的免疫原性。该疫苗可以广泛应用于早期免疫，避免雏鹅发病带来的严重经济损失。
上述内容仅为本发明创造的较佳实施例而已，不能以此限定本发明创造的实施范围，即凡是依本发明创造权利要求及发明创造说明内容所做出的简单的等效变化与修饰，皆仍属于本发明创造涵盖的范围。
序列表
<110> 浙江鼎持生物制品有限公司 北京鼎持生物技术有限公司
<120> 高效表达鹅星状病毒可溶性衣壳蛋白的ORF2基因的方法及其应用
<141> 2020-04-26
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2115
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atggctgatc gtgcggttgc accgcgtgaa aaagtgacca aaaaagttac caaagttgtg 60
accgttaaaa agaaacaccc gaaaaagaaa ccgaaacaga aagtttataa accgcagaaa 120
ctgccgatga aagctgaacg taaactggaa aaagaagtta aaggtctgaa aaaacgtgtg 180
gctggcccgc cggtgaacga taaaatgacc accaccatta ccctgggtca gatcaccggt 240
aactccaccg ataccctgga ccgtaaacat aaatacttca ccaacccgct gatgatgaaa 300
aaccaagaaa acggccagac cgctaccccg ctgtctatcc gtgcttctca gtacaacctg 360
tggcgtatcc gtaaactgca catccgtctg gtgccgctgg ctggtcgtgc aaacatcctg 420
ggttctgttg ttttcctgga catcgaacag gaagcaaaca ccgcgggccc ggaatctatc 480
gataccatca aagctcgtcc gcacctggaa ctgccgatcg gcagcaaaca cctgtggcgt 540
gttcagccgc gtctgatgca gggcccgcgc cagggttggt ggaacgtaga ccctggtgat 600
tctccgaccg attctctggg cccggccatt aacatgtgga cctacctgaa aactgttaac 660
gcgctgtcca cccgcgctca ggcacagcag gtgccgtaca ctagcgcact cttcctggtt 720
gaagcaactg tgacctacga attctccaac tacggcccga aaccgggtct gagtctgatg 780
acctctgaaa cactgagcgc gagcggtaaa actgcgaccc tggttaacac ccaggacggt 840
gccctggcgc tgaccgttag cggtgcgctg cagcgtttcc tggatgaaaa agaacagcac 900
cgccgtgtta gcaacgccca aacctctggc gttggtgaag ttttctgggc tgttagcacc 960
gaagttgttg aaaccgttgc gtctgcactg ggtggctggg gctggctgct gaaaggtggc 1020
tggttcgtta tccgtaaact gtttggcgcg gcttccaaca gcggtagcac gtacctgatc 1080
tactcctccg taagcgatgc acagatcgat agccgtatct accagaccgt tccgccgaac 1140
accccgctgc agctggcggc gaacactgtt aaactggtgc agctgaccca gccgaacgtt 1200
aacaccactg gtcagggcac taccgttctg agccgcgatg ctgattacct gccgctgccg 1260
gttgcaccga tgcaggtgac cccgagcctg gtatataact ttcagggtga acgccagagc 1320
actaccgaat cctgtagctt cctggtcttt ggcattccac aggcggaaag ccgtagccgt 1380
tacaatgcta acatcacctt caacgtcggt tatcgcggcc gtacaagcac ctctttcacc 1440
ctgggcaccc ataactggtg ggcggttatg accctgagcc agaccggcgt gattttcgcg 1500
cctccggcgg tgggcaccgg cgtttgtaac accctggcca ccgcgatcca acatctgaac 1560
ccggaactgg aaaccgcagt gctgcgtgtt aacaccagca ccacctccac cggcggtctg 1620
atcaccgaac tgcgtaaccg cctgaacatc gctgatggtg attacgtgat ctctatgggt 1680
gatccgcagg gcaaccgtag cgctctgtac tttcgtaact ccgaccagaa atgggtatgg 1740
ctgtgggccg gtgattccaa tccgggtgaa accttccagt ctttcaaaat gccggtgctg 1800
atcaactggt ctgtgtccga tagccaggaa cagtacaatg cgcgcgtacg tatggttcag 1860
tacgctaacg cgcagcagca gactctgact gatcctgaag aagatgatga tccgctgtct 1920
gatgttacca gcctgtttga cccgaccgct gaagatgaaa ccgacttcca cctggcggtg 1980
agcctgaaaa ccagcgatta tctgaaagaa gaagctgaat attggaaagc taaagcgcag 2040
gctctgctga tggaaaaagc tctgtctgcg ccgcaggctg gtaccgttcg ttttgaaaaa 2100
ggcggtcacg aataa 2115
<210> 2
<211> 1104
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
cagaaactgc cgatgaaagc tgaacgtaaa ctggaaaaag aagttaaagg tctgaaaaaa 60
cgtgtggctg gcccgccggt gaacgataaa atgaccacca ccattaccct gggtcagatc 120
accggtaact ccaccgatac cctggaccgt aaacataaat acttcaccaa cccgctgatg 180
atgaaaaacc aagaaaacgg ccagaccgct accccgctgt ctatccgtgc ttctcagtac 240
aacctgtggc gtatccgtaa actgcacatc cgtctggtgc cgctggctgg tcgtgcaaac 300
atcctgggtt ctgttgtttt cctggacatc gaacaggaag caaacaccgc gggcccggaa 360
tctatcgata ccatcaaagc tcgtccgcac ctggaactgc cgatcggcag caaacacctg 420
tggcgtgttc agccgcgtct gatgcagggc ccgcgccagg gttggtggaa cgtagaccct 480
ggtgattctc cgaccgattc tctgggcccg gccattaaca tgtggaccta cctgaaaact 540
gttaacgcgc tgtccacccg cgctcaggca cagcaggtgc cgtacactag cgcactcttc 600
ctggttgaag caactgtgac ctacgaattc tccaactacg gcccgaaacc gggtctgagt 660
ctgatgacct ctgaaacact gagcgcgagc ggtaaaactg cgaccctggt taacacccag 720
gacggtgccc tggcgctgac cgttagcggt gcgctgcagc gtttcctgga tgaaaaagaa 780
cagcaccgcc gtgttagcaa cgcccaaacc tctggcgttg gtgaagtttt ctgggctgtt 840
agcaccgaag ttgttgaaac cgttgcgtct gcactgggtg gctggggctg gctgctgaaa 900
ggtggctggt tcgttatccg taaactgttt ggcgcggctt ccaacagcgg tagcacgtac 960
ctgatctact cctccgtaag cgatgcacag atcgatagcc gtatctacca gaccgttccg 1020
ccgaacaccc cgctgcagct ggcggcgaac actgttaaac tggtgcagct gacccagccg 1080
aacgttaaca ccactggtca gggc 1104
<210> 3
<211> 368
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Gln Lys Leu Pro Met Lys Ala Glu Arg Lys Leu Glu Lys Glu Val Lys
1 5 10 15
Gly Leu Lys Lys Arg Val Ala Gly Pro Pro Val Asn Asp Lys Met Thr
20 25 30
Thr Thr Ile Thr Leu Gly Gln Ile Thr Gly Asn Ser Thr Asp Thr Leu
35 40 45
Asp Arg Lys His Lys Tyr Phe Thr Asn Pro Leu Met Met Lys Asn Gln
50 55 60
Glu Asn Gly Gln Thr Ala Thr Pro Leu Ser Ile Arg Ala Ser Gln Tyr
65 70 75 80
Asn Leu Trp Arg Ile Arg Lys Leu His Ile Arg Leu Val Pro Leu Ala
85 90 95
Gly Arg Ala Asn Ile Leu Gly Ser Val Val Phe Leu Asp Ile Glu Gln
100 105 110
Glu Ala Asn Thr Ala Gly Pro Glu Ser Ile Asp Thr Ile Lys Ala Arg
115 120 125
Pro His Leu Glu Leu Pro Ile Gly Ser Lys His Leu Trp Arg Val Gln
130 135 140
Pro Arg Leu Met Gln Gly Pro Arg Gln Gly Trp Trp Asn Val Asp Pro
145 150 155 160
Gly Asp Ser Pro Thr Asp Ser Leu Gly Pro Ala Ile Asn Met Trp Thr
165 170 175
Tyr Leu Lys Thr Val Asn Ala Leu Ser Thr Arg Ala Gln Ala Gln Gln
180 185 190
Val Pro Tyr Thr Ser Ala Leu Phe Leu Val Glu Ala Thr Val Thr Tyr
195 200 205
Glu Phe Ser Asn Tyr Gly Pro Lys Pro Gly Leu Ser Leu Met Thr Ser
210 215 220
Glu Thr Leu Ser Ala Ser Gly Lys Thr Ala Thr Leu Val Asn Thr Gln
225 230 235 240
Asp Gly Ala Leu Ala Leu Thr Val Ser Gly Ala Leu Gln Arg Phe Leu
245 250 255
Asp Glu Lys Glu Gln His Arg Arg Val Ser Asn Ala Gln Thr Ser Gly
260 265 270
Val Gly Glu Val Phe Trp Ala Val Ser Thr Glu Val Val Glu Thr Val
275 280 285
Ala Ser Ala Leu Gly Gly Trp Gly Trp Leu Leu Lys Gly Gly Trp Phe
290 295 300
Val Ile Arg Lys Leu Phe Gly Ala Ala Ser Asn Ser Gly Ser Thr Tyr
305 310 315 320
Leu Ile Tyr Ser Ser Val Ser Asp Ala Gln Ile Asp Ser Arg Ile Tyr
325 330 335
Gln Thr Val Pro Pro Asn Thr Pro Leu Gln Leu Ala Ala Asn Thr Val
340 345 350
Lys Leu Val Gln Leu Thr Gln Pro Asn Val Asn Thr Thr Gly Gln Gly
355 360 365
<210> 4
<211> 816
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
cagctgaccc agccgaacgt taacaccact ggtcagggca ctaccgttct gagccgcgat 60
gctgattacc tgccgctgcc ggttgcaccg atgcaggtga ccccgagcct ggtatataac 120
tttcagggtg aacgccagag cactaccgaa tcctgtagct tcctggtctt tggcattcca 180
caggcggaaa gccgtagccg ttacaatgct aacatcacct tcaacgtcgg ttatcgcggc 240
cgtacaagca cctctttcac cctgggcacc cataactggt gggcggttat gaccctgagc 300
cagaccggcg tgattttcgc gcctccggcg gtgggcaccg gcgtttgtaa caccctggcc 360
accgcgatcc aacatctgaa cccggaactg gaaaccgcag tgctgcgtgt taacaccagc 420
accacctcca ccggcggtct gatcaccgaa ctgcgtaacc gcctgaacat cgctgatggt 480
gattacgtga tctctatggg tgatccgcag ggcaaccgta gcgctctgta ctttcgtaac 540
tccgaccaga aatgggtatg gctgtgggcc ggtgattcca atccgggtga aaccttccag 600
tctttcaaaa tgccggtgct gatcaactgg tctgtgtccg atagccagga acagtacaat 660
gcgcgcgtac gtatggttca gtacgctaac gcgcagcagc agactctgac tgatcctgaa 720
gaagatgatg atccgctgtc tgatgttacc agcctgtttg acccgaccgc tgaagatgaa 780
accgacttcc acctggcggt gagcctgaaa accagc 816
<210> 5
<211> 272
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Gln Leu Thr Gln Pro Asn Val Asn Thr Thr Gly Gln Gly Thr Thr Val
1 5 10 15
Leu Ser Arg Asp Ala Asp Tyr Leu Pro Leu Pro Val Ala Pro Met Gln
20 25 30
Val Thr Pro Ser Leu Val Tyr Asn Phe Gln Gly Glu Arg Gln Ser Thr
35 40 45
Thr Glu Ser Cys Ser Phe Leu Val Phe Gly Ile Pro Gln Ala Glu Ser
50 55 60
Arg Ser Arg Tyr Asn Ala Asn Ile Thr Phe Asn Val Gly Tyr Arg Gly
65 70 75 80
Arg Thr Ser Thr Ser Phe Thr Leu Gly Thr His Asn Trp Trp Ala Val
85 90 95
Met Thr Leu Ser Gln Thr Gly Val Ile Phe Ala Pro Pro Ala Val Gly
100 105 110
Thr Gly Val Cys Asn Thr Leu Ala Thr Ala Ile Gln His Leu Asn Pro
115 120 125
Glu Leu Glu Thr Ala Val Leu Arg Val Asn Thr Ser Thr Thr Ser Thr
130 135 140
Gly Gly Leu Ile Thr Glu Leu Arg Asn Arg Leu Asn Ile Ala Asp Gly
145 150 155 160
Asp Tyr Val Ile Ser Met Gly Asp Pro Gln Gly Asn Arg Ser Ala Leu
165 170 175
Tyr Phe Arg Asn Ser Asp Gln Lys Trp Val Trp Leu Trp Ala Gly Asp
180 185 190
Ser Asn Pro Gly Glu Thr Phe Gln Ser Phe Lys Met Pro Val Leu Ile
195 200 205
Asn Trp Ser Val Ser Asp Ser Gln Glu Gln Tyr Asn Ala Arg Val Arg
210 215 220
Met Val Gln Tyr Ala Asn Ala Gln Gln Gln Thr Leu Thr Asp Pro Glu
225 230 235 240
Glu Asp Asp Asp Pro Leu Ser Asp Val Thr Ser Leu Phe Asp Pro Thr
245 250 255
Ala Glu Asp Glu Thr Asp Phe His Leu Ala Val Ser Leu Lys Thr Ser
260 265 270
Claims (7)
1.高效表达鹅星状病毒可溶性衣壳蛋白的ORF2基因的方法，其特征在于，将鹅星状病毒可溶性衣壳蛋白的ORF2基因进行分段表达，包括以下步骤，
(1)ORF2基因的合成，参照鹅星状病毒株ORF2基因序列，对序列进行大肠杆菌的密码子偏嗜性优化，人工合成ORF2基因，如SEQ ID NO.1所示，连入载体得pUC57-ORF2；
(2)克隆基因的设计及PCR扩增，将ORF2基因根据编码的蛋白的功能不同将其分为衣壳蛋白39nt～406nt和刺突蛋白394nt～665nt两段分别进行载体构建和表达；所述衣壳蛋白的氨基酸序列如SEQ ID NO.3所示，核酸序列如SEQ ID NO.2所示，所述刺突蛋白的氨基酸序列如SEQ ID NO.5所示，核酸序列如SEQ ID NO.4所示；
(3)扩增片段分别连接pET28a载体，将衣壳蛋白DNA片段和刺突蛋白DNA片段切胶后用DNA纯化回收试剂盒回收，从重组菌株DH5α-pET28a中提取质粒pET28a，将提取的pET28a载体质粒与纯化回收的衣壳蛋白DNA片段和刺突蛋白DNA片段均分别用BamHI和NotI进行双酶切；将回收后的pET28a质粒、衣壳蛋白DNA片段和刺突蛋白DNA片段用T4 DNA Ligase连接质粒和片段，构建重组质粒pET28a-cap和pET28a-spike；
(4)重组质粒的转化，衣壳蛋白DNA片段和刺突蛋白DNA片段分别与pET28a的连接产物转化至大肠杆菌JM109感受态细胞，将转化菌液涂布于固体LB琼脂平板，再将LB琼脂平板放于37℃温箱中过夜培养；待LB平板上长出单克隆菌体后，挑出单菌落活化于LB液体，取微量菌液进行PCR鉴定，余下部分菌液用于提取重组质粒进行双酶切鉴定；
(5)重组菌株的构建与表达，将鉴定正确的重组质粒pET28a-cap和pET28a-spike分别转化至大肠杆菌BL21-DE3-pLysS，构建重组表达菌株BL21-DE3-pLysS-pET28a-cap和BL21-DE3-pLysS-pET28a-spike，将BL21-DE3-pLysS-pET28a-cap和BL21-DE3-pLysS-pET28a-spike分别活化后，按1：100的比例接种至液体TB培养基中，置于37℃摇床培养3h，加入IPTG至终浓度0.5mM，过夜诱导后将菌液离心，去掉TB上清，余下菌体用PBS重悬超声破碎，将超声菌体离心分离上清和沉淀，利用SDS-PAGE和Western Blot进行可溶性分析。
2.根据权利要求1所述的高效表达鹅星状病毒可溶性衣壳蛋白的ORF2基因的方法，其特征在于，所述步骤(2)中克隆基因的设计及PCR扩增的方法为，根据核苷酸序列分别设计引物进行基因扩增，引物序列为Seg1-F CGCGGATCCCAGAAACTGCCGATGAAAGCTGAAC；Seg1-RAAAAGGAAAAGCGGCCGCGCCCTGACCAGTGGTGTTAACGTTC用于扩增368个氨基酸的衣壳蛋白；Seg2-F CGCGGATCCATGCAGGTGACCCCGAGCCT；Seg2-R AAAAGGAAAAGCGGCCGCGCTGGTTTTCAGGCTCACCGCCA用于扩增272个氨基酸的刺突蛋白；在上游引物Seg1-F和Seg2-F的5’端引入限制性酶切位点BamHI，在下游引物Seg1-R和Seg2-R的5’端引入限制性酶切位点NotI；分别以Seg1-F，Seg1-R为引物，以pUC57-ORF2为模板对衣壳蛋白的基因片段进行扩增；并以Seg2-F，Seg2-R为引物，以pUC57-ORF2为模板对刺突蛋白的基因片段进行扩增。
3.根据权利要求1所述的高效表达鹅星状病毒可溶性衣壳蛋白的ORF2基因的方法，其特征在于，所述方法还包括通过Ni-NTA层析柱对蛋白进行纯化，操作步骤如下：(1)填装镍料：取10mL镍填料装填柱子中，用ddH2O缓慢流过镍柱，将20％乙醇溶液流出，反复灌柱清洗；(2)平衡镍柱：20mL Binding Buffer平衡柱子；(3)样品上样：向镍柱中分别加入10mLcap蛋白或spike蛋白，重复上样3次；(4)洗涤蛋白：50mL Washing Buffer持续洗涤杂蛋白；(5)洗脱蛋白：10mL Elution Buffer洗脱目的蛋白。
4.根据权利要求3所述的高效表达鹅星状病毒可溶性衣壳蛋白的ORF2基因的方法，其特征在于，重组菌株BL21-DE3-pLysS-pET28a-cap和BL21-DE3-pLysS-pET28a-spike分别能够可溶性高效表达衣壳蛋白和刺突蛋白，二者分别形成鹅星状病毒的衣壳蛋白结构域和刺突蛋白结构域。
5.一种鹅星状病毒的亚单位疫苗，其特征在于，所述亚单位疫苗利用权利要求4所述的方法制得的衣壳蛋白和刺突蛋白与免疫佐剂进行配比后进行乳化制得。
6.根据权利要求5所述的鹅星状病毒的亚单位疫苗，其特征在于，所述免疫佐剂为油佐剂、铝佐剂、蜂胶佐剂、油水双相佐剂中的一种。
7.如权利要求4所述的方法制得的衣壳蛋白和刺突蛋白在制备基于预防鹅星状病毒感染的卵黄抗体中的应用。
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