CN111440815A - 一种新型鸭呼肠孤病毒的复合疫苗及卵黄抗体制备方法 - Google Patents
一种新型鸭呼肠孤病毒的复合疫苗及卵黄抗体制备方法 Download PDFInfo
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Abstract
本发明的目的是提供一种新型鸭呼肠孤病毒的复合疫苗及卵黄抗体制备方法，即将含新型鸭呼肠孤病毒σB蛋白基因的重组PVAX1‑σB质粒与新型鸭呼肠孤病毒σC蛋白按一定比例混合后与白油佐剂乳化制备复合疫苗，三免后7～150日所采集鸡蛋的平均抗体效价达1:1024以上，最高抗体效价可达1:4096。高免蛋提取纯化制备的卵黄抗体产品可对感染新型鸭呼肠孤病毒的鸭群提供完全保护。本发明方法制备的新型鸭呼肠孤病毒卵黄抗体效果确实、成本低廉，具有显著的经济与社会效益。
Description
技术领域
本发明属于生物技术领域，具体涉及一种新型鸭呼肠孤病毒的复合疫苗及卵黄抗体制备方法。
背景技术
新型鸭呼肠孤病毒(Novel duck reovirus，NDRV)，为呼肠孤病毒科正呼肠孤病毒属成员，主要引起雏鸭肝脾出血和坏死，临床上称之为“鸭新肝病”、“鸭白点病”、“鸭出血性坏死性肝炎”、“鸭脾坏死病”等。新型鸭呼肠孤病毒在血清学上完全不同于之前的番鸭呼肠孤病毒，未见明显交叉保护性。2005年前后该病先在我国南方一些省份，如福建省、广东省、浙江省等鸭群中开始出现，后蔓延至全国主要养鸭区，给我国养鸭业造成了巨大经济损失。
新型鸭呼肠孤病毒含双层衣壳，其中σC蛋白位于病毒外衣壳，由S1基因节段编码。在病毒感染过程中，σC蛋白活化后形成同源三聚体与受体分子相互作用，介导病毒侵入易感细胞。此外，σC蛋白还能诱导机体产生保护性中和抗体，可作为基因工程疫苗的开发的候选蛋白。张云等(番鸭呼肠孤病毒MW9710σC蛋白基因克隆及其在E.coli中表达，畜牧兽医学报，2005，36(4)，376-380)、王锦祥等(新型鸭呼肠孤病毒σC基因原核表达及其抗原性的初步研究，福建农业学报，2014,29(4)：310-313)在原核表达系统实现了σC蛋白的表达，Kuntz等(Baculovirus-expressed muscovy duck reovirus「C protein induces serumneutralizing antibodies and protection against challenge,2002,Vaccine 20:3113–3122)、毕庄莉等(Induction of a robust immunity response against novelduck reovirus in ducklings using a subunit vaccine of sigma C protein,Scientific Reports,2016,6:1-11)则在杆状病毒表达系统实现了σC蛋白的基因工程表达，并证明可诱导动物机体产生一定的保护力。σB蛋白是呼肠孤病毒S3基因编码的病毒外衣壳蛋白，是群特异性抗原，同时含有病毒的多个中和表位。Lin等(Baculovirus SurfaceDisplay of sigmaC and sigmaB Proteins of Avian Reovirus and Immunogenicity ofthe Displayed Proteins in a Mouse Model,2008,Vaccine,26(50):6361-7)用杆状病毒表达系统分别表达了禽呼肠孤病毒σB蛋白与σC蛋白并在小鼠模型中进行试验，结果表明小鼠产生了血清中和抗体。吴晓平等(原核表达番鸭呼肠孤病毒σC与σB蛋白对雏番鸭的免疫保护，2013，中国农业科学，2013，46(14)：3040-3045年)则用原核表达系统表达了番鸭呼肠孤病毒的σB蛋白与σC蛋白并乳化后制备疫苗免疫番鸭，结果σB蛋白与σC蛋白联合制备的疫苗的攻毒保护率可达70％，而单个蛋白的保护率仅为40～60％。该方法的每个重组蛋白用量达到250μg/只，生产成本较高，且未进一步进行卵黄抗体产品研究。
唐熠等(专利申请号：CN201810494056.5、CN201810911067.9)、张立武等(专利申请号：CN201810720764.6)进行了鸭呼肠孤病毒卵黄抗体产品的研究，这些方法均采用鸡胚或鸭培养的方式制备免疫蛋鸡用的抗原，病毒生产效价低，易引入其他胚源病原，同时抗原免疫蛋鸡后制备的高免鸡蛋效价较低，相对来说生产成本也较高。
发明内容
本发明的目的是提供一种新型鸭呼肠孤病毒的复合疫苗及卵黄抗体制备方法。该制备方法优选的新型鸭呼肠孤病毒复合疫苗免疫蛋鸡后可显著提高蛋黄中新型鸭呼肠孤病毒抗体效价，提取纯化制备的卵黄抗体性质稳定、纯度高，可大大降低新型鸭呼肠孤病毒卵黄抗体的生产成本，对新型鸭呼肠孤病毒病的预防及治疗极具应用价值。
首先根据GenBank中新型鸭呼肠孤病毒σB蛋白的氨基酸序列，设计一种新型鸭呼肠孤病毒σB蛋白，氨基酸序列为SEQ ID NO:1。进一步根据密码子使用偏好性，设计获得新型鸭呼肠孤病毒σB蛋白基因，其中一种核苷酸序列为SEQ ID NO:2。
制备一种重组PVAX1-σB质粒，其是由上述获得的新型鸭呼肠孤病毒σB蛋白基因与真核表达载体PVAX1通过酶切连接而成。该重组PVAX1-σB质粒转化大肠杆菌DH5α后经发酵、提取纯化等步骤进行大量制备。
根据GenBank中新型鸭呼肠孤病毒σC蛋白的氨基酸序列，设计一种新型鸭呼肠孤病毒σC蛋白，氨基酸序列为SEQ ID NO:3。将新型鸭呼肠孤病毒σC蛋白进行大肠杆菌稀有密码子优化，获得的一种新型鸭呼肠孤病毒σC蛋白核苷酸序列SEQ ID NO:4。
将新型鸭呼肠孤病毒σC蛋白基因与表达载体pET28a连接并转化大肠杆菌表达菌株BL21(DE3)后进行高密度发酵、菌体破碎、蛋白纯化，制备一种重组新型鸭呼肠孤病毒σC蛋白。
将重组PVAX1-σB质粒与重组新型鸭呼肠孤病毒σC蛋白混合制备水相，将水相与白油佐剂按1:2比例乳化制备成新型鸭呼肠孤病毒复合疫苗。
优选的水相中重组质粒终浓度是10～100μg/ml，重组新型鸭呼肠孤病毒σC蛋白终浓度为0.2～0.8mg/ml。
更优选的水相中重组PVAX1-σB质粒终浓度是45μg/ml，重组新型鸭呼肠孤病毒σC蛋白终浓度为0.5mg/ml。
将该复合疫苗三次免疫蛋鸡，三免后7日所产鸡蛋的抗体效价即达到1:1024，免疫期中鸡蛋抗体效价最高可达1:4096，三免后5个月依然可达1:1024，符合高免蛋要求(不低于1:1024)。
将高免蛋经过蛋黄分离、灭活、萃取、过滤、分装等生产工艺，制备卵黄抗体成品，终产品抗体效价达1:1024。
本发明由新型鸭呼肠孤病毒σB蛋白基因与真核表达载体PVAX1通过酶切连接得到一种重组PVAX1-σB质粒。该重组PVAX1-σB质粒转化大肠杆菌DH5α后经发酵、提取纯化等方法可进行大量制备。采用高密度发酵、菌体破碎、蛋白纯化制备重组新型鸭呼肠孤病毒σC蛋白生产菌株。将重组PVAX1-σB质粒与重组新型鸭呼肠孤病毒σC蛋白混合制备水相。将水相与白油佐剂乳化制备成新型鸭呼肠孤病毒复合疫苗。制备的新型鸭呼肠孤病毒复合疫苗免疫蛋鸡后，三免后7日即可达到收蛋要求(1:1024)，最高可达1:4096，且三免后5个月进行检测，卵黄抗体效价仍符合要求。
新型鸭呼肠孤病毒卵黄抗体预防与治疗组的鸭群对强毒攻毒达到100％保护，生理盐水对照组的鸭群则80％发病，证明本发明制备的卵黄抗体对感染新型鸭呼肠孤病毒的鸭群具有良好的临床保护效果，可进行临床推广和应用。
与常规制备方法相比，本发明的新型鸭呼肠孤病毒卵黄抗体制备方法，在卵黄抗体产生时间、抗体产生高度及抗体持续期方面，新型鸭呼肠孤病毒复合疫苗组均远优于仅含σB基因DNA疫苗组、单σC蛋白亚单位疫苗组、新型鸭呼肠孤全病毒疫苗组。本发明的实施一方面可以降低卵黄抗体的制备成本，另一方面也可减少蛋鸡的免疫应激，极具推广与应用价值。
具体实施方式
下面结合具体实施方式来进一步描述本发明，但本领域技术人员应该理解的是，在不偏离本发明的技术方案的情况下可以对本发明的技术方案的细节和形式进行修改或替换，这些修改和替换均落入本发明保护范围内。
实施例1
重组PVAX1-σB质粒的构建与制备
1重组PVAX1-σB质粒的载体构建
1.1将GenBank中新型鸭呼肠孤病毒σB蛋白的氨基酸序列进行同源比对，并导入在线MOSAIC疫苗设计软件筛选更多潜在9-mer表位覆盖的σB蛋白，最终获得一种新型鸭呼肠孤病毒σB蛋白，氨基酸序列为SEQ ID NO:1。
1.2根据密码子使用偏好性，设计获得新型鸭呼肠孤病毒σB蛋白基因，其中一种核苷酸序列为SEQ ID NO:2。
1.3将获得的新型鸭呼肠孤病毒σB基因两端分别加上Kpn I和Xho I酶切位点后进行全基因合成。
1.4合成的新型鸭呼肠孤病毒σB蛋白基因用Kpn I和Xho I双酶切后连入PVAX1载体相应的酶切位点，CaCl2法转化大肠杆菌DH5α，提取质粒进行Kpn I和Xho I双酶切鉴定，正确后送上海生工测序，测序结果表明新型鸭呼肠孤病毒σB蛋白基因连入PVAX1载体相应酶切位点，成功构建重组组PVAX1-σB质粒。
2重组PVAX1-σB质粒的体外表达与鉴定
2.1选取生长良好的PK-15细胞接种6孔细胞培养板，37℃、5％二氧化碳培养箱培养至细胞85％～90％汇合度时，进行重组PVAX1-ORF C1质粒转染。
2.2细胞转染操作按LipofectamineTM2000试剂盒说明书进行，步骤如下：
2.2.1取4μg重组PVAX1-σB质粒DNA，用无血清细胞培养基稀释至250μl，轻轻混匀。
2.2.2取10μl的Lipo2000，用无血清细胞培养基稀释至250μl，轻轻混匀，室温静置5min。
2.2.3将DNA悬液和Lipo2000悬液混合后轻轻混匀，室温静置20min。
2.2.4将PK-15细胞已长好的6孔板中培养液弃掉，无血清细胞培养液洗2次，吸弃培养液，加入上述混合液，轻轻混匀后，补加1ml无血清细胞培养基，置于37℃、5％CO2培养箱中培养。
2.2.5转染6h后，弃掉6孔板细胞液，每孔加入2ml含10％新生牛血清的DMEM培养基。
2.2.6设转染pVAX1空载体的PK-15细胞做为阴性对照。
2.3转染48小时后，用pH7.2的PBS液洗涤接毒后细胞板1遍，注意动作轻缓，防止细胞脱落，预冷的甲醇4℃固定细胞15～20min，PBS洗3遍，加入鸡抗新型鸭呼肠孤病毒阳性血清100μl，37℃孵育1h，PBS洗3遍，加入100倍稀释的FITC标记羊抗鸡二抗100μl，37℃避光孵育45min，PBS洗3遍，荧光显微镜观察结果。
结果重组PVAX1-σB质粒转染PK-15细胞后可观察到明显的荧光信号，而对照组无明显荧光信号，证明构建的重组PVAX1-σB质粒可正确表达新型鸭呼肠孤病毒σB蛋白。
3重组PVAX1-σB质粒的大量制备
3.1重组菌的高密度发酵与菌体裂解 含重组PVAX1-σB质粒DNA的大肠杆菌DH5α经50L发酵罐高密度发酵培养后，5000r/min离心10min收集菌体，按每克湿菌体5ml的比例加入Solution I，再按1:2:1.5的比例分别加入Solution II和Solution III，室温孵育15min后，10000r/min室温离心10min，收集上清，加入0.7倍体积异丙醇，-20℃沉淀30min，10000r/min室温离心10min，弃上清，沉淀溶解于10mmol/L TE缓冲液。
3.2内毒素的去除 将纯化的质粒DNA溶液加入10％的TritonX-114，使其终浓度为1％，混匀后冰浴10min，然后42℃继续孵育10min，10000r/min室温离心10min，小心吸取上清液至无热原容器中，必要时重复操作1次。
3.3定量分装 用10mmol/L TE缓冲液稀释至500μg/ml，定量分装，即为重组PVAX1-σB质粒。
3.4重组PVAX1-σB质粒的检测
3.4.1重组PVAX1-σB质粒的浓度测定 超微量核酸分析仪检测质粒浓度，应不低于500μg/ml。
3.4.2酶切鉴定 将纯化的重组PVAX1-σB质粒用Kpn I和Xho I双酶切后进行琼脂糖凝胶电泳分析。结果应看到约3000bp与1104bp大小的两条带。
3.4.3宿主蛋白检测 按照BCA蛋白检测试剂盒说明书，以已知浓度的BSA制备标准曲线。用无菌水将纯化的重组PVAX1-σB质粒进行梯度稀释，在同样条件下对纯化质粒DNA中的菌体蛋白进行定量检测，重组质粒的菌体蛋白含量应低于10μg/mg。
3.4.4内毒素检测 按鲎试剂方法进行内毒素检测，内毒素含量应低于1000EU/mg。
实施例2
重组新型鸭呼肠孤病毒σC蛋白的制备
1生产菌株
1.1将GenBank中的新型鸭呼肠孤病毒σC蛋白的氨基酸序列进行同源比对，并导入在线MOSAIC疫苗设计软件筛选更多潜在9-mer表位覆盖的σC蛋白，最终获得一种新型鸭呼肠孤病毒σC蛋白，氨基酸序列为SEQ ID NO:3。
1.2利用在线生物软件DNAWorks，将新型鸭呼肠孤病毒σC蛋白进行大肠杆菌稀有密码子优化，获得的一种核苷酸序列SEQ ID NO:4。
1.3将获得的新型鸭呼肠孤病毒σC蛋白核苷酸序列两端分别加上Nde I和HindIII酶切位点后进行全基因合成。
1.4全基因合成的新型鸭呼肠孤病毒σC蛋白基因用Nde I和Hind III双酶切后连入pET28a载体相应的酶切位点，构建表达载体。
1.5用CaCl2法将表达载体转化入大肠杆菌BL21(DE3)，涂布于含50μg/ml卡那霉素的琼脂平板，37℃过夜培养。选取10个单菌落提取质粒，Nde I和Hind III双酶切验证阳性的菌落进一步测序鉴定。将测序验证后的阳性克隆在LB培养基中发酵培养至0.6～0.8时加入0.2～0.5mM IPTG诱导4～9小时，离心收集菌体后超声破碎，离心取上清，SDS-PAGE电泳检测蛋白表达，同时设立未诱导菌体作为对照。结果诱导后阳性克隆比对照菌在35kD处多出一条蛋白条带，与重组蛋白理论分子量一致，表达量约为20％以上。
1.6表达菌株破碎上清跑电泳，用新型鸭呼肠孤病毒阳性血清做Western-blot鉴定，结果显示阳性反应。
以上结果证明获得的阳性克隆为新型鸭呼肠孤病毒σC蛋白工程菌，命名为NA株。
2重组新型鸭呼肠孤病毒σC蛋白的制备与检验
2.1生产用种子制备
选取典型菌落10个混合于少量LB培养液中，接种于含卡那霉素的LB培养液中，37℃培养8～10小时，作为种子液。
2.2发酵培养基为改良LB培养基，每1000ml培养基中含胰蛋白胨10g，酵母浸粉5g，氯化钠10g，葡萄糖5g，MgSO4·7H2O 5g。
2.3重组新型鸭呼肠孤病毒σC蛋白制备
2.3.1菌液培养 用培养罐通气培养，按培养罐容积装入适量培养基(70％左右)及消泡剂，灭菌后按培养基量的2％接种种子菌液，37℃通气培养，待菌液的OD600值达到7.0时，加入2～10g/L的α-乳糖诱导，再继续培养6～8小时。通过转速关联控制溶氧。当溶氧迅速上升时，流加补料。
2.3.2破菌 培养结束后，离心收集菌体。收集的菌体清洗后用高压匀浆机破碎细菌。破碎后的菌液，8000r/min，离心15分钟，收集上清液。
2.3.3镍柱层析纯化 常规镍柱纯化，收集的重组蛋白洗脱液放入透析袋，PBS液作为透析外液，透析脱盐后即得重组蛋白液。
2.3.4灭活 将纯化的上清液中按比例加入10％的甲醛溶液，甲醛溶液的最终浓度为0.2％，37℃灭活12小时。
2.3.5定量分装 用无菌生理盐水将蛋白液稀释至终浓度2.0mg/ml，无菌过滤，待用。
2.4检验
2.4.1蛋白含量检测 用BCA法检测上清液蛋白浓度，应不低于2.0mg/ml。
2.4.2无菌检验 按现行《中国兽药典》进行检验，应无菌生长。
2.4.3内毒素含量检测 按鲎试剂方法进行内毒素检测，内毒素含量应不高于1000EU/ml。
2.4.4甲醛、汞类防腐剂残留量测定 按现行《中国兽药典》进行，均应符合规定。
实施例3
新型鸭呼肠孤病毒复合疫苗的制备与免疫
1新型鸭呼肠孤病毒复合疫苗的制备
1.1半成品制备 将实施例1的3.3中制备的重组PVAX1-σB质粒DNA与实施例2的2.3.5中制备的重组新型鸭呼肠孤病毒σC蛋白适当稀释，使质粒DNA终含量为45μg/ml，σC蛋白终含量为0.5mg/ml，充分混匀，即为半成品。
1.2疫苗制备
1.2.1油相制备 取优质注射用白油94份，硬脂酸铝2份。在油相罐中混合均匀，加热融化至半透明状，再加司本-80 6份，至温度达到125～130℃时维持30分钟，冷却至室温备用。
1.2.2水相制备 取灭菌的吐温-80 4份，加检验合格的半成品96份，充分搅拌至吐温-80完全溶解。
1.2.3乳化取油相2份置于高速剪切机内，开动电机低速搅拌，同时缓缓加入水相1份，再以3600r/min乳化40分钟，在终止搅拌前加入1％硫柳汞溶液，终浓度达到0.01％。乳化后，取样10ml加入离心管，以3000r/min离心15分钟，管底应无水相析出。
1.2.4分装 定量分装，密封瓶口。
2新型鸭呼肠孤病毒复合疫苗的检验
2.1物理性状
外观 为乳白色乳剂。
剂型 为油包水型。取一清洁吸管，吸取少量疫苗滴于冷水中，除第1滴外，均应不扩散。
稳定性 吸取疫苗10ml加入离心管中，经3000r/min离心15分钟，管底应无水相析出。
黏度 按现行《中国兽药典》进行，符合规定。
装量检查 按现行《中国兽药典》进行，符合规定。
2.2无菌检验 按现行《中国兽药典》进行，无菌生长。
2.3甲醛、汞类防腐剂残留量测定 按现行《中国兽药典》进行，均符合规定。
3新型鸭呼肠孤病毒复合疫苗对蛋鸡免疫程序研究
选取180日龄蛋鸡100只，平均分为5组，每组20只。DNA疫苗组免疫实施例1制备的重组PVAX1-σB质粒，用10mmol/L TE缓冲液稀释至10μg/ml；亚单位疫苗组免疫重组新型鸭呼肠孤病毒亚单位疫苗，由实施例2制备的重组新型鸭呼肠孤病毒σC蛋白与白油佐剂按1:2比例乳化制成，其中水相中Cap蛋白的终浓度为0.5mg/ml；复合疫苗组免疫实施例3制备的新型鸭呼肠孤病毒复合疫苗；全病毒疫苗组免疫新型鸭呼肠孤病毒NC株灭活后与白油佐剂按1:2比例乳化制成的灭活疫苗，其中灭活前新型鸭呼肠孤病毒NC株的毒价不低于105.0ELD50/0.1ml；不免疫对照组注射同等体积的生理盐水。各组蛋鸡分别进行相应疫苗免疫，皮下注射，0.5ml/只，免疫间隔14日，共三次免疫。分别于免疫前，三免前(二免后14日)，三免后7、14、21、28日收集鸡蛋测定卵黄琼扩抗体效价，之后每30日收集高免蛋鸡蛋测定卵黄琼扩抗体效价，确定蛋鸡的卵黄抗体产生期和持续期。
结果：制备的新型鸭呼肠病毒复合疫苗免疫蛋鸡后，三免后7日抗体效价可达1:1024，达到收蛋要求(不低于1:1024)(见表1)，最高可达1:4096，且三免后5个月进行检测，鸡蛋的卵黄抗体效价仍符合要求(见表2)。结果表明不论是卵黄抗体产生时间、抗体产生高度还是抗体持续期，复合疫苗组均远优于DNA疫苗组、亚单位疫苗组与全病毒疫苗组，显示了良好的应用前景。
表1各疫苗组免疫后蛋鸡的卵黄抗体产生期
表2各疫苗组免疫后蛋鸡的卵黄抗体持续期
实施例4
新型鸭呼肠孤病毒卵黄抗体的制备与效力实验
1新型鸭呼肠孤病毒卵黄抗体的制备
按实施例3中3部分的方法将新型鸭呼肠孤病毒复合疫苗免疫蛋鸡制备高免蛋，收集合格的高免蛋(抗体效价不低于1:64)，蛋壳消毒后进行卵黄分离、萃取、灭活、过滤除菌、稀释分装等方法，终产品抗体效价应不低于1:16。
2新型鸭呼肠孤病毒卵黄抗体的安全性实验
将7日龄健康易感鸭20只平均分为两组，每组10只。第一组腿部肌肉注射本发明制备的卵黄抗体，1.0ml/只，第二组腿部肌肉注射同体积生理盐水。免疫后21日观察鸭群，应全部健活，无任何不良反应。
3新型鸭呼肠孤病毒卵黄抗体的效力试验
将2日龄健康易感雏鸭60只平均分为三组，每组20只。第一组为卵黄抗体治疗组，肌肉注射新型鸭呼肠孤强毒NC株0.5ml，24h后肌肉注射本发明制备的新型鸭呼肠病毒卵黄抗体，0.5ml/只。第二组为卵黄抗体与预防组，肌肉注射本发明制备的新型鸭呼肠病毒卵黄抗体，0.5ml/只，24h后肌肉注射新型鸭呼肠孤强毒NC株0.5mll。第三组为生理盐水对照组，肌肉注射新型鸭呼肠孤强毒NC株0.5ml，24h后肌肉注射无菌生理盐水，0.5ml/只。攻毒后连续观察7日，7日后剖检观察脾脏情况，统计结果。发病标准：攻毒后7日剖检，攻毒组鸭脾脏表面有出血斑或坏死灶、肿胀等特异性病变。
结果：卵黄抗体预防组与卵黄抗体治疗组的鸭群均获得100％保护，生理盐水对照组的鸭群则80％发病。证明本发明制备的新型鸭呼肠孤病毒卵黄抗体对感染新型鸭呼肠孤的鸭群具有良好的临床保护效果，可用于新型鸭呼肠孤病毒病的预防与治疗。
表3新型鸭呼肠孤病毒卵黄抗体的效力试验
序列表
<110> 潍坊华英生物科技有限公司
<120> 一种新型鸭呼肠孤病毒的复合疫苗及卵黄抗体制备方法
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<211> 321
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Met Asp Arg Asn Glu Val Ile Arg Leu Ile Leu Ser Leu Leu Pro Tyr
1 5 10 15
Gln Ser Ser Asp Val Asp His Leu Lys Thr Gln Ile Lys Ser Leu Gln
20 25 30
Ser Ala Val Asp Ser Leu Lys Glu Ser Gln Val Val Val Leu Arg Arg
35 40 45
Leu Thr Thr Ile Thr Ser Thr Leu Ala Asp Leu Gln Ser Thr Thr Glu
50 55 60
Leu Leu Thr Ser Gln Val Ala Gly Leu Ser Ser Arg Val Ala Ser Val
65 70 75 80
Thr Asp Glu Val Val Arg Val Asn Ser Val Ile Gly Ser Thr Ile Thr
85 90 95
Asn Leu Asp Asn Val Arg Ser Glu Pro Ser Ser Leu Ser Ser Gln Val
100 105 110
Ser Ser Gln Thr Ser Thr Leu Thr Asn Leu Thr Ser Thr Val Ser Ser
115 120 125
Gln Ser Leu Ser Ile Ser Asp Leu Gln Gln Arg Val Thr Ala Leu Glu
130 135 140
Arg Ser Gly Gly Ala Pro Thr Arg Phe Glu Ala Pro Leu Arg Leu Gln
145 150 155 160
Asn Gly Val Val Ser Leu Gln Ala Ser Pro Ser Phe Cys Ser Leu Ser
165 170 175
Pro Ile Leu Ser Gly Pro Ser Asp Ala Ala Val Phe Lys Val Gly Glu
180 185 190
Trp Leu Gly Thr Val Ile Ser Gly Gln Ser Gln Ser Ser Ala Ile Met
195 200 205
Asn Val Arg Ile His Ser Phe Gly Gln Arg Thr Met Leu Leu Met Ser
210 215 220
Ser Gln Asn Val Phe Thr Ile Pro Pro Gly Ser Gly Ala Ser Leu Gln
225 230 235 240
Leu Asp Val Asn Arg Ile Thr Thr Pro Ala Ile Asp Val Ala Met Val
245 250 255
Thr Pro Ser Ala Ala Phe Ala Ser Ala Ser Phe Met Ala Asp Ile Ala
260 265 270
Phe Lys Asp Ser Lys Thr Gly Glu Val His Ala Leu His Thr Thr Gly
275 280 285
Ser Phe Arg Ser Pro Ser Phe Ser Ile Ala Trp Val Pro Val Ala Ser
290 295 300
Glu Thr Arg Asn Tyr Gln Ile Met Ala Leu Arg Phe Thr Val Ala Thr
305 310 315 320
Gly
<210> 4
<211> 963
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
atggaccgta acgaagttat ccgtctgatc ctgtctctgc tgccgtacca gtcttctgac 60
gttgaccacc tgaaaaccca gatcaaatct ctgcagtctg ctgttgactc tctgaaagaa 120
tctcaggttg ttgttctgcg tcgtctgacc accatcacct ctaccctggc tgacctgcag 180
tctaccaccg aactgctgac ctctcaggtt gctggtctgt cttctcgtgt tgcttctgtt 240
accgacgaag ttgttcgtgt taactctgtt atcggttcta ccatcaccaa cctggacaac 300
gttcgttctg aaccgtcttc tctgtcttct caggtttctt ctcagacctc taccctgacc 360
aacctgacct ctaccgtttc ttctcagtct ctgtctatct ctgacctgca gcagcgtgtt 420
accgctctgg aacgttctgg tggtgctccg acccgtttcg aagctccgct gcgtctgcag 480
aacggtgttg tttctctgca ggcttctccg tctttctgct ctctgtctcc gatcctgtct 540
ggtccgtctg acgctgctgt tttcaaagtt ggtgaatggc tgggtaccgt tatctctggt 600
cagtctcagt cttctgctat catgaacgtt cgtatccact ctttcggtca gcgtaccatg 660
ctgctgatgt cttctcagaa cgttttcacc atcccgccgg gttctggtgc ttctctgcag 720
ctggacgtta accgtatcac caccccggct atcgacgttg ctatggttac cccgtctgct 780
gctttcgctt ctgcttcttt catggctgac atcgctttca aagactctaa aaccggtgaa 840
gttcacgctc tgcacaccac cggttctttc cgttctccgt ctttctctat cgcttgggtt 900
ccggttgctt ctgaaacccg taactaccag atcatggctc tgcgtttcac cgttgctacc 960
ggt 963
Claims (8)
1.一种重组PVAX1-σB质粒，由新型鸭呼肠孤病毒σB蛋白基因与真核表达载体PVAX1通过酶切连接而成，所述的新型鸭呼肠孤病毒σB蛋白的编码蛋白氨基酸序列为SEQ ID NO:1，所述的新型鸭呼肠孤病毒σB蛋白基因的一种核苷酸序列为SEQ ID NO:2。
2.一种权利要求1所述的重组PVAX1-σB质粒的大量制备方法，将重组PVAX1-σB质粒转化大肠杆菌DH5α后进行大肠杆菌高密度发酵、菌体裂解、质粒DNA提取纯化制备而成。
3.一种新型鸭呼肠孤病毒复合疫苗，复合疫苗由水相与佐剂制备而成，水相中抗原由权利要求1所述的重组PVAX1-σB质粒与重组新型鸭呼肠孤病毒σC蛋白混合而成，所述的新型鸭呼肠孤病毒σC蛋白的编码蛋白氨基酸序列为SEQ ID NO:3，所述的新型鸭呼肠孤病毒σC蛋白基因的一种核苷酸序列为SEQ ID NO:4。
4.根据权利要求3所述的复合疫苗，其特征在于，所述的水相中重组PVAX1-σB质粒终浓度是10～100μg/ml，重组新型鸭呼肠孤病毒σC蛋白终浓度为0.2～0.8mg/ml。
5.根据权利要求4所述的复合疫苗，其特征在于，所述的水相中重组PVAX1-σB质粒终浓度是45μg/ml，重组新型鸭呼肠孤病毒σC蛋白终浓度为0.5mg/ml。
6.根据权利要求3所述的复合疫苗，其特征在于，所述的佐剂为白油。
7.根据权利要求3所述的复合疫苗，其特征在于，所述的水相与佐剂经过乳化制备而成。
8.一种新型鸭呼肠孤病毒的卵黄抗体制备方法，用权利要求3-7所述的新型鸭呼肠孤病毒复合疫苗对产蛋鸡进行免疫，然后从高免鸡蛋的蛋黄中提取纯化得到。
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| CN112980802A (zh) * | 2021-03-25 | 2021-06-18 | 浙江省农业科学院 | 一种分泌鸭新型呼肠孤病毒σB蛋白单抗的杂交瘤细胞、单抗及应用 |
| CN113832113A (zh) * | 2021-09-14 | 2021-12-24 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | 新型鸭呼肠孤病毒弱毒株及其应用 |
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| CN108676092A (zh) * | 2018-05-22 | 2018-10-19 | 山东农业大学 | 一种防治新型鸭呼肠孤病毒的卵黄抗体及其制备方法 |
| CN108794627A (zh) * | 2018-07-03 | 2018-11-13 | 重庆永健生物技术有限责任公司 | 一种鸭呼肠孤病毒精制卵黄抗体的制备方法 |
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| CN108676092A (zh) * | 2018-05-22 | 2018-10-19 | 山东农业大学 | 一种防治新型鸭呼肠孤病毒的卵黄抗体及其制备方法 |
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| CN112980802A (zh) * | 2021-03-25 | 2021-06-18 | 浙江省农业科学院 | 一种分泌鸭新型呼肠孤病毒σB蛋白单抗的杂交瘤细胞、单抗及应用 |
| CN112980802B (zh) * | 2021-03-25 | 2022-03-22 | 浙江省农业科学院 | 一种分泌鸭新型呼肠孤病毒σB蛋白单抗的杂交瘤细胞、单抗及应用 |
| CN113832113A (zh) * | 2021-09-14 | 2021-12-24 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | 新型鸭呼肠孤病毒弱毒株及其应用 |
| CN113832113B (zh) * | 2021-09-14 | 2023-09-22 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | 新型鸭呼肠孤病毒弱毒株及其应用 |
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