CN116350764A - 一种鸭细小病毒vp2蛋白重组杆状病毒载体灭活疫苗及其制备方法 - Google Patents
一种鸭细小病毒vp2蛋白重组杆状病毒载体灭活疫苗及其制备方法 Download PDFInfo
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Abstract
本发明根据鸭细小病毒VP2基因核苷酸序列，利用昆虫细胞‑杆状病毒表达系统进行鸭细小病毒VP2蛋白的高效表达，经灭活、纯化后配以适当佐剂制备灭活疫苗，并使用琼扩试验进行疫苗免疫后鸭体内抗体评价。安全、效力试验结果显示：使用本发明所述的灭活疫苗免疫鸭后无不良反应，且均能产生良好的保护力。通过琼扩试验可快速测定鸭细小病毒抗体效价。结果说明利用本发明制备出的鸭细小病毒VP2蛋白重组杆状病毒载体灭活疫苗能够有效预防鸭细小病的发生，可为市场提供安全、高效的新型疫苗与更方便的检测方法，以满足市场对鸭细小疫苗的需求与应用。
Description
技术领域
本发明涉及一种鸭细小病毒VP2蛋白重组杆状病毒载体灭活疫苗及其制备方法，属于兽用生物制品技术领域。
背景技术
鸭细小病毒病又称鸭短喙-侏儒综合征、“鸭大舌病”，由鸭细小病毒(Muscovyduck parvovirus,DPV)引起。DPV的基因组为单链线状DNA，大小约5000nt，主要编码非结构蛋白(NS)和结构蛋白(VP)。结构蛋白包括VP1、VP2、VP3，由同一段基因通过可变剪接形成不同的翻译模板所产生，三种蛋白的大小分别为82kDa、65kDa、58kaDa，虽然起始位点不同，但在同一位点终止。其中VP1包含VP2和VP3的全部氨基酸序列，而VP2又包含VP3蛋白的全部氨基酸序列。VP2和VP3是病毒最重要的免疫性蛋白成分。
该病主要症状为鸭喙畸形、萎缩变短，舌头肿大伸出并垂在喙外，两腿无力、不愿行走，生长发育障碍，翅腿易折、羽毛凌乱。发病率10％～30％，部分鸭群发病率可达50％，但死亡率相对较低。病毒感染雏鸭可造成大量残次鸭，成年鸭虽不表现临床症状，但可将病毒垂直传播给下一代，给水禽养殖业造成极大的安全隐患。
防控该病的最有效途径是疫苗免疫，但目前市场上的商品化疫苗较少，研制安全、高效的疫苗迫在眉睫。
杆状病毒表达系统(Baculovirus expression system)又称昆虫细胞表达系统，是以杆状病毒(主要是昆虫核型多角体病毒)作为外源基因载体，昆虫细胞或幼虫作为受体的真核表达系统。是一种安全、高效的新型疫苗载体，广泛用于各种重组蛋白的表达。该表达系统具有如下诸多优点：表达的蛋白具有完整的生物学功能，如蛋白的正确折叠、二硫键的搭配；蛋白翻译后的加工修饰；表达水平高，可达总蛋白量的30％；可容纳大分子的插入片段；能同时表达多个基因。
发明内容
本发明的目的在于根据鸭细小病毒VP2基因核苷酸序列，利用昆虫细胞-杆状病毒表达系统进行鸭细小病毒VP2蛋白的高效表达，经灭活、纯化后配以适当佐剂制备灭活疫苗，并使用琼脂扩散沉淀试验(琼扩试验)进行疫苗免疫后鸭体内抗体评价，为市场提供安全、高效的新型疫苗与更方便的检测方法，以满足市场对鸭细小疫苗的需求与应用。
本发明的技术方案
1.本发明所述的一种鸭细小病毒VP2蛋白重组杆状病毒载体灭活疫苗，其特征在于，所述疫苗的主要成分为重组杆状病毒rBac-HBM-VP2株高效表达的鸭细小病毒VP2蛋白；
其所述的重组杆状病毒rBac-HBM-VP2株的构建步骤包括：
(1)真核表达载体的选择：选用真核表达载体pFastbac1进行鸭细小病毒VP2蛋白的表达；
(2)真核表达载体的构建：将合成的优化序列克隆至pUC19载体，获得质粒(pUC-VP2)后与真核表达载体pFastbac1同步进行BamHI/XhoI双酶切，连接后转化DH5α感受态细胞，获得真核表达载体pFastbac1-HBM-VP2质粒；
(3)重组杆粒的制备：将pFastbac1-HBM-VP2质粒转化DH10bac菌株，利用Tn7转座子优势，实现目的基因在宿主菌内的大量繁殖；用传统碱裂解法获得重组杆粒(Bac-HBM-VP2)；
(4)转染与纯化：将重组杆粒Bac-HBM-VP2转染sf9昆虫细胞，27℃培养，收获上清；将上清接种sf9昆虫细胞进行蚀斑纯化，获得重组杆状病毒rBac-HBM-VP2；该毒株已于2023年02月20日送交北京市朝阳区北辰西路1号院3号中国科学院微生物研究所中国微生物菌种保藏管理委员会普通微生物中心保藏，保藏号为CGMCC No.45418。
2.本发明所述一种鸭细小病毒VP2蛋白重组杆状病毒载体灭活疫苗，其特征在于该疫苗制备包括：使用细胞培养罐进行sf9昆虫细胞的培养；将重组杆状病毒rBac-HBM-VP2接种sf9昆虫细胞，收获细胞培养物并使用BEI进行灭活；使用层析柱进行层析纯化后用水性佐剂配制灭活疫苗。
3.本发明所述一种鸭细小病毒VP2蛋白重组杆状病毒载体灭活疫苗的应用，其特征在于，用本发明制备的鸭细小病毒VP2蛋白重组杆状病毒载体灭活疫苗免疫鸭，免疫后28日采血；使用琼扩试验对鸭的血清样品进行抗体测定。
本发明有益效果
本发明根据鸭细小病毒VP2基因核苷酸序列，利用昆虫细胞-杆状病毒表达系统进行鸭细小病毒VP2蛋白的高效表达，经灭活、纯化后配以适当佐剂制备灭活疫苗，并使用琼扩试验进行疫苗免疫后鸭体内抗体评价。安全、效力试验结果显示：使用本发明所述的灭活疫苗免疫鸭后无不良反应，且均能产生良好的保护力。通过琼扩试验可快速测定鸭细小病毒抗体效价。结果说明利用本发明制备出的鸭细小病毒VP2蛋白重组杆状病毒载体灭活疫苗能够有效预防鸭细小病的发生，可为市场提供安全、高效的新型疫苗与更方便的检测方法，以满足市场对鸭细小疫苗的需求与应用。
本发明涉及的生物材料资源信息
本发明所述疫苗制备毒株为鸭细小病毒VP2蛋白重组杆状病毒rBac-HBM-VP2株，该毒株已于2023年02月20日送交北京市朝阳区北辰西路1号院3号中国科学院微生物研究所中国微生物菌种保藏管理委员会普通微生物中心保藏，保藏号为CGMCC No.45418。
本发明具体实施方式
1.构建重组杆状病毒
(1)构建HBM-VP2：
1)合成引物HBM-F和HBM-R扩增蜂素信号肽(HBM)：
HBM-F 5’-cgcggatcca tgaaattctt agtcaacgtt gcccttgttt ttatggtcg-3’49(序列1)HBM-R 5’-tccgcataga tgtaagaaat gtatacgacc ataaaaacaa gggc-3’44(序列2)
2)采用引物VP2-F和VP2-R扩增VP2基因并通过HBM-R与VP2-F之间的互补碱基获得HBM-VP2：
VP2-F 5’-tacatctatg cggatatggc tcccgccaag aaga-3’34(序列3)
VP2-R 5’-ccgctcgagt tagtgatggt gatggtgatg gccttgaaag tacaagttttcttagtggtg gtggtggtgg tg-3’72(序列4)
(2)构建真核表达载体pFastbac1-HBM-VP2：将生物公司合成的质粒pUC-HBM-VP2与真核表达载体pFastbac1同步进行双酶切(BamHI/XhoI)，并对酶切产物进行凝胶电泳，分别使用凝胶回收纯化试剂盒纯化酶切的HBM-VP2基因片段和pFastbac 1载体。反应体系如下(表1、表2)。
表1VP2基因片段酶切反应体系
表2pFastbac 1载体酶切反应体系
将完成双酶切的HBM-VP2基因片段和pFastbac 1载体使用T4 DNA连接酶16℃水浴连接过夜。反应体系如下(表3)。
表3连接体系
将10μL连接成功的连接产物加入100μL的DH5α感受态细胞中混匀，42℃水浴热休克90s，再冰浴2min，加入900μL不含Amp的LB液体培养基，37℃培养1h。取1.0mL菌液浓缩成100μL涂布于含有Amp的LB固体培养基上，37℃培养16h。
挑取平板上的单菌落分别接种LB液体培养基，37℃培养2h，以菌液作为模板，VP2-F和VP2-R作为引物进行菌落PCR鉴定，将PCR产物进行凝胶电泳验证目的基因的大小。将鉴定正确的菌液送测序公司进行测序，选择测序正确的菌液保存，获得pFastbac1-HBM-VP2真核表达载体，即杆状病毒重组质粒。
(3)制备重组杆粒Bac-HBM-VP2：取1μL构建成功的杆状病毒重组质粒pFastbac1-HBM-VP2，加入到100μL的DH10Bac感受态细胞中，混匀，冰浴30min，42℃水浴热休克90s，再冰浴2min，加入900μL不含Amp的LB液体培养基，37℃培养5h。取100μL菌液稀释81倍后，取100μL稀释的菌液涂布到含有庆大霉素、卡那霉素、四环素、X-gal以及IPTG的LB固体培养基上，37℃培养48h后挑取大的白色菌落，然后在含有庆大霉素、卡那霉素、四环素、X-gal以及IPTG的LB固体培养基上划线，37℃培养48h，挑取单菌落接种含有庆大霉素、卡那霉素、四环素的LB液体培养基培养，保存菌种，抽提质粒，PCR鉴定后获得含有重组Bacmid的重组杆粒Bac-HBM-VP2。
(4)重组杆状病毒的转染与纯化：在6孔板中加入生长状态良好的Sf9细胞(约4.0×105个细胞/mL)，待细胞贴壁。取40μL Cellfectin Reagent转染试剂加入到Grace’s培养基中，终体积为1mL，混匀作为A液，取10μg重组Bacmid加入到Grace’s培养基中，终体积为1mL，混匀作为B液。将A液和B液混合均匀，室温静置15～30min。Sf9细胞贴壁后弃去培养基，补加无血清的Grace’s培养基，2.0mL/孔，然后每孔滴加DNA-脂质复合物，200μL/孔，27℃孵育5h，移除转染复合物，细胞培养基洗三次，添加新鲜培养基，2.0ml/孔，27℃培养，当出现细胞病变时，将上层培养基转移到无菌EP管中，收获重组杆状病毒病毒液，置-70℃以下保存。在直径为6cm的细胞培养皿中接种2.0×106～2.5×106个Sf9细胞，室温下放置5min。梯度稀释(稀释比例：10-4～10-7)上述共转染病毒上清，每个培养皿添加1.0mL不同稀释度的病毒液，室温放置1h，每15min适当震荡，使病毒充分感染。
无菌水配置2％琼脂糖，微波加热到60℃，充分融化。然后放置42℃恒温水浴，添加1体积的2×Grace’s培养基，混合均匀。弃细胞培养皿中培养液上清，在细胞表面覆盖4.0mL1％琼脂糖，同时吸走所有气泡，10～15min后琼脂糖凝固。将培养皿放置于湿润环境中，27℃培养7日，观察蚀斑。在培养皿上标记蚀斑，挑取蚀斑，接种SF-SFM培养基中，2～8℃放置过夜，收获病毒悬液，接种Sf9细胞，27℃培养96～120h，收获细胞培养物，获得重组杆状病毒rBac-HBM-VP2。
2.鸭细小病毒VP2蛋白的制备
(1)细胞接种及放大培养：选择生长旺盛的Sf9细胞，接种细胞培养罐，接种密度为1.5×106～2.0×106个/mL，27℃培养，待细胞密度高于3.0×106个/mL时，补加培养基，继续培养至细胞密度高于3.0×106个/mL时，将细胞培养罐内细胞悬液转移至新细胞培养罐培养，接种密度为1.5×106～2.0×106个/mL。
(2)接毒及收获：Sf9细胞密度达到1.5×106～2.0×106个/ml时按MOI＝0.5～2.0接种rBac-HBM-VP2，27℃培养72～96h，收获细胞培养物。
(3)灭活：向rBac-HBM-VP2细胞培养物中加入2％的BEI溶液，使其终浓度为0.2％，37℃灭活24h。灭活结束后，再向rBac-HBM-VP2细胞培养物中添加50％的硫代硫酸钠溶液，使其终浓度为0.2％，搅拌1h终止灭活。2～8℃保存备用。
(4)抗原纯化：将灭活后的rBac-HBM-VP2细胞培养物离心、过滤后使用层析柱进行层析纯化。
(5)疫苗制备：按水相:油相＝9:1的比例向灭活检验合格的rBac-HBM-VP2细胞培养物中加入国产水性佐剂，边加边搅拌，充分搅拌后制备出鸭细小病毒VP2蛋白重组杆状病毒载体灭活疫苗。
3、鸭细小病毒VP2蛋白重组杆状病毒载体灭活疫苗的应用
(1)疫苗免疫：将疫苗免疫鸭，免疫后28日采血。
(2)抗体测定：使用琼扩的方法对鸭的血清样品进行抗体测定。
实施例
以下实施例为进一步说明本发明，不对本发明构成限制。
实施例1
鸭细小病毒VP2蛋白重组杆状病毒载体灭活疫苗的制备
1.细胞罐接毒、收毒工艺
将sf9健康细胞接种于荷兰Applikon公司30L细胞培养罐，并最终逐级放大至2000L细胞培养罐。sf9细胞在2000L细胞培养罐培养至密度为2.0×106个/mL时，按感染复数MOI＝1.0接种rBac-HBM-VP2后置27℃培养96h进行收获，测定的病毒滴度为108.5～ 9.0TCID50/mL。当病毒滴度为108.5TCID50/mL时，VP2蛋白表达量为80～120mg/L。表明利用荷兰Applikon公司的细胞培养罐制备鸭细小病毒VP2蛋白重组杆状病毒载体灭活疫苗的半成品是可行的。
2.灭活
向rBac-HBM-VP2细胞培养物中加入2％的BEI溶液，使其终浓度为0.2％，37℃灭活24h，灭活结束后，向rBac-VP2细胞培养物中添加50％的硫代硫酸钠溶液，使其终浓度为0.2％，搅拌1h终止灭活。2～8℃保存备用。
3.疫苗制备
按水相:油相＝9:1的比例向灭活检验合格的rBac-HBM-VP2细胞培养物中加入国产水性佐剂，边加边搅拌，待水性佐剂添加完毕后，常温低速(400r/min)搅拌15min，配制成鸭细小病毒VP2蛋白重组杆状病毒载体灭活疫苗。
实施例2
疫苗的安全性试验
1.对雏鸭的安全性试验
选取2～3日龄健康易感雏鸭15只，随机分为3组，5只/组。第1组进行单剂量重复安全性试验(颈背侧皮下注射，0.2mL/只，二次接种)；第2组进行超剂量安全性试验(颈背侧皮下注射，0.4mL/只)；第3组不免疫作为空白对照。疫苗免疫后连续观察14日。除接种当日超剂量免疫组2/5的鸭出现轻微厌食外，其余的精神、饮食、粪便、注射部位等均正常，证明疫苗对雏鸭安全。
2.对产蛋鸭的安全性试验
选取健康易感产蛋鸭15只，随机分为3组，5只/组。第1组进行单剂量重复安全性试验(颈背侧皮下注射，0.2mL/只，二次接种)；第2组进行超剂量安全性试验(颈背侧皮下注射，0.4mL/只)；第3组不免疫作为空白对照。免疫后连续观察14日，观察不良反应发生情况，并分别于免疫后1～7日、8～14日、15～21日、22～28日共4个时间段收集鸭蛋，人工孵化。结果疫苗免疫后，10/10的鸭均未表现明显的不良反应。单剂量重复组和超剂量组于免疫后1～7日、8～14日、15～21日、22～28日的孵化率分别为95.2％、96.5％、96.3％、96.0％和94.7％、93.8％、96.2％、95.8％，与对照组相比，无明显差异，证明疫苗对产蛋鸭安全。
实施例3
疫苗的效力试验
选取1日龄健康易感雏鸭15只，随机分为3组，5只/组。第1组颈背侧皮下注射疫苗，0.2ml/只；第2、3组不免疫作为空白对照。免疫后7日，对第1、2组的鸭使用DPV-QL株攻击，每只鸭口服病毒液1.0mL(含107.5TCID50)。攻毒后3、5、7、14、21日对2组试验鸭采集肛门拭子进行PCR，检测排毒情况；攻毒后5、7、14、21日对2组试验鸭测量喙长；攻毒后7、14、21日对2组试验鸭测量体重。第1组(疫苗免疫组)与第2组(空白对照组)相比，第1组攻毒后无排毒现象，第2组自攻毒后5日开始排毒；第2组的喙长短于第1组；第1组体重增长显著高于第2组。具体结果如下(表4)。
表4效力试验数据
第1、3组的鸭于免疫后7日(攻毒前)采血，分离血清，使用琼扩的方法测定抗体效价。第1组(疫苗免疫组)抗体效价分别为1:8、1:8、1:16、1:16、1:16，第3组(空白对照组)均无抗体效价。
DPV-QL株攻击易感鸭后可产生临床症状，且排毒，说明攻毒模型成立；免疫疫苗7日后，使用琼扩的方法测得鸭的抗体效价高于1:8，能够后抵抗强毒株的攻击，说明疫苗免疫鸭后可使用琼扩的方法检测抗体水平，当抗体效价不低于1:8时可产生足够的保护力。
Claims (3)
1.本发明所述的一种鸭细小病毒VP2蛋白重组杆状病毒载体灭活疫苗，其特征在于，所述疫苗的主要成分为重组杆状病毒rBac-HBM-VP2株高效表达的鸭细小病毒VP2蛋白；
其所述的重组杆状病毒rBac-HBM-VP2株的构建步骤包括：
(1)真核表达载体的选择：选用真核表达载体pFastbac1进行鸭细小病毒VP2蛋白的表达；
(2)真核表达载体的构建：将合成的优化序列克隆至pUC19载体，获得质粒(pUC-VP2)后与真核表达载体pFastbac1同步进行BamHI/XhoI双酶切，连接后转化DH5α感受态细胞，获得真核表达载体pFastbac1-HBM-VP2质粒；
(3)重组杆粒的制备：将pFastbac1-HBM-VP2质粒转化DH10bac菌株，利用Tn7转座子优势，实现目的基因在宿主菌内的大量繁殖；用传统碱裂解法获得重组杆粒(Bac-HBM-VP2)；
(4)转染与纯化：将重组杆粒Bac-HBM-VP2转染sf9昆虫细胞，27℃培养，收获上清；将上清接种sf9昆虫细胞进行蚀斑纯化，获得重组杆状病毒rBac-HBM-VP2；该毒株已于2023年02月20日送交北京市朝阳区北辰西路1号院3号中国科学院微生物研究所中国微生物菌种保藏管理委员会普通微生物中心保藏，保藏号为CGMCCNo.45418。
2.本发明所述一种鸭细小病毒VP2蛋白重组杆状病毒载体灭活疫苗，其特征在于该疫苗制备包括：使用细胞培养罐进行sf9昆虫细胞的培养；将重组杆状病毒rBac-HBM-VP2接种sf9昆虫细胞，收获细胞培养物并使用BEI进行灭活；使用层析柱进行层析纯化后用水性佐剂配制灭活疫苗。
3.本发明所述一种鸭细小病毒VP2蛋白重组杆状病毒载体灭活疫苗的应用，其特征在于，用本发明制备的鸭细小病毒VP2蛋白重组杆状病毒载体灭活疫苗免疫鸭，免疫后28日采血；使用琼扩试验对鸭的血清样品进行抗体测定。
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