CN114591920A - 猪流行性腹泻病毒毒株、灭活疫苗及其应用 - Google Patents

猪流行性腹泻病毒毒株、灭活疫苗及其应用 Download PDF

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CN114591920A
CN114591920A CN202210245577.3A CN202210245577A CN114591920A CN 114591920 A CN114591920 A CN 114591920A CN 202210245577 A CN202210245577 A CN 202210245577A CN 114591920 A CN114591920 A CN 114591920A
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porcine epidemic
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秦立廷
王潇博
吕闯
陈婷
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Shandong New Hope Liuhe Group Co Ltd
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Qingdao Jiazhi Biotechnology Co Ltd
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Abstract

本发明提出了猪流行性腹泻病毒毒株、灭活疫苗及其应用，该毒株命名为猪流行性腹泻病毒PEDV‑CH/GM2018，该毒株于2021年10月20日保藏于中国典型培养物保藏中心，保藏号为CCTCC NO:V202174。该毒株为优化分离培养所得的流行PEDV变异株，能够在细胞上稳定传代，为有效的疫苗制备和猪流行性腹泻病防控提供了基础，利用该毒株制备灭活疫苗能对仔猪产生良好的保护作用。

Description

猪流行性腹泻病毒毒株、灭活疫苗及其应用
技术领域
本发明属于病毒疫苗技术领域，具体涉及猪流行性腹泻病毒毒株、灭活疫苗及其应用。
背景技术
猪流行性腹泻病毒（Porcine epidemic diarrhea virus，PEDV）是目前在全球范围内广泛流行的冠状病毒。PEDV主要侵害各日龄猪，尤其可感染仔猪小肠，并迅速导致仔猪产生水样腹泻，伴随呕吐、脱水等临床症状，致其死亡率可达100%。成年猪不发生腹泻，但常伴有精神萎靡不振，食欲下降等症状。PEDV于1971年首次在英国发现。在我国，PEDV于1984年首次在猪小肠原代细胞中分离。在2010年以前，PEDV在我国猪场的感染情况具有散发和区域性特征，并未爆发大规模流行。2010年10月，在南方诸省开始出现PEDV的急性爆发并迅速蔓延至全国，即使接种了疫苗的哺乳仔猪也未能幸免。仔猪症状主要表现为黄色水样腹泻，体重下降，最后死于严重的脱水。经确认此次流行的毒株为变异毒株，应用荧光定量PCR检测各省平均的感染率为58.32% 。进一步实验表明，该毒株感染仔猪后具有PEDV的典型症状，3-5日龄哺乳仔猪2-3天后出现症状，死亡率为100%。随着灭活疫苗和二联弱毒疫苗的使用，PED疫情在我国得到了一定的控制。但毒株的变异导致其传染性急剧增加，给我国养猪业造成重大损失。
PEDV的病毒粒子呈多形性，在囊膜外表面排列类似“日冕”样，长约20 nm的纤突突起，直径95—190 nm。长度约为28kb，其基因组为单股正链RNA，包括 5′端帽和3′端多聚腺苷酸尾，从5′到3′端依次是Pol基因、S基因、ORF3基因、sM/E 基因、M基因和N基因。针对PEDVS基因序列可将 PEDV 分为 G1与 G2基因亚型。其中， G1型包含 PEDV 经典毒株 CV777 疫苗株，G2 型为PEDV流行毒株，由于毒株的突变而使得目前可用的疫苗对变异毒株未能完全保护。
另外，PEDV变异毒株在细胞上分离困难，并且在细胞上增殖获得病毒的毒价低，也是影响猪流行性腹泻疫苗的保护效果的重要原因。通常猪场利用反饲和制备组织灭活疫苗来预防该病的感染，反饲风险大，猪场制备的组织灭活疫苗杂蛋白多、每批苗批间差异较大，导致组织苗免疫后副反应大，保护效果不确实。
发明内容
本发明提供一株猪流行性腹泻病毒毒株、灭活疫苗及其应用，该毒株为优化分离培养所得的流行PEDV变异株，能够在细胞上稳定传代，为有效的疫苗制备和猪流行性腹泻病防控提供了基础，利用该毒株制备灭活疫苗能对仔猪产生良好的保护作用。
为了达到上述目的，本发明采用的技术方案为：
本发明第一方面公开了一株猪流行性腹泻病毒毒株，命名为猪流行性腹泻病毒PEDV-CH/GM2018，该毒株于2021年10月20日保藏于中国典型培养物保藏中心，保藏号为CCTCC NO: V202174。
作为优选，所述猪流行性腹泻病毒PEDV-CH/GM2018于2018年从山东省高密市PEDV阳性猪场病猪肠道采集分离所得。
作为优选，所述的猪流行性腹泻病毒毒株的S基因全长4158bp，属于GII-b基因亚型，相对于经典疫苗株CV777，氨基酸序列在58-59位有4个aa插入，135-136位有1个aa；在158位有2个aa缺失，1194位有1个缺失，所述猪流行性腹泻病毒PEDV-CH/GM2018与GI型CV777毒株同源性为92.8%，与GII型AH2012同源性最高为98.3%。
作为优选，所述猪流行性腹泻病毒PEDV-CH/GM2018毒株为GII型现地流行毒株，其的分离鉴定方法，包括以下步骤：
病料处理：将经RT-PCR检测为PEDV阳性的具有腹泻症状的病仔猪的肠道内容物用DMEM制成悬液，涡旋震荡混匀，冻融一次后高速离心，取上清液过滤后得病毒悬液，冷冻备用；
分离筛选：将所得的病毒悬液接种到形成单层的Vero细胞培养层中，在培养箱中感作后弃去接种物，加入含有胰蛋白示磷酸肉汤及胰酶的DMEM培养基培养，每日观察细胞是否产生病变CPE，若无病变则于第5天收集培养液冻融一次后继续盲传；所得的病毒中命名为猪流行性腹泻病毒PEDV-CH/GM2018的毒株传至第五代可见细胞产生明显的CPE，收集每代次病毒液应用qPCR试剂盒对病毒核酸拷贝数进行监测，结果发现随着代次升高，核酸拷贝数也随之升高；
鉴定：采用Western blot与间接免疫荧光法对分离所得猪流行性腹泻病毒PEDV-CH/GM2018进行鉴定。
作为优选，所述病料处理步骤包括：将经RT-PCR检测为PEDV阳性的具有腹泻症状的病仔猪的肠道内容物与DMEM按体积比1:5制成悬液，涡旋震荡混匀，-80℃冻融一次，4℃条件下12000r/min离心30min，取上清液用0.22μm的滤器进行过滤后得病毒悬液，-80℃冰箱冷冻备用。
作为优选，所述分离鉴定方法还包括蚀斑纯化步骤：在分离所得病毒传至第10代时及进行毒株的蚀斑纯化，通过两次纯化获得性状相对一致的病毒粒子，作为猪流行性腹泻病毒PEDV-CH/GM2018。
本发明第二方面公开了所述猪流行性腹泻病毒PEDV-CH/GM2018在制备预防猪流行性腹泻病的灭活疫苗中的应用。
本发明第三方面公开了所述的灭活疫苗的制备方法，包括以下步骤：
1）增殖所述猪流行性腹泻病毒PEDV-CH/GM2018，收获病毒液；
2）将病毒液加入灭活剂中进行灭活；
3）将灭活的病毒液与佐剂混合，乳化即得灭活疫苗。
作为优选，所述步骤1）用Vero细胞增殖所述猪流行性腹泻病毒PEDV-CH/GM2018。
本发明第三方面公开了所述的制备方法制得的猪流行性腹泻病灭活疫苗。
本发明的有益效果为：提出了一株猪流行性腹泻病毒毒株、灭活疫苗及其应用，该毒株为优化分离培养所得的流行PEDV变异株，通过对该毒株S基因的测定，其与国内经典疫苗株CV777与常用的商品化疫苗株AH2012株均存在差异，经测序该毒株为流行毒株，其序列特征与大部分中国流行毒株相一致，具备制备疫苗价值，且致病性实验证明该毒株致病力很强，具有良好的免疫原性，利用该毒株制备灭活疫苗能对仔猪产生良好的保护作用。该毒株能够在细胞上稳定传代，为有效的疫苗制备和猪流行性腹泻病防控提供了基础，利用该毒株制备灭活疫苗能对仔猪产生良好的保护作用。
附图说明
图1为本发明的猪流行性腹泻病毒PEDV-CH/GM2018的Western Blot检测结果图，其中，Control表示未感染病毒的Vero细胞样本。
图2为实施例4的免疫荧光检测结果图。
图3为实施例5的基于S基因系统进化树分析结果图。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
分离出近年来流行的PEDV变异株，并将其在细胞上稳定传代，是有效的疫苗制备及PEDV防控的基础。发明人于2018年从山东省高密市PEDV阳性猪场病猪仔肠道采集分离得到一株猪流行性腹泻病毒毒株，将其命名为猪流行性腹泻病毒PEDV-CH/GM2018，该毒株于2021年10月20日保藏于中国典型培养物保藏中心，保藏号为CCTCC NO: V202174。该毒株能够在Vero细胞上稳定传代，利用该毒株制备灭活疫苗能对仔猪产生良好的保护作用。
实施例1 毒株分离和鉴定
1.1 细胞和病料
Vero细胞购自ATCC公司，病料于2018年采集自山东省高密市PEDV阳性猪场病仔猪肠道内容物。
1.2 病料处理
将经RT-PCR检测为PEDV阳性的具有腹泻症状的病仔猪的肠道内容物用DMEM 按体积比1：5制成悬液，涡旋震荡混匀，-80℃冻融一次，4℃条件下 12000r/min离心30min，取上清液用0.22μm的滤器进行过滤后得病毒悬液，放入-80℃冰箱冷冻保存备用。在分离病毒时将肠内容物悬液置于-80℃速冻，加快了病毒从肠组织中释放出来的同时减轻冷冻过程中活病毒的损失，提高了病毒分离成功的几率。
1.3 药品和试剂
细胞培养基DMEM、胰酶、胎牛血清购自Gibco公司；胰蛋白示磷酸肉汤(TPB)为Sigma公司产品。病毒基因组DNA/RNA快速抽提试剂盒为天根生化科技有限公司产品；反转录试剂盒PrimeScript™ IV 1st strand cDNA Synthesis Mix购自TakaRa公司；质粒纯化试剂盒及DNA片段琼脂糖凝胶回收试剂盒为爱思进公司产品；E.Coli DH5a感受态细胞购自北京全式金生物公司。
1.4 基因合成、测序和克隆方法
基因合成、测序和克隆采用常规方法进行，委托生工生物工程（上海）股份有限公司（以下简称“上海生工公司”）完成。
1.5 病毒分离筛选
待6孔板中Vero细胞长满单层，用PBS洗细胞一次，随后加入病料处理所得的病毒悬液1mL，在5%CO2、37℃培养箱中感作1h，随后弃去接种物，加入含有0.3wt%胰蛋白示磷酸肉汤（TPB）及 10μg/mL胰酶的DMEM培养基中培养，每日观察细胞是否产生病变（CPE），若无病变则于第5天收集培养液冻融一次后继续盲传，结果，筛选的暂命名为猪流行性腹泻病毒PEDV-CH/GM2018毒株传至第五代可见细胞产生明显的CPE，收集每代次病毒液应用商品化qPCR试剂盒对病毒核酸拷贝数进行监测，发现随着代次升高，核酸拷贝数也随之升高。在病毒感染Vero细胞维持液中添加TPB，有助于稳定细胞贴壁生长，从而利于病毒在细胞上增殖。
1.6 病毒的蚀斑纯化
采用Vero细胞对分离筛选所得的病毒毒株传至第10代时进行毒株的蚀斑纯化，纯化方法为弃去6孔板中已经长成单层的细胞培养液，用维持液清洗2次，再利用维持液稀释病毒，稀释倍数设置为102，103，104。每孔400μL，置于培养箱吸附2h；配胶：2%的凝胶与2×DMEM等体积混匀后，每孔加入2mL，室温静置30min，待胶凝固后倒置于37℃，5% CO2培养箱中培养，每日观察，记录蚀斑生长情况，挑取细胞孔内小且孤立的空斑，以维持液稀释后接种至培养瓶。按以上方法进行一次纯化，以获得性状相对一致的病毒粒子，随后对克隆株在Vero细胞上继续传代，选择出适应细胞较好，传代后毒价较高的克隆株作为候选株，将其正式命名为猪流行性腹泻病毒PEDV-CH/GM2018。
实施例2 病毒毒价的标定
待实施例1的1.5分离筛选所得的猪流行性腹泻病毒PEDV-CH/GM2018毒株在Vero细胞上传至第十代开始测定病毒半数感染剂量（TCID50）。取已长成良好铺满单层的Vero细胞96孔细胞培养板，弃去细胞培养液，并用PBS洗涤两次。将分离毒株第10、12、14、16、18、20代病毒液用含10μg/mL胰酶的无血清DMEM培养基作10倍系列稀释，分别将10-1-10-6稀释度病毒液接种到96孔细胞培养板，每个稀释度接种8孔，同时设立阳性对照与阴性对照。将培养板置于37℃、5%CO2培养箱中培养3-5天，逐日观察CPE并记录。病毒滴度按Reed-Muench法计算。实验结果分离毒株第10、12、14、16、18、20代毒毒价分别为104.0TCID50/mL、104.5TCID50/mL、104.2TCID50/mL、105.0TCID50/mL、106.0TCID50/mL、105.8TCID50/mL。
实施例3 Western blot 鉴定试验
将实施例1的1.5分离筛选所得的猪流行性腹泻病毒PEDV-CH/GM2018毒株的第十代病毒液1mL接种于6孔板中长满的单层Vero细胞上，感作1h后加入维持液，48h收集细胞用哺乳动物细胞裂解液100μL 4℃旋转混匀作用20 min，然后4℃ 12000rpm离心30 min，收集上清液。加入25μL 5×蛋白上样buffer，混匀后100℃水浴煮10min，进行SDS-PAGE电泳。随后转膜至NC膜上。应用Western blot方法对：PEDV N蛋白表达情况进行验证，先将NC膜置于5%脱脂乳中封闭2 h，用PBST洗膜3次，5min每次；加入1:500体积比稀释的抗PEDV N蛋白单克隆抗体，作用2h，后用PBST洗膜3次，5min每次。加入1:5000体积比稀释的抗小鼠红外荧光二抗，室温避光作用45 min。用PBST洗膜3次，5min每次，用红外扫膜仪检测蛋白表达。测试结果如图1所示，由图1的测试结果可以看出:接毒组可见明显的N蛋白条带，而对照组未出现，说明病毒接种细胞后在细胞内复制产生了N蛋白。
实施例4 间接免疫荧光鉴定
将Vero细胞传代，铺于12孔细胞培养板中，约48 h细胞长成单层，用 PBS洗细胞后，加入1mL第10代病毒液，感作1 h后用PBS洗细胞后加入维持液感染24 h。 PBS洗细胞3次，加入4v%多聚甲醛 4℃孵育 30 min，PBS 洗 3遍后用0.2v% TritonX-100 37℃透膜20min，PBS洗3遍，加入 1wt% BSA 溶液 37℃封闭2 h，PBS洗3遍，分别加入1:500体积比稀释的抗PEDV N蛋白单克隆抗体，37℃作用1 h，PBS洗细胞3次，加入1:500体积比稀释的抗小鼠荧光二抗，37℃温箱避光孵育45 min，PBS洗3次后观察细胞内荧光产生情况，所得实验结果如图2所示，图2中，a、b、c为对照组，分别为DAPI染色、病毒N蛋白检测与两组荧光融合图。d、e、f为病毒感染组，分别为DAPI染色、病毒N蛋白检测与两组荧光融合图。由图2的测试结果可以看出:该病毒感染后可在细胞内检测到大量的N蛋白，说明分离毒株可以很好的在宿主细胞内复制。
实施例5 病毒全基因组测试与序列分析
按照病毒基因组DNA/RNA快速抽提试剂盒使用方法提取猪流行性腹泻病毒PEDV-CH/GM2018毒株的第十代病毒液总RNA。按PrimeScript™ IV 1st strand cDNA SynthesisMix方法对病毒RNA进行反转录。针对病毒基因组序列设计全基因组扩增引物进行PCR扩增，对扩增片段胶回收连接pMD-18T载体后，送上海生工公司测序并将结果拼接成完整序列。针对PEDV S基因的测序结果进行序列分析，并绘制系统进化树，如图3所示。结果发现猪流行性腹泻病毒PEDV-CH/GM2018毒株S基因全长4158bp，属于GII-b基因亚型，与商品化疫苗株CV777、AH2012分属不同基因亚型。相对于经典疫苗株CV777，氨基酸序列在58-59位有4个aa插入，135-136位有1个aa；在158位有2个aa缺失，1194位有1个缺失。GI型CV777毒株同源性为92.8%，与GII型AH2012同源性最高为98.3%。
实施例6 病毒灭活疫苗的制备
采用本发明筛选所得的猪流行性腹泻病毒PEDV-CH/GM2018毒株可按常规方法制得灭活疫苗，用于预防猪流行性腹泻病，如该灭活疫苗的制备方法可包括以下步骤：
1）增殖猪流行性腹泻病毒PEDV-CH/GM2018，收获病毒液；
2）将病毒液加入灭活剂中进行灭活；
3）将灭活的病毒液与佐剂混合，乳化即得灭活疫苗。
具体的，步骤1）用Vero细胞增殖所述猪流行性腹泻病毒PEDV-CH/GM2018。
猪流行性腹泻病毒PEDV-CH/GM2018毒株为流行PEDV变异株，且该毒株能够在细胞上稳定传代，能够为有效的疫苗制备和猪流行性腹泻病防控提供了基础，利用该毒株制备灭活疫苗能对仔猪产生良好的保护作用。
以上所述，仅是本发明的实施例证而已，并非是对本发明作其它形式的限制，任何熟悉本专业的技术人员可能利用上述揭示的技术内容加以变更或改型为等同变化的等效实施例应用于其它领域，但是凡是未脱离本发明的技术方案内容，依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化与改型，仍属于本发明技术方案的保护范围。
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<110> 青岛嘉智生物技术有限公司，山东新希望六和集团有限公司，新希望六和股份有限公司
<120> 猪流行性腹泻病毒毒株、灭活疫苗及其应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 27929
<212> RNA
<213> 猪流行性腹泻病毒(PEDV-CH/GM2018)
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cgcaccucaa ggccugcugu uuuuacauac aguacuugua ccaggugacu uuguaaaugu 24060
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gcagcgcuca gagucucucc guaauacuac agaagagcuc caaagucuua uauauaauau 24480
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gccaacuugu cuucggacgc uguccaagag uuggagcuca auguaguucc aauuagacaa 24840
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acacuuauug gcaggcucuu uuuagucugc uuuuauuccu ggcgcuauaa aaaugcgcuc 25080
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ggcaaaucca uugugauucu agaagguggu gaucauuaca ucacuuuugg caacucuuuc 25200
guugcuuucg uuaguagcau uaacuuguau cuagcuauac gugggcggca agaagcugac 25260
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acgguucuau ucccguugau gaggugauuc aacaccuuag aaacuggaau uucacaugga 25680
auaucauacu gacgauacua cuuguagugc uucaguaugg ccauuacaag uacucugcgu 25740
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ggcgcaggac acauucuugg uggucuuuca auccugaaac agacgcgcuu cucacuacuu 25980
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gucaauuacc uaauuucguc acagucgcca aggccacuac aacaauuguc uacggacgug 26160
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acggguucca uuaucccucu augccccucu uaggguuacu aaugacaaac cccuuucuaa 26400
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cuggaaugag caaauucgcu ggcgcaugcg ccguggugag cgaauugaac aaccuuccaa 26520
uuggcauuuc uacuaccucg gaacaggacc ucaugccgac cuccguuaua ggacucguac 26580
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ugguaauggc aacaacaggu ccagaucucc aaguaacaac agaggcaaua accagucccg 26820
cgguaauuca cagaaucgug gaaauaacca gggucgugga gcuucucaga acagaggagg 26880
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ccgugguggu guaacaucac gcgaugaucu gguggcugcu gucaaggaug cccuuaaauc 27000
uuugggcauu ggcgaaaacc cugacaagcu uaagcaacag cagaagccca aacaggaaag 27060
gucugacagc agcggcaaaa auacaccuaa gaagaacaaa uccagagcca cuucgaaaga 27120
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agcuugcuuc ggacccaggg gaggcuucaa aaauuuugga gaugcggaau uugucgaaaa 27240
agguguugau gccucaggcu augcccagau cgccaguuua gcaccaaaug uugcagcauu 27300
gcucuuuggu gguaaugugg cuguucguga gcuagcggac ucuuacgaga uuacauauaa 27360
uuauaaaaug acugugccaa agucugaccc aaauguagag cuucuuguuu cacaggugga 27420
ugcauuuaaa acugggaaug caaaacccca gagaaagaag gaaaagaaga acaagcguga 27480
aaccgcgcag cagcugaaug aagaggccau cuacgaugau gugggugugc caucugaugu 27540
gacucaugcc aauuuggaau gggacacagc uguugauggu ggugacacgg ccguugaaau 27600
uaucaacgag aucuucgaua caggaaauua aacaauguuu gacuggcuua uccuggcuau 27660
gucccagggu auugccauua cacuguuauu acugaguguu uuucuagcga cuuggcugcu 27720
gggcuauggc uuugcccucu aacuagcggu cuuggucuug cacacaacgg uaagccagug 27780
guaaugucag ugcaagaagg auauuaccau agcacuguca cgaggggaac gcagcaccuu 27840
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gugccaggua uugacucaag acguagggc 27929

Claims (10)

1.一株猪流行性腹泻病毒毒株，其特征在于，命名为猪流行性腹泻病毒PEDV-CH/GM2018，该毒株于2021年10月20日保藏于中国典型培养物保藏中心，保藏号为CCTCC NO:V202174。
2.根据权利要求1所述的猪流行性腹泻病毒毒株，其特征在于，所述猪流行性腹泻病毒PEDV-CH/GM2018于2018年从山东省高密市PEDV阳性猪场病猪肠道采集分离所得。
3.根据权利要求1所述的猪流行性腹泻病毒毒株，其特征在于，其S基因全长4158bp，属于GII-b基因亚型，相对于经典疫苗株CV777，氨基酸序列在58-59位有4个aa插入，135-136位有1个aa；在158位有2个aa缺失，1194位有1个缺失，所述猪流行性腹泻病毒PEDV-CH/GM2018与GI型CV777毒株同源性为92.8%，与GII型HM2017同源性最高为98.3%。
4.根据权利要求1所述的猪流行性腹泻病毒毒株，其特征在于，其分离鉴定方法，包括以下步骤：
病料处理：将经RT-PCR检测为PEDV阳性的具有腹泻症状的病仔猪的肠道内容物用DMEM制成悬液，涡旋震荡混匀，冻融一次后高速离心，取上清液过滤后得病毒悬液，冷冻备用；
分离筛选：将所得的病毒悬液接种到形成单层的Vero细胞培养层中，在培养箱中感作后弃去接种物，加入含有胰蛋白示磷酸肉汤及胰酶的DMEM培养基培养，每日观察细胞是否产生病变CPE，若无病变则于第5天收集培养液冻融一次后继续盲传；所得的病毒中命名为猪流行性腹泻病毒PEDV-CH/GM2018的毒株传至第五代可见细胞产生明显的CPE，收集每代次病毒液应用qPCR试剂盒对病毒核酸拷贝数进行监测，结果发现随着代次升高，核酸拷贝数也随之升高；
鉴定：采用Western blot与间接免疫荧光法对分离所得猪流行性腹泻病毒PEDV-CH/GM2018进行鉴定。
5.根据权利要求4所述的猪流行性腹泻病毒毒株，其特征在于，所述病料处理步骤包括：将经RT-PCR检测为PEDV阳性的具有腹泻症状的病仔猪的肠道内容物与DMEM按体积比1:5制成悬液，涡旋震荡混匀，-80℃冻融一次，4℃条件下 12000r/min离心30min，取上清液用0.22μm的滤器进行过滤后得病毒悬液，放入-80℃冰箱冷冻备用。
6.根据权利要求4所述的猪流行性腹泻病毒毒株，其特征在于，所述分离鉴定方法还包括蚀斑纯化步骤：在分离所得病毒传至第10代时进行毒株的蚀斑纯化，通过一次纯化获得性状相对一致的病毒粒子，作为猪流行性腹泻病毒PEDV-CH/GM2018。
7.权利要求1所述的猪流行性腹泻病毒在制备预防猪流行性腹泻病的灭活疫苗中的应用。
8.根据权利要求7所述的灭活疫苗的制备方法，包括以下步骤：
增殖所述猪流行性腹泻病毒PEDV-CH/GM2018，收获病毒液；
将病毒液加入灭活剂中进行灭活；
将灭活的病毒液与佐剂混合，乳化即得灭活疫苗。
9.根据权利要求8所述的灭活疫苗的制备方法，其特征在于，所述步骤1）用Vero细胞增殖所述猪流行性腹泻病毒PEDV-CH/GM2018，在增殖过程中，在病毒感染Vero细胞维持液中添加胰蛋白示磷酸胨肉汤。
10.权利要求8或9所述的制备方法制得的猪流行性腹泻病灭活疫苗。
CN202210245577.3A 2022-03-14 2022-03-14 猪流行性腹泻病毒毒株、灭活疫苗及其应用 Pending CN114591920A (zh)

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Cited By (1)

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Publication number Priority date Publication date Assignee Title
CN115253144A (zh) * 2022-08-09 2022-11-01 青岛嘉智生物技术有限公司 针对戊二醛类消毒剂的中和剂及其制备方法

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115253144A (zh) * 2022-08-09 2022-11-01 青岛嘉智生物技术有限公司 针对戊二醛类消毒剂的中和剂及其制备方法

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