CN108159409A - 一种猪圆环病毒3型Cap蛋白疫苗及其制备方法和应用 - Google Patents
一种猪圆环病毒3型Cap蛋白疫苗及其制备方法和应用 Download PDFInfo
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- CN108159409A CN108159409A CN201711420604.1A CN201711420604A CN108159409A CN 108159409 A CN108159409 A CN 108159409A CN 201711420604 A CN201711420604 A CN 201711420604A CN 108159409 A CN108159409 A CN 108159409A
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Abstract
一种猪圆环病毒3型Cap蛋白疫苗及其制备方法和应用，属于兽医生物制品技术领域。本发明所述的猪圆环病毒3型Cap蛋白抗原，包含可溶性蛋白，该可溶性蛋白为大肠杆菌表达猪圆环病毒3型Cap蛋白基因得到。其中该Cap蛋白去除N端1~30aa（核定位序列），同时增加了抗原增效结构片段基因，经过大肠杆菌的密码子优化，不仅能在大肠杆菌中大量表达，而且可以形成可溶性蛋白。应用本发明的制备的猪圆环病毒3型Cap蛋白亚单位疫苗，具有抗原纯度高、免疫原性强的特点，同时制备疫苗过程简单，生产成本较低。
Description
技术领域
本发明涉及兽用生物制品技术领域，涉及一种猪圆环病毒3型Cap蛋白疫苗及其制备方法和应用。
背景技术
猪圆环病毒(porcine circovirus，PCV)是圆环病毒科圆环病毒属的重要成员之一。该病毒包括两个基因型(genotypes)：1型和2型，即PCVl和PCV2。PCVl是从猪肾上皮细胞(PK-15)中分离出来的非致病性病毒粒子，而PCV2则是目前对世界养猪业危害极大的病原体之一，给全球养殖业带来了巨大的经济损失。
2016年10月，美国Kansas州立大学的R.Palinski等和加州大学San Francisco分校T.G.Phan等几乎在同一时间报道一个新的PCV基因型，又称为PCV3。该病毒从出现病症的母猪或仔猪中分离得到，同时PCV2检测为阴性。基因组序列分析发现，PCV3基因组包含2000碱基，具有与PCV1和PCV2相似的基因组结构，主要编码cap和rep两个基因。
与PCV相关的疾病(PCV2 associated diseases，PCVAD)包括断奶仔猪多系统衰竭综合征(postweaning multisystemic wasting syndrome，PMWS)、猪皮炎肾病综合征(porcine dermatitis and nephropathy syndrome，PDNS)、呼吸系统疾病(respiratorydisease)、繁殖障碍(reproductive failure)和肠道疾病(enteric disease)等。
猪圆环病毒3型在我国的猪场病例中被检测到，从流产母猪及发生皮炎肾病的小猪上发现大量的病毒数量，而且与猪圆环病毒2型同时感染发病，引起较大的危害。因此，在临床上和实际应用中，需要能够防治猪圆环病毒3型的新型疫苗，采用基因工程的方法，将猪圆环病毒3型的Cap蛋白基因表达出来，制备安全高效的疫苗是未来的重要需求。
发明内容
解决的技术问题：本发明提供一种猪圆环病毒3型Cap蛋白疫苗及其制备方法和应用。
技术方案：一种猪圆环病毒3型Cap蛋白疫苗的制备方法，包括以下步骤：（1）合成目的基因：根据猪圆环病毒3型ORF2的Cap蛋白氨基酸序列，去除N端1~30aa，同时C端加入linker加入抗原增效片段，并在整个基因片段两端加入酶切位点，如SEQ ID NO:2所示的氨基酸序列，再用大肠杆菌密码子优化，反向编译为核苷酸序列如SEQ ID NO:3所示，命名为rPCV3-Cap；（2）重组表达质粒的构建：将步骤（1）获得的rPCV3-Cap与载体PGEX-4T-1进行双酶切，然后回收，连接过夜，转化；获得阳性克隆后，提取质粒，进行PCR鉴定及双酶切鉴定，鉴定正确的质粒保存备用，命名为PGEX-4T-1-rPCV3-Cap；（3）重组菌株的筛选：将步骤（2）获得的PGEX-4T-1-rPCV3-Cap质粒转化大肠杆菌表达菌株BL21（DE3），获得阳性克隆后，进行PCR鉴定，鉴定正确再进行SDS-PAGE分析，，鉴定正确的重组菌株为BL21（DE3）-PGEX-4T-1-rPCV3-Cap；（4）重组蛋白的表达：将步骤（3）获得的表达菌株BL21（DE3）-PGEX-4T-1-rPCV3-Cap进行培养，利用IPTG诱导蛋白表达；（5）重组蛋白的纯化、酶切去标签：离心收集步骤（4）获得的重组菌，进行超声波破碎，破碎后离心，弃沉淀物质，将上清液通过0.45μm滤器过滤，再将上清液用GST融合蛋白亲和层析柱纯化，最后用凝血酶再酶切20小时，去除GST-TAG，最后得到纯化的融合蛋白PCV3-Cap；（6）疫苗的制备：将制备好的抗原蛋白PCV3-Cap无菌过滤，进行浓度测定，按照浓度剂量设定稀释好抗原蛋白PCV3-Cap，然后与佐剂混合、乳化，获得猪圆环病毒3型亚单位疫苗。
步骤（1）中，两端加入的双酶切位点为BamH I和Xho I。
步骤（4）中的培养基主要成分为：葡萄糖、胰蛋白胨、酵母浸出粉、酪蛋白胨、维生素B1、NaCl、MgSO4。
步骤（6）采用的佐剂为法国赛比克GEL01佐剂。
步骤（6）佐剂在配制体系中的体积比为25%。
上述制备方法获得的猪圆环病毒3型亚单位疫苗。。
上述疫苗在制备断奶仔猪多系统衰竭综合征、猪皮炎肾病综合征药物中的应用。
有益效果：1、本发明提供一种猪圆环病毒3型Cap蛋白疫苗及其制备方法和应用，采用去除Cap蛋白N端核定位序列的方法，同时在C端加入了linker以及增强免疫原性的氨基酸序列，增加了猪圆环病毒3型Cap蛋白的可溶性，在细胞上清中，获得了表达量较高的可溶性蛋白，同时也增强了免疫原性。
2、使用PGEX-4T-1载体作为表达载体，形成的融合蛋白，用凝血酶酶切去除GST载体的部分，最后得到的纯化蛋白接近猪圆环病毒3型天然结构，达到较高的免疫原性。
3、应用此方法制备猪圆环病毒3型亚单位疫苗，不涉及到强毒，没有致病性，能够在临床使用后产生较高的免疫原性，同时安全性得到了保证。
4、猪圆环病毒3型Cap蛋白抗原制备及其应用，有制备方法简单、方便，低成本、疫苗效价高的特点。
附图说明
图1为构建好的表达质粒PGEX-4T-1-rPCV3-Cap双酶切示意图，M1泳道表示DNAmaker（100-2000bp），M2泳道表示DNA maker（1000-10000bp）,1,2泳道为PCV3-Cap的目的基因600bp，同时含有5000 bp左右PGEX-4T-1载体片段。
图2 PGEX-4T-1-rPCV3-Cap表达蛋白示意图， M泳道表示蛋白质maker（15-170KDa），1泳道表示诱导前的菌体，2泳道表示诱导后的菌体（49 kDa出现明显表达带），3泳道表示超声破碎后的菌体沉淀（未出现明显表达带），4泳道表示超声破碎后的菌体上清液（49kDa出现明显表达带）。
图3纯化的Cap-GST融合蛋白与GST单抗的western blotting图片， M泳道表示蛋白质maker（20-80 KDa），1泳道表示纯化的Cap-GST融合蛋白与GST单抗反应（49 kDa出现明显反应带），2泳道表示GST蛋白对照与GST单抗反应（26 kDa出现明显反应带）。
图4为猪圆环病毒3型亚单位疫苗免疫后抗体水平变化图。
具体实施方式
下面结合具体实施例进一步描述本发明。这些实施例仅是举例说明，不应被视为对发明总体范围的任何限制。本领域技术人员在不偏离本发明的精神和范围下对本发明技术方案的细节和形式进行的任何修改和替换，均落入本发明的保护范围内。
本发明实施例中用于猪圆环病毒3型Cap蛋白抗原制备及其应用为例说明本发明。
实施例1 猪圆环病毒3型Cap蛋白抗原制备
1 材料与方法
1.1 菌株与载体 PGEX-4T-1载体，引物，序列合成，大肠杆菌DH5α感受态和大肠杆菌BL21（DE3）感受态购自于上海生工。
1.2 试验动物 体质量约25g的BALB/c雌性小鼠，购自南京医科大学动物实验中心，实验用猪来源于江苏省农科院兽医研究所。
1.3 主要试剂 DNA质粒小提试剂盒，DNA胶回收试剂盒，T4连接酶，鼠抗组氨酸标签(His)IgG，GST亲和层析填料，BCA蛋白定量试剂盒，HRP标记的山羊抗鼠IgG，辣根过氧化物酶（HRP），BamHⅠ和XholⅠ限制性内切酶等购自上海生工生物工程（上海）股份有限公司。
1.4 猪圆环病毒3型Cap蛋白表达载体构建
1.4.1猪圆环病毒3型Cap蛋白基因合成
以Porcine circovirus 3 strain CN/Hubei-618/2016（GenBank: KY354039.1）的Cap蛋白的氨基酸序列为基础，如SEQ ID NO:1，通过序列分析，去除N端1~30aa（核定位序列），同时C端加入linker加入抗原增效片段，最后得到需求的氨基酸序列如SEQ ID NO:2的序列，再通过大肠杆菌密码子优化反向编译成核苷酸序列，并在整个片段两端加入酶切位点，使其适宜在大肠杆菌中表达，委托上海生工公司合成，最后的基因序列如：SEQ ID NO:3，命名为rPCV3-Cap，连接于pMD-18T，转化大肠杆菌DH5α菌种内。
1.4.2 目的片段与载体的双酶切回收
用质粒提取试剂盒提取含有rPCV3-Cap基因的质粒与PGEX-4T-1空载体，同时进行BamHⅠ和XholⅠ双酶切，50μL反应体系为限制性内切酶BamHⅠ1μL；限制性内切酶XholⅠ1μL；质粒4μL；10x K buffer 5μL；ddH2O 39μL；置于37℃反应3小时，跑胶后发现有600bp的rPCV3-Cap基因片段，以及载体的4000bp的PGEX-4T-1片段，用胶回收实际盒进行切胶回收。
1.4.2 目的片段与载体的连接转化
将rPCV3-Cap片段与PGEX-4T-1片段，进行连接，按照以下20μL反应体系配置：rPCV3-Cap片段8μL；PGEX-4T-1片段2μL；T4连接酶2μL；10 x T4 buffer 2μL；ddH2O 6μL，至于16℃反应12小时。之后将连接产物转化大肠杆菌DH5α感受态内。反应程序如下：无菌条件下，将10μL连接产物加入大肠杆菌DH5α感受态内，冰浴30min;取出置于42℃，热激90s；再置于冰浴10min；加入700μL LB液体培养基, 37℃恒温摇床复苏培养45min；6000r/min离心5min,去掉上清600ul；剩余重悬菌体后均匀涂布氨苄平板，置于37℃培养12小时。挑取单菌落，接种于含氨苄青霉素的LB液体培养基，培养6小时，（T7启动子上下游序列）进行PCR鉴定及提取质粒BamHⅠ和XholⅠ双酶切鉴定。
1.4.3进行BamHⅠ和XholⅠ双酶切鉴定，50μL反应体系为限制性内切酶HindⅢ1μL；限制性内切酶XholⅠ1μL；质粒4μL；10x K buffer 5μL；ddH2O 39μL；置于37℃反应3小时，产物经凝胶电泳，发现有600bp目的片段及4000bp左右的载体片段。
1.4.4进行PCR鉴定，50μL扩增体系为ddH2O 20.5μL；2xPCRmix 25μL；引物1（50mmol/Ｌ）2μL；引物2（50mmol/Ｌ）2μL；质粒模板（100mmol/L）0.5μL；反应条件为94℃5min；94℃45ｓ，60℃45s，72℃90s，循环30次；72℃10min，产物经凝胶电泳，扩增出600bp左右目的条带。
最后，经PCR及双酶切鉴定正确的菌液及质粒，送上海生工测序正确，重组质粒构建完成，命名为PGEX-4T-1-rPCV3-Cap。
1.5 猪圆环病毒3型Cap蛋白表达
将重组表达载体PGEX-4T-1-rPCV3-Cap质粒转化大肠杆菌BL21(DE3)，挑取阳性转化子培养至OD600达到0.5，加入终浓度为1mmol/L IPTG，分别于37℃诱导5小时；28℃诱导5小时；16℃诱导10小时，4℃8000 r/min离心10min收集菌体。加入6mL生理盐水重悬菌体，超声破碎菌悬液至溶液清亮，12000r/min离心10min，所收集的上清和沉淀液采用SDS-PAGE分析检测目的蛋白的表达部位。
随后，将表达蛋白转印硝酸纤维素膜，并在室温下先后孵育鼠抗GST抗体（一抗，1:2000）和羊抗鼠IgG-HRP抗体（二抗，1:2000）各2小时；随后将硝酸纤维素膜置于DAB染色液中浸泡3-15min，进行western blotting检测。
1.6 猪圆环病毒3型Cap蛋白纯化
1.6.1含有猪圆环病毒3型Cap蛋白的大肠杆菌细胞裂解上清与50%谷胱甘肽-琼脂糖树脂匀浆混合，每100mL上清加2mL树脂，于室温下轻摇8-12小时;
1.6.2细胞裂解上清与树脂混合物于4℃以500g(2100r/min)离心5min，小心去掉上清并留样少许进行SDS-PAGE;
1.6.3留下的沉淀中加入10倍标准体积的PBS，颠倒离心管数次混匀，洗去未与树脂结合的蛋白；
1.6.4 4℃以500g(2100r/min)离心5min，小心去掉上清并留样少许进行SDS-PAGE;
1.6.5重复步骤1.6.3和1.6.4两次；
1.6.6 猪圆环病毒3型Cap蛋白与GST融合蛋白可用凝血酶因子切割，每毫升树脂加入50单位1mL PBS的凝血酶，颠倒离心管数次混匀，室温下震荡4～6h。
1.6.7 4℃以500g(2100r/min)离心5min，最后得到上清小心移至新管中，即为猪圆环病毒3型Cap蛋白；
1.6.8 10%SDS-PAGE分析每一步洗涤、洗脱样品的蛋白质组成。
2 结果与分析
2.1 猪圆环病毒3型Cap蛋白表达载体构建
构建好的PGEX-4T-1-rPCV3-Cap重组质粒，经BamHⅠ和XholⅠ双酶切鉴定，产物经凝胶电泳，发现有600bp目的片段及4000bp左右的载体片段。（见图1）
2.2 猪圆环病毒3型Cap蛋白表达
取诱导后的培养液12000 rpm离心5 min，去除上清液，加入PBS液重悬沉淀，最后加入SDS-PAGE上样缓冲液于100℃下加热样品10 min，然后离心取上清电泳。电泳前10 min 时，100 V稳压电泳，待溴酚蓝指示剂进入分离胶后200 V稳压电泳至溴酚蓝带迁移至离凝胶底部1 cm，取出凝胶用考马斯亮兰染色液染色，随后转入脱色液中，脱色至背景清晰。发现破碎的细胞上清，全菌中出现49KD的目的条带，细胞沉淀及空质粒对照未出现，与预期相符合。（见图2）
2.3 猪圆环病毒3型Cap蛋白纯化
使用GST亲和层析在方法，纯化猪圆环病毒3型Cap蛋白，摸索大肠杆菌细胞裂解上清与50%谷胱甘肽-琼脂糖树脂的结合条件及浓度。最后摸索用凝血酶切割GST融合蛋白的浓度及反应时间、温度等参数，最后获得纯化的猪圆环病毒3型Cap蛋白。
将纯化好的蛋白，进行SDS-PAGE电泳，切下后，附在硝酸纤维素膜上，用石墨电极转印硝酸纤维素膜，并在室温下先后孵育鼠抗GST抗体（一抗，1:2000）和羊抗鼠IgG-HRP抗体（二抗，1:2000）各2小时；随后将硝酸纤维素膜置于DAB染色液中浸泡3-15min显色，出现目的条带大小的反应，分析结果（见图3）。
实施例2 猪圆环病毒3型Cap蛋白亚单位疫苗制备
用BCA蛋白定量试剂盒测定由实施例1制备好的纯化猪圆环病毒3型Cap蛋白原液浓度，将蛋白浓度调节终浓度为到组1：0.2mg/mL，组2：0.1mg/mL，组3：0.05 mg/mL，组4：0.01 mg/mL再与法国赛匹克佐剂GEL01配比，佐剂添加体积占总体积的25%，进行混合均匀搅拌后，按照现行版中国兽药典附录要求无菌检验、粘度测定、稳定性测定合格后，放置于2-8℃备用。
实施例3 猪圆环病毒3型Cap蛋白亚单位疫苗应用
用实施例2制备的猪圆环病毒3型Cap蛋白亚单位疫苗，按照梯度浓度进行分组免疫仔猪程序见表1。每2周采血一次（第2、4、6、8周），评估抗体水平变化规律，不同浓度的猪圆环病毒3型Cap蛋白亚单位疫苗，免疫后产生抗体水平相较于对照组有显著差异，用猪圆环病毒3型Cap蛋白包被ELISA板，建立间接ELISA方法测定抗体水平，见图4。
表1仔猪免疫猪圆环病毒3型Cap蛋白亚单位疫苗程序
| 分组 | 动物数量 | 副反应 | 免疫日龄 | 免疫程序 |
| 组1 | 5头 | 无 | 21天 | 2mL/次 |
| 组2 | 5头 | 无 | 21天 | 2mL/次 |
| 组3 | 5头 | 无 | 21天 | 2mL/次 |
| 组4 | 5头 | 无 | 21天 | 2mL/次 |
| 空白组 | 5头 | 无 | 21天 | 2mL/次 |
涉及到基因序列如下：
Porcine circovirus 3 strain CN/Hubei-618/2016（GenBank: KY354039.1）Cap蛋白氨基酸序列
SEQ ID NO:1
MRHRAIFRRRPRPRRRRRHRRRYARRRLFIRRPTAGTYYTKKYSMNVISVGTPQNNKPWHANHFITRLNEWETAISFEYYKILKMKVTLSPVISPAQQTKTMFGHTAIDLDGAWTTNTWLQDDPYAESSTRKVMTSKKKHSRYFTPKPILAGTTSAHPGQSLFFFSRPTPWLNTYDPTVQWGALLWSIYVPEKTGMTDFYGTKEVWIRYKSVL
去除核定位序列以及后端加入linker及增效氨基酸序列
SEQ ID NO:2
AGTYYTKKYSTMNVISVGTPQNNKPWHANHFITRLNEWETAISFEYYKILKMKVTLSPVISPAQQTKTMFGHTAIDLDGAWTTNTWLQDDPYAESSTRKVMTSKKKHSRYFTPKPILAGTTSAHPGQSLFFFSRPTPWLNTYDPTVQWGALLWSIYVPEKTGMTDFYGTKEVWIRYKSVLGGGSMAGCKNFFWKTFTSC
大肠杆菌密码子优化后的cap蛋白基因序列
SEQ ID NO:3
酶切位点：BamHI/XhoI (XhoI前加终止子 )
GGATCCGCGGGCACCTACTACACTAAAAAATATAGCACCATGAACGTGATTAGCGTAGGCACCCCACAGAATAATAAACCGTGGCACGCTAACCACTTTATAACCCGTCTGAATGAATGGGAAACCGCGATCAGCTTCGAATACTACAAAATCCTGAAAATGAAAGTTACCCTGTCTCCGGTTATCAGCCCGGCGCAGCAGACCAAAACCATGTTCGGCCACACCGCGATCGATCTGGATGGCGCGTGGACCACCAACACCTGGCTGCAGGATGATCCGTACGCGGAAAGCAGCACCCGTAAAGTTATGACCAGCAAAAAGAAACACAGCCGTTACTTCACCCCGAAACCGATCCTGGCCGGTACCACCAGCGCTCATCCGGGCCAGAGCCTGTTCTTCTTCAGCCGTCCGACCCCGTGGCTGAACACCTACGATCCGACCGTTCAGTGGGGCGCACTGCTGTGGAGCATCTACGTTCCGGAAAAAACCGGCATGACCGATTTCTACGGCACCAAAGAAGTTTGGATTCGTTACAAAAGCGTTCTGGGCGGCGGCAGCATGGCGGGTTGCAAAAACTTTTTCTGGAAAACCTTCACCAGCTGCTAACTCGAG
猪圆环病毒3型与GST融合蛋白表达氨基酸序列及分子量预测
SEQ ID NO:4
Protein Length=425 MW=49044.0 Predicted pI=8.92
MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLVPRGSAGTYYTKKYSTMNVISVGTPQNNKPWHANHFITRLNEWETAISFEYYKILKMKVTLSPVISPAQQTKTMFGHTAIDLDGAWTTNTWLQDDPYAESSTRKVMTSKKKHSRYFTPKPILAGTTSAHPGQSLFFFSRPTPWLNTYDPTVQWGALLWSIYVPEKTGMTDFYGTKEVWIRYKSVLGGGSMAGCKNFFWKTFTSC。
序列表
<110> 南京大爻网络科技有限公司
<120> 一种猪圆环病毒3型Cap蛋白疫苗及其制备方法和应用
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Claims (7)
1.一种猪圆环病毒3型Cap蛋白疫苗的制备方法，其特征在于包括以下步骤：
（1）合成目的基因：根据猪圆环病毒3型ORF2的Cap蛋白氨基酸序列，去除N端1~30aa，同时C端加入linker加入抗原增效片段，并在整个基因片段两端加入酶切位点，如SEQ ID NO:2所示的氨基酸序列，再用大肠杆菌密码子优化，反向编译为核苷酸序列如SEQ ID NO:3所示，命名为rPCV3-Cap；
（2）重组表达质粒的构建：将步骤（1）获得的rPCV3-Cap与载体PGEX-4T-1进行双酶切，然后回收，连接过夜，转化；获得阳性克隆后，提取质粒，进行PCR鉴定及双酶切鉴定，鉴定正确的质粒保存备用，命名为PGEX-4T-1-rPCV3-Cap；
（3）重组菌株的筛选：将步骤（2）获得的PGEX-4T-1-rPCV3-Cap质粒转化大肠杆菌表达菌株BL21（DE3），获得阳性克隆后，进行PCR鉴定，鉴定正确再进行SDS-PAGE分析，，鉴定正确的重组菌株为BL21（DE3）-PGEX-4T-1-rPCV3-Cap；
（4）重组蛋白的表达：将步骤（3）获得的表达菌株BL21（DE3）-PGEX-4T-1-rPCV3-Cap进行培养，利用IPTG诱导蛋白表达；
（5）重组蛋白的纯化、酶切去标签：离心收集步骤（4）获得的重组菌，进行超声波破碎，破碎后离心，弃沉淀物质，将上清液通过0.45μm滤器过滤，再将上清液用GST融合蛋白亲和层析柱纯化，最后用凝血酶再酶切20小时，去除GST-TAG，最后得到纯化的融合蛋白PCV3-Cap；
（6）疫苗的制备：将制备好的抗原蛋白PCV3-Cap无菌过滤，进行浓度测定，按照浓度剂量设定稀释好抗原蛋白PCV3-Cap，然后与佐剂混合、乳化，获得猪圆环病毒3型亚单位疫苗。
2.根据权利要求1所述一种猪圆环病毒3型Cap蛋白疫苗的制备方法，其特征在于：步骤（1）中，两端加入的双酶切位点为BamH I和Xho I。
3.根据权利要求1所述一种猪圆环病毒3型Cap蛋白疫苗的制备方法，其特征在于：步骤（4）中的培养基主要成分为：葡萄糖、胰蛋白胨、酵母浸出粉、酪蛋白胨、维生素B1、NaCl、MgSO4。
4.根据权利要求1所述一种猪圆环病毒3型Cap蛋白疫苗的制备方法，其特征在于：步骤（6）采用的佐剂为法国赛比克GEL01佐剂。
5.根据权利要求1所述一种猪圆环病毒3型Cap蛋白疫苗的制备方法，其特征在于：步骤（6）佐剂在配制体系中的体积比为25%。
6.权利要求1-5任一制备方法获得的猪圆环病毒3型亚单位疫苗。
7.权利要求6所述的疫苗在制备断奶仔猪多系统衰竭综合征、猪皮炎肾病综合征药物中的应用。
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