CN116855543A - 一种猪细小病毒病、猪丹毒二联亚单位疫苗及制备方法 - Google Patents
一种猪细小病毒病、猪丹毒二联亚单位疫苗及制备方法 Download PDFInfo
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- CN116855543A CN116855543A CN202310888889.0A CN202310888889A CN116855543A CN 116855543 A CN116855543 A CN 116855543A CN 202310888889 A CN202310888889 A CN 202310888889A CN 116855543 A CN116855543 A CN 116855543A
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了一种猪细小病毒病、猪丹毒二联亚单位疫苗及制备方法,从而制备猪细小病毒病、猪丹毒二联亚单位疫苗。本发明使用的昆虫‑杆状病毒真核表达系统安全、高效,实现了PPV VP2和Ery SpaA高水平、高纯度的表达,得到的蛋白结构状态与天然结构状态完全一致,最大程度地保留了蛋白的功能和免疫原性;所制备的猪细小病毒病、猪丹毒二联亚单位疫苗具有高度的免疫原性和安全性,能够有效激活机体的免疫反应,刺激免疫靶动物猪产生高水平的保护性中和抗体,同时对猪细小病毒病、猪丹毒发挥优异的免疫保护效果;与商业化的猪细小病毒病、猪丹毒灭活疫苗(NADL‑2株+2型R32E11株)相比,具有更好的免疫保护效果。
Description
技术领域
本发明涉及亚单位疫苗领域,更具体涉及一种猪细小病毒病、猪丹毒二联亚单位疫苗及制备方法。
背景技术
猪细小病毒(Porcine parvovirosis,PPV)是引起猪繁殖障碍的重要病原,不同品系和日龄的家猪、野猪均可感染。猪细小病毒呈季节性流行,春夏产仔季节多发,母猪感染后主要表现为流产,产弱仔、死胎、木乃伊胎,仔猪感染后表现为消瘦、生长受阻、皮炎、非化脓性心肌炎等。PPV属于细小病毒科、细小病毒属,为无囊膜单股DNA病毒,基因组大小为4.0~6.3kb,编码的衣壳蛋白包括VP1、VP2和VP3。其中VP2是PPV主要结构蛋白,在聚集和包装子代单链DNA中发挥重要作用,且其N端肽段可诱导机体产生中和抗体,对PPV的免疫原性和毒力有较大影响。该病毒外界抵抗力较强,对常见消毒剂、强碱、强酸、热等均具有较高的抵抗力,但2%火碱液、0.5%漂白粉5分钟即可使猪细小病毒失活。
研究显示,一些猪群中猪细小病毒病的检出率可达85%,对我国养猪业造成巨大损失。猪群一旦感染则很难清除,目前尚无有效治疗方法,因此猪细小病毒病的防治仍以疫苗接种为主。我国考虑到生物安全等问题,目前使用的疫苗均为灭活疫苗,但灭活疫苗诱导产生抗体所需时间长、抗体水平不如基因工程疫苗。此外,灭活疫苗免疫后,猪群仍可能持续排毒、不断感染,不利于养殖业发展。目前我国基因工程PPV疫苗仅限于实验室研究,尚未商品化应用。
猪丹毒(Swine Erysipelas)是猪丹毒杆菌(Erysipelothrix rhusiopathiae)引起的,急性型表现为败血症并伴有皮肤菱形损害,慢性型表现为非化脓性关节炎及增生性心内膜炎。猪丹毒易发于夏秋高温潮湿季节,2~6月龄猪最为易感。猪丹毒杆菌为革兰氏阳性菌,形状细长或略弯曲,两端钝圆,无荚膜,不形成芽孢,不能运动。猪丹毒杆菌对热敏感,但在腌制、熏制、冷冻和干燥肉中稳定存活。猪丹毒杆菌可通过皮肤伤口感染人,引起局部性蜂窝组织炎,局限或弥漫性丹毒样皮肤病变、进一步发展为感染性心内膜炎及菌血症。
目前疫苗免疫是防控猪丹毒的主要手段,《中国兽药典》2020年版收录的猪丹毒相关疫苗中减毒活疫苗3个、灭活疫苗2个。由于减毒活疫苗对猪体健康状态有相对严苛的要求,且免疫效果很大程度上受到抗生素的影响,从而给免疫程序和用药治疗的制订实施带来一定困难。因此,饲养端更倾向于猪丹毒灭活疫苗。但是,猪丹毒灭活疫苗在使用过程中也暴露一些缺陷,如免疫后易引起动物局部炎症及毒性反应,且在制备过程中存在毒素外泄或灭活不彻底等生物安全隐患。因此,开发成分明确单一、安全性佳、免疫原性强的猪丹毒杆菌亚单位疫苗迫在眉睫。
目前,针对这两种疾病的二联苗仅有西班牙海博莱生物大药厂生产的猪细小病毒病、猪丹毒二联灭活疫苗,灭活疫苗由于失活过程中某些抗原特性的改变导致疫苗效力降低,且不能在体内繁殖,因此对机体的保护效果和持续时间有限,所以该疫苗存在诱导抗体产生时间长、抗体水平不稳定等缺点。
发明内容
为解决上述问题,本发明通过基因工程方法制备的亚单位疫苗可避免上述问题,为猪细小病毒病、猪丹毒二联苗提供新方法。即选择具备生物安全性高、易规模放大、成本低等优点的杆状病毒-昆虫细胞表达系统制备了高效表达猪细小病毒VP2和猪丹毒SpaA蛋白的重组病毒,以及利用重组杆状病毒制备猪细小病毒病、猪丹毒二联亚单位疫苗。
本发明的目的之一是提供一种猪细小病毒病、猪丹毒二联亚单位疫苗的制备方法,基于重组杆状病毒表达系统制备猪细小病毒VP2和猪丹毒杆菌SpaA蛋白。
所述重组杆状病毒表达载体的制备步骤包括:(1)根据杆状病毒的密码子偏爱性对猪细小病毒VP2和猪丹毒杆菌SpaA密码子序列进行优化;(2)构建重组杆状病毒载体pFASTBac-PPV VP2、pFASTBac-Ery SpaA和重组穿梭杆粒rBac-PPV VP2和rBac-Ery SpaA;和(3)在昆虫细胞中表达猪细小病毒VP2和猪丹毒杆菌SpaA蛋白。
所述步骤(1)为,在优化设计的猪细小病毒VP2和猪丹毒杆菌SpaA的密码子序列的氨基端引入AcMNPV GP64信号肽序列,在羧基端引入8×His标签肽序列,得到PPV VP2的2条密码子优化序列(如SEQ ID NO.5和SEQ ID NO.6所示),得到Ery SpaA的2条密码子优化序列(如SEQ ID NO.7和SEQ ID NO.8所示)。
所述步骤(2)为,将所述步骤(1)获得的目标基因片段1,插入到经BamHⅠ/SalⅠ双酶切后进行胶回收得到的杆状病毒载体pFastBac1中,分别获得重组转移质粒pFASTBac-PPVVP2和pFASTBac-Ery SpaA,再进行PCR鉴定,筛选出阳性克隆,测序鉴定正确,获得重组杆状病毒载体pFASTBac-PPV VP2和pFASTBac-Ery SpaA;
所述步骤(2)还包括,将重组杆状病毒载体pFASTBac-PPV VP2和pFASTBac-ErySpaA再分别转入感受态DH10Bac中,冰浴30分钟后,42℃热激30秒,再冰浴5分钟,加入500ul无抗性的SOC,37℃复苏4小时,涂布含卡那霉素、庆大霉素和四环素的LB平板中,37℃继续培养24-48小时,通过蓝白斑筛选阳性菌落,提取获得重组穿梭杆粒rBac-PPV VP2和rBac-Ery SpaA。
所述步骤(3)为,将步骤(2)提取的重组穿梭杆粒rBac-PPV VP2和rBac-Ery SpaA转染到sf9昆虫细胞中,置27℃培养,待48-72小时细胞病变后,收集细胞培养上清即可获得重组杆状病毒Ac-PPV VP2和重组杆状病毒Ac-Ery SpaA;
所述步骤(3)还包括,将上述细胞培养上清获得的重组杆状病毒,接毒至昆虫细胞High Five,取接毒后72-96小时的细胞上清液,经离心和亲和层析法得到猪细小病毒VP2和猪丹毒杆菌SpaA蛋白。
本发明的目的之二是提供一种猪细小病毒和猪丹毒杆菌二联亚单位疫苗及其制备方法。
所述疫苗包括重组杆状病毒表达系统制备猪细小病毒VP2和猪丹毒杆菌SpaA蛋白以及佐剂,所述PPV VP2的优化序列如SEQ ID NO.5和SEQ ID NO.6所示,所述Ery SpaA优化序列如SEQ ID NO.7和SEQ ID NO.8所示。所述佐剂为无菌201佐剂。
所述疫苗的制备方法包括以下步骤,将所述纯化得到的PPV VP2和Ery SpaA蛋白等质量混合,混合后的蛋白与无菌佐剂按照(2~5):1的质量比乳化混合制备成二价亚单位疫苗,使每毫升疫苗中的PPV VP2和Ery SpaA抗原含量均为40μg。
相对于现有技术,本发明的有益效果是:
1.本发明根据昆虫细胞密码子偏好性,在对猪细小病毒PPV VP2基因和猪丹毒杆菌SpaA基因密码子优化的基础上,利用昆虫-杆状病毒表达系统对猪细小病毒VP2和猪丹毒杆菌SpaA蛋白进行表达,为猪细小病毒VP2与猪丹毒杆菌SpaA蛋白二联基因工程疫苗的制备提供技术支持。同时昆虫细胞大规模悬浮培养技术成熟与应用,使得杆状病毒表达系统的应用更加广泛。杆状病毒载体表达系统使用P10启动子或PH启动子,将外源目的基因以单拷贝或多拷贝形式插入到启动子下游,通过酶切酶连或酶切重组的方法获得重组杆状病毒,这种重组杆状病毒在昆虫细胞或虫体内感染的同时,使外源基因得到高效表达。即本发明利用昆虫-杆状病毒表达系统对PPV VP2和Ery SpaA蛋白进行表达,为猪细小病毒病、猪丹毒二联亚单位疫苗的制备提供技术基础。
2.本发明使用的昆虫-杆状病毒真核表达系统安全、高效,且通过人工密码子优化,猪细小病毒VP2和猪丹毒杆菌SpaA基因的氨基端AcMNPV GP64信号肽序列的引入,有效提高两者表达水平;羧基端8×His标签肽序列的引入,不仅构成表位有利于后续检测,同时构成独特的结构特征,有利于重组蛋白的纯化。实现了PPV VP2和Ery SpaA高水平、高纯度的表达,得到的蛋白结构状态与天然结构状态完全一致,最大程度地保留了蛋白的功能和免疫原性。
3.本发明制备的猪细小病毒病、猪丹毒二联亚单位疫苗具有高度的免疫原性和安全性,能够有效激活机体的免疫反应,刺激免疫靶动物猪产生高水平的保护性中和抗体,同时对猪细小病毒病、猪丹毒发挥优异的免疫保护效果。
4.本发明提供的猪细小病毒病、猪丹毒二联亚单位疫苗,与单苗相比,可减少人工成本、减少疫苗的接种次数,降低动物因免疫产生的应激反应,同时降低生产成本等。
5.本发明提高的猪细小病毒病、猪丹毒二联亚单位疫苗,与商业化的猪细小病毒病、猪丹毒灭活疫苗(NADL-2株+2型R32E11株)相比,具有更好的免疫保护效果。同时避免了毒素外泄、灭活不彻底等一系列安全隐患。
附图说明
图1为实施例1中重组转移载体的质粒PCR鉴定图。其中,图1A:pFASTBac-PPV VP2重组质粒PCR鉴定图M:DL2000;1:pFASTBac-PPV VP2-1;2:pFASTBac-PPV VP2-2。图1B:pFASTBac-Ery SpaA重组质粒PCR鉴定图M:DL2000;1:pFASTBac-Ery SpaA-1;2:pFASTBac-Ery SpaA-2。
图2为实施例2中Ac-PPV VP2表达纯化蛋白的SDS-PAGE检测结果图;其中,M:彩色预染蛋白marker(10-180kDa);1:Ac-PPV VP2-1;2:Ac-PPV VP2-2。
图3为实施例2中Ac-Ery SpaA表达纯化蛋白的SDS-PAGE检测结果图;其中,M:彩色预染蛋白marker(10-180kDa);1:Ac-Ery SpaA-1;2:Ac-Ery SpaA-2。
图4为实施例2中Ac-PPV VP2和Ac-Ery SpaA表达纯化蛋白的Western Blotting检测结果图;其中,M:彩色预染蛋白marker(10-180kDa);1:Ac-Ery SpaA;2:Ac-PPV VP2。
图5为实施例4中PPV VP2抗体水平检测结果。
图6为实施例5中PPV VP2抗体水平检测结果。
具体实施方式
以下结合具体实施例来进一步描述本发明,本发明的优点和特点将会随着描述而更为清楚。但这些实施例仅是范例性的,并不对本发明的范围构成任何限制。本领域技术人员应该理解的是,在不偏离本发明的精神和范围下可以对本发明的细节和形式进行修改或替换,但这些修改和替换均落入本发明的保护范围内。
实施例1构建携带PPV VP2或Ery SpaA蛋白编码基因的重组杆状病毒表达系统
(一)构建重组转移载体pFASTBac-PPV VP2和pFASTBac-Ery SpaA
1.PPV VP2和Ery SpaA基因的密码子优化和合成
为实现PPV VP2和Ery SpaA蛋白的高效表达,本发明首先根据杆状病毒的密码子偏爱性对相关序列进行优化。本发明发现,采用常规的密码子优化软件或简单遵循昆虫-杆状病毒表达系统密码子的偏爱性进行PPV VP2和Ery SpaA基因的密码子优化,得到蛋白的表达水平和纯度并不能满足大量疫苗生产制备的要求,且不同程度的密码子优化得到的编码序列的表达水平差异较大,而密码子优化位置和优化程度与表达水平之间并无明显规律。
本实施例针对表达PPV VP2和Ery SpaA的基因,分别设计不同密码子,以优化序列中的2条序列为例,进行说明。其中针对PPV VP2的2条密码子优化序列分别如SEQ ID NO.1和SEQ ID NO.2所示;针对Ery SpaA的2条密码子优化序列分别如SEQ ID NO.3和SEQ IDNO.4所示。
分别在上述密码子优化序列的氨基端引入AcMNPV GP64信号肽序列,以提高蛋白的表达量;在羧基端引入8×His标签肽序列,便于后续检测和重组蛋白的纯化。
针对PPV VP2的2条密码子优化序列(SEQ ID NO.1和SEQ ID NO.2)分别引入信号肽和标签得到如SEQ ID NO.5和SEQ ID NO.6所示的序列。针对Ery SpaA的2条密码子优化序列(SEQ ID NO.3和SEQ ID NO.4)分别引入信号肽和标签得到的SEQ ID NO.7和SEQ IDNO.8所示的序列。
人工合成SEQ ID NO.5、SEQ ID NO.6所示的片段,以及SEQ ID NO.7、SEQ ID NO.8所示的片段,得到PPV VP2-1、PPV VP2-2和Ery SpaA-1、Ery SpaA-2。将合成的上述目的基因连接质粒pUC18-T上,获得pUC18-PPV VP2-1、pUC18-PPV VP2-2和pUC18-Ery SpaA-1、pUC18-Ery SpaA-2。
2.构建重组转移载体pFASTBac-PPV VP2和pFASTBac-Ery SpaA
采用酶切酶连的方法构建重组转移载体pFASTBac-PPV VP2和pFASTBac-ErySpaA,方法如下:
(1)酶切:采用BamHⅠ和SalⅠ(购自TAKARA)分别对上述试验1中得到的pUC18-PPVVP2-1、pUC18-PPV VP2-2、pUC18-Ery SpaA-1和pUC18-Ery SpaA-2进行双酶切,获得人工合成基因PPV VP2-1、PPV VP2-2、Ery SpaA-1和Ery SpaA-2;同时,用相同的内切酶对转移载体pFAST进行酶切。双酶切反应体系如表1所示。
表1双酶切反应体系
载体 | 3ug |
BamHⅠ | 2ul |
SalⅠ | 2ul |
10×Buffer | 4ul |
水 | 补充至40ul |
37℃酶切3小时。酶切后用琼脂糖核酸电泳分离片段,并切胶回收。
(2)连接:将上述得到的PPV VP2-1、PPV-VP2-2、Ery SpaA-1和Ery SpaA-2基因片段分别与载体pFAST进行连接,连接反应体系如表2所示。
表2连接反应体系
片段 | 12ul |
载体 | 5ul |
T4 Ligase | 1ul |
10×T4 Ligase Bufer | 2ul |
22℃连接4小时。
(3)转化:将连接产物转入大肠杆菌感受态DH5α中,涂平板。
(4)鉴定重组质粒pFASTBac-PPV VP2-1、pFASTBac-PPV VP2-2、pFASTBac-ErySpaA-1和pFASTBac-Ery SpaA-2,从平板上挑取单克隆菌落,采用质粒抽提试剂盒提取质粒,用PCR进行鉴定(引物如表3),结果表明重组质粒构建成功,如图1A、图1B所示,A为pFASTBac-PPV VP2质粒PCR鉴定图,其中,M:DNA Marker DL2000,1:pFASTBac-PPV VP2-1质粒,2:pFASTBac-PPV VP2-2质粒;B为pFASTBac-Ery SpaA质粒PCR鉴定图,其中,M:DNAMarker DL2000;1:pFASTBac-Ery SpaA质粒,2:pFASTBac-Ery SpaA-2质粒。
表3PCR鉴定引物序列
(二)构建重组杆状病毒Ac-PPV VP2和Ac-Ery SpaA
1.构建重组穿梭载体
取实施例1中试验2的4ul重组转移质粒pFASTBac-PPV VP2-1、pFASTBac-PPV VP2-2、pFASTBac-Ery SpaA-1和pFASTBac-Ery SpaA-2分别转入感受态DH10Bac中,冰浴30分钟后,42℃热激30秒,再冰浴5分钟,加入500ul无抗性的SOC,37℃复苏4小时,涂布三抗(卡那霉素、庆大霉素和四环素)LB平板中,37℃继续培养24-48小时,通过蓝白斑筛选阳性菌落,提取重组杆粒rBac-PPV VP2和rBac-Ery SpaA,立即使用或-20℃保存。
2.获得重组杆状病毒
利用脂质体转染法,将提取的重组穿梭载体转染到sf9昆虫细胞中,置27℃培养。待48-72小时细胞病变后,收集细胞培养上清即可获得重组杆状病毒Ac-PPV VP2-1、Ac-PPVVP2-2、Ac-Ery SpaA-1和Ac-Ery SpaA-2,立即使用或将收获的重组杆状病毒置-80℃避光保存。
实施例2目的蛋白PPV VP2和Ery SpaA的表达与纯化
1.目的蛋白PPV VP2和Ery SpaA的表达
将实施例1收获的重组杆状病毒接种于悬浮培养的昆虫细胞High Five中,接毒剂量为0.01MOI,细胞密度为0.8×106cells/ml,细胞体积为400ml。72-96小时后收获细胞培养上清进行Western blotting检测目的蛋白PPV VP2和Ery SpaA的表达,图2、图3的结果表明,重组杆状病毒Ac-PPV VP2-1、Ac-PPV VP2-2、Ac-Ery SpaA-1和Ac-Ery SpaA-2均可以表达。
2.目的蛋白的纯化
纯化方法采用常规镍柱亲和层析法。具体操作步骤如下:取接毒后72-96小时的细胞上清液,10000rpm离心取出细胞碎片,用0.45μm的滤膜过滤除去细小杂质;过滤后的上清与镍柱进行结合过柱;洗杂缓冲液(50mM咪唑,20mM Tris,200mM NaCl)过柱洗杂;洗脱缓冲液(300mM咪唑,20mM Tris,200mM NaCl)过柱洗脱;洗脱液用透析缓冲液(20mM Tris,200mMNaCl)4℃透析过夜,得到目的蛋白。
将重组杆状病毒Ac-PPV VP2-1、Ac-PPV VP2-2、Ac-Ery SpaA-1和Ac-Ery SpaA-2表达蛋白经上述镍柱亲和层析纯化后进行SDS-PAGE检测纯化后的蛋白。Ac-PPV VP2-1和Ac-PPV VP2-2的SDS-PAGE检测结果如图2所示,其中,M:彩色预染蛋白marker(10-180kDa);1:Ac-PPV VP2-1;2:Ac-PPV VP2-2。Ac-Ery SpaA-1和Ac-Ery SpaA-2的SDS-PAGE检测结果如图3所示,其中,M:彩色预染蛋白marker(10-180kDa);1:Ac-Ery SpaA-1;2:Ac-Ery SpaA-2。
如图2的结果显示,Ac-PPV VP2-2表达的VP2蛋白得率为50μg/ml,而Ac-PPV VP2-1表达的VP2蛋白得率提高至180μg/ml;同样地,如图3的结果显示,Ac-Ery SpaA-2表达的VP2蛋白得率为20μg/ml,而Ac-Ery SpaA-1表达的VP2蛋白得率提高至100μg/ml。以上结果表明,本发明经特异性的人工密码子优化、添加信号肽和标签肽序列的得到的如SEQ ID NO.5所示的PPV VP2序列和如SEQ ID NO.7所示的SpaA序列能够显著提高蛋白的表达水平,实现高水平分泌表达。
表达效果最优的Ac-PPV VP2-1和Ac-Ery SpaA-1的表达纯化蛋白的WesternBlotting检测结果如图4所示,其中,M:彩色预染蛋白marker(10-180kDa);1:Ac-Ery SpaA;2:Ac-PPV VP2。
后续二联亚单位疫苗或对照的单价亚单位疫苗的制备均采用Ac-PPV VP2-1和Ac-Ery SpaA-1表达纯化的VP2和SpaA蛋白。
实施例3猪细小病毒病、猪丹毒二联亚单位疫苗的制备
将实施例2纯化得到的PPV VP2和Ery SpaA蛋白测定浓度后,等质量混匀,再与无菌201佐剂(购自SEPPIC)按照(2~5):1的质量比乳化制备成二价亚单位疫苗,使每毫升疫苗中的PPV VP2和Ery SpaA抗原含量均为40μg,置4℃保存备用。
实施例4猪细小病毒病、猪丹毒二联亚单位疫苗对小鼠的安全性和免疫效果试验
1.猪细小病毒病、猪丹毒二联亚单位疫苗对小鼠的安全性试验
购买16~18g雌性Balb/C小鼠10只,随机分为A、B组,每组5只。A组每只小鼠皮下注射0.3ml实施例3制备的二联亚单位疫苗;B组每只小鼠注射0.3ml透析缓冲液(20mM Tris,200mM NaCl);连续观察14日,两组小鼠采食、饮水均正常,且无临床反应。说明实施例3制备的二联亚单位疫苗对小鼠是安全的。
2.猪细小病毒病、猪丹毒二联亚单位疫苗对小鼠的免疫原性试验
取6~8周龄的雌性Balb/C小鼠20只,随机分为A、B组,各10只。A、B组每只小鼠皮下注射0.2ml实施例3制备的二联亚单位疫苗。另取10只同条件小鼠分为C组和D组,不接种疫苗作为对照组。免疫14日后,A组10只小鼠和C组5只对照组小鼠各皮下注射1000MLD的猪丹毒1型和2型(各1株)强毒菌的混合菌液,D组5只对照小鼠各皮下注射1MLD的上述混合菌液,连续观察28日,A、B组小鼠均10/10健活,C组注射1000MLD的对照小鼠全部死亡,D组注射1MLD的对照小鼠4/5死亡。
在免疫后0日、14日、28日采血分离血清进行血凝效价测定,结果表明二联亚单位疫苗可诱导小鼠产生较高水平PPV HI抗体效价(图5)。由图5可知,B组试验结果说明A组攻猪丹毒后,对小鼠体内猪细小病毒病抗体水平的产生没有影响,可知猪细小病毒抗体水平主要是猪细小病毒病、猪丹毒二联亚单位疫苗免疫导致的结果,与攻猪丹毒无关。虽然28天后B组小鼠体内猪细小病毒抗体水平略高,但经生物统计分析,其与A组无显著性差异,说明A组攻猪丹毒后,不影响猪细小病毒病、猪丹毒二联亚单位疫苗对猪细小病毒抗体的产生。综上所述,本发明的二联亚单位疫苗对小鼠具有良好的免疫原性。
实施例5猪细小病毒病、猪丹毒二联亚单位疫苗对靶动物猪的安全性和免疫效果试验
1.猪细小病毒病、猪丹毒二联亚单位疫苗对靶动物猪的安全性试验
购买5~6周龄阴性猪10头,随机分为A、B两组,每组5头。A组每头猪免疫4.0ml实施例3制备的二联亚单位疫苗,皮下注射;B组不免疫,作为阴性对照组。连续观察14日,观察期内,所有猪只精神、食欲均正常,未出现任何局部或全身不良反应,说明该疫苗对靶动物猪是安全的。
2.猪细小病毒病、猪丹毒二联亚单位疫苗对靶动物猪的免疫原性试验
购买5~6周龄阴性猪25头,其中将20头随机分为2组,每组10头,另5头不接种作为对照。第1组各皮下注射1.0ml(1头份)实施例3制备的二联亚单位疫苗,第2组各颈部肌肉注射1头份猪细小病毒病、猪丹毒灭活疫苗(NADL-2株+2型R32E11株),对照组不接种疫苗。接种后14日,第1组、第2组以及对照组的所有猪均静脉注射1MLD的猪丹毒杆菌1型和2型(各1株)强毒混合菌液。
连续观察28日,第1组猪只10/10健活;第2组猪只10/10健活,但个别猪只体温反应,且2/10稽留时间超过1日;对照组猪只5/5发病,出现典型的猪丹毒症状,有明显的体温反应,4/5稽留时间超过1日,且2/5死亡。在免疫后0日、14日、28日采血分离血清进行血凝效价测定,结果表明猪细小病毒病、猪丹毒二联亚单位疫苗可诱导仔猪产生较高水平PPV HI抗体效价,并显著高于市面使用的猪细小病毒病、猪丹毒灭活疫苗(NADL-2株+2型R32E11株)(图6)。综上所述,本发明的二联亚单位疫苗具有良好的免疫原性,优于猪细小病毒病、猪丹毒灭活疫苗(NADL-2株+2型R32E11株)的免疫效果。
Claims (7)
1.一种猪细小病毒病、猪丹毒二联亚单位疫苗的制备方法,基于重组杆状病毒表达载体制备猪细小病毒VP2和猪丹毒杆菌SpaA蛋白,其特征在于,包括以下步骤:
所述重组杆状病毒表达载体的制备步骤包括:(1)根据杆状病毒的密码子偏爱性对猪细小病毒VP2和猪丹毒杆菌SpaA密码子序列进行优化;(2)构建重组杆状病毒载体pFASTBac-PPV VP2、pFASTBac-Ery SpaA和重组穿梭杆粒rBac-PPV VP2和rBac-Ery SpaA;和(3)在昆虫细胞中表达猪细小病毒VP2和猪丹毒杆菌SpaA蛋白。
2.根据权利要求1所述的方法,其特征在于,所述步骤(1)为,在优化设计的猪细小病毒VP2和猪丹毒杆菌SpaA的密码子序列的氨基端引入AcMNPV GP64信号肽序列,在羧基端引入8×His标签肽序列,得到PPV VP2的2条密码子优化序列如SEQ ID NO.5和SEQ ID NO.6所示,得到Ery SpaA的2条密码子优化序列如SEQ ID NO.7和SEQ ID NO.8所示。
3.根据权利要求1所述的方法,其特征在于,所述步骤(2)为,将所述步骤(1)获得的目标基因片段1,插入到经BamHⅠ/SalⅠ双酶切后进行胶回收得到的杆状病毒载体pFastBac1中,分别获得重组转移质粒pFASTBac-PPV VP2和pFASTBac-Ery SpaA,再分别进行PCR鉴定,筛选出阳性克隆,测序鉴定正确,获得重组杆状病毒载体pFASTBac-PPV VP2和pFASTBac-Ery SpaA;
所述步骤(2)还包括,将重组杆状病毒载体pFASTBac-PPV VP2和pFASTBac-Ery SpaA分别转入感受态DH10Bac中,冰浴30分钟后,42℃热激30秒,再冰浴5分钟,加入500ul无抗性的SOC,37℃复苏4小时,涂布含有卡那霉素、庆大霉素和四环素的LB平板中,37℃继续培养24-48小时,通过蓝白斑筛选阳性菌落,提取获得重组穿梭杆粒rBac-PPV VP2和rBac-ErySpaA。
4.根据权利要求1所述的方法,其特征在于,所述步骤(3)为,将步骤(2)提取的重组穿梭杆粒rBac-PPV VP2和rBac-Ery SpaA转染到sf9昆虫细胞中,置27℃培养,待48-72小时细胞病变后,收集细胞培养上清即可获得重组杆状病毒Ac-PPV VP2和重组杆状病毒Ac-ErySpaA;
所述步骤(3)还包括,将上述细胞培养上清获得的重组杆状病毒,接毒至昆虫细胞HighFive,取接毒后72-96小时的细胞上清液,经离心和亲和层析法得到猪细小病毒VP2和猪丹毒杆菌SpaA蛋白。
5.根据权利要求1所述的方法,其特征在于,步骤(3)之后还包括将所述纯化得到的PPVVP2和Ery SpaA蛋白等质量混合,混合后的蛋白与无菌佐剂按照(2~5):1的质量比乳化混合制备成二价亚单位疫苗,使每毫升疫苗中的PPV VP2和Ery SpaA抗原含量均为40μg。
6.一种猪细小病毒和猪丹毒杆菌二联亚单位疫苗,其特征在于,包括权利要求1所述重组杆状病毒表达系统制备猪细小病毒VP2和猪丹毒杆菌SpaA蛋白以及佐剂,所述PPV VP2的优化序列如SEQ ID NO.5和SEQ ID NO.6所示,所述Ery SpaA优化序列如SEQ ID NO.7和SEQID NO.8所示。
7.根据权利要求6所述的猪细小病毒和猪丹毒杆菌二联亚单位疫苗,其特征在于,所述佐剂为无菌201佐剂。
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