CN111793650A - 嵌合南非2型口蹄疫病毒多表位基因猪细小病h毒样颗粒的制备方法及应用 - Google Patents
嵌合南非2型口蹄疫病毒多表位基因猪细小病h毒样颗粒的制备方法及应用 Download PDFInfo
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Abstract
本发明根据计算机软件模拟结果，在猪细小病毒PPV VP2蛋白Loop2区和Loop4区后嵌入SAT2FMDV B细胞表位(VYTKAAAAIRGDRAALAAKYAD TNHTLPPTFNFGYVTVDK)，N端嵌入两个T细胞表位(NVQEGRRKHTDVAFLLDRST)串联。根据昆虫细胞嗜性优化合成嵌合基因GS1927OP，克隆到载体pFastBacTMDual上，转化感受态细胞DH10Bac，获得重组杆粒rBacmidN(T1)2L2B4B，转染sf9细胞收获重组杆状病毒rBacN(T1)2L2B4B，用其感染HF细胞，使用间接免疫荧光IFA针对嵌合表位和VP2蛋白进行检测，具有特异性荧光。Western‑blot针对B、T细胞表位和VP2蛋白进行检测，在72KDa出现一条特异性条带。透射电镜TEM观察，重组蛋白N(T1)2L2B4B能够自我组装成病毒样颗粒。本发明旨在为南非2型口蹄疫制备储备疫苗，并能在此基础上达到同时预防南非2型口蹄疫和猪细小病的目的。
Description
技术领域
本发明属于生物工程技术领域，尤其是一种嵌合南非2型口蹄疫病毒多表位基因猪细小病毒样颗粒的组装及应用。
背景技术
南非型(Southern Africa Territories，SAT)FMDV是口蹄疫病毒属的成员。从历史上 看，SAT FMD从未蔓延过赤道，一些学者认为南非的口蹄疫传播需要非洲特殊的生活环 境。目前，SAT FMD尚未在中国发生，中国的FMD研究仅限于O，A，C和Asia 1型，SAT FMD的研究包括检测技术，疫苗研究处于空白状态。
猪细小病毒(Porcine parvovirus，PPV)属于细小病毒科，细小病毒属，能导致初产母猪及血清学阴性的经产母猪发生流产、不孕、产死胎、木乃伊胎及弱仔等。PPV一般是地方性流行，其产生抗体的过程如下：大多数母猪在分娩之前接种过疫苗，通过初乳将高水平的PPV抗体传递给仔猪，这种被动免疫在4-6个月的时间内持续处于较低水平。随着抗体的减少，仔猪被感染获得主动免疫的可能性越来越大。反复接触病毒可能会导致主动免疫持续终生。然而，由于各种各样的原因，一些母猪在初产时才会接触PPV，发生胎盘感染和生殖功能衰竭。经产母猪由于环境污染也会导致PPV再次感染。学者们认为PPV的水平传播是健康猪通过摄入或吸入携带病毒的分泌物和排泄物引起的。在大多数情况下，PPV感染不会在未怀孕的成年猪或仔猪中引起明显的临床症状，其菌株毒力的定义是它导致生殖失败的严重程度。实验和流行病学研究表明，妊娠前期PPV感染可导致生殖失败。妊娠70天后感染的仔猪会产生抗体，通常在感染后存活下来。成年公猪感染PPV时几乎没有临床症状，只有急性感染时，可以从其阴囊淋巴结和精液中分离到PPV。公猪可以作为PPV的携带者，在感染和未感染的母猪之间传播。PPV病毒粒子呈二十面体对称，直径介于20～25nm。基因组为单股负链DNA，5000bp左右，编码结构蛋白VP1、VP2、VP3。VP2拥有4个Loop环，能自组装形成病毒样颗粒(Virus-like Particles， VLPs)。Hurta-do等人分别使犬细小病毒(CPV)VP2Loop1、2、3、4内的相应基因缺失，再用杆状病毒表达系统表达，结果Loop1、3、4的缺失突变株均不能表达出VLPs，而Loop2 (217～234)的缺失突变株则能装配出规则的病毒样颗粒，进一步研究证明Loop2能容纳外源抗原决定簇，Loop2区域作为外源基因的主要插入位点组装嵌合型VLPs。现有的研究表明，VP2的N端含有甘氨酸富集区，Chen等人将其缺失，并不影响VLPs的组装，且免疫原性也不受影响。Fan将O型口蹄疫病毒(Foot and Mouth Disease Virus，FMDV)的 B细胞表位按照不同的设计缺失并插入到Loop2中，成功制备了嵌合型VLPs。Pan等人将 O型FMDV的多表位同时缺失插入到VP2的非必需区，成功构建了VLPs。他们还在VP2 的N端插入PCV2主要T细胞抗原表位，在腺病毒表达载体中成功表达出嵌合VLPs，证明了PPVVP2的N端能够容纳60个氨基酸不影响VLPs的装配。各种研究表明，PPV VP2 可以作为一种表位载体携带展示外源表位，诱导体液和细胞免疫，成为研制多表位基因工程疫苗给力工具。
发明内容
为解决上述技术问题，本发明提供一种嵌合南非2型口蹄疫病毒多表位基因猪细小病毒样颗粒的制备方法，包括重组杆状病毒rBacN(T1)2L2B4B的制备和 N(T1)2L2B4B-VLPs的组装；
所述重组杆状病毒rBacN(T1)2L2B4B的制备方法，包括以下步骤:
(1)N(T1)2L2B4B基因的扩增及重组供体质粒的构建
根据优化合成含有嵌合序列的质粒GS1927OP，设计引物P1/P2，扩增得到嵌合基因N(T1)2L2B4B；回收纯化目的条带N(T1)2L2B4B，将目的片段和pFastBacTMDual载体分别进行BamH I、Pst I双酶切，回收清洁后连接，化学法转DH5α感受态细胞，筛选阳性克隆，提取重组供体质粒pFastN(T1)2L2B4B；
表1引物序列及酶切位点
注：下划线处为保护碱基和酶切位点
(2)N(T1)2L2B4B重组穿梭质粒的构建与鉴定
将pFastN(T1)2L2B4B供体质粒转化至DH10Bac感受态细胞，在含有庆大霉素(7 μg/m)，卡那霉素(50μg/mL)，四环素(10μg/mL)，IPTG(40μg/mL)和X-gal(100μg/mL) 的固体LB平板上37℃倒置培养48h进行蓝白斑筛选，挑取白色菌落，置于三种抗生素液体培养基中摇菌；抽提DNA，以M13F/R载体通用引物进行PCR检测，筛选阳性克隆杆粒，命名为rBacmidN(T1)2L2B4B，并提取rBacmidN(T1)2L2B4B重组杆粒，用于细胞转染；
(3)N(T1)2L2B4B重组杆状病毒的获得与鉴定
将rBacmidN(T1)2L2B4B杆粒转染生长状态良好的sf9细胞，28℃恒温培养箱培养36～72h，待出现细胞病变后，收集上清作为种毒，命名为rBacN(T1)2L2B4B。
优选地，使用毒株rBacN(T1)2L2B4B感染HF细胞进行重组蛋白N(T1)2L2B4B表达，27℃无CO2细胞摇床125r/min震荡培养72h；离心收集细胞，超声破碎得到重组蛋白后，使用蔗糖密度梯度离心纯化后收取样品，得到N(T1)2L2B4B-VLPs病毒样颗粒。
嵌合南非2型口蹄疫病毒多表位基因猪细小病毒样颗粒在制备SAT2 FMDV储备疫苗的应用。
嵌合南非2型口蹄疫病毒多表位基因猪细小病毒样颗粒在制备PPV的新型疫苗的应用。
本发明根据计算机软件模拟结果，在猪细小病毒PPV VP2蛋白Loop2区和Loop4 区后嵌入SAT2 FMDV B细胞表位(VYTKAAAAIRGDRAALAAKYADTNHTLPPTFNF GYVTVDK)，N端嵌入两个T细胞表位(NVQEGRRKHTDVAFLLDRST)串联。根据昆虫细胞嗜性优化合成嵌合基因GS1927OP，克隆到载体pFastBacTMDual上，转化感受态细胞DH10Bac，获得重组杆粒rBacmidN(T1)2L2B4B，转染sf9细胞收获重组杆状病毒 rBacN(T1)2L2B4B，用其感染HF细胞，使用间接免疫荧光(indirect immunofluorescenc e assay，IFA)针对嵌合表位和VP2蛋白进行检测，具有特异性荧光。Western-blot针对 B、T细胞表位和VP2蛋白进行检测，在72KDa出现一条特异性条带。透射电镜(Tra nsmission Electron Micrograph，TEM)观察，重组蛋白N(T1)2L2B4B能够自我组装成病毒样颗粒。本发明可以应用在为南非2型口蹄疫制备储备疫苗，并能在此基础上达到一针两防的目的。
附图说明
下面结合附图对本发明作进一步详细的描述。
图1是PPV结构蛋白VP2简单示意图；
图2是VP2Loop结构的空间结构图。
图3是N(T1)2L2B4B嵌合基因插入位置示意图。
图4是N(T1)2L2B4B序列扩增结果图。
图5是重组供体质粒pFastN(T1)2L2B4B的双酶切鉴定结果图
图6是重组穿梭质粒的PCR引物鉴定结果图。
图7是rBacN(T1)2L2B4B感染HF细胞产生的CPE情况图。
图8是VP2蛋白表达的Western-bolt检测结果图。
图9是SAT2 FMDV B细胞表位表达的Western-bolt检测结果图。
图10是SAT2 FMDV T细胞表位表达的Western-bolt检测结果图。
图11是IFA检测N(T1)2L2B4B蛋白在感染细胞中的表达图(400×)。
图12是PPV病毒粒子(25.0k×)、VP2-VLPs(40.0k×)、N(T1)2L2B4B-VLPs(25.0k×)透射电镜观察结果图。
具体实施方式
1.材料与方法
1.1嵌合基因N(T1)2L2B4B设计如图1-3所示：
1.2材料
PPV毒株、pFastBacTMDual载体、猪细小阳性血清和sf9、HF细胞由本实验室保存。感受态细胞DH5a、DH10Bac、限制性内切酶BamH I、Pst I均购自TaKaRa公司(大连)。Sf-900TM III SFM(1×)、EXPRESS
SFM(1×)、L-Glutamine 200mM(100×)及转染试剂盒Cellfectin@II Reagent购自Gibco公司。HRP标记的兔二抗、猪二抗，Anti-Swine IgG(H+L)购自Sigma公司。FITC标记山羊抗兔购自康为世纪。抗南非2型口蹄疫病毒B、T细胞表位的多克隆兔抗(简称：VYT、NVQ)由北京三卿生物科技有限公司制备。扩增引物P1/2和鉴定引物M13F/R均由西安擎科公司合成，具体序列见表1。
1.3N(T1)2L2B4B基因的扩增及重组供体质粒的构建
根据优化合成含有嵌合序列的质粒GS1927OP，运用生物信息学软件Primer 5.0设计引物P1/P2，扩增得到嵌合基因N(T1)2L2B4B。回收纯化目的条带N(T1)2L2B4B，将目的片段和pFastBacTMDual载体分别进行BamH I、Pst I双酶切，回收清洁后连接，化学法转 DH5α感受态细胞，筛选阳性克隆，提取重组供体质粒pFastN(T1)2L2B4B，并进行酶切、测序鉴定。
表1引物序列及酶切位点
Table 1Primer sequence and enzyme cutting site
注：下划线处为保护碱基和酶切位点，其中前面CG和AA为保护碱基。
1.4N(T1)2L2B4B重组穿梭质粒的构建与鉴定
将pFastN(T1)2L2B4B供体质粒转化至DH10Bac感受态细胞，在含有庆大霉素(7 μg/m)，卡那霉素(50μg/mL)，四环素(10μg/mL)，IPTG(40μg/mL)和X-gal(100μg/mL) 的固体LB平板上37℃倒置培养48h进行蓝白斑筛选，挑取白色菌落，置于三种抗生素液体培养基中摇菌。抽提DNA，以M13F/R(表1)载体通用引物进行PCR检测，筛选阳性克隆杆粒，命名为rBacmidN(T1)2L2B4B，并提取rBacmidN(T1)2L2B4B重组杆粒，用于细胞转染。
1.5N(T1)2L2B4B重组杆状病毒的获得与鉴定
参照脂质体转染试剂盒Cellfectin@II Reagent的说明书，将rBacmidN(T1)2L2B4B杆粒转染生长状态良好的sf9细胞，28℃恒温培养箱培养36～72h，待出现细胞病变后，收集上清，获得阳性重组杆状病毒，命名为rBacN(T1)2L2B4B。
1.6Western-blot鉴定N(T1)2L2B4B蛋白的表达
将重组杆状病毒rBacN(T1)2L2B4B接种HF细胞，感染72h后，离心收集细胞沉淀。加入PMSF并做超声处理，10,000r/min离心10min收集上清，用于Western-blot分析。用5％脱脂奶粉按1:100稀释PPV阳性血清(1:100稀释VYT兔多抗、1:100稀释NVQ兔多抗)为一抗，以5％脱脂奶粉按1:2000稀释HRP标记的抗猪IgG(1:300稀释HRP标记山羊抗兔)作为二抗，同样的方法处理正常HF细胞蛋白样品作为阴性对照。
1.7IFA鉴定NT1L2B4B蛋白的表达
将重组杆状病毒rBacN(T1)2L2B4B接种于生长状况良好的HF细胞，感染12h后，用PBS洗3次。4℃预冷的4％多聚甲醛室温固定15min后PBS洗3次，0.1％Triton-100X通透细胞膜10min后PBS洗3次。使用5％NBS 37℃封闭1h。5％NBS 1:200稀释PPV阳性血清(1:100稀释VYT、NVQ兔多抗)为一抗，37℃孵育1h，用PBST洗涤5次。5％NBS 1:500稀释Anti-Swine IgG(H+L)(1:300稀释FITC标记山羊抗兔)为二抗，37℃孵育1h，用PBST洗涤5次，置于荧光倒置显微镜下观察结果，以同样方法处理HF细胞为阴性对照。
1.8TEM观察N(T1)2L2B4B-VLPs的组装
使用重组杆状病毒rBacN(T1)2L2B4B感染HF细胞进行重组蛋白N(T1)2L2B4B表达，27℃无CO2细胞摇床125r/min震荡培养72h。离心收集细胞，超声破碎得到重组蛋白后，使用蔗糖密度梯度离心纯化后收取样品，经磷钨酸负染色处理，TEM观察病毒样颗粒的组装情况。
2结果
2.1N(T1)2L2B4B基因的扩增及重组供体质粒pFastN(T1)2L2B4B的鉴定
以质粒GS1927OP为模板，P1/P2为引物扩增N(T1)2L2B4B基因，获得约为1965bp 的目的条带(见图4)，与预计大小相符。筛选获得重组供体质粒pFastN(T1)2L2B4B，进行BamHI和Pst I双酶切验证，结果(见图5)显示，双酶切后有2条特异条带，其中一条为N(T1)2L2B4B基因约为1965bp，另一条为载体片段约为6000bp，均与预计大小相符。进一步对pFastN(T1)2L2B4B重组质粒进行测序验证，结果表明插入序列没有碱基突变，完全正确。
2.2N(T1)2L2B4B重组穿梭质粒及杆状病毒的获得
将pFastN(T1)2L2B4B重组供体质粒转化DH10Bac感受态细胞，筛选白色菌落纯化后摇菌。提取重组杆粒，经M13F/R(表1)引物PCR检测可以扩增到一约4525bp的目的条带，结果见图6。将重组杆粒rBacmidN(T1)2L2B4B脂质体法转染sf9细胞，28℃培养，细胞病表现为：细胞胀大变圆，细胞核不明显，结果见图7。
2.3Western-blot鉴定重组蛋白的表达
湿转法对重组蛋白N(T1)2L2B4B进行Western-blot鉴定(见图8、9、10)。检测结果显示，重组病毒感染的HF细胞样品中，有一约72KDa左右的特异条带，而正常细胞未出现该条带。表明嵌合蛋白N(T1)2L2B4B在HF细胞中成功表达，且表达产物具有一定的反应原性。
2.4IFA鉴定重组蛋白的表达
rBactN(T1)2L2B4B杆状病毒感染HF细胞，针对VP2蛋白进行免疫荧光检测后，rBactN(T1)2L2B4B感染的HF细胞出现红色荧光(见图11B)，针对SAT2 FMDV B细胞表位检测后，rBactN(T1)2L2B4B感染的HF细胞出现绿色荧光(见图11C)，针对SAT2 FMDV T细胞表位检测后，rBactN(T1)2L2B4B感染的HF细胞出现绿色荧光(见图11D)，而正常的HF细胞未出现荧光(见图11A)，表明嵌合的SAT2 FMDV B、T细胞表位基因与VP2 蛋白均在细胞中表达。
2.5.TEM观察N(T1)2L2B4B-VLPs
用TEM观察纯化的蛋白，结果显示直径为27nm左右，重组蛋白形态大小均与本实验室之前纯化的PPV病毒粒子(图12A)及组装的VP2-VLPs相似(图12B)，表明嵌合了SAT2 FMDVB、T细胞表位的VP2蛋白在HF细胞中成功折叠表达组装成VLPs，并且将其呈现在VLPs表面。
实施例1
嵌合南非2型口蹄疫病毒多表位基因猪细小病毒样颗粒的制备方法，包括重组杆状病毒N(T1)2L2B4B的制备和N(T1)2L2B4B-VLPs的组装；
所述重组杆状病毒N(T1)2L2B4B的制备方法，包括以下步骤:
(1)N(T1)2L2B4B基因的扩增及重组供体质粒的构建
根据优化合成含有嵌合序列的质粒GS1927OP，设计引物P1/P2，扩增得到嵌合基因N(T1)2L2B4B；回收纯化目的条带N(T1)2L2B4B，将目的片段和pFastBacTMDual载体分别进行BamH I、Pst I双酶切，回收清洁后连接，化学法转DH5α感受态细胞，筛选阳性克隆，提取重组供体质粒pFastN(T1)2L2B4B；
表1引物序列及酶切位点
下划线处为保护碱基和酶切位点
(2)N(T1)2L2B4B重组穿梭质粒的构建与鉴定
将pFastN(T1)2L2B4B供体质粒转化至DH10Bac感受态细胞，在含有庆大霉素(7 μg/m)，卡那霉素(50μg/mL)，四环素(10μg/mL)，IPTG(40μg/mL)和X-gal(100μg/mL) 的固体LB平板上37℃倒置培养48h进行蓝白斑筛选，挑取白色菌落，置于三种抗生素液体培养基中摇菌；抽提DNA，以M13F/R载体通用引物进行PCR检测，筛选阳性克隆杆粒，命名为rBacmidN(T1)2L2B4B，并提取rBacmidN(T1)2L2B4B重组杆粒，用于细胞转染；
(3)N(T1)2L2B4B重组杆状病毒的获得与鉴定
将rBacmidN(T1)2L2B4B杆粒转染生长状态良好的sf9细胞，28℃恒温培养箱培养36～72h，待出现细胞病变后，收集上清，获得阳性重组杆状病毒，命名为rBacN(T1)2L2B4B。
使用重组杆状病毒rBacN(T1)2L2B4B感染HF细胞进行重组蛋白N(T1)2L2B4B表达，27℃无CO2细胞摇床125r/min震荡培养72h；离心收集细胞，超声破碎得到重组蛋白后，使用蔗糖密度梯度离心纯化后收取样品，得到N(T1)2L2B4B-VLPs病毒样颗粒。
嵌合南非2型口蹄疫病毒多表位基因猪细小病毒样颗粒在制备SAT2 FMDV储备疫苗的应用。
嵌合南非2型口蹄疫病毒多表位基因猪细小病毒样颗粒在制备PPV的新型疫苗的应用。
本发明采用Bac-to-Bac表达系统，构建并表达根据昆虫细胞嗜性优化的嵌合蛋白，命名为N(T1)2L2B4B。Western-blot和间接免疫荧光(Indirect immunofluores-cenceassay， IFA)分析其反应原性，说明嵌合的B、T细胞表位在VP2蛋白中表达。透射电镜(Transmission Electron Micrograph，TEM)观察，其形成了与病毒粒子相似的颗粒，表明嵌合B细胞表位、T细胞表位没有影响VLPs的形成，并且成功展示在VLPs的表面。此N(T1)2L2 B4B-VLPs的成功组装，为SAT2 FMDV制备储备疫苗奠定了基础，并且为PPV的新型疫苗研制提供了理论基础。
最后应说明的是：以上所述仅为本发明的优选实施例而已，并不用于限制本发明，尽管参照前述实施例对本发明进行了详细的说明，对于本领域的技术人员来说，其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
序列表
<110> 中国农业科学院兰州兽医研究所
<120> 嵌合南非2型口蹄疫病毒多表位基因猪细小病H毒样颗粒的制备方法及应用
<130> 2020.7.6
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Claims (4)
1.嵌合南非2型口蹄疫病毒多表位基因猪细小病毒样颗粒的制备方法，其特征在于，包括重组杆状病毒rBacN(T1)2L2B4B的制备和N(T1)2L2B4B-VLPs的组装；
所述重组杆状病毒rBacN(T1)2L2B4B的制备方法，包括以下步骤:
(1)N(T1)2L2B4B基因的扩增及重组供体质粒的构建
根据昆虫细胞嗜性优化合成含有嵌合序列的质粒GS1927OP，设计引物P1/P2，扩增得到嵌合基因N(T1)2L2B4B；回收纯化目的条带N(T1)2L2B4B，将目的片段和pFastBacTMDual载体分别进行BamH I、PstI双酶切，回收清洁后连接，化学法转DH5α感受态细胞，筛选阳性克隆，提取重组供体质粒pFastN(T1)2L2B4B；
表1 引物序列及酶切位点
注：下划线处为保护碱基和酶切位点
(2)N(T1)2L2B4B重组穿梭质粒的构建与鉴定
将pFastN(T1)2L2B4B供体质粒转化至DH10Bac感受态细胞，在含有庆大霉素(7μg/m)，卡那霉素(50μg/mL)，四环素(10μg/mL)，IPTG(40μg/mL)和X-gal(100μg/mL)的固体LB平板上37℃倒置培养48h进行蓝白斑筛选，挑取白色菌落，置于三种抗生素液体培养基中摇菌；抽提DNA，以M13F/R载体通用引物进行PCR检测，筛选阳性克隆杆粒，命名为rBacmidN(T1)2L2B4B，并提取rBacmidN(T1)2L2B4B重组杆粒，用于细胞转染；
(3)N(T1)2L2B4B重组杆状病毒的获得与鉴定
将rBacmidN(T1)2L2B4B杆粒转染生长状态良好的sf9细胞，28℃恒温培养箱培养36～72h，待出现细胞病变后，收集上清，获得阳性重组杆状病毒，命名为rBacN(T1)2L2B4B。
2.根据权利要求1所述的N(T1)2L2B4B-VLPs病毒样颗粒的制备方法，其特征在于：使用毒株rBacN(T1)2L2B4B感染HF细胞进行重组蛋白N(T1)2L2B4B表达，27℃无CO2细胞摇床125r/min震荡培养72h；离心收集细胞，超声破碎得到重组蛋白后，使用蔗糖密度梯度离心纯化后收取样品，得到N(T1)2L2B4B-VLPs病毒样颗粒。
3.嵌合南非2型口蹄疫病毒多表位基因猪细小病毒样颗粒在制备SAT2 FMDV储备疫苗的应用。
4.嵌合南非2型口蹄疫病毒多表位基因猪细小病毒样颗粒在制备PPV的新型疫苗的应用。
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