CN114854789A - 一种纳米晶体包裹CyHv-2核酸亚单位疫苗的制备方法 - Google Patents
一种纳米晶体包裹CyHv-2核酸亚单位疫苗的制备方法 Download PDFInfo
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Abstract
本发明公开了一种纳米晶体包裹CyHv‑2核酸亚单位疫苗的制备方法，重组病毒基因转染家蚕培养细胞，再培养至细胞发病，然后收集细胞培养上清，获重组病毒，再将重组病毒接种家蚕幼虫或家蚕培养细胞，通过收集感染家蚕的血淋巴或家蚕培养细胞，离心处理即可获得纳米晶体包裹的疫苗，为纳米晶体包裹CyHv‑2核酸亚单位疫苗；所述重组病毒基因含有CyHv‑2核酸序列以及部分多角体基因序列。现有技术公开的多角体CyHv‑2疫苗都为微米尺寸，本发明采用新的方法，得到纳米晶体包裹CyHv‑2核酸亚单位疫苗。
Description
技术领域
本发明涉及病毒基因工程技术领域，具体涉及一种纳米晶体包裹CyHv-2核酸亚单位疫苗的制备方法。
背景技术
异育银鲫鳃出血病由鲤疱疹病毒II型（Cyprinid herpesvirus II，CyHV-2）感染引起，一般认为免疫预防是鱼类病毒性疾病防治最有效的方法之一。现有技术CN2017101096414公开了基于杆状病毒表达系统制备包裹鲤疱疹病毒II型抗原的多角体的方法，用该病毒接种家蚕或家蚕培养细胞，重组病毒表达的鲤疱疹病毒II型的抗原蛋白可包裹进家蚕质型多角体内，形成的多角体可以通过简单的差速离心实现纯化；纯化的多角体在碱性条件下裂解后，可以通过离心使多角体蛋白沉淀，而鲤疱疹病毒II型抗原留存在上清中，从而可以快速、方便地获得鲤疱疹病毒II型抗原；显微镜镜检可观察到感染发病的蚕的血淋巴中存在多角体，感染发病的蚕的血淋巴通过1000转/分和3000转/分的反复差速离心，可纯化到质型多角体，说明质型多角体蛋白基因已正确表达，多角体为2～3微米左右的蛋白微晶。现有技术CN 2021114991781公开了一种制备包裹外源蛋白的多角体的方法，通过基因工程方法利用杆状病毒表达系统在家蚕培养细胞或家蚕中共表达家蚕质型多角体蛋白、带有家蚕质型多角体病毒结构蛋白VP5标签以及蛋白酶特异性断裂位点的融合外源蛋白，从而形成包裹外源蛋白质的多角体，多角体为二十面体或八面体、大小为2～3µm左右的蛋白微晶，不仅对包裹的蛋白有防降解作用，而且有缓控释作用，因此包裹多肽药物的多角体可作为缓释药物开发。现有技术CN 2022100903243公开了一种基于多角体纳米结构的SARS冠状病毒-2疫苗及其制备方法和应用，通过对家蚕核型多角体病毒（BmNPV）多角体基因进行选择，使其形成的多孔状蛋白晶体由微米结构改造成为纳米结构，与SARS-Cov-2刺突蛋白受体结合域（RBD）融合，插入杆状病毒穿梭载体中，通过转座构建重组杆状病毒，最终获得具有纳米晶体包裹的RBD肽段。可以看出，现有技术针对CyHV-2制备的杆状病毒表达系统多为微米级，因此需要研发纳米晶体包裹CyHv-2核酸亚单位疫苗。
发明内容
本发明的目的是提供一种纳米晶体包裹CyHv-2核酸亚单位疫苗的制备方法，将多角体晶体结构作为基础材料，包埋固定外源蛋白；常规家蚕核型多角体病毒(Bombyx morinucleopolyhedrovirus, BmNPV)是一种双链环状DNA病毒，基因组为128 kb左右，最外层包裹有多孔状的蛋白晶体，呈多面体，直径1～10微米，由晶状排列的碱溶性蛋白质组成，本发明制备的多角体晶体为纳米级，此晶体内部呈多空状、耐酸、耐强碱、耐高温和紫外线，由多角体包埋的外源蛋白在高温干燥的环境中依然有很高的稳定性，且能保持良好的生物学活性。
为达到上述目的，本发明采用的技术方案是：
一种纳米晶体包裹CyHv-2核酸亚单位疫苗的制备方法，包括以下步骤，将重组病毒基因转染家蚕培养细胞，再常规培养至细胞发病，然后收集细胞培养上清，获重组病毒，再将重组病毒接种家蚕幼虫或家蚕培养细胞，通过收集感染家蚕的血淋巴或家蚕培养细胞，离心处理即可获得纳米晶体包裹的疫苗，为纳米晶体包裹CyHv-2核酸亚单位疫苗；所述重组病毒基因含有CyHv-2核酸序列以及部分多角体基因序列。
本发明中，常规培养的培养温度为26～27℃。
本发明中，将重组病毒接种家蚕幼虫，发病后，收集家蚕血淋巴，通过离心纯化多角体，获纳米晶体包裹CyHv-2核酸亚单位疫苗；家蚕幼虫为家蚕为4～5龄幼虫或蚕蛹。离心纯化的转速为6000～10000转/分钟，纯化时采用SDS缓冲液清洗，可得到纯化的纳米晶体包裹的疫苗。
本发明中，将重组病毒接种家蚕培养细胞，26～27℃培养3～6天，收集细胞，破碎细胞后通过离心获纯化的纳米晶体包裹的疫苗；离心的转速为14000～15000转/分钟，同时采用SDS缓冲液清洗。
本发明中，接种家蚕幼虫或家蚕培养细胞的重组病毒为二代重组病毒，可提高接种成功率、提高蚕的发病率；将重组病毒基因转染家蚕培养细胞，再常规培养至细胞发病，然后收集细胞培养上清，获重组病毒，将所述重组病毒再次转染家蚕培养细胞，再常规培养至细胞发病，然后收集细胞培养上清，获二代重组病毒，用于接种家蚕幼虫或家蚕培养细胞。
本发明中，通过将重组质粒转化感受态细胞，得到重组病毒基因，所述重组质粒含有CyHv-2核酸序列以及部分多角体基因序列；优选的，将重组质粒转化DH10/Bac感受态细胞，琼脂培养后挑取白色菌落，提取DNA得到重组病毒基因；进一步优选的，琼脂培养的温度为37℃，采用含有常规添加剂的LB 琼脂培养基平板，常规添加剂为四环素、卡那霉素、庆大霉素、IPTG 和X-gal。
本发明中，将SEQ ID NO:1序列、SEQ ID NO:2序列分别克隆入载体，得到重组质粒；载体为pFSATBacDual载体（Invitrogen公司），属于Bac-to-Bac （Bacteria toBaculovirus）表达系统载体。
本发明还公开了上述纳米晶体包裹CyHv-2核酸亚单位疫苗的制备方法制备的纳米晶体包裹CyHv-2核酸亚单位疫苗以及纳米晶体包裹CyHv-2核酸亚单位疫苗在制备异育银鲫鳃出血病免疫预防药物中的应用。进一步的，本发明公开了一种异育银鲫鳃出血病免疫预防药物，包括上述纳米晶体包裹CyHv-2核酸亚单位疫苗。
本发明首次公开了纳米晶体包裹CyHv-2核酸亚单位疫苗，解决了现有技术都是微米结构的蛋白晶体包裹病毒粒子的问题，得到的粒子为纳米级，免受环境因素的破坏，能够抗高温，抗强碱，抗强酸，抗紫外线等。
具体实施方式
本发明将SEQ ID NO:1序列、SEQ ID NO:2序列分别克隆入pFSATBacDual载体，得到pFSATBacDual-PH330-CyHv质粒；然后质粒转化DH10/Bac感受态细胞，然后涂布于LB 琼脂培养基平板上，培养，再挑取白色菌落，得到重组Bacmid-PH330-CyHv DNA；将重组Bacmid-PH330-CyHv DNA转染家蚕培养细胞，培养至细胞发病，然后收集细胞培养上清，获重组病毒Bacmid-ph-CyHv，再将重组病毒Bacmid-ph-CyHv接种家蚕幼虫或家蚕培养细胞，通过收集感染家蚕的血淋巴或家蚕培养细胞，离心即可获得纳米晶体包裹的疫苗，为纳米晶体包裹CyHv-2核酸亚单位疫苗。优选的，将重组病毒Bacmid-ph-CyHv感染家蚕培养细胞，发病后，收集家蚕培养细胞上清，得到二代重组病毒Bacmid-ph-CyHv；再将二代重组病毒Bacmid-ph-CyHv接种家蚕幼虫或家蚕培养细胞，通过收集感染家蚕的血淋巴或家蚕培养细胞，离心即可获得纳米晶体包裹的疫苗。具体的，将SEQ ID NO:1序列克隆进pFSATBacDual的BamHⅠ/SalⅠ位点；将SEQ ID NO:2序列克隆进pFSATBacDual的SalⅠ/Hind III位点。
本发明用杆状病毒作为疫苗载体，可以通过注射、口服和浸泡等方式实现免疫，增加了免疫途径的选择，通过此法制备的疫苗，制备过程简单、实现疫苗常温保存和运输。本发明采用的原料都是本领域常规原料，具体操作方法比如克隆、酶切、转染、感染、离心纯化等，都是本领域常规方法。下面结合实施例对本发明作进一步描述，其中一些常规方法所示简略。
实施例一
（1）将SEQ ID NO:1序列克隆进pFSATBacDual的SalⅠ/Hind III位点，获pFSATBacDual-CyHv载体，再将SEQ ID NO:2序列克隆进pFSATBacDual-CyHv的BamHⅠ/SalⅠ位点，获pFSATBacDual-PH330-CyHv质粒；
（2）将pFSATBacDual-PH330-CyHv质粒转化感受态DH5ɑ，并涂布于含四环素（8 µg/ml）、卡那霉素（50 µg/ml）、庆大霉素（6µg/ml）、IPTG（40µg/ml）、X-gal（100 µg/ml）的LB 琼脂培养平板上于37℃培养48小时，挑取白色菌落培养，提取重组病毒Bacmid-PH330-CyHvDNA；
（3）将2×108个BmN细胞用含有10% FBS 的TC-100 培养基吹起，平铺在培养皿上，待细胞贴壁时将重组Bacmid-PH330-CyHv DNA转染(FuGENE HD 转染试剂，Roche公司)BmN细胞，具体的，在一管中加入无血清培养基和Roche脂质体，另一管中加入无血清培养基和质粒，然后将两管中的液体混合到一起，静置60 min，形成脂质体-质粒混合物；然后去除培养基，加入无血清培养基混匀，在26℃培养箱中培养6 h后更换新的含血清培养基，待细胞发病（发病症状：细胞变圆，从半贴壁状态转变为悬浮状态），此时收集细胞培养基上清获得第一代（P1）病毒，将P1代重组病毒再次感染BmN细胞，收集细胞培养液上清得到P2代病毒；
（4）取P2代病毒，按1∶100（V/V）接种家蚕培养细胞，27℃培养5天，发病后，收集细胞培养上清，1000转/分离心15分钟，去除细胞碎片，上清以15000转/分4度离心30分钟，弃上清，沉淀用1*SDS缓冲液溶解后，1500转/分离心5分钟，上清以15000转/分4度离心50分钟，沉淀用1*SDS缓冲液清洗，得到纳米晶体包裹CyHv-2核酸亚单位疫苗。
用纳米晶体包裹CyHv-2核酸亚单位疫苗感染家蚕培养细胞，离心收集感染4天的BmN细胞，常规切片并透射电镜观察发现，细胞核内出现致密的黑色颗粒，大部分尺寸在100～150 nm，未观察到300nm以上的粒子，为多角体纳米晶体。常规裂解后进行Westernblotting检测，说明纳米晶体包裹CyHv-2核酸亚单位疫苗构建成功。
杆状病毒表达系统作为一个商业化的真核表达系统，可以用来表达多种候选疫苗和生物活性蛋白。杆状病毒具有特异的感染宿主（鳞翅目昆虫），对哺乳动物没有感染性，此类病毒最主要的特征是病毒粒子外包裹一个多孔状蛋白晶体，此蛋白晶体是由病毒基因编码，且在病毒感染的后期，超量表达。鲤疽疹病毒II型(CyHV-2,Cyprinid herpesvirus II)是一种DNA病毒，其主要感染鲤科鱼类，如金鱼、锦鲤、螂鱼等其他相近鱼类的变种，且对鱼的各个发育阶段均有危害，给相关淡水鱼类的养殖业带来了很大的损失，鲤疽疹病毒II型属于鲤疽疹病毒属，与其同一种属的病毒还有鲤疽疹病毒I型和III型，目前己有很多报道表明，鲤疽疹病毒II型是一种致死率很高的病毒株，而且其感染区域在逐年扩大。本发明利用多角体纳米结构包裹CyHV-2，制备CyHV-2疫苗，通过此法制备的疫苗，首次得到纳米结构的多孔状蛋白晶体，制备过程简单、实现疫苗常温保存和运输。
本发明中，序列SEQ ID NO:1（CyHV-2）为：
ATGTACGGTCTGAACAACGCTCAGGGTTTCATCGATACCGAGTGGATCGACAGACAGTCAATCGCTATGACCGCTCAGGAAACAAGCAGACTGTTGAACCCGTACCTGGCTACAAAAGGTCAGAGAGTGGACCCTTCAAACCTGTACATCCCAGACGGTATCTTCGTGACATACACACCTACCGGTTCAAGACACCCTATGGTGGAATACGAACGCATCGAATCACAACTGGAACCAGACACAGGAGCTTCAAGCTTCGTGGACAAACTGGTGGAAAAGTCAGACCTGTCAGCTTGCTTGTCACACACAGACGGTACAATCGAACTGCCTTCATCTTGGGTGGACCCTAGAGCTAAACAGTTCCACGATCTGGACGACCTGATGATCTACGGTTCAGCAGATTACATGGACTCAGACGACGAAGATTACGATTCATACGCCGGAGCAGCAGAAACACTGCAAAGAAGCATGCGCGACTACGCTGACGAAGAAGATAGCGACATGGACGATAGCACATCACTGCTGAACAGCGTGAGAAAGATGAGCAGCAAGTTCAAGAGCACCGTGTACGAAGACGACCAAACACAGAGCAGCAACACAACAGCTACAAGCGACCGCTACAAGTACTACACAAGCAGAGGTACAATCCAACGCGCTAACAGACCGGGTTTGTGA
序列SEQ ID NO:2为：
ATGCCGAATTATTCATACACCCCCACCATCGGGCGTACTTACGTGTACGACAATAAATATTACAAAAACTTGGGCTGTCTTATCAAAAACGCCAAGCGCAAGAAGCACCTAGTCGAACATGAACAAGAGGAGAAGCAATGGGATCTTCTAGACAACTACATGGTTGCCGAAGATCCCTTTTTAGGACCGGGCAAAAACCAAAAACTTACCCTTTTTAAAGAAATTCGCAGTGTGAAACCCGATACCATGAAGTTAATCGTCAACTGGAGCGGCAAAGAGTTTTTGCGTGAAACTTGGACCCGTTTTGTTGAGGACAGCTTCCCCATTGTA。
序列表
<110> 苏州大学
<120> 一种纳米晶体包裹CyHv-2核酸亚单位疫苗的制备方法
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 675
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atgtacggtc tgaacaacgc tcagggtttc atcgataccg agtggatcga cagacagtca 60
atcgctatga ccgctcagga aacaagcaga ctgttgaacc cgtacctggc tacaaaaggt 120
cagagagtgg acccttcaaa cctgtacatc ccagacggta tcttcgtgac atacacacct 180
accggttcaa gacaccctat ggtggaatac gaacgcatcg aatcacaact ggaaccagac 240
acaggagctt caagcttcgt ggacaaactg gtggaaaagt cagacctgtc agcttgcttg 300
tcacacacag acggtacaat cgaactgcct tcatcttggg tggaccctag agctaaacag 360
ttccacgatc tggacgacct gatgatctac ggttcagcag attacatgga ctcagacgac 420
gaagattacg attcatacgc cggagcagca gaaacactgc aaagaagcat gcgcgactac 480
gctgacgaag aagatagcga catggacgat agcacatcac tgctgaacag cgtgagaaag 540
atgagcagca agttcaagag caccgtgtac gaagacgacc aaacacagag cagcaacaca 600
acagctacaa gcgaccgcta caagtactac acaagcagag gtacaatcca acgcgctaac 660
agaccgggtt tgtga 675
<210> 2
<211> 330
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
atgccgaatt attcatacac ccccaccatc gggcgtactt acgtgtacga caataaatat 60
tacaaaaact tgggctgtct tatcaaaaac gccaagcgca agaagcacct agtcgaacat 120
gaacaagagg agaagcaatg ggatcttcta gacaactaca tggttgccga agatcccttt 180
ttaggaccgg gcaaaaacca aaaacttacc ctttttaaag aaattcgcag tgtgaaaccc 240
gataccatga agttaatcgt caactggagc ggcaaagagt ttttgcgtga aacttggacc 300
cgttttgttg aggacagctt ccccattgta 330
Claims (10)
1.一种纳米晶体包裹CyHv-2核酸亚单位疫苗的制备方法，包括以下步骤，将重组病毒基因转染家蚕培养细胞，再培养至细胞发病，然后收集细胞培养上清，获重组病毒，再将重组病毒接种家蚕幼虫或家蚕培养细胞，通过收集感染家蚕的血淋巴或家蚕培养细胞，离心处理即可获得纳米晶体包裹的疫苗，为纳米晶体包裹CyHv-2核酸亚单位疫苗；所述重组病毒基因含有CyHv-2核酸序列以及部分多角体基因序列。
2.根据权利要求1所述纳米晶体包裹CyHv-2核酸亚单位疫苗的制备方法，其特征在于，培养的培养温度为26～27℃。
3.根据权利要求1所述纳米晶体包裹CyHv-2核酸亚单位疫苗的制备方法，其特征在于，将重组病毒接种家蚕幼虫，发病后，收集家蚕血淋巴，通过离心纯化多角体，获纳米晶体包裹CyHv-2核酸亚单位疫苗。
4.根据权利要求1所述纳米晶体包裹CyHv-2核酸亚单位疫苗的制备方法，其特征在于，将重组病毒接种家蚕培养细胞，26～27℃培养3～6天，收集细胞，破碎细胞后通过离心获纯化的纳米晶体包裹的疫苗；离心的转速为14000～15000转/分钟，同时采用SDS缓冲液清洗。
5.根据权利要求1所述纳米晶体包裹CyHv-2核酸亚单位疫苗的制备方法，其特征在于，接种家蚕幼虫或家蚕培养细胞的重组病毒为二代重组病毒。
6.根据权利要求5所述纳米晶体包裹CyHv-2核酸亚单位疫苗的制备方法，其特征在于，将重组病毒基因转染家蚕培养细胞，再常规培养至细胞发病，然后收集细胞培养上清，获重组病毒，将所述重组病毒再次转染家蚕培养细胞，再常规培养至细胞发病，然后收集细胞培养上清，获二代重组病毒。
7.根据权利要求1所述纳米晶体包裹CyHv-2核酸亚单位疫苗的制备方法，其特征在于，将SEQ ID NO:1序列、SEQ ID NO:2序列分别克隆入载体，得到重组质粒；将重组质粒转化感受态细胞，得到重组病毒基因，所述重组质粒含有CyHv-2核酸序列以及部分多角体基因序列。
8.根据权利要求1所述纳米晶体包裹CyHv-2核酸亚单位疫苗的制备方法制备的纳米晶体包裹CyHv-2核酸亚单位疫苗。
9.权利要求8所述纳米晶体包裹CyHv-2核酸亚单位疫苗在制备异育银鲫鳃出血病免疫预防药物中的应用。
10.一种异育银鲫鳃出血病免疫预防药物，包括权利要求8所述纳米晶体包裹CyHv-2核酸亚单位疫苗。
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