CN117384962A - 一种牛副流感病毒3型和牛病毒性腹泻病毒二联腺病毒载体疫苗及其应用 - Google Patents
一种牛副流感病毒3型和牛病毒性腹泻病毒二联腺病毒载体疫苗及其应用 Download PDFInfo
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Abstract
本发明提供了一种牛副流感病毒3型和牛病毒性腹泻病毒二联腺病毒载体疫苗,它是将BPIV3C F基因、BVDV‑1E2基因克隆至复制缺陷型人5型腺病毒载体上制备得到,其中,BPIV3C F基因序列如SEQ ID No.1所示,BVDV‑1E2基因序列如SEQ ID No.2所示。本发明二联疫苗可刺激机体产生较强的细胞免疫和体液免疫,免疫效果好,安全性高。
Description
技术领域
本发明属于兽用生物制品领域,具体涉及一种牛副流感病毒3型和牛病毒性腹泻病毒二联腺病毒载体疫苗及其应用。
背景技术
牛副流感3型(Bovine parainfluenza virus type 3,BPIV3)和牛病毒性腹泻病毒(Bovine viral diarrhea virus,BVDV)是导致牛呼吸道疾病综合征(bovinerespiratory disease complex,BRDC)的重要病原体。它们在牛群中广泛存在并呈世界流行性趋势,是造成全球养牛业经济损失的主要原因之一。目前尚无特异性治疗方法,疫苗接种是预防该病的方法之一。然而目前,国内并没有针对这两种病原开发的二联商品化疫苗,因此,加快对安全、高效新型基因工程疫苗的研发,对更好的防控BRDC具有重要的意义。
BPIV3属于副黏病毒科呼吸道病毒属(Respirovirus),是单股负链RNA病毒,BPIV3分可分为A、B、C三个基因型,其中基因型C在中国首次被鉴定。流行病学调查显示在中国BPIV3C已成为牛群中的主要流行基因型之一。BVDV属于黄病毒科瘟病毒属(Pestivirus),是单股正链RNA病毒。流行病学调查显示BVDV分为三种基因型即BVDV-(1、2、3)。国内BVDV病原学监测结果表明,BVDV-1已成为牛群中的主要流行基因型之一。
F蛋白和E2蛋白分别是BPIV3和BVDV的主要结构蛋白,同时也是诱导中和抗体产生的主要抗原蛋白。在国内外的大量研究中已经证实F蛋白和E2蛋白是开发亚单位疫苗和基因工程疫苗研究的重要抗原靶位点,目前仅有分别针对这两个蛋白开发的亚单位疫苗和基因工程疫苗,未见同时预防两种病原体的二联疫苗。
发明内容
为了解决上述技术问题,本发明提供了一种牛副流感3型病毒(BPIV3C)和牛病毒性腹泻病毒(BVDV-1)的二联重组腺病毒载体疫苗及其制备方法和应用。
本发明提供了一种牛副流感病毒3型和牛病毒性腹泻病毒二联腺病毒载体疫苗,它是将BPIV3C F基因、BVDV-1E2基因克隆至复制缺陷型人5型腺病毒载体上制备得到,其中BPIV3C F基因序列如SEQ ID No.1所示,BVDV-1E2基因序列如SEQ ID No.2所示。
其中,BPIV3C F基因和BVDV-1E2基因以融合蛋白的形式克隆至复制缺陷型人5型腺病毒载体,其中融合蛋白的核苷酸序列如SEQ ID No.3所示。
其中,二联疫苗的制备方法如下:
a.合成权利要求1或2的序列,将其克隆到腺病毒穿梭质粒pDC316上,获得重组穿梭质粒pDC316-F-P2A-E2;
b.将重组穿梭载体pDC316-F-P2A-E2与腺病毒骨架质粒pBHGlox E1,3Cre共转染HEK293细胞,重组并包装,即可。
本发明还提供了上述二联疫苗在用于制备防疫牛副流感3型和牛病毒性腹泻的生物制品中的应用。
其中,所述二联疫苗被制备为注射剂、滴鼻剂或吸入剂。
进一步地,所述注射剂为肌肉注射剂。
本发明构建了能同时表达BPIV3C型F蛋白全长和BVDV-1型E2蛋白全长的二联重组腺病毒载体疫苗,将二联疫苗通过肌注及滴鼻免疫接种小鼠,均能够诱导小鼠产生较强的体液免疫和细胞免疫反应,能诱导机体产生高滴度抗体,效果优于商业疫苗,未出现疫苗同时免疫而引起的免疫抑制,可同时防控牛副流感病毒3型和牛病毒性腹泻病毒,免疫效果好,安全性高,具有良好的应用前景。
以下结合说明书附图和实施例形式的具体实施方式,来进一步说明本发明,但实施例并不对本发明做任何形式的限定。对于本领域技术人员来说,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
附图说明
图1重组腺病毒构建示意图。
图2pDC316-F-P2A-E2双酶切鉴定。
图3在HEK293细胞进行重组腺病毒的包装。
图4琼脂糖凝胶电泳鉴定重组腺病毒。
图5目标抗原的IFA结果,其中A1/A2:HAd5-F-P2A-E2感染细胞;A3:空白细胞;B1/B2:有明显绿色荧光;B3:无绿色荧光。
图6目标抗原的WB结果,其中M:预染Marker;1:HAd5-F-P2A-E2感染细胞;2:空白细胞。
图7目标抗原的WB结果,其中M:预染Marker;1:HAd5-F感染细胞;2:空白细胞。
图8目标抗原的WB结果,其中M:预染Marker;1:HAd5-E2感染细胞;2:空白细胞。
图9ELISA法检测血清中BPIV3C F蛋白的IgG抗体滴度。
图10ELISA法检测血清中BVDV-1E2蛋白的IgG抗体滴度。
图11细胞免疫反应检测图。
具体实施方式
以下结合实施例对本发明作进一步详细说明,但本发明并不限于此。下述实施方案中所述实验方法,如无特殊说明,均为常规方法,所述试剂和材料,如无特殊说明,均可从商业途径获得。
实施例1疫苗的制备
一、F蛋白和E2蛋白基因优化及合成
重组腺病毒疫苗的目标抗原为BPIV3C毒株CH/SC20/2020(GenBank号:OM621819.1)的F蛋白和BVDV-1毒株CH/SWU-Z6/2016(GenBank号:MF693403.1)的E2蛋白。通过对F蛋白基因和E2蛋白基因进行优化,提高F蛋白和E2蛋白的表达水平,从而提高了疫苗的免疫原性。
基因优化前,F蛋白基因的GC含量为35.7%。基因优化后GC含量提升至55.5%,调整密码子使用偏差以适应目标宿主的最高表达谱,CAI(密码子适应指数)从0.64上调至0.97。基因优化前,E2蛋白基因的GC含量为44.2%。基因优化后GC含量提升至61.9%,CAI(密码子适应指数)从0.71上调至0.99(0.8-1.0的CAI被认为有利于高表达)。
F和E2蛋白基因优化之后,在翻译起始密码子前面加入Kozak序列,并在整个序列上游插入酶切位点EcoRI,在F基因和E2基因中间引入P2A多肽,下游插入酶切位点SalⅠ,对基因序列进行合成。F和E2蛋白基因优化后融合蛋白序列(酶切位点为EcoRI和SalⅠ)见SEQID NO.3。
F蛋白基因优化后(SEQ ID NO.1):
ATGATCACCATCGTGGCCACCACCGTGATCCTGCTGCTGCCCCTGAGCCTGTGCCAGATCGACATCACCAAGCTGCAGAGAGTGGGCGTGCTGGTGAATAACCCCAAGGGCATGAAGATCTCCCAGAACTTCGAGACCAGATACCTGATCCTGTCCCTGATCCCCAAGATTGAGAACCCTCAGTCCTGCGGCGACCAGCAGATCAGCCAGTACAAGAAGCTGCTGGACAGACTGATCATCCCCCTGTACGACGGCCTGAAGCTGCAGAAGGACGTGATTGTGGTGAGGCACGAGTCTAATAACAGCACCAGCTCTAGGAAGAAGAGGTTCTTCGGCGAGATCATCGGCACCATCGCCATCGGCATCGCCACCAGCGCCCAGATCACCGCAGCCGTGGCCCTGGTGGAGGCTAAGCAGGCCAGGGCCGACATTGACAAGCTGAAGGAGGCCATTAGAGACACCAACAAGGCCGTCCAGAGCATCCAGTCCAGCGTGGGCAATCTGATCGTGGCCGTGAAGAGCGTGCAGGATTACGTGAATAATGAGATCGTGCCCTCCATTGCCAGACTGGGCTGCGAGGCCGCCGGCCTGCAGCTGGGCATCGCCCTGACCCAGCACTATAGCGAGCTGACCAACATCTTCGGCGATAATATCGGCACACTGAAGGAGAAGGGCATCAAGCTGCAGGGAATCGCCAGTCTGTACCACACCAACATCACCGAAATCTTCACCACCTCCACCGTGGACCAGTACGACATCTACGATCTGCTGTTCACAGAGAGCATTAAGATGAGAGTGATCGACGTGGATCTGAATGACTACTCCATCACCCTGCAGGTGAGGCTCCCCCTGCTGACCAAGCTGAGCAACACCCAGATCTACCGGGTGGACTCCATCAGCTACAATATCCAGGGCAAAGAGTGGTACATCCCTCTGCCCAATCACATTATGACCAAGGGGGCCTTCCTGGGCGGGGCCGACATTAAGGAGTGCATTGAAGCCTTTAGCTCTTATATTTGCCCCTCCGACCCTGGCTTTATCCTGAATAGGGAGATTGAGAATTGCCTGTCCGGCAATATCACCCAGTGCCCAAAGACCGTGGTGACCTCCGACATCGTGCCCAGGTACGCCTTCGTGAACGGAGGAGTGATCGCCAACTGCATCCCCACCACCTGCACCTGCGATGGCATCGACAATAGAATTAATCAGGCCCCCGACCAGGGCATCAGAGTGATCACACACAAGGAGTGCCAGGTTATCGGAATCAACGGGATGCTGTTCAGGCCCAATAAGGAGGGCACCCTGGCCACATACACCTACGATGACATCGTGCTGAATAATAGCGTGGCCCTGAATCCCATCGACATCTCCATGGAGCTGAATAAGGCCAAGCTGGAGCTGGAGAAGAGCAAGGAGTGGATCAAGAAGAGCAATCAGAAGCTGGACTCTGTGGGCGGGTGGTACCAGTCTAACATCACCATCACCATTATGATCGTGATGATCATCATCCTGTTTATCATCAATATGATCATCATCATTATCATCTTCAAGCAGTTCAAGATCCGCAACAGGCACCCTATCGACAACAGCAACGAGCCCTACGTGCTGACCAGAAAGCAG
E2蛋白基因优化后(SEQ ID NO.2):
ATGCACAAGTTCCAGACCGGTATCCTGATCTGCCACTCCAAGGAGTGCTACGAGTCCAACCACTACGAGCTCAAGACCAGCGCCCTGTTCGGCCGTATCACCCACACCAAGTGCCACCTGTACACTCAGTGGAGCAGCTTCGGCGCCTGGATGGTGAAGTTCGTCTACCTGGCTCGTTGTACCCGCGAAACCCGTTACCTGGCCATCCTGCACAGCCGCGCTCTGCACACACGCGTGGTGTTCGAAAAGCTGTTCGACGGTCTGCGCCAGTGGGACACCGTTGAGATGGACGACAACTTCGAGTTCGGCCTGTGTCCTTGTGACGCTAAGCCTATCGTCCGTGGAAAGTTCAACACCACCCTGGTGAACGGCCGCGCCTTCCAGATGGTGTGCCCTATCGGTTGGACAGGCACCGTGAGCTGCATGCTGGCTAACCGTGACACTCTGGACACCGCTGTGGGCCGTAGCTACCGTCGCAGCAAGCCTTTCCTGTACCGCCAGGGTTGTATCACCCAGAAGATCCTGGGTGAGGACCTGTACAACTGTGACCTGGGTGGTAACTGGACATGTGTGACCGGCGACCAGCTGCAGTACACCGGTGGCTCCATCGAATCCTGTAAGTGGTGTGGCTTCAAGTTCCAGAAGAGCGAAGGTCTGCCCCACTACCCTATCGGCAAGTGTCGTCTGGAGAACGAGACCGGCTACCGCTTCGTGGACGACACTAGCTGTAACCGTGAAGGTGTGGCCATCGTGCCTACCGGTCAGATCAAGTGTAAGATCGGTGACACAATCGTGCAGGTGCTGGCCCTGGACACCAAGCTGGGCCCTATGCCTTGCAAGCCCCACGAAATCATCAGCAGCGAAGGACCCGTGGAAAAGACCGCCTGCACCTTCAACTACACCAAGACCCTGAAGAACAAGTACTTCGAACCTCGCGACTCCTACTTCCAGCAGTACATGCTGAAGGGTGAATACCAGTACTGGTTCGACCTGGAGGTGACTGACCACCACCGTGACTACTTCGCTGAGTCCATCCTGCTGGTGGTGGTGCCTCTGCTGGGTGGTCGCTACGTGCTGTGGCTGCTGCTGACATACATGGTGCTGAGCGAACACCGTGCTTCCGGCTAA
融合蛋白序列(SEQ ID NO.3):
GAATTCGCCACCATGATCACCATCGTGGCCACCACCGTGATCCTGCTGCTGCCCCTGAGCCTGTGCCAGATCGACATCACCAAGCTGCAGAGAGTGGGCGTGCTGGTGAATAACCCCAAGGGCATGAAGATCTCCCAGAACTTCGAGACCAGATACCTGATCCTGTCCCTGATCCCCAAGATTGAGAACCCTCAGTCCTGCGGCGACCAGCAGATCAGCCAGTACAAGAAGCTGCTGGACAGACTGATCATCCCCCTGTACGACGGCCTGAAGCTGCAGAAGGACGTGATTGTGGTGAGGCACGAGTCTAATAACAGCACCAGCTCTAGGAAGAAGAGGTTCTTCGGCGAGATCATCGGCACCATCGCCATCGGCATCGCCACCAGCGCCCAGATCACCGCAGCCGTGGCCCTGGTGGAGGCTAAGCAGGCCAGGGCCGACATTGACAAGCTGAAGGAGGCCATTAGAGACACCAACAAGGCCGTCCAGAGCATCCAGTCCAGCGTGGGCAATCTGATCGTGGCCGTGAAGAGCGTGCAGGATTACGTGAATAATGAGATCGTGCCCTCCATTGCCAGACTGGGCTGCGAGGCCGCCGGCCTGCAGCTGGGCATCGCCCTGACCCAGCACTATAGCGAGCTGACCAACATCTTCGGCGATAATATCGGCACACTGAAGGAGAAGGGCATCAAGCTGCAGGGAATCGCCAGTCTGTACCACACCAACATCACCGAAATCTTCACCACCTCCACCGTGGACCAGTACGACATCTACGATCTGCTGTTCACAGAGAGCATTAAGATGAGAGTGATCGACGTGGATCTGAATGACTACTCCATCACCCTGCAGGTGAGGCTCCCCCTGCTGACCAAGCTGAGCAACACCCAGATCTACCGGGTGGACTCCATCAGCTACAATATCCAGGGCAAAGAGTGGTACATCCCTCTGCCCAATCACATTATGACCAAGGGGGCCTTCCTGGGCGGGGCCGACATTAAGGAGTGCATTGAAGCCTTTAGCTCTTATATTTGCCCCTCCGACCCTGGCTTTATCCTGAATAGGGAGATTGAGAATTGCCTGTCCGGCAATATCACCCAGTGCCCAAAGACCGTGGTGACCTCCGACATCGTGCCCAGGTACGCCTTCGTGAACGGAGGAGTGATCGCCAACTGCATCCCCACCACCTGCACCTGCGATGGCATCGACAATAGAATTAATCAGGCCCCCGACCAGGGCATCAGAGTGATCACACACAAGGAGTGCCAGGTTATCGGAATCAACGGGATGCTGTTCAGGCCCAATAAGGAGGGCACCCTGGCCACATACACCTACGATGACATCGTGCTGAATAATAGCGTGGCCCTGAATCCCATCGACATCTCCATGGAGCTGAATAAGGCCAAGCTGGAGCTGGAGAAGAGCAAGGAGTGGATCAAGAAGAGCAATCAGAAGCTGGACTCTGTGGGCGGGTGGTACCAGTCTAACATCACCATCACCATTATGATCGTGATGATCATCATCCTGTTTATCATCAATATGATCATCATCATTATCATCTTCAAGCAGTTCAAGATCCGCAACAGGCACCCTATCGACAACAGCAACGAGCCCTACGTGCTGACCAGAAAGCAGCTCGAGGGCTCCGGCGCCACCAATTTCAGCCTGCTGAAGCAGGCCGGCGACGTGGAGGAGAACCCCGGCCCCAAGCTTGCCACCATGCACAAGTTCCAGACCGGTATCCTGATCTGCCACTCCAAGGAGTGCTACGAGTCCAACCACTACGAGCTCAAGACCAGCGCCCTGTTCGGCCGTATCACCCACACCAAGTGCCACCTGTACACTCAGTGGAGCAGCTTCGGCGCCTGGATGGTGAAGTTCGTCTACCTGGCTCGTTGTACCCGCGAAACCCGTTACCTGGCCATCCTGCACAGCCGCGCTCTGCACACACGCGTGGTGTTCGAAAAGCTGTTCGACGGTCTGCGCCAGTGGGACACCGTTGAGATGGACGACAACTTCGAGTTCGGCCTGTGTCCTTGTGACGCTAAGCCTATCGTCCGTGGAAAGTTCAACACCACCCTGGTGAACGGCCGCGCCTTCCAGATGGTGTGCCCTATCGGTTGGACAGGCACCGTGAGCTGCATGCTGGCTAACCGTGACACTCTGGACACCGCTGTGGGCCGTAGCTACCGTCGCAGCAAGCCTTTCCTGTACCGCCAGGGTTGTATCACCCAGAAGATCCTGGGTGAGGACCTGTACAACTGTGACCTGGGTGGTAACTGGACATGTGTGACCGGCGACCAGCTGCAGTACACCGGTGGCTCCATCGAATCCTGTAAGTGGTGTGGCTTCAAGTTCCAGAAGAGCGAAGGTCTGCCCCACTACCCTATCGGCAAGTGTCGTCTGGAGAACGAGACCGGCTACCGCTTCGTGGACGACACTAGCTGTAACCGTGAAGGTGTGGCCATCGTGCCTACCGGTCAGATCAAGTGTAAGATCGGTGACACAATCGTGCAGGTGCTGGCCCTGGACACCAAGCTGGGCCCTATGCCTTGCAAGCCCCACGAAATCATCAGCAGCGAAGGACCCGTGGAAAAGACCGCCTGCACCTTCAACTACACCAAGACCCTGAAGAACAAGTACTTCGAACCTCGCGACTCCTACTTCCAGCAGTACATGCTGAAGGGTGAATACCAGTACTGGTTCGACCTGGAGGTGACTGACCACCACCGTGACTACTTCGCTGAGTCCATCCTGCTGGTGGTGGTGCCTCTGCTGGGTGGTCGCTACGTGCTGTGGCTGCTGCTGACATACATGGTGCTGAGCGAACACCGTGCTTCCGGCTAAGTCGAC
F和E2蛋白原始基因序列(SEQ ID NO.4,其中,第13-1632位序列为F基因,第1717-2844为序列为E2基因):
GAATTCGCCACCATGATCACTATAGTTGCAACAACAGTTATATTATTATTACCTTTGTCACTATGTCAGATAGATATAACAAAGTTGCAACGAGTAGGGGTGCTAGTCAACAATCCAAAAGGCATGAAGATTTCACAAAATTTTGAAACAAGATACTTAATATTAAGTTTGATACCAAAGATAGAGAACCCACAATCATGTGGAGACCAACAAATAAGTCAATATAAGAAATTATTAGATAGATTAATTATTCCTTTATATGATGGGTTAAAATTACAGAAAGATGTAATCGTAGTAAGGCATGAGTCTAATAACAGTACTAGTTCCAGGAAGAAACGGTTCTTCGGAGAAATAATTGGGACAATTGCGATAGGGATAGCCACATCAGCTCAAATAACTGCAGCAGTTGCCCTTGTTGAAGCCAAACAAGCAAGAGCAGATATAGACAAGCTCAAGGAGGCCATCAGAGATACAAACAAGGCAGTACAGTCAATTCAAAGCTCAGTAGGAAATCTAATTGTTGCAGTTAAGTCAGTTCAAGATTATGTCAACAATGAGATTGTACCATCAATAGCAAGACTAGGCTGTGAAGCAGCAGGATTACAACTAGGAATTGCATTGACACAACATTACTCAGAGTTAACAAATATATTTGGTGATAACATAGGAACACTAAAGGAGAAGGGAATAAAATTACAAGGGATAGCATCATTATATCACACAAACATAACAGAGATATTCACTACTTCAACAGTTGATCAATATGACATTTATGATCTATTGTTCACTGAGTCAATCAAAATGAGAGTTATAGATGTAGACTTAAATGACTATTCAATTACACTACAAGTTAGACTACCTTTATTAACAAAATTATCAAACACTCAGATTTATAGAGTGGATTCTATATCATATAATATCCAAGGTAAGGAATGGTACATTCCTCTTCCCAATCACATCATGACAAAGGGGGCATTTTTAGGTGGTGCTGATATAAAAGAGTGTATAGAAGCATTCAGCAGCTACATATGTCCCTCTGACCCAGGTTTTATATTAAATCGTGAGATAGAAAACTGTCTATCAGGGAATATCACGCAGTGTCCGAAGACCGTCGTTACATCAGACATAGTACCAAGATATGCATTTGTTAATGGCGGAGTAATTGCCAATTGTATACCAACCACTTGTACATGTGATGGGATTGACAATAGGATCAACCAGGCACCCGACCAAGGGATAAGGGTGATAACACATAAAGAATGTCAAGTGATAGGCATAAACGGGATGTTATTCAGACCTAACAAAGAAGGGACTTTGGCAACTTATACATATGATGACATTGTTTTAAACAATTCTGTCGCACTTAACCCAATTGACATATCAATGGAACTCAACAAAGCTAAATTGGAACTAGAAAAATCAAAAGAATGGATAAAGAAGTCAAATCAAAAGTTGGACTCTGTTGGAGGTTGGTATCAGTCAAATATAACAATTACTATAATGATAGTGATGATAATAATTCTGTTTATAATCAACATGATAATCATTATAATAATCTTTAAACAATTCAAGATTCGCAATAGACACCCAATTGACAATAGCAATGAACCCTATGTCCTGACCAGGAAACAGCTCGAGGGAAGCGGAGCCACGAACTTCTCTCTGTTAAAGCAAGCAGGAGATGTTGAAGAAAACCCCGGGCCTAAGCTTGCCACCATGCACAAGTTCCAGACCGGCATCCTGATCTGCCACAGCAAGGAGTGCTACGAGAGCAACCACTACGAGCTCAAGACCTCCGCCCTGTTCGGCAGGATCACCCACACCAAGTGCCACCTGTACACCCAGTGGTCCTCCTTCGGCGCCTGGATGGTGAAGTTCGTGTACCTGGCCCGGTGCACCAGGGAGACCAGATACCTGGCCATCCTGCACAGCAGGGCCCTGCACACTCGGGTGGTGTTCGAGAAGCTGTTCGACGGCCTGAGACAGTGGGACACCGTGGAGATGGACGACAATTTCGAGTTCGGCCTGTGCCCTTGCGACGCCAAGCCCATCGTGAGGGGCAAGTTCAACACCACCCTGGTGAACGGCAGAGCCTTCCAGATGGTGTGCCCCATCGGCTGGACCGGCACCGTGAGCTGCATGCTGGCCAACCGGGACACCCTGGACACCGCCGTGGGCAGGAGCTACAGGAGGAGCAAGCCCTTCCTGTACCGGCAGGGCTGCATCACCCAGAAGATCCTGGGCGAGGATCTGTATAACTGCGACCTGGGCGGCAACTGGACCTGCGTGACCGGCGACCAGCTGCAGTACACCGGCGGCTCCATCGAGAGCTGCAAGTGGTGCGGCTTCAAGTTCCAGAAGTCCGAGGGCCTGCCCCACTACCCCATCGGCAAGTGCAGGCTGGAGAACGAGACCGGCTACAGGTTCGTGGACGACACCAGCTGCAACAGAGAGGGCGTGGCCATCGTGCCCACCGGCCAGATCAAGTGCAAGATCGGCGACACCATCGTGCAGGTGCTGGCCCTGGACACCAAGCTGGGCCCAATGCCTTGCAAGCCCCACGAGATCATCAGCTCCGAGGGCCCCGTGGAGAAAACCGCCTGCACCTTCAACTACACCAAGACCCTGAAGAACAAGTACTTCGAGCCCAGAGACAGCTACTTCCAGCAGTACATGCTGAAGGGCGAGTACCAGTACTGGTTCGACCTGGAGGTGACCGACCACCACAGAGACTACTTCGCCGAGAGCATCCTGCTGGTGGTGGTGCCCCTGCTGGGCGGCAGATACGTGCTGTGGCTGCTGCTGACCTACATGGTGCTGAGCGAGCACAGAGCCAGCGGCTGAGTCGAC
二、载体构建
以上合成的基因序列,分别用EcoRI和SalⅠ酶切,回收目的基因片段,将其连接至AdMax腺病毒系统(加拿大Microbix Biosystems Inc.)的穿梭质粒pDC316上,转化DH5α感受态,涂布Amp LB平板,挑单克隆进行菌落PCR鉴定,并对PCR鉴定为阳性的克隆进行测序验证。F和E2蛋白基因优化后的质粒记为pDC316-F-P2A-E2。重组腺病毒构建示意图如图1所示,pDC316-F-P2A-E2双酶切鉴定如图2所示。
三、重组腺病毒包装、制备和鉴定
3.1重组腺病毒包装
将以上构建好的载体pDC316-F-P2A-E2与AdMax腺病毒系统的骨架质粒pBHGlox_E1,3Cre共转染HEK293细胞进行重组腺病毒的包装。过程如下:
a.转染前一天,将HEK293细胞接种于六孔板中,每孔7×105个细胞,培养基为DMEM+10%FBS,置37℃含5%CO2细胞培养箱中培养过夜。
b.待细胞生长至底面积的80-90%时,取骨架质粒(pBHGlox_E1,3Cre)和穿梭质粒,用jetPRIME转染试剂按其所附说明书进行转染。具体步骤为:
(1)每个转染孔取骨架质粒1.6μg,穿梭质粒0.4μg,混和均匀;质粒用200μLbuffer培养基进行稀释。
(2)取2μL jetPRIME转染试剂加入到稀释于buffer中混匀。
(3)将转染试剂和质粒混合物室温放置10min,然后加入到细胞中。
c.转染后第二天,将长满的细胞传代于25cm2细胞培养瓶中,用含5%FBS的DMEM培养基继续培养,每天观察,待细胞长满瓶底时,再传入75cm2细胞培养瓶中,每天观察细胞出毒迹象。出毒现象为细胞变大变圆,呈葡萄状,并开始出现明显噬斑(图3)。待细胞大部分病变并从底部脱落时进行收毒。
d.将出毒的细胞先后置于70℃冰箱和37℃水浴锅中反复冻融三次。
12000g离心10分钟,收集含病毒的上清液,弃沉淀。将表达F-P2A-E2全长蛋白基因优化序列的毒种标记为HAd5-F-P2A-E2。
3.2重组腺病毒鉴定
3.2.1PCR扩增F-P2A-E2基因全序列及测序鉴定
使用pDC316载体的通用引物,扩增F-P2A-E2蛋白的全序列,引物序列如下:
pDC316-F:ACGTGGGTATAAGAGGCG
pDC316-R:CGATGCTAGACGATCCAG
取20μL疫苗候选株毒种液,加入10μL蛋白酶K,58℃消化30min释放病毒基因组,再100℃加热5min,以此为模版扩增F-P2A-E2蛋白基因序列。
琼脂糖凝胶电泳结果显示能扩增到单一目的条带,片段大小正确(图4),其中1,2,3分别为HAd5-F-P2A-E2的P1,P2,P3,即HAd5-F-P2A-E2第一代到第三代毒,-为阴性对照。将目的条带进行胶回收并测序,比对结果表明,测序结果序列完全正确。
3.2.2目标抗原表达鉴定
将重组腺病毒感染HEK293细胞,48小时后进行目标抗原的IFA和Western blot检测,IFA可检测到明显的绿色荧光,WB可检测到目标蛋白的明显表达。
用种毒HAd5-F-P2A-E2感染HEK293细胞,以不接种病毒的HEK293细胞做阴性对照继续培养48h。弃去培养液,80%丙酮固定,加入5%脱脂奶粉37℃封闭1h,分别加入兔抗BVDV-1E2蛋白血清作(1:100)和兔抗BPIV3C多克隆血清(1:100)为一抗,后避光再以羊抗兔IgG抗体(IgG-FITC)作为二抗(1﹕5000),于荧光倒置显微镜下观察目的蛋白的表达情况。
样品制备:HAd5-F-P2A-E2接毒48小时后,小心地吸弃培养基,用PBS重悬细胞,500g离心5分钟,弃上清。细胞用200μL RIPA缓冲液(Thermo Scientific,Prod89900),另外添加适量蛋白酶抑制剂和核酸酶重悬,冰浴15分钟,4℃12000rpm离心5分钟,取上清,加入1/4体积含有200mmol/L DTT的5×SDS-PAGE上样缓冲液,95℃加热5分钟,冻存,用于WB检测。
Western blot检测:使用10孔的12%SDS-PAGE胶进行SDS-PAGE,上样量每孔20μL。电泳条件:80V,30min;120V,直到溴酚蓝刚好从凝胶中出来为此。将SDS-PAGE胶上的蛋白质通过电转仪转移到聚偏二氟乙烯膜(PVDF膜)上,电转条件为200mA,1.5小时。电转完成后,将PVDF膜用5%脱脂奶粉封闭1小时,然后分别以兔抗BVDV-1E2蛋白多克隆血清作(1:1000)和兔抗BPIV3C全病毒多克隆血清(1:1000)为一抗,4℃放置过夜。将膜用WB洗涤液洗涤4次,每次于摇床上摇动10分钟。然后加入以1:5000稀释于5%脱脂奶粉的HRP标记的羊抗兔IgG抗体(Bioss,bs-0296G-HRP),室温孵育2小时。用WB洗涤液将膜洗涤4次,使用BeyoECL Star(Beyotime,Cat.No.P0018AFT)进行化学发光反应,使用化学发光成像仪采集不同曝光时间的图像。IFA结果、WB结果分别见图5-8。
3.2.3重组腺病毒培养
HEK293细胞在37℃、5%CO2条件下贴壁培养。毒种接种48小时后,当细胞活度下降到40%以下时,将细胞瓶先后置于-70℃冰箱和37℃水浴锅中反复冻融三次。12000g离心10分钟,收集含病毒的上清液,弃沉淀。
3.2.4重组腺病毒纯化
采用Diamond Layer 700BA(博格隆)分离纯化腺病毒颗粒。层析柱用A液(PBS,PH7.4)平衡;缓慢的将样品推入loop管中;上样结束,0.6mL/min,分管收集洗脱峰,平衡至Uv基线;100%B洗杂,B液为1M NaOH+1M NaCl pH7.5。
3.3HAd5-F-P2A-E2的鉴定和滴度测定
3.3.1PCR扩增目标蛋白基因全序列和测序鉴定
实验方法和过程同3.2.1。琼脂糖凝胶电泳结果显示能扩增到单一目的条带,片段大小正确。将目的条带进行胶回收并测序,比对结果表明,测序结果序列完全正确。
3.3.2感染滴度测定
待HEK293细胞生长密度约为80-90%时,消化细胞并计数细胞量;用含有5% FBS的DMEM培养液制备细胞悬液,每板需要10mL浓度为1×105/mL的细胞悬液;按每孔100μL(即1×104个细胞)加入3个96孔板;制备感染样品。
接种样品:将96孔板中的11、12两列各加入100μL 5% FBS的DMEM,做阴性对照;依次加入96孔板中的A-H排,各加100μL标记为8个连续梯度稀释的样品溶液;盖上第一个板并在37℃ CO2培养箱中培养;同样步骤操作第二、三块板。
通过计算可得HAd5-F-P2A-E2的TCID50为1×105.8/mL。
3.3.3病毒颗粒数测定
病毒样品经SDS处理后测定A260nm值(UVSDS法)测定病毒颗粒数:取HAd5-F-P2A-E2注射液200μL加等体积0.2%SDS溶液裂解,振荡混匀。56℃水浴中放置10min。等温度降至室温时瞬时离心,12000rpm离心5分钟,取上清。以病毒稳定液与0.2%SDS溶液等体积混匀后的溶液作空白对照,测定波长260nm与280nm处的吸光值。平行实验2次。
计算公式:病毒颗粒数=A260nm×稀释倍数×1.1×1012。
经纯化后的HAd5-F-P2A-E2的病毒颗粒数为到9.2×1010VP/mL。
以下通过试验例具体说明本发明的有益效果:
试验例1重组腺病毒HAd5-F-P2A-E2免疫小鼠试验
一、疫苗体液免疫反应检测
35只SPF级雌性BALB/c小鼠(6-8周龄),随机分成7组,每组5只。根据表1所示分组情况对小鼠进行HAd5-F-P2A-E2免疫。
其中,HAd5-F肌注组和HAd5-E2肌注组均是使用优化后序列,构建过程同实施例1。
肌肉注射方式为后大腿内侧注射200μL,滴鼻免疫方式为将小鼠用异氟烷麻醉,经鼻腔滴入200μL。并分别于一免14天后按照同样剂量进行二免。
表1小鼠免疫分组情况表
小鼠于免疫后特定时间点采血,分离血清,使用ELISA法检测血清中BPIV3C F蛋白和BVDV-1E2蛋白的IgG抗体滴度。检测结果如图9-10所示。
ELISA抗体水平检测结果显示,小鼠肌肉注射HAd5-F-P2A-E2可产生较高的血清IgG抗体。
针对BPIV3C-F蛋白抗体效价在二免14天后达到最高,HAd5-F-P2A-E2肌注组和HAd5-F肌注组无显著性差异,但都显著高于HAd5-F-P2A-E2滴鼻组和BPIV3C灭活苗组(ns、p<0.01、p<0.001);HAd5-F-P2A-E2滴鼻组与BPIV3C灭活苗组无显著性差异(ns)(图9)。
针对BVDV-E2蛋白抗体效价在二免14天后达到最高,HAd5-F-P2A-E2肌注组和HAd5-E2肌注组无显著性差异,但都显著高于HAd5-F-P2A-E2滴鼻组和BVDV-1商业灭活苗组(ns、p<0.01、p<0.01),其中HAd5-F-P2A-E2滴鼻免疫组抗体水平显著高于BVDV-1商业灭活苗组(p<0.05)(图10)。
因此,本发明制备的二联疫苗,免疫后能够诱导产生较高的血清IgG抗体,未出现免疫抑制,抗体水平显著高于商业灭活疫苗。
二、细胞免疫反应检测
免疫方式同上,于二免后14天处死小鼠,分离脾淋巴细胞。机械法处理脾脏组织呈单细胞悬液,用PBS调节细胞浓度为4×106个/mL。使用体外刺激培养基,RPMI1640培养基(含10%fetal bovine serum简称FBS)含100ng/mL PMA、1μg/mL Ionomysin和3μMMonomysin于37℃培养箱刺激培养6小时,同时加入蛋白分泌阻断剂阻断细胞因子分泌。
IL-4和IFN-γ流式检测,取100μl的细胞悬液于流式管中,向每个管中加入1mLFoxp3固定/破膜工作溶液并脉冲涡旋4℃或室温孵育40分钟。每管加入2mL 1X破膜液,在室温下500g离心样品5分钟,弃上清,重复此步骤一次。加入IL-4、IFN-γ荧光抗体1ug,4℃孵育30min,加入2ml的PBS液重悬细胞,500g离心弃上清,加入400μl的PBS液重悬细胞。立即上机检测,用Everest软件对结果进行分析。
结果如图11所示,HAd5-F-P2A-E2肌注能产生较高的IL-4和IFN-γ表达,HAd5-F-P2A-E2肌注组与对照组(PBS组)具有显著差异(p<0.01)。
CD4+T淋巴细胞、CD8+T淋巴细胞检测,取100μl的细胞悬液于流式管中,分别加入CD4+T、CD8+T荧光抗体各1ug,4℃孵育30min。加入2ml的PBS液重悬细胞,300g离心弃上清。重复此步骤一次。加入400μl的PBS液重悬细胞,立即上机检测。用Everest软件对结果进行分析。
结果如图11所示,HAd5-F-P2A-E2肌注能产生较高的CD4+T淋巴细胞和CD8+T淋巴细胞表达,其中HAd5-F-P2A-E2肌注组诱导CD4+T淋巴细胞和CD8+T淋巴细胞显著高于对照组(p<0.01)、(p<0.05)。
因此,本发明制备的二联疫苗,免疫后能够诱导产生较高的细胞免疫反应,显著高于对照组。
综上,本发明利用重组腺病毒载体制备的牛副流感病毒3型和牛病毒性腹泻病毒二联疫苗,免疫后能够诱导产生良好的体液免疫和细胞免疫效应,未出现疫苗同时免疫而引起的免疫抑制,可解决实际生产中不同疫苗无法同时免疫的问题,是一种较为理想的二联腺病毒载体疫苗。
Claims (6)
1.一种牛副流感病毒3型和牛病毒性腹泻病毒二联腺病毒载体疫苗,其特征在于:它是将BPIV3C F基因、BVDV-1E2基因克隆至复制缺陷型人5型腺病毒载体上制备得到,其中,BPIV3C F基因序列如SEQ ID No.1所示,BVDV-1E2基因序列如SEQ ID No.2所示。
2.根据权利要求1所述的二联疫苗,其特征在于:BPIV3C F基因和BVDV-1E2基因以融合蛋白的形式克隆至复制缺陷型人5型腺病毒载体,其中,融合蛋白的核苷酸序列如SEQ IDNo.3所示。
3.根据权利要求1或2所述的二联疫苗,其特征在于:制备方法如下:
a.合成权利要求1或2的序列,将其克隆到腺病毒穿梭质粒pDC316上,获得重组穿梭质粒pDC316-F-P2A-E2;
b.将重组穿梭载体pDC316-F-P2A-E2与腺病毒骨架质粒pBHGlox E1,3Cre共转染HEK293细胞,重组并包装,即可。
4.权利要求1-3任意一项所述的二联疫苗在用于制备防疫牛副流感3型和牛病毒性腹泻的生物制品中的应用。
5.根据权利要求4所述的应用,其特征在于:所述二联疫苗被制备为注射剂、滴鼻剂或吸入剂。
6.根据权利要求5所述的应用,其特征在于:所述注射剂为肌肉注射剂。
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