CN116785420A - 一种牛病毒性腹泻病毒的mRNA疫苗及其应用 - Google Patents
一种牛病毒性腹泻病毒的mRNA疫苗及其应用 Download PDFInfo
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Abstract
本发明公开一种牛病毒性腹泻病毒的mRNA疫苗及其应用，属于核酸疫苗技术领域。为了提高牛病毒性腹泻病毒mRNA疫苗的免疫保护作用。本发明提供一种牛病毒性腹泻病毒（Bovine viral diarrhea Virus）的mRNA疫苗，所述mRNA疫苗编码牛病毒性腹泻病毒的E2蛋白或牛病毒性腹泻病毒截短E2蛋白。实现了对牛病毒性腹泻病毒的免疫效果的提升。
Description
技术领域
本发明属于病毒疫苗技术领域，具体涉及一种牛病毒性腹泻病毒的mRNA疫苗及其应用。
背景技术
牛病毒性腹泻病毒（Bovine Viral Diarrhea Virus, BVDV）可感染牛引起病毒性腹泻——急性、热性、接触性传染病。BVDV呈世界性分布，感染情况复杂，隐性感染率极高。据估计，其导致的生产损失达687.8美元/头，给全球养牛业造成了巨大损失。BVDV可引起牛生长缓慢、繁殖紊乱、产能降低等多种症状。妊娠50~150天母畜感染后病毒可通过胎盘垂直传播至胎儿，此时由于胎儿免疫***尚未发育完全，未能识别BVDV而引起免疫耐受，出生后即成为BVDV持续感染牛，且终生带毒，并通过鼻涕、唾液、***等不断向外界排毒，成为畜群重要的传染源。此病给全球养殖业造成经济损失与生物安全问题，危害巨大。
目前使用的国外使用的BVDV疫苗主要包括改良性减毒活疫苗、灭活疫苗、E2亚单位疫苗。在美国，BVDV疫苗主要与牛传染性鼻气管炎病毒、牛呼吸道合胞体病毒等组成多联传统疫苗进行应用。国内商品化BVDV疫苗仅为灭活疫苗，种类单一；可分为BVDV单联灭活疫苗与BVDV、牛传染性鼻气管炎二联灭活疫苗。
亚单位疫苗与灭活疫苗存在共有缺点，即需要佐剂，并且需多次免疫，在单剂至抗体水平达到峰值间往往存在免疫空白易感期，且亚单位疫苗存在无法保持天然免疫原构象等缺点。
发明内容
本发明的目的是为了提高牛病毒性腹泻病毒mRNA疫苗的免疫保护作用。
本发明提供一种牛病毒性腹泻病毒（Bovine viral diarrhea Virus）的mRNA疫苗，所述mRNA疫苗编码牛病毒性腹泻病毒的E2蛋白或牛病毒性腹泻病毒截短E2蛋白。
进一步地限定，牛病毒性腹泻病毒的E2蛋白的序列如SEQ ID NO.9所示；牛病毒性腹泻病毒截短E2蛋白的序列如SEQ ID NO.10所示。
进一步地限定，mRNA疫苗由以下成分组成：5’端非翻译区、信号肽序列、牛病毒性腹泻病毒的E2蛋白的编码RNA基因或牛病毒性腹泻病毒截短E2蛋白的的编码RNA基因、3’端非翻译区和多聚腺苷酸。
进一步地限定，5’端非翻译区的核苷酸序列如SEQ ID NO.1所示；3’端非翻译区的核苷酸序列如SEQ ID NO.2所示；多聚腺苷酸的核苷酸序列如SEQ ID NO.3所示；信号肽的核苷酸序列如SEQ ID NO.5所示；牛病毒性腹泻病毒的E2蛋白的编码RNA基因序列如SEQ IDNO.15所示；牛病毒性腹泻病毒截短E2蛋白的编码RNA基因序列如SEQ ID NO.16所示。
本发明提供牛病毒性腹泻病毒的E2蛋白的序列或牛病毒性腹泻病毒截短E2蛋白的序列在制备预防或治疗牛呼吸道疾病综合征的mRNA疫苗中的应用，牛病毒性腹泻病毒的E2蛋白的氨基酸序列如SEQ ID NO.9所示；牛病毒性腹泻病毒截短E2蛋白的氨基酸序列如SEQ ID NO.10所示。
本发明提供一种含有编码上述的mRNA疫苗的DNA分子的重组质粒或重组微生物细胞在制备预防或治疗牛呼吸道疾病综合征的mRNA疫苗中的应用。
本发明提供一种含有上述的mRNA疫苗的脂质纳米颗粒在制备预防或治疗牛呼吸道疾病综合征的mRNA疫苗中的应用。
进一步地限定，引发牛呼吸道疾病综合征的病毒是牛病毒性腹泻病毒。
本发明提供一种上述的mRNA疫苗在制备牛病毒性腹泻病毒的抗体中的应用。
有益效果：
1.疫苗设计上
本研究以中国BVDV流行亚型1a基因亚型毒株E2蛋白基因序列为基础，通过密码子优化以提高翻译效率。在编码E2蛋白的RNA序列前加上信号肽以提高蛋白分泌量，并且在信号肽序列前端添加T7启动子、5’UTR、“cap 1”结构，在编码E2蛋白的RNA序列后端增加3’UTR和多聚腺苷酸尾（poly A）等元件以提高mRNA疫苗翻译效率和稳定性，构建表达E2靶抗原候选mRNA疫苗。并且通过表达截短后的E2蛋白构建高分泌水平的候选mRNA疫苗。
通过体外细胞转染实验与Western Blot实验验证了构建的候选mRNA疫苗在HEK-293T细胞内的表达以及胞外分泌表达，并验证了E2蛋白的二聚体形式， 表达截短E2蛋白的BVDV tE2候选mRNA疫苗表达更多的分泌E2单体蛋白与E2二聚体蛋白。
2.免疫效果
将验证表达的质粒通过体外转录制备mRNA。通过脂质纳米颗粒包装、电镜检测等质量分析，制备成BVDV E2 mRNA疫苗。将构建好的候选mRNA免疫小鼠和犊牛，均可诱导产生针对BVDV的高水平IgG抗体和中和抗体，并且其对于BVDV-1多种不同基因亚型毒株具有交叉保护性。安全性实验表明制备的mRNA候选疫苗具备安全性。牛的攻毒实验表明，BVDV E2mRNA疫苗对国内流行基因亚型BVDV-1b JL毒株具有良好的保护作用，表明了该mRNA疫苗对异源现地流行毒株具有交叉保护作用。
附图说明
图1抗原表达载体质粒图谱；
图2是抗原表达载体质粒表达验证实验中的Western Blot检测结果；
图3是体外转录 mRNA蛋白表达验证实验中的Western Blot检测结果；
图4是体外转录mRNA琼脂糖凝胶电泳结果图；
图5是LNP-mRNA包装后的电镜分析结果图；图5（A）是牛病毒性腹泻病毒的E2蛋白的LNP-mRNA包装后，图5（B）是牛病毒性腹泻病毒截短E2蛋白的LNP-mRNA包装后；
图6是BVDV mRNA疫苗与灭活疫苗免疫小鼠的E2特异性IgG抗体-时间曲线图；
图7是BVDV mRNA疫苗与灭活疫苗免疫小鼠的NADL特异性中和抗体抗体-时间曲线图；
图8是BVDV mRNA疫苗与灭活疫苗免疫小鼠2免后两周的E2特异性IgG抗体、NADL特异性中和抗体结果；
图9是BVDV mRNA疫苗免疫鼠血清的交叉中和实验结果；
图10是 BVDV mRNA疫苗免疫牛的NADL特异性中和抗体结果；
图11是BVDV mRNA疫苗与BVDV灭活疫苗免疫牛的NADL特异性中和抗体结果。
具体实施方式
实施例1. 牛病毒性腹泻病毒mRNA疫苗的抗原表达载体的构建
一、抗原表达载体质粒的构建方法如下：
（1）交由金斯瑞生物公司合成以下基因序列：从5’至3’端分别为T7启动子（SEQ IDNO .4）、5’UTR（SEQ ID NO .4）、kozak序列、tPA信号肽序列（SEQ ID NO .5）、E2序列（SEQID NO .6），构建至pCAGGS载体上，合成质粒命名为pCAGGS-1a-E2；
（2）使用New England Biolabs公司XhoⅠ和KpnⅠ限制性内切酶进行双酶切，通过琼脂糖凝胶电泳回收包含E2基因的DNA片段与pCAGGS载体片段。
（3）在哈尔滨睿博兴科公司合成条引物（F: ATATAAGAGCCACCGCTAGCCTCGAGGCCACCATGGACGCCATGAA；R-tE2: GAGGCTCCAGCCTATTATCAGATATCGGTACCTCAAAAGTAGTCCCGGTGG；）
利用TOYOBO公司的KOD酶以回收包含E2基因的DNA片段为模板进行PCR扩增反应得到截短E2蛋白（tE2）基因序列（SEQ ID NO .7）。
在哈尔滨睿博兴科公司合成引物（F: ATATAAGAGCCACCGCTAGCCTCGAGGCCACCATGGACGCCATGAA；R-EGFP：GAGGCTCCAGCCTATTATCAGATATCGGTACCCTACTTGTACAGCTCGTC），以实验室保存的EGFP质粒为模板通过TOYOBO公司的KOD酶进行PCR扩增反应得到EGFP基因序列（SEQ ID NO .8）。
（4）将PCR产物进行1%琼脂糖凝胶电泳，利用Axygen公司凝胶回收试剂盒回收PCR片段DNA。
（5）通过Takara公司infusion酶进行同源重组，分别将（2）中回收的pCAGGS载体片段与（4）回收的截短tE2 DNA片段与EGFP DNA片段进行同源重组反应，50℃反应15min后取2.5uL产物加入置擎科公司的感受态DH5α感受态细胞中，冰浴30min，42℃热激90s，冰浴2min，加入1mL LB培养液， 37℃摇床 200rpm振摇30min后，2000rpm离心10min后，用100uLLB重悬菌体后涂布与卡纳抗性固体琼脂培养平板上，待第二天挑取菌落。
（6）通过AxyPrep公司的质粒DNA小量试剂盒分别提取2mL菌液中的质粒，质粒分别命名为pCAGGS-1a-tE2（连接有tE2 DNA片段的pCAGGs载体）；pCAGGS-EGFP（连接EGFP的pCAGGs载体）。使用New England Biolabs公司XhoⅠ和KpnⅠ限制性内切酶进行双酶切鉴定，通过哈尔滨睿博兴科公司测序鉴定重组质粒序列正确。重组质粒pCAGGS-EGFP 、pCAGGS-1a-E2、pCAGGS-1a-tE2如图1所示。
实施例2. mRNA转录验证试验
将实施例1中所获得的质粒（pCAGGS-EGFP ；pCAGGS-1a-E2；pCAGGS-1a-tE2）分别用New England Biolabs公司的BsaI限制性核酸内切酶进行酶切，使质粒线性化。下一步进行线性化DNA的纯化，加入 1/10 体积3 M醋酸钠 (pH=5.2)以及3体积无水乙醇，将体系放置于–20°C 1h，通过离心机15000rpm转速4℃条件下离心30min，弃去上清，用DEPC水重悬DNA，测定DNA浓度。
使用Thermofisher公司的mMESSAGE mMACHINE™ T7 Transcription Kit产品进行体外转录。在PCR仪器中37℃ 反应2h。加入试剂盒中的TURBO DNase 1uL，混匀后在PCR仪器中37℃反应15min。使用Qiagen公司的RNA纯化试剂盒进行纯化，使用DEPC水洗脱吸附柱上的RNA，通过Nanodrop仪器测RNA浓度，并将RNA储存于-80℃。
配制1%琼脂糖凝胶，通过琼脂糖凝胶电泳验证体外转录的RNA质量。结果如图3所示，体外转录的EGFP mRNA、1a-E2 mRNA与1a-tE2 mRNA条带清晰单一。体外转录的EGFPmRNA序列如SEQ ID NO.20所示，1a-E2 mRNA序列如SEQ ID NO.18所示，1a-tE2 mRNA序列如SEQ ID NO.19所示。
5’端非翻译区（5’UTR）的DNA序列如SEQ ID NO.1所示：GAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCGCTAGCCTCGAG。
3’端非翻译区（3’UTR）的DNA序列如SEQ ID NO.2所示GATATCTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTG。
多聚腺苷酸（poly A）其序列如SEQ ID NO.3所示，含有104个碱基A：AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA。
T7启动子的DNA序列如SEQ ID NO.4所示：TAATACGACTCACTATAGG
信号肽tPA序列的DNA序列如SEQ ID NO.5所示：ATGGACGCCATGAAGAGGGGGCTGTGCTGCGTGCTGCTGCTGTGCGGAGCCGTGTTCGTGAGCGCCTCC。
EGFP核苷酸编码基因序列如SEQ ID NO.8所示：ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAG。
密码子优化后的E2抗原DNA序列如SEQ ID NO.6所示：ATGCACCTGGACTGTAAGCCTGAGTTCAGCTACGCCATCGCCAAAGACGAGCGGATCGGCCAGCTGGGAGCTGAAGGCCTCACCACCACATGGAAGGAATACAGCCCCGGAATGAAACTGGAAGATACCATGGTCATCGCCTGGTGCGAGGATGGCAAGCTTATGTACCTGCAGCGGTGCACCAGAGAAACAAGATACCTGGCCATCCTGCACACCCGGGCTCTGCCTACCAGCGTGGTTTTCAAGAAGCTGTTCGATGGAAGAAAGCAGGAGGACGTGGTGGAAATGAACGACAATTTTGAGTTCGGCCTGTGCCCTTGTGACGCCAAGCCAATAGTGCGGGGCAAGTTCAACACCACGCTGCTGAACGGCCCCGCCTTCCAGATGGTGTGCCCTATCGGCTGGACCGGCACCGTGTCTTGCACCAGCTTCAACATGGATACACTGGCAACCACAGTGGTTAGAACCTACCGGAGAAGCAAGCCCTTCCCTCACAGACAGGGCTGCATCACACAGAAGAACCTGGGCGAGGACCTGCACAACTGCATCCTGGGCGGCAATTGGACCTGTGTGCCTGGCGACCAGCTGCTGTATAAGGGCGGCAGCATCGAGAGCTGCAAGTGGTGCGGCTACCAATTTAAAGAAAGCGAAGGCCTGCCCCACTACCCCATTGGAAAATGCAAGCTGGAAAACGAGACAGGCTACAGACTGGTGGACAGCACCAGCTGTAATAGAGAAGGTGTGGCCATCGTGCCACAGGGCACCCTGAAGTGCAAGATCGGCAAAACAACAGTGCAGGTGATCGCCATGGACACAAAGCTGGGCCCAATGCCTTGCAGACCCTACGAGATCATCAGCAGCGAGGGCCCTGTGGAAAAGACAGCCTGCACTTTTAACTACACCAAAACCCTGAAGAATAAGTACTTCGAGCCTAGAGACTCCTACTTCCAGCAGTACATGCTGAAGGGCGAGTATCAGTACTGGTTTGACCTGGAAGTGACAGACCACCACCGGGACTACTTTGCCGAGAGCATCCTGGTGGTGGTGGTCGCCCTGCTCGGAGGCAGATACGTGCTGTGGCTGCTCGTGACCTACATGGTGCTCAGCGAGCAGAAAGCCCTGGGC。
密码子优化后的截短E2蛋白tE2抗原DNA序列如SEQ ID NO.7所示：ATGCACCTGGACTGTAAGCCTGAGTTCAGCTACGCCATCGCCAAAGACGAGCGGATCGGCCAGCTGGGAGCTGAAGGCCTCACCACCACATGGAAGGAATACAGCCCCGGAATGAAACTGGAAGATACCATGGTCATCGCCTGGTGCGAGGATGGCAAGCTTATGTACCTGCAGCGGTGCACCAGAGAAACAAGATACCTGGCCATCCTGCACACCCGGGCTCTGCCTACCAGCGTGGTTTTCAAGAAGCTGTTCGATGGAAGAAAGCAGGAGGACGTGGTGGAAATGAACGACAATTTTGAGTTCGGCCTGTGCCCTTGTGACGCCAAGCCAATAGTGCGGGGCAAGTTCAACACCACGCTGCTGAACGGCCCCGCCTTCCAGATGGTGTGCCCTATCGGCTGGACCGGCACCGTGTCTTGCACCAGCTTCAACATGGATACACTGGCAACCACAGTGGTTAGAACCTACCGGAGAAGCAAGCCCTTCCCTCACAGACAGGGCTGCATCACACAGAAGAACCTGGGCGAGGACCTGCACAACTGCATCCTGGGCGGCAATTGGACCTGTGTGCCTGGCGACCAGCTGCTGTATAAGGGCGGCAGCATCGAGAGCTGCAAGTGGTGCGGCTACCAATTTAAAGAAAGCGAAGGCCTGCCCCACTACCCCATTGGAAAATGCAAGCTGGAAAACGAGACAGGCTACAGACTGGTGGACAGCACCAGCTGTAATAGAGAAGGTGTGGCCATCGTGCCACAGGGCACCCTGAAGTGCAAGATCGGCAAAACAACAGTGCAGGTGATCGCCATGGACACAAAGCTGGGCCCAATGCCTTGCAGACCCTACGAGATCATCAGCAGCGAGGGCCCTGTGGAAAAGACAGCCTGCACTTTTAACTACACCAAAACCCTGAAGAATAAGTACTTCGAGCCTAGAGACTCCTACTTCCAGCAGTACATGCTGAAGGGCGAGTATCAGTACTGGTTTGACCTGGAAGTGACAGACCACCACCGGGACTACTTT。
密码子优化后的E2抗原氨基酸序列如SEQ ID NO.9所示：
MHLDCKPEFSYAIAKDERIGQLGAEGLTTTWKEYSPGMKLEDTMVIAWCEDGKLMYLQRCTRETRYLAILHTRALPTSVVFKKLFDGRKQEDVVEMNDNFEFGLCPCDAKPIVRGKFNTTLLNGPAFQMVCPIGWTGTVSCTSFNMDTLATTVVRTYRRSKPFPHRQGCITQKNLGEDLHNCILGGNWTCVPGDQLLYKGGSIESCKWCGYQFKESEGLPHYPIGKCKLENETGYRLVDSTSCNREGVAIVPQGTLKCKIGKTTVQVIAMDTKLGPMPCRPYEIISSEGPVEKTACTFNYTKTLKNKYFEPRDSYFQQYMLKGEYQYWFDLEVTDHHRDYFAESILVVVVALLGGRYVLWLLVTYMVLSEQKALG。
密码子优化后的截短E2蛋白 tE2抗原氨基酸序列如SEQ ID NO.10所示：
MHLDCKPEFSYAIAKDERIGQLGAEGLTTTWKEYSPGMKLEDTMVIAWCEDGKLMYLQRCTRETRYLAILHTRALPTSVVFKKLFDGRKQEDVVEMNDNFEFGLCPCDAKPIVRGKFNTTLLNGPAFQMVCPIGWTGTVSCTSFNMDTLATTVVRTYRRSKPFPHRQGCITQKNLGEDLHNCILGGNWTCVPGDQLLYKGGSIESCKWCGYQFKESEGLPHYPIGKCKLENETGYRLVDSTSCNREGVAIVPQGTLKCKIGKTTVQVIAMDTKLGPMPCRPYEIISSEGPVEKTACTFNYTKTLKNKYFEPRDSYFQQYMLKGEYQYWFDLEVTDHHRDYF。
5’端非翻译区（5’UTR）的RNA序列如SEQ ID NO.11所示
GAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCGCUAGCCUCGAG。
3’ 端非翻译区（3’UTR）的RNA序列如SEQ ID NO.12所示：GAUAUCUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGA。
多聚腺苷酸（poly A）的RNA序列如SEQ ID NO.13所示：
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA。
tPA信号肽的RNA序列如SEQ ID NO.14所示：
AUGGACGCCAUGAAGAGGGGGCUGUGCUGCGUGCUGCUGCUGUGCGGAGCCGUGUUCGUGAGCGCCUCC。
密码子优化后的E2抗原RNA序列如SEQ ID NO.15所示：
AUGCACCUGGACUGUAAGCCUGAGUUCAGCUACGCCAUCGCCAAAGACGAGCGGAUCGGCCAGCUGGGAGCUGAAGGCCUCACCACCACAUGGAAGGAAUACAGCCCCGGAAUGAAACUGGAAGAUACCAUGGUCAUCGCCUGGUGCGAGGAUGGCAAGCUUAUGUACCUGCAGCGGUGCACCAGAGAAACAAGAUACCUGGCCAUCCUGCACACCCGGGCUCUGCCUACCAGCGUGGUUUUCAAGAAGCUGUUCGAUGGAAGAAAGCAGGAGGACGUGGUGGAAAUGAACGACAAUUUUGAGUUCGGCCUGUGCCCUUGUGACGCCAAGCCAAUAGUGCGGGGCAAGUUCAACACCACGCUGCUGAACGGCCCCGCCUUCCAGAUGGUGUGCCCUAUCGGCUGGACCGGCACCGUGUCUUGCACCAGCUUCAACAUGGAUACACUGGCAACCACAGUGGUUAGAACCUACCGGAGAAGCAAGCCCUUCCCUCACAGACAGGGCUGCAUCACACAGAAGAACCUGGGCGAGGACCUGCACAACUGCAUCCUGGGCGGCAAUUGGACCUGUGUGCCUGGCGACCAGCUGCUGUAUAAGGGCGGCAGCAUCGAGAGCUGCAAGUGGUGCGGCUACCAAUUUAAAGAAAGCGAAGGCCUGCCCCACUACCCCAUUGGAAAAUGCAAGCUGGAAAACGAGACAGGCUACAGACUGGUGGACAGCACCAGCUGUAAUAGAGAAGGUGUGGCCAUCGUGCCACAGGGCACCCUGAAGUGCAAGAUCGGCAAAACAACAGUGCAGGUGAUCGCCAUGGACACAAAGCUGGGCCCAAUGCCUUGCAGACCCUACGAGAUCAUCAGCAGCGAGGGCCCUGUGGAAAAGACAGCCUGCACUUUUAACUACACCAAAACCCUGAAGAAUAAGUACUUCGAGCCUAGAGACUCCUACUUCCAGCAGUACAUGCUGAAGGGCGAGUAUCAGUACUGGUUUGACCUGGAAGUGACAGACCACCACCGGGACUACUUUGCCGAGAGCAUCCUGGUGGUGGUGGUCGCCCUGCUCGGAGGCAGAUACGUGCUGUGGCUGCUCGUGACCUACAUGGUGCUCAGCGAGCAGAAAGCCCUGGGC。
截短E2蛋白 tE2抗原RNA序列如SEQ ID NO.16所示：
AUGCACCUGGACUGUAAGCCUGAGUUCAGCUACGCCAUCGCCAAAGACGAGCGGAUCGGCCAGCUGGGAGCUGAAGGCCUCACCACCACAUGGAAGGAAUACAGCCCCGGAAUGAAACUGGAAGAUACCAUGGUCAUCGCCUGGUGCGAGGAUGGCAAGCUUAUGUACCUGCAGCGGUGCACCAGAGAAACAAGAUACCUGGCCAUCCUGCACACCCGGGCUCUGCCUACCAGCGUGGUUUUCAAGAAGCUGUUCGAUGGAAGAAAGCAGGAGGACGUGGUGGAAAUGAACGACAAUUUUGAGUUCGGCCUGUGCCCUUGUGACGCCAAGCCAAUAGUGCGGGGCAAGUUCAACACCACGCUGCUGAACGGCCCCGCCUUCCAGAUGGUGUGCCCUAUCGGCUGGACCGGCACCGUGUCUUGCACCAGCUUCAACAUGGAUACACUGGCAACCACAGUGGUUAGAACCUACCGGAGAAGCAAGCCCUUCCCUCACAGACAGGGCUGCAUCACACAGAAGAACCUGGGCGAGGACCUGCACAACUGCAUCCUGGGCGGCAAUUGGACCUGUGUGCCUGGCGACCAGCUGCUGUAUAAGGGCGGCAGCAUCGAGAGCUGCAAGUGGUGCGGCUACCAAUUUAAAGAAAGCGAAGGCCUGCCCCACUACCCCAUUGGAAAAUGCAAGCUGGAAAACGAGACAGGCUACAGACUGGUGGACAGCACCAGCUGUAAUAGAGAAGGUGUGGCCAUCGUGCCACAGGGCACCCUGAAGUGCAAGAUCGGCAAAACAACAGUGCAGGUGAUCGCCAUGGACACAAAGCUGGGCCCAAUGCCUUGCAGACCCUACGAGAUCAUCAGCAGCGAGGGCCCUGUGGAAAAGACAGCCUGCACUUUUAACUACACCAAAACCCUGAAGAAUAAGUACUUCGAGCCUAGAGACUCCUACUUCCAGCAGUACAUGCUGAAGGGCGAGUAUCAGUACUGGUUUGACCUGGAAGUGACAGACCACCACCGGGACUACUUU。
EGFP蛋白RNA序列如SEQ ID NO.17所示：
AUGGUGAGCAAGGGCGAGGAGCUGUUCACCGGGGUGGUGCCCAUCCUGGUCGAGCUGGACGGCGACGUAAACGGCCACAAGUUCAGCGUGUCCGGCGAGGGCGAGGGCGAUGCCACCUACGGCAAGCUGACCCUGAAGUUCAUCUGCACCACCGGCAAGCUGCCCGUGCCCUGGCCCACCCUCGUGACCACCCUGACCUACGGCGUGCAGUGCUUCAGCCGCUACCCCGACCACAUGAAGCAGCACGACUUCUUCAAGUCCGCCAUGCCCGAAGGCUACGUCCAGGAGCGCACCAUCUUCUUCAAGGACGACGGCAACUACAAGACCCGCGCCGAGGUGAAGUUCGAGGGCGACACCCUGGUGAACCGCAUCGAGCUGAAGGGCAUCGACUUCAAGGAGGACGGCAACAUCCUGGGGCACAAGCUGGAGUACAACUACAACAGCCACAACGUCUAUAUCAUGGCCGACAAGCAGAAGAACGGCAUCAAGGUGAACUUCAAGAUCCGCCACAACAUCGAGGACGGCAGCGUGCAGCUCGCCGACCACUACCAGCAGAACACCCCCAUCGGCGACGGCCCCGUGCUGCUGCCCGACAACCACUACCUGAGCACCCAGUCCGCCCUGAGCAAAGACCCCAACGAGAAGCGCGAUCACAUGGUCCUGCUGGAGUUCGUGACCGCCGCCGGGAUCACUCUCGGCAUGGACGAGCUGUACAAGUAG。
mRNA疫苗中编码E2抗原的1a-E2 mRNA分子序列如SEQ ID NO.18所示：
5’cap-GAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCGCUAGCCUCGAGGCCACCAUGGACGCCAUGAAGAGGGGGCUGUGCUGCGUGCUGCUGCUGUGCGGAGCCGUGUUCGUGAGCGCCUCCAUGCACCUGGACUGUAAGCCUGAGUUCAGCUACGCCAUCGCCAAAGACGAGCGGAUCGGCCAGCUGGGAGCUGAAGGCCUCACCACCACAUGGAAGGAAUACAGCCCCGGAAUGAAACUGGAAGAUACCAUGGUCAUCGCCUGGUGCGAGGAUGGCAAGCUUAUGUACCUGCAGCGGUGCACCAGAGAAACAAGAUACCUGGCCAUCCUGCACACCCGGGCUCUGCCUACCAGCGUGGUUUUCAAGAAGCUGUUCGAUGGAAGAAAGCAGGAGGACGUGGUGGAAAUGAACGACAAUUUUGAGUUCGGCCUGUGCCCUUGUGACGCCAAGCCAAUAGUGCGGGGCAAGUUCAACACCACGCUGCUGAACGGCCCCGCCUUCCAGAUGGUGUGCCCUAUCGGCUGGACCGGCACCGUGUCUUGCACCAGCUUCAACAUGGAUACACUGGCAACCACAGUGGUUAGAACCUACCGGAGAAGCAAGCCCUUCCCUCACAGACAGGGCUGCAUCACACAGAAGAACCUGGGCGAGGACCUGCACAACUGCAUCCUGGGCGGCAAUUGGACCUGUGUGCCUGGCGACCAGCUGCUGUAUAAGGGCGGCAGCAUCGAGAGCUGCAAGUGGUGCGGCUACCAAUUUAAAGAAAGCGAAGGCCUGCCCCACUACCCCAUUGGAAAAUGCAAGCUGGAAAACGAGACAGGCUACAGACUGGUGGACAGCACCAGCUGUAAUAGAGAAGGUGUGGCCAUCGUGCCACAGGGCACCCUGAAGUGCAAGAUCGGCAAAACAACAGUGCAGGUGAUCGCCAUGGACACAAAGCUGGGCCCAAUGCCUUGCAGACCCUACGAGAUCAUCAGCAGCGAGGGCCCUGUGGAAAAGACAGCCUGCACUUUUAACUACACCAAAACCCUGAAGAAUAAGUACUUCGAGCCUAGAGACUCCUACUUCCAGCAGUACAUGCUGAAGGGCGAGUAUCAGUACUGGUUUGACCUGGAAGUGACAGACCACCACCGGGACUACUUUGCCGAGAGCAUCCUGGUGGUGGUGGUCGCCCUGCUCGGAGGCAGAUACGUGCUGUGGCUGCUCGUGACCUACAUGGUGCUCAGCGAGCAGAAAGCCCUGGGCUGAGGUACCGAUAUCUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA。
mRNA疫苗中编码截短E2蛋白的1a-tE2 mRNA分子序列如SEQ ID NO.19所示：
5’cap-GAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCGCUAGCCUCGAGGCCACCAUGGACGCCAUGAAGAGGGGGCUGUGCUGCGUGCUGCUGCUGUGCGGAGCCGUGUUCGUGAGCGCCUCCAUGCACCUGGACUGUAAGCCUGAGUUCAGCUACGCCAUCGCCAAAGACGAGCGGAUCGGCCAGCUGGGAGCUGAAGGCCUCACCACCACAUGGAAGGAAUACAGCCCCGGAAUGAAACUGGAAGAUACCAUGGUCAUCGCCUGGUGCGAGGAUGGCAAGCUUAUGUACCUGCAGCGGUGCACCAGAGAAACAAGAUACCUGGCCAUCCUGCACACCCGGGCUCUGCCUACCAGCGUGGUUUUCAAGAAGCUGUUCGAUGGAAGAAAGCAGGAGGACGUGGUGGAAAUGAACGACAAUUUUGAGUUCGGCCUGUGCCCUUGUGACGCCAAGCCAAUAGUGCGGGGCAAGUUCAACACCACGCUGCUGAACGGCCCCGCCUUCCAGAUGGUGUGCCCUAUCGGCUGGACCGGCACCGUGUCUUGCACCAGCUUCAACAUGGAUACACUGGCAACCACAGUGGUUAGAACCUACCGGAGAAGCAAGCCCUUCCCUCACAGACAGGGCUGCAUCACACAGAAGAACCUGGGCGAGGACCUGCACAACUGCAUCCUGGGCGGCAAUUGGACCUGUGUGCCUGGCGACCAGCUGCUGUAUAAGGGCGGCAGCAUCGAGAGCUGCAAGUGGUGCGGCUACCAAUUUAAAGAAAGCGAAGGCCUGCCCCACUACCCCAUUGGAAAAUGCAAGCUGGAAAACGAGACAGGCUACAGACUGGUGGACAGCACCAGCUGUAAUAGAGAAGGUGUGGCCAUCGUGCCACAGGGCACCCUGAAGUGCAAGAUCGGCAAAACAACAGUGCAGGUGAUCGCCAUGGACACAAAGCUGGGCCCAAUGCCUUGCAGACCCUACGAGAUCAUCAGCAGCGAGGGCCCUGUGGAAAAGACAGCCUGCACUUUUAACUACACCAAAACCCUGAAGAAUAAGUACUUCGAGCCUAGAGACUCCUACUUCCAGCAGUACAUGCUGAAGGGCGAGUAUCAGUACUGGUUUGACCUGGAAGUGACAGACCACCACCGGGACUACUUUUGAGGUACCGAUAUCUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA。
体外转录完整EGFP mRNA其RNA序列如SEQ ID NO.20所示：
5’cap-GAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCGCUAGCCUCGAGGCCACCAUGGACGCCAUGAAGAGGGGGCUGUGCUGCGUGCUGCUGCUGUGCGGAGCCGUGUUCGUGAGCGCCUCCAUGGUGAGCAAGGGCGAGGAGCUGUUCACCGGGGUGGUGCCCAUCCUGGUCGAGCUGGACGGCGACGUAAACGGCCACAAGUUCAGCGUGUCCGGCGAGGGCGAGGGCGAUGCCACCUACGGCAAGCUGACCCUGAAGUUCAUCUGCACCACCGGCAAGCUGCCCGUGCCCUGGCCCACCCUCGUGACCACCCUGACCUACGGCGUGCAGUGCUUCAGCCGCUACCCCGACCACAUGAAGCAGCACGACUUCUUCAAGUCCGCCAUGCCCGAAGGCUACGUCCAGGAGCGCACCAUCUUCUUCAAGGACGACGGCAACUACAAGACCCGCGCCGAGGUGAAGUUCGAGGGCGACACCCUGGUGAACCGCAUCGAGCUGAAGGGCAUCGACUUCAAGGAGGACGGCAACAUCCUGGGGCACAAGCUGGAGUACAACUACAACAGCCACAACGUCUAUAUCAUGGCCGACAAGCAGAAGAACGGCAUCAAGGUGAACUUCAAGAUCCGCCACAACAUCGAGGACGGCAGCGUGCAGCUCGCCGACCACUACCAGCAGAACACCCCCAUCGGCGACGGCCCCGUGCUGCUGCCCGACAACCACUACCUGAGCACCCAGUCCGCCCUGAGCAAAGACCCCAACGAGAAGCGCGAUCACAUGGUCCUGCUGGAGUUCGUGACCGCCGCCGGGAUCACUCUCGGCAUGGACGAGCUGUACAAGUAGGGUACCGAUAUCUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA。
实施例3. E2蛋白表达验证试验
（1）将纯化后的体外转录RNA通过Mirus公司TransIT™-mRNA Transfection Kits试剂转染HEK-293T细胞进行验证表达。将293T细胞均匀铺于六孔板中，使用Mirus公司TransIT™-mRNA Transfection Kits试剂盒进行RNA的转染，每孔转染2 μg mRNA。转染48h后，收集细胞上清液，使用Millipore公司的10kDa浓缩管进行上清液的浓缩，分别加入含DDT的5×loading buffer与不含DTT的5×loading buffer进行样品处理，通过还原性SDS-PAGE与非还原性SDS-PAGE电泳后使用proteintech公司的抗His标签抗体得到WesternBlot结果；
（2）结果如图4所示，与载体对照组pCAGGs-tPA-EGFP mRNA相比，转染了1a-E2mRNA与1a-tE2 mRNA的试验组中均出现了与大小与预期相符的蛋白条带。并且1a-tE2 mRNA试验组上清中的蛋白含量远高于pCAGGs-tPA-E2 mRNA试验组，表明携带tPA信号肽的tE2a靶抗原序列表达蛋白具有高效分泌特征。
实施例4.制备mRNA疫苗
通过BsaI限制性核酸内切酶对实施例1中构建成功得到的质粒pCAGGS-1a-E2；pCAGGS-1a-tE2进行单酶切，使其环状DNA线性化。通过3 M醋酸钠 (pH=5.2)、无水乙醇、0.5M EDTA纯化DNA分子：加入 1/10 体积3 M醋酸钠 (PH=5.2)以及3体积无水乙醇，1/20体积0.5 M EDTA，混匀，放置-20℃，15000rpm离心后得到沉淀的DNA分子。使用蛋白酶K进行下一步纯化，去除杂质蛋白。通过苯酚/氯仿方法提取线性化DNA分子，DNA溶解至无核酸酶水以待体外转录。
通过诺唯赞公司的T7 High Yield RNA Transcription Kit(N1 -Me-PseudoUTP)产品对上述线性化DNA分子进行体外转录，该反应可获得大量带N1 -Me-Pseudo UTP修饰的mRNA，N1 -Me-Pseudo UTP 修饰目的是为了降低mRNA疫苗诱导的天然免疫反应。接着对体外转录mRNA进行加帽修饰，按近岸公司的Cap 1 Capping System产品说明书操作。通过酚氯仿法提取含帽结构的体外转录mRNA分子，并对其包装制备为LNP-mRNA疫苗。
具体实验步骤为：首先是醇相的制备，将脂质以阳离子脂质（SM102）:二硬酯酰磷脂酰胆碱（DSPC） : 胆固醇：DMG-2000（购自赛诺邦格公司）=50:10:38.5:1.5 （质量比）比例溶解在无水乙醇中；其次制备水相，pH4.0的柠檬酸缓冲液（50 mM）作为溶解mRNA的溶质；通过微流控设备以醇相:水相(体积比为1:3)进行mRNA的包装。接着以无RNase的PBS缓冲液进行稀释，使用30 KDa超滤管对其进行浓缩换液。加入等体积蔗糖浓度为20%的PBS溶液，将mRNA浓度调整后为60 μg/ml，蔗糖浓度为10%，最后使用0.22 μm滤膜对其过滤，即为制备好的1a-E2、1a-tE2LNP-mRNA疫苗，分装后置于-20℃储存。
所述的质体纳米颗粒包含阳离子脂质、二硬酯酰磷脂酰胆碱（DSPC）、 胆固醇和DMG-2000中的至少一种。
通过电镜检测结果（图5）验证制备的制备后的1a-E2、1a-tE2 LNP-mRNA疫苗颗粒均匀一致。
实施例5.小鼠免疫评价实验
将实施例4制备好的1a-E2 mRNA疫苗与1a-tE2 mRNA疫苗免疫BALB/c小鼠进行免疫效果的评价。将20只购于辽宁长生生物技术股份有限公司的8周龄SPF BALB/c小鼠随机分成44组，5只/组，分别免疫20ug （100uL ）1a-E2 mRNA疫苗、20ug （100uL）1a-tE2 mRNA疫苗、60uL 华威特公司的BVDV（1a-NM01毒株）灭活疫苗；100uL PBS。免疫3周后，使用和首次免疫相同剂量体积的疫苗进行加强免疫，之后每隔两周进行小鼠血液的采集并分离血清进行抗体检测。
（1）抗E2 IgG抗体和中和抗体的检测
通过基于E2蛋白为包被抗原的间接ELISA方法分析免疫血清中特异性抗E2抗体效价。将纯化后的E2蛋白（33ng/孔）包被ELISA板，将加强免疫后不同时间点所分离的血清进行倍比稀释，加入对应ELISA板中，依次加入HRP-anti-mouse IgG 二抗，及TMB，显色终止后A450检测OD值，测定抗体效价。结果如图6所示，结果表明mRNA疫苗二免后二周的小鼠血清特异性抗E2抗体效价最高，与BVDV灭活疫苗相比，mRNA疫苗可诱导高水平特异性抗E2 IgG抗体，可以有效地诱导机体产生体液免疫应答（图6，表1）。
通过中和试验分析免疫血清中的中和抗体效价。将等体积报告病毒NADL-mcherry与等体积不同稀释度小鼠血清在37℃中和作用1h后，加入至MDBK细胞中，感染2h后换为含2%马血清的DMEM培养液。3天后通过蔡司公司高通量活细胞分析仪分析NADL-mcherry感染范围及细胞数，计算IC50，中和抗体效价定义为中和50% NADL-mcherry感染时的血清稀释倍数。结果如图7，表2所示，mRNA疫苗二免后二周的小鼠anti-NADL nAb效价最高，与BVDV灭活疫苗相比，mRNA疫苗可诱导高水平特异性anti-NADL nAb。
进一步比较免疫峰值加强免疫后两周的抗体（图8），相较BVDV灭活疫苗，BVDVmRNA疫苗可诱导显著升高的E2特异性IgG抗体、NADL特异性中和抗体。
表1 BVDV E2 mRNA疫苗与灭活疫苗免疫接种小鼠诱导的E2特异性IgG抗体滴度
表2BVDV E2 mRNA疫苗与灭活疫苗免疫接种小鼠诱导的NADL特异性中和抗体滴度
（2）交叉保护性的检测
通过其他不同基因亚型BVDV毒株进行中和试验，分析免疫BVDV E2 mRNA疫苗小鼠血清中的交叉中和抗体效价。分别将等体积不同基因亚型BVDV毒株VEDEVAC、3877等与等体积最终稀释度为200的小鼠血清在37℃中和作用1h后，加入至MDBK细胞中，感染2h后换为含2%马血清的DMEM培养液。3天后使用VMRD公司的BVDV多抗血清以及invitrogen的488标记anti-goat IgG抗体通过间接免疫荧光实验观察BVDV感染细胞，通过蔡司公司高通量活细胞分析仪分析BVDV感染范围及细胞数，计算200稀释倍数下的BVDV E2 mRNA疫苗免疫小鼠血清对不同基因亚型BVDV毒株的抑制率。结果如图9所示，mRNA疫苗免疫的小鼠血清中对于BVDV-1多种不同基因亚型毒株具有高达90%的抑制率，对不同基因亚型BVDV毒株具有交叉保护性，其可诱导小鼠产生交叉中和抗体。
（3）细胞免疫反应的检测
通过细胞因子染色（ICCS）实验检测抗原特异性T细胞以评估细胞免疫，取疫苗免疫后鼠淋巴细胞接种到培养板中，使用E2蛋白肽库刺激。将细胞与抗 CD28抗体在 37 ℃和 5% CO2条件下共刺激。DMSO用作阴性对照。使用PMA/ionomycin作为阳性对照。随后，使用固定/透化试剂盒进行实验，使用以下细胞因子抗体：抗IFN-γ，抗TNF-α，抗IL-2和抗IL-4与脾细胞作用，最后通过流式细胞术分析细胞因子特异性淋巴细胞数量。
结果在加强疫苗接种后数周，实验结果表明，BVDV E2 mRNA疫苗疫苗可诱导表达IFN-γ、IL-2 和 TNF-a 的抗原特异性、多功能 CD8+T 细胞；相比BVDV灭活疫苗，BVDV E2mRNA疫苗诱导了更高的CD4+和CD8+记忆T细胞。
实施例6.mRNA疫苗安全性实验
对2月龄犊牛进行免疫接种。共10只犊牛，每组各5头犊牛，一组肌肉接种高剂量mRNA疫苗，600μg/头；另一组注射PBS作为对照。首次接种后3周，相同剂量和途径进行第二次接种。观察犊牛身体健康状况。PBS组、mRNA疫苗组犊牛中所有牛健康状态良好、体温正常，表明疫苗的安全性良好。
实施例7.mRNA疫苗免疫牛实验
用3～4月龄健康易感牛8头，每组4只牛。其中一组颈部肌肉注射1a-E2 mRNA疫苗200ug、另一组颈部肌肉注射107TCID50BVDV 灭活病毒疫苗。BVDV 灭活病毒疫苗为实验室制备：BVDV病毒在MDBK细胞中培养，四天后收获细胞并于 -80℃ 冻融 3 次，BVDV通过sigma公司的Binary Ethyleneimine（BEI）灭活，按BVDV灭活病毒与MONTANIDETMISA 15A VG佐剂体积比为85%:15%比例制备BVDV灭活疫苗。21日后进行二免，接种剂量同首免。BVDV 灭活病毒疫苗免疫牛组在上一次免疫后4周分别进行了第三次、第四次免疫，接种剂量同首免。
1a-E2 mRNA免疫组在免疫前、第一次免疫后3周，加强免疫后1周分别进行了血液采集并且分离血清。BVDV 灭活疫苗免疫组在第四次免疫1周后进行血液采集并且分离血清。
通过中和试验分析免疫牛血清中的中和抗体效价。将等体积报告病毒NADL-mcherry与等体积不同稀释度牛血清在37℃中和作用1h后，加入至MDBK细胞中，感染2h后换为含2%马血清的DMEM培养液。3天后通过蔡司公司高通量活细胞分析仪分析NADL-mcherry感染范围及细胞数，计算IC50，中和抗体效价定义为中和50% NADL-mcherry感染时的血清稀释倍数。结果如图10所示，mRNA疫苗在加强免疫后可诱导高水平anti-NADL nAb效价。与BVDV 灭活疫苗相比，mRNA疫苗可诱导约2倍的更高水平anti-NADL nAb（图11、表3）所示。
表3 BVDV E2 mRNA疫苗与BVDV灭活疫苗免疫接种牛诱导的NADL特异性中和抗体滴度
实施例8.mRNA疫苗攻毒实验
用3～4月龄健康易感牛4头，每组2只牛。其中一组颈部肌肉注射1a-E2 mRNA疫苗200ug、另一组作为对照注射PBS。21日后进行二免，接种剂量同首免。二免后21日，每头牛鼻内喷雾接种1b - JL株（病毒含量≥106.5FAID50/ml）6ml。攻毒前观察牛只并无任何咳嗽、发烧等临床症状，并通过PCR检测判定牛只为BVDV阴性，攻毒前测量牛只直肠温度作为牛基础体温数值；攻毒前采取牛的血液并检测其白细胞数量作为牛只的白细胞基础数值。攻毒后每日测量牛只体温并隔日采血，进行血液的白细胞数量测定、并进行BVDV病毒分离。
牛的攻毒实验结果为，注射PBS的对照牛在攻毒后皆出现发烧现象，而免疫1a-E2mRNA疫苗牛只体温正常；白细胞计数结果显示，PBS对照组牛攻毒后白细胞数目下降、1a-E2mRNA接种疫苗组白细胞计数显着高于对照组 。
实验结果表明BVDV E2 mRNA疫苗对国内流行基因亚型BVDV-1b JL毒株具有良好的保护作用，表明了该mRNA疫苗对异源现地流行毒株具有交叉保护作用。
实施例9.mRNA疫苗制备抗体的方法
将制备好的1a-E2 mRNA疫苗与1a-tE2 mRNA疫苗免疫BALB/c小鼠进行单抗的制备。将10只购于辽宁长生生物技术股份有限公司的8周龄SPF BALB/c小鼠随机分成2组，5只/组，分别免疫20ug1a-E2 mRNA疫苗、20ug 1a-tE2 mRNA疫苗。免疫3周后，使用和首次免疫相同剂量体积的疫苗进行加强免疫，之后进行小鼠血液的采集并测定血清抗体效价。取免疫鼠脾细胞与 SP2/0 细胞融合。通过ELISA 方法、间接免疫荧光等筛选阳性细胞克隆，获得稳定分泌特异性单克隆抗体的杂交瘤细胞株，所制备的单抗可用于建立检测 BVDV 病原及抗体的诊断方法。
Claims (9)
1.一种牛病毒性腹泻病毒（Bovine viral diarrhea Virus）的mRNA疫苗，其特征在于，所述mRNA疫苗编码牛病毒性腹泻病毒的E2蛋白或牛病毒性腹泻病毒截短E2蛋白；牛病毒性腹泻病毒的E2蛋白的序列如SEQ ID NO.9所示；牛病毒性腹泻病毒截短E2蛋白的序列如SEQID NO.10所示。
2.根据权利要求1所述的mRNA疫苗，其特征在于，mRNA疫苗由以下成分组成：5’端非翻译区、信号肽序列、牛病毒性腹泻病毒的E2蛋白的编码RNA基因或牛病毒性腹泻病毒E2截短蛋白的的编码RNA基因、3’端非翻译区和多聚腺苷酸。
3.根据权利要求2所述的mRNA疫苗，其特征在于，5’端非翻译区的核苷酸序列如SEQ IDNO.1所示；3’端非翻译区的核苷酸序列如SEQ ID NO.2所示；多聚腺苷酸的核苷酸序列如SEQ ID NO.3所示；信号肽的核苷酸序列如SEQ ID NO.5所示；牛病毒性腹泻病毒的E2蛋白的编码RNA基因序列如SEQ ID NO.15所示；牛病毒性腹泻病毒截短E2蛋白的编码RNA基因序列如SEQ ID NO.16所示。
4.牛病毒性腹泻病毒的E2蛋白的氨基酸序列或牛病毒性腹泻病毒截短E2蛋白的氨基酸序列在制备预防或治疗牛呼吸道疾病综合征的mRNA疫苗中的应用，其特征在于，牛病毒性腹泻病毒的E2蛋白的氨基酸序列如SEQ ID NO.9所示；牛病毒性腹泻病毒截短E2蛋白的氨基酸序列如SEQ ID NO.10所示。
5.含有编码权利要求1所述的mRNA疫苗的DNA分子的重组质粒或重组微生物细胞在制备预防或治疗牛呼吸道疾病综合征的mRNA疫苗中的应用。
6.含有编码权利要求1所述的mRNA疫苗的DNA分子的重组微生物细胞在制备预防或治疗牛呼吸道疾病综合征的mRNA疫苗中的应用。
7.含有权利要求1所述的mRNA疫苗的脂质纳米颗粒在制备预防或治疗牛呼吸道疾病综合征的mRNA疫苗中的应用。
8.根据权利要求4-7任一项所述的应用，其特征在于，引发牛呼吸道疾病综合征的病毒是牛病毒性腹泻病毒。
9.权利要求1-3任一项所述的mRNA疫苗在制备牛病毒性腹泻病毒的抗体中的应用。
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Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6060457A (en) * | 1996-06-20 | 2000-05-09 | Universite De Montreal | DNA plasmid vaccine for immunization of animals against BVDV |
| AU3510001A (en) * | 1996-07-19 | 2001-07-12 | Merial | Polynucleotide vaccine formula, particularly for treating bovine respiratory disease |
| US20030129744A1 (en) * | 2000-06-27 | 2003-07-10 | Tobias Schlapp | Bvdv virus-like particles |
| CN110167587A (zh) * | 2016-11-11 | 2019-08-23 | 摩登纳特斯有限公司 | 流感疫苗 |
| CN110545839A (zh) * | 2017-05-05 | 2019-12-06 | 伊兰科美国公司 | 牛呼吸道疾病疫苗 |
| CN113230394A (zh) * | 2021-04-30 | 2021-08-10 | 广州源博医药科技有限公司 | 一种用于牛病毒性腹泻的rna疫苗及其构建方法 |
| CN113388587A (zh) * | 2021-07-06 | 2021-09-14 | 天康制药(苏州)有限公司 | 表达牛病毒性腹泻e2基因的重组牛结节疹病毒及其应用 |
| CN113637695A (zh) * | 2021-10-14 | 2021-11-12 | 深圳湾实验室 | 靶向刺激体液免疫和细胞免疫的新冠病毒mRNA疫苗 |
| CN115521942A (zh) * | 2022-06-14 | 2022-12-27 | 天津市农业科学院 | 一种bvdv表位基因疫苗的构建方法及其应用 |
| CN116240222A (zh) * | 2022-12-29 | 2023-06-09 | 北京华夏兴洋生物科技有限公司 | 一种密码子优化的牛病毒性腹泻病毒1型e2蛋白基因及其应用 |
| CN116284272A (zh) * | 2023-05-15 | 2023-06-23 | 南京澄实生物科技有限公司 | 一种广谱抗牛科病毒性腹泻病毒的mRNA疫苗及其应用 |
-
2023
- 2023-08-21 CN CN202311047976.XA patent/CN116785420B/zh active Active
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6060457A (en) * | 1996-06-20 | 2000-05-09 | Universite De Montreal | DNA plasmid vaccine for immunization of animals against BVDV |
| AU3510001A (en) * | 1996-07-19 | 2001-07-12 | Merial | Polynucleotide vaccine formula, particularly for treating bovine respiratory disease |
| US20030129744A1 (en) * | 2000-06-27 | 2003-07-10 | Tobias Schlapp | Bvdv virus-like particles |
| CN110167587A (zh) * | 2016-11-11 | 2019-08-23 | 摩登纳特斯有限公司 | 流感疫苗 |
| CN110545839A (zh) * | 2017-05-05 | 2019-12-06 | 伊兰科美国公司 | 牛呼吸道疾病疫苗 |
| CN113230394A (zh) * | 2021-04-30 | 2021-08-10 | 广州源博医药科技有限公司 | 一种用于牛病毒性腹泻的rna疫苗及其构建方法 |
| CN113388587A (zh) * | 2021-07-06 | 2021-09-14 | 天康制药(苏州)有限公司 | 表达牛病毒性腹泻e2基因的重组牛结节疹病毒及其应用 |
| CN113637695A (zh) * | 2021-10-14 | 2021-11-12 | 深圳湾实验室 | 靶向刺激体液免疫和细胞免疫的新冠病毒mRNA疫苗 |
| CN115521942A (zh) * | 2022-06-14 | 2022-12-27 | 天津市农业科学院 | 一种bvdv表位基因疫苗的构建方法及其应用 |
| CN116240222A (zh) * | 2022-12-29 | 2023-06-09 | 北京华夏兴洋生物科技有限公司 | 一种密码子优化的牛病毒性腹泻病毒1型e2蛋白基因及其应用 |
| CN116284272A (zh) * | 2023-05-15 | 2023-06-23 | 南京澄实生物科技有限公司 | 一种广谱抗牛科病毒性腹泻病毒的mRNA疫苗及其应用 |
Non-Patent Citations (5)
| Title |
|---|
| COLETT, M.S. ET AL.: "structural protein E2 [Bovine viral diarrhea virus 1], NCBI Reference Sequence: NP_776263.1", 《GENPEPT》, vol. 2, pages 908 * |
| RONG LIANG ET AL.: "Priming with DNA encoding E2 and boosting with E2 protein formulated with CpG oligodeoxynucleotides induces strong immune responses and protection from Bovine viral diarrhea virus in cattle", 《JOURNAL OF GENERAL VIROLOGY》, vol. 87, pages 2971 * |
| VENTZISLAV B. VASSILEV ET AL.: "Microparticle-mediated RNA immunization against bovine viral diarrhea virus", 《VACCINE》, vol. 19, pages 2012, XP004316941, DOI: 10.1016/S0264-410X(00)00438-2 * |
| 李世芳 等: "牛病毒性腹泻疫苗的研究进展", 《中国兽医学报》, vol. 38, no. 4, pages 828 - 832 * |
| 苏畅: "BVDV E2基因mRNA疫苗的研究", 《中国博士学位论文全文数据库》, no. 6, pages 1 - 42 * |
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