CN108904793B - 预防流行性乙型脑炎的dna疫苗及其构建方法 - Google Patents
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Abstract
本发明公开了一种预防流行性乙型脑炎的DNA疫苗。其中，该DNA疫苗以真核表达质粒融合SEQ ID No.1所示的抗原编码基因构建得到。本发明优化后得到的prM/E同义序列，符合适宜人体高表达且利于DNA疫苗的合成和应用。本发明所构建的JEV DNA疫苗与现有DNA疫苗相比，该质粒携带抗原序列精简，保护性位点集中，不含神经毒性位点，安全性高，经密码子优化后更适于人类细胞的高表达，引入JEV信号序列，可进一步提高表达效率和目的蛋白的正确折叠和定向转运。免疫效率更高，免疫时效更持久，可尽量少的避免因疫苗诱导的免疫力衰减而造成的发病率上升的现象，以进一步缓解我国公共卫生压力。
Description
技术领域
本发明涉及一种DNA疫苗，具体地说是一种用于预防流行性乙型脑炎的DNA疫苗及其构建方法。
背景技术
流行性乙型脑炎病毒，又称日本脑炎病毒（Japanese encephalitis virus, JEV）在亚洲和西太平洋国家和地区广泛流行，对当地公共卫生事业造成严重威胁。据世界卫生组织估计，全球约60%的人口生活在JEV流行区域，每年约有67900例流行性乙型脑炎（Japanese encephalitis, JE）病例，其中75%的患者为15岁以下的儿童，该病的致死率高达20～30%，约一半幸存者留有严重的神经系统后遗症。
疫苗被认为是预防JE较为有效的途径之一。近年来，虽然随着各类JEV疫苗（主要为减毒活疫苗和灭活疫苗）的研究和广泛应用，JE患病率有所下降，但在疫苗普遍接种的国家中，也尚存在接种者体内特异性抗体逐年衰减这一难以规避的现象，局部地区仍有小范围流行或偶有散发病例报道。Pan等的研究指出，在JE广泛流行的区域内，不同年龄组血清中的JEV抗体阳性率程度与该年龄组JE发病率呈负相关。这提示目前正使用的疫苗的长效免疫效果仍有待改进，有望克服这一问题的新型疫苗亟待研发及推广，以进一步降低发病率，缓解公共卫生压力。
现今广泛使用的JEV疫苗主要有灭活疫苗和减毒活疫苗两种。日本、韩国和我国台湾地区等普遍使用灭活疫苗，但因其需要多次接种（一般为四次及以上）、价格昂贵、免疫效力持续时间短、免疫反应类型偏向体液免疫等限制因素，WHO已建议相关国家和地区以其它类型疫苗逐步替代灭活疫苗。我国自主研发的JEV减毒活疫苗SA14-14-2，可以诱导高强度的中和抗体反应，还能通过T细胞反应产生高水平的IFN-γ，增强抗病毒反应，但SA14-14-2本身除了可能发生“毒力返祖”这一减毒活疫苗的“原罪”之外，依然存在特异性抗体逐渐衰减的现象。
发明内容
本发明所要解决的技术问题在于提出一种免疫保护效率高、免疫保护作用时效长的预防流行性乙型脑炎的DNA疫苗pVAX1-SA14ME。
本发明所要解决的另一技术问题在于提供一种上述DNA疫苗pVAX1-SA14ME的构建方法。
为了实现上述发明目的，本发明采用如下的技术方案：
一种预防流行性乙型脑炎的DNA疫苗，其中，所述DNA疫苗以真核表达质粒融合SEQID No .1所示的抗原编码基因构建得到。
优选地，所述真核表达质粒为pVAX1载体质粒。
一种上述预防流行性乙型脑炎的DNA疫苗的构建方法，其中，所述方法步骤如下：
(1) 对SEQ ID No .2所示的JEV SA14-14-2株的prM/E基因进行优化获得SEQ IDNo .3所示的优化基因prM/E；
(2) 在优化基因prM/E前插入SEQ ID No .4所示的prM信号序列；
(3) 在步骤(2)制备序列上游引入ATG起始密码子，下游引入TGA终止密码子；
(4) 在步骤(3)制备序列上下游引入SEQ ID No .5所示的Kozak序列、限制性内切酶识别序列BamH I和限制性内切酶识别序列Xho I；
(5) 将步骤(4)制备的目的基因亚克隆至载体上，即得。
SEQ ID No .1所示的抗原编码基因在制备预防流行性乙型脑炎的DNA疫苗中的用途。
本发明的优点及有益效果：
原始JEV SA14-14-2株的prM/E基因不利于克隆和质粒扩增，甚至影响投产，增加产品成本，经发明人再次优化后得到的同义序列，符合适宜人体高表达且利于DNA疫苗的合成和应用。本发明所构建的JEV DNA疫苗与现有DNA疫苗相比，该质粒携带抗原序列精简，保护性位点丰富集中，不含神经毒性位点，安全性高，经密码子优化后更适于人类细胞的高表达，引入JEV信号序列，可进一步提高表达效率和目的蛋白的正确折叠和定向转运。免疫效率更高，免疫时效更持久，可尽量少的避免因疫苗诱导的免疫力衰减而造成的发病率上升的现象，以进一步缓解我国公共卫生压力。
附图说明
图1为pVAX1-SA14ME/pVAX1体外和体内水平表达；
图2对免疫pVAX1-SA14ME小鼠进行初步体液免疫反应(短效)及保护效果评价 (n＝6)；
图3为发明人对免疫后3周(短效)及35周(长效)小鼠进行IgG效价及中和抗体效价（PRNT50）的测定及对比图；
图4对免疫pVAX1-SA14ME小鼠进行初步细胞免疫反应(短效)评价 (n＝8)；
图5对免疫pVAX1-SA14ME小鼠进行细胞因子产生情况(长效)检测 (n＝8)；
图6为本发明DNA疫苗构建方法示意图。
具体实施方式
为了使本领域技术人员对本发明有清楚的了解，下面利用具体实施例进行详细说明，实施例中所使用的方法如无特殊说明均为常规方法。
实施例1 流行性乙型脑炎的DNA疫苗pVAX1-SA14ME真核表达质粒的构建
1. 首先进行抗原编码目的基因的制备：以SEQ ID No.2所示的原始JEV SA14-14-2株的prM/E基因为模板进行优化，优化主要是针对人源种属的偏嗜性以及序列合理降低GC含量。原始序列与优化后序列翻译成的氨基酸序列同义，但原始序列和优化后序列的核苷酸序列差异大。优化后获得SEQ ID No.3所示的优化基因prM/E，对该基因进行人工合成，人工合成方法为公知技术。在优化基因prM/E前插入SEQ ID No.4所示的prM信号序列得到prM信号序列优化prM/E基因。在信号序列优化基因的基础上在上游引入ATG起始密码子，下游引入TGA终止密码子，然后连接限制性内切酶识别序列BamH I和XhoI和Kozak序列。
2. 将目的基因连接进pVAX1载体质粒：通过BamH I和XhoI限制性内切酶将目的基因和pVAX1载体分别进行剪切。再用DNA 连接酶将目的基因和pVAX1载体（预先去磷酸化）相连，制备得到重组质粒，将之命名为“pVAX1-SA14ME”。
上述方法图示见图6所示。
实施例2 本发明构建的流行性乙型脑炎的DNA疫苗pVAX1-SA14ME的体内、体外水平表达
为验证实施例1构建质粒pVAX1-SA14ME的免疫原性，需在确保测序完全匹配后，进行靶基因在体外和体内水平的验证。为此，我们将本发明构建质粒pVAX1-SA14ME或pVAX1空载体转染至BHK-21细胞中，48h后，以小鼠JEV抗血清为一抗，FITC标记的羊抗鼠抗体为二抗，进行间接免疫荧光染色（图1AB）；将质粒pVAX1-SA14ME或pVAX1空质粒免疫小鼠腿部肌肉，一周后，免疫部位肌肉切片，以小鼠JEV抗血清为一抗，HRP标记的羊抗鼠抗体为二抗，免疫组化染色（图1CD）。
结果如图1所示，图1A为转染pVAX1-SA14ME的BHK-21细胞；图1B为转染pVAX1的BHK-21细胞；图1C为免疫pVAX1-SA14ME的小鼠腿部肌肉切片染色；图1D为免疫pVAX1的小鼠腿部肌肉切片染色。从图中可见转染质粒pVAX1-SA14ME的BHK-21细胞有特异性绿色荧光，而对照组则无；免疫pVAX1-SA14ME的腿部肌肉，有特异性抗原着色，而对照组则无。以上实验证明，本发明构建质粒的抗原基因可在体外和体内水平成功表达。
实施例3 本发明构建的流行性乙型脑炎的DNA疫苗pVAX1-SA14ME的免疫原性评价及保护作用评价
1．为保证pVAX1-SA14ME可用于后续应用，需验证其免疫原性及保护作用，因为JEV诱导的免疫保护作用以体液免疫为主，发明人对免疫后3周(短效)及35周(长效)小鼠进行IgG效价及中和抗体效价（PRNT50）的测定，并对免疫后小鼠攻毒，初步评价其保护作用。具体步骤如下：
对免疫后小鼠进行攻毒试验（腹腔注射1×107 PFU Beijing-1株JEV病毒，破坏血脑屏障）。图2A为动态抗JEV IgG效价；图2B为动态抗JEV中和抗体效价；图2C为攻毒后体重动态变化；图2D为生存率；图2E为攻毒后10天体型个体间区别（典型代表图）。
每日测量小鼠体重并记录生存率（图2CDE）。结果显示，疫苗组小鼠在观察期（21天）内仅有小范围体重下降，并达到100%生存率（6/6），而对照组体重逐渐下降，观察末期已下降39.2%，生存率为0%（0/6）。无论体重变化（**P < 0.01, 图2C）和生存率（**P < 0.01,图2D），两组均有显著差异。
图3A和图3B显示的是经过免疫次数及末次免疫后时间的上升，IgG及PRNT50变化的趋势。较基线水平、第一次和第二次免疫相比，在三次免疫后3周达到最高，体液免疫反应达到相对较高水平，即使在此之后35周，抗体效价下降幅度不大，依然维持在较高水平，说明pVAX1-SA14ME诱导出的JEV特异性体液免疫反应即可在短效发挥重要保护作用，亦可在长效维持在相对较高的水平。
上述结果表明，本发明制备疫苗三次DNA疫苗免疫可对小鼠提供完全的保护作用，小鼠可完全抵抗致死量JEV的攻击。已证明该疫苗保护作用良好。
2. 本发明疫苗抗体效价与现有技术对比
（1）以JEV浓缩蛋白为包被抗原，检测免疫前及每次免疫后动态IgG及IgG终点抗体效价。结果显示，随免疫次数增加，抗JEV抗体效价逐渐上升，末次免疫后3周，即终点效价高达1:60887，明显高于初次免疫前水平（*** P < 0.001, 图2A）；与现有文献比较，Sheng等所用质粒pCAG-JME三次50μg免疫后诱导小鼠产生的IgG效价仅为1:25600。说明本发明抗体效价显著优于现有技术。
（2）两组免疫后血清由1:10起2倍倍比稀释至1:2048，分别与等体积JEV混合，37℃孵育1 h后，将血清/病毒混合物接种至Vero细胞，37℃孵育1 h后，洗去混合物，覆盖1.2%甲基纤维素，8 d后去除甲基纤维素并以结晶紫染色，噬斑减少50%（较单纯病毒感染孔）孔所对应血清稀释度为PRNT50，PRNT50 ≥1:10时，认为具有中抗体活性。结果显示，随免疫次数增加，疫苗组中和抗体效价逐渐上升，末次免疫后3周(短效)，终点PRNT50高达1:830，不仅显著高于初始免疫前水平（** P < 0.01），且高于末次免疫前水平（* P < 0.05, 图2B）；与现有文献比较，Sheng等所用质粒pCAG-JME三次免疫后诱导小鼠产生的PRNT50效价为1:160。本发明三次免疫后抗体效价显著优于现有技术。
上述（1）和（2）的结果表明，三次DNA疫苗免疫可诱导小鼠产生良好的体液免疫反应，且显著优于现有技术。
3. 本发明制备的疫苗长效性体液免疫反应评价实验
如图3A及图3B右栏显示，免疫后35周(长效)，测定小鼠体内IgG效价仍高达1:23475，PRNT50效价仍高达1:698。说明本发明长效免疫效果佳，抗体衰减慢，且程度低。目前尚未有JEV DNA疫苗35周(长效)免疫反应评价的相关报道。
4. 免疫pVAX1-SA14ME小鼠进行短效细胞免疫反应评价 (n＝8)
活化的CD8+T细胞在抗JEV感染中也起到关键作用，本发明运用流式细胞术的方法对活化的CD8+T细胞进行标记染色，主要标记细胞群为CD3+CD8+CD44HighCD62LLow的T细胞，该群细胞为效应型记忆T细胞，在长效细胞免疫反应中发挥重要作用，在末次免疫后3周(短效)时，对该群细胞的比例进行测定，可一定程度上预测疫苗组与对照组的长效细胞免疫反应水平。如图4所示，图4A为pVAX1-SA14ME组活化的CD8+T细胞比例（典型代表图）；图4B为pVAX1组活化的CD8+T细胞比例（典型代表图）；图4C为组间统计分析。有图可知，pVAX1-SA14ME免疫后的小鼠脾细胞中，活化的CD8+T细胞比例较pVAX1组更多（**P < 0.01），说明pVAX1-SA14ME成功诱导了具有杀伤功能的T细胞的产生。
5. 对免疫pVAX1-SA14ME小鼠进行长效细胞因子产生情况评价 (n＝8)
Th1类型细胞因子代表，IL-2和IFN-γ，和Th2类型细胞因子代表，IL-4和IL-5，对免疫后小鼠的抗病毒免疫反应中发挥重要作用。在末次免疫后35周时，以JEV特异性抗原蛋白分别刺激pVAX1-SA14ME组小鼠和pVAX1组小鼠脾细胞，检测上述四种细胞因子的产生情况，可以反应两组小鼠在特异性抗原刺激下，长效产生细胞因子的能力，以及疫苗的长效细胞免疫反应强弱。如图5所示，同等脾细胞数（5×105个）下，pVAX1-SA14ME组经JEV特异性抗原刺激后，可产生IL-2、IL-4、IL-5和IFN-γ的脾细胞数均明显高于pVAX1组。此结果说明，末次免疫后35周（长效），pVAX1-SA14ME仍可诱导小鼠产生较高水平的Th1和Th2类型细胞因子，对小鼠发挥重要的抗病毒免疫作用。
综上所述，pVAX1-SA14ME在小鼠体内诱导了良好的体液免疫和细胞免疫反应，且具有很好的长效免疫性。
序列表
<110> 首都医科大学附属北京友谊医院
<120> 预防流行性乙型脑炎的DNA疫苗及其构建方法
<140> 2018108506433
<141> 2018-07-29
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2094
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
ggatccgcca ccatgggtaa gaggtctgct ggatcaatca tgtggctggc ttcactcgcc 60
gtcgttattg cttgtgctgg cgctatgaag ctcagtaact ttcagggcaa actcctcatg 120
accatcaata acacagacat cgccgacgtc atcgtgatcc caacatccaa gggcgagaac 180
cgctgctggg ttcgggctat agatgtcgga tacatgtgtg aggacacaat cacctacgag 240
tgccctaaac tgactatggg aaatgatccc gaggatgtcg attgttggtg cgacaatcaa 300
gaggtctacg tgcaatatgg tcggtgcaca cgcaccagac atagtaagag gtcacgcagg 360
tctgtgtctg tgcagacaca cggcgagtcc tctctggtca ataagaaaga ggcctggctg 420
gactccacca aggccactcg ctacctgatg aagaccgaga actggattat ccgcaaccct 480
ggatatgctt tcctggcagc agtcctggga tggatgctcg ggtcaaacaa tgggcagaga 540
gttgtgttca ccattctgct cctcctggtt gctccagcat atagctttaa ctgcctggga 600
atgggaaaca gagacttcat agaaggagcc tccggagcta catgggtgga tctcgtcctc 660
gaaggagact cctgtctcac aatcatggct aacgacaagc caactctcga cgttagaatg 720
atcaatatcg aggcctcaca gctggccgag gttagatcat actgctacca cgcctccgtg 780
acagacattt ccacagtcgc cagatgtcca acaactggcg aggcacacaa cgagaagaga 840
gccgactctt cctatgtgtg caaacagggc ttcacagata gaggatgggg gaatgggtgc 900
ggcttcttcg gcaagggctc cattgatacc tgcgctaagt tctcttgtac tagcaaagcc 960
ataggaagga caatccagcc tgagaacatc aaatacaaag ttggcatatt cgtccacggc 1020
accacaacct cagagaacca cggaaactat tctgcccagg ttggagctag tcaggctgcc 1080
aagtttaccg tcacacccaa tgcacctagc gtggccctga aactcggtga ttatggcgaa 1140
gtcaccctgg attgtgagcc acggtccgga ctgaacacag aagccttcta cgtgatgact 1200
gtgggctcca agagcttcct ggtgcaccgc gaatggttcc acgacctggc actgccttgg 1260
acatcaccct caagtaccgc ctggcgcaat agagagctgc tgatggaatt tgaaggagcc 1320
cacgccacaa agcaatccgt ggttgctctg ggctctcagg agggaggact gcatcatgct 1380
ctcgcaggag ccatcgtggt tgagtattca tcatccgtga tgctcacatc cggacacctg 1440
aagtgcagac tcaagatgga caagctcgca ctgaaaggca caacatacgg gatgtgcaca 1500
gagaaattca gtttcgccaa gaatcccgtt gacactggac acggtactgt ggttatcgaa 1560
ctgtcttata gtggaagcga tgggccttgt aagatcccaa tcgtgtccgt tgcttctctc 1620
aatgacatga cacccgtggg acggctcgtg actgtgaacc ctttcgtggc taccagcagc 1680
gctaattcca aagttctcgt tgaaatggag cctcccttcg gcgattccta catagtcgtt 1740
ggaagaggtg ataaacagat caaccaccac tggcataagg caggctccac cctgggtaag 1800
gctttctcca ccactctcaa aggcgcacag cggctggcag ccctgggaga cactgcctgg 1860
gatttcggct ctatcggtgg agtctttaac agtataggaa gagctgtgca tcaggtgttt 1920
ggaggtgcat tcaggacact gttcggaggc atgagctgga ttactcaggg cctcatgggt 1980
gcactgctgc tgtggatggg cgtgaacgca agggaccgga gtattgccct ggcattcctg 2040
gccacaggcg gagtgctggt ctttctggca actaacgtgc acgcatgact cgag 2094
<210> 2
<211> 3246
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
atgaagttgt cgaatttcca ggggaagctt ttgatgacca tcaacaacac ggacattgca 60
gacgttatcg tgattcccac ctcaaaagga gagaacagat gctgggtccg ggcaatcgac 120
gtcggctaca tgtgtgagga cactatcacg tacgaatgtc ctaagcttac catgggcaat 180
gatccagagg atgtggattg ctggtgtgac aaccaagaag tctacgtcca atatggacgg 240
tgcacgcgga ccaggcattc caagcgaagc aggagatccg tgtcggtcca aacacatggg 300
gagagttcac tagtgaataa aaaagaggct tggctggatt caacgaaagc cacacgatat 360
ctcatgaaaa ctgagaactg gatcataagg aatcctggct atgctttcct ggcggcggta 420
cttggctgga tgcttggcag taacaacggt caacgcgtgg tatttaccat cctcctgctg 480
ttggtcgctc cggcttacag ttttaattgt ctgggaatgg gcaatcgtga cttcatagaa 540
ggagccagtg gagccacttg ggtggacttg gtgctagaag gagacagctg cttgacaatc 600
atggcaaacg acaaaccaac attggacgtc cgcatgatta acatcgaagc tagccaactt 660
gctgaggtca gaagttactg ctatcatgct tcagtcactg acatctcgac ggtggctcgg 720
tgccccacga ctggagaagc ccacaacgag aagcgagctg atagtagcta tgtgtgcaaa 780
caaggcttca ctgaccgtgg gtggggcaac ggatgtggat ttttcgggaa gggaagcatt 840
gacacatgtg caaaattctc ctgcaccagt aaagcgattg ggagaacaat ccagccagaa 900
aacatcaaat acaaagttgg catttttgtg catggaacca ccacttcgga aaaccatggg 960
aattattcag cgcaagttgg ggcgtcccag gcggcaaagt ttacagtaac acccaatgct 1020
ccttcggtag ccctcaaact tggtgactac ggagaagtca cactggactg tgagccaagg 1080
agtggactga acactgaagc gttttacgtc atgaccgtgg ggtcaaagtc atttctggtc 1140
catagggagt ggtttcatga cctcgctctc ccctggacgt ccccttcgag cacagcgtgg 1200
agaaacagag aactcctcat ggaatttgaa ggggcgcacg ccacaaaaca gtccgttgtt 1260
gctcttgggt cacaggaagg aggcctccat catgcgttgg caggagccat cgtggtggag 1320
tactcaagct cagtgatgtt aacatcaggc cacctgaaat gtaggctgaa aatggacaaa 1380
ctggctctga aaggcacaac ctatggcatg tgtacagaaa aattctcgtt cgcgaaaaat 1440
ccggtggaca ctggtcacgg aacagttgtc attgaactct cctactctgg gagtgatggc 1500
ccctgcaaaa ttccgattgt ttccgttgcg agcctcaatg acatgacccc cgttgggcgg 1560
ctggtgacag tgaacccctt cgtcgcgact tccagtgcca actcaaaggt gctggtcgag 1620
atggaacccc ccttcggaga ctcctacatc gtagttggaa ggggagacaa gcagatcaac 1680
caccattggc acaaagctgg aagcacgctg ggcaaggcct tttcaacaac tttgaaggga 1740
gctcaaagac tggcagcgtt gggcgacaca gcctgggact ttggctctat tggaggggtc 1800
ttcaactcca taggaagagc cgttcaccaa gtgtttggtg atgccttcag aacactcttt 1860
gggggaatgt cttggatcac acaagggcta atgggtgccc tactgctctg gatgggcgtc 1920
aacgcacgag accgatcaat tgctttggcc ttcttagcca caggaggtgt gctcgtgttc 1980
ttagcgacca atgtgcatgc tgacactgga tgtgccattg acatcacaag aaaagagatg 2040
agatgtggaa gtggcatctt cgtgcacaac gacgtggaag cctgggtgga taggtataaa 2100
tatttgccag aaacgcccag atccctagcg aagatcgtcc acaaagcgca caaggaaggc 2160
gtgtgcggag tcagatctgt cactagactg gagcaccaaa tgtgggaagc cgtaagggac 2220
gaattgaacg tcctgctcaa agagaatgca gtggacctca gtgtggttgt gaacaagccc 2280
gtgggaagat atcgctcagc ccctaaacgc ctatccatga cgcaagagaa gtttgaaatg 2340
ggctggaaag catggggaaa aagcatcctc tttgccccgg aattggctaa ctccacattt 2400
gtcgtagatg gacctgagac aaaggaatgc cctgatgagc acagagcttg gaacagcatg 2460
caaatcgaag acttcggctt tggcatcaca tcaacccgtg tgtggctgaa aattagagag 2520
gagagcactg acgagtgtga tggagcgatc ataggcacgg ctgtcaaagg acatgtggca 2580
gtccatagtg acttgtcgta ctggattgag agtcgctaca acgacacatg gaaacttgag 2640
agggcagtct ttggagaggt caaatcttgc acttggccag agacacacac cctttgggga 2700
gatgatgttg aggaaagtga actcatcatt ccgcacacca tagccggacc aaaaagcaag 2760
cacaatcgga gggaagggta taagacacaa aaccagggac cttgggatga gaatggcata 2820
gtcttggact ttgattattg cccagggaca aaagtcacca ttacagagga ttgtagcaag 2880
agaggccctt cggtcagaac cactactgac agtggaaagt tgatcactga ctggtgctgt 2940
cgcagttgct cccttccgcc cctacgattc cggacagaaa atggctgctg gtacggaatg 3000
gaaatcagac ctgttatgca tgatgaaaca acactcgtca gatcacaggt tcatgctttc 3060
aaaggtgaaa tggttgaccc ttttcagctg ggccttctgg tgatgtttct ggccacccag 3120
gaagtccttc gcaagaggtg gacggccaga ttgaccattc ctgcggtttt gggggtccta 3180
cttgtgctga tgcttggggg tatcacttac actgatttgg cgaggtatgt ggtgctagtc 3240
gctgct 3246
<210> 3
<211> 2001
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atgaagctca gtaactttca gggcaaactc ctcatgacca tcaataacac agacatcgcc 60
gacgtcatcg tgatcccaac atccaagggc gagaaccgct gctgggttcg ggctatagat 120
gtcggataca tgtgtgagga cacaatcacc tacgagtgcc ctaaactgac tatgggaaat 180
gatcccgagg atgtcgattg ttggtgcgac aatcaagagg tctacgtgca atatggtcgg 240
tgcacacgca ccagacatag taagaggtca cgcaggtctg tgtctgtgca gacacacggc 300
gagtcctctc tggtcaataa gaaagaggcc tggctggact ccaccaaggc cactcgctac 360
ctgatgaaga ccgagaactg gattatccgc aaccctggat atgctttcct ggcagcagtc 420
ctgggatgga tgctcgggtc aaacaatggg cagagagttg tgttcaccat tctgctcctc 480
ctggttgctc cagcatatag ctttaactgc ctgggaatgg gaaacagaga cttcatagaa 540
ggagcctccg gagctacatg ggtggatctc gtcctcgaag gagactcctg tctcacaatc 600
atggctaacg acaagccaac tctcgacgtt agaatgatca atatcgaggc ctcacagctg 660
gccgaggtta gatcatactg ctaccacgcc tccgtgacag acatttccac agtcgccaga 720
tgtccaacaa ctggcgaggc acacaacgag aagagagccg actcttccta tgtgtgcaaa 780
cagggcttca cagatagagg atgggggaat gggtgcggct tcttcggcaa gggctccatt 840
gatacctgcg ctaagttctc ttgtactagc aaagccatag gaaggacaat ccagcctgag 900
aacatcaaat acaaagttgg catattcgtc cacggcacca caacctcaga gaaccacgga 960
aactattctg cccaggttgg agctagtcag gctgccaagt ttaccgtcac acccaatgca 1020
cctagcgtgg ccctgaaact cggtgattat ggcgaagtca ccctggattg tgagccacgg 1080
tccggactga acacagaagc cttctacgtg atgactgtgg gctccaagag cttcctggtg 1140
caccgcgaat ggttccacga cctggcactg ccttggacat caccctcaag taccgcctgg 1200
cgcaatagag agctgctgat ggaatttgaa ggagcccacg ccacaaagca atccgtggtt 1260
gctctgggct ctcaggaggg aggactgcat catgctctcg caggagccat cgtggttgag 1320
tattcatcat ccgtgatgct cacatccgga cacctgaagt gcagactcaa gatggacaag 1380
ctcgcactga aaggcacaac atacgggatg tgcacagaga aattcagttt cgccaagaat 1440
cccgttgaca ctggacacgg tactgtggtt atcgaactgt cttatagtgg aagcgatggg 1500
ccttgtaaga tcccaatcgt gtccgttgct tctctcaatg acatgacacc cgtgggacgg 1560
ctcgtgactg tgaacccttt cgtggctacc agcagcgcta attccaaagt tctcgttgaa 1620
atggagcctc ccttcggcga ttcctacata gtcgttggaa gaggtgataa acagatcaac 1680
caccactggc ataaggcagg ctccaccctg ggtaaggctt tctccaccac tctcaaaggc 1740
gcacagcggc tggcagccct gggagacact gcctgggatt tcggctctat cggtggagtc 1800
tttaacagta taggaagagc tgtgcatcag gtgtttggag gtgcattcag gacactgttc 1860
ggaggcatga gctggattac tcagggcctc atgggtgcac tgctgctgtg gatgggcgtg 1920
aacgcaaggg accggagtat tgccctggca ttcctggcca caggcggagt gctggtcttt 1980
ctggcaacta acgtgcacgc a 2001
<210> 4
<211> 69
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
ggtaagaggt ctgctggatc aatcatgtgg ctggcttcac tcgccgtcgt tattgcttgt 60
gctggcgct 69
<210> 5
<211> 6
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
gccacc 6
Claims (4)
1.一种预防流行性乙型脑炎的DNA疫苗，其特征在于：所述DNA疫苗以真核表达质粒融合SEQ ID No .1所示的抗原编码基因构建得到。
2.如权利要求1所述的DNA疫苗，其特征在于：所述真核表达质粒为pVAX1载体质粒。
3.一种权利要求1或2所述的预防流行性乙型脑炎的DNA疫苗的构建方法，其特征在于所述方法步骤如下：
(1)对SEQ ID No .2所示的JEV SA14-14-2株的prM/E基因进行优化获得SEQ ID No .3所示的优化基因prM/E；
(2)在优化基因prM/E前插入SEQ ID No .4所示的prM信号序列；
(3)在步骤(2)制备序列上游引入ATG起始密码子，下游引入TGA终止密码子；
(4)在步骤(3)制备序列上下游引入SEQ ID No .5所示的Kozak序列、限制性内切酶识别序列BamH I和限制性内切酶识别序列XhoI；
(5)将步骤(4)制备的目的基因亚克隆至载体上，即得。
4.SEQ ID No .1所示的抗原编码基因在制备预防流行性乙型脑炎的DNA疫苗中的用途。
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