CN114533862B - 一种鱼用dna疫苗及其制备方法与应用 - Google Patents
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Abstract
本发明公开了一种鱼用DNA疫苗及其制备方法与应用,属于生物医药技术领域。所述的鱼用DNA疫苗为含有Z‑DNA序列的重组质粒与含有PKZ开放阅读框序列的重组质粒的混合物;其中所述的含有Z‑DNA序列的重组质粒与所述的含有PKZ开放阅读框序列的重组质粒之间的质量比为1:1;所述的含有Z‑DNA序列的重组质粒的序列如SEQ ID NO.1所示;所述的含有PKZ开放阅读框序列的重组质粒的序列如SEQ ID NO.2所示。本发明制备的DNA疫苗能显著激活鱼体先天免疫反应,对GCRV病毒引起的鱼体死亡具有良好的保护效果,免疫保护率达60.2%,为后期DNA疫苗的应用及产业化提供理论基础和科学依据。
Description
技术领域
本发明属于生物医药技术领域,具体地说,涉及一种鱼用DNA疫苗及其制备方法与应用。
背景技术
我国是淡水经济鱼养殖大国。集约化、高密度养殖模式容易导致鱼病爆发。疾病的暴发,往往会导致一些药品的过量使用。这种行为一方面可能造成鱼体产生严重耐药性、药物残留,造成食用者的二次污染,而且也会对生态环境造成严重影响。因此疫苗是解决疫病,尤其是病毒性疾病的优选方法。目前常用的鱼用疫苗主要有传统疫苗和DNA疫苗。DNA疫苗因其具有生产成本低、生化性能稳定、用量少、安全性能好等优点而成为研究热点。
目前常用的鱼用核酸疫苗通常都是选择单一病原体的某个基因作为免疫原,因此这种核酸疫苗仅能适用于预防单一病原体感染,而且有些病原体尤其是病毒,经常发生变异,使得疫苗的免疫效果下降。另一方面,注射核酸疫苗进鱼体内可能会引起自身免疫、过敏反应和炎症反应等,对鱼本身造成伤害。
因此迫切需要开发一种广谱、低毒性的,同时又不会使鱼体产生自身免疫反应的DNA疫苗。
发明内容
、要解决的问题
针对上述检测技术存在的问题,本发明提供一种鱼用DNA疫苗及其制备方法与应用,克服现有DNA疫苗的缺点与不足,得到一种鱼类广谱、低毒的DNA疫苗。
、技术方案
为解决上述问题,本发明采用如下的技术方案。
一种鱼用DNA疫苗,所述的鱼用DNA疫苗为含有Z-DNA序列的重组质粒与含有PKZ开放阅读框序列的重组质粒的混合物;其中所述的含有Z-DNA序列的重组质粒与所述的含有PKZ开放阅读框序列的重组质粒之间的质量比为1:1;
所述的含有Z-DNA序列的重组质粒的序列如SEQ ID NO.1所示;
所述的含有PKZ开放阅读框序列的重组质粒的序列如SEQ ID NO.2所示。
上述所述的鱼用DNA疫苗中,
所述的Z-DNA序列通过人工合成来获取;
所述的PKZ开放阅读框序列通过PCR扩增来获取。
上述所述的鱼用DNA疫苗中,
所述的含有Z-DNA序列的重组质粒通过将Z-DNA序列插入到真核表达载体上来获取;
所述的含有PKZ开放阅读框序列的重组质粒通过将PKZ开放阅读框序列插入到真核表达载体上来获取;
所述的真核表达载体为含有Flag标签的真核表达载体。
上述所述的鱼用DNA疫苗中,
所述的含有Flag标签的真核表达载体为pCMV-Flag。
一种鱼用DNA疫苗的制备方法,包括以下步骤:
(1)构建含有Z-DNA序列的重组质粒:在Poly (GC)10的一端添加Hind III限制性内切酶位点,其另一端添加Kpn I限制性内切酶位点,将添加限制性内切酶位点的Poly (GC)10用退火缓冲液并经95℃处理30min,然后自然冷却至室温形成双链DNA,接着将pCMV-Flag质粒与退火后的Poly (GC)10分别经Hind III限制性内切酶与Kpn I限制性内切酶的双酶切反应,回收产物,利用T4 DNA连接酶将双酶切后的Poly (GC)10连接至pCMV-Flag质粒上,最后提取pCMV-Flag-GC重组质粒;
(2)构建含有PKZ开放阅读框序列的重组质粒:获取含有酶切位点的草鱼PKZ开放阅读框的基因序列,设计并合成特异性引物,取草鱼cDNA模板,进行PCR扩增,得到草鱼PKZ开放阅读框的全长基因序列,接着通过将pCMV-Flag质粒与草鱼PKZ开放阅读框的全长基因序列分别经EcoR I限制性内切酶与Kpn I限制性内切酶的双酶切反应,回收产物,利用T4DNA连接酶将双酶切后的PKZ开放阅读框序列连接至pCMV-Flag质粒上,最后提取pCMV-Flag-PKZ重组质粒;
(3)将步骤(1)构建的含有Z-DNA序列的重组质粒与步骤(2)构建的含有PKZ开放阅读框序列的重组质粒,进行混合,即可。
上述所述的鱼用DNA疫苗的制备方法中,
其中构建含有Z-DNA序列的重组质粒的步骤中:
退火缓冲液的浓度为1倍。
上述所述的鱼用DNA疫苗的制备方法中,
其中构建含有PKZ开放阅读框序列的重组质粒的步骤中PCR扩增反应体系如下:
草鱼cDNA模板2 µl,上下游引物各0.5 µl,LA Taq DNA聚合酶1 µl,Taq酶buffer2.5 µl,ddH2O补至50 µl。
上述所述的鱼用DNA疫苗的制备方法中,
其中构建含有PKZ开放阅读框序列的重组质粒的步骤中PCR扩增反应条件如下:
95℃预变性5 min,95℃变性30 s,58℃退火30 s,72℃延伸2min,35个循环,72℃延伸10 min。
一种鱼用DNA疫苗在草鱼养殖中GCRV病毒感染防治的应用。
上述所述的鱼用DNA疫苗在草鱼养殖中GCRV病毒感染防治的应用中,所述的鱼用DNA疫苗通过腹腔注射方式给药,给药浓度为30 µg-50 µg/尾。
、有益效果
相比于现有技术,本发明的有益效果为:
本发明制备的DNA疫苗能显著激活鱼体先天免疫反应,对GCRV病毒引起的鱼体死亡具有良好的保护效果,免疫保护率达60.2%,为后期DNA疫苗的应用及产业化提供理论基础和科学依据。
附图说明
图1为寡聚核苷酸Z-DNA(poly GC)构建至pCMV-Flag质粒的测序结果。
图2为CIK细胞中转染pCMV-Flag-GC与pCMV-Flag-PKZ后检测Ⅰ型干扰素的表达图,图中,A为qRT-PCR检测结果图,B为双荧光素酶报告分析结果图。
图3为在草鱼体内注射疫苗质粒后,利用qRT-PCR检测先天免疫相关基因在各组织中的表达情况,A图为注射四天后的表达,B图为注射八天后的表达。
图4为在草鱼体内注射疫苗质粒后,利用western blot检测疫苗质粒(Flag标签)在体内各组织表达4天、8天、12天、16天、20天、24天情况。
图5为攻毒实验结果,A图为CIK细胞中转染疫苗质粒后,对细胞病毒滴度的影响。B图为草鱼注射疫苗质粒后感染GCRV病毒,个体的致死率。
具体实施方式
下面结合具体实施例对本发明进一步进行描述。
实施例1
DNA疫苗的制备(其他质粒也能制备,但是用含有flag标签的真核表达质粒能够在体内表达标签蛋白,能够监测疫苗在体内的表达情况,具有一定优势。(flag标签质粒并非本发明的创新点之处,这种载体仅仅只是一种工具而已,本发明的重要创新点在于应用Z-DNA作为免疫原,PKZ作为免疫增强剂。)
Z-DNA和PKZ的选择依据:Z-DNA是指具有左手螺旋Z型构象的DNA分子,其糖磷酸骨架排列呈Z型。在Z-DNA结构中,嘌呤与嘧啶的交替排列,嘌呤是顺式构象,而嘧啶是反式构象,这样交替的顺反式构象就是Z-DNA的骨架。有研究表明,Z-DNA能够模拟病毒激活机体的免疫反应,这是因为在胞质中存在大量的DNA识别受体,他们通过特异性识别DNA后启动先天免疫反应。
有研究报道DNA识别受体ZBP1作为核酸疫苗的固有免疫刺激成分,能够增强核酸疫苗的免疫效果(DeFilippis VR, Alvarado D, Sali T, Rothenburg S, Früh K. Humancytomegalovirus induces the interferon response via the DNA sensor ZBP1. JVirol, 2010, 84:585–598.)。我们发现鱼类特有的蛋白激酶Z(PKZ)能够作为胞质中DNA识别受体,促进Oligo DNA形成并稳定为Z-DNA(Xu X, Li M, Wu C, Li D, Jiang Z, Liu C,Cheng B, Mao H, Hu C. The fish-specific protein kinase (PKZ) initiates innateimmune responses via IRF3- and ISGF3-like mediated pathways. Font Immunol,2019, 10: 582.;Lu P, Deng S, Wu C, Zhu Y, Liu Y, Lin G, Yan Y, Hu C. et al.The Zalpha domain of fish PKZ converts DNA hairpin with d(GC)(n) inserts toZ-conformation. Acta Biochim Biophys Sin (Shanghai), 2013, 45: 1062-1068.;夏玉洁, 吴初新, 马梅生, 胡成钰. Poly d(GC)在负超螺旋下形成的 Z-DNA. 细胞生物学杂志, 2009, 31: 867-870.)。这就表明PKZ的另一个功能是能够作为维持Z-DNA疫苗活性的佐剂。同时,由于寡聚Z-DNA(d(GC)n)的分子量很小,没有编码功能,在进入细胞后不产生任何病原体蛋白,故不具有毒性。因此,基于小分子Z-DNA制作的疫苗有非常好的安全性和稳定性。
(1)构建pCMV-Flag-GC与pCMV-Flag-PKZ的重组质粒
Poly (GC)10序列由上海生工生物公司合成,序列设计时,在5’与3’端分别添加Hind III和Kpn I限制性酶切位点:
5-aagcttTAACTGATACTACATTGAATTCGCGCGCGCGCGCGCGCGCGCGAATTCAATGTAGTATCAGAggatcc-3 (aagctt为Hind III酶切位点,ggatcc为Kpn I酶切位点)
将合成的带有双酶切位点的poly (GC)10用1×退火缓冲液,经95℃处理30 min,自然冷却至室温形成双链DNA。将pCMV-Flag质粒与退火后的poly (GC)10经Hind III与Kpn I双酶切,T4 DNA连接酶将poly (GC)10连接至pCMV-Flag质粒上,经上海生工生物测序,测序结果如图1所示,poly (GC)10序列已构建至载体上,如SEQ ID NO.3所示。随后用去内毒素质粒抽提试剂盒提取pCMV-Flag-GC质粒,测定浓度后,置于-20℃冰箱备用。
根据NCBI上草鱼PKZ的基因序列(Genbank ID: GU299765),其中PKZ开放阅读框序列如SEQ ID NO.4所示,设计并合成特异性引物,引物由上海生工生物公司合成,引物序列如表1所示:
表1 PCR获取PKZ基因开放阅读框片段的引物序列
目的基因 | 上游引物(5’-3’) | 下游引物(5’-3’) |
PKZ | CGGAATTCAATGTCTGCCGAAACTCAAATG | CGGGTACCTCAAATCGTTTTCTGGCTTAACA |
取草鱼cDNA模板,通过PCR技术扩增PKZ开放阅读框全长序列。PCR反应体系:草鱼cDNA模板2 μl,引物(上/下游引物)各0.5 μl,LA Taq DNA聚合酶1 μl,Taq酶buffer 2.5 μl,ddH2O补至50 μl。PCR反应条件为:95℃预变性5 min,95℃变性30 s,58℃退火30 s,72℃延伸2min,35个循环,72℃延伸10 min。通过EcoR I与Kpn I限制性内切酶对扩增得到的PKZ开放阅读框片段与pCMV-Flag质粒双酶切,回收产物后,利用T4 DNA连接酶将PKZ开放阅读框片段连接至pCMV-Flag载体上。
将构建好的重组质粒在草鱼肾细胞(CIK)中表达分析:
将抽提得到的去内毒素重组质粒2 μg用lip6000转染试剂(质粒:转染试剂=1:3)转染至草鱼肾细胞,转染24 h后,通过qRT-PCR检测Ⅰ型IFN的表达,检测引物的序列如表2所示,检测结果如图2A所示,结果表明单转(3.7倍)或共转重组质粒(6.2倍)能够显著提升Ⅰ型IFN的表达水平。同样的,相似结论通过双荧光素酶报告系统也得到了证实,单转提升1.4倍,共转提升2.4倍,如图2B所示。其中qRT-PCR的反应体系如下:10 μl TB green mastmix,2 μl 10×模板,上游/下游引物0.4 μl,dd H2O补足至20 μl;95℃预变性5min,95℃变性30 s,55℃退火30 s,72℃延伸30 s,40个循环,72℃延伸10 min。双荧光素酶报告系统体系如下:0.25 μg重组质粒/空载体,0.25 μg pGL-IFN I-promoter质粒,0.025 μg pRL-TK质粒。
表2 qRT-PCR引物序列(5’-3’)
目的基因 | 上游引物 | 下游引物 |
IFN I | GTCAATGCTCTGCTTGCGAAT | CAAGAAACTTCACCTGGTCCT |
β-actin | CACTGTGCCCATCTACGA | CCATCTCCTGCTCGAAGTC |
重组质粒疫苗在鱼体内的表达分析:
于实验前两周,选取长度在16-20 cm,体重在80-120 g,健壮无病的同一批次草鱼于19-22℃水温种充氧饲养。随机分成四组,每组30尾,分别为PBS组,pCMV-Flag(空载)组,pCMV-Flag-GC组,pCMV-Flag-GC+pCMV-Flag-PKZ组。采用胸鳍基部注射法进行免疫,pCMV-Flag(空载)组:质粒30 μg(溶解于100 μl无菌PBS);pCMV-Flag-GC组:质粒30 μg(溶解于100 μl无菌PBS);pCMV-Flag-GC+pCMV-Flag-PKZ组:pCMV-Flag-GC质粒30 μg+pCMV-Flag-PKZ质粒30 μg(溶解于100 μl无菌PBS);PBS组:100 μl无菌PBS。分别在免疫4天(A)和8天(B)收集鱼各组织(眼、脑、肠、鳃、皮肤、脾、肝、肾),鉴定组织中先天免疫相关基因IRF7、IRF3、ISG15、PKR及TLR3的表达,表3为检测先天免疫相关基因的定量引物序列。结果如图3所示,在肠、脾、肝、肾等组织中,注射DNA疫苗质粒(pCMV-Flag-GC和pCMV-Flag-PKZ)组能够在免疫4天和8天后促进免疫基因(IRF3、IRF7、ISG15、PKR及TLR3)显著上调,表明DNA疫苗能够有效激活鱼体免疫能力。
表3 先天免疫相关基因的定量引物序列
目的基因 | 上游引物(5’-3’) | 下游引物(5’-3’) |
IRF7 | GTTCCTGCTCTCTGCGACCTG | GCCGTAGATGCCATACTGGTT |
IRF3 | TCCAGGCCAAGCATACGAA | CCATTTGCAACAGCCATCAT |
ISG15 | GGTGAAAGTTGATGCCACAGTTG | TTGGAAAGGGGGGTTCGTG |
PKR | TTCGTGAGGTCCGTGCTTTG | TCTCAATCCAGGCACGCAGT |
TLR3 | GAAACTCATACTGTTGCCCC | GTGGCAGAGAAAGACATAGATC |
对注射有pCMV-Flag-GC+pCMV-Flag-PKZ的实验组分别在4天、8天、12天、16天、20天、24天提取各组织蛋白,利用western blot和SOD试剂盒分别检测各组织中PKZ-Flag和SOD酶的表达。如图4所示,重组质粒在注射的4天和8天后,肠、肝、脾、肾、鳃组织中,可以被检测到表达,随着时间增加,能被检测到的组织变少,最后在24天时各检测组织不再被检测到。结果表明重组质粒能够被鱼体内吸收,并持续表达20天,在第24天表达急剧减弱,说明DNA疫苗免疫能在体内高效表达进行免疫,并在免疫结束后不会在体内残留,不会对鱼体产生毒性,具有较好的安全性。
攻毒实验:
(1)细胞攻毒:将CIK细胞接种在6孔板中,12 h后转染2 μg pCMV-Flag、2 μgpCMV-Flag-GC、2 μg pCMV-Flag-GC+2 μg pCMV-Flag-PKZ质粒。转染24 h后,50 μl 5×107TCID50/ml GCRV病毒感染细胞,感染2天后,收集样品,用CIK细胞测每个样品的病毒滴度。结果如图5A所示,转染pCMV-Flag-GC+pCMV-Flag-PKZ重组质粒组能抑制病毒的扩增,相较于空载体对照组下降7倍。
(2)个体攻毒:实验分为三组,每组60尾:1) pCMV-Flag(空载)组;2)pCMV-Flag-GC组;3) pCMV-Flag-GC+pCMV-Flag-PKZ组。免疫14天后,对草鱼进行攻毒,攻毒用GCRV-097的浓度为3.5×107 TCID50/ml,攻毒剂量为300 μl/尾。逐日观察鱼体的死亡情况,及时记录死亡数,各组草鱼的存活率比较分析结果如图5B所示。在鱼体死亡趋于平缓时,计算免疫保护率=(1-免疫组死亡率/对照组死亡率)。结果表明,免疫组(pCMV-Flag-GC+pCMV-Flag-PKZ)死亡率为33%,对照组空载体(pCMV-Flag)死亡率为83%,因此pCMV-Flag-GC+pCMV-Flag-PKZ组(DNA疫苗组)对草鱼的免疫保护率达60.2%,具有较好的免疫保护效果。
本发明制备的DNA疫苗使用了广谱性免疫原Z-DNA,免疫增强剂PKZ。一方面,由于Z-DNA(d(GC)n)的分子量很小,没有编码功能,在进入细胞后不产生任何病原体蛋白,因此在产生免疫原性的时候不会对机体造成伤害;另一方面PKZ蛋白是鱼类特有的DNA识别受体,能够识别Z-DNA后激活免疫反应。上述结果已充分证明该鱼用DNA疫苗(pCMV-Flag-GC+pCMV-Flag-PKZ)进入鱼体后,能够在体内表达,并能够启动免疫反应。免疫14天后的能够较好保护草鱼免受GCRV病毒的侵染,提升存活率。因此该鱼用DNA疫苗对草鱼具有较好的免疫保护能力。
以上内容是结合具体实施方式对本发明作进一步详细说明,不能认定本发明具体实施只局限于这些说明,对于本发明所属技术领域的普通技术人员来说,在不脱离本发明的构思的前提下,还可以做出若干简单的推演或替换,都应当视为属于本发明所提交的权利要求书确定的保护范围。
序列表
<110> 南昌大学
<120> 一种鱼用DNA疫苗及其制备方法与应用
<141> 2022-01-06
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 4763
<212> DNA
<213> 草鱼(Ctenopharyngodon idella)
<400> 1
ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct 60
attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg 120
gttttcccag tcacgacgtt gtaaaacgac ggccagtgcc aagctgatct atacattgaa 180
tcaatattgg caattagcca tattagtcat tggttatata gcataaatca atattggcta 240
ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc 300
aatatgaccg ccatgttgac attgattatt gactagttat taatagtaat caattacggg 360
gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc 420
gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat 480
agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc 540
ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga 600
cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg 660
gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac 720
caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt 780
caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaataaccc 840
cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc 900
tcgtttagtg aaccgtcaga attaaccatg gactacaaag accatgacgg tgattataaa 960
gatcatgaca tcgattacaa ggatgacgat gacaagcttt aactgatact acattgaatt 1020
cgcgcgcgcg cgcgcgcgcg cgaattcaat gtagtatcag aggatccgaa caaaaactca 1080
tctcagaaga ggatctgtga cccgggtggc atccctgtga cccctcccca gtgcctctcc 1140
tggccctgga agttgccact ccagtgccca ccagccttgt cctaataaaa ttaagttgca 1200
tcattttgtc tgactaggtg tccttctata atattatggg gtggaggggg gtggtatgga 1260
gcaaggggca agttgggaag acaacctgta gggcctgcgg ggtctattgg gaaccaagct 1320
ggagtgcagt ggcacaatct tggctcactg caatctccgc ctcctgggtt caagcgattc 1380
tcctgcctca gcctcccgag ttgttgggat tccaggcatg catgaccagg ctcagctaat 1440
ttttgttttt ttggtagaga cggggtttca ccatattggc caggctggtc tccaactcct 1500
aatctcaggt gatctaccca ccttggcctc ccaaattgct gggattacag gcgtgaacca 1560
ctgctccctt ccctgtcctt ctgattttaa aataactata ccagcaggag gacgtccaga 1620
cacagcatag gctacctggc catgcccaac cggtgggaca tttgagttgc ttgcttggca 1680
ctgtcctctc atgcgttggg tccactcagt agatgcctgt tgaattgggt acgcggccag 1740
cttggctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca 1800
gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct 1860
ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc 1920
ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg 1980
gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc 2040
agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc ctcgaggaac 2100
tgaaaaacca gaaagttaat tccctatagt gagtcgtatt aaattcgtaa tcatgtcata 2160
gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag 2220
cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg 2280
ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca 2340
acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc 2400
gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg 2460
gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa 2520
ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga 2580
cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag 2640
ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct 2700
taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg 2760
ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc 2820
ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt 2880
aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta 2940
tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac 3000
agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc 3060
ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat 3120
tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc 3180
tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt 3240
cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta 3300
aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct 3360
atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg 3420
cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga 3480
tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt 3540
atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt 3600
taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt 3660
tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat 3720
gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc 3780
cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc 3840
cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat 3900
gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag 3960
aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt 4020
accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc 4080
ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa 4140
gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg 4200
aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa 4260
taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg cgccctgtag 4320
cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag 4380
cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt 4440
tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca 4500
cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata 4560
gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca 4620
aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc 4680
gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa 4740
caaaatatta acgcttacaa ttt 4763
<210> 2
<211> 6277
<212> DNA
<213> 草鱼(Ctenopharyngodon idella)
<400> 2
ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct 60
attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg 120
gttttcccag tcacgacgtt gtaaaacgac ggccagtgcc aagctgatct atacattgaa 180
tcaatattgg caattagcca tattagtcat tggttatata gcataaatca atattggcta 240
ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc 300
aatatgaccg ccatgttgac attgattatt gactagttat taatagtaat caattacggg 360
gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc 420
gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat 480
agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc 540
ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga 600
cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg 660
gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac 720
caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt 780
caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaataaccc 840
cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc 900
tcgtttagtg aaccgtcaga attaaccatg gactacaaag accatgacgg tgattataaa 960
gatcatgaca tcgattacaa ggatgacgat gacaagcttg cggccgcgaa ttcaatgtct 1020
gccgaaactc aaatggagag gaagatcatt gatttcttga gacaaaatgg gaaaagcata 1080
gctttgacaa ttgctaaaga aatcggactt gacaaatcca ccgtgaacag acatttgtac 1140
aacctacaga gatcaaacca ggtgtttaac tctaacgaaa agcctcctgt ttgggatcta 1200
atggagaaga cgaatgggat caaacaaaca gtcaaaacac cagagcaaaa gtctctgacg 1260
acgacagcag aaacatgtga agagaaacac gtaagggatc tgctgaaatc aggaggttta 1320
aaagcctgtc agatcgctaa agacttggga caaccaagaa aaaccgtaaa caaacagctg 1380
tacagtatga tgcagacggg taaagtaaag aaatgtgaaa taagtagctt gtggcttctg 1440
gaaggcgagg agagcaatga aagtcactcc caagagagtg atcatagact ggggtcagtc 1500
gcagggttgt ctcaaagttt tgatgtgatc acaaagcttg gtgaaggagg atttggctgt 1560
gtttttaaag taaagcataa atttgatggc aagatctacg ctgtaaagaa agtcgtctta 1620
actggggaag ctgattccga ggtgaaggca ttggccagac tagaccatcc aaacatagtg 1680
cgctacatta catgttggcc ggattctgag agctgcacat caaaccaaga cagaaaccaa 1740
gtgtccaaca catcaggttc ttcatcatgt ggagtgacct ttgatagagc tggctgtgaa 1800
gagaggaatg atgaagacga tgaagacgat gatgatgacg ttgatgtcag tgatgtcaca 1860
tcaagaatgg aaagtctggg ttcgacagca gaattagcat ctgctgctgg gccctccgga 1920
aacctggacc cattaaatca cagcaggatg tatttgttta ttcagatgga gttctgtgag 1980
ggaggaacac tgaccacgtg gatacgggtg agaaatcgta tgaacaaaca gagaatcgcg 2040
gtggaaatcc ataaaatatt ttatgaaatt gtcagtggag tggaatacat ccattcaaac 2100
agtctcatcc acagagactt gaagcctgat aacatattgt ttggcatgga tggcaaagtg 2160
aagatcggag actttgggct ggtggcggca cagaccaatc acagcggtgg tcctgtagag 2220
aggacaaaga gaagaggaac actgcaatat atgagtcctg aacaggaaaa taagaggaat 2280
tatgatgaaa aaacagacat tttccctctt ggactcatat ggtttgagat gctttggaaa 2340
ttgtccaccg gtatggagag agcagagctg ttgaaagatc tgagaaatca aaggtttccg 2400
gaaggttttt gtgacagtta tccaactgaa aataaattca tcgagaagat gctgtccttt 2460
gcaccggagg acaggccacc tgcaaaagac atcaaagaaa aacttgagaa gtttttctct 2520
ctggatgagg atctgttaag ccagaaaacg atttgaggta ccagtcgact ctagaggatc 2580
cgaacaaaaa ctcatctcag aagaggatct gtgacccggg tggcatccct gtgacccctc 2640
cccagtgcct ctcctggccc tggaagttgc cactccagtg cccaccagcc ttgtcctaat 2700
aaaattaagt tgcatcattt tgtctgacta ggtgtccttc tataatatta tggggtggag 2760
gggggtggta tggagcaagg ggcaagttgg gaagacaacc tgtagggcct gcggggtcta 2820
ttgggaacca agctggagtg cagtggcaca atcttggctc actgcaatct ccgcctcctg 2880
ggttcaagcg attctcctgc ctcagcctcc cgagttgttg ggattccagg catgcatgac 2940
caggctcagc taatttttgt ttttttggta gagacggggt ttcaccatat tggccaggct 3000
ggtctccaac tcctaatctc aggtgatcta cccaccttgg cctcccaaat tgctgggatt 3060
acaggcgtga accactgctc ccttccctgt ccttctgatt ttaaaataac tataccagca 3120
ggaggacgtc cagacacagc ataggctacc tggccatgcc caaccggtgg gacatttgag 3180
ttgcttgctt ggcactgtcc tctcatgcgt tgggtccact cagtagatgc ctgttgaatt 3240
gggtacgcgg ccagcttggc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg 3300
ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg 3360
aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc 3420
aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca 3480
ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc 3540
ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa 3600
gctcctcgag gaactgaaaa accagaaagt taattcccta tagtgagtcg tattaaattc 3660
gtaatcatgt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac 3720
atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca 3780
ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat 3840
taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc 3900
tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca 3960
aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca 4020
aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg 4080
ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg 4140
acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt 4200
ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt 4260
tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc 4320
tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt 4380
gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt 4440
agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc 4500
tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa 4560
agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt 4620
tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct 4680
acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta 4740
tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa 4800
agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc 4860
tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact 4920
acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc 4980
tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt 5040
ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta 5100
agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg 5160
tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt 5220
acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc 5280
agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt 5340
actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc 5400
tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc 5460
gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa 5520
ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac 5580
tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa 5640
aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt 5700
tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa 5760
tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct 5820
gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc 5880
gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc 5940
acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt 6000
agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg 6060
ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt 6120
ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta 6180
taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt 6240
aacgcgaatt ttaacaaaat attaacgctt acaattt 6277
<210> 3
<211> 61
<212> DNA
<213> 草鱼(Ctenopharyngodon idella)
<400> 3
taactgatac tacattgaat tcgcgcgcgc gcgcgcgcgc gcgaattcaa tgtagtatca 60
g 61
<210> 4
<211> 1542
<212> DNA
<213> 草鱼(Ctenopharyngodon idella)
<400> 4
atgtctgccg aaactcaaat ggagaggaag atcattgatt tcttgagaca aaatgggaaa 60
agcatagctt tgacaattgc taaagaaatc ggacttgaca aatccaccgt gaacagacat 120
ttgtacaacc tacagagatc aaaccaggtg tttaactcta acgaaaagcc tcctgtttgg 180
gatctaatgg agaagacgaa tgggatcaaa caaacagtca aaacaccaga gcaaaagtct 240
ctgacgacga cagcagaaac atgtgaagag aaacacgtaa gggatctgct gaaatcagga 300
ggtttaaaag cctgtcagat cgctaaagac ttgggacaac caagaaaaac cgtaaacaaa 360
cagctgtaca gtatgatgca gacgggtaaa gtaaagaaat gtgaaataag tagcttgtgg 420
cttctggaag gcgaggagag caatgaaagt cactcccaag agagtgatca tagactgggg 480
tcagtcgcag ggttgtctca aagttttgat gtgatcacaa agcttggtga aggaggattt 540
ggctgtgttt ttaaagtaaa gcataaattt gatggcaaga tctacgctgt aaagaaagtc 600
gtcttaactg gggaagctga ttccgaggtg aaggcattgg ccagactaga ccatccaaac 660
atagtgcgct acattacatg ttggccggat tctgagagct gcacatcaaa ccaagacaga 720
aaccaagtgt ccaacacatc aggttcttca tcatgtggag tgacctttga tagagctggc 780
tgtgaagaga ggaatgatga agacgatgaa gacgatgatg atgacgttga tgtcagtgat 840
gtcacatcaa gaatggaaag tctgggttcg acagcagaat tagcatctgc tgctgggccc 900
tccggaaacc tggacccatt aaatcacagc aggatgtatt tgtttattca gatggagttc 960
tgtgagggag gaacactgac cacgtggata cgggtgagaa atcgtatgaa caaacagaga 1020
atcgcggtgg aaatccataa aatattttat gaaattgtca gtggagtgga atacatccat 1080
tcaaacagtc tcatccacag agacttgaag cctgataaca tattgtttgg catggatggc 1140
aaagtgaaga tcggagactt tgggctggtg gcggcacaga ccaatcacag cggtggtcct 1200
gtagagagga caaagagaag aggaacactg caatatatga gtcctgaaca ggaaaataag 1260
aggaattatg atgaaaaaac agacattttc cctcttggac tcatatggtt tgagatgctt 1320
tggaaattgt ccaccggtat ggagagagca gagctgttga aagatctgag aaatcaaagg 1380
tttccggaag gtttttgtga cagttatcca actgaaaata aattcatcga gaagatgctg 1440
tcctttgcac cggaggacag gccacctgca aaagacatca aagaaaaact tgagaagttt 1500
ttctctctgg atgaggatct gttaagccag aaaacgattt ga 1542
Claims (3)
1.一种鱼用DNA疫苗,其特征在于:
所述的鱼用DNA疫苗为含有Z-DNA序列的重组质粒与含有PKZ开放阅读框序列的重组质粒的混合物;其中所述的含有Z-DNA序列的重组质粒与所述的含有PKZ开放阅读框序列的重组质粒之间的质量比为1:1;
所述的含有Z-DNA序列的重组质粒的核苷酸序列如SEQ ID NO.1所示;
所述的含有PKZ开放阅读框序列的重组质粒的核苷酸序列如SEQ ID NO.2所示;
所述的Z-DNA序列通过人工合成来获取;
所述的PKZ开放阅读框序列通过PCR扩增来获取;
所述的含有Z-DNA序列的重组质粒通过将Z-DNA序列插入到真核表达载体上来获取;
所述的含有PKZ开放阅读框序列的重组质粒通过将PKZ开放阅读框序列插入到真核表达载体上来获取;
所述的真核表达载体为含有Flag标签的真核表达载体;
所述的含有Flag标签的真核表达载体为pCMV-Flag;
上述鱼用DNA疫苗的制备方法,包括以下步骤:
(1)构建含有Z-DNA序列的重组质粒:在Poly (GC)10的一端添加Hind III限制性内切酶位点,其另一端添加Kpn I限制性内切酶位点,将添加限制性内切酶位点的Poly (GC)10用退火缓冲液并经95℃处理30min,然后自然冷却至室温形成双链DNA,接着将pCMV-Flag质粒与退火后的Poly (GC)10分别经Hind III限制性内切酶与Kpn I限制性内切酶的双酶切反应,回收产物,利用T4 DNA连接酶将双酶切后的Poly (GC)10连接至pCMV-Flag质粒上,最后提取pCMV-Flag-GC重组质粒;
(2)构建含有PKZ开放阅读框序列的重组质粒:获取含有酶切位点的草鱼PKZ开放阅读框的基因序列,设计并合成特异性引物,取草鱼cDNA模板,进行PCR扩增,得到草鱼PKZ开放阅读框的全长基因序列,接着通过将pCMV-Flag质粒与草鱼PKZ开放阅读框的全长基因序列分别经EcoR I限制性内切酶与Kpn I限制性内切酶的双酶切反应,回收产物,利用T4 DNA连接酶将双酶切后的PKZ开放阅读框序列连接至pCMV-Flag质粒上,最后提取pCMV-Flag-PKZ重组质粒;
(3)将步骤(1)构建的含有Z-DNA序列的重组质粒与步骤(2)构建的含有PKZ开放阅读框序列的重组质粒,进行混合,即可;
其中构建含有Z-DNA序列的重组质粒的步骤中:
其中构建含有PKZ开放阅读框序列的重组质粒的步骤中PCR扩增反应体系如下:
草鱼cDNA模板2 µl,上下游引物各0.5 µl,LA Taq DNA聚合酶1 µl,Taq酶buffer 2.5µl,ddH2O补至50 µl;
其中构建含有PKZ开放阅读框序列的重组质粒的步骤中PCR扩增反应条件如下:
95℃预变性5 min,95℃变性30 s,58℃退火30 s,72℃延伸2min,35个循环,72℃延伸10 min。
2.一种鱼用DNA疫苗在制备草鱼养殖中GCRV病毒感染防治药物中的应用。
3.根据权利要求2所述的鱼用DNA疫苗在制备草鱼养殖中GCRV病毒感染防治药物中的应用,其特征在于:
所述的鱼用DNA疫苗通过腹腔注射方式给药,给药浓度为30 µg-50 µg/尾。
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