CN112342212B - 一种用于日本对虾抗WSSV感染的AMO-miRNA - Google Patents
一种用于日本对虾抗WSSV感染的AMO-miRNA Download PDFInfo
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Abstract
本发明公开了一种用于日本对虾抗WSSV感染的AMO‑miRNA,所述AMO‑miRNA的核苷酸序列为5’‑CATCAAAGCTGGCTGTGATA‑3’。本发明能够显著抑制WSSV在日本对虾体内的增殖,有效解决了养殖对虾感染WSSV致死的现象。
Description
技术领域
本发明涉及一种miRNA反义核酸,特别涉及一种用于日本对虾抗白斑综合症病毒(white spot syndrome virus,WSSV)感染的AMO-miRNA。
背景技术
对虾广泛生长在浅海地区,具有种类繁多、繁殖力强、生长迅速、肉味鲜美等特点。因此,对虾养殖业正在蓬勃发展并迅速地成为了一个重要产业。然而,对虾白斑综合症病毒(white spot syndrome virus,WSSV)自20世纪90年代首次爆发以来,给对虾养殖业造成了重大的经济损失,并在全球范围内引起水产养殖户的密切关注。它致病力极强,被感染的对虾虾壳内侧会显现毫米级别的白色斑点,一周内死亡率高达90%以上。迄今为止,仍然没有防治白斑综合症病毒的药物。因此,寻找不仅能够有利于水产养殖业的可持续发展,又能有效防治病害的药物十分重要。
目前抗WSSV的药物发明主要包括由多种材料合成的药物,如防治对虾白斑病的复方中草药,该药物的原材料包括:葛根、黄连、金银花、板蓝根、黄芪、甘草、柴胡、当归、山楂、陈皮和茯苓,混合拌匀成粉末状后添加至饲料中给对虾喂食。其所需的原材料种类繁多,所包含的材料分子质量相对较大,降解难易程度未知,是否会影响养殖生态平衡也未知。此外,中草药虽源于自然,副作用小,但长期服用,对于对虾自身来说也是一种刺激和应激,且中草药没有具体的作用靶点。因此,寻找方法简单,高效环保的防治方法极为重要。
miRNA是一类由内源基因编码的长度约为20-24个核苷酸的非编码单链RNA分子,它们在动植物中参与转录后基因表达调控。到目前为止,在动植物以及病毒中已经发现有约3万个miRNA分子,大多数miRNA基因以单拷贝、多拷贝或基因簇的形式存在于基因组中。最新研究表明,miRNA在宿主与病毒的互作过程中发挥着关键的作用,miRNA作为抗病毒治疗的候选药物具备良好的应用前景。
发明内容
本发明的目的在于提供一种用于日本对虾抗WSSV感染的AMO-miRNA,能够显著抑制WSSV在日本对虾体内的增殖,有效解决了养殖对虾感染WSSV致死的现象。
本发明解决其技术问题所采用的技术方案是:
一种用于日本对虾抗WSSV感染的AMO-miRNA,其特征在于,所述AMO-miRNA的核苷酸序列为5’-CATCAAAGCTGGCTGTGATA-3’(SEQ ID No.1)。
一种用于日本对虾抗WSSV感染的制剂,其特征在于,所述制剂包含AMO-miRNA。
AMO-miRNA的核苷酸序列为5’-CATCAAAGCTGGCTGTGATA-3’。
本发明通过研究miRNA在WSSV感染过程中的表达情况,发现日本对虾受到白斑综合症病毒感染后其体内的miRNA表达明显上调,这表明miRNA可能参与了宿主的病毒感染过程,是潜在的WSSV防治新靶点。本发明进一步表明当上调日本对虾体内miRNA的表达,对虾体内的病毒拷贝数明显增加,显示该miRNA能够促进病毒在宿主体内的感染,是病毒在宿主体内增殖的关键miRNA。基于上述发现,本发明设计了反义核酸AMO-miRNA抑制其表达量。将WSSV和AMO-miRNA同时注射到日本对虾体内后,宿主体内的病毒拷贝数下降显著。此外,细胞内验证miRNA与Rab10的3’UTR发生相互作用,通过靶向调控Rab10基因的表达。miRNA的过表达促进自噬在对虾体内的发生,自噬被病毒利用有利于感染增值。因此,AMO-miRNA抵抗病毒的作用机制主要是抑制宿主细胞发生自噬进而抑制WSSV在日本对虾体内的复制。
一种用于日本对虾抗WSSV感染的AMO-miRNA作为制备抗WSSV感染药物的应用。
本发明的有益效果是:本发明的miRNA反义核酸AMO-miRNA见效快,对WSSV抑制效率高;AMO-miRNA分子量极小,安全易降解,污染小。
附图说明
图1是WSSV处理后日本对虾血淋巴细胞中miRNA的表达量。
图2是日本对虾体内miRNA的表达量随miRNA模拟物及miRNA-scrambled(miRNA模拟物打乱的序列)处理的变化情况。
图3是日本对虾体内WSSV拷贝数随miRNA和miRNA-scrambled处理的变化情况;对WSSV感染后的日本对虾进行上述处理,检测WSSV感染0h、24h、36h和48h后日本对虾体内WSSV的拷贝数。
图4是日本对虾的累积死亡率随miRNA、miRNA-scrambled及PBS处理的变化情况;对WSSV感染后的日本对虾进行上述处理,记录WSSV感染一周的日本对虾累积死亡率。
图5是日本对虾体内miRNA的表达量随AMO-miRNA及AMO-miRNA-scrambled(AMO-miRNA-scrambled打乱的序列)处理的变化情况。
图6是日本对虾体内WSSV拷贝数随AMO-miRNA和AMO-miRNA-scrambled处理的变化情况;对WSSV感染后的日本对虾进行上述处理,检测WSSV感染0h、24h、36h和48h后日本对虾体内WSSV的拷贝数。
图7是日本对虾的累积死亡率随AMO-miRNA、AMO-miRNA-scrambled及PBS处理的变化情况;对WSSV感染后的日本对虾进行上述处理,记录WSSV感染一周的日本对虾累积死亡率。
图8是High Five细胞内验证miRNA与Rab10 3’UTR的相互作用。
图9是对虾体内过表达miRNA检测Rab10的表达。
图10是对虾体内AM0-miRNA抑制miRNA检测Rab10的表达。
图11是miRNA的过表达对自噬在对虾体内发生的影响。
图12是在对虾体内AM0-miRNA抑制miRNA表达对自噬的影响。
具体实施方式
下面通过具体实施例,对本发明的技术方案作进一步的具体说明。
本发明中,若非特指,所采用的原料和设备等均可从市场购得或是本领域常用的。下述实施例中的方法,如无特别说明,均为本领域的常规方法。
实施例1:
WSSV感染后我们提取对虾miRNA测序,结果表明miRNA表达上调,通过体外验证日本对虾体内miRNA表达量的变化
将体重约为15g,体长约为10-12cm,RT-PCR检测WSSV为阴性的日本对虾置于70L水箱内有气泵充氧、盐度为26、温度为22℃的人工海水中暂养1-2天后,使用1mL注射器,在对虾倒数第三节尾部注射100μL浓度为105copies/mL的病毒稀释液(104个病毒粒子)。分别在注射后的0h、24h、36h及48h收集对虾血淋巴细胞,提取总RNA后进行cDNA反转录,所用的Stem-loop Primer序列如下:
最后利用qPCR技术检测miRNA表达量,以U6作为内参。同时采用Northern blot检测miRNA表达量。
定量引物名称 | 定量引物序列 |
U6-F | 5’-TTCACGAATTTGCGTGTCAT-3’(SEQ ID No.4) |
U6-R | 5’-CGCTTCGGCAGCACATATAC-3’(SEQ ID No.5) |
miRNA-F | 5’-CGCCGTATCACAGCCAGC-3’(SEQ ID No.6) |
miRNA-R | 5’-TGCAGGGTCCGAGGTCACTG-3’(SEQ ID No.7) |
结果见图1,数据显示日本对虾受到WSSV感染48h后其血淋巴细胞中miRNA的表达显著升高,表明miRNA可能参与了宿主病毒间的互作过程,因此,miRNA是潜在的WSSV防治的新靶点。
实施例2:
日本对虾体内miRNA的过表达试验
按实施例1的方法随机将日本对虾分成3组,分别注射100μL/只的miRNA模拟物(体外合成,与日本对虾miRNA序列一致)或100μL/只的miRNA-mimic-scrambled(体外合成,与miRNA模拟物相同核苷酸组成但顺序随机打乱)或不处理。注射48h后参照实施例1的方法检测对虾血淋巴细胞中miRNA的表达量。
名称 | miRNA-mimic序列 |
miRNA-mimic | 5’-TATCACAGCCAGCTTTGATG-3’(SEQ ID No.8) |
miRNA-mimic-scrambled | 5’-ATTCAGTGCGTCCAAACTAG-3’(SEQ ID No.9) |
结果见图2,数据显示注射miRNA模拟物可以显著上调日本对虾体内miRNA的表达量,可用于后续功能试验。
实施例3:
miRNA过表达促进WSSV在日本对虾体内增殖的试验
按实施例1的方法随机将日本对虾分成3组,分别注射100μL/只的miRNA模拟物或100μL/只的miRNA-scrambled或不处理。对照组注射WSSV与miRNA-scrambled的混合液。分别提取注射0h、24h、36h及48h后日本对虾鳃组织的基因组DNA,然后通过qPCR技术进行病毒拷贝数的检测,所用引物和探针序列如下:
引物或探针名称 | 引物或探针序列 |
WSSV-specific-F | 5’-TTGGTTTCATGCCCGAGATT-3’(SEQ ID No.10) |
WSSV-specific-R | 5’-CCTTGGTCAGCCCCTTGA-3’(SEQ ID No.11) |
WSSV-specific TaqMan probe | 5’-FAM-TGCTGCCGTCTCCAA-TAMRA-3’(SEQ ID No.12) |
结果见图3,数据显示日本对虾被WSSV感染后,注射miRNA模拟物能够促进病毒在宿主体内的复制。
实施例4:miRNA过表达提高日本对虾累积死亡率的试验
按实施例1的方法随机将日本对虾分成4组,实验组注射WSSV与miRNA的混合液,对照组注射WSSV与miRNA-scrambled的混合液或仅注射WSSV或仅注射PBS。观察并记录WSSV感染一周的日本对虾累积死亡率。
结果见图4,数据显示日本对虾被WSSV感染后,注射miRNA模拟物能够促进病毒在宿主体内的复制,从而使病毒感染导致的对虾累积死亡率显著升高。
实施例5:
按实施例1的方法随机将日本对虾分成3组,分别注射100μL/只的AMO-miRNA或AMO-miRNA-scrambled或不处理。注射48h后参照实施例1的方法检测对虾血淋巴细胞中miRNA的表达量。序列如下:
AMO-miRNA抑制日本对虾体内miRNA表达的试验
名称 | AMO-miRNA序列 |
AMO-miRNA | 5’-CATCAAAGCTGGCTGTGATA-3’(SEQ ID No.1) |
AMO-miRNA-scrambled | 5’-GTAGTTTCGACCGACACTAT-3’(SEQ ID No.13) |
结果见图5,数据显示注射AMO-miRNA后日本对虾体内miRNA的表达量相较于对照组显著降低,即AMO-miRNA可以显著抑制对虾体内miRNA的表达量,可用于后续功能试验。
实施例6:AMO-miRNA抑制对虾体内WSSV复制的试验
按实施例3的方法,实验组注射WSSV(1×104)与AMO-miRNA(50nM)的混合液,对照组注射WSSV与AMO-miRNA-scrambled的混合液,分别于注射0h、24h、36h和48h后按实施例3的方法检测病毒拷贝数。
结果见图6,结果表明日本对虾被WSSV感染后,注射AMO-miRNA能够显著抑制病毒在体内的复制,增加日本对虾的抗病毒能力。
实施例7:AMO-miRNA降低日本对虾累积死亡率的试验
按实施例4的方法,实验组注射WSSV与AMO-miRNA的混合液,对照组注射WSSV与AMO-miRNA-scrambled的混合液或仅注射WSSV或仅注射PBS。观察并记录WSSV感染一周的日本对虾累积死亡率。
结果见图7,数据显示日本对虾被WSSV感染后,注射AMO-miRNA能够抑制病毒在宿主体内的复制,从而使病毒感染导致的对虾累积死亡率显著降低。
实施例8:High Five细胞内验证miRNA与Rab10 3’UTR相互作用的试验首先将Rab10基因的3’UTR克隆到pIZ/V5-EGFP载体上,即野生型重组质粒。同时,将miRNA与Rab10基因结合的位点进行突变后构建到pIZ/V5-EGFP载体上,即突变型重组质粒。将构建好的野生型重组质粒和突变型重组质粒分别与miRNA模拟物或模拟物对照共转染到High Five昆虫细胞内,48h后检测昆虫细胞的荧光强度。
结果见图8,共转染野生型重组质粒和miRNA模拟物的细胞荧光强度与其他组相比要显著减弱,表明miRNA可与Rab10 mRNA 3’UTR互作。
实施例9:对虾体内过表达miRNA降低Rab10的表达。
在对虾体内通过注射miRNA模拟物(miRNA-mimic)过表达miRNA,同时以注射miRNA模拟物对照(miRNA-mimic-scrambled)作为对照组。随后在其感染后的48h收集对虾的肌肉组织,检测Rab10的表达量。
结果见图9,当miRNA过表达时,对虾体内Rab10的表达水平被显著抑制。
实施例10:对虾体内抑制miRNA上调Rab10的表达。
在对虾体内采用反义核酸(AMO-miRNA)抑制miRNA表达,同样以注射AMO-miRNA-scrambled作为对照。随后在其感染48h后,取其肌肉组织检测,结果见图10,当miRNA的表达被抑制时,对虾体内Rab10的表达水平显著增加。这表明,miRNA与Rab10基因在对虾体内可发生互作。
实施例11:对虾体内过表达miRNA促进自噬在对虾体内的发生。
在对虾体内通过注射miRNA模拟物(miRNA-mimic)过表达miRNA。随后在其感染后的0h、24h、36h和48h收集对虾的肌肉组织,检测p62和LC-3Ⅱ/Ⅰ的表达量。在自噬发生过程中,LC3-Ⅰ通过活化酶Atg7与结合酶Atg3与磷脂酰乙醇胺(PE)结合而成为LC3-Ⅱ,LC-3Ⅱ/Ⅰ的比例上升表明自噬发生。而p62则通过与短的LC3相互作用区域(LIR)直接与LC3和GABARAP(Atg8直向同源物)家族蛋白结合,以通过自噬降解的机制来用作递送选择性自噬货物。p62蛋白本身会被自噬降解,并作为研究自噬通量的标记。当自噬被抑制时,p62会积累,而自噬被诱导时,p62数量会减少。结果见图11,当miRNA过表达时,可促进自噬在对虾体内的发生。
实施例12:对虾体内抑制miRNA的表达抑制自噬在对虾体内的发生。在对虾体内通过注射反义核酸(AMO-miRNA)以抑制miRNA的表达。随后在其感染后的0h、24h、36h和48h收集对虾的肌肉组织,检测p62和LC-3Ⅱ/Ⅰ的表达量。结果见图12,当AMO-miRNA注射时,可以抑制自噬的发生,进而抑制WSSV的增值。
以上所述的实施例只是本发明的一种较佳的方案,并非对本发明作任何形式上的限制,在不超出权利要求所记载的技术方案的前提下还有其它的变体及改型。
SEQUENCE LISTING
<110> 浙江理工大学
<120> 一种用于日本对虾抗WSSV感染的AMO-miRNA
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Claims (3)
1.一种用于日本对虾抗WSSV感染的AMO-miRNA,其特征在于,所述AMO-miRNA的核苷酸序列为5’- CATCAAAGCTGGCTGTGATA-3’。
2.一种用于日本对虾抗WSSV感染的制剂,其特征在于,所述制剂包含AMO-miRNA,所述AMO-miRNA的核苷酸序列为5’- CATCAAAGCTGGCTGTGATA-3’。
3.一种如权利要求1所述的用于日本对虾抗WSSV感染的AMO-miRNA作为制备抗WSSV感染药物的应用。
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Temporal regulation of microRNA expression in Drosophila melanogaster mediated by hormonal signals and Broad-Complex gene activity;Lorenzo F. Sempere等;《DEVELOPMENTAL BIOLOGY》;20030701;第259卷(第1期);第9-18页 * |
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