CN106957847A - 有效抑制仔猪流行性腹泻病毒的siRNA及用途 - Google Patents
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Abstract
本发明提供有效抑制仔猪流行性腹泻病毒的siRNA及用途,能够抑制PEDV复制及增殖,可以用于针对猪流行性腹泻病毒的生物制剂的研发,同时可以结合基因编辑技术来制备和培育出携带有抗病基因的转基因动物,减少猪流行性腹泻病毒给养猪业造成的经济损失。
Description
技术领域
本发明提供有效抑制仔猪流行性腹泻病毒的siRNA及用途,能够抑制PEDV复制及增殖,属于生物技术领域。
背景技术
猪流行性腹泻是由仔猪流行性腹泻病毒(PEDV)引起的猪的一种高度接触性肠道传染病,以水样腹泻、呕吐、脱水和食欲下降为主要特征。不同年龄和不同品种的猪对本病都易感,但对哺乳仔猪的危害最为严重。尽管研制出了抗PEDV的灭活疫苗和弱毒疫苗,但由于近几年PED的流行特点和发病情况发生很大的变化,给防治工作带来了许多困难,对养猪业造成巨大的经济损失。因此,建立新的抗仔猪流行性腹泻病毒方法,加快仔猪流行性腹泻抗病育种研究具有重要的现实意义。
RNA干扰(RNAi)是一种由内源性或外源性双链RNA(double stranded RNA,dsRNA)分子引发的转录后基因沉默(posttranscriptional gene silencing, PTGS)现象,是生物体本身存在的对抗侵入的病毒、转座子、转基因等外源基因,抑制其循环复制从而减弱其基因毒性作用的一种保护自身的天然机制。由于其作用的特异性、高效性,已广泛应用于抗病毒等方面的的研究。
本发明利用RNAi、SCNT和CRISPR/Cas9基因组定点编辑技术,制备基因组定点整合抗PEDV的shRNA表达元件的转基因克隆猪。项目的顺利实施,有望获得稳定表达抑制PEDV增殖的转基因克隆猪,从而提高猪对PEDV的抵抗能力,在源头上有效预防仔猪流行性腹泻病毒病的发生。
发明内容
本发明提供了能显著抑制仔猪流行性腹泻病毒复制及增殖的siRNA的序列。
本发明所提供的能抑制仔猪流行性腹泻病毒复制及增殖的siRNA,是下述双链RNA序列之一:
1)正义链为序列表中的SEQ ID 1所示的RNA序列,反义链为序列表中 SEQ ID 2所示的RNA序列;
2)正义链为序列表中的SEQ ID 3所示的RNA序列,反义链为序列表中 SEQ ID 4所示的RNA序列;
3)正义链为序列表中的SEQ ID 5所示的RNA序列,反义链为序列表中 SEQ ID 6所示的RNA序列;
将上述双链RNA序列分别命名为siRNA PE-1-1、siRNA PE-4-1、 siRNA PE-4-2。
编码上述能抑制仔猪流行性腹泻病毒复制及增殖的siRNA的DNA序列也属于本发明的保护范围内,是下述DNA序列之一:
1)正义链为序列表中的SEQ ID 7所示的DNA序列,反义链为序列表中SEQ ID 8所示的DNA序列;
2)正义链为序列表中的SEQ ID 9所示的DNA序列,反义链为序列表中SEQ ID 10所示的DNA序列;
3)正义链为序列表中的SEQ ID 11所示的DNA序列,反义链为序列表中SEQ ID 12所示的DNA序列。
本发明的积极效果在于:
本发明所述的两种能有效抑制猪流行性腹泻病毒复制和增殖的siRNA,其可以用于针对猪流行性腹泻病毒的生物制剂的研发,同时可以结合基因编辑技术来制备和培育出携带有抗病基因的转基因动物,减少猪流行性腹泻病毒给养猪业造成的经济损失。
附图说明
图1为本发明评估各siRNA抗仔猪流行性腹泻病毒的能力的IFA荧光显微镜成像图;
图2为本发明评估各siRNA抗仔猪流行性腹泻病毒的能力的Hoechest 33342细胞核染色图;
图3为本发明进一步评估各siRNA抗仔猪流行性腹泻病毒的能力的RT-PCR电泳图。
具体实施方式
下面结合附图对本发明进行详细地解释说明。
实施例1
siRNA序列的设计及抗仔猪流行性腹泻病毒能力的验证
通过NCBI数据库(https://www.ncbi.nlm.nih.gov/nuccore/)查询PEDV的RNA信息,根据siRNA设计原则,设计了针对PEDV的siRNA序列,同时设计了一条无关的对照序列(control)。上述设计完成的siRNA序列送予苏州吉玛有限公司合成。9条siRNA分别靶向PEDV的RNA基因组的9个不同区域,长为22个碱基。
其中,本发明所涉及的
siRNA-PE-1-1的RNA序列为:5-AUUAACAACAAAAUCACAUGGA-3;
siRNA-PE-4-1的RNA序列为:5-UAUUAAAACUAAAACCUUCUGG-3 ;
siRNA-PE-4-2的RNA序列为:5-UAUUAAACCUCAGAGCCUCUGG-3;
针对上述3种siRNA的作用位点的序列设计成相应的siRNA正义链及反义链序列及Control-siRNA的RNA序列:
siRNA-PE-1-1正义链序列:5-CCCAUGUGAUUUUGUUGUUAAU-3 (SEQ ID NO.1);
siRNA-PE-1-1反义链序列:5-AUUAACAACAAAAUCACAUGGA-3(SEQ ID NO.2);
siRNA-PE-4-1正义链序列:5-ACAGAAGGUUUUAGUUUUAAUA-3(SEQ ID NO.3);
siRNA-PE-4-1反义链序列:5-UAUUAAAACUAAAACCUUCUGG-3(SEQ ID NO.4);
siRNA-PE-4-2正义链序列:5-ACAGAGGCUCUGAGGUUUAAUA-3 SEQ ID NO.5);
siRNA-PE-4-2反义链序列:5-UAUUAAACCUCAGAGCCUCUGG-3(SEQ ID NO.6);
Control-siRNA正义链序列: 5-UAGCUAUACUCACCUUAUCAUA-3;
Control-siRNA反义链序列: 5-UAUGAUAAGGUGAGUAUAGCUA-3;
其中,本发明所涉及的
siRNA-PE-1-1的DNA序列为:5-ATTAACAACAAAATCACATGGA-3;
siRNA-PE-4-1的DNA序列为:5-TATTAAAACTAAAACCTTCTGG-3;
siRNA-PE-4-2的DNA序列为:5-TATTAAACCTCAGAGCCTCTGG-3;
针对上述3种siRNA的作用位点的序列设计成相应的siRNA正义链及反义链及Control-siRNA的DNA序列为:
siRNA-PE-1-1正义链序列:5-CCCATGTGATTTTGTTGTTAAT-3 (SEQ ID NO.7);
siRNA-PE-1-1反义链序列:5-ATTAACAACAAAATCACATGGA-3(SEQ ID NO.8);
siRNA-PE-4-1正义链序列:5-ACAGAAGGTTTTAGTTTTAATA-3(SEQ ID NO.9);
siRNA-PE-4-1反义链序列:5-TATTAAAACTAAAACCTTCTGG-3(SEQ ID NO.10);
siRNA-PE-4-1正义链序列:5-ACAGAGGCTCTGAGGTTTAATA-3 SEQ ID NO.11);
siRNA-PE-4-1反义链序列:5-TATTAAACCTCAGAGCCTCTGG-3(SEQ ID NO.12);
Control-siRNA正义链序列: 5-TAGCTATACTCACCTTATCATA-3;
Control-siRNA反义链序列: 5-TATGATAAGGTGAGTATAGCTA-3。
实施例2
将这些退火后的ds-oligo以无DNA酶,无RNA酶水溶解成浓度为100uM的工作液,添加到电穿孔的转染体系中,混匀后,以电穿孔的方式将这些ds-oligo引入到PK-15细胞系中,37℃,培养6小时后,接种仔猪流行性腹泻病毒,1~2h后换液,继续培养72小时后,PBST清洗2~3遍后,加入80%冷丙酮,放入-20冰箱中进行固定。后进行IFA检测、染核分析及RT-PCR检测。
检测
1)吸出个各培养孔中的固定液,各孔加入500ul PBST,放到摇床上,清洗10min,清洗2~3次;
2)用抗体稀释液按1:100的比例稀释一抗,加入到各细胞培养孔中,37℃,孵育1~2小时;
3)各孔加入500ul PBST,放到摇床上,清洗10min,清洗2~3次;
4)用抗体稀释液按1:100的比例稀释荧光二抗,加入到各细胞培养孔中,37℃,孵育30分钟;
5)各孔加入500ul PBST,放到摇床上,清洗10min,清洗2~3次;
6)各孔加入500ulPBST,在倒置荧光显微镜显微镜下进行分析。
图1为本发明评估各siRNA抗仔猪流行性腹泻病毒的能力的IFA荧光显微镜成像图。
细胞核染色分析
1)按1:200的比例稀释Hochest33342储存液,充分混匀;
2)加入适量的PBS清洗各孔2~3遍;
3)各孔中加入适量的Hochest33342的稀释液,37℃,孵育5~6分钟后,再加入适量的PBS清洗各孔2~3遍;
4)各孔加入适量的PBS后,倒置荧光显微镜下观察细胞核的形态。
图2为本发明评估各siRNA抗仔猪流行性腹泻病毒的能力的Hoechest 33342细胞核染色图。
检测抗PEDV能力
1)各siRNA转染组分的细胞接种PEDV 72小时后,对各组分分别进行总RNA的提取及浓度的测量;
2)反转录体系的计算及反转录反应;
3) RT-PCR,反应的条件为95℃,4分钟;(94℃ 30秒,55℃ 30秒,72℃ 1分钟)循环数为:35个;72℃ 5分钟;4℃,1小时。
4)琼脂糖凝胶电泳及条带分析。图3为本发明一步评估各siRNA抗仔猪流行性腹泻病毒的能力的RT-PCR电泳图。
<110> 吉林大学
<120> 有效抑制仔猪流行性腹泻病毒的siRNA及其序列
<160> 8
<210> 1
<211> 22
<212> RNA
<213> 人工序列
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<223> siRNA cs2-1正义链序列
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CCCUGUACAUUCAACUACGCAA 22
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<213> 人工序列
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<223> siRNA cs2-1反义链序列
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UUGCGUAGUUGAAUGUACAGGA 22
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<212> RNA
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<223> siRNA cs3-2正义链序列
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AACACUGUGACAGACUAUGUAA 22
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<223> siRNA cs3-2反义链序列
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TTACATAGTCTGTCACAGTGTC 22
Claims (4)
1.能有效抑制PEDV复制与感染的siRNA,是下述双链RNA序列之一:
正义链为序列表中的SEQ ID 1,反义链为序列表中 SEQ ID 2的双链RNA序列;
正义链为序列表中的SEQ ID 3,反义链为序列表中 SEQ ID 4的双链RNA序列;
正义链为序列表中的SEQ ID 5,反义链为序列表中 SEQ ID 6的双链RNA序列。
2.权利要求1所述的能有效抑制PEDV复制及增殖的siRNA的编码序列,其特征在于所述能抑制PEDV复制与感染的siRNA的编码序列是下述双链DNA序列之一:
正义链为序列表中的SEQ ID 7所示的DNA序列,反义链为序列表中SEQ ID 8所示的DNA序列;
正义链为序列表中的SEQ ID 9所示的DNA序列,反义链为序列表中SEQ ID 10所示的DNA序列;
正义链为序列表中的SEQ ID 11所示的DNA序列,反义链为序列表中SEQ ID 12所示的DNA序列。
3.权利要求1或权利要求2所述的siRNA在细胞及个体水平中抗PEDV的应用,其特征在于该siRNA在细胞水平及个体水平中能有效的抑制PEDV增殖和复制。
4.一种权利要求3所述的能有效抑制PEDV复制和增殖的siRNA序列的应用,其特征在于,所述的siRNA及其序列在猪流行性腹泻治疗生物制剂、制备抗PEDV转基因细胞系以及抗PEDV转基因猪中的应用。
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CN111454954A (zh) * | 2020-04-22 | 2020-07-28 | 深圳市金新农科技股份有限公司 | 抑制猪流行性腹泻病毒M基因表达的shRNA和重组表达载体及其应用 |
CN112760320A (zh) * | 2020-12-02 | 2021-05-07 | 扬州大学 | 有效抑制猪流行性腹泻病毒复制的外源性人工miRNA及其用途 |
CN112760320B (zh) * | 2020-12-02 | 2023-11-28 | 扬州大学 | 有效抑制猪流行性腹泻病毒复制的外源性人工miRNA及其用途 |
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