CN106957846A - 有效抑制猪瘟病毒复制和增殖的siRNA及用途 - Google Patents
有效抑制猪瘟病毒复制和增殖的siRNA及用途 Download PDFInfo
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Abstract
本发明提供了有效抑制猪瘟病毒复制和增殖的siRNA及用途,涉及抑制猪瘟病毒复制及增殖的2条siRNA及其序列,其可以用于针对猪瘟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,减少猪瘟病毒给养猪业造成的经济损失。
Description
技术领域
本发明提供了有效抑制猪瘟病毒复制和增殖的siRNA及其序列,涉及抑制猪瘟病毒复制及增殖的2条siRNA及其序列,属于生物技术领域。
技术背景
猪瘟(classic swine fever, CSF)是由猪瘟病毒(classic swine fever virus,CSFV)引起的一种猪高度接触性传染病,以高热、出血、淋巴细胞减少和免疫抑制为主要特征,是国际兽医局(OIE)所列的疫病目录必须通报的传染病之一,我国将其定为1类烈性传染病,在世界范围内给养猪业造成重大经济损失。1833年该病首先发现于美国的俄亥俄州,后来欧洲、中南美洲、非洲和亚洲的部分国家和地区都陆续发现并在一定范围内广泛流行。许多发达国家都采取了屠杀和强制免疫等控制措施之后宣布消灭了猪瘟,但是该病在不发达国家至今仍没有得到有效控制,特别是亚洲地区。此外,一些宣布消灭猪瘟的国家近年来也不时有暴发猪瘟的报道。我国长期贯彻预防为主的指导方针,坚持全面接种兔化弱毒疫苗,该疫苗的应用对我国猪瘟的防控做出了重大贡献,使该病在我国大规模暴发基本停止。但是该病仍在全国各地不间断的散发流行,多呈温和、慢性、非典型、隐性感染和持续性感染等新特征,同时多种原因引起的疫苗免疫失败时有发生,这些对猪瘟的预防提出了严峻的考验"除了疫苗免疫之外,人们一直在探索其它的防治手段。
RNA干扰(RNA interference, RNAi)技术发现为人类对抗病毒病提供了新的途径。RNAi 是由双链RNA所引起的序列特异性基因沉默现象。大量实验证明,小干扰RNA(small interfering RNA,siRNA)能对抗病毒感染,作用于病毒或宿主细胞受体,抑制病毒复制,阻抑病毒感染。随着转基因技术的发展,可以把有利于改善动物性状的外源基因添加到动物的基因组中,使其在体内表达,从而培育出具有新遗传特性和优良性状的转基因动物,不受传统育种方法的限制。将RNAi和转基因克隆技术结合起来进行抗病毒转基因育种,可以培育出携带有抗病基因的转基因动物,减少病毒病带来的经济损失。目前RNAi技术和克隆动物制备技术结合已经成功用于构建人类疾病动物模型,正应用于家畜抗病育种的研究中。
发明内容
本发明公开了有效抑制猪瘟病毒复制和增殖的siRNA及其序列,能显著抑制猪瘟病毒复制及增殖。
本发明所述的能有效抑制猪瘟病毒复制与感染的siRNA,是下述双链RNA序列之一:
1)正义链为序列表中的SEQ ID 1,反义链为序列表中 SEQ ID 2的双链RNA序列;
2)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。
本发明所述的能有效抑制猪瘟病毒复制及增殖的siRNA的序列,其特征在于所述能抑制猪瘟病毒复制与感染的siRNA的编码序列是下述双链DNA序列之一:
1)正义链为序列表中的SEQ ID 5所示的DNA序列,反义链为序列表中SEQ ID 6所示的DNA序列;
2)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。
本发明所述的siRNA及其序列在猪瘟治疗生物制剂、抗猪瘟病毒细胞系的构建及抗猪瘟转基因猪制备中的应用。
本发明所述的抑制猪瘟病毒复制和增殖的siRNA及其序列的应用,其特征在于,所述的siRNA及其序列在猪瘟治疗生物制剂、抗猪瘟病毒细胞系的构建及抗猪瘟转基因猪制备中的应用。
将上述双链RNA序列分别命名为siRNAcs2-1和siRNAcs3-2。
本发明的积极效果在于:
本发明所述的两种能有效抑制猪瘟病毒复制和增殖的siRNA,其可以用于针对猪瘟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,减少猪瘟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抑制猪瘟病毒复制和增殖的能力的IFA荧光显微镜成像图;
图2为本发明进一步验证各siRNA抗猪瘟病毒复制和增殖的能力的RT-PCR电泳图。
具体实施方式
通过以下实施例进一步举例描述本发明,并不以任何方式限制本发明,在不背离本发明的技术解决方案的前提下,对本发明所作的本领域普通技术人员容易实现的任何改动或改变都将落入本发明的权利要求范围之内。
实施例1
siRNA序列的设计及抗猪瘟能力的验证
通过NCBI数据库(https://www.ncbi.nlm.nih.gov/nuccore/)查询猪瘟病毒的RNA信息,根据siRNA设计原则,设计了针对猪瘟病毒的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。2条siRNA分别靶向猪瘟病毒RNA基因组(GenBank登录号:AF092448)的3371位点和7502位点,长为22个碱基。
其中,本发明所涉及的
siRNA cs2-1(3371位点)的RNA序列为:5-UCCUGUACAUUCAACUACGCAA-3;
siRNA cs3-2(7502位点)的RNA序列为:5-GACACUGTGACAGACUAUGUAA-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 cs2-1正义链序列:5-CCCUGUACAUUCAACUACGCAA-3(SEQ ID NO.1);
siRNA cs2-1反义链序列:5-UUGCGUAGUUGAAUGUACAGGA-3(SEQ ID NO.2);
siRNA cs3-2正义链序列:5-AACACUGUGACAGACUAUGUAA-3(SEQ ID NO.3);
siRNA cs3-2反义链序列:5-UUACAUAGUCUGUCACAGUGUC-3(SEQ ID NO.4);
Control-siRNA正义链序列:5-AACGAAUACUCACCUUAUGUUA-3;
Control-siRNA反义链序列:3-UAACAUAAGGUGAGUAUUCGUU-3;
其中,本发明所涉及的
siRNA cs2-1(3371位点)的DNA序列为:5-TCCTGTACATTCAACTACGCAA-3;
siRNA cs2-1(7502位点)的DNA序列为:5-GACACTGTGACAGACTATGTAA-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-shRNA的DNA序列:
siRNA cs2-1正义链DNA序列:5-CCCTGTACATTCAACTACGCAA-3(SEQ ID NO.5);
siRNA cs2-1反义链DNA序列:5-TTGCGTAGTTGAATGTACAGGA-3(SEQ ID NO.6);
siRNA cs3-2正义链DNA序列:5-AACACTGTGACAGACTATGTAA-3(SEQ ID NO.7);
siRNA cs3-2反义链DNA序列:5-TTACATAGTCTGTCACAGTGTC-3(SEQ ID NO.8);
Control- siRNA正义链DNA序列:5-AACGAATACTCACCTTATGTTA-3;
Control- siRNA反义链DNA序列:3-TAACATAAGGTGAGTATTCGTT-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,接种猪瘟病毒,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检测和RT-PCR分析。
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抑制猪瘟病毒复制和增殖的能力的IFA荧光显微镜成像图。
分析
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,52℃ 30秒,72℃ 1分钟)循环数为:32个;72℃ 5分钟;4℃,1小时;
4)琼脂糖凝胶电泳及条带分析;
图2为本发明进一步验证各siRNA抗猪瘟病毒复制和增殖的能力的RT-PCR电泳图。
<110> 吉林大学
<120> 有效抑制猪瘟病毒复制和增殖的siRNA及其序列
<160> 22
<210> 1
<211> 22
<212> RNA
<213> 人工序列
<220>
<223> siRNA cs2-1正义链序列
<400> 1
CCCUGUACAUUCAACUACGCAA 22
<210> 2
<211> 22
<212> RNA
<213> 人工序列
<220>
<223> siRNA cs2-1反义链序列
<400> 2
UUGCGUAGUUGAAUGUACAGGA 22
<210> 3
<211> 22
<212> RNA
<213> 人工序列
<220>
<223> siRNA cs3-2正义链序列
<400> 3
AACACUGUGACAGACUAUGUAA 22
<210> 4
<211> 22
<212> RNA
<213> 人工序列
<220>
<223> siRNA cs3-2反义链序列
<400> 4
UUACAUAGUCUGUCACAGUGUC 22
<210> 5
<211> 22
<212> DNA
<213> 人工序列
<220>
<223> siRNA cs2-1正义链序列
<400> 5
CCCTGTACATTCAACTACGCAA 22
<210> 6
<211> 22
<212> DNA
<213> 人工序列
<220>
<223> siRNA cs2-1反义链序列
<400> 6
TTGCGTAGTTGAATGTACAGGA 22
<210> 7
<211> 22
<212> DNA
<213> 人工序列
<220>
<223> siRNA cs3-2正义链序列
<400> 7
AACACTGTGACAGACTATGTAA 22
<210> 8
<211> 22
<212> DNA
<213> 人工序列
<220>
<223> siRNA cs3-2反义链序列
<400> 8
TTACATAGTCTGTCACAGTGTC 22
Claims (4)
1.抑制猪瘟病毒复制与感染的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。
2.权利要求1所述的能有效抑制猪瘟病毒复制及增殖的siRNA的序列,其特征在于所述能抑制猪瘟病毒复制与感染的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 5所示的DNA序列,反义链为序列表中SEQ ID 6所示的DNA序列;
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。
3.权利要求1或权利要求2所述的siRNA在猪瘟治疗生物制剂、抗猪瘟病毒细胞系的构建及抗猪瘟转基因猪制备中的应用。
4.一种权利要求3所述的抑制猪瘟病毒复制和增殖的siRNA及其序列的应用,其特征在于,所述的siRNA及其序列在猪瘟治疗生物制剂、抗猪瘟病毒细胞系的构建及抗猪瘟转基因猪制备中的应用。
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