CN101748125B - 用于治疗和/或预防猪繁殖与呼吸综合征的siRNA片段及其应用 - Google Patents
用于治疗和/或预防猪繁殖与呼吸综合征的siRNA片段及其应用 Download PDFInfo
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Abstract
本发明提供一种治疗和/或预防猪繁殖与呼吸综合征的siRNA片段及其应用。所述siRNA片段及包含该siRNA片段的载体均对猪繁殖与呼吸综合征病毒(PRRSV)感染的Marc145细胞系具有保护作用，通过实时定量PCR和免疫印迹检测，发现该siRNA片段能使PRRSV-M蛋白mRNA水平降低约30％-50％。因此，本发明的RNAi片段及包含该RNAi片段的载体可用于制备治疗和/或预防猪繁殖与呼吸综合征的药物，对基因治疗猪繁殖与呼吸综合征具有重要价值。
Description
技术领域
本发明涉及一种siRNA片段及其应用，特别涉及一种用于治疗和/或预防猪繁殖与呼吸综合征的siRNA片段及其应用。
背景技术
RNA干扰(RNA interference，RNAi)是由双链RNA介导的序列特异性的基因沉默。1998年2月由华盛顿卡耐基研究院的Fire与马萨诸塞大学癌症中心的Mello首次在对秀丽新小杆线虫(Caenorhabditis elegan)的研究中提出这一概念，其与在植物中发现的共表达和在粗糙脉孢霉菌中发现的消除(quelling)现象都属于转录后基因沉默机制。这一机制已被证实在真菌、拟南芥、锥虫、水螅、涡虫、斑马鱼等几乎所有真核生物细胞中都存在，甚至在大肠杆菌中也存在类似机制。在发现哺乳动物细胞中也存在这一机制后，RNAi作为一种快速、有效、特异性抑制基因表达的工具被广泛应用于基因功能的研究以及肿瘤和病毒性疾病的治疗研究中。
猪繁殖与呼吸综合征病毒(porcine reproductive and respiratorysyndrome virus，PRRSV)是尼多病毒目动脉炎病毒科成员，其基因组全长约15kb，不分节，含9个开放性阅读框架(ORF)，即ORF1a、ORF1b、ORF2a、ORF2b、ORF3、ORF4、ORF5、ORF6和ORF7。对PRRSV的基因组序列分析表明，PRRSV至少含有7个结构蛋白，即糖蛋白GP2、GP3、GP4、GP5、膜基质蛋白(M蛋白)、核衣壳蛋白(N蛋白)以及新发现的由ORF2b编码的73个氨基酸的非糖基化蛋白。其中，M蛋白由ORF6编码，分子量为19kd，为非糖基化的膜蛋白。亲水性氨基酸序列分析表明，M蛋白有3个疏水性跨膜区。M蛋白聚集于粗面型内质网，并与GP5形成异源二聚体，北美洲和欧洲分离株分别含有174和173个氨基酸。虽然M蛋白可能定位于病毒囊膜并含有相对大的扩展区域，但该蛋白为PRRSV最保守的结构蛋白，其对于PRRSV的复制是必需的。M蛋白具有很强的免疫原性，感染10天即可检测到抗体应答，故体外表达的重组M蛋白可以作为血清学试验的靶抗原。
PRRSV主要引起母猪早产、流产等繁殖障碍，仔猪、育肥猪呼吸困难以及育成猪类的流感疾病，这些疾病统称为猪繁殖与呼吸综合征。目前猪繁殖与呼吸综合征在许多养猪国家爆发流行，成为威胁养猪业安全的重要病原之一。虽然预防PRRSV的疫苗有灭活苗和弱毒苗，然而这些疫苗的使用只能够提供部分保护，仅使机体不再出现临床症状，但不能阻止再次感染。而且，弱毒苗毒株在自然状态下还有可能发生毒力返强现象，并经过胎盘传播给胎儿，造成感染猪群的持续感染，使得一些常规的疫苗很难取得理想的效果。因此，迫切需要开发一种新型的能对所有PRRSV病毒株提供更有效保护的抗病毒措施。
发明内容
因此，本发明的目的在于克服现有治疗和/或预防猪繁殖与呼吸综合征方法的上述缺点和不足，提供一种基于RNAi技术的抑制猪繁殖与呼吸综合征病毒的基因表达，从而治疗和/或预防猪繁殖与呼吸综合征的特异性siRNA片段。
本发明的上述目的是采用以下技术方案来实现的：
一种用于治疗和/或预防猪繁殖与呼吸综合征的siRNA片段，其序列为下列序列：
M-229：
5’-GCAGUAGUUGCACUCCUUU-3’
3’-CGUCAUCAACGUGAGGAAA-5’。
本发明采用化学合成的方法合成上述siRNA片段。在优选实施方案中，在所述siRNA片段的3’端进一步加上2-6个dT或2-6个U的修饰序列，以减少siRNA在细胞内降解，增强其稳定性。
按照以下方法将合成的siRNA片段分别转染至Marc145细胞系(上皮样细胞，来源于猴肾细胞)：
1、Marc145细胞的培养：用含10％胎牛血清的DMEM培养基(GIBCO)，于37℃、5％CO2条件下培养；
2、转染：采用脂质体Hiperfect(Qiagen公司生产)进行细胞转染，用1μg siRNA转染Marc145细胞(6孔板)。
完成转染后24h，接种PRRSV(MOI＝0.01)进行攻毒。
攻毒24h后，提取细胞总RNA进行反转录，以GAPDH基因作为内参，通过实时定量PCR(Real-time PCR)检测PRRSV-M蛋白的mRNA水平，并通过免疫印迹(Western Blot)检测PRRSV-M蛋白的表达水平。
本发明还提供一种用于治疗和/或预防猪繁殖与呼吸综合征的载体，其中包含序列为上述序列的siRNA片段。所述载体优选为逆转录病毒载体(包括慢病毒载体)、腺病毒载体、腺相关病毒载体(AAV载体)以及质粒载体等，例如pSuper、pBabe-Super、pRNA-U6.1/Neo或pSilencer载体等。
本发明所设计的siRNA片段及其载体，可应用于制备治疗和/或预防猪繁殖与呼吸综合征的药物。该药物为抑制猪繁殖与呼吸综合征病毒的药物。具体来说，该药物为抑制猪繁殖与呼吸综合征病毒M蛋白表达的药物。
本发明根据siRNA设计策略，从GenBank中查找PRRSV S1株基因组序列(GenBank登陆号：AF090173)，设计出多对siRNA片段，并通过转染Marc145细胞系试验对这些siRNA片段进行活性筛选。筛选结果表明，本发明筛选出的siRNA片段能够降低Marc145细胞中PRRSV-M蛋白的表达，即M-229降低了PRRSV-M蛋白的mRNA水平约50％。将所述siRNA片段插入到载体并转染Vero细胞系的试验表明，包含该siRNA片段的载体能使PRRSV-M蛋白的mRNA水平降低30％。由于PRRSV-M蛋白对于PRRSV的复制是必需的，抑制PRRSV-M蛋白的表达就抑制了PRRSV在细胞内的生长与繁殖。因此本发明所设计的siRNA片段M-229使PRRSV-M蛋白表达量下降，可以应用于制备治疗和/或预防猪繁殖与呼吸综合征的药物。
综上所述，本发明基于RNAi技术，提供了一种抑制PRRSV-M蛋白表达的特异性RNAi片段，通过抑制PRRSV复制必需的M蛋白表达，从而实现了抑制PRRSV的生长与繁殖，可应用于制备治疗和/或预防猪繁殖与呼吸综合征的药物。
附图说明
图1为本发明的siRNA对PRRSV-M蛋白的mRNA水平影响结果示意图。
图2为本发明的siRNA对PRRSV-M蛋白的表达水平影响结果示意图。
图3为本发明的重组质粒对PRRSV-M蛋白的mRNA水平影响结果示意图。
图4为本发明的重组质粒对PRRSV-M蛋白的表达水平影响结果示意图。
具体实施方式
以下通过具体实施例对本发明作进一步说明。应理解，以下买施例仅用于说明本发明，而不用于限定本发明的范围。
以下实施例中所使用的技术，包括基因测序、合成、细胞转染等分子生物学技术，以及细胞培养、检测技术等，除非特别说明，均为本领域的技术人员已知的常规技术；所使用的仪器设备、试剂、细胞系等，除非是本说明书特别说明，均为本领域的研究和技术人员可以通过公共途径获得的。
实施例1：RNAi序列的设计
siRNA设计策略：1)从靶基因的起始密码子AUG下游50～100个核苷酸开始搜寻理想的siRNA序列，越靠近靶基因的3’端，其基因沉默效果可能越好；2)siRNA序列最好为AA(Nn)UU(N代表任意碱基；n为碱基数目，在19～29nt之间)，NA(Nn)UU和NA(Nn)NN序列也可以。3)具有均衡的碱基含量(即G/C含量在30％～70％)。依据PRRSV S1株基因组序列(GenBank登陆号：AF090173)设计出2对siRNA片段，其序列分别为：
M-229(SEQ ID NO.1)：
5’-GCAGUAGUUGCACUCCUUU-3’
3’-CGUCAUCAACGUGAGGAAA-5’；
M-379(SEQ ID NO.2)：
5’-GCAAAUGAUAACCACGCAU-3’
3’-CGUUUACUAUUGGUGCGUA-5’
M-167(SEQ ID NO.3)：
5’-CCUUCGGGUACAUGACUUU-3’
3’-GGAAGCCCAUGUACUGAAA-5’。
上述RNAi片段长度均为21nt，负链及其任意一个位置的突变体与已知人类基因和基因表达片段无同源性。
在所述siRNA片段的3’端进一步加上2-6个dT或2-6个U的修饰序列，以减少siRNA在细胞内降解，增强其稳定性。
另外，使用的阴性及阳性对照序列分别为：
阴性对照(SEQ ID NO.4)：
5’-UUCUCCGAACGUGUCACGUTT-3’
3’-TTAAGAGGCUUGCACAGUGCA-5’
GAPDH阳性对照(SEQ ID NO.5)：
5’-GUAUGACAACAGCCUCAAGTT-3’
3’-TTCAUACUGUUGUCGGAGUUC-5’
所述序列均委托上海吉玛生物技术有限公司生物公司合成。
实施例2：siRNA转染Marc145细胞系并进行PRRSV感染
实验材料：Marc145细胞、美洲型PRRSV S1株(MOI＝0.01)、美洲型PRRSV多克隆抗体。
1、转染：Marc145细胞用含10％胎牛血清的RPMI 1640培养基(GIBCO)，于37℃、5％CO2条件下培养。采用脂质体Hiperfect(Qiagen)进行细胞转染，操作方法可完全根据厂家说明。转染时，用1μgsiRNA转染Marc145细胞(6孔板)。
2、PRRSV感染：转染24h后，每个培养板中加入PRRSV细胞毒(MOI＝0.1)，继续培养24h。
实施例3：siRNA干扰效果的检测
I.实时定量PCR(Real-time RCR)分析：
1.提取总RNA
使用Trizol试剂(Invitrogen)提取细胞总RNA，步骤参考Invitrogen公司说明书。
2.实时定量PCR分析
1)引物与探针设计
以狒狒(Papio anubis)GAPDH基因和长尾猴(C.aethiops)beta-actin基因作为实时定量PCR反应的阳性对照，分别设计引物和探针序列如表1所示。
表1 GAPDH基因、beta-actin基因和PRRSV-M基因引物和探针序列
2)试剂与仪器
试剂：铁克曼(TaqMan)实时定量PCR通用试剂(上海吉玛生物技术有限公司)；
仪器：FTC-2000A实时定量PCR仪(枫岭，中国)。
3)逆转录cDNA
PCR管中混合以下试剂：
Oligo-dT 4μl
RNA 20μl
70℃下保温10min，迅速在冰上冷却2min以上，瞬时离心，加入以下试剂：
5×M-MLV Buffer 8μl
dNTP(0.25mM) 4μl
RNasin 2μl
M-MLV 2μl
42℃下保温2小时后，再在70℃下保温15分钟，而后冰上冷却，得到cDNA。
4)建立PCR反应体系，如表2所示：
表2 PCR反应体系
| 成分 | 终浓度 | 体积 |
| 2×实时定量PCR Master MixF引物(10μM)R引物(10μM)探针(10μM)cDNA模板Taq DNA聚合酶(5U/μl)dd H2O | 1×0.2μM0.2μM0.1μM—0.5U/μl | 10μl0.4μl0.4μl0.2μl2μl0.2μl达20μl |
5)实时定量PCR反应条件：95℃，5min变性；40个循环：95℃，15秒；55℃，30秒；72℃，30秒。
6)实验结果：
图1为本发明的实时定量PCR检测PRRSV-M蛋白的mRNA水平的结果示意图，其中野生型为未导入siRNA处理的细胞样品，其它处理与样品均一致，其具体实验数据及数据处理分别如表3和表4所示：
表3 siRNA(M-229)干扰组的实验结果
表4 野生型组的实验结果
在上述表3及表4中，CT值代表每个反应管内荧光信号达到设定的阈值时所进行的循环数，Mean CT是三个重复样的CT值平均值，该项“±”后的数值为三个重复样的标准偏差，其计算公式为：
标准差
ΔCT指同一样品中，待检基因与内参基因GAPDH的平均CT值的差值，即ΔCT＝CT待测基因-CT内参基因。ΔΔCT指其余样品的ΔCT值和对照组相应基因的ΔCT值之差，即ΔΔCT＝(CT待测基因-CT内参基因)实验组-(CT待测基因-CT内参基因)对照组。2-ΔΔC T是按照ΔΔCT计算的，括号内为其置信区间，也是根据ΔΔCT的范围计算的。
可见，以2-ΔΔC T值表示实验组待测基因的表达相对于对照组的变化倍数，两者差异为：1.00-0.51＝0.49＝49％。
M-229干扰组与对照组的相对表达量差约为50％，即M-229干扰组相对野生型组表达量的差异百分比达到约50％。
经过三次重复实验进行T检验，标准差小于0.05，为显著性差异。
II.免疫印迹检验
1、总蛋白抽提步骤：
1)PBS清洗细胞3次；
2)加入裂解缓冲液100μl，冰上放置20min；
裂解液配方：50mmol/L Tris-Cl(pH8.0)：0.07882g；
150mmol/L氯化钠：0.08775g；
0.2g/L叠氮钠：0.002g；
1g/L SDS：0.01g；
100mg/L Aprotin；0.001g；
10g/L NP-40：0.1g；
5g/L去氧胆酸钠：0.05g；
100mg/L苯甲基磺酰氟(PMSF)0.001g；
3)收集裂解液；
4)离心，12000rpm，2～10min；
5)吸取上清，蛋白定量，分装，于-80℃保存备用；
2.蛋白质的聚丙烯酰胺凝胶电泳
按所需分离的蛋白质分子大小选择30％的丙烯酰胺百分比浓度。在微量离心管中，用2×SDS加样缓冲液按1:1(v/v)稀释待测蛋白质样品，于100℃煮沸5-10min。用带平嘴针头的50μl注射器将同样浓度的蛋白质样品等体积加入到样品孔中。连接电源，将电压调至120V继续电泳2h，至溴酚蓝到达凝胶底部为止。
3.蛋白质从SDS聚丙烯酰胺凝胶转移至硝酸纤维素滤膜
准备转移缓冲液。切割与胶尺寸相符的硝酸纤维素薄膜，并用转移缓冲液浸湿，放置15min直到没有气泡。切割8张普通滤纸，其大小与胶尺寸大小相符，并将其浸泡在有转移缓冲液的培养皿中(与硝酸纤维素薄膜分开浸泡)。电泳后，切取有用部分的胶，并很快地在转移缓冲液中洗涤。打开蛋白质转移槽的盖板，依次放入：
①4张用转移缓冲液浸泡过的滤纸；
②用转移缓冲液洗过的胶，并小心地赶走滤纸和胶之间的所有气泡；
③放上硝酸纤维素膜；
④另4张用转移缓冲液浸泡过的滤纸；
⑤小心地合上转移槽的盖板；
⑥插入电极，注意正负极方向(硝酸纤维素膜面向阳极)，打开电泳仪开关，300mA，2h。
转移结束后打开盖板取出硝酸纤维素薄膜，用铅笔标出作为分子量标准的参照蛋白的位置。
4.免疫印迹膜的处理
用TBST缓冲液洗膜5～10min。将膜用8％的脱脂牛奶作为封闭溶液封闭，用摇床轻摇(37℃)60min。用TBST缓冲液洗膜3次，每次10min。
加入一抗：
将膜置于第一抗体溶液(PRRSV阳性血清，1:200)中，置摇床上轻摇，37℃摇动1h。去掉第一抗体溶液，并用PBS洗膜3次，每次10min。
加入二抗：
将膜置于二抗辣根过氧化酶兔抗猪IgG溶液(1:200)中，37℃轻摇40min。去掉第二抗体溶液，并用TBST洗膜3次，每次10min。最后用TBST溶液洗。
5.ECL化学发光底物检测
ECL溶液中反应5min，用去离子水洗涤，以终止反应。用放射自显影法在X-光片上留下清晰的图像。
结果如图2所示，图2为本发明的免疫印迹检测PRRSV-M蛋白表达水平的结果示意图，其中对照为未导入siRNA处理的细胞样品，其它处理与样品均一致。由图可知，M-229、M-379与M-167样品的M蛋白表达量均少于对照组M蛋白的表达量，表明这三组siRNA均对PRRSV-M蛋白表达造成了抑制效果。
实施例4：将siRNA导入载体
I.M-229siRNA设计
选用干扰效果明显的M-229，在原siRNA序列(转换为DNA序列)的基础上进行设计，在其第一链5’端加入BglII酶切位点，第二链5’端加入HindIII酶切位点，得到两段互补的寡核苷酸片段，其序列为：
5’-GATCCCCGCAGTAGTTGCACTCCTTTTTCAAGAGAAAAGGAGTGCAACTACTGCTTTTTA-3’
3’-GGGCGTCATCAACGTGAGGAAAAAGTTCTCTTTTCCTCACGTTGATGACGAAAAATTCGA-5’
两段寡核苷酸经过退火，会形成互补的双链，然后经过酶切，再连接到pBabe-Super载体中。
上述寡核苷酸片段均委托Invitrogen公司合成。
II.siRNA载体的构建
将pBabe-Puro载体上的H1-RNA启动子克隆入无启动子的穿梭载体pSuper载体(Oligoengine公司)中，得到新的穿梭载体命名为pBabe-Super。
上述合成的寡核苷酸序列退火并连接到pBabe-Super的Bgl II和HindIII位点，得到重组质粒pBabe-M-229，经EcoRI酶切鉴定、测序鉴定正确。
实施例5：重组质粒干扰效果的检测
I.用重组质粒转染Marc145细胞，并感染PRRSV，具体方法参见实施例2。
II.实时定量PCR分析，具体方法参见实施例3。
结果如图3所示。图3为实时定量PCR分析本发明的重组质粒对PRRSV-M蛋白的mRNA水平影响结果示意图，其中阴性对照为未导入siRNA处理的细胞样品，其它处理与样品均一致。结果表明，重组质粒pBabe-M-229的mRNA水平为对照样本的70％。
III.免疫印迹检验，具体方法参见实施例3。
结果如图4所示。图4为免疫印迹检测本发明的重组质粒对PRRSV-M蛋白表达水平的影响结果示意图，其中阴性对照为未导入siRNA处理的细胞样品，其它处理与样品均一致。由图可知，重组质粒pBabe-M-229的M蛋白表达量少于对照组M蛋白的表达量，表明该pBabe-M-229对PRRSV的M基因转录蛋白表达具有抑制效果。
序列表
<110>中国科学院动物研究所
<120>用于治疗和/或预防猪繁殖与呼吸综合征的siRNA片段及其应用
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Claims (9)
1.一种用于治疗和/或预防猪繁殖与呼吸综合征的siRNA片段，其序列为下列序列：
5’-GCAGUAGUUGCACUCCUUU-3’
3’-CGUCAUCAACGUGAGGAAA-5’。
2.根据权利要求1所述的siRNA片段，其特征在于，所述siRNA片段的3’端还添加的序列为2-6个dT或2-6个U的修饰序列。
3.一种用于治疗和/或预防猪繁殖与呼吸综合征的载体，其中包含序列为下列序列的siRNA片段：
5’-GCAGUAGUUGCACUCCUUU-3’
3’-CGUCAUCAACGUGAGGAAA-5’。
4.根据权利要求3所述的载体，其特征在于，所述siRNA片段的3’端还添加的序列为2-6个dT或2-6个U修饰序列。
5.根据权利要求3或4所述的载体，其特征在于，所述载体为逆转录病毒载体、腺病毒载体、腺相关病毒载体及质粒载体。
6.根据权利要求5所述的载体，其特征在于，所述逆转录病毒载体为慢病毒载体。
7.根据权利要求5所述的载体，其特征在于，所述载体为pSuper、pBabe-Super、pRNA-U6.1/Neo或pSilencer载体。
8.根据权利要求1或2所述的siRNA片段在制备抑制猪繁殖与呼吸综合征病毒M蛋白表达的药物中的应用。
9.根据权利要求3至7中任一项所述的载体在制备抑制猪繁殖与呼吸综合征病毒M蛋白表达的药物中的应用。
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| CN102115731B (zh) * | 2010-12-01 | 2012-10-17 | 北京济福霖生物技术有限公司 | 制备抗蓝耳病转基因猪的方法 |
| CN102676519B (zh) * | 2012-05-07 | 2013-11-27 | 北京诺派生物科技有限公司 | 一种抑制猪繁殖与呼吸综合征病毒增殖的microRNA及其应用 |
| CN102698289A (zh) * | 2012-05-24 | 2012-10-03 | 南京农业大学 | Pabp抑制剂在制备抗猪繁殖与呼吸综合征病毒药物中的应用 |
| CN103667297B (zh) * | 2012-06-04 | 2015-09-23 | 中国农业科学院兰州兽医研究所 | 一种用于抑制猪繁殖与呼吸综合征病毒复制的1010shRNA及其制备方法 |
| CN102796181A (zh) * | 2012-06-20 | 2012-11-28 | 韩健宝 | 猪繁殖与呼吸综合征病毒的肽核酸及其应用 |
| CN107828788B (zh) * | 2017-11-02 | 2020-03-27 | 中国农业科学院兰州兽医研究所 | 一种PRRSV感染相关的lncRNA及其siRNA在抑制病毒复制中的应用 |
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| WO2000053787A1 (en) * | 1999-03-08 | 2000-09-14 | Id-Lelystad, Instituut Voor Dierhouderij En Dierg Ezondheid B.V. | Prrsv vaccines |
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| WO2000053787A1 (en) * | 1999-03-08 | 2000-09-14 | Id-Lelystad, Instituut Voor Dierhouderij En Dierg Ezondheid B.V. | Prrsv vaccines |
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