CN101659953A - 抗猪圆环病毒病shRNA及其设计合成法和用途 - Google Patents
抗猪圆环病毒病shRNA及其设计合成法和用途 Download PDFInfo
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Abstract
本发明公开了一种设计合成抗猪圆环病毒shRNA的方法,包括以下步骤:1)根据PCV-2型病毒的基因组序列,以Rep基因或Cap基因为目的基因,选取一段19bp长的核酸序列;2)选取同时满足以下3个条件的核酸序列为候选核酸序列:该核酸序列的GC含量为40%-60%,在15-19的位点中至少有3个A或T核酸残基,检查该19bp核酸序列,保证其不会形成二级结构;3)将所得的候选核酸序列与猪圆环病毒基因组进行比对;以确定该候选核酸序列是特异性的只针对目的基因。本发明的抗猪圆环病毒shRNA,通过抑制猪圆环病病毒在细胞内的复制,从而发挥抗病毒作用。
Description
技术领域
本发明涉及利用RNA干扰技术进行抗猪圆环病毒病的关键技术和方法。
背景技术
RNA干扰(RNA interference,RNAi)是指在进化过程中高度保守的、由双链RNA(double-stranded RNA,dsRNA)诱发的、同源mRNA高效特异性降解的现象。由于使用RNAi技术可以特异性剔除或关闭特定基因的表达,所以该技术已被广泛用于探索基因功能和传染性疾病及恶性肿瘤的基因治疗领域。
RNAi首先由Fire等发现于秀丽隐杆线虫(C.elegans)中,他们发现将dsRNA注入线虫体内后可抑制序列同源基因的表达,并证实这种抑制主要作用于转录之后,所以又称RNAi为转录后基因沉默(postt ranscriptional gene silencing,PTGS)。随后人们陆续在果蝇(Drosophila)、锥虫(Trypanosomes)、涡虫(Planaria)、斑马鱼(Zebrafish)、拟南芥(Arabidopsis thaliana)、大小鼠和人体内发现了RNAi现象,遗传学研究表明RNAi是真核生物中一种普遍存在且非常保守的机制,与真核细胞中许多重要生物学过程密切相通过对RNAi现象的遗传学与生物化学研究,其作用机制已日渐清晰(见图1)。
猪圆环病毒病是最近10年来才发现的一种新的猪病。研究表明,II型猪圆环病毒(PCV-2)不仅是断奶仔猪多系统衰竭综合症(PMWS)的病原,而且能够引起多种猪病。近年来,由PCV-2引起的疾病主要有PMWS、猪皮炎及肾病综合征、猪增生性坏死性肺炎等,另外,猪呼吸与繁殖障碍综合征、猪细小病毒、传染性先天性震颤等均与PCV-2感染有重要关联。
猪圆环病毒(PCV)属于圆环病毒科、圆环病毒属,为已知的最小的动物病毒之一。PCV以滚环方式复制,呈2O面体对称,无囊膜,直径17nm~22nm,含有1.76kb~2.3kb的单链环状DNA,相对分子质量58ku,沉降系数为52S。根据PCV的致病性、抗原性及核苷酸序列,将其分为PCV-1和PCV-2两个型,其中PCV-1无致病性,广泛存在于猪体内及猪源传代细胞系。PCV-2则具有致病性,在临床上主要引起断奶仔猪多系统衰竭综合征(PMWS)。目前尚无有效的疫苗可以用来预防PCV-2的感染。
发明内容
本发明要解决的技术问题是提供一种抗猪圆环病毒病shRNA及其设计合成法和用途。
为了解决上述技术问题,本发明提供一种设计合成抗猪圆环病毒shRNA的方法,包括以下步骤:
1)、根据PCV-2型病毒的基因组序列,以Rep基因或Cap基因为目的基因,选取一段19bp长的核酸序列;
2)、选取同时满足以下3个条件的核酸序列为候选核酸序列:
A、该核酸序列的GC含量为40%-60%;
B、在15-19的位点中至少有3个A或T核酸残基;
C、检查该19bp核酸序列,保证其不会形成二级结构;
3)、将所得的候选核酸序列与猪圆环病毒基因组进行比对;以确定该候选核酸序列是特异性的只针对目的基因,与其他基因没有太大的相似性。
本发明还同时提供了按照上述方法所得的抗猪圆环病毒shRNA,其为下述任意一种,其中的核苷酸序列为5′→3′,Rep为猪圆环病毒复制基因,CAP为猪圆环病毒结构基因:
pSIREN-Rep-1:
正向:
GATCCGTACGGGAGCTTCCAATCTCTTCAAGAGAGAGATTGGAAGCTCCCGTATTTTT
TACGCGTG (SEQ ID NO:1)
反向:
GCATGCCCTCGAAGGTTAGAGAAGTTCTCTCTCTAACCTTCGAGGGCATAAAAAATG
CGCACTTAA (SEQ ID NO:2)
pSIREN-Rep-2:
正向:
GATCCAGTGAGCGGGAAAATGCAGTTCAAGAGACTGCATTTTCCCGCTCACTTTTTT
TACGCGTG (SEQ ID NO:3)
反向:
GTCACTCGCCCTTTTACGTCAAGTTCTCTGACGTAAAAGGGCGAGTGAAAAAAATGC
GCACTTAA (SEQ ID NO:4)
pSIREN-Rep-3:
正向:
GATCCGTGGTACTCCTCAACTGCTGTTCAAGAGACAGCAGTTGAGGAGTACCATTTT
TTACGCGTG (SEQ ID NO:5)
反向:
GCACCATGAGGAGTTGACGACAAGTTCTCTGTCGTCAACTCCTCATGGTAAAAAATG
CGCACTTAA (SEQ ID NO:6)
pSIREN-Rep-4:
正向:
GATCCAATGCAGAAGCGTGATTGGTTCAAGAGACCAATCACGCTTCTGCATTTTTTTT
ACGCGTG (SEQ ID NO:7)
反向:
GTTACGTCTTCGCACTAACCAAGTTCTCTGGTTAGTGCGAAGACGTAAAAAAAATGC
GCACTTAA (SEQ ID NO:8)
pSIREN-Rep-5:
正向:
GATCCAAGCAAATGGGCTGCTAATTTCAAGAGAATTAGCAGCCCATTTGCTTTTTTTT
ACGCGTG (SEQ ID NO:9)
反向:
GTTCGTTTACCCGACGATTAAAGTTCTCTTAATCGTCGGGTAAACGAAAAAAAATGC
GCACTTAA (SEQ ID NO:10)
pSIREN-Rep-6:
正向:
GATCCACCACATACTGGAAACCACTTCAAGAGAGTGGTTTCCAGTATGTGGTTTTTTT
ACGCGTG (SEQ ID NO:11)
反向:
GTGGTGTATGACCTTTGGTGAAGTTCTCTCACCAAAGGTCATACACCAAAAAAATGC
GCACTTAA (SEQ ID NO:12)
pSIREN-Rep-7:
正向:
GATCCAAATGGCATCTTCAACACCTTCAAGAGAGGTGTTGAAGATGCCATTTTTTTTT
ACGCGTG (SEQ ID NO:13)
反向:
GTTTACCGTAGAAGTTGTGGAAGTTCTCTCCACAACTTCTACGGTAAAAAAAAATGC
GCACTTAA (SEQ ID NO:14)
pSIREN-Rep-8:
正向:
GATCCGGTTGAATTCTGGCCCTGCTTCAAGAGAGCAGGGCCAGAATTCAACCTTTTT
TACGCGTG (SEQ ID NO:15)
反向:
GCCAACTTAAGACCGGGACGAAGTTCTCTCGTCCCGGTCTTAAGTTGGAAAAAATGC
GCACTTAA (SEQ ID NO:16)
pSIREN-Rep-9:
正向:
GATCCACTACTCCTCCCGCCATACTTCAAGAGAGTATGGCGGGAGGAGTAGTTTTTTT
ACGCGTG (SEQ ID NO:17)
反向:
GTGATGAGGAGGGCGGTATGAAGTTCTCTCATACCGCCCTCCTCATCAAAAAAATGC
GCACTTAA (SEQ ID NO:18)
pSIREN-Rep-10:
正向:
GATCCGCCAGCAAGAAGAATGGAAGTTCAAGAGACTTCCATTCTTCTTGCTGGTTTT
TTG (SEQ ID NO:19)
反向:
GCGGTCGTTCTTCTTACCTTCAAGTTCTCTGAAGGTAAGAAGAACGACCAAAAAACT
TAA (SEQ ID NO:20)
pSIREN-Rep-11:
正向:
GATCCGTCGAGAAAGCGAAAGGAACTTCAAGAGAGTTCCTTTCGCTTTCTCGATTTT
TTG (SEQ ID NO:21)
反向:
GCAGCTCTTTCGCTTTCCTTGAAGTTCTCTCAAGGAAAGCGAAAGAGCTAAAAAACT
TAA (SEQ IDNO:22)
pSIREN-Rep-12:
正向:
GATCCGTGCAGAAGCGTGATTGGAATTCAAGAGATTCCAATCACGCTTCTGCATTTTT
TG (SEQ ID NO:23)
反向:
GCACGTCTTCGCACTAACCTTAAGTTCTCTAAGGTTAGTGCGAAGACGTAAAAAACT
TAA (SEQ ID NO:24)
pSIREN-Rep-13:
正向:
GATCCGCTGGGATGATCTACTGAGATTCAAGAGATCTCAGTAGATCATCCCAGTTTTT
TG (SEQ ID NO:25)
反向:
GCGACCCTACTAGATGACTCTAAGTTCTCTAGAGTCATCTAGTAGGGTCAAAAAACTT
AA (SEQ ID NO:26)
pSIREN-Rep-14:
正向:
GATCCGCCCGCAGTATTCTGATTACTTCAAGAGAGTAATCAGAATACTGCGGGTTTTT
TG (SEQ ID NO:27)
反向:
GCGGGCGTCATAAGACTAATGAAGTTCTCTCATTAGTCTTATGACGCCCAAAAAACTT
AA (SEQ ID NO:28)
pSIREN-Rep-15:
正向:
GATCCGACCAATGTACACGTCATTTTCAAGAGAAATGACGTGTACATTGGTCTTTTTT
G (SEQ ID NO:29)
反向:
GCTGGTTACATGTGCAGTAAAAGTTCTCTTTACTGCACATGTAACCAGAAAAAACTT
AA (SEQ ID NO:30)
pSIREN-Rep-16:
正向:
GATCCGCGTGATTGGAAGACCAATTTCAAGAGAATTGGTCTTCCAATCACGCTTTTTT
G (SEQ ID NO:31)
反向:
GCGCACTAACCTTCTGGTTAAAGTTCTCTTAACCAGAAGGTTAGTGCGAAAAAACTT
AA (SEQ ID NO:32)
pSIREN-Rep-17:
正向:
GATCCAACAGATCAGCAGAATAAATTCAAGAGATTTATTCTGCTGATCTGTTTTTTTTG
(SEQ ID NO:33)
反向:
GTTGTCTAGTCGTCTTATTTAAGTTCTCTAAATAAGACGACTAGACAAAAAAAACTTA
A (SEQ ID NO:34)
pSIREN-CAP-1:
正向:
GATCCGGTGGACATGATGAGATTCTTCAAGAGAGAATCTCATCATGTCCACCTTTTTT
G (SEQ ID NO:35)
反向:
GCCACCTGTACTACTCTAAGAAGTTCTCTCTTAGAGTAGTACAGGTGGAAAAAACTT
AA (SEQ ID NO:36)
pSIREN-CAP-2:
正向:
GATCCGTCGGCATTGCGTTCGAAAATTCAAGAGATTTTCGAACGCAATGCCGATTTTT
TG (SEQ ID NO:37)
反向:
GCAGCCGTAACGCAAGCTTTTAAGTTCTCTAAAAGCTTGCGTTACGGCTAAAAAACT
TAA (SEQ ID NO:38)
pSIREN-CAP-3:
正向:
GATCCGCGGATATACTATCAAGCGATTCAAGAGATCGCTTGATAGTATATCCGTTTTTT
G (SEQ ID NO:39)
反向:
GCGCCTATATGATAGTTCGCTAAGTTCTCTAGCGAACTATCATATAGGCAAAAAACTTA
A (SEQ ID NO:40)
pSIREN-CAP-4:
正向:
GATCCAAGCGAACCACAGTCAAAATTCAAGAGATTTTGACTGTGGTTCGCTTTTTTTT
G (SEQ ID NO:41)
反向:
GTTCGCTTGGTGTCAGTTTTAAGTTCTCTAAAACTGACACCAAGCGAAAAAAAACTT
AA (SEQ ID NO:42)
pSIREN-CAP-5:
正向:
GATCCGCGACCAGGAATACAATATCTTCAAGAGAGATATTGTATTCCTGGTCGTTTTTT
G (SEQ ID NO:43)
反向:
GCGCTGGTCCTTATGTTATAGAAGTTCTCTCTATAACATAAGGACCAGCAAAAAACTT
AA (SEQ ID NO:44)
pSIREN-CAP-6:
正向:
GATCCGCGTTACCGGAGAAGAAGATTCAAGAGATCTTCTTCTCCGGTAACGCTTTTTT
G (SEQ ID NO:45)
反向:
GCGCAATGGCCTCTTCTTCTAAGTTCTCTAGAAGAAGAGGCCATTGCGAAAAAACTT
AA (SEQ ID NO:46)
pSIREN-CAP-7:
正向:
GATCCAGGTTAAGGTTGAATTCTGTTCAAGAGACAGAATTCAACCTTAACCTTTTTTT
G (SEQ ID NO:47)
反向:
GTCCAATTCCAACTTAAGACAAGTTCTCTGTCTTAAGTTGGAATTGGAAAAAAACTT
AA (SEQ ID NO:48)
pSIREN-CAP-8:
正向:
GATCCGCAGTATATACGACCAGGAATTCAAGAGATTCCTGGTCGTATATACTGTTTTTT
G (SEQ ID NO:49)
反向:
GCGTCATATATGCTGGTCCTTAAGTTCTCTAAGGACCAGCATATATGACAAAAAACTT
AA (SEQ ID NO:50)。
本发明还提供上述抗猪圆环病毒shRNA的用途:将含有抗猪圆环病毒病shRNA的RNA干扰载体转到细胞内,通过抑制猪圆环病病毒在细胞内的复制,从而发挥抗病毒作用。
在本发明中,选择RNAi-Ready pSIREN-RetroQ-ZsGreen Vector作为RNA干扰载体,将上述设计好的shRNA序列经退火反应(95℃/30sec,72℃/2min,37℃/2min,25℃/2min)形成具有粘性末端的双链,可以直接连接到线性化的RNAi-Ready pSIREN-RetroQ-ZsGreen载体上。本发明的抗猪圆环病毒病的RNA干扰载体,该载体含有经检测具有很好抑制病毒复制的shRNA。
综上所述,本发明的优点是通过RNA干扰技术,将抗猪圆环病毒病的shRNA克隆到RNA干扰载体中,并转入细胞中,利用表达的shRNA来抑制猪圆环病毒的复制,从而起到预防作用。由于目前还没有有效的疫苗可以用来预防PCV-2,而本发明能通过RNA干扰这种新的途径来预防PCV-2,从而降低猪圆环病毒病对养猪业的危害。
附图说明
下面结合附图对本发明的具体实施方式作进一步详细说明。
图1是RNA干扰作用机制的示意图;
图2本发明的shRNA序列的设计示意图;
图3是转染24小时后荧光检测效果图。
具体实施方式
实施例1、抗猪圆环病毒病shRNA的选取
1.根据PCV-2型病毒的基因组序列,以Rep基因和Cap基因为目的基因,选取一段19bp长的核酸序列。该序列不要在5’和3’非翻译区,以及起始密码子后75bp,因为这些区域富含调控蛋白,可能会影响RNA干扰效果。
2.计算该19bp核酸序列的GC含量,GC含量必须在40%-60%之间。GC含量大约在45%时最佳。
3.在15-19的位点中至少有3个A或T核酸残基,可以增强RNA干扰活性。
4.检查该19bp核酸序列,保证其不会形成二级结构。
5.将同时满足上述步骤2、步骤3和步骤4这3个条件的19bp核酸序列作为候选核酸序列,与相应的猪圆环病毒病基因组进行比对。以确定该候选核酸序列是特异性的只针对猪圆环病毒病目的基因,与其他基因没有太大的相似性。
实施例2、抗猪圆环病毒病shRNA的设计和合成
根据我们所用的RNA干扰载体RNAi-Ready pSIREN-RetroQ-ZsGreen Vector,将设计好的shRNA序列经退火反应(95℃/30sec,72℃/2min,37℃/2min,25℃/2min)形成具有粘性末端的双链,可以直接连接到线性化的RNAi-Ready pSIREN-RetroQ-ZsGreen载体(公知载体)上。
实施例3、设计和合成的抗猪圆环病毒病shRNA
PSIREN-REP-1:
5’-GATCCGTACGGGAGCTTCCAATCTCTTCAAGAGAGAGATTGGAAGCTCCCGTATTTTTTACGCGTG-3’
3’-GCATGCCCTCGAAGGTTAGAGAAGTTCTCTCTCTAACCTTCGAGGGCATAAAAAATGCGCACTTAA-5’
PSIREN-REP-2:
5’-GATCCAGTGAGCGGGAAAATGCAGTTCAAGAGACTGCATTTTCCCGCTCACTTTTTTTACGCGTG-3’
3’-GTCACTCGCCCTTTTACGTCAAGTTCTCTGACGTAAAAGGGCGAGTGAAAAAAATGCGCACTTAA-5’
PSIREN-REP-3:
5’-GATCCGTGGTACTCCTCAACTGCTGTTCAAGAGACAGCAGTTGAGGAGTACCATTTTTTACGCGTG-3’
3’-GCACCATGAGGAGTTGACGACAAGTTCTCTGTCGTCAACTCCTCATGGTAAAAAATGCGCACTTAA-5’
PSIREN-REP-4:
5’-GATCCAATGCAGAAGCGTGATTGGTTCAAGAGACCAATCACGCTTCTGCATTTTTTTTACGCGTG-3’
3’-GTTACGTCTTCGCACTAACCAAGTTCTCTGGTTAGTGCGAAGACGTAAAAAAAATGCGCACTTAA-5’
PSIREN-REP-5:
5’-GATCCAAGCAAATGGGCTGCTAATTTCAAGAGAATTAGCAGCCCATTTGCTTTTTTTTACGCGTG-3’
3’-GTTCGTTTACCCGACGATTAAAGTTCTCTTAATCGTCGGGTAAACGAAAAAAAATGCGCACTTAA-5’
PSIREN-REP-6:
5’-GATCCACCACATACTGGAAACCACTTCAAGAGAGTGGTTTCCAGTATGTGGTTTTTTTACGCGTG-3’
3’-GTGGTGTATGACCTTTGGTGAAGTTCTCTCACCAAAGGTCATACACCAAAAAAATGCGCACTTAA-5’
PSIREN-REP-7:
5’-GATCCAAATGGCATCTTCAACACCTTCAAGAGAGGTGTTGAAGATGCCATTTTTTTTTACGCGTG-3’
3’-GTTTACCGTAGAAGTTGTGGAAGTTCTCTCCACAACTTCTACGGTAAAAAAAAATGCGCACTTAA-5’
PSIREN-REP-8:
5’-GATCCGGTTGAATTCTGGCCCTGCTTCAAGAGAGCAGGGCCAGAATTCAACCTTTTTTACGCGTG-3’
3’-GCCAACTTAAGACCGGGACGAAGTTCTCTCGTCCCGGTCTTAAGTTGGAAAAAATGCGCACTTAA-5’
PSIREN-REP-9:
5’-GATCCACTACTCCTCCCGCCATACTTCAAGAGAGTATGGCGGGAGGAGTAGTTTTTTTACGCGTG-3’
3’-GTGATGAGGAGGGCGGTATGAAGTTCTCTCATACCGCCCTCCTCATCAAAAAAATGCGCACTTAA-5’
PSIREN-REP-10
5′-GATCCGCCAGCAAGAAGAATGGAAGTTCAAGAGACTTCCATTCTTCTTGCTGGTTTTTTG-3′
3′-GCGGTCGTTCTTCTTACCTTCAAGTTCTCTGAAGGTAAGAAGAACGACCAAAAAACTTAA-5′
PSIREN-REP-11
5′-GATCCGTCGAGAAAGCGAAAGGAACTTCAAGAGAGTTCCTTTCGCTTTCTCGATTTTTTG-3′
3′-GCAGCTCTTTCGCTTTCCTTGAAGTTCTCTCAAGGAAAGCGAAAGAGCTAAAAAACTTAA-5′
PSIREN-REP-12
5′-GATCCGTGCAGAAGCGTGATTGGAATTCAAGAGATTCCAATCACGCTTCTGCATTTTTTG-3′
3′-GCACGTCTTCGCACTAACCTTAAGTTCTCTAAGGTTAGTGCGAAGACGTAAAAAACTTAA-5′
PSIREN-REP-13
5′-GATCCGCTGGGATGATCTACTGAGATTCAAGAGATCTCAGTAGATCATCCCAGTTTTTTG-3′
3′-GCGACCCTACTAGATGACTCTAAGTTCTCTAGAGTCATCTAGTAGGGTCAAAAAACTTAA-5′
PSIREN-REP-14
5′-GATCCGCCCGCAGTATTCTGATTACTTCAAGAGAGTAATCAGAATACTGCGGGTTTTTTG-3′
3′-GCGGGCGTCATAAGACTAATGAAGTTCTCTCATTAGTCTTATGACGCCCAAAAAACTTAA-5′
PSIREN-REP-15
5′-GATCCGACCAATGTACACGTCATTTTCAAGAGAAATGACGTGTACATTGGTCTTTTTTG-3′
3′-GCTGGTTACATGTGCAGTAAAAGTTCTCTTTACTGCACATGTAACCAGAAAAAACTTAA-5′
PSIREN-REP-16
5′-GATCCGCGTGATTGGAAGACCAATTTCAAGAGAATTGGTCTTCCAATCACGCTTTTTTG-3′
3′-GCGCACTAACCTTCTGGTTAAAGTTCTCTTAACCAGAAGGTTAGTGCGAAAAAACTTAA-5′
PSIREN-REP-17
5′-GATCCAACAGATCAGCAGAATAAATTCAAGAGATTTATTCTGCTGATCTGTTTTTTTTG-3′
3′-GTTGTCTAGTCGTCTTATTTAAGTTCTCTAAATAAGACGACTAGACAAAAAAAACTTAA-5′
PSIREN-CAP-1
5′-GATCCGGTGGACATGATGAGATTCTTCAAGAGAGAATCTCATCATGTCCACCTTTTTTG-3′
3′-GCCACCTGTACTACTCTAAGAAGTTCTCTCTTAGAGTAGTACAGGTGGAAAAAACTTAA-5′
PSIREN-CAP-2
5′-GATCCGTCGGCATTGCGTTCGAAAATTCAAGAGATTTTCGAACGCAATGCCGATTTTTTG-3′
3′-GCAGCCGTAACGCAAGCTTTTAAGTTCTCTAAAAGCTTGCGTTACGGCTAAAAAACTTAA-5′
PSIREN-CAP-3
5′-GATCCGCGGATATACTATCAAGCGATTCAAGAGATCGCTTGATAGTATATCCGTTTTTTG-3′
3′-GCGCCTATATGATAGTTCGCTAAGTTCTCTAGCGAACTATCATATAGGCAAAAAACTTAA-5′
PSIREN-CAP-4
5′-GATCCAAGCGAACCACAGTCAAAATTCAAGAGATTTTGACTGTGGTTCGCTTTTTTTTG-3′
3′-GTTCGCTTGGTGTCAGTTTTAAGTTCTCTAAAACTGACACCAAGCGAAAAAAAACTTAA-5′
PSIREN-CAP-5
5′-GATCCGCGACCAGGAATACAATATCTTCAAGAGAGATATTGTATTCCTGGTCGTTTTTTG-3′
3′-GCGCTGGTCCTTATGTTATAGAAGTTCTCTCTATAACATAAGGACCAGCAAAAAACTTAA-5′
PSIREN-CAP-6
5′-GATCCGCGTTACCGGAGAAGAAGATTCAAGAGATCTTCTTCTCCGGTAACGCTTTTTTG-3′
3′-GCGCAATGGCCTCTTCTTCTAAGTTCTCTAGAAGAAGAGGCCATTGCGAAAAAAC TTAA-5′
PSIREN-CAP-7
5′-GATCCAGGTTAAGGTTGAATTCTGTTCAAGAGACAGAATTCAACCTTAACCTTTTTTTG-3′
3′-GTCCAATTCCAACTTAAGACAAGTTCTCTGTCTTAAGTTGGAATTGGAAAAAAACTTAA-5′
PSIREN-CAP-8
5′-GATCCGCAGTATATACGACCAGGAATTCAAGAGATTCCTGGTCGTATATACTGTTTTTTG-3′
3′-GCGTCATATATGCTGGTCCTTAAGTTCTCTAAGGACCAGCATATATGACAAAAAACTTAA-5′。
实施例4、抗猪圆环病毒病shRNA的鉴定
将连接上如实施例3所示的shRNA的RNAi-Ready pSIREN-RetroQ-ZsGreen载体转化大肠杆菌。涂板后,挑取单克隆过夜培养,并将培养的菌液提取质粒。由于在shRNA的设计时在终止位点后加入了一个特异的酶切位点,因此这个酶切位点可以用来鉴定shRNA是否已经成功连接到载体上。
由于突变或合成等可能的原因,应该将样品测序,以保证shRNA序列百分之百的正确。
实施例5、抗猪圆环病毒病shRNA活性的检测
将上述实施例4提取的质粒DNA,分别在转染试剂介导下导入猪PK15细胞。将质粒DNA与转染试剂按1∶1混合,铺在细胞上。3小时后给细胞换上新鲜的完全培养基继续培养24小时。由于RNAi-Ready pSIREN-RetroQ-ZsGreen载体可以表达绿色荧光蛋白,24小时后进行荧光观察,确定转染效率(如图3所示)。
转染24小时后对细胞接种PCV-2病毒,进行攻毒实验。接毒48-72小时后将上清悬液和细胞分开收集,通过免疫荧光的方法分别测上清和细胞中病毒的TCID50。分析所设计的shRNA对PCV-2病毒的抑制作用。
表1是根据猪圆环病毒病Rep和Cap基因设计的shRNA对病毒复制和增殖的抑制作用结果。
表1
序列名 | 相对病毒拷贝数 |
pSIREN-Rep1 | 10-0.92 |
pSIREN-Rep2 | 10-0.76 |
pSIREN-Rep3 | 10-1.59 |
pSIREN-Rep4 | 10-0.93 |
pSIREN-Rep5 | 10-1.38 |
pSIREN-Rep6 | 10-0.2 |
pSIREN-Rep7 | 10-1.2 |
pSIREN-Rep8 | 10-0.98 |
pSIREN-Rep9 | 10-0.8 |
pSIREN-Rep10 | 10-1.8 |
pSIREN-Rep11 | 10-1.57 |
pSIREN-Rep13 | 10-1.16 |
pSIREN-Cap1 | 100 |
pSIREN-Cap2 | 10-1.17 |
pSIREN-Cap3 | 10-1.8 |
pSIREN-Cap4 | 10-0.3 |
pSIREN-Cap5 | 10-2.57 |
pSIREN-Cap6 | 10-0.83 |
pSIREN-Cap7 | 100 |
pSIREN-Cap8 | 10-0.23 |
根据表1我们可以得知:大部分设计的shRNA对PCV-2病毒的复制增殖具有抑制作用,其中pSIREN-Rep10、pSIREN-Cap3和pSIREN-Cap5的抑制率分别达到66.6、66.6和500倍,显示出较好的保护作用。
注:抑制率是指相对于对照的病毒拷贝数比率。即对照病毒拷贝数为1,pSIREN-Rep10、pSIREN-Cap3和pSIREN-Cap5的相对病毒拷贝数分别为10-1.8=0.015、10-1.8=0.015和10-2.57=0.002,则其pSIREN-Rep10、pSIREN-Cap3的抑制率分别为1/0.015=66.6倍;pSIREN-Cap5为1/0.002=500倍。
最后,还需要注意的是,以上列举的仅是本发明的若干个具体实施例。显然,本发明不限于以上实施例,还可以有许多变形。本领域的普通技术人员能从本发明公开的内容直接导出或联想到的所有变形,均应认为是本发明的保护范围。
序列表
SEQ ID NO:1
gatccgtacgggagcttccaatctcttcaagagagagattggaagctcccgtattttttacgcgtg 66
SEQ ID NO:2
gcatgccctcgaaggttagagaagttctctctctaaccttcgagggcataaaaaatgcgcacttaa 66
SEQ ID NO:3
gatccagtgagcgggaaaatgcagttcaagagactgcattttcccgctcactttttttacgcgtg 65
SEQ ID NO:4
gtcactcgcccttttacgtcaagttctctgacgtaaaagggcgagtgaaaaaaatgcgcacttaa 65
SEQ ID NO:5
gatccgtggtactcctcaactgctgttcaagagacagcagttgaggagtaccattttttacgcgtg 66
SEQ ID NO:6
gcaccatgaggagttgacgacaagttctctgtcgtcaactcctcatggtaaaaaatgcgcacttaa 66
SEQ ID NO:7
gatccaatgcagaagcgtgattggttcaagagaccaatcacgcttctgcattttttttacgcgtg 65
SEQ ID NO:8
gttacgtcttcgcactaaccaagttctctggttagtgcgaagacgtaaaaaaaatgcgcacttaa 65
SEQ ID NO:9
gatccaagcaaatgggctgctaatttcaagagaattagcagcccatttgcttttttttacgcgtg 65
SEQ ID NO:10
gttcgtttacccgacgattaaagttctcttaatcgtcgggtaaacgaaaaaaaatgcgcacttaa 65
SEQ ID NO:11
gatccaccacatactggaaaccacttcaagagagtggtttccagtatgtggtttttttacgcgtg 65
SEQ ID NO:12
gtggtgtatgacctttggtgaagttctctcaccaaaggtcatacaccaaaaaaatgcgcacttaa 65
SEQ ID NO:13
gatccaaatggcatcttcaacaccttcaagagaggtgttgaagatgccatttttttttacgcgtg 65
SEQ ID NO:14
gtttaccgtagaagttgtggaagttctctccacaacttctacggtaaaaaaaaatgcgcacttaa 65
SEQ ID NO:15
gatccggttgaattctggccctgcttcaagagagcagggccagaattcaaccttttttacgcgtg 65
SEQ ID NO:16
gccaacttaagaccgggacgaagttctctcgtcccggtcttaagttggaaaaaatgcgcacttaa 65
SEQ ID NO:17
gatccactactcctcccgccatacttcaagagagtatggcgggaggagtagtttttttacgcgtg 65
SEQ ID NO:18
gtgatgaggagggcggtatgaagttctctcataccgccctcctcatcaaaaaaatgcgcacttaa 65
SEQ ID NO:19
gatccgccagcaagaagaatggaagttcaagagacttccattcttcttgctggttttttg 60
SEQ ID NO:20
gcggtcgttcttcttaccttcaagttctctgaaggtaagaagaacgaccaaaaaacttaa 60
SEQ ID NO:21
gatccgtcgagaaagcgaaaggaacttcaagagagttcctttcgctttctcgattttttg 60
SEQ ID NO:22
gcagctctttcgctttccttgaagttctctcaaggaaagcgaaagagctaaaaaacttaa 60
SEQ ID NO:23
gatccgtgcagaagcgtgattggaattcaagagattccaatcacgcttctgcattttttg 60
SEQ ID NO:24
gcacgtcttcgcactaaccttaagttctctaaggttagtgcgaagacgtaaaaaacttaa 60
SEQ ID NO:25
gatccgctgggatgatctactgagattcaagagatctcagtagatcatcccagttttttg 60
SEQ ID NO:26
gcgaccctactagatgactctaagttctctagagtcatctagtagggtcaaaaaacttaa 60
SEQ ID NO:27
gatccgcccgcagtattctgattacttcaagagagtaatcagaatactgcgggttttttg 60
SEQ ID NO:28
gcgggcgtcataagactaatgaagttctctcattagtcttatgacgcccaaaaaacttaa 60
SEQ ID NO:29
gatccgaccaatgtacacgtcattttcaagagaaatgacgtgtacattggtcttttttg 59
SEQ ID NO:30
gctggttacatgtgcagtaaaagttctctttactgcacatgtaaccagaaaaaacttaa 59
SEQ ID NO:31
gatccgcgtgattggaagaccaatttcaagagaattggtcttccaatcacgcttttttg 59
SEQ ID NO:32
gcgcactaaccttctggttaaagttctcttaaccagaaggttagtgcgaaaaaacttaa 59
SEQ ID NO:33
gatccaacagatcagcagaataaattcaagagatttattctgctgatctgttttttttg 59
SEQ ID NO:34
gttgtctagtcgtcttatttaagttctctaaataagacgactagacaaaaaaaacttaa 59
SEQ ID NO:35
gatccggtggacatgatgagattcttcaagagagaatctcatcatgtccaccttttttg 59
SEQ ID NO:36
gccacctgtactactctaagaagttctctcttagagtagtacaggtggaaaaaacttaa 59
SEQ ID NO:37
gatccgtcggcattgcgttcgaaaattcaagagattttcgaacgcaatgccgattttttg 60
SEQ ID NO:38
gcagccgtaacgcaagcttttaagttctctaaaagcttgcgttacggctaaaaaacttaa 60
SEQ ID NO:39
gatccgcggatatactatcaagcgattcaagagatcgcttgatagtatatccgttttttg 60
SEQ ID NO:40
gcgcctatatgatagttcgctaagttctctagcgaactatcatataggcaaaaaacttaa 60
SEQ ID NO:41
gatccaagcgaaccacagtcaaaattcaagagattttgactgtggttcgcttttttttg 59
SEQ ID NO:42
gttcgcttggtgtcagttttaagttctctaaaactgacaccaagcgaaaaaaaacttaa 59
SEQ ID NO:43
gatccgcgaccaggaatacaatatcttcaagagagatattgtattcctggtcgttttttg 60
SEQ ID NO:44
gcgctggtccttatgttatagaagttctctctataacataaggaccagcaaaaaacttaa 60
SEQ ID NO:45
gatccgcgttaccggagaagaagattcaagagatcttcttctccggtaacgcttttttg 59
SEQ ID NO:46
gcgcaatggcctcttcttctaagttctctagaagaagaggccattgcgaaaaaacttaa 59
SEQ ID NO:47
gatccaggttaaggttgaattctgttcaagagacagaattcaaccttaacctttttttg 59
SEQ ID NO:48
gtccaattccaacttaagacaagttctctgtcttaagttggaattggaaaaaaacttaa 59
SEQ ID NO:49
gatccgcagtatatacgaccaggaattcaagagattcctggtcgtatatactgttttttg 60
SEQ ID NO:50
gcgtcatatatgctggtccttaagttctctaaggaccagcatatatgacaaaaaacttaa 60
Claims (3)
1、一种设计合成抗猪圆环病毒shRNA的方法,其特征是包括以下步骤:
1)、根据PCV-2型病毒的基因组序列,以Rep基因或Cap基因为目的基因,选取一段19bp长的核酸序列;
2)、选取同时满足以下3个条件的核酸序列为候选核酸序列:
A、该核酸序列的GC含量为40%-60%;
B、在15-19的位点中至少有3个A或T核酸残基;
C、检查该19bp核酸序列,保证其不会形成二级结构;
3)、将所得的候选核酸序列与猪圆环病毒基因组进行比对;以确定该候选核酸序列是特异性的只针对目的基因。
2、如权利要求1所述方法获得的抗猪圆环病毒shRNA,其特征是为下述任意一种,其中的核苷酸序列为5′→3′,:
pSIREN-Rep-1:
正向:
GATCCGTACGGGAGCTTCCAATCTCTTCAAGAGAGAGATTGGAAGCTCCCGTATTTTTTA
CGCGTG
反向:
GCATGCCCTCGAAGGTTAGAGAAGTTCTCTCTCTAACCTTCGAGGGCATAAAAAATGCG
CACTTAA
pSIREN-Rep-2:
正向:
GATCCAGTGAGCGGGAAAATGCAGTTCAAGAGACTGCATTTTCCCGCTCACTTTTTTTA
CGCGTG
反向:
GTCACTCGCCCTTTTACGTCAAGTTCTCTGACGTAAAAGGGCGAGTGAAAAAAATGCGC
ACTTAA
pSIREN-Rep-3:
正向:
GATCCGTGGTACTCCTCAACTGCTGTTCAAGAGACAGCAGTTGAGGAGTACCATTTTTT
ACGCGTG
反向:
GCACCATGAGGAGTTGACGACAAGTTCTCTGTCGTCAACTCCTCATGGTAAAAAATGCG
CACTTAA
pSIREN-Rep-4:
正向:
GATCCAATGCAGAAGCGTGATTGGTTCAAGAGACCAATCACGCTTCTGCATTTTTTTTAC
GCGTG
反向:
GTTACGTCTTCGCACTAACCAAGTTCTCTGGTTAGTGCGAAGACGTAAAAAAAATGCGC
ACTTAA
pSIREN-Rep-5:
正向:
GATCCAAGCAAATGGGCTGCTAATTTCAAGAGAATTAGCAGCCCATTTGCTTTTTTTTAC
GCGTG
反向:
GTTCGTTTACCCGACGATTAAAGTTCTCTTAATCGTCGG GTAAACGAAAAAAAATGCGC
ACTTAA
pSIREN-Rep-6:
正向:
GATCCACCACATACTGGAAACCACTTCAAGAGAGTGGTTTCCAGTATGTGGTTTTTTTAC
GCGTG
反向:
GTGGTGTATGACCTTTGGTGAAGTTCTCTCACCAAAGGTCATACACCAAAAAAATGCGC
ACTTAA
pSIREN-Rep-7:
正向:
GATCCAAATGGCATCTTCAACACCTTCAAGAGAGGTGTTGAAGATGCCATTTTTTTTTAC
GCGTG
反向:
GTTTACCGTAGAAGTTGTGGAAGTTCTCTCCACAACTTCTACGGTAAAAAAAAATGCGC
ACTTAA
pSIREN-Rep-8:
正向:
GATCCGGTTGAATTCTGGCCCTGCTTCAAGAGAGCAGGGCCAGAATTCAACCTTTTTTA
CGCGTG
反向:
GCCAACTTAAGACCGGGACGAAGTTCTCTCGTCCCGGTCTTAAGTTGGAAAAAATGCGC
ACTTAA
pSIREN-Rep-9:
正向:
GATCCACTACTCCTCCCGCCATACTTCAAGAGAGTATGGCGGGAGGAGTAGTTTTTTTAC
GCGTG
反向:
GTGATGAGGAGGGCGGTATGAAGTTCTCTCATACCGCCCTCCTCATCAAAAAAATGCGC
ACTTAA
pSIREN-Rep-10:
正向:
GATCCGCCAGCAAGAAGAATGGAAGTTCAAGAGACTTCCATTCTTCTTGCTGGTTTTTT
G
反向:
GCGGTCGTTCTTCTTACCTTCAAGTTCTCTGAAGGTAAGAAGAACGACCAAAAAACTTA
A
pSIREN-Rep-11:
正向:
GATCCGTCGAGAAAGCGAAAGGAACTTCAAGAGAGTTCCTTTCGCTTTCTCGATTTTTT
G
反向:
GCAGCTCTTTCGCTTTCCTTGAAGTTCTCTCAAGGAAAGCGAAAGAGCTAAAAAACTTA
A
pSIREN-Rep-12:
正向:
GATCCGTGCAGAAGCGTGATTGGAATTCAAGAGATTCCAATCACGCTTCTGCATTTTTTG
反向:
GCACGTCTTCGCACTAACCTTAAGTTCTCTAAGGTTAGTGCGAAGACGTAAAAAACTTA
A
pSIREN-Rep-13:
正向:
GATCCGCTGGGATGATCTACTGAGATTCAAGAGATCTCAGTAGATCATCCCAGTTTTTTG
反向:
GCGACCCTACTAGATGACTCTAAGTTCTCTAGAGTCATCTAGTAGGGTCAAAAAACTTAA
pSIREN-Rep-14:
正向:
GATCCGCCCGCAGTATTCTGATTACTTCAAGAGAGTAATCAGAATACTGCGGGTTTTTTG
反向:
GCGGGCGTCATAAGACTAATGAAGTTCTCTCATTAGTCTTATGACGCCCAAAAAACTTAA
pSIREN-Rep-15:
正向:
GATCCGACCAATGTACACGTCATTTTCAAGAGAAATGACGTGTACATTGGTCTTTTTTG
反向:
GCTGGTTACATGTGCAGTAAAAGTTCTCTTTACTGCACATGTAACCAGAAAAAACTTAA
pSIREN-Rep-16:
正向:
GATCCGCGTGATTGGAAGACCAATTTCAAGAGAATTGGTCTTCCAATCACGCTTTTTTG
反向:
GCGCACTAACCTTCTGGTTAAAGTTCTCTTAACCAGAAGGTTAGTGCGAAAAAACTTAA
pSIREN-Rep-17:
正向:
GATCCAACAGATCAGCAGAATAAATTCAAGAGATTTATTCTGCTGATCTGTTTTTTTTG
反向:
GTTGTCTAGTCGTCTTATTTAAGTTCTCTAAATAAGACGACTAGACAAAAAAAACTTAA
pSIREN-CAP-1:
正向:
GATCCGGTGGACATGATGAGATTCTTCAAGAGAGAATCTCATCATGTCCACCTTTTTTG
反向:
GCCACCTGTACTACTCTAAGAAGTTCTCTCTTAGAGTAGTACAGGTGGAAAAAACTTAA
pSIREN-CAP-2:
正向:
GATCCGTCGGCATTGCGTTCGAAAATTCAAGAGATTTTCGAACGCAATGCCGATTTTTTG
反向:
GCAGCCGTAACGCAAGCTTTTAAGTTCTCTAAAAGCTTGCGTTACGGCTAAAAAACTTA
A
pSIREN-CAP-3:
正向:
GATCCGCGGATATACTATCAAGCGATTCAAGAGATCGCTTGATAGTATATCCGTTTTTTG
反向:
GCGCCTATATGATAGTTCGCTAAGTTCTCTAGCGAACTATCATATAGGCAAAAAACTTAA
pSIREN-CAP-4:
正向:
GATCCAAGCGAACCACAGTCAAAATTCAAGAGATTTTGACTGTGGTTCGCTTTTTTTTG
反向:
GTTCGCTTGGTGTCAGTTTTAAGTTCTCTAAAACTGACACCAAGCGAAAAAAAACTTAA
pSIREN-CAP-5:
正向:
GATCCGCGACCAGGAATACAATATCTTCAAGAGAGATATTGTATTCCTGGTCGTTTTTTG
反向:
GCGCTGGTCCTTATGTTATAGAAGTTCTCTCTATAACATAAGGACCAGCAAAAAACTTAA
pSIREN-CAP-6:
正向:
GATCCGCGTTACCGGAGAAGAAGATTCAAGAGATCTTCTTCTCCGGTAACGCTTTTTTG
反向:
GCGCAATGGCCTCTTCTTCTAAGTTCTCTAGAAGAAGAGGCCATTGCGAAAAAACTTAA
pSIREN-CAP-7:
正向:
GATCCAGGTTAAGGTTGAATTCTGTTCAAGAGACAGAATTCAACCTTAACCTTTTTTTG
反向:
GTCCAATTCCAACTTAAGACAAGTTCTCTGTCTTAAGTTGGAATTGGAAAAAAACTTAA
pSIREN-CAP-8:
正向:
GATCCGCAGTATATACGACCAGGAATTCAAGAGATTCCTGGTCGTATATACTGTTTTTTG
反向:
GCGTCATATATGCTGGTCCTTAAGTTCTCTAAGGACCAGCATATATGACAAAAAACTTAA。
3、如权利要求2所述抗猪圆环病毒shRNA的用途:通过抑制猪圆环病病毒在细胞内的复制,从而发挥抗病毒作用。
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---|---|---|---|---|
CN102925987A (zh) * | 2012-11-19 | 2013-02-13 | 杨国栋 | 一种随机shRNA文库的构建方法 |
CN103468733A (zh) * | 2013-07-26 | 2013-12-25 | 吴珍芳 | 抗猪圆环病毒2型的表达载体和转基因猪及其构建方法 |
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2009
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102925987A (zh) * | 2012-11-19 | 2013-02-13 | 杨国栋 | 一种随机shRNA文库的构建方法 |
CN103468733A (zh) * | 2013-07-26 | 2013-12-25 | 吴珍芳 | 抗猪圆环病毒2型的表达载体和转基因猪及其构建方法 |
CN103468733B (zh) * | 2013-07-26 | 2016-01-27 | 华南农业大学 | 抗猪圆环病毒2型的表达载体和转基因猪及其构建方法 |
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