CN108456692A - 一种抗口蹄疫病毒感染的四联miRNA及构建方法 - Google Patents
一种抗口蹄疫病毒感染的四联miRNA及构建方法 Download PDFInfo
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Abstract
本发明公开了一种抗口蹄疫病毒miRNA载体,载体上有针对FMDV 3C蛋白的3C1片段序列、3C2片段序列及针对FMDV 3D蛋白的3D1片段序列、3D2片段序列。该载体可以突破血清型的壁垒,解决传统疫苗免疫血清型不能交叉保护的问题;可有效的激活细胞免疫;而使用减毒鼠伤寒沙门氏菌作为疫苗活载体具有能够激发宿主产生针对其所携带外源抗原的特异性免疫应答,增强粘膜、细胞免疫的功能,制备简单,易于储存运输,抗原无需提纯、成本低廉等优点。
Description
技术领域
本发明分子生物学领域,特别涉及一种抗口蹄疫病毒miRNA载体的构建方法及应用。
背景技术
口蹄疫(Foot and Mouth Disease,FMD)又称口疮热(aphthous fever),是由口蹄疫病毒(Foot-and-mouth disease virus,FMDV)引起的一种急性、热性、高度接触性及高度传染性的疾病。口蹄疫的爆发带来的经济损失和对农业、政治的不良影响极其恶劣。因此,世界动物卫生组织(OIE)将口蹄疫列为必须上报的动物类A类传染病。口蹄疫易感动物为牛、绵羊、山羊和猪等30多种偶蹄目动物。其传播特征具有传播范围广、易感动物多、感染剂量低、感染后排毒量高、传播速度快等,因此曾多次在世界上爆发及流行。患病动物潜伏期36h至7d,病初体温升高、精神抑郁、食欲低下,继而在口腔粘膜、唇舌部、趾间、蹄冠、乳房出现水疱,水疱破裂后常发生融合,形成弥漫性损害。口蹄疫可引起患病动物继发乳腺炎从而降低产奶量,生产性能下降,甚至造成幼畜死亡。另外,口蹄疫的爆发还可引起国家和地区之间的贸易限制如禁止动物及其产品的出口,继而造成更巨大的损失。因此,FMD的爆发与流行已经上升为影响国际关系、国家声誉和国家经济发展的问题,在国际上素有“政治经济病”之称。近年,FMD在南亚及德法等欧洲国家相继传播,并带来巨大的经济损失。
口蹄疫病毒(Foot and mouth disease virus,FMDV)属于小RNA病毒科,口蹄疫病毒属,是单股正链RNA病毒。目前有7个血清主型,分别为O、A、C、SAT 1、SAT 2、SAT 3和AsiaI,各型之间无交叉保护反应。每一主型又分若干亚型,迄今为止已发现的亚型有65个且仍在不同的地区出现新的变异株或新的亚型。FMDV病毒粒子直径为(23±2)nm,大致呈圆形,无囊膜。FMDV的结构和成分均较简单,成熟病毒粒子约含30%的RNA,其余70%为蛋白质。FMDV的基因组全长约8500nt,由5’非编码区(untranslated region,UTR)、开放阅读框架(open reading frame,ORF)和3’UTR构成。5’端共价连接一种特殊的小蛋白VPg,接着是约1300nt的5’UTR,包括S片段(small fragment)、聚胞嘧啶区(Poly C)和内部核糖体进入位点(Internal ribosome entry site,IRES)等。3’UTR由Poly(A)尾及ORF和Poly(A)尾之间的92nt组成,全长为172nt。基因组中部是一大开放阅读框用于编码一多聚蛋白,该多聚蛋白经三级裂解后,形成3-4种结构蛋白:VP0或VP4、VP2、VP3、VP1,和8-9种非结构蛋白:Lab、Lb、2A、2B、2C、3A、3B、3C、和3D。
FMDV侵染细胞的必要条件为吸附宿主细胞,而吸附过程依赖于宿主细胞受体。当FMDV颗粒表面的宿主识别位点与靶细胞胞膜上的受体位点结合后,即通过吞噬作用进入宿主细胞胞质内。当宿主胞内溶酶体因病毒介入而释放后,随即将FMDV生存环境转变为酸性,从而导致FMDV颗粒周围酶类降解。由于酶类降解导致FMDV衣壳蛋白构象发生变化,衣壳蛋白迅速膨胀,从而导致FMDV基因RNA经由吞噬小泡膜上的通道释放,并随之进行自身RNA复制、翻译及蛋白装配。
在FMDV感染宿主细胞之后,导致其发生细胞病变或死亡的机制有两个原因:其一为病毒产生的特异性蛋白直接作用于细胞,导致细胞的mRNA翻译受到抑制。同时,宿主细胞的翻译启动子失活,核糖体被病毒RNA侵占等也是细胞死亡的重要原因;其二为病毒感染机体后,可通过刺激机体细胞产生细胞免疫从而间接诱发细胞凋亡。
RNA干扰(RNA interference,RNAi)是20世纪90年代末发现的1种真核生物细胞在转录后引发基因沉默的分子机制。其广泛定义为RNA引起的同源依赖性基因沉默现象(homology-dependent gene silencing,HDGs),狭义定义为外源双链RNA(double-stranded RNA,dsRNA)介导的转录后基因沉默(post-transcriptional gene silencing,PTGS)。RNAi广泛存在于从真菌到高等植物、从无脊椎动物到哺乳动物的各种生物中。因此,可以尝试利用RNAi技术对FMDV进行研究和防控。
发明内容
本发明的目的在于一种抗口蹄疫病毒miRNA载体的构建方法及应用。
本发明所采取的技术方案是:
一种抗口蹄疫病毒miRNA载体,载体上有针对FMDV 3C蛋白3C1片段的干扰序列、3C2片段的干扰序列及针对FMDV 3D蛋白3D1片段的干扰序列、3D2片段的干扰序列,其中:
3C1片段的编码序列是:
TGCTGATCTCAAACTCAAACA CTCTGGTTTTGGCCAC(SEQ ID NO.9);
3C2片段的编码序列是:
TGCTGAACACTCTGTAGTC ACTGTCTGTTTTGGCCAC(SEQ ID NO.10);
3D1片段的编码序列是:
TGCTGATCAAAGGCCGAATAGTCCACGTTTTGGCCACTGACTGACGTGGACTACGGCCTTTGAT(SEQID NO.11);
3D2片段的编码序列是:
TGCTGAGATCATGGTGTAAGTGTCCAGTTTTGGCCACTGACTGACTGGACACTCACC ATGATCT(SEQID NO.12)。
优选的,载体由目的片段序列串联后连接pcDNA6.2-GW/EmGFP-miR载体构建而成。
优选的,构建3C1片段序列的引物是:
F:5’-ATCTCAAACTCAAACACTCTGGTTTTGGCCACTGACTGACCAGATTGTGAGTTTGAGATCAGGA-3’(SEQ ID NO.1);
R:5’-ATCTCAAACTCACACTCTGGTCAGTCAGTGGCCAAAACCAGAGTGTTTGAGTTTGAGATCAGCA-3’(SEQ ID NO.2)。
优选的,构建3C2片段序列的引物是:
F:5’-AACACTCTGTAGTCACTGTCTCTTTTGGCCACTGACTGACAGACAGTGTACAGAGTGTTCAGGA-3’(SEQ ID NO.3);
R:5’-AACACTCTGTACACTGTCTGTCAGTCAGTGGCCAAAACAGACAGTGACTACAGAGTGTTCAGCA-3’(SEQ ID NO.4)。
优选的,构建3D1片段序列的引物是:
F:5’-TGC TGA TCA AAG GCC GAA TAG TCC ACG TTT TGG CCA CTG ACT GAC GTGGAC TAC GGC CTT GTA T-3’(SEQ ID NO.5);
R:5’-CCT GAT CTC AAA CTC ACA CTC TGG TCA GTC AGT GGC CAA AAC CAG AGTGTT TGA GTT TGA GAT C-3’(SEQ ID NO.6)。
优选的,构建3D2片段序列的引物是:
F:5’-AGATCATGGTGTAAGTGTCCAGTTTTGGCCACTGACTGACTGGACACTCACCATGATCTCAGGA-3’(SEQ ID NO.7);
R:5’-CCT GAG ATC ATG GTG AGT GTC CAG TCA GTC AGT GGC CAA AAC TGG ACACTT ACA CCA TGA TCT C-3’(SEQ ID NO.8)。
一种制备抗口蹄疫病毒miRNA载体的方法,其步骤是:将目的片段FMDV的3C1片段引物序列、3C2片段引物序列、3D1片段引物序列、3D2片段引物序列分别进行扩增制备双链RNA;将双链RNA与pcDNA6.2-GW/EmGFP-miR载体的连接,连接后转化感受态细胞,提取质粒,酶切,将目的片段串联在一起,检测挑选阳性质粒。
本发明的有益效果是:该载体可以突破血清型的壁垒,解决传统疫苗免疫血清型不能交叉保护的问题;可有效的激活细胞免疫;而使用减毒鼠伤寒沙门氏菌作为疫苗活载体具有能够激发宿主产生针对其所携带外源抗原的特异性免疫应答,增强粘膜、细胞免疫的功能,制备简单,易于储存运输,抗原无需提纯、成本低廉等优点。
附图说明
图1dsRNA与pcDNA6.2-GW/EmGFP-miR载体连接示意图。
图2位空白质粒转染BHK-21细胞接毒48h后观测图。
图3重组miRNA质粒转染BHK-21细胞接毒48h后观测图
具体实施方式
引物设计
分别为针对FMDV 3C蛋白的3C1片段、3C2片段及针对FMDV 3D蛋白的3D1片段、3D2片段(见表1)。
表1单链引物RNA序列
3C1片段的编码序列是:
TGCTGATCTCAAACTCAAACACTCTGGTTTTGGCCAC(SEQ ID NO.9);
3C2片段的编码序列是:
TGCTGAACACTCTGTAGTCACTGTCTGTTTTGGCCAC(SEQ ID NO.10);
3D1片段的编码序列是:
TGCTGATCAAAGGCCGAATAGTCCACGTTTTGGCCACTGACTGACGTGGACTACGGCCTTTGAT(SEQID NO.11);
3D2片段的编码序列是
TGCTGAGATCATGGTGTAAGTGTCCAGTTTTGGCCACTGACTGACTGGACACTCACCATGATCT(SEQID NO.12)。
双链RNA(dsRNA)的构建
(1)在PCR反应管中加入下列反应体系:
(2)将样品置于金属水浴锅中,95℃热冲击反应4min。
(3)取出样品,静置5-10min,待反应物温度降至室温。
(4)将样品放入低速微型离心机中,轻柔混匀后低速离心5s。
(5)取出1μL dsRNA放入PCR管中待稀释并将剩余产物放置于-20℃保存。
(6)采用梯度稀释,先将dsRNA稀释100倍,在1.5mL离心管中加入下列反应体系:
(7)再将已稀释100倍dsRNA稀释50倍,在上述1.5mL离心管中加入下列反应体系:
dsRNA与pcDNA6.2-GW/EmGFP-miR载体的连接及转化
按照图1的方式将dsRNA与pcDNA6.2-GW/EmGFP-miR载体进行连接、转化。
(1)在PCR反应管中加入下列连接反应体系:
(2)将样品充分摇匀,短暂离心3-5s,室温孵育5min。
(3)将样品置于冰上,以待继续使用Top-10化学感受态细胞进行转化。
连接产物转化Top-10化学感受态细胞
(1)将2μL连接样品加入至1管Top-10化学感受态细胞中,并轻柔震荡充分混匀。
(2)冰浴5-30min。
(3)42℃热休克细胞30s,期间禁止晃动。
(4)立即将感受态细胞置于冰上。
(5)向感受态细胞中加入250μL室温S.O.C培养基。
(6)37℃振荡培养1h。
(7)将20-100μL细胞悬液均匀涂布于含有50μg/mL壮观霉素的LB琼脂培养基上,倒置静置37℃培养过夜。
将转化后平板置于37℃温箱培养12~16h后取出,挑取单个菌落,在加有壮观霉素抗性的LB液体培养基内扩大培养,菌液送测序。将测序反馈回来的阳性菌液利用无内毒素质粒小提试剂盒提取质粒。
将鉴定阳性的3C1、3C2质粒分别利用:BamHⅠ/BglⅠ及BamHⅠ/XhoⅠ双酶切后进行串联,制备成串联质粒;用同样的方法,将3C1、3C2、3D1、3D2串联在同一在载体上,送测序,挑选3C1、3C2、3D1、3D2串联成功的阳性质粒备用。
口蹄疫病病毒TCID50测定
将10-1-10-10连续稀释的病毒接种到96孔细胞培养板上的BHK-21单层细胞。每个稀释度做1列,共6个细胞孔,设2孔对照(用无菌Hanks液代替病毒液)。逐日检查细胞是否出现细胞病变,记录出现细胞病变的孔数,停止出现细胞病变时停止观察。按Reed—Muench法计算TCID50。
siRNA干扰效果实验
将适量的BHK-21细胞接种24孔细胞培养板,待细胞长至单层后,按Lipofectamine2000转染试剂盒按比例转染质粒,每组3个重复。同时设立细胞对照孔、病毒对照及pcDNA6.2-GW/EmGFP-miR negative control对照。12h后,各孔分别接种1000TCID50、100TCID50、10TCID50病毒,24h后观察细胞病变情况。CPE观察:致密的单层细胞局部出现细胞间隙增大,疏松。细胞多角形的轮廓逐渐失去棱角,细胞变圆、收缩,聚集成岛状。有的细胞破裂,从瓶壁上脱落,呈灶状空斑。
将抽提的不含内毒素的siRNA表达质粒(即)转染BHK-21细胞,同时设立相应对照。12h后,各孔分别加入100个TCID50的O/Mya98/BY/CHA/2010毒株病毒液,48h后显微镜下观察细胞病变(CPE)出现情况。发现:pcDNA6.2-GW/EmGFP-miR negative control载体对照孔和接毒对照孔大部细胞死亡脱落(图2),重组质粒对照孔有少量细胞死亡,细胞对照状态良好,转染的重组质粒孔的细胞状态明显优于病毒对照,表明质粒对毒株O/Mya98/BY/CHA/2010的复制均有显著的抑制效果,其中3C1、3C2、3D1、3D2串联载体效果最为显著(图3)。
SEQUENCE LISTING
<110> 广东省农业科学院动物卫生研究所
<120> 一种抗口蹄疫病毒感染的四联miRNA及构建方法
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Claims (8)
1.一种抗口蹄疫病毒miRNA载体,其特征在于,载体上有针对FMDV 3C蛋白3C1片段的干扰序列、3C2片段的干扰序列及针对FMDV 3D蛋白3D1片段的干扰序列、3D2片段的干扰序列,其中
3C1片段的编码序列是:
TGCTGATCTCAAACTCAAACACTCTGGTTTTGGCCAC(SEQ ID NO.9);
3C2片段的编码序列是:
TGCTGAACACTCTGTAGTCACTGTCTGTTTTGGCCAC(SEQ ID NO.10);
3D1片段的编码序列是:
TGCTGATCAAAGGCCGAATAGTCCACGTTTTGGCCACTGACTGACGTGGACTACGGCCTTTGAT(SEQ IDNO.11);
3D2片段的编码序列是:
TGCTGAGATCATGGTGTAAGTGTCCAGTTTTGGCCACTGACTGACTGGACACTCACCATGATCT(SEQ IDNO.12)。
2.根据权利要求1所述的载体,其特征在于,所述载体由目的片段序列串联后连接pcDNA6.2-GW/EmGFP-miR载体构建而成。
3.根据权利要求1所述的载体,其特征在于,构建3C1片段序列的引物是:
F:5’-ATCTCAAACTCAAACACTCTGGTTTTGGCCACTGACTGACCAGATTGTGAGTTTGAGATCAGGA-3’(SEQ ID NO.1);
R:5’-ATCTCAAACTCACACTCTGGTCAGTCAGTGGCCAAAACCAGAGTGTTTGAGTTTGAGATCAGCA-3’(SEQ ID NO.2)。
4.根据权利要求1所述的载体,其特征在于,构建3C2片段序列的引物是:
F:5’-AACACTCTGTAGTCACTGTCTCTTTTGGCCACTGACTGACAGACAGTGTACAGAGTGTTCAGGA-3’(SEQ ID NO.3);
R:5’-AACACTCTGTACACTGTCTGTCAGTCAGTGGCCAAAACAGACAGTGACTACAGAGTGTTCAGCA-3’(SEQ ID NO.4)。
5.根据权利要求1所述的载体,其特征在于,构建3D1片段序列的引物是:
F:5’-TGC TGA TCA AAG GCC GAATAG TCC ACG TTT TGG CCACTG ACT GAC GTG GACTAC GGC CTT GTA T-3’(SEQ ID NO.5);
R:5’-CCT GAT CTC AAA CTC ACA CTC TGG TCA GTC AGT GGC CAA AAC CAG AGT GTTTGA GTT TGA GAT C-3’(SEQ ID NO.6)。
6.根据权利要求1所述的载体,其特征在于,构建3D2片段序列的引物是:
F:5’-AGATCATGGTGTAAGTGTCCAGTTTTGGCCACTGACTGACTGGACACTCACCATGATCTCAGGA-3’(SEQ ID NO.7);
R:5’-CCT GAG ATC ATG GTG AGT GTC CAG TCA GTC AGT GGC CAA AAC TGG ACA CTTACA CCA TGA TCT C-3’(SEQ ID NO.8)。
7.一种制备抗口蹄疫病毒miRNA载体的方法,其步骤是:将目的片段FMDV的3C1片段引物序列、3C2片段引物序列、3D1片段引物序列、3D2片段引物序列分别进行扩增制备双链RNA;将双链RNA与pcDNA6.2-GW/EmGFP-miR载体的连接,连接后转化感受态细胞,提取质粒,酶切,将目的片段串联在一起,检测挑选阳性质粒。
8.根据权利要求1~6任一项所述的载体在制备抑制口蹄疫病病毒药物中的应用。
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