CN116665779B - 一种筛选家禽RNA病毒源siRNA方法和应用 - Google Patents
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Abstract
本发明公开了一种筛选家禽RNA病毒源siRNA的方法及其应用。本发明筛选家禽病毒源siRNA方法是基于NCBI GEO数据库中GSE数据集的小RNA与相应参考文献的病毒毒株序列进行比对。其次,通过基因组可视化软件IGV比对可知,IBVM41病毒源siRNA富集区为IBV M41 NSP10siRNA富集区序列。我们将IBVM41 NSP10siRNA富集区序列克隆至能高效表达siRNA的pSilencer4.1载体中,构建成PS‑M10质粒。最后,将PS‑M10质粒转染至巨噬细胞,再进行IBVM41感染试验,荧光定量PCR以及蛋白质印迹结果表明,PS‑M10可高效抑制IBV M41的复制,限制病毒的增殖。
Description
技术领域
本发明属于分子生物检测技术领域,具体涉及一种筛选家禽RNA病毒源siRNA的方法和应用,具体涉及基于病毒源siRNA富集区的选择和IBV-siRNA富集片段质粒的构建与应用。
背景技术
近年来,随着在我国家禽养殖业的快速发展,家禽病毒性传染病的频繁爆发也给我国家禽养殖业造成了巨大经济损失。值得注意的是,RNA病毒造成的影响尤为严重,如鸡传染性支气管炎病毒(Infectious Bronchitis Virus,IBV)中QX与M41亚型、甲型流感病毒(Influenza A virus,AIV)的H9N2以及鸡新城疫病毒(Newcastle disease virus,NDV)中的F48E9以及LaSota毒株等。其中,RNA病毒的高频突变重组使得疫苗的研发和使用存在滞后性和保护力不足。然而,近年来研究者在家禽RNA干扰(RNA interference,RNAi)***的研究给我们对家禽RNA病毒的防控提供了新的思路。
RNAi是真核生物中存在的高度保守的一种基因沉默机制,主要由21~23nt小非编码RNA(microRNA和siRNA)特异性沉默mRNA,抑制靶基因表达。RNAi介导的抗病毒免疫途径主要分为3个阶段:dsRNA(Double strand RNA,dsRNA)的产生、Dicer的切割以及RNA诱导的沉默复合体(RNA-induced silencing complex,RISC)降解作用。第一阶段是病毒基因组RNA复制产生dsRNA。病毒进入宿主细胞后,病毒的基因组RNA首先作为mRNA翻译出病毒的RNA依赖的RNA聚合酶(RNA dependent RNA polymerase,RdRP),然后以病毒的基因组RNA作为模板在RdRP的作用下合成新的基因组RNA。在RdRP对模板进行复制期间,旧链和新链会形成中间体dsRNA,这时dsRNA诱发第二阶段的切割。第二阶段是Dicer将dsRNA切割成21-23nt的病毒源siRNA。第三阶段是切割后的小RNA与Argonaute2蛋白(Ago2)装配形成RISC,靶向降解病毒的基因组RNA。
目前,家禽RNAi抗病毒***的优势在于其保留着两个具有催化酶活性的RNaseIII成员Drosha和Dicer,有着完善的RNAi抗病毒***。其中,核糖核酸内切酶Dicer可对病毒复制过程中产生的dsRNA切割成病毒源siRNA。直至现阶段,国内外仅有少量相关研究表明家禽的禽流感病毒和传染性法氏囊病毒均可以调控siRNA的生成抑制病毒复制。其痛点在于,家禽病毒源siRNA数据库的未建立使得我们对家禽RNA病毒感染产生的siRNA研究仍然处于早期阶段。
发明内容
发明目的:鉴于此,本发明的所要解决的技术问题是提供了一种筛选家禽RNA病毒源siRNA的方法。
本发明还要解决的技术问题是提供了IBV,AIV和NDV的siRNA富集区片段以及他们对应的siRNA数据库。
本发明还要解决的技术问题是提供了上述制备不同禽类病毒的富集片段质粒的方法以及其应用。
技术方案:为了解决上述技术问题,本发明提供了一种家禽筛选RNA病毒源siRNA的方法,包括以下步骤:在NCBI的GEO数据库对GSE数据集中的siRNA与相应病毒的序列比对,匹配上病毒的序列以及长度为21-23nt的siRNA,即为病毒源siRNA。
其中,所述病毒源包括IBV病毒毒株、AIV病毒毒株或NDV病毒毒株;优选地,所述IBV病毒毒株包括QX亚型或M41亚型。
本发明内容还包括一种家禽RNA病毒源的siRNA富集区基因片段的筛选方法,包括筛选家禽RNA病毒源siRNA的方法,还包括将收集得到的病毒源siRNA序列数据,通过snapgene软件与相应病毒基因组序列进行分析比对,再将分析比对的数据使用IGV软件进行数据可视化,得到相应病毒基因组序列上siRNA最多的区域,即为RNA病毒源的siRNA富集区片段。
其中,IBV病毒毒株siRNA富集区根据对应的QX毒株或M41毒株的核苷酸序列进行筛选;AIV病毒毒株siRNA富集区根据H9N2毒株中的PB2、PB1、PA、HA、NA、M、NEP和/或NS1核苷酸筛选获得;NDV病毒毒株siRNA富集区根据LaSota或F48E9毒株的核苷酸序列筛选获得。
本发明内容还包括一种家禽RNA病毒源的siRNA富集区基因片段,所述siRNA富集区基因片段包括IBV组病毒源siRNA富集区、AIV组病毒源siRNA富集区或NDV组病毒源siRNA富集区;作为优选地,包括IBV M41亚型病毒源siRNA富集区为NSP10,所述IBV M41 NSP10siRNA富集区核苷酸序列如SEQ ID NO.1所示,优选的,AIV组H9N2病毒源siRNA富集区核苷酸序列如SEQ ID NO.2所示,优选的,NDV组F48E9病毒源siRNA富集区核苷酸序列如SEQ IDNO.3所示。
本发明内容还包括一种家禽RNA病毒源的siRNA富集区重组质粒或细胞系,其特征在于,所述重组质粒或细胞系包括将所述的基因片段。
其中,所述重组质粒是将所述的基因片段导入质粒中获得,作为优选,所述质粒包括psliencer 4.1。
本发明内容还包括一种家禽RNA病毒源的siRNA富集区重组质粒的构建方法,包括以下步骤:将所述的RNA病毒源的siRNA富集区基因片段导入质粒中即得。
本发明内容还包括siRNA、所述的基因片段或所述的重组质粒或细胞系或在制备预防或治疗RNA病毒源感染疾病的药物或疫苗中的应用。
优选的,IBV组进行siRNA序列比对的毒株为QX与M41,对应的QX毒株的QX GenBank号:ON350837.1,M41毒株IBV M41 GenBank号:MK937830.1。
优选的,AIV组进行siRNA序列比对的毒株为H9N2,H9N2毒株片段1(PB2)核苷酸序列Genbank号为KP865892.1,片段2(PB1)核苷酸序列Genbank号为KP865845.1,片段3(PA)核苷酸序列Genbank号为KP865799.1,片段4(HA)核苷酸序列Genbank号为KP865958.1,片段6(NA)核苷酸序列Genbank号为KP866002.1,片段7(M)核苷酸序列Genbank号为KP866046.1,片段8(NEP、NS1)核苷酸序列Genbank号为KP866088.1。
优选的,NDV组进行siRNA序列比对的毒株为LaSota和F48E9,LaSota毒株核苷酸序列Genbank号为JF950510.1,F48E9毒株核苷酸序列Genbank号为MG456905.1。
本发明所述的以IBV、AIV和NDV为主的家禽病毒源siRNA数据库,是以IBV组、AIV组和NDV组病毒源siRNA所形成。
优选的,AIV组H9N2病毒源siRNA富集区核苷酸序列如SEQ ID NO.2所示。
本发明内容还包括一种IBV-siRNA富集片段质粒(PS-M10),所用的载体为赛默飞世尔科技(中国)有限公司商品化载体pSilencer4.1,连接片段为IBV M41 NSP10 siRNA富集区核苷酸序列如SEQ ID NO.1所示,得到的质粒为PS-M10质粒图谱为图4,核苷酸序列为如SEQ ID NO.4所示。
有益效果:本发明聚焦于家禽RNA病毒性传染病防控这一技术难点问题,从家禽细胞RNAi抗病毒***的角度出发,建立一种筛选家禽RNA病毒源siRNA方法以及其数据库。另外,本发明在研发增强家禽细胞抗病毒能力产品的前提下,基于病毒源siRNA的富集区构建相应的抗病毒质粒,在细胞层面上探讨其抗病毒效果,以期为临床实践上防控家禽呼吸道病原奠定坚实理论依据和技术基础。
附图说明
图1为本发明IBV组病毒源siRNA检测结果;IBV感染巨噬细胞所产生21-23ntsiRNA在IBV基因组的分布,实线上方代表QX组,下方代表M41组;紫色虚线框内圈住的siRNA区域片段用于后续质粒的构建;
图2为本发明AIV组和NDV组病毒源siRNA检测结果;A.NDV组病毒源siRNA的数量,分为LaSota组、F48E9组以及Control组;B.AIV组病毒源siRNA的数量,分为Mock组和H9N2组;差异不显著表示为P≥0.05(ns);显著则表示为P<0.001(***)、P<0.0001(****);
图3为本发明AIV组和NDV组靶向病毒基因的siRNA;A.NDV组病毒源siRNA的数量,分为LaSota组、F48E9组以及Control组;B.AIV组病毒源siRNA的数量,分为Mock组和H9N2组;
图4为本发明IBV M41 NSP10 siRNA富集区质粒图谱;
图5为本发明IBV-siRNA富集区质粒抗病毒结果。
具体实施方式
本发明提供一种筛选家禽RNA病毒源siRNA的方法和建立以IBV、AIV和NDV为主的家禽病毒源siRNA数据库,病毒源siRNA富集区的选择以及IBV-siRNA富集区质粒的构建方法和应用。
本发明所述的筛选家禽RNA病毒源siRNA的方法,具体如下,首先是登陆美国国家生物技术信息中心(National Center for Biotechnology Information,NCBI)官网,在GEO数据库输入相应病毒的全称或简写,点击http下载其GSE数据集,并下载与参考文献中相应病毒的序列。将数据集小RNA中的bam文件与病毒的序列使用IGV可视化软件以及Snapgene基因序列比对软件进行比对分析,获取相应病毒源siRNA。
本发明的以IBV、AIV和NDV为主的家禽病毒源siRNA数据库,是以IBV组、AIV组以及NDV组所筛选出的病毒源siRNA构建而成。
本发明的家禽病毒源siRNA富集区是根据IGV可视化软件以及Snapgene基因序列比对软件进行比对分析,初步筛选出病毒源siRNA较多的核苷酸序列片段。其中,IBV组病毒源siRNA富集区根据对应的IBV QX GenBank号:ON350837.1,IBV M41 GenBank号:MK937830.1。AIV组病毒源siRNA富集区根据H9N2毒株PB2核苷酸序列Genbank号KP865892.1,PB1核苷酸序列Genbank号KP865845.1,PA核苷酸序列Genbank号KP865799.1,HA核苷酸序列Genbank号KP865958.1,NA核苷酸序列Genbank号KP866002.1,M核苷酸序列Genbank号为KP866046.1,NEP、NS1核苷酸序列Genbank为KP866088.1筛选所得。NDV组病毒源siRNA富集区根据LaSota毒株核苷酸序列Genbank号JF950510.1,F48E9毒株核苷酸序列Genbank号MG456905.1筛选所得。
本发明提供的IBV M41病毒源siRNA富集区包括IBV M41 NSP10 siRNA富集区核苷酸序列如SEQ ID NO.1所示。
本发明提供AIV组H9N2病毒源siRNA富集区核苷酸序列如SEQ ID NO.2所示。
本发明提供的NDV组F48E9病毒源siRNA富集区核苷酸序列如SEQ ID NO.3所示。
本发明所提供的IBV-siRNA富集片段质粒(PS-M10)的构建方法,所用的载体为赛默飞世尔科技(中国)有限公司商品化载体pSilencer4.1,连接片段为IBV M41 NSP10siRNA富集区核苷酸序列如SEQ ID NO.1所示,PS-M10质粒图谱为图4,核苷酸序列为如SEQID NO.4所示,PS-M10构建方法是同源重组。
本发明所提供的IBV-siRNA富集片段质粒(PS-M10)的应用,是在鸡巨噬细胞上转染PS-M10质粒,再感染IBV-M41毒株,利用荧光定量PCR和蛋白质印迹验证其抗病毒效果。
下面结合具体的实施例,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解在阅读了本发明讲授的内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于申请所附权利要求书所限定的范围。
实施例1家禽病毒源siRNA的筛选方法
首先登陆美国国家生物技术信息中心(National Center for BiotechnologyInformation,NCBI)官网,在GEO数据库输入H9N2查询高通量数据,并且是与小RNA相关的数据集,点击http下载其GSE数据集,并根据GEO中涉及的参考文献查找相应病毒的序列。其中,H9N2毒株PB2核苷酸序列Genbank号KP865892.1,PB1核苷酸序列Genbank号KP865845.1,PA核苷酸序列Genbank号KP865799.1,HA核苷酸序列Genbank号KP865958.1,NA核苷酸序列Genbank号KP866002.1,M核苷酸序列Genbank号为KP866046.1,NEP、NS1核苷酸序列Genbank为KP866088.1。将数据集小RNA中的bam文件与病毒的序列使用IGV可视化软件以及Snapgene基因序列比对软件进行比对分析,获取相应病毒源siRNA,主要筛选21-23个碱基的siRNA。AIV组病毒源siRNA结果如图2和图3所示。H9N2感染后可产生约200条病毒源siRNA,其分布区域主要为HA、M1和M2。
实施例2 IBV-siRNA基因富集区质粒的构建
根据图1的IBV组病毒源siRNA可视化结果,初步选定NSP10基因片段(SEQ IDNO.1)和NSP16(SEQ ID NO.7)。IBV组抗病毒质粒的构建方法如下,以PS-M10为例。首先,以上游引物F(核苷酸序列SEQ ID NO.5,5’端添加BamHI酶切位点及其保护碱基),下游引物R(核苷酸序列SEQ ID NO.6,5’端添加HindIII酶切位点及其保护碱基)扩增IBV M41 NSP10siRNA富集区核苷酸序列如SEQ ID NO.1,并在两端添加了酶切位点BamHI和HindIII。具体如下,使用诺唯赞公司的2×Taq Plus Master Mix II进行扩增,体系为2×Taq PlusMaster Mix II 25μL,10μM上游引物F与10μM下游引物R各2μL,模板DNA(提取病毒IBV M41的RNA后进行反转录得到的cDNA产物)5μL,灭菌ddH2O补充至50μL。反应条件如下,95℃,3min;95℃,15s,60℃,30s,72℃,30s,35个循环;72℃,10min。
另外,使用TAKARA公司的QuickCutTM BamHI和QuickCutTM Hind III限制性内切酶对pSliencer 4.1质粒进行双酶切,具体如下,10x QuickCut Green Buffer 1μL,pSilencer4.1质粒0.5μg,QuickCutTM BamHI和QuickCutTM Hind III限制性内切酶各1μL,灭菌ddH2O补充至10μL,轻轻混匀后瞬时离心,并在37℃恒温孵育15min。进行1%核酸凝胶电泳试验,并使用OMEGA公司的胶回收试剂盒获取线性化的pSilencer4.1片段。
将线性化的pSilencer4.1片段200ng,***片段IBV M41 NSP10 siRNA富集区核苷酸片段40ng,5x CE II Buffer 4μL,ExnaseII 2μL,灭菌ddH2O补充至20μL,混匀在37℃恒温孵育20min,4℃连接12h。将连接产物转化至诺唯赞公司的DH5α感受态细胞中,进行转化试验。具体如下,将加入连接产物的感受态静置在冰盒里30min,然后马上42℃水浴锅进行热激45s,立即置于冰上冷却3min,随后加入900μL无抗性LB液体培养基37℃、220rpm摇菌45min,将菌液涂布在氨苄抗性(Amp+)的固体LB平板上,倒置于37℃培养箱中,培养12h。挑取单菌落加入至1mL LB液体培养基(Amp+)进行6h的扩大培养,然后吸取40μL菌液送生工生物工程(上海)股份有限公司进行测序验证。选出测序正确的菌液,使用OMEGA公司的无内毒素质粒小提试剂盒进行质粒提取,得到相应的质粒PS-M10。
采用同PS-M10相同的方法,对富集区片段NSP16(SEQ ID NO.7)构建得到质粒PS-M16。
实施例3 IBV-siRNA富集区质粒的应用
将本实验室培养的巨噬细胞铺于6孔板中,待巨噬细胞长至80%,使用翌圣生物科技(上海)股份有限公司的脂质体转染试剂将IBV-siRNA富集区质粒(PS-M10)转染至6孔板中,并使用pSilencer4.1作为对照,转染24h后,接入江苏省农科院兽医所提供的本实验室保存的病毒株IBV M41 40μL,感染2h后,更换新的2%Sigma公司的1640细胞培养基培养至24h进行荧光定量PCR以及48h进行蛋白质印迹检验质粒的抗病毒效果。
实验结果由图5可知,荧光定量PCR以及蛋白质印迹结果表明,pSilencer4.1组(PS)和PS-M16组中IBV M41的病毒含量并没有下降,而PS-M10组中,M41的mRNA表达量以及蛋白表达量都显著下降,表明PS-M10可高效抑制IBV M41的复制,限制病毒的增殖。因此,该质粒可广泛应用于养殖场中IBV M41的预防、治疗以及净化。
Claims (7)
1.一种家禽RNA病毒源的siRNA富集区基因片段,其特征在于,所述siRNA富集区基因片段包括IBV M41亚型病毒源siRNA富集区为NSP10,所述IBV M41 NSP10 siRNA富集区核苷酸序列如SEQ ID NO.1所示。
2.一种家禽RNA病毒源的siRNA富集区重组质粒,其特征在于,所述重组质粒包括权利要求1所述的基因片段。
3.一种家禽RNA病毒源的siRNA富集区细胞系,其特征在于,所述家禽RNA病毒源的siRNA富集区细胞系包括权利要求1所述的基因片段。
4.根据权利要求2所述的重组质粒,其特征在于,所述重组质粒是将权利要求1所述的基因片段导入质粒中获得。
5.一种家禽RNA病毒源的siRNA富集区重组质粒的构建方法,其特征在于,包括以下步骤:将权利要求1所述的RNA病毒源的siRNA富集区基因片段导入质粒中即得。
6.根据权利要求5所述的一种家禽RNA病毒源的siRNA富集区重组质粒的构建方法,其特征在于,所述质粒包括psliencer 4.1。
7.权利要求1所述的基因片段或权利要求2所述的重组质粒或权利要求3所述的细胞系在制备预防或治疗RNA病毒源感染疾病的药物或疫苗中的应用。
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