CN111110828A - 一种鸭源天然免疫调节蛋白ddx3x的应用 - Google Patents
一种鸭源天然免疫调节蛋白ddx3x的应用 Download PDFInfo
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Abstract
本发明属于生物技术和免疫学领域,具体涉及一种鸭源天然免疫调节蛋白DDX3X在鸭胚成纤维细胞中调控天然免疫信号通路、参与抗病毒反应及在鸭抗病毒感染中的作用。本发明在鸭胚成纤维细胞(DEF)内证明了过表达鸭源DDX3X能通过激活转录因子IRF1和Nf‑κB诱导鸭I型干扰素的产生;干扰掉DDX3X的本底表达后,poly(I:C)和DTMUV诱导的I型干扰素的活性也显著降低;超表达DDX3X能显著抑制鸭坦布苏病毒(Duck tembusu virus,DTMUV)的增殖。本发明揭示,鸭源DDX3X有望成为治疗鸭炎症性疾病的新靶点,可以在制备具有抑制DTMUV增殖功效的药物中应用;也可以作为鸭病毒病的免疫调节剂应用。
Description
技术领域
本发明属于生物技术和免疫学领域,具体涉及一种鸭源天然免疫调节蛋白DDX3X的应用。
背景技术
鸭在我国是重要的经济动物,我国鸭的养殖量占据世界第一。但与其它哺乳动物相比,关于其机体天然免疫系统的相关研究起步较晚。已有研究表明,家禽的天然免疫系统具有特殊性,例如鸭拥有完整的RIG-I受体从未使它在对抗病毒感染方面比没有RIG-I受体的鸡更具有优势。
细胞内抗病毒因子介导的抗病毒反应是宿主抵抗病毒感染的第一道防线,模式识别受体 (pattern recognition receptors,PRRs)率先识别病原相关分子模式(pathogenassociated molecular patterns,PAMPs),启动宿主的天然免疫反应。对于病毒感染而言,不论体外还是体内,干扰素(IFN)通路都发挥着主要的抗病毒作用。病原微生物入侵后其保守的病原相关分子模式(pathogen associated molecular patterns,PAMPs)能被相应的PRRs识别,从而通过级联反应诱导I型干扰素和其他先天性免疫基因的表达。而旁分泌或自分泌的I型干扰素又可与细胞表面的I型干扰素受体结合激活下游的JAK-STAT信号通路,此通路下游包含大量可以在多个步骤中抑制病毒复制的因子,即干扰素诱导基因(IFN-stimulated gene,ISG)表达产物,从而使机体维持抗病毒的状态。而有关鸭的天然免疫应答反应、信号转导过程和干扰素下游ISG的表达及调节机制尚不十分清楚。
DEAD-box RNA解旋酶3(DEAD-box RNA helicase,DDX3X)属于DExD/H-box解旋酶家族,该家族蛋白在进化上具有保守序列,有2个大的结构域:N-末端和C末端,由至少12 个保守的motif构成。其定位于细胞核或细胞质内,参与多种RNA的代谢过程。近年的研究表明,一些DExD/H-box解旋酶家族蛋白具有调节天然免疫信号通路和抗病毒功能,目前仅部分DExD/H-box解旋酶家族蛋白在天然免疫信号通路中的功能得到阐述。
发明内容
本发明针对现有技术的不足,目的在于提供一种鸭源DDX3X在鸭天然免疫调节方面的新用途。
为实现上述发明目的,本发明所采用的技术方案为:
鸭源DDX3X在鸭胚成纤维细胞中调控天然免疫信号通路、参与抗病毒天然免疫、制备鸭抗病毒感染药物中的应用。
本发明所述鸭源天然免疫调节蛋白DDX3X为申请人首次从鸭脾脏中解析,上传至美国国家生物信息中心NCBI(http://www.ncbi.nlm.nih.gov/),登录号为MH360982,全长cDNA 为1959bp。
本发明的有益效果:本发明借助自主构建的鸭IFN-β启动子双荧光素酶报告系统,通过 RNA病毒的模拟物——双链RNA类似物poly(I:C)和鸭坦布苏病毒(Duck tembusuvirus, DTMUV)来研究鸭源DDX3X与DTMUV病毒间的关系,在鸭胚成纤维细胞(DEF)内证明了过表达鸭源DDX3X能促进鸭自身I型干扰素的活性,干扰掉DDX3X的本底表达后, DTMUV感染后I型干扰素的活性也显著降低;本发明揭示,DDX3X有望成为治疗鸭炎症性疾病的新靶点,可以在制备具有抑制DTMUV增殖功效的药物中应用;也可以作为鸭病毒病的免疫调节剂应用。
附图说明
图1为DTMUV感染DEF细胞后DDX3X mRNA转录变化。
图2为duDDX3X随着DTMUV感染剂量的增加被降解的情况。
图3A)为鸭源DDX3X的PCR凝胶电泳图,1为duDDX3X,2为阴性对照;B)为真核表达载体pCAGGS-duDDX3X-flag的双酶切鉴定图,1~2为pDDX3X-flag/XhoI+KpnI双酶切。
图4为duDDX3X对于鸭源IFN-β启动子活性的影响及在DEF细胞中过表达的WesternBlot图。将pCAGGS-Flag-DDX3X与鸭源IFN-β启动子的荧光素酶报告质粒pGL-IFNB-Luc 共转染DEF细胞,pTK作为内参同时转染,24小时后,使用poly(I:C)刺激16小时,然后收集细胞,使用双荧光素酶报告基因检测试剂盒进行检测,图中显示值为萤火虫荧光素酶与海肾荧光素酶的比值,Western Blot检测转染质粒蛋白的表达。
图5为DDX3X干扰分子能抑制poly(I:C)激活的IFN-β的启动子活性。(A)DEF细胞中分别按转染duDDX3X干扰分子阴性对照siNegative、siduDDX3X-1、siduDDX3X-2和siDDX3X-3 36小时后,提取细胞总RNA,进行荧光定量PCR检测内源性duDDX3X的转录水平,筛选出干扰效率最高的干扰分子;(B)DEF细胞中分别将干扰分子阴性对照NC和干扰效率最高的siduDDX3X-2与pGL-IFNB-Luc和内参pRL-TK共转染DEF细胞,24小时后用poly(I:C)刺激,12小时后收集细胞,使用双荧光素酶报告基因检测试剂盒进行检测。
图6为DDX3X过表达可以抑制DTMUV的复制。在DEF细胞中过表达DDX3X后,感染DTMUV,24小时后收获细胞上清,进行TCID50试验检测病毒复制。
具体实施方式
为了更好地理解本发明,下面结合实施例进一步阐明本发明的内容,但本发明的内容不仅仅局限于下面的实施例。
实施例1病毒感染后DDX3X转录水平的变化
DTMUV感染DEF细胞,分别在感染后24h、48h时收获细胞,提取细胞总RNA,经逆转录后进行荧光定量PCR,检测duDDX3X的mRNA水平。具体的操作如下所示,duDDX3X 的mRNA水平的结果如图1所示,结果显示,DTMUV感染后,duDDX3X的mRNA转录水平上调。
DTMUV和DEF传代细胞系均由本实验室保存。
荧光定量PCR引物:
目的基因duDDX3X
上游引物duDDX3X-F:ATACTATGCCACCGAAAG
下游引物duDDX3X-R:GAACCTACTCTGCCAACA
内参基因GAPDH
上游引物qGAPDH-f:ATGAGAAGTATGACAAGTCC
下游引物qGAPDH-r:ACTGTCTTCGTGTGTGGCT
荧光定量PCR方法检测目的基因的表达
(1)细胞总RNA提取:
弃掉培养皿中的培养基,加入1ml TRIzol,室温放置5min;加入200μl氯仿,剧烈振荡15s后室温放置3min,使其自然分相;4℃12,000rpm离心10min,将上清小心移至新的Eppendorf管(无RNAase)中,加入等体积的异丙醇,混匀后室温放置10min;4℃12,000rpm离心10min,小心弃上清;向RNA沉淀中加入1ml 75%乙醇(用RNase-free水配制),温和悬浮沉淀;4℃8,000rpm离心5min,小心弃上清,室温干燥沉淀;向沉淀中加入50μl RNase-free水,置于55℃10min,以充分溶解RNA,测定浓度,分装于-80℃保存备用。
(2)逆转录反应(RT)
用商品化的试剂盒,参考罗氏反转录试剂盒说明书进行。对不同样品的RNA进行等质量的反转录。1μL oligo(dT)18引物、1μg RNA样品,再补加DEPC水至13μL,置于PCR 仪65℃10min,反应结束后将产物置于冰上。随后加入4μL的5×RT缓冲液、2μL的dNTPs、 0.5μL的RNA酶抑制剂、0.5μL的反转录酶,混匀后,置于PCR仪,55℃30分钟、85℃5 min,得到cDNA样品。
(3)实时荧光定量PCR(RT-qPCR)
使用SYBR Green试剂盒(Toyobo公司)检测样品中的RNA水平。配制的反应体系如下:10.0μL的SYBR Green I Master、0.5μL的上游引物、0.5μL的下游引物、8μL的灭菌水、1μL的cDNA模板。同时设置GAPDH作为内参。PCR扩增条件为:94℃ 10min进行预变性;94℃15s,56℃ 30s,72℃ 40s进行40个循环的扩增并收集荧光信号。应用ΔΔCt 法计算每个基因的相对mRNA表达水平。
实施例2鸭源DDX3X的克隆及真核表达载体pCAGGS-duDDX3X-flag的构建
(1)取黄豆大小的鸭脾脏组织,研磨后用TRIZOL法,按实施例1中的操作步骤(1)提取总RNA。
(2)将步骤(1)中提取的RNA反转录成cDNA,用商品化的试剂盒,参考罗氏反转录试剂盒说明书进行,具体操作按照实施例1中的操作步骤(2)进行。
(3)根据预测的鸭源DDX3X基因全长cDNA序列,选择KpnI和XhoI双酶切位点设计引物,其中上游引物为duDDX3X-f:5`-GCGGTACCATGAGTCATGTGGCGGTGG-3`;下游引物为duDDX3X-r:5`-GCCTCGAGTCAGTTGCCCCACCAGTCA-3`。以步骤(2)中制备的脾脏cDNA为模板,进行PCR扩增,得到特异性扩增产物大小在2000bp左右,详见图 3A。将带有酶切位点的duDDX3X连接至已用KpnI和XhoI双酶切及纯化后的pCAGGS-flag 真核表达载体中,得到重组表达载体,重组表达载体经PCR验证的凝胶电泳结果见图3B,经测序验证序列正确性、无移码突变,证明pCAGGS-duDDX3X-flag载体(简写为 pduDDX3X-flag)构建完成。
实施例3 DDX3X对IFN-β启动子活性的影响
通过荧光素酶检测系统,将pduDDX3X-flag与含有IFN-β启动子的荧光素酶报告质粒 pGL-IFNB-Luc(实验室已构建,具体方法参照实施例2)共转染DEF细胞。24小时后使用poly(I:C)刺激,刺激16小时后检测荧光素酶活化情况,具体的操作过程如下,结果如图4所示。其中图4A柱形图显示,IFN-β启动子的激活与duDDX3X呈剂量依赖性,表明超表达 DDX3X可以激活IFN-β启动子的活化;NF-κB和IRF1是调控IFN-β表达的两个重要转录因子,图4B和4C的柱形图表明,duDDX3X是通过激活NF-κB和IRF1从而诱导IFN-β的表达。具体操作步骤如下:
真核细胞的基因转染,转染试剂用jetTransfection Reagent,按照说明书进行。
以24孔板为例。将适量的DEF细胞接种到24孔板中,次日细胞待长至50%~60%时即可转染。转染前换为无血清和无抗生素的培养液。将鸭源IFN-β启动子的荧光素酶报告质粒0.1μg/孔、内参海参荧光素酶TK质粒(购自promega公司)0.01μg/孔,Flag-DDX3X等真核表达质粒(对照组用空载体质粒代替)总量1.0μg/孔加入到jetTransfectionReagent试剂盒配套的转染buffer中,转染的质粒总量相等为1.11μg/孔(不足的用空载体质粒补足)涡旋混匀,再加入2倍质粒剂量的转染试剂,涡旋混匀后瞬时离心,静止10min 后即可加入到24孔板中,4h后换上含2%FBS的无双抗细胞维持液。每个目的片段的质粒浓度设立3个重复。
报告基因转录活性检测
转染后24~36h,收取细胞。弃细胞培养液,加入1×被动裂解液(Promega,120μl/孔),室温放置在摇床上震动30min,待细胞充分裂解后,按照双荧光素酶报告基因检测试剂盒说明书进行检测,本实验室一般将试剂盒中的LAR II液以50μl/管分装至1.5mL EP管,吸10μl 细胞裂解液于含LAR II液的EP管。混匀后检测萤火虫荧光素酶活性,随后加入50μl现配的Stop&Reagent,再次混匀后检测海肾荧光素酶活性。为去除转染效率的影响,所得数据为萤火虫荧光素酶活性值除以海肾荧光素酶活性值。所测报告基因的活化情况用相对于对照组的倍数来表示。剩余的全细胞裂解液,用于Western Blot检测蛋白表达,如图4ABC 的Western Blot图表明,带有FLAG标签的鸭源DDX3X质粒在DEFs中成功过表达
Western Blot
(1)瞬时转染后48h,收获细胞,弃培养基,用预冷的PBS洗1~2次。每个平皿加入200~300 μL Western裂解液,摇匀,刮细胞,用一次性1mL注射器吸出裂解的细胞液,注入1.5mL 离心管中,并反复吹打至清亮。加入相应体积的5×SDS-PAGE上样缓冲液,沸水煮10min, -20℃保存。
(2)将蛋白样品用10%聚丙烯酰胺凝胶电泳(SDS-PAGE)分离,然后经2mA/cm2恒流将蛋白质转移至硝酸纤维素(PVDF)薄膜上;具体操作如下:戴上手套,剪2张7层滤纸和1张0.45μm PVDF膜,PVDF膜的大小要略大于凝胶。在一托盘中加入少量转膜缓冲液,把滤纸和海绵浸泡于其中。PVDF膜在甲醇中浸泡2min后也移入转膜缓冲液。依次在玻璃板上铺海绵,滤纸,用玻璃棒赶走气泡,将电泳后的胶轻放在滤纸上,玻璃棒赶走气泡,贴上PVDF 膜,再铺上滤纸及海绵,玻璃棒赶走气泡。将其整体转移到转膜夹上,放入电转装置中(黑色负极一面对应凝胶,红色正极一面对应PVDF膜)
(3)13v电压恒压转膜12h,期间在电转装置四周放上冰袋。
(4)封闭:配制封闭液,在20mL 1×TBST中加入1g脱脂奶粉或BSA(一块膜的用量),充分混匀倒入保鲜盒中。将膜用镊子夹出平铺在封闭液中,蛋白面朝下,置摇床上室温孵育2h。
(5)一抗与靶蛋白结合:把膜从封闭液中夹出剪去多余的边缘,平铺入一抗溶液中(1:1000稀释),蛋白面朝下,摇床上室温孵育4h。
(6)二抗与抗原抗体复合物结合:在保鲜盒内加入8mL 1×TBST洗3次,每次5min。然后倒掉冲洗液,加入二抗溶液(1:5000稀释),摇床上室温孵育2h。
(7)显色:在保鲜盒内加入10mL 1×TBST洗3次,每次5min。取出PVDF膜保持蛋白面朝上,将化学发光底物的A和B液的等量混合覆盖PVDF膜进行避光显色,用化学发光成像系统进行图像采集。
实施例4下调duDDX3X的表达抑制内源性IFN-β的表达
siRNA干扰duDDX3X
duDDX3X的siRNA(small interference RNA,siRNA),三对靶序列由吉玛公司合成),
将siRNA配成20μM的母液-20℃储存备用,工作母液为2μM,全程严格无菌,用无RNA酶的枪头进行操作。
siRNA干扰效率的检测
转染试剂用jetTransfection Reagent,按照说明书进行。分别将siduDDX3X-1、 siduDDX3X-2和siduDDX3X-3以及阴性对照siNegative control按1.0μg/孔,加入到试剂盒配套的转染buffer中,涡旋混匀后加入2倍siRNA剂量的转染试剂,每个干扰分子做3次重复,再次涡旋混匀,瞬时离心后静置10min,即可加入DEF细胞中。4h后换2%细胞维持液, 36h后在无菌操作台中用TRIZOL吹打收样,提取细胞中的总RNA反转成cDNA后,用荧光定量PCR技术检测细胞中内源性DDX3X的表达情况,从而筛选出干扰效率最高的干扰分子。结果表明siduDDX3X-2的干扰效率最高(图5A)。
siRNA的转染如下:
转染试剂用jetTransfection Reagent,按照说明书进行。将鸭源IFN-β启动子的荧光素酶报告质粒0.1μg/孔、内参海参荧光素酶TK质粒0.01μg/孔依次加入到jet Transfection Reagent试剂盒配套的转染buffer中,涡旋混匀后分别加入筛选出的干扰分子 siduDDX3X-2和阴性对照siNegative control,涡旋混匀后再加入2倍质粒剂量的转染试剂,涡旋混匀后瞬时离心,静止10min后即可加入到24孔板中,4h后换上含2%FBS的无双抗细胞维持液。24h后用poly(I:C)刺激或感染DTMUV,过夜培养后收集用细胞裂解液收集样品,然后按照双荧光素酶报告基因检测试剂盒说明书进行检测荧光素酶的活性。结果如图5B 所示,结果表明DDX3X的表达被降低后可以使poly(I:C)刺激的IFN-β表达显著降低。
实施例5过表达duDDX3X可以抑制DTMUV的复制
表达duDDX3X的载体和空载体分别转染DEF细胞,转染24小时后,感染DTMUV(MOI=1),感染24小时后,分别收取细胞培养上清即病毒上清。将收获的病毒上清按10倍梯度在离心管中逐级稀释(10-1~10-8),用DEF细胞进行病毒半数组织培养物感染剂量(TCID50)实验,检测上清中病毒的滴度,具体的操作过程如下,结果如图6所示,结果显示过表达DDX3X 24h和48h后,与mock组(空白对照)和空载组(pCAGGS-Flag组)相比,细胞总培养物的病毒TCID50下降了1~2个滴度,表明duDDX3X具有抑制DTMUV增殖的作用。
TCID50实验
(1)前一天将DEF细胞以2×105/mL细胞悬液,传代于96孔细胞培养板中,每孔100μL,置于37℃的5%CO2细胞培养箱中培养过夜。使细胞长成单层(铺满80%以上)即可用于病毒含量测定。
(2)取出-80℃冻存的DTMUV病毒液,融化后用含2%FBS的DMEM培养基在灭菌的1.5mL离心管内作10倍数量级系列稀释(10-1~10-8),涡旋混匀,另取1管加入900μL含 2%FBS的DMEM细胞培养液作为对照孔培养基。
(3)将不同稀释度的病毒液加入到96孔板,每个稀释度接种8个平行孔,每孔100μL。加样时先加空白对照,再由高稀释度到低稀释度依次加入。
(4)将96孔板放入5%CO2细胞培养箱中37℃培养,逐日观察,第5天观察细胞病变并记录结果,根据Reed-Muench法计算TCID50。
显然,上述实施例仅仅是为清楚地说明所作的实例,而并非对实施方式的限制。对于所属领域的普通技术人员来说,在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而因此所引申的显而易见的变化或变动仍处于本发明创造的保护范围之内。
序列表
<110> 湖北省农业科学院畜牧兽医研究所
<120> 一种鸭源天然免疫调节蛋白DDX3X的应用
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<213> 人工序列
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acgugacacg uucggagaat t 21
Claims (1)
1.鸭源天然免疫调节蛋白DDX3X在鸭胚成纤维细胞中调控天然免疫信号通路、参与抗病毒反应及在鸭抗病毒感染中的应用。
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Cited By (1)
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CN112481263A (zh) * | 2020-12-09 | 2021-03-12 | 福建省农业科学院畜牧兽医研究所 | 抑制鸭RIG-I基因表达的siRNA |
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