CN105861559B - 一种能直接反应i型干扰素应答的慢病毒载体及其制备方法与应用 - Google Patents
一种能直接反应i型干扰素应答的慢病毒载体及其制备方法与应用 Download PDFInfo
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Abstract
本发明提供了一种能直接反应I型干扰素应答的慢病毒载体，通过克隆技术将pDonor‑IRF3全长的穿梭质粒同源重组克隆到慢病毒BiLC表达载体中；所述慢病毒BiLC表达载体由高斯荧光素酶从109位氨基酸的位置分为N端和C端，记为GlucN和GlucC，形成慢病毒表达载体IRF3‑BiLC。本发明的有益效果：第一：与目前是用的I型干扰素应答报告系统相比，IRF3‑BiLC的报告系统具有特异的反应I型干扰素的应答。第二：IRF3‑BiLC的报告系统能够直接且快速的反应瞬时的或者是持续性的I型干扰素的应答。
Description
技术领域
本发明涉及一种能直接反应I型干扰素应答的慢病毒载体及其制备方法与应用，属于医药技术领域。
背景技术
人体在遭受病毒如流感病毒、丙肝病毒和疱疹病毒等以及细菌感染会引起天然免疫(innate immunity)和获得性免疫反应(adaptive immunity)(1)。其中，天然免疫反应主要是通过诱导干扰素(Interferon,IFN)的产生，激活下游的干扰素诱导基因(ISGs)，从而发挥抗病毒作用(2,3)。对于RNA病毒，如流感病毒和丙肝病毒，病毒侵染进入细胞，释放出来的RNA能被细胞内的RNA识别受体如RIG-I、MDA5等，然后招募线粒体上的IPS-1蛋白以及磷酸化TBK1蛋白，被磷酸化的TBK1蛋白能够引起IRF3蛋白的磷酸化和二聚体的形成，从而进入细胞核引起干扰素的产生(4)。对于DNA病毒，如疱疹病毒和腺病毒，其进入体内后释放出来的DNA能被DDX41、cGAS以及IFI16等DNA识别受体识别，并诱导STING的多聚，从而引起TBK1的磷酸化以及IRF3二聚的活化，进而使干扰素转录翻译(5-7)。对于细菌侵染，主要是通过细胞膜上的TLR4识别受体识别细菌的LPS物质，从而引起下游TRIF、TRAF6、TBK1以及IRF3的二聚体的激活，从而使干扰素应答反应。产生的干扰素能够通过诱导ISGs的表达，从而使细胞能够清除侵染进入的RNA病毒或者DNA病毒。因此，干扰素应答在病毒和细菌侵染过程中异常重要。为了更好和有效地抵抗病毒以及细菌的侵染以及清除，对干扰素应答的调节机制的研究尤为重要。
目前，干扰素激活反应元件的荧光素酶报告(ISRE-Luc)做为研究干扰素产生机制研究的主要报告系统。ISRE-Luc是一个模拟细胞基因组的反应过程，其拥有一个与基因组一样序列的ISRE启动子，启动子后接有一个全长的人源化的萤火虫荧光素酶的表达基因。当细胞遭受病毒或者细菌感染时，其能引起细胞内的干扰素应答反应，从而是ISRE启动子转录活化，从而引起荧光素酶基因的转录翻译和表达。因此，通过检测萤火虫荧光素酶的活性，可以反应细胞内干扰素应答反应的强度。目前，其主要用于一些蛋白分子或者是药物对干扰素应答调节机制的研究和筛选。然后，ISRE的转录激活能被IRF3二聚体、IRF7二聚体识别并发生转录(8)；同时转录翻译产生的干扰素能够二次激活放大通过干扰素识别受体(IFNAR1等),引起STAT1/STAT2/IRF3(ISGF3)三聚体的形成，并能识别ISRE并引起转录(9,10)。因此，ISRE-Luc不能特异及精确地反应细胞内干扰素调节机制；其次，由ISRE的二次放大需要依赖细胞的干扰素识别受体(IFNAR1等)，因此，其不适合用于敲除IFNAR1或者是STAT1等活性收到影响等体系的条件下检测细胞的干扰素调节机制；第三，ISRE-Luc需要转录和翻译水平，其不能很好地反应对于一些分子或者药物在转录水平或者是翻译水平对干扰素应答的调节。
综上所述，一个特异、灵敏、直接且被能广泛应用的干扰素应答反应的报告系统具有广阔的应用前景。
以上背景技术中提及的文献具体分别参考于以下：
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2.Yan,N.,and Z.J.Chen.2012.Intrinsic antiviral immunity.Nat Immunol13:214-222.
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5.Zhang,Z.Q.,B.Yuan,M.S.Bao,N.Lu,T.Kim,and Y.J.Liu.2012.The helicaseDDX41senses intracellular DNAmediated by the adaptor STING in dendritic cells(vol 12,pg 959,2011).Nat Immunol 13:196-196.
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发明内容
本发明的目的在于解决上述的技术问题，提供一种灵敏特异直接反应I型干扰素应答的IRF3-BiLC报告系统。
本发明的目的通过以下技术方案来实现：
一种能直接反应I型干扰素应答的慢病毒载体，通过克隆技术将pDonor-IRF3全长的穿梭质粒同源重组克隆到慢病毒BiLC表达载体中；所述慢病毒BiLC表达载体由高斯荧光素酶从109位氨基酸的位置分为N端和C端，记为GlucN和GlucC，形成慢病毒表达载体IRF3-BiLC。
优选地，所述慢病毒载体可以构建能直接反应I型干扰素应答的细胞系。
优选地，所述细胞系为THP-1(IRF3-BiLC)细胞系或THP-1-Dual细胞系。
优选地，所述细胞系构建方法包括如下步骤：
S1、质粒的构建：构建pEntry-IRF3质粒:通过特异性引物将IRF3基因进行扩增，并对扩增产物进行双酶切，然后通过T4连接酶连接至载体，提取pEntry-IRF3全长的穿梭质粒；
S2、慢病毒表达载体构建：采用GlucN和GlucC部分，分别克隆到慢病毒载体上；
S3、慢病毒载体构建：通过克隆技术，将构建好的pEntry-IRF3全长的穿梭质粒同源到可诱导型的慢病毒表达载体，形成pBiLC-IRF3-GlucN和pBiLC-IRF3-GlucC分别表达IRF3-GlucN融合蛋白、及IRF3-GlucC融合蛋白；
S4、稳定表达IRF3-BiLC的细胞的构建：表达IRF3-GlucN融合蛋白载体、表达IRF3-GlucC融合蛋白载体通过pMDLg/pRRE/，pRSV-Rev,pMD2.G三质粒系统整合于相对应的细胞内；IRF3基因用于扩增的特异性引物，所述特异性引物的序列为SEQ ID NO:1、SEQ ID NO:2。优选地，所述S1中IRF3基因的扩增条件为95℃预变性2min；再进行30个循环，循环所遵循条件为95℃变性20s，56℃退火30s，72℃延伸1min；最后采用72℃延伸5min。
优选地，所述的IRF3-BiLC报告系统的IRF3-GlucN和IRF3-GlucC是基于慢病毒载体系统作为稳转使用。
本发明的有益效果：
第一：与目前是用的I型干扰素应答报告系统相比，IRF3-BiLC的报告系统具有特异的反应I型干扰素的应答。
第二：IRF3-BiLC的报告系统能够直接且快速的反应瞬时的或者是持续性的I型干扰素的应答。
附图说明
图1是构建IRF3-BiLC质粒结构示意图。
图2是检测THP-1(IRF3-BiLC)在不同的刺激条件下诱导的IRF3二聚体信号值。
图3是检测不同IFN刺激对ISRE-Luc的影响。
图4是检测不同IFN刺激对IRF3-BiLC的影响。
图5是检测CHX对IRF3-BiLC的影响。
图6是检测CHX对ISRE-Luc的影响。
具体实施方式
本发明具体揭示了一种灵敏特异直接反应I型干扰素应答的IRF3-BiLC报告细胞系以及构建方法。其具体包括：
1、IRF3-BiLC慢病毒表达载体的构建
BiLC的原理为：将高斯荧光素酶(Gaussia luciferase)在某些特定的位点切开，形成没有荧光素酶活性的N和C端两个多肽，称为N片段(N-fragment)和C片段(C-fragment)(Remy andMichnick,2006；Cassonnet et al.,2011；Tannous et al.,2005)。这两个片段在细胞内共表达或体外混合时，不能自发地组装成具有活性的荧光素酶蛋白。但是，当这两个荧光素酶蛋白的片段分别连接到一组有相互作用的目标蛋白上，在细胞内共表达或体外混合这两个融合蛋白时，由于目标蛋白质的相互作用，荧光素酶蛋白的两个片段在空间上互相靠近互补，重新构建成完整的具有活性的荧光素酶蛋白分子，在底物腔肠素(CTZ)的条件下，以CTZ为底物发光。简言之，如果目标蛋白质之间有相互作用，则会有荧光素酶以CTZ为底物产生的光。反之，若蛋白质之间没有相互作用，则不有荧光素酶的活性。
将Gaussialuciferase从109位氨基酸的位置分为N端和C端，并将N端的16个氨基酸去掉，记为GlucN和GlucC。在上海捷锐公司合成人源化的GlucC和GlcuN基因片段，分别通过AscI and RsrII(购自NEB)酶切位点，通过T4链接酶(购自NEB)进入慢病毒慢病毒载体上(US20120201794A1)，标记为pBiLC1-2。
IRF3(NM_001197122.1)基因则是通过设计特异的引物已人源细胞的cDNA作为模板扩增，所诉扩增条件95℃预变性2min；再进行30个循环，循环所遵循条件为95℃变性20s，56℃退火30s，72℃延伸1min；最后采用72℃延伸5min。以上所述引物序列见表1。
表1：引物序列表
SEQ ID NO:1PA-F ATAGCGGCCGCAATGGGAACCCCAAAGCC
SEQ ID NO:2PA-R GGCGCGCCCTTGGTTGAGGTGGTGGGG
用NotI、AscI(购自NEB公司)双酶切PCR产物和pEntry载体(购自Invitrogen公司)，用T4连接酶(购自NEB公司)连接片段到pEntry载体上，测序鉴定正确后提质粒保存记为pEntry-IRF3。通过Gateway cloning技术，将pEntry-IRF3克隆到慢病毒BiLC表达载体即pBiLC-IRF3-GlucN和pBiLC-IRF3-GlucC，分别表达IRF3-GlucN和IRF3-GlucC的融合蛋白。构建的质粒如图1所示。
检测IRF3-BiLC对于不同刺激的应答
构建具有稳定表达IRF3-BiLC的THP-1细胞系。具体操作步骤如下：
a，慢病毒包装：转染的前一天铺HEK293T细胞(ATCC：CRL-11268)于24孔板(购自Thermo公司)，500uLDMEM(购自Invitrogen)全培养基(10％FBS,购自Gibco)；待细胞长至50％-60％密度时转染；每孔转染的质粒共约1ug，其中pMDLg/pRRE:pRSV-Rev:pMD2.G:IRF3-GlucN/IRF3-GlucC＝4:2:1:2。转染8h后，去除培养基，补充1mL新鲜的培养基。48h后收取上清放入EP管中，2500rpm离心4min。取上清转移到新的EP管中以用于病毒侵染。
b，病毒侵染：在收取病毒前18h，用100uL的DMEM铺10,000个THP-1细胞(Invivogen)于96孔板(购自Thermo公司)中。在侵染前，移去约50uL的RMPI 1640。在收取的上清病毒液中加入6ug/mL的polybrene(购自sigma)，混匀，每个96孔中加入约100uL的病毒液。待侵染6-8h后，移去50uL96孔中的部分培养基，加入100uL新鲜的培养基。72h后即可从96孔板中将细胞传出筛选并扩大培养，即得到THP-1(IRF3-BiLC)细胞系。
得到的THP-1(IRF3-BiLC)细胞系用于测试对于不同刺激下IRF3的二聚化形成的荧光素酶活性。分别用TNF、IL-1B、LPS、polyI:C、polydA:dT以及VSV-eGFP刺激。用80uL海肾荧光素酶裂解液(购自Promega，E2820)，冰上裂解10min，用移液器吹匀，转移50uL细胞裂解液到荧光素酶检测板(购自PE),每孔加入20uL海肾荧光素酶底物(购自Promega，E2820)，用酶标仪来检测(购自Bio-Tek，SynergyH1)荧光素酶活性。结果如图2所示，TNFa、IL-1B不能引起IRF3二聚信号的形成，而LPS、polyI:C、polydA:dT以及VSV-eGFP能不同程度的引起IRF3二聚信号。
检测IRF3-BiLC反应干扰素应答反应的特异性
铺THP-1-Dual(具有稳定表达的ISRE-Luc报告系统，购于Invitogen公司)或THP-1(IRF3-BiLC)细胞,以1000,000个细胞每mL的浓度铺到24孔板中。14小时后，转染。分为8组。其中Dual的四组中，一组记为NT组，其他三组分别加入IFNα(终浓度10ng/mL)、IFNβ(终浓度10ng/mL)、IFNγ(终浓度20ng/mL)；IRF3-BiLC的四组中做与ISRE-Luc的四组同样的处理。24小时，分别检测萤火虫荧光素酶(以海肾荧光素酶作为内参)和高斯荧光素酶(异萤火虫荧光素酶作为内参)。
结果如图3-图4所示，IRF3-BiLC组不受干扰素二次放大激活的影响，而ISRE-Luc组能被很强的影响，且不能特异的反应I型干扰素的诱导。
检测IRF3-BiLC应用的广泛性
为了验证IRF3-BiLC广泛性应用，设置了如下实验：
1.转录抑制剂存在的条件下应用
THP-1-Dual(Invivogen)具有稳定表达的ISRE-Luc报告系统。将THP-1-Dual和THP-1(IRF3-BiLC)细胞，以1000,000个细胞每mL的浓度铺到24孔板中，分别设置4个小组，共8组。12小时后，对于THP-1-Dual细胞用Lipo2000转染5mg/mL的polydA:dT刺激其中2组细胞，另外两组不做处理。对于THP-1(IRF3-BiLC)也同样的处理。转染4小时候后，对于THP-1-Dual细胞转染了polydA:dT刺激的2组，一组加入终浓度为100ng/mL的CHX，对于不做处理的两组中的一组加入终浓度为100ng/mL的CHX，其他两组则不做处理。对于THP-1(IRF3-BiLC)也同样的处理。加入CHX处理10h后，对于THP-1-Dual细胞则分别取40uL上清检测荧光素酶活性(购自Promega，E2820)，对于THP-1(IRF3-BiLC)用80uL海肾荧光素酶裂解液(购自Promega，E2820)，冰上裂解10min，用移液器吹匀，转移50uL细胞裂解液到荧光素酶检测板(购自PE),每孔加入20uL海肾荧光素酶底物(购自Promega，E2820)，用酶标仪来检测(购自Bio-Tek，SynergyH1)荧光素酶活性。将检测的荧光素酶的值以未处理组做归一化处理。
结果如图5-图6所示，IRF3-BiLC能在存在CHX的情况下，其能反应细胞内I型干扰素的应答反应。ISRE-Luc则受到了明显的影响。
Claims (4)
1.一对能直接反应I型干扰素应答的慢病毒载体pBiLC-IRF3-GlucN和pBiLC-IRF3-GlucC，其特征在于由以下方法构建得到：
步骤1、通过特异性引物将IRF3基因进行扩增，并对扩增产物进行双酶切，然后通过T4连接酶连接至pEntry载体，提取得到的pEntry-IRF3全长的穿梭质粒；
步骤2、高斯荧光素酶从109位氨基酸的位置分为N端和C端，记为GlucN和GlucC，采用GlucN和GlucC部分分别克隆到慢病毒载体上；
步骤3、通过克隆技术，将构建好的pEntry-IRF3全长的穿梭质粒同源重组克隆到步骤2中得到的可诱导型的慢病毒表达载体，形成能够分别表达IRF3-GlucN融合蛋白和IRF3-GlucC融合蛋白的慢病毒载体pBiLC-IRF3-GlucN和pBiLC-IRF3-GlucC；
用于IRF3基因扩增的特异性引物的序列为SEQ ID NO:1和SEQ ID NO:2。
2.如权利要求1所述的一对能直接反应I型干扰素应答的慢病毒载体的应用，其特征在于：用于构建能直接反应I型干扰素应答的细胞系。
3.如权利要求2所述的一对能直接反应I型干扰素应答的慢病毒载体的应用，其特征在于：所述细胞系为THP-1(IRF3-BiLC)细胞系。
4.如权利要求2所述的一对能直接反应I型干扰素应答的慢病毒载体的应用，其特征在于：所述细胞系的构建方法包括如下步骤：
将慢病毒载体pBiLC-IRF3-GlucN和pBiLC-IRF3-GlucC通过pMDLg/pRRE, pRSV-Rev,pMD2.G三质粒系统整合于相对应的细胞内，得到能够稳定表达IRF3-GlucN融合蛋白和IRF3-GlucC融合蛋白的细胞。
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| CN105861559B (zh) | 2016-04-11 | 2018-05-01 | 苏州奥特铭医药科技有限公司 | 一种能直接反应i型干扰素应答的慢病毒载体及其制备方法与应用 |
| CN114350615B (zh) * | 2021-12-20 | 2024-04-16 | 北京镁伽科技有限公司 | Stat2基因缺失细胞株及其制备方法和应用 |
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| RU2015133046A (ru) * | 2009-07-15 | 2018-12-24 | Кэлиммьюн Инк. | Двойной вектор для подавления вируса иммунодефицита человека |
| CN105861559B (zh) | 2016-04-11 | 2018-05-01 | 苏州奥特铭医药科技有限公司 | 一种能直接反应i型干扰素应答的慢病毒载体及其制备方法与应用 |
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Non-Patent Citations (4)
| Title |
|---|
| A highly sensitive protein-protein interaction assay based on Gaussia luciferase;Ingrid Remy等;《NATURE METHODS》;20061231;第3卷(第12期);977-979 * |
| Benchmarking a luciferase complementation assay for detecting protein complexes;Patricia Cassonnet等;《NATURE METHODS》;20111231;第8卷(第12期);990-992 * |
| Complex Regulation Pattern of IRF3 Activation Revealed by a Novel Dimerization Reporter System;Zining Wang等;《JOURNAL OF IMMUNOLOGY》;20160404;第196卷(第10期);4322-4330，具体参见摘要，材料与方法部分 * |
| 干扰素调节因子3研究进展;陈建忠等;《国外医学-流行病学传染病学分册》;20030831;第30卷(第4期);223-226 * |
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| Publication number | Publication date |
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| US20190119700A1 (en) | 2019-04-25 |
| CN105861559A (zh) | 2016-08-17 |
| WO2017177826A1 (zh) | 2017-10-19 |
| US11384366B2 (en) | 2022-07-12 |
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