CN109536492B - 人cGAS基因启动子区转录调控元件及其应用 - Google Patents
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Abstract
本发明公开了人cGAS基因启动子区转录调控元件及其应用。人cGAS基因启动子转录调控区域,核苷酸序列如SEQ ID NO:1所示。第765bp~第1254bp所示的为cGAS基因启动子区主要转录活性调控区段。这段序列中有一个CREB结合位点,两个SP1结合位点。一种载体,它含有所述的人cGAS基因启动子区转录调控元件或人cGAS基因启动子区主要转录活性调控区段。所述的启动子区转录调控元件可操作地连接的外源基因。本发明首次鉴定了人cGAS基因启动子关键调控元件,能够在构建含有人cGAS基因启动子的重组表达载体中应用。
Description
技术领域
本发明涉及基因表达调控,具体地说,涉及人cGAS基因启动子区的转录调控元件及其应用。
背景技术
艾滋病、严重急性呼吸综合症(SARS)、肝炎、流感等传染病的广泛存在表明人类与感染性疾病的斗争远未停止,SARS、流感的大流行已经引起了国际社会对病毒感染性疾病的普遍担忧和高度关注。病毒感染已经对人类的健康构成了严重威胁。人体对病毒感染的反应如感染的严重程度、是否慢性化等表现出较大的个体差异,深入研究造成个体易感性差异的相关基因的结构和调控则有利于充分认识病毒感染性疾病的过程和找到有效的治疗方法[1]。
抗病毒固有免疫反应是机体抵抗病毒感染的第一道防线,在抗病毒免疫过程中具有关键作用[2]。抗病毒固有免疫反应涉及多个重要蛋白分子,其中环磷酸鸟苷-腺苷合成酶(Cyclic guanosine monophosphate-adenosine monophosphate synthase,cGAS)在抗病毒固有免疫反应中的关键性作用近年已经得到了越来越多的重视[3]。cGAS属于核苷酸转移酶家族的成员,可以识别胞质DNA,催化第二信使环磷酸鸟苷-腺苷(cyclic guanosinemonophosphate-adenosine monophosphate,cGAMP)的合成,并通过STING(stimulator ofinterferon genes)依赖的方式活化转录因子IRF3,介导Ⅰ型干扰素的产生,启动机体的抗病毒固有免疫反应。过度表达cGAS后,IRF3则被激活,I型IFN及其依赖基因的表达增强;cGAS被敲低后,IRF3激活途径明显受到抑制,I型IFN及其依赖基因的表达明显降低。cGAS基因敲除小鼠对DNA病毒感染和DNA转染缺乏有效地应答,不能产生I型IFN[3-11]。
目前对cGAS的研究主要集中在其活化下游STING和转录因子IRF3的分子机制及其在不同病原相关分子模式诱导宿主产生固有免疫应答过程中的功能方面。而cGAS自身的表达调控机制尚不清楚。该基因表达调控机制的阐明对进一步揭示抗病毒固有免疫信号通路及防治病毒感染性疾病具有重要意义。
在基因表达调控方面启动子和mRNA的可变剪接起十分关键的作用。启动子包含了核心启动子和近端启动子。核心启动子指引RNA聚合酶Ⅱ的精确转录起始,它包含TATA盒、启动因子(initiator,Inr)、下游启动子成分(downstream promoter element,DPE)、转录因子ⅡB识别成分(TFⅡB recognition element,BRE)和基序十要素(motif ten element,MTE),这些不同成分的组合可形成不同类型的核心启动子[12]。不同核心启动子可指引产生不同的剪接异构体。近端启动子则包含了转录因子结合位点。功能转录因子结合位点的发现有助于进一步揭示靶基因上游的信号通路,Ishikawa等就是通过使用含IRF3调控元件的IFN-β启动子作为报告子,成功筛选出位于上游的具有活化IRF3功能的重要蛋白分子STING。功能转录因子结合位点的确定也为阐明转录因子结合位点变异相关疾病的发病机制提供依据[13]。最近研究证实14%-58%的人类基因具有多个可变的启动子[11],而这些可变启动子的特性以及生物学功能还未得到充分认识。可变剪接是从一个mRNA前体中选择不同的剪接位点而组合产生出不同的mRNA剪接异构体的过程。目前已知至少74%的人类多外显子基因被可变剪接,不同剪接异构体结构不同而功能多样[14]。与蛋白翻译后修饰相比剪接异构体对基因表达的调控更加精细和持久。研究发现与抗病毒固有免疫相关的多个基因,如TBK-1、TRAM25、MAVS和IRF3包含了多个剪接异构体,这些剪接异构体在抗病毒固有免疫信号的调控中发挥了重要作用[15-19]。
目前,尚未见到有关cGAS启动子和剪接异构体的研究报道。
发明内容
本发明的目的在于鉴定人cGAS基因启动子区。
本发明的另一个目的在于确定人cGAS基因的启动子区转录调控元件并提供含有上述基因启动子区转录调控元件的载体。
本发明提供的人cGAS基因启动子转录调控区域,该基因得核苷酸序列包含SEQDNO:1中第765bp~第1254bp所示的cGAS基因启动子区
主要转录活性调控区段。
所述的人cGAS基因启动子转录调控区域优选自以下任意一种:
(1)核苷酸序列如SEQ ID NO:1所示,位于人cGAS基因5'上游-1178~+76的片段;SEQ ID NO:1中第1179bp为转录起始位点,即为+1;
(2)SEQDNO:1所示的核苷酸序列中发生一处或多处点突变,但依然具备人cGAS基因启动子功能的核苷酸序列。
为了获得本发明的cGAS基因的表达调控元件,正如在下述实施所详细描述的,对人cGAS基因的启动子区进行缺失,插入到pGL3-Basic载体,得到一系列序列长度不同的表达质粒,利用萤火虫荧光素酶报告基因,检测这些质粒在Hela和HEK 293T细胞中的转录活性。为减少细胞培养微环境所造成的误差,以表达海肾荧光素酶的质粒pRL-TK作为内参照,与上述缺失质粒共同转染Hela和HEK 293T细胞,测定萤火虫荧光素酶与海肾荧光素酶的酶活性,用二者的比值,即相对荧光素酶活性表示启动子转录活性。经过多次实验结果验证:-414~+76之间即SEQDNO:1中第765bp~第1254bp所示的核苷酸序列区段为主要转录活性调控区段。
对-414~+76这段的序列分析表明:这段序列中有一个CREB结合位点(831bp-838bp),SP1一个结合位点(840bp-853bp),SP1另一个结合位点(989bp-999bp)。
本发明针对CREB、SPI结合位点作进一步修饰,即点突变,检测修饰调控元件的重组质粒的表达能力,分析上述调控元件对cGAS启动子转录活性的影响。结果显示,-414~+76这段的序列是基因启动子关键调控序列,CREB、SP1结合位点是启动子转录活性的关键调控元件。
一种载体,它含有所述的人cGAS基因启动子区转录调控元件或人cGAS基因启动子区主要转录活性调控区段。
所述的载体,还优选含有所述的启动子区转录调控元件可操作地连接的外源基因。
所述外源基因优选报告基因。
一种宿主细胞,含有本发明所述的载体。
所述的宿主细胞优选不具遗传功能的哺乳动物细胞。
本发明所述的人cGAS基因启动子区转录调控元件在构建含有人cGAS基因启动子的重组表达载体中的应用。
与现有技术相比,本发明具有如下有益效果:本发明首次鉴定了人cGAS基因启动子关键调控元件,并将5'端序列逐渐缺失和部分调控元件被修饰的启动子序列插入报告基因上游构建了一系列真核表达质粒,转染哺乳动物细胞瞬时表达,确定了人cGAS基因启动子在转录调控的关键序列。本发明对研究cGAS基因的表达调控机制具有重要意义。对今后进一步深入研究cGAS的生物学功能和揭示抗病毒固有免疫信号通路有重要意义。
附图说明
图1为5’RACE产物凝胶电泳图;
在cGAS第3外显子上设计两条反义引物作RACE的巢式PCR引物,用Hela细胞的RNA为模板,得到如图1-1所示的4个条带,测序后发现A条带为野生型(与已发表的cGAS mRNA序列一致);B条带用了第1外显子下游36bp的序列作为外显子;C条带用了位于第1内含子内即第一外显子下游的406bp的序列作为外显子;D条带用了位于第1内含子内即第一外显子下游的450bp的序列作为外显子
其中M:DNA Marker 5000;从上到下依次为5000、3000、2000、1500、1000、700、500、200、100;泳道1:5’RACE产物;
图2为转染HELA细胞及HEK293细胞,检测相对荧光素酶活性;
图3为cGAS启动子缺失突变体在HEK293T细胞中相对荧光素酶活性分析。
图4为ChIP实验结果,证实转录因子SP1和CREB和cGAS启动子在siha细胞中的结合。
具体实施方式
实施例1确定cGAS的mRNA的起始和终止点,寻找新的剪接异构体
5'和3'快速cDNA末端扩增法(Rapid Amplification of cDNA Ends,RACE),可以获得不同异构体5'端和3'端的大致位置、各自启动子的大致位置以及结合RT-PCR测序结果获得新的剪接异构体的信息。利用CLONTECH公司的SMART 5'RACE反转录酶PowerScriptTMReverse Transcriptase特殊的反转录能力进行5'末端的扩增。首链cDNA的合成将2μl的RNA与1μl的5'-CDS Primer、1μl的SMART II oligo和0.5μl的RNase inhibitor混合均匀,70℃放置5min后冰浴2min,短暂离心;随后加入2μl的5×First-Strand Buffer,1μl的dNTPMix(各10mmol/L)和2μl的DTT(100mmol/L),混匀后再加入1μl的PowerScriptTM ReverseTranscriptase,55℃下反应90min,加入50μl的Tricine-EDTA Buffer稀释,再72℃处理7min后,短暂离心。将前一步获得的反应液为模板,以DK 01和UPM为引物,用Ex Taq酶PCR,反应条件如下:94℃变性5min;94℃反应30s,72℃反应3min,进行5次循环;94℃反应30s,70℃反应30s,72℃反应2min,进行5次循环;94℃反应30s,68℃反应30s,72℃反应2min,进行25次循环;然后72℃延伸10min。将第一次PCR的反应液用Tricine-EDTA Buffer稀释100倍为模板,以DK09与NUP为引物,用Ex Taq酶进行第二次PCR,反应条件如下:94℃变性5min;94℃反应30s,68℃反应30s,72℃反应2min,进行20次循环;然后72℃延伸10min。将第二次PCR产物电泳观察结果。
简而言之,根据全长cGAS的mRNA序列设计RACE引物。抽提人的经病毒感染的或非感染的培养细胞的RNA,逆转录总RNA成cDNA。用接头引物和RACE引物作巢式PCR,亚克隆至载体并测序。
测序序列用美国国家医学图书馆国立生物技术信息中心的BLAST分析软件判断新的剪接异构体的结构及各自启动子的位置。
在cGAS第3外显子上设计两条反义引物作RACE的巢式PCR引物,两条反义引物序如下:GATTACGCCAAGCTTCTTGGGTGCTAGCAGGCCAGCTAC(SEQ ID NO.2);GATTACGCCAAGCTTGTAGCTGGCCTGCTAGCACCCAAGAAGG(SEQ ID NO.3)。
用Hela细胞的RNA为模板,得到如图1所示的4个条带,测序后发现A条带为野生型(与已发表的cGAS mRNA序列一致);B条带用了第一外显子即野生型转录起始位点下游36bp作为转录起始位点;C条带用了位于第1内含子内即第一外显子下游的406bp的序列作为外显子;D条带用了位于第1内含子内即第一外显子下游的450bp的序列作为外显子。
实施例2克隆不同长度的5'侧翼和可能的下游启动子元件区域做荧光素酶功能分析,确定核心启动子的范围和类型。
根据引物延伸法获得的转录起始位点信息,克隆其5'上游不同长度的基因组DNA片断,亚克隆至含荧光素酶的pGL3-Basic载体,先作初步荧光素酶测定,如果确定为启动子部位,则作进一步的缺失变异功能分析,确定核心启动子的范围、是否有TATA、InR和DPE。
(1)对cGAS野生型启动子进行特征分析。首先在NCBI网站及UCSC网站获得cG AS的5’端序列,利用Promoter 2.0Prediction Serve(http://www.cbs.dtu.dk/services/Promoter/)对野生型转录起始位点上游约1500bp的序列进行预测,结果显示该序列具有启动子活性。同时利用Neural Network Promoter Pre diction(http://fruitfly.org/cgi-bin/seq_tools/prom-oter.pl)对该序列进行预测,得分为0.82。将此区域作为启动子的候选区域,并将其序列亚克隆至无启动子活性的pGL3-Basic质粒中。启动子引物序列如下:
F 5'-GGGGTACCCAGTGGCTCATGCCTACAAT-3’(SEQ ID NO.4)
R 5'-GAAGATCTCTGTTGGAAACCAAGCACTACT-3'(SEQ ID NO.5)
(2)构建野生型启动子:
以Hela细胞的全基因组DNA为模板,根据设计的引物,利用PCR技术扩增cGAS 5’端序列,PCR产物进行核酸凝胶电泳,可见一条介于1000和1500bp大小的条带,与目的条带(1254bp)大小相同。将PCR产物及pGL3-Basic质粒进行双酶切,双酶切产物切胶回收,利用T4连接酶进行连接构建重组报告质粒。转化大肠杆菌后挑取单克隆进行菌落扩增及质粒小提,将获得的质粒进行测序验证,测序结果明质粒构建成功。
(3)cGAS野生型启动子在Hela和HEK 293T细胞中具有较强的活性
HEK293T细胞用含10%灭活胎牛血清的高糖DMEM培养基培养,置于37℃,5%CO2培养箱中,2d传代一次。实验操作均采用对数期生长细胞。
荧光素酶报告基因实验检测采用Promega公司的Dual-Luciferase ReporterAssay System进行分析。将转染24h后的HEK293T和Hela细胞用1×PBS洗涤一次,去尽残液,加入20ul新鲜配制的I×PLB,室温下,摇床上剧烈振荡15min以裂解细胞。取20ul上述细胞裂解液加入含100ulLARII溶液的1.5mlEP管中,混匀,测定荧火虫荧光素酶活性,再向同一EP管中加入100ul新鲜的1×STOP液,混匀,测定海肾荧光素酶(内参照)活性,计算出两种荧光素酶活性比值,即相对荧光素酶活性,每次结果重复至少3次。
对构建的cGAS野生型启动子报告质粒进行荧光素酶活性分析。结果表明启动子在Hela和HEK293T细胞中具有较强活性(图2)。
(4)cGAS野生型启动子特征的初步分析
本发明以siha细胞cDNA为模板,表1所示引物通过PCR对cGAS野生型启动子的5'端进行逐步缺失,5个缺失突变体,并将其克隆至PGL3-Basic质粒。缺失突变体1序列如SEQDNO:1中第1bp-1254bp所示;缺失突变体2如SEQDNO:1中第765bp-第1254bp所示;缺失突变体3如SEQDNO:1中第975bp-第1254bp所示;缺失突变体4如SEQDNO:1中第1133bp-第1254bp所示;缺失突变体5如SEQDNO:1中第1179bp-第1254bp所示。
表1
引物名称 | 引物序列 |
缺失突变体1上游引物 | 5'GGGGTACCCAGTGGCTCATGCCTACAAT 3'(SEQ ID NO.6) |
缺失突变体2上游引物 | 5'GGGGTACCCGCCTAGCTAATATTTGTATTT 3'(SEQ ID NO.7) |
缺失突变体3上游引物 | 5'GGGGTACCCTGGTCTCAAACTCCTGAGCT 3'(SEQ ID NO.8) |
缺失突变体4上游引物 | 5'GGGGTACCGCGGCCAGCCTCTTCGCGGC3'(SEQ ID NO.9) |
缺失突变体5上游引物 | 5'GGGGTACCAGCCTGGGGTTCCCCTTCGG3'(SEQ ID NO.10) |
共用下游引物 | 5'GAAGATCTCTGTTGGAAACCAAGCACTACT 3'(SEQ ID NO.11) |
将它们分别转染入HEK293T细胞中,24小时后检测其荧光素酶活性,结果表明启动子的最小功能启动子范围位于相对于转录起始位点的-414~+76bp之间(见图3),缺失突变体2为核心区域。生物信息学预测该区域包含了潜在的Sp1、Sp3和CREB等转录因子结合位点。
(5)基因启动子转录调控元件的初步鉴定
针对基因启动子转录活性调控区域进行的定点修饰结果表明:在cGAS启动子—414~+76区间的CREB和SP1结合位点发生不同碱基的突变时,启动子转录活性下降,初步表明CREB和SP1是转录活性的关键调控元件。
实施例3染色质免疫沉淀法(ChIP)证实启动子与转录因子的结合
体内实验用染色质免疫沉淀法(chromatin immunoprecipitation,ChIP)论证转录因子与启动子区DNA的结合。
染色质免疫沉淀法(ChIP):使用Magna CHIPTM kit(Millipore)试剂盒,参照其说明书进行实验,具体步骤如下:
1、体外交联与裂解
1.1待Siha细胞在15cm的培养皿中长到约90%时,此时细胞数量约1×107个,向20ml培养基中加550ul 37%的甲醛,轻轻摇晃使其混匀。室温下孵育10min。
1.2取2ml用冰预冷的1×PBS,加入5ul蛋白酶抑制剂II混合后放置冰上备用。
1.3孵育完成后向培养基中加入2ml 10×甘氨酸溶液,轻轻摇晃混匀,室温下孵育5min,中和未反应的甲醛。
1.4将皿置于冰上,轻轻吸弃培养皿中全部的液体。加入10ml冰浴的1×PBS,轻轻摇晃进行冲洗。吸尽PBS,并用同样的方法重复清洗一次。
1.5将步骤1.2中配制的2ml含有1×蛋白酶抑制剂的冰浴PBS加入细胞培养皿中,轻轻摇晃使其充分接触,刮下培养皿中的细胞,并全部转移至EP管中。
1.6在4℃下800g,离心5min,弃上清并保留沉淀。
1.7离心时准备0.5ml细胞裂解液进行重悬。
1.8向沉淀中加入步骤7准备的细胞裂解液进行重悬。
1.9重悬冰上孵育15min,期间每隔5min震荡一次,促进细胞裂解。
2、DNA的超声波裂解
2.1超声破碎前取5ul的重悬液备用,将余下的细胞重悬液使用超声破碎仪进行超声破碎,置冰上,仪器参数设置如下:工作时间15s,间隔50s,次数20-30次。
2.2超声完成后取5ul裂解液加入新的EP管中,使用1%琼脂糖凝胶进行核酸电泳,观察超声破碎后核酸大小,200~1000bp为宜(200~500bp最佳),如条带过大,适当增加工作次数。
2.3超声破碎完成后,13000g 4℃离心10min,将上清按每50ul一管进行分装,-80℃储存备用。
3、免疫沉淀
3.1准备含有蛋白酶抑制剂的Dilution buffer,置于冰上备用。每个反应需450ul含有2.25ul蛋白酶抑制剂II的Dilution buffer。
3.2取出50ul超声裂解液,置于冰上溶解后加入步骤3.1中配制的Dilutionbuffer。
3.3取出5ul(1%)上清作为Input,-20℃下暂存以备用。
3.4每管加入适量免疫沉淀的抗体和20ul protein A magnetic beads(使用前混匀)。充分混匀后,4℃下50~200rpm旋转孵育过夜。
3.5将含有protein A magnetic beads沉淀的EP管置于磁力架上,弃尽上清只保留沉淀。
4、洗脱蛋白/DNA复合物
4.1向上述沉淀中加入500ul Low salt immune complex wash buffer,轻轻混匀后室温孵育3~5min,然后置于磁力架上,小心吸弃上清。
4.2依次加入500ul High salt immune complex wash buffer、Lic1 immunecomplex wash buffer及TE buffer,按照步骤4.1中的操作各洗一次。
5、逆转录交联的蛋白质/DNA复合物,释放DNA
5.1为每个样品准备100ul Elution buffer,加入1ul蛋白酶K。
5.2向样品中加入100ul配制好的Elution buffer,62℃下进行震荡孵育2h,然后95℃孵育10min并冷却至室温。
5.3将样品置于磁力架上并将上清转移至新的EP管。
6、DNA纯化
6.1为每个样品准备一个收集管,将Spin Filter置入收集管中。
6.2将500ul Bind Reagent A加入到100ul DNA样品管中,将其充分混匀,并转移至已放入收集管的Spin Filter中。
6.3室温下,10000g离心30s,取出Spin Filter,弃收集管中液体,并将SpinFilter套回收集管中。
6.4取出500ul wash Reagent B加入Spin Filter中,室温下10000g离心30s。弃废液后再套回。
6.5加入50ml Elution Buffer C至Spin Filter中心的白膜上。室温静置1min后,10000g离心30s。
6.6丢弃Spin Filter,收集管中的液体即为纯化的DNA。
7、纯化DNA的检测
根据转录因子结合位点所在位置设计PCR引物。
CREB结合位点和SP1第一个结合位点引物序列:
F 5'-AAGTGATTCTCCAGCCTCAGCCTC-3’(SEQ ID NO.12)
R 5'-TGACTCACGCCTGTAATCCCAGCA-3'(SEQ ID NO.13)
SP1第二个结合位点引物序列:
F 5'-AGGCAACACACACACACACATATA-3’(SEQ ID NO.14)
R 5'-TCCGTGCCCCTGTCTTAAGAAGAT-3'(SEQ ID NO.15)
用实时荧光定量PCR方法进行检测。ChIP实验结果如图4,转录因子SP1富集倍数为18.4,CREB富集倍数为15.5。体外实验表明cGAS启动子与转录因子SP1、CREB的结合。
序列表
<110> 江苏省人民医院
<120> 人cGAS基因启动子区转录调控元件及其应用
<160> 15
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1254
<212> DNA
<213> 人类(Homo sapiens)
<400> 1
tgcctacaat cccaacactt tgggaggttg aggagggaga atcgcttgag tctaggagtt 60
cgagaccagc ctgggcaaca tagtgagacc tccccatgta tacaaaaaaa ttaaaaagaa 120
aagaaaaagc tgggcatggt gtccgcacct gtagtaacag ctactcagga tgctgaggtg 180
ggaggatcgc ttgagccctg gtggctgagg ttgcagtgag ccgagattgc gcccccctaa 240
ctcaaaccaa aattaaattt aaaaaaactt tcacttgaaa gagaaaagtt tctctgtcaa 300
tgtttttgtt ttgttttgtt ttgttttgtg atggactctt tttctgttgc ccaggctgga 360
gtgcagtggc gcgatctcgg ctcactgcaa cctctgcctc ccgggttcaa gcagttctct 420
gtctcggcct cccgagtaga tgggactaca ggcgccacca agcccggcta attttttgta 480
tttttagtag agacggggtt tcaccatgtt ggccaggaag gtctcgatgt cttggcctcg 540
tgatccgccc gcctcggcct cccaaactgc tgagattaca ggcgtgagcc accacgcctg 600
gcctcttttt cttttttgag acagagtctc gctctgtcgc ccaggctgga gtgaagtggc 660
acgtctcagc tcactgcaac ctccgcctcc cgggttcaag tgattctcca gcctcagcct 720
cccgagtagc tgggattaca ggtgcccgcc accacgccta gctaatattt gtatttttta 780
gtagagacgg ggtttctttc gccttgttgg ccaggctggt cttgaactcc tgacctcagg 840
tgatccgccc acctcggtct cccagattgc tgggattaca ggcgtgagtc agtgtgccca 900
ggcaacacac acacacacat atatttaatt ataagaaaga ccaggtcttg ctgggttgcc 960
caggctggtc tcaaactcct gagctcaagc gacccgcctg cctcggcctc tcggatgctg 1020
aggttacagg cgtgagccac cgcgcccggc cctacctcat cttcttaaga caggggcacg 1080
gattgcctgg agagttagaa acttcgagac ttttgtagcc tcaggaaagg ccgcggccag 1140
cctcttcgcg gcatgggcgt ggctcccagc gacttcccag cctggggttc cccttcgggt 1200
cgcagactct tgtgtgcccg ccagtagtgc ttggtttcca acagctgctg ctgg 1254
<210> 2
<211> 39
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
gattacgcca agcttcttgg gtgctagcag gccagctac 39
<210> 3
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
gattacgcca agcttgtagc tggcctgcta gcacccaaga agg 43
<210> 4
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
ggggtaccca gtggctcatg cctacaat 28
<210> 5
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
gaagatctct gttggaaacc aagcactact 30
<210> 6
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
ggggtaccca gtggctcatg cctacaat 28
<210> 7
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
ggggtacccg cctagctaat atttgtattt 30
<210> 8
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
ggggtaccct ggtctcaaac tcctgagct 29
<210> 9
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
ggggtaccgc ggccagcctc ttcgcggc 28
<210> 10
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
ggggtaccag cctggggttc cccttcgg 28
<210> 11
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
gaagatctct gttggaaacc aagcactact 30
<210> 12
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
aagtgattct ccagcctcag cctc 24
<210> 13
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
tgactcacgc ctgtaatccc agca 24
<210> 14
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
aggcaacaca cacacacaca tata 24
<210> 15
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
tccgtgcccc tgtcttaaga agat 24
Claims (7)
1.人cGAS基因启动子区转录调控元件,其特征在于,该基因的核苷酸序列如SEQ IDNO.1所示。
2.一种载体,其特征在于,它含有权利要求1所述的人cGAS基因启动子区转录调控元件。
3.根据权利要求2所述的载体,其特征在于,它还含有所述的启动子区转录调控元件可操作地连接的外源基因。
4.根据权利要求3所述的载体,其特征在于,所述外源基因是报告基因。
5.一种宿主细胞,其特征在于,它含有权利要求4所述的载体;所述的宿主细胞是不具遗传功能的哺乳动物细胞。
6.权利要求1所述的人cGAS基因启动子区转录调控元件在构建含有人cGAS基因启动子的重组表达载体中的应用。
7.权利要求1所述的人cGAS基因启动子区转录调控元件作为抗病毒药物作用靶点在筛选抗病毒药物中的应用。
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