CN111593068B - 新型基因治疗载体pIRES-Rsirt2/4-Tet-nap的制备方法及应用 - Google Patents
新型基因治疗载体pIRES-Rsirt2/4-Tet-nap的制备方法及应用 Download PDFInfo
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Abstract
本发明公开了一种pIRES‑Rsirt2/4‑Tet‑nap重组质粒,其沿载体转录方向依次连接有R‑ Sirt2基因、IRES序列、R‑Sirt4基因、HSV TK polyA序列、tTA序列、TRE序列和nap序列,或者反向连接。其中,R‑Sirt2和R‑Sirt4基因的功能是重构线粒体网络并逆转衰老细胞表型;IRES序列则是连接sirtuin2基因和sirtuin4基因,使两者反向共表达;tTA和TRE是四环素诱导表达元件,调控DNA结合和分子伴侣介导自噬的肽段nap序列表达。本发明所述的自噬重组载体,采用IRES序列连接sirtuin2和sirtuin4基因,能在同一载体中共表达Sirtuin2和Sirtuin4蛋白,表达后由四环素元件调控质粒DNA细胞内自噬。故该重组质粒可用于基因治疗线粒体功能障碍相关疾病的药物中的应用。
Description
技术领域
本发明属于生物技术领域,特别涉及一种能重构线粒体网络并逆转衰老细胞表型的重组载体及其制备方法和应用。
背景技术
随着人们对IRES结构和功能研究的不断深入,利用IRES结构研究疾病治疗药物、构建可转入细胞内的病毒模型,以及在基因治疗方面的应用越来越多。经过查询专利,我们发现如今IRES在构建载体上得到了非常广泛的应用。IRES (internal ribosome entrysite)序列是1988年分别由Nahum Sonenberg和Eckard Wimmer实验室在脊髓灰质炎病毒(PV)和脑心肌炎病毒(EMCV)的RNA基因组中发现的。IRES通常位于RNA病毒的5'UTR中,允许RNA以5'帽子非依赖性方式翻译。利用IRES连接两个基因,在上游启动子的控制下,IRES和两个基因转录成为同一条mRNA。翻译时,IRES上游基因翻译起始遵循真核生物基因的帽依赖性方式,而下游基因则通IRES招募核糖体进入起始基因的翻译,实现了IRES序列连接的两个基因共用同一个转录单位表达。
目前利用IRES为靶点进行的药物研究主要集中在丙型肝炎病毒感染的治疗药物。Dash等证实IFN.II、IFN.B和IFN-t能够靶向作用于HCV的5’端IRES,抑制蛋白的翻译。Prabhu等证实靶向于IRES颈环结构II的siRNA能够在体外抑制HCV的六个基因型,因此颈环结构II可以作为抑制HCV的靶点研究药物。Ray等找到一种与IRES颈环结构11I相对应的的RNA分子,能够剂量依赖的抑制IRES介导的翻译,而对帽结构依赖的翻译无作用。颈部和第四环(SLrV)区域、起始密码子附近的GCAC序列,对于介导核糖体在HCV RNA上的聚集具有重要功能,Subramanian等实验证实了短发夹结构sh.SLIV在Huh7细胞中对HCV RNA的复制起到显著的抑制作用。Das等已经发现一种小的酵母RNA能够特异地抑制脊髓灰质炎病毒(PV)IRES介导的翻译。同时这种IRNA能够在体内和体外特异抑制HCV IRES介导的翻译,能够竞争La自身抗原与HCV IRES的结合,这为抗HCV感染研究药物提供了新的基础。根据IRES结构的特点,针对结构域II、结构域III-IV和结构域IV设计了不同的RNA适配子,均证实具有抑制IRES介导翻译的活性。另外,能够靶向性抑制IRES活性的还有人工核糖核酸酶、DNAzyme(Dz)分子、反义寡核苷酸、合成肽(LAP)、肽核酸(PNA)等,这些分子的研究为抗HCV病毒药物研究奠定了基础。
四环素(tetracycline,Tet)诱导调控表达系统的基本原理是由诱导药物如Tet改变调控蛋白质的构象,从而控制目标蛋白质的表达。最初Tet诱导调控基因表达系统是以大肠杆菌Tn10转座子上Tet抗性操纵子为基础而建立的。Tet阻遏蛋白(Tet repressorprotein,TetR)与Tet操纵基因(Tet operator,TetO) DNA序列有特异的亲和能力,当细胞内无Tet存在时,TetR会与TetO结合,从而阻断下游的抗性基因表达。当有Tet存在时,药物使TetR的构象发生改变,导致TetR与TetO分离,从而引起抗性基因的抑制解除,抗性蛋白表达使细菌产生耐药性。利用TetR和TetO特异结合的特性,研究人员发展了多种类型的Tet调控系统,但根据其表达特点可以归为两大类:抑制型系统Tet-off和激活性系统Tet-on。
Tet-off基因调控表达系统是Gossen等最早建立起的诱导表达系统,四环素的转录激活子(tetracycline transcrip-tional activator,tTA)是一种融合蛋白。四环素的应答元件(Tet-responsive element,TRE)是由人巨细胞的病毒IE启动子微小启动序列(Pcmv)、目的基因和大肠杆菌的tet纵子序列三部分连接而成。当不加入强力霉素(Doxycycline,Dox)或四环素时,目的基因转录。Tet-on系统与Tet-off系统的区别在于其调节蛋白质为反义四环素转录活化因子(reverse tetracy-cline transcriptionalactivator,rtTA)。它的转录调节元件rtTA由反义的Tet阻遏蛋白(reverse TetR,rTetR)与VP16融合而成。应答元件TRE与正义四环素诱导表达调控系统的相似,但是其作用机制与正义四环素诱导表达调控系统相反。
为使得目的基因在细胞内的表达得到有效控制,我们构建一个新型基因治疗载体pIRES-Rsirt2/4-Tet-nap,将分子伴侣自噬的含有五肽基序的核酸自噬肽引入载体内,通过外源给予4-表强力霉素诱导载体发生其自噬,再通过检测目基因在细胞内的表达水平,以此来评估新型表达载体的作为控制目的基因有效可控表达的可行性。
发明内容
本发明的首要目的在于克服现有技术的缺点与不足,提供一种pIRES-Rsirt2/4-Tet-nap重组质粒。该重组质粒转染入细胞内,能有效重构线粒体网络并逆转衰老哺乳动物细胞衰老表型。
本发明的另一目的在于提供所述的pIRES-Rsirt2/4-Tet-nap重组质粒的构建方法。
本发明的再一目的在于提供所述的pIRES-Rsirt2/4-Tet-nap重组质粒的应用。
pIRES-Rsirt2/4-Tet-nap双基因共表达自噬载体,其沿载体转录方向依次连接有R-Sirt2基因、IRES序列、R-Sirt4基因、HSV TK polyA序列、tTA序列、TRE序列和nap序列,或者相反连接。其中,R-Sirt2和R-Sirt4基因的功能是重构线粒体网络并逆转衰老细胞表型;IRES序列则是连接sirtuin2基因和sirtuin4基因,使两者反向共表达;tTA和TRE是四环素诱导表达元件,调控DNA结合和分子伴侣介导自噬的肽段nap序列表达。
本发明所述的自噬重组载体,采用IRES序列连接sirtuin2和sirtuin4基因,能在同一载体中共表达Sirtuin2和Sirtuin4蛋白,表达后由四环素元件诱导调控质粒DNA载体的细胞内自噬,实现外源质粒DNA载体在哺乳类细胞的高效可控表达。pIRES-Rsirt2/4-Tet-nap双基因共表达自噬重组载体在重构线粒体网络、激活细胞代谢并逆转细胞表型中的作用明显,因此其在预防和治疗衰老相关疾病或代谢综合征等线粒体功能障碍相关疾病药物中具有应用广阔。故该重组质粒DNA载体可用于基因治疗线粒体功能障碍相关疾病相关的载体、药物、食品、保健品中药物中的应用。
附图说明
图1为实施例1pAcGFP1-C3载体信息。
图2为实施例1 pTRE-Tight载体信息。
图3为实施例1 pTet-On载体信息。
图4为实施例1构建的pIRES-Rsirt2/4-Tet-nap载体信息。
图5为实施例1粘红酵母sirtuin2和sirtuin4基因的PCR扩增结果(泳道1:DL2000Marker;泳道2:sirtuin2基因扩增产物;泳道3:sirtuin4基因扩增产物)。
图6为实施例1 pMD18-T-Rsirt2/4载体酶切结果(M:DL5000 Marker;泳道1:EcoRⅠ和EcoRⅤ双酶切pMD18-T-Rsirt2/4结果)。
图7为实施例1 pIRES-Rsirt2/4-C3载体酶切结果。A、DL2000 Marker鉴定双酶切结果(M:DL2000 Marker;泳道1:EcoRⅤ和EcoRⅠ双酶切结果);B、DL5000 Marker鉴定双酶切结果(M:DL5000 Marker;泳道1:EcoRⅤ和EcoRⅠ双酶切结果)。
图8为实施例1启动子调控区片段PCR扩增结果(M:DL2000 Marker;1:启动子调控区片段扩增产物)。
图9为实施例1转录元件片段PCR扩增结果(M:DL2000 Marker;1:转录元件片段扩增产物)。
图10为实施例1重组质粒pIRES-Rsirt2/4-Tet-nap双酶切鉴定(M1:DL2000Marker;1:NheⅠ和BglⅡ双酶切重组质粒pIRES-Rsirt2/4-Tet-nap结果;2:BglⅡ和HindⅢ双酶切重组质粒pIRES-Rsirt2/4-Tet-nap结果;3:HindⅢ和PstⅠ双酶切重组质粒pIRES-Rsirt2/4-Tet-nap结果;4:KpnⅠ和BamHⅠ双酶切重组质粒pIRES-Rsirt2/4-Tet-nap结果;M2:DL10000 Maker)。
图11为实施例1 SDS-PAGE鉴定表达蛋白(M:蛋白Marker;泳道1:表达Rt-sirt2/4质粒的大肠杆菌菌株;泳道2:表达空质粒的大肠杆菌菌株;泳道3:未转染的大肠杆菌菌株)。
图12为实施例1 Western blot检测目的基因的表达。A、在转染后第1天检测HEK293T细胞中目的基因的表达;B、在转染后第2天检测HEK293T细胞中目的基因的表达;C、在转染后第4天检测HEK293T细胞中目的基因的表达;D、在转染后第8天检测HEK293T细胞中目的基因的表达(泳道1:表达Rt-sirt2/4质粒的HEK293T细胞;泳道2:表达空质粒的HEK293T细胞;泳道3:未转染的HEK293T细胞)。
图13为实施例1自噬后目的基因的表达。A、给予4-表强力霉素后第6 h检测HEK293T细胞中目的基因的表达;B、给予4-表强力霉素后第12 h检测HEK293T细胞中目的基因的表达;C、给予4-表强力霉素后第24 h检测HEK293T细胞中目的基因的表达;D、给予4-表强力霉素后第48 h检测HEK293T细胞中目的基因的表达;E、给予4-表强力霉素后第72 h检测HEK293T细胞中目的基因的表达(1:未给予4-表强力霉素的质粒Rt-sirt2/4的表达;2:给予4-表强力霉素的质粒Rt-sirt2/4的表达;3:转染空载质粒的HEK293T细胞)。
具体实施方式
下述实施例中,所用到的实验技术,例如PCR技术、引物设计技术、载体构建技术、检测技术、电泳技术等均为基因工程中的常规技术,本领域技术人员可根据现有技术实现(例如参考J. 萨姆布鲁克等著,黄培堂等译的《分子克隆实验指南》,科学出版社第三版;或者按照产品说明书进行)。在操作过程中所用到的设备、试剂、载体、菌株等,均为通过市场购买得到的常规产品。以下结合实施例,对本发明进行进一步详细说明。
实施例一:载体pIRES-Rsirt2/4-Tet-nap的构建、鉴定及表达
1 菌种、细胞与质粒
菌种:所用的菌株大肠杆菌(E. coli) DH5α和粘红酵母(R. glutinis) CCTCC M2012203均冻存于本实验室。
细胞:HEK293T细胞为实验室保藏。
质粒:pMD18-T载体购自大连宝生物公司,pTet-On和pTRE-Tight两个诱导载体分别购自优宝生物和拓飞生物科技有限公司。表达空质粒pAcGFP1-C3购自Clontech公司(USA),pAcGFP1-C3质粒大小为4.7 kb,含有AcGFP1报告基因,绿色荧光蛋白AcGFP1与EGFP相比,亮度更高,细胞毒性更低。目的基因片段nap由北京擎科新业生物技术有限公司合成。
实验方法
2.1 重组质粒pIRES-Rsirt2/4-C3的构建与鉴定
2.1.1 粘红酵母sirtuin2和sirtuin4基因的克隆与测序
从培养48 h的R.glutinis CCTCC M 2012203中提取提取总RNA,按反转录试剂盒说明书取1 μg总RNA为模板进行反转录合成第一条cDNA,-20℃冻存,备用。同时,基于已获得的部分序列的相似性搜索结果,以NCBI GenBank中Rhodotorula toruloides NP11的sirtuin2基因序列作为参考,设计合成引物,在引物两端分别引入NheⅠ和BglⅠ酶切位点。以Rhodotorula toruloides NP11的基因组为模板,S1和A1,对目的片段进行PCR扩增。PCR扩增条件为:94 ℃变性2 min,然后按94 ℃ 1 min,56 ℃ 1 min,72 ℃ 2 min,30个循环,最后72 ℃ 10 min。将PCR产物回收并克隆到pMD18-T载体上,转化E. coli DH5α,蓝白斑筛选和菌落PCR鉴定阳性克隆,挑取正确的阳性克隆进行测序。同理进行sirtuin4基因克隆,酶切位点为HindⅢ和PstⅠ,引物为S2和A2。
表1 引物
2.1.2 表达质粒pMD18-T-Rsirt2/4的构建
将PCR产物和表达载体分别用HindIII和EcoRI双酶切、回收、连接并转化E. coli DH5α。E. coli感受态细胞的制备与转化、载体构建和转化、重组质粒的鉴定均按常规方法进行,所得重组质粒命名为pMD18-T-Rsirt2/4。同理构建获得质粒pIRES-C3和pMD18-T-nap,其中所使用的载体为pAcGFP1-C3,图谱如图1所示。
载体的构建与鉴定
将pMD18-T-Rsirt2/4载体和pIRES-C3载体分别用限制性内切酶EcoRⅤ和EcoRⅠ双酶切,37℃过夜,酶切体系:EcoRⅠ8 μL,EcoRⅤ8 μL,10×H buffer 10 μL,H2O 34 μL,质粒40 μL。用1%琼脂糖凝胶电泳检测,切下需要的条带进行胶回收。将回收纯化得到的产物用SolutionⅠ连接,连接转化后,提取质粒pIRES-Rsirt2/4-C3,酶切鉴定并测序。
重组质粒的构建与鉴定
2.2.1 pMD18-T-PTight-MCS载体的构建与鉴定
pTRE-Tight载体(图谱如图2所示)中含有Tet真核基因表达系统的启动子调控区,即PTight+MCS片段。根据PTight+MCS基因片段,设计引物S3和A3(引物序列见表1),将上游引物5’端引入Bam HⅠ酶切位点,然后下游引物扩增到载体上的XbaⅠ酶切位点。以pTRE-Tight质粒为模板,S3和A3为引物,进行PCR扩增。PCR扩增体系为20 μL,PCR反应结束后,用1﹪琼脂糖凝胶电泳检测,胶回收试剂盒回收纯化DNA片度。将回收的DNA片度连接于pMD18-T载体上,连接体系:pMD18-T Vector (50 ng/μL) 0.5 μL,SolutionⅠ 5μL,DNA 20~40 ng,加ddH2O至10 μL。4℃过夜连接。转化,提质粒,酶切鉴定并送往北京擎科新业生物技术有限公司测序。
Tight载体的构建与鉴定
将pMD18-T-PTight-MCS载体和pMD18-T-nap载体分别用限制性内切酶EcoRⅤ和EcoRⅠ双酶切,37℃过夜,酶切体系:EcoRⅠ8 μL,EcoRⅤ8 μL,10×H buffer 10 μL,H2O 34 μL,质粒40 μL。用1%琼脂糖凝胶电泳检测,切下需要的条带进行胶回收。将回收纯化得到的产物用SolutionⅠ连接,连接转化后,提取质粒pMD18-T-PTight-nap,酶切鉴定并测序。
Tight真核表达载体的构建
将pMD18-T-PTight-nap载体和真核表达载体pIRES-Rsirt2/4-C3分别用限制性内切酶SalⅠ和BamHⅠ双酶切,37℃过夜。酶切体系:SalⅠ8 μL,BamHⅠ8 μL,10 × K buffer 10 μL,H2O 34 μL,质粒40 μL。酶切后用1%琼脂糖凝胶电泳检测,切下需要的条带进行胶回收。将回收纯化得到的产物用SolutionⅠ连接,连接转化后,提取质粒pIRES-Rsirt2/4-PTight-nap,酶切鉴定并测序。
含有转录元件的pMD18-T-rtTA载体的构建及鉴定
pTet-On载体(如图3)中含有Tet真核基因表达系统的转录元件,即rTetR-minVP16-sv40polyA片段(766-1988 bp)。根据此片段设计两对引物S4和A4、S5和A5(引物序列见表1),在引物两端引入BamHⅠ和HindⅢ两个酶切位点,使用重叠PCR方法,除去BamHⅠ酶切位点。以pTet-On质粒为模板,进行PCR扩增,胶回收纯化后,将PCR产物连接于pMD18-T载体上,转化后再挑选阳性单克隆菌株,摇菌,提质粒pMD18-T-rtTA,酶切鉴定并测序。
重组质粒pIRES-Rsirt2/4-Tet-nap的构建与鉴定
将pMD18-T-rtTA载体和pIRES-Rsirt2/4-PTight-nap载体分别用限制性内切酶BamHⅠ和HindⅢ双酶切,37℃过夜。BamHⅠ8 μL,SalⅠ8 μL,10×K buffer 10 μL,H2O 34 μL,质粒40 μL。酶切后用1%琼脂糖凝胶电泳检测,切下需要的条带进行胶回收。将回收纯化得到的产物用SolutionⅠ连接,连接转化后,提取重组质粒pIRES-Rsirt2/4-Tet-nap(重命名为pIRES-Rsirt2/4-Tet-nap,如图4),酶切鉴定并测序。
E. coliα感受态细胞制备
(1)取-80℃冻存的E. coli DH5α菌种接种于LB平板中,37℃培养过夜。
(2)挑取单菌落接种于50 mL LB液体培养基中,37℃培养,至OD值为0.4-0.6左右。
(3)移取25 mL菌液至预冷过的50 mL EP管中,并在冰上放置30 min,使培养物冷却至4℃,低温4000 rpm离心10 min,弃上清,回收菌体。
(4)加入5 mL预冷的CaCl2(0.1 mol/L),重悬沉淀后,冰浴30 min;4℃、4000 rpm离心10 min,弃上清,回收菌体。
(5)加入1 mL预冷过的0.1 mol/L的CaCl2(含15%甘油)重悬菌体沉淀。
(6)在冰上将细胞分装成小份,100 μL/份,-80℃冻存备用。
重组质粒的转化
(1)取100 μL E. coli DH5α感受态细胞悬液于1.5 mL离心管中,再加入10 μL重组质粒,轻弹管壁混匀,在冰上静置30 min;
(2)于42℃水浴热激90 s后,迅速置于冰上冷却2~3 min;
(3)向上述管中加入500 μL的LB液体培养基(不含抗性),37℃、200 rpm振荡培养1h,使受体菌活化;
(4)将活化的菌液3000 rpm离心3 min,弃500 μL上清液,用剩余的培养液将菌体重悬,并涂布在Kan+-LB固体培养基上,晾干后,37℃正置培养1 h后,倒置培养过夜;
(5)挑取白色单菌落,接种于5 mL Kan+-LB液体培养基上,37℃培养过夜。
重组质粒的提取
根据EndoFree Maxi Plasmid Kit质粒提取试剂盒的说明,具体操作如下:
(1)取100 mL过夜培养的菌液(含pIRES-Rsirt2/4-Tet-nap质粒)加入离心管,室温8000 rpm离心3 min收集菌体,尽量吸除上清(干净吸水纸吸去管壁水滴);
(2)向菌体沉淀中加入8 mL溶液P1,移液器或涡旋振荡器彻底悬浮菌体沉淀;
(3)再加入8 mL溶液P2,立即温和地上下翻转6~8次,使菌体充分裂解,室温放置5min,待菌液变得清亮粘稠;
(4)又加入8 mL溶液P4,立即温和地上下翻转6-8次,充分混匀,至溶液出现白色分散絮状沉淀。然后室温放置10 min。8000 rpm离心10 min,使白色沉淀离至管底,将全部溶液小心倒入过滤器CS1中,慢慢推动推柄过滤,滤液收集在干净的50 ml的管中;
(5)柱平衡步骤:向吸附柱CP6中(吸附柱放入50ml收集管中)加入2.5 ml的平衡液BL,8000 rpm离心2 min,倒掉收集管中的废液,将吸附柱重新放回收集管中;
(6)向滤液中加入0.3倍滤液体积的异丙醇,上下颠倒混匀后转移到吸附柱CP6中;
(7)室温8000 rpm离心2 min,倒掉收集管中的废液,将吸附柱CP6重新放回收集管中,第5步中所得溶液分3次过柱,每次条件相同。
(8)向吸附柱CP6中加入10 mL漂洗液PW,8000 rpm离心2 min,弃掉收集管中的废液,将吸附柱重新放回收集管中。
(9)重复操作步骤(8)。
(10)向吸附柱CP6中加入3 mL无水乙醇,室温8,000 rpm离心2 min,弃废液。
(11)将吸附柱CP6重新放回收集管中,8000 rpm离心5 min,将吸附柱中残余的漂洗液去除,然后将吸附柱CP6开盖,置于室温放置数分钟,以彻底晾干残余的漂洗液;
(12)将吸附柱CP6置于一个干净的50 mL收集管中,向吸附膜的中间部位悬空滴加2 mL洗脱缓冲液TB,室温放置5 min,然后室温8000 rpm离心2 min,将滤液重新加入到吸附柱中8000 rpm离心2 min。将50 mL离心管中的洗脱液全部移入一个干净的1.5 mL离心管,-20℃保存。
重组质粒的鉴定
(1)把收集的pIRES-Rsirt2/4-Tet-nap重组质粒用EcoRI和BamHI双酶切,37℃水浴;
组分 | 用量/μL |
10×2 Buffer | 3.0 |
pIRES-Rsirt2/4-Tet-nap | 3.0 |
EcoRI | 0.1 |
BamHI | 0.1 |
DEPC水 | 43.8 |
总体积 | 50.0 |
(2)制备1.5%琼脂糖胶液:取1.5 g琼脂溶于100 mL TAE电泳缓冲液中,接着微波炉加热至琼脂糖完全融化,待溶液冷却至70℃时,加入1.5 μL 10 mg/mL的EB;
(3)将温热琼脂糖倒入胶槽中,冷却直至成凝胶,凝固约30 min;
(4)在凝胶完全凝固之后,小心移去梳子,放入电泳槽内,加样孔一侧靠近阴极,注入适量的1×TAE电泳缓冲液,通常缓冲液高于胶面1 cm;
(5)将适量质粒样品与上样缓冲液(溴酚蓝)混合,用移液枪将样品加入加样孔,之后连接电泳槽和电源,设定电压100 V,当溴酚蓝移动至距离胶板下沿1 cm处时,停止电泳;
(6)取出凝胶,用0.5 μg/mL的EB/1×TAE溶液染色30 min,再用蒸馏水清洗15min;
(7)在紫外灯(310 nm波长)下观察染色后的凝胶,并用凝胶成像系统拍照保存。
重组质粒的纯度和含量测定
用紫外分光光度法测定所提质粒的纯度和浓度:将一定体积的质粒DNA溶液用移液器吸取,在紫外分光光度计上分别检测230 nm、260 nm和280 nm波长下的吸光度A230、A260和A280,计算A260/A280和A260/A230比值,并计算样品的浓度与纯度。
DNA样品的浓度(μg/μL):A260 × 稀释位数 × 50/1000。
细胞培养
将293T细胞按9 × 104个/ml的密度接种于六孔板,用含10%FBs的高糖DMEM培养基于5% CO2、37℃培养箱中培养24 h。
质粒的构建
质粒pFlag-IRES-Rsirt2/4-Tet-nap由上海吉凯基因公司构建。
质粒大肠杆菌感受态细胞的制备,质粒转化,提取,鉴定等参照3.3~3.8。
脂质体转染
根据Lipofectamine 2000脂质体转染试剂盒,具体操作如下:
(1)在24孔板中每孔0.5-2 × 105个细胞接种于500 μL不含抗生素和血清的DMEM培养基中,在转染时细胞可长至90-95%融合。
(2)用50 μL Opti-MEI无血清培养基稀释质粒DNA,轻轻混匀。取适量Lipofectamine 2000在50 μL Opti-MEI培养基中稀释,室温孵育5分钟。
(3)将前两步所稀释的DNA和Lipofectamine 2000混合(使总体积为100 μL),轻轻混匀,室温放置20 min。
(4)每孔细胞中加入100 μL转染液,轻轻摇匀。
(5)37℃ 5% CO2培养转染4-6 h后可更换培养基,于1天、2天、4天、8天后裂解细胞,以备检测基因表达。
目的蛋白的鉴定
(1)总蛋白提取
a、细胞用PBS溶液清洗3遍;
b、在37℃下,量取2 mL胰蛋白水解酶(0.025%)处理5~8 min,直至大部分细胞可以浮动时,消化终止,900 rpm离心5min,弃上清;
c、取RIPA裂解酶20μL,涡旋仪中完全震荡裂解,冰浴10 min,每隔5 min震荡30s。
d、将离心管放入95℃水浴锅中加热5 min,随后重复步骤c和d 2 次;
e、4℃、12000 g高速离心15 min,吸取上清液到EP管中,即为总蛋白。
(2)Western blotting检测目的蛋白的表达情况,具体操作步骤如下:
a、制胶:将分离胶倒入电泳玻璃板的间隙,直到距离上边缘1.5 ~2 cm时停止添加,再用75%乙醇封层;等到胶变为固体状态后,将乙醇倒掉后添加配置好的浓缩胶,插入梳子等待胶干透。
b、上样:待测蛋白沸水浴3 min,将新现配的电泳液倒入电泳槽,量取8 μL和5 μL蛋白Maker,分别倒入两边泳道,再量取1×蛋白上样缓冲液2 μL和5 μL,分别加入刚才倒入蛋白Marker的泳道,填补到10 μL,剩余泳道加10 μL样品,电泳时电压设为50 V。
c、转膜:停止后,将滤纸剪成11 cm × 8cm的形状,另取0.22 μm的PVDF膜,用一定量的甲醇溶液将膜处理5 min,组装转印夹层的顺序为“海绵-6层滤纸-凝胶-PVDF膜-6层滤纸-海绵”,之后转移至转膜槽,电流调为200 mA处理60 min。
d、孵育抗体:停止转膜后,膜用TBS液清洗5 min,再封闭1 h,TBST液洗三次膜,每次5 min,加入一抗稀释液,4℃条件下过夜孵育;洗三次膜,加二抗稀释液室温孵育1 h。
e、发光检测:膜用TBST液清洗后用发光液处理3 min,吸干,将胶片和膜一起压片,持续1 min再显影30 s,用水清洗干净,等待烘干再进一步扫描。用ImajeJ2x测定内参照基因和目的基因的灰度值。
质粒自噬的诱导
在转染的第9代更换培养基后培养基。向培养皿中加入5 μg/mL的四表强力霉素,继续培养,于6 h、12 h、24 h、48 h、72 h裂解细胞,以备检测基因的表达。
结果
3.1 粘红酵母sirtuin2和sirtuin4基因克隆
以粘红酵母的基因组为模板,带有EcoRⅠ和EcoRⅤ酶切位点的两对引物(S1和A1、S2和A2)为引物,退火温度分别为76℃和72℃进行PCR扩增,扩增粘红酵母sirtuin2和sirtuin4基因的全部编码区。结果分别扩增出两条约1179 bp和1104 bp的特异性带(如图5),与预计大小一致。
重组克隆质粒pMD18-T-Rsirt2/4的酶切鉴定
将PCR产物回收纯化后连接到pMD18-T载体上,制备重组质粒DNA,用EcoRⅠ和EcoRⅤ双酶切鉴定阳性重组质粒,酶切结果见图6。结果表明,扩增出2283 bp的完整CDS序列,与目的片段大小完全一致。初步认定为粘红酵母sirtuin2和sirtuin4基因的完整编码区。
载体的酶切鉴定
将pMD18-T-Rsirt2/4载体和pIRES-C3载体分别用限制性内切酶EcoRⅤ和EcoRⅠ双酶切,胶回收,连接转化,提取质粒pIRES-Rsirt2/4-C3,通过PCR进行酶切鉴定,结果如图7所示。结果表明,扩增出2283 bp和584 bp的特异性条带,与目的片段大小一致。
载体的构建
3.4.1 启动子调控区扩增结果
pTRE-Tight 载体中含有Tet真核基因表达系统的启动子调控区,即PTight+MCS片段。以pTRE-Tight质粒为模板,S2和A2为引物,上游引物5′端带有BamHⅠ酶切位点,下游引物扩增到载体上的XbaⅠ酶切位点结束。退火温度为58℃进行PCR扩增,扩增Tet真核基因表达系统的启动子调控区。扩增出一条约410 bp的特异性带(如图8),与预计大小一致,将产物回收纯化后备用。测序结果进行拼接比对,测序结果完全正确。
转录元件rTetR片段扩增结果
pTet-On载体中含有Tet真核基因表达系统的转录元件,即rTetR-minVP16-sv40polyA片段(766-1988bp)。使用重叠PCR方法,设计两对引物S4和A4、S5和A5,目的除去BamHⅠ酶切位点,在引物两端引入BamHⅠ和HindⅢ两个酶切位点,以pTet-On质粒为模板,退火温度为60℃进行PCR扩增,扩增Tet真核基因表达系统的转录元件rTetR片段。扩增出一条约1220 bp的特异性带(如图9),与预计大小一致,将产物回收纯化后备用。测序结果进行拼接比对,测序结果完全正确。
载体的酶切鉴定
将pMD18-T-PTight-MCS载体和pMD18-T-nap载体分别用限制性内切酶EcoRⅤ和EcoRⅠ双酶切,胶回收,连接转化,提取质粒pMD18-T-PTight-nap。将pMD18-T-PTight-nap载体和真核表达载体pIRES-Rsirt2/4-C3分别用限制性内切酶SalⅠ和BamHⅠ双酶切,胶回收。将回收纯化得到的产物用SolutionⅠ连接,连接转化后,提取质粒pIRES-Rsirt2/4-PTight-nap。将pMD18-T-rtTA载体和pIRES-Rsirt2/4-PTight-nap载体分别用限制性内切酶BamHⅠ和HindⅢ双酶切,胶回收,连接转化,提取质粒pIRES-Rsirt2/4-Tet-nap,酶切鉴定。用限制性内切酶NheⅠ和BglⅡ酶切质粒,得到1179 bp的条带,分别为sirtuin2基因片段;BglⅡ和HindⅢ双酶切,下方得到584 bp的条带,为IRES片段大小;HindⅢ和PstⅠ双酶切,得到1104 bp的条带,为sirtuin4基因片段大小;KpnⅠ和BamHⅠ双酶切,得到1681 bp的条带,为rtTA-TRE-nap片段大小(如图10)。上述结果,均与预计大小一致。
目的蛋白的表达鉴定
将重组质粒pFlag-IRES-Rsirt2/4-Tet-nap转入E. coli,重组菌培养发酵后使用SDS-PAGE鉴定蛋白的表达,如图11,我们在重组大肠杆菌中的39 kDa和44 kDa附近发现清晰的条带,提示Rt-sirt4和Rt-sirt2蛋白正常表达。
我们构建了带有Flag标签的质粒pFlag-IRES-Rsirt2/4-Tet-nap,以真核表达质粒和空质粒分别转染HEK239T细胞,并在转染后1天、2天、4天、8天后裂解细胞经过Westernblot分析。由图12可得,在anti-Flag特异性抗体检测下,发现Rt-sirt2在45 kDa有目的条带,Rt-sirt4在40 kDa有目的条带,而且转染后的1、2、4、8天目的基因的表达均趋于稳定状态。因此质粒pFlag-IRES-Rsirt2/4-Tet-nap可以在HEK239T细胞内能稳定表达。
自噬后细胞内目的基因的表达水平
为使得目的基因的表达得到有效控制,我们引入了分子伴侣介导的自噬元件,我们在转染的第9天我们外源给予4-表强力霉素,于6 h、12 h、24 h、48 h、72 h裂解细胞检测目的蛋白的表达情况。实验结果表明:在给予4-表强力霉素的第24 h目的蛋白表达显著降低,72 h基本完全消失。而对照组中目的基因的表达量无明显变化(图13)。
小结
本研究成功构建了pIRES-Rsirt2/4-Tet-nap表达载体。我们主要通过HEK239T细胞体外实验观察四环素调控的自噬诱导元件是否会诱导载体自噬的作用。首先将构建新型表达载体转染,再转入细胞中,在转染后1天、2天、4天、8天后检测目的基因的表达,在第9天通过外源给予4-表强力霉素启动分子伴侣诱导的自噬,于第6 h、12 h、24 h、48 h、72 h检测目的基因的表达,发现在给予4-表强力霉素的72 h,目的蛋白基本完全消失,成功实现了目的基因在细胞内可控表达。
Claims (3)
1.一种pIRES-Rsirt2/4-Tet-nap双基因共表达自噬载体,其特征在于:其沿载体转录方向依次连接有CMV启动子、粘红酵母sirt2基因(Sirtuin2 of Rhodosporidium toruloides,R-Sirt2)、IRES序列、粘红酵母sirt4基因(Sirtuin4 of Rhodosporidium toruloides,R-Sirt4)、HSV TK polyA序列、rtTA序列、TRE序列和nap序列、Kana抗性基因以及PUC质粒骨架元件;
所述nap序列为分子伴侣介导自噬的含五肽基序的核酸肽段,所述五肽基序为“KFERQ”;
将分子伴侣介导自噬的含有五肽基序的核酸肽段引入载体内,通过外源给予4-表强力霉素启动分子伴侣介导的自噬,诱导载体发生自噬,实现目的基因的外源质粒DNA载体在哺乳类细胞的高效可控表达;
nap肽段的诱导表达启动分子伴侣介导的自噬,从而使外源的质粒DNA分子在细胞内自噬消失。
2.权利要求1所述的pIRES-Rsirt2/4-Tet-nap双基因共表达自噬载体的构建方法,其特征在于,步骤包括:
(1)获得含有特异性酶切位点的sirtuin2基因片段和sirtuin4基因片段;
(2)将步骤(1)得到的sirtuin2基因片段和sirtuin4基因片段连接于载体,构建含有sirtuin2-IRES-sirtuin4基因的重组载体;
(3)将步骤(2)构建的含有sirtuin2-IRES-sirtuin4基因的重组载体与pTet-On和pTRE-Tight载体通过双酶切连接,获得含有sirtuin2-IRES-sirtuin4-rtTA-TRE的载体;
(4)将合成的nap序列插入含有sirtuin2-IRES-sirtuin4-rtTA-TRE的载体,构建pIRES-Rsirt2/4-Tet-nap双基因共表达自噬重组载体。
3.权利要求1所述的pIRES-Rsirt2/4-Tet-nap双基因共表达自噬载体在制备预防和/或治疗线粒体功能障碍相关疾病的药物中的应用。
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"Autophagic plasmid expressing SIRT3 and SIRT4 to improve Alzheimer’s disease";Shiyu Li 等;《Research Square》;第1-28页 * |
"Enterococcus faecalis sir2-like gene enhances aerobic metabolism of themselves and mitochondrial respiration of mammal cells to bring about improving metabolic syndrome through the PGC-1 alpha pathway";Shiyu Li 等;《Journal of Tissue Engineering and Regenerative Medicine》;第13卷;第1-26页 * |
"自噬在米诺环素保护PC12细胞氧糖剥夺-复糖复氧中的作用";肖世庚 等;《中国临床药理学与治疗学》;第20卷(第2期);第145-150页 * |
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