CN116410999A - 含有ires及新型启动子的核酸载体及其应用 - Google Patents
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Abstract
本发明公开了一种使用新型启动子的核酸载体。该核酸载体是能够在体外转录mRNA的质粒载体。本发明构建的核酸载体,包括T7或VSW3启动子、目标蛋白基因、内部核糖体进入位点(IRES)以及位于IRES之后的外源基因3’‑端的长度为120‑200的多聚腺苷脱氧核酸poly(A)片段,转录出的mRNA直接拥有poly(A),不需要额外的加尾操作。VW3启动子体外转录所得mRNA在转染细胞后,呈现出更强的mRNA稳定性和更高的蛋白表达能力。另外,使用本发明构建的核酸载体体外转录出的mRNA含有IRES序列,可同时表达两种蛋白。含有CVB3 IRES序列的核酸载体体外转录出的mRNA蛋白表达量高于含有ECMV等其他IRES序列的核酸载体。
Description
技术领域
本发明涉及分子生物学技术、细胞培养、免疫生物学和图像分析等技术领域。具体而言,包括通过酶切、连接等方式将包含5'端内切酶位点、VSW-3启动子、UTR序列、内部核糖体进入位点(IRES序列)、polyA序列以及3'端内切酶位点的DNA片段连接入多克隆位点,构建为一种可用于体外转录生成mRNA的质粒载体PGEM-3zf-ORF1-IRES-ORF2;将此示例质粒转化入适当的大肠杆菌菌株、培育后提取质粒、在体外进行酶切线性化、转录得到带有对应多聚腺苷核酸尾的mRNA;并将此示例mRNA转染细胞,最后通过检测细胞中不同时间点此mRNA所表达的两种蛋白含量定量评估此类载体所表达的mRNA稳定性和翻译表达能力。
背景技术
信使核糖核酸(mRNA)是真核生物基因表达的重要一环,在DNA-(转录)-mRNA、mRNA-(翻译)蛋白的中心法则中处于居中的重要地位。成熟mRNA由5’-帽、包含RNA聚合酶启动子的5’-非翻译区域(5’-UTR)、蛋白编码基因开放阅读框(ORF)、3’-非翻译区域(3’-UTR)、3’-多聚腺苷核酸(3’-poly(A))尾部组成。其中5’-帽和3’-poly(A)尾部对mRNA在体内或培养细胞系内的稳定性起到重要作用,进而对mRNA翻译为蛋白或多肽的效率存在影响。目前在分子生物学领域,大量DNA质粒载体设计为可在体外转录为信使核糖核酸(mRNA,随后这些mRNA可用于生物医学研究、临床应用领域等。体外人工转录所得mRNA在一定程度上模拟了真核生物体内转录的mRNA,其5’-帽通常来自于体外转录试剂盒,而3’-poly(A)尾部则来自于DNA载体自身所包含的多聚腺苷脱氧核糖核酸序列(同样简称为3’-poly(A))模板。天然或人工合成的mRNA的3’-poly(A)尾部在生物体或细胞内均起到保护mRNA、防止核酸外切酶降解的作用。目前学术界和工业界绝大部分使用的体外转录用载体使用T7或SP6RNA聚合酶启动子,仅包含长度为30的3’-poly(A)模板,并且大多数人工合成的mRNA仅编码一个基因,翻译形成一种蛋白。
psychrophilic phage VSW-3是分离自plateau lake的一种噬菌体,该噬菌体编码一种新型单亚基RNA聚合酶(RNAP),这种聚合酶可以在较低温度(4-25℃)催化体外转录反应,低温反应条件可以减少转录过程中RNA降解,无需使用RNA酶抑制剂。与常用的T7RNAP相比,其转录产量相当,但对II类转录终止子不敏感,合成的RNA不具有额外的3’-cis延伸。更重要的是,该酶的体外转录产物中双链RNA含量极低,甚至检测不出。而在T7催化的IVT产物中双链RNA含量较高,会引起细胞免疫反应,从而降低翻译效率。
发明内容
目前大多数mRNA体外转录采用T7或SP6 RNA聚合酶,其中T7应用更为普遍。T7 RNA聚合酶转录过程中除了单链mRNA产物以外,会产生一定数量的双链RNA副产品。双链RNA进入细胞后会激活细胞的免疫反应,导致mRNA的翻译效率下降。所以合成后需要通过HPLC等方法对IVT产物进行纯化以去除双链mRNA。本发明的一个目的是构建具有psychrophilicphage VSW-3RNA聚合酶启动子的mRNA体外转录用载体。VSW-3RNA聚合酶能够识别该启动子并启动模板质粒的转录。使用VSW-3RNA聚合酶的体外转录产物中双链RNA含量极少,可大大提高mRNA的纯度,进而提高翻译效率。
目前通过体外转录的方式人工合成的mRNA大多编码一个基因,翻译形成一种蛋白。在科学研究中,需要对mRNA在体内外的翻译情况进行检测。但检测mRNA编码的目的蛋白多有不便,需要添加额外的报告基因,或将目的基因与报告基因融合表达成融合蛋白。融合蛋白因蛋白组成、分子量大小都与目的蛋白不同,并且有可能具有与目的蛋白不同的二级结构,从而影响目的蛋白的功能。既能同时表达目的基因和报告基因,又不影响各自功能的方法之一是在两个基因的ORF之间添加内部核糖体进入位点(IRES)序列。该位点能够不依赖5’帽结构而独立启始翻译过程,即独立介导核糖体与RNA结合,起始蛋白质翻译。本发明的另一个目的是构建具有IRES序列,能够同时编码两种基因的mRNA体外转录用载体。
在科学研究中,可以在体外转录之后使用poly(A)Polymerase在转录之后向mRNA添加poly(A)尾,但这一额外的步骤不仅增加了试验的复杂性,而且不能保证添加的poly(A)长度的均一性,也使试验的重复性变差。在工业化生产中,额外添加加尾步骤即使生产程序更加繁琐,又不能保证产品均一性,因而不具有现实意义。本发明的另一个目的是构建具有更长3’-poly(A)尾部模板的mRNA体外转录用载体。这一载体能够在转录同时添加长度为60-200的poly(A)尾,已有研究证实具有更长3’-poly(A)尾部的mRNA更加稳定、翻译效率也大幅提升。
具体来说,本发明使用限制性内切酶EcoRI和XbaI分别将PGEM-3zf(+)载体切开,采用同源重组的方式连接插入一段带有VSW3启动子、5'UTR、基因1ORF、IRES、基因2ORF、3'UTR和poly(A)区段的人工合成序列,构建为PGEM-IRES质粒载体。
本发明的第一方面在于提供一种体外表达mRNA的质粒载体,包括启动子以及3’-端的长度60-200的多聚腺苷脱氧核酸poly(A)片段。
在本发明的一些实施方式中,所述多聚腺苷脱氧核酸poly(A)的长度为60。在本发明的另外一些实施方式中,所述多聚腺苷脱氧核酸poly(A)的长度为120-150。
在本发明的一些实施方式中,还包括启动子序列。在本发明的一些实施方式中,启动子为T7启动子。在本发明的另外一些实施方式中,启动子为VW3启动子。
本发明的一些实施方式中,还包括目标蛋白基因。
在本发明的一些实施方式中,所述目标蛋白基因为萤火虫荧光素酶基因等报告基因或参比蛋白基因。在本发明的另外一些实施方式中,目标蛋白基因为WT1等肿瘤抗原基因等编码功能蛋白的基因。
在本发明的一些实施方式中,还包括内部核糖体结合位点(IRES)。
在本发明的一些实施方式中,所述IRES为以脑炎心肌炎病毒encephalomyocarditis virus(EMCV)IRES为代表的I型IRES、以柯萨奇病毒B3Coxsackievirus B3(CVB3)IRES为代表的II型IRES,以甲型肝炎病毒(HAV)IRES为代表的III型IRES以及以丙型肝炎病毒(HCV)和猪瘟病毒(CSFV)为代表的IV型IRES中的一种。
本发明的第二方面在于提供构建第一方面所述的体外表达mRNA的质粒载体的方法,可同时表达2种蛋白。包括以下步骤:S1,使用限制性内切酶EcoRI和XbaI将pGEM-3zf(+)切开,将编码两端带有同样酶切位点的人工合成基因序列连入,构建为pGEM-ORF1-IRES-ORF2质粒载体;该人工合成基因序列包括:T7或VSW-3启动子、5’-UTR、ORF1、IRES、ORF2、3’-UTR及poly A序列。
S2,使用EcoRI和XbaI限制性内切酶对构建好的质粒进行双酶切,验证外源片段的成功连入。
在本发明的一些实施方式中,包括以下设计:T7启动子序列:TAATACGACTCACTATAGGGAGA;VW3启动子序列:5’-TTAATTGGGCCACCTATA-3’。在本发明的一些实施方式中,包括以下步骤:EcoRI、XbaI酶切pGEM-3zf(+)质粒并回收长片段:将切开的线性pGEM质粒与人工合成的基因片段以1:1的摩尔比共转化StbL3感受态菌。PCR扩增连入的基因片段进行筛选;阳性质粒用EcoRI和XbaI双酶切验证。
本发明的第三方面在于提供第一方面所述的体外表达mRNA的质粒载体的应用。本发明的有益效果在于:1、采用新型启动子VW3构建体外转录mRNA的载体,该启动子可在较低温度催化转录反应,基本没有双链RNA转录副产物,转录出的mRNA转入细胞内后,在细胞内蛋白表达水平更高。2、本发明构建的体外表达mRNA的质粒载体,包括表达目标蛋白基因和60-200个poly(A),表达的mRNA直接拥有poly(A),不需要额外的加尾操作。另外,体外转录所得mRNA在转染细胞后,呈现出更强的mRNA稳定性和更高的蛋白表达能力检测。
3、本发明构建的体外表达mRNA的质粒载体,可同时独立表达两种蛋白,适用于制备多价mRNA疫苗、多价mRNA药物等,或用于添加报告基因如EGFP或其他辅助蛋白如IFNγ的研究及mRNA药物应用。
附图说明图1为人工合成的转录本(transcript)构成:包括VSW3启动子序列、UTR序列、基因ORF序列、CVB3 IRES序列以及poly(A)区段;图2为点杂交法检测VSW3 RNA聚合酶体外转录产物以及T7 RNA聚合酶转录产物中的双链RNA;图3为使用VW3和T7两种不同启动子转录的luciferase mRNA在动物体内的蛋白表达水平的比较。
具体实施方式
以下通过特定的具体实例说明本发明的实施方式,本领域技术人员可由本说明书所揭露的内容轻易地了解本发明的其他优点与功效。本发明还可以通过另外不同的具体实施方式加以实施或应用,本说明书中的各项细节也可以基于不同观点与应用,在没有背离本发明的精神下进行各种修饰或改变。在进一步描述本发明具体实施方式之前,应理解,本发明的保护范围不局限于下述特定的具体实施方案;还应当理解,本发明实施例中使用的术语是为了描述特定的具体实施方案,而不是为了限制本发明的保护范围。当实施例给出数值范围时,应理解,除非本发明另有说明,每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义,本发明中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外,根据本技术领域的技术人员对现有技术的掌握及本发明的记载,还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。
实施例1(一)构建体外转录载体pGEM-Luc-IRES-EGFP为了克服现有体外转录用启动子效率不高、poly A序列长度不足从而导致所转录得到的mRNA稳定性不够强和翻译表达能力不够高的缺陷,本发明采用了新型VW3启动子以及长度为120的3’-poly(A)区段来解决。具体来说,本发明使用限制性内切酶EcoRI和XbaI分别酶切pGEM-3zf质粒。将合成的luciferase基因及EGFP基因连入,构建为pGEM-Luc-IRES-EGFP质粒载体。(二)分别构建带有T7启动子和VW3启动子的pGEM-Luc-IRES-EGFP质粒。两者的区别在于启动子分别为T7和VW3。本发明在大肠杆菌中扩增pGEM-Luc-IRES-EGFP质粒后,提取纯化此质粒,使用3’端polyA尾后的IIS型限制性内切酶位点将其线性化,分别使用T7 RNA聚合酶以及VSW3 RNA聚合酶将其在体外转录为携带有luciferase及EGFP编码序列和长度为120的3’-poly(A)尾部的mRNA。将mRNA肌肉注射到小鼠体内,采用小动物成像方法检测luciferase基因的表达情况。
(一)主要试剂和仪器
本发明涉及的主要仪器及供应商PCR仪(BioRad),凝胶电泳仪(六一),CO2细胞培养箱(Thermo Fisher),-20℃冰箱(中科美菱),超低温冰箱(海尔),小动物成像仪(PerkinElmer)。
(二)实验方法
1.构建体外转录表达载体pGEM-Luc-IRES-EGFP
1.1酶切pUC19质粒并回收长片段首先,将pUC19质粒和XbaI内切酶、FlyCutBuffer、水按如下比例混合:pGEM-3zf 1μg,EcoRI 1μL,XbaI 1ul,FlyCut Buffer 2μL加水至20μL混合物在37℃放置2小时。随后在1.5%琼脂糖凝胶上进行电泳分离,使用DNA凝胶回收试剂盒回收长度约为2300bp的片段,溶于20μL去离子水中。
1.2合成携带有T7或VW3启动子、5’UTR、luciferase基因、ECMV或CVB3 IRES、EGFP基因、3’UTR以及长度120的poly(A)区段的插入序列。合成基因序列两端分别带有EcoRI和XbaI酶切位点。
1.3将酶切后合成基因片段与pGEM-3zf载体按照5:1摩尔比混合加入T4连接酶进行连接反应。将连接后的质粒转化到TOP10大肠杆菌感受态细胞。取适当转化细胞涂抹于LB固体培养基平板,于37℃培养14-16小时。挑取6个单菌落置于2mL LB液体培养基中,于37℃培养14-16小时后进行菌落PCR扩增合成基因。挑取PCR阳性克隆分别提取质粒,使用T7或VSW3引物进行测序,选择序列正确的质粒储存。即得到pGEM-Luc-IRES-EGFP载体。
1.4经测序验证后得到pGEM(T7)-Luc-ECMV IRES-EGFP质粒(简写为T7-ECMV)、pGEM(T7)-Luc-CVB3 IRES-EGFP质粒(简写为T7-CVB3)以及pGEM(VSW3)-Luc-CVB3 IRES-EGFP质粒(简写为VSW3-CVB3)。
2.载体pGEM-Luc-IRES-EGFP的应用示例:
测试其体外转录产物特性。
2.1酶切pUC19质粒并回收长片段首先,使用polyA序列3’端的内切酶将T7-ECMV质粒、T7-CVB3质粒及VSW3–CVB3质粒线性化。
2.2酚氯仿抽提纯化线性化质粒。
2.3取1ug线性化质粒作为模板,分别使用T7、耐热T7和VSW3 RNA聚合酶进行体外转录反应。得到包含5’UTR、Luciferase、IRES、EGFP、3’UTR及poly A序列的单链mRNA。
2.3将该mRNA分别用无核酶去离子水溶解后取少量进行点杂交。取双链RNA标准品作为对照。将标准品及mRNA样品点样于NC膜上,使用能够特异性识别双链RNA的J2单克隆抗体孵育膜,室温1h后,使用TBS-T buffer洗膜2次,加入HRP标记的兔抗鼠二抗孵育膜,室温0.5h,之后加入ECL检测试剂进行检测,结果如图2所示。两种T7 RNA聚合酶转录产物中双链RNA含量明显高于VSW3转录产物。
2.4将耐热T7转录的T7-ECMV及T7-CVB3 mRNA产物用LiCL沉淀后,用生理盐水溶解。分别取1ug mRNA,使用Lipofectamine 3000试剂转染293T细胞。48h后使用荧光显微镜观察EGFP的表达情况,并进行比较。
2.5结果显示T7-CVB3 mRNA转染的293细胞中EGFP表达水平较高,绿色荧光强,明显高于T7-ECMV,说明CVB3 IRES介导的蛋白表达水平高于ECMV IRES。成像结果如图3所示,A为T7-ECMV mRNA,B为T7-CVB3 mRNA。
2.6分别取30ug在BalB-C小鼠后腿进行局部肌肉注射,24h后使用PerkinELmer小动物成像仪检测Luciferase的表达情况。
2.7成像结果如图4所示,A为T7 RNA聚合酶转录产物,B为VSW3转录产物。B产物的荧光强度明显高于A,说明pUC19(VW3)-LUCIFERASE质粒与pUC19(T7)-LUCIFERASE质粒相比,体外转录所得mRNA在小鼠体内,呈现出更高的蛋白表达能力(小动物成像检测LUCIFERASE信号更强)(见图4)。
以上对本发明优选的具体实施方式和实施例作了详细说明,但是本发明并不限于上述实施方式和实施例,在本领域技术人员所具备的知识范围内,还可以在不脱离本发明构思的前提下作出各种变化。
Claims (9)
1.一种用于mRNA体外转录的质粒载体,包括启动子序列。
2.根据权利要求1所述的质粒载体,其特征在于,所述启动子序列为T7、SP6、T3或VSW3,优选地,所述启动子序列为VSW3。
3.根据权利要求1-2所述的质粒载体,其特征在于,还包括位于待插入表达的基因尾部3’-端的长度大于30的多聚腺苷脱氧核酸poly(A)片段。
4.根据权利要求1-3任一所述的质粒载体,其特征在于,根据权利要求1所述的质粒载体,其特征在于,所述多聚腺苷脱氧核酸poly(A)的长度为60-200,优选地,所述多聚腺苷脱氧核酸poly(A)的长度为 120。
5.根据权利要求1-4任一所述的质粒载体,其特征在于,还包括位于启动子下游两个插入外源基因ORF之间的核糖体进入位点(IRES)。
6.根据权利要求1-5任一所述的质粒载体,其特征在于,所述IRES 为脑炎心肌炎病毒(EMCV) IRES或柯萨奇病毒B3 (CVB3) IRES,优选地,所述IRES为 CVB3 IRES。
7.根据权利要求1-6任一所述的质粒载体,其特征在于,还包括目标蛋白基因,所述目标蛋白基因包括但不限于绿色荧光蛋白基因、zsGreen基因、mCherry基因、萤火虫荧光素酶基因、其他编码荧光蛋白的基因、WT1、GP2、KRAS、NY-ESO-1等肿瘤抗原基因的全部或部分序列。
8.一种根据权利要求1-7任一所述的体外表达mRNA的质粒载体的构建方法,包括以下步骤: 使用限制性内切酶EcoRI和XbaI将PGEM-3zf(+)质粒切成线性,并连接插入一段人工合成序列,该合成序列包括启动子序列、5'UTR、信号肽序列、基因1-ORF、内部核糖体进入位点IRES序列、基因2-ORF、其他辅助序列、3'UTR以及长度为60-200bp的-poly(A) 区段,该序列3'末端带有IIS型的BsiI内切酶位点,构建为pGEM-ORF1-IRES-ORF2质粒载体。
9.一种根据权利要求1-8任一所述的体外表达mRNA的质粒载体的应用。
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