CN112226435A - 一组降低尿酸酶基因表达的核苷酸序列及应用 - Google Patents
一组降低尿酸酶基因表达的核苷酸序列及应用 Download PDFInfo
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Abstract
本发明公开了一组降低尿酸酶基因表达的RNA序列，与该组序列相对应的DNA序列，修饰的核苷酸序列，双链核苷酸序列，包含上述核苷酸序列的递送载体，以及应用上述核苷酸序列或递送载体构建高尿酸血症小鼠模型的方法。本发明的优点在于：本发明公开了一组新的抑制尿酸酶基因表达的核苷酸序列，利用本发明公开的核苷酸序列或包含其的递送载体构建的高尿酸血症小鼠的尿酸水平升高比较稳定，发病机制与人类相似，小鼠存活时间长，并且构建动物模型的周期比较短且操作方便，从而为后续评价治疗高尿酸及相关痛风等疾病药物提供了更好的动物模型。
Description
技术领域
本发明涉及基因治疗领域，尤其涉及一组降低尿酸酶基因表达的核苷酸序列及应用。
背景技术
高尿酸血症是一类因嘌呤代谢紊乱或尿酸排泄减少，进而导致血清尿酸水平过高的一类疾病，是指正常嘌呤饮食状态下，非同日两次空腹血尿酸水平男性高于420μmol/L，女性高于360μmol/L，即称为高尿酸血症。高尿酸血症是痛风、肾功能损伤和多种心血管疾病的重要发病基础，近年来，高尿酸血症和痛风的患病率越来越高，并且高尿酸血症作为诸多代谢紊乱因素之一，常与高血压、糖尿病、肥胖、动脉粥样硬化等疾病的发展密切相关，是威胁人类健康的重要疾病。控制高尿酸血症对这些疾病具有较好的防治作用。
目前治疗高尿酸血症的药物根据作用机制不同可分为：抑制尿酸生成的药物；促进尿酸排泄的药物；促进尿酸分解的药物。常见的有别嘌呤醇、非布索坦、丙磺舒、苯溴马隆等。但这些药物大多副作用明显，对肝功能、肾功能及心肌功能造成一定损伤，患者易出现皮肤过敏、腹泻、肝功能异常等不良反应。目前寻找副作用小、安全经济的降尿酸药物的需求十分迫切，但此类药物的筛选和开发依赖于合适的高尿酸血症动物模型，因此，确立一种高效筛选药物的高尿酸血症动物模型显得至关重要。
常用的高尿酸血症动物模型的造模方法主要有三种，一是由动物直接摄入尿酸、高嘌呤食物或尿酸前体物质，如黄嘌呤或次黄嘌呤，促进尿酸的形成，形成高尿酸血症模型。二是通过给药抑制尿酸排泄，增加血尿酸浓度，形成高尿酸血症，常用的药物为腺嘌呤、烟酸等。三是抑制尿酸酶(urate oxidase，Uox)的活性，常用的尿酸酶抑制剂为氧嗪酸等。目前大多数高尿酸血症多以两种或三种造模物质同时使用，获得稳定持久的高尿酸血症模型。但是以上方法诱导的高尿酸动物模型中的血尿酸水平存在波动，并且与人高尿酸发病机制存在不一致的情况。
尿酸酶可将尿酸分解为更易溶于水的小分子尿囊素排出体外，这是许多包括小鼠在内的低级动物维持尿酸稳态的主要途径，但是人类在进化过程中由于基因突变，导致该基因不能表达尿酸酶，这是人类易患高尿酸血症的主要原因。因此有研究采用敲除尿酸酶基因法制备高尿酸血症小鼠模型，但是此种方法制备的动物模型血尿酸会升高很多，达到致死水平，可以存活到成熟的小鼠很少，并且试验周期比较长。
基于上述问题，亟需开发出一种有效构建稳定和长期有效的高尿酸模型动物的方法。
RNA干扰(RNA interference，RNAi)是由双链RNA(double-stranded RNA，dsRNA)诱发的基因沉默现象，当细胞中导入与内源性mRNA编码区同源的双链RNA时，该mRNA发生降解而导致基因沉默。RNA干扰是一种有力的基因沉默工具，在微生物学、基因表达调控机制研究等领域广泛应用。当外源性dsRNA进入细胞后，可被Dicer酶识别加工成21-23个核苷酸的短链RNA，即为小干扰RNA(siRNA)。siRNA进入细胞后，可与细胞质中的蛋白质形成RNA诱导沉默核酸蛋白复合物(RNA-induced silencing complex，RISC)，与序列互补的靶mRNA结合，对mRNA进行酶切，阻断相应蛋白的翻译。siRNA具有特异性、高效性，是特异性抑制基因表达的强效方法。
但是，体外化学合成的siRNA在体内易被核酸酶快速酶解，其跨细胞膜转运能力弱、半衰期短、体内基因沉默效率低。目前多项研究正尝试通过利用化学手段对构成siRNA的多聚核苷酸链进行结构修饰，来增强siRNA的稳定性，修饰法包括：末端修饰，例如：5’-端修饰(磷酸化、接合、反向键联)或3’-端修饰(接合、DNA核苷酸、反向键联，等等)；碱基修饰，例如使用稳定的碱基、去稳定化的碱基或会与对象的扩张区的碱基配对的碱基置换、排除碱基(去碱基核苷酸)、或接合碱基；糖修饰(例如：位于2’-位置或4’-位置上)或置换糖；和/或主链修饰，包括修饰或置换磷酸二酯键联体。但是，此类修饰一般会导致siRNA的干扰活性降低。
与直接使用siRNA相比，载体介导的shRNA在体内表达的方法具有一定的优势。将shRNA对应的dsRNA序列克隆到到相应载体中，这些载体包含依赖RNA polymerase III启动子，如U6、7SK或Hl启动子，有一个明确的转录起始点和一个由连续5个胸腺嘧啶组成的转录终止信号(T5)。其中一对特定的寡核苷酸来源于目标基因mRNA的一段长度为19-21个碱基的独特序列。当将正向和反向的DNA寡核苷酸退火，并克隆至载体的两个限制性内切酶位点之间时，正向DNA寡核苷酸将正确定位于H1启动子的下游。重组载体的转录产物即发夹型RNA(small hairpin RNA，shRNA)可以自身折叠配对形成长为19-21个碱基的茎环结构，这种茎环结构的前体在细胞内很快被切割形成有功能的siRNA。这种载体表达的shRNA经剪切形成的siRNA具有表达量稳定、持续时间长的特点，从而可引起目标基因表达的长期有效抑制。
常用的载体包括腺相关病毒(adeno-associated virus,AAV)、逆转录病毒、慢病毒。与后两者相比,腺相关病毒(AAV)的突出优势在于安全性高，免疫原性低,宿主范围广，携带的转染基因能长期稳定表达,体外和体内均可应用,具有多种血清型,能满足针对不同组织的转染要求,具有广阔的应用范围和前景。
目前尚无利用RNA干扰技术构建高尿酸模型小鼠的报道，因此，寻找一种针对尿酸酶基因的特异RNA干扰序列，并将此干扰序列构建于腺相关病毒载体，病毒载体进入体内，从而抑制尿酸酶的表达，这为有效构建一种高尿酸模型动物提供了新思路。
发明内容
本发明的目的是提供一组降低尿酸酶基因表达的核苷酸序列，包括核糖核酸(RNA)和脱氧核糖核酸(DNA)。
本发明的另一个目的是提供上述核苷酸序列的应用，利用上述核苷酸序列，通过RNA干扰技术抑制尿酸酶的表达。
为实现上述发明目的，本发明采用以下设计方案：
一种降低尿酸酶基因表达的RNA序列，所述RNA序列选自：
1)AUACCACAGCAUCAAAGAGGU(SEQ ID NO：1)；
2)AAGUGUACUGCAAGUGGCGCU(SEQ ID NO：2)；
3)CAGACACCAUCAAGAACACUG(SEQ ID NO：3)或
4)CAGACACCAUCAAGAACACAG(SEQ ID NO：4)。
所述的RNA序列和与之反向互补的序列杂交形成的双链RNA序列。该RNA双链具有RNA干扰活性，可以使特异的mRNA降解。
所述的RNA序列和与之反向互补的序列由中间的非互补连接序列共价连接后获得的可形成发夹样结构的RNA序列。
所述的可形成发夹样结构的RNA序列和与之反向互补的序列杂交形成的双链RNA序列，在细胞内可通过载体表达发夹样双链，具有干扰RNA活性，促使特异mRNA降解。
所述的RNA序列的5’端和/或3’端进行核苷酸修饰获得的序列。这种修饰可以是在合成RNA的3’端和/或5’端加入UU修饰。
一种与前述的降低尿酸酶基因表达的RNA序列相对应的DNA序列，所述DNA序列选自：
1)ATACCACAGCATCAAAGAGGT(SEQ ID NO：5)；
2)AAGTGTACTGCAAGTGGCGCT(SEQ ID NO：6)；
3)CAGACACCATCAAGAACACTG(SEQ ID NO：7)或
4)CAGACACCATCAAGAACACAG(SEQ ID NO：8)。
所述的DNA序列和与之反向互补的序列杂交形成的双链DNA序列。
所述的DNA序列和与之反向互补的序列由中间的非互补连接序列共价连接后获得的可形成发夹样结构的DNA序列。
所述的可形成发夹样结构的DNA序列和与之反向互补的序列杂交形成的双链DNA序列。
在所述DNA序列的5’端和/或3’端进行核苷酸修饰获得的序列。其用途如在DNA两端加上酶切位点。
包含前述的RNA序列或DNA序列的递送载体，所述递送载体选自病毒载体和非病毒载体。优选地，所述非病毒载体选自脂质体、质粒载体、噬菌体载体组成的组。优选地，所述病毒载体选自腺病毒载体、腺相关病毒载体、慢病毒载体和杂合病毒载体组成的组。更优选地，所述病毒载体选自腺相关病毒载体。
一种构建高尿酸血症模型动物的方法，包括如下步骤：利用前述的序列或前述的递送载体降低目的动物中尿酸酶基因(Uox)的表达，得到的所述尿酸酶表达降低的动物即为高尿酸血症模型动物。
一种特异性降低尿酸酶基因表达的shRNA，其碱基序列如SEQ ID NO：13所示，其针对的靶序列为尿酸酶编码基因的第108-128位，如SEQ ID NO：5所示，该shRNA通过如SEQ IDNO：21所示的DNA寡核苷酸链转录而成。或通过包含如SEQ ID NO：21所示的DNA寡核苷酸链的质粒转录而成，该shRNA可剪切形成含如SEQ ID NO：1和SEQ ID NO：9所示的siRNA正义链和反义链。
一种与前述shRNA相对应的DNA序列，其碱基序列如SEQ ID NO：21所示。
所述的DNA序列和与之反向互补的序列杂交形成的双链DNA序列。
一种包含前述特异性降低尿酸酶基因表达的shRNA或对应DNA序列的载体。
前述的特异性降低尿酸酶基因表达的shRNA或对应DNA序列或包含前述shRNA或对应DNA序列的载体在制备治疗高尿酸血症模型动物中的应用。
一种构建高尿酸血症模型动物的方法，包括利用前述的特异性降低尿酸酶基因表达的shRNA或对应DNA序列或包含前述shRNA或对应DNA序列的载体降低目的动物中尿酸酶基因(Uox)的表达，从而得到的所述尿酸酶表达降低的动物即为高尿酸血症模型动物。
本发明与现有技术相比具有以下优点：
1)与现有利用化学试剂诱导的高尿酸动物模型相比，利用本发明的核苷酸序列和方法构建的高尿酸模型动物尿酸水平升高比较稳定，持续时间长久，为后续药物在动物水平上的活性评价提供了有力的帮助。
2)与完全敲除尿酸酶的转基因小鼠相比，利用本发明所产生的模型动物存活时间长，且构建模型动物的时间周期比较短，操作方便。
下面结合附图和具体实施方式对本公开做进一步说明，并非对本公开的限制。凡是依照本专利申请公开内容所进行的任何本领域等同替换，均属于本专利的保护范围。
附图说明
图1示出了BglII和HindIII双酶切T载体所产生的不同片段，其中M1为15K DNAMarker，M2为2K DNA Marker。
图2示出了BglII和HindIII双酶切公司自建质粒pSC-H1-shRNA-HPRT intron2，其中M1为15K DNA Marker，M2为2K DNA Marker。
图3示出了不同干扰组Uox蛋白表达水平，其中图3A表示蛋白WB结果，图3B表示蛋白WB灰度扫描结果，对照组为注射PBS组。
图4示出了rAAV-shRNA#1干扰组2周时血尿酸检测水平，对照组为注射PBS组。
图5示出了rAAV-shRNA#1干扰组5周时血尿酸检测水平，对照组为注射PBS组。
图6示出了rAAV-shRNA#1干扰组2周、5周时血尿酸检测水平，对照组为注射PBS组。
图7示出了rAAV-shRNA#1干扰组小鼠的存活率，对照组为注射PBS组。
具体实施方式
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所使用的材料、试剂等，如无特殊说明，均可从商业途径得到。
下面的实施例仅仅是说明性的，而非意在限制本发明。
实施例1载体构建
根据小鼠尿酸酶基因(Uox)序列(Genebank：NM_009474.5)，利用https://www.sigmaaldrich.com/life-science/functional-genomics-and-rnai/sirna/mission-predesigned-sirna.html网站设计针对小鼠尿酸酶基因的RNA干扰片段，具体序列见表1所示。
表1
| 名称 | 靶序列(SEQ ID NO:5-8) | 正义链(5’-3’)(SEQ ID NO:1-4) | 反义链(5’-3’)(SEQ ID NO:9-12) |
| siRNA#1 | ATACCACAGCATCAAAGAGGT | AUACCACAGCAUCAAAGAGGU | ACCUCUUUGAUGCUGUGGUAU |
| siRNA#2 | AAGTGTACTGCAAGTGGCGCT | AAGUGUACUGCAAGUGGCGCU | AGCGCCACUUGCAGUACACUU |
| siRNA#3 | CAGACACCATCAAGAACACTG | CAGACACCAUCAAGAACACUG | CAGUGUUCUUGAUGGUGUCUG |
| siRNA#4 | CAGACACCATCAAGAACACAG | CAGACACCAUCAAGAACACAG | CUGUGUUCUUGAUGGUGUCUG |
将上述RNA干扰片段设计成能形成发夹结构的序列见表2所示，其相应的DNA序列见表3所示，在3’端增加shRNA转录终止信号5个连续的T，两端分别增加BglII酶切位点和HindIII酶切位点，将所述序列合成，并插入到T载体(Promega,A1360)。
表2
表3
将分别含有上述不同shRNA序列相对应DNA序列的T载体命名为T1-T4，用BglII和HindIII双酶切T1，酶切结果如图1所示，胶回收小片段；T2-T4载体也采用同样的操作。同样用BglII和HindIII双酶切载体pSC-H1-shRNA-HPRT intron(公司自建载体)，酶切结果如图2所示，回收载体大片段；将回收的小片段与载体大片端连接，转化，测序。携带外源基因shRNA的AAV包装所需要的穿梭质粒构建成功，用于后续AAV病毒包装。
实施例2病毒包装及基因组滴度检测
本实施例采用HEK293T细胞(购于ATCC，其编号为CRL-11268)作为生产细胞系，常规三质粒包装系统生产重组AAV病毒载体。所使用实验方法均为本领域常规方法(参见XiaoXiao,Juan Li,and Richard Jude Samulski.Production of high-titer recombinantadeno-associated virus vectors in the absence of helperadenovirus.J.Virol.1998,72(3):2224)。
取适量纯化AAV样品，配制DNase I消化反应混合液，37℃孵育30min，75℃孵育10min，失活DNase I。
处理后的AAV纯化样品稀释适当的倍数后，参照下表(表4)配置Q-PCR反应体系，并按照下列程序进行检测。
表4
其中使用的引物参见下表(表5)：
表5
| 上游引物(5’-3’) | ACCCGCTCCAAGGAATCG |
| 下游引物(5’-3’) | AAATATTGCAGGGCGCCAC |
包装产量结果参见下表(表6)：
表6
| 病毒载体 | 基因组滴度(vg/ml) |
| rAAV-shRNA#1 | 2E+12 |
| rAAV-shRNA#2 | 1E+12 |
| rAAV-shRNA#3 | 4E+12 |
| rAAV-shRNA#4 | 5E+11 |
实施例3含有不同干扰片段的重组病毒在小鼠体内降低尿酸酶基因表达的效率检测
选择6-8周龄的C57小鼠，分成5组，4种给药组(rAAV-shRNA#1、rAAV-shRNA#2、rAAV-shRNA#3、rAAV-shRNA#4)及PBS空白对照组，尾静脉注射,每种给药组设为一种特定的干扰组。4种给药组注射剂量为1×1012vg/kg，空白对照组PBS注射剂量为200μl，注射2两周后处死，同时留取血清和肝组织，用于血尿酸和Uox蛋白的检测。
首先通过离心(4,000rpm，4℃，5分钟)分离血清，然后用Uric Acid Assay Kit(sigma，MAK077)检测不同组中血尿酸水平，具体操作参照试剂盒说明书。首先配制标准品和反应体系，绘制标准曲线。采用荧光检测血尿酸，根据标准曲线计算出各组的血尿酸值，检测结果见表7所示。从表7中，可以看出注射rAAV-shRNA#1重组病毒后，小鼠的血尿酸水平明显高于对照组50μM左右。
表7不同干扰组血尿酸水平
采用RIPA裂解液(普利莱，C1053)提取小鼠的肝组织蛋白，具体提取方法参照试剂说明书，首先取100mg肝组织在冰上剪成碎片，用预冷的PBS洗涤2次离心弃去PBS，然后加入0.5mL预冷RIPA裂解缓冲液，用玻璃匀浆器在冰上匀浆，直到95％的细胞被破碎。最后，离心(12000g，4℃，10分钟)，上清即为肝组织总蛋白。
然后采用Western-Blot检测尿酸酶的含量，具体方法如下：将提取的蛋白应用BCA蛋白定量试剂盒(Thermo Scientific，23225)进行蛋白定量后，各组样品用RIPA裂解液统一调整浓度至30mg/mL，制备好样品后，SDS-PAGE电泳。电泳完成后，转模，封闭，清洗，一抗孵育过夜(抗-UOX抗体，Santa Cruze，sc-166214；抗-actin抗体，proteintech，66009-1-Ig)，清洗。第二天，孵育山羊抗小鼠IgG(碧云天，A0216)，清洗，曝光显色，结果见图3A所示。并采用Image J软件对WB结果进行灰度分析，结果见图3B所示。从图3A和图3B可知，rAAV-shRNA#1干扰效果最好，干扰效果在90％左右。
实施例4高尿酸血症动物模型的建立与评价
根据上述实验结果，我们选取rAAV-shRNA#1进行动物模型制造实验。选取6-8周龄的C57小鼠，分成给药组和空白对照组，每组10只小鼠，尾静脉注射。给药组注射rAAV-shRNA#1病毒,注射剂量为1×1012vg/kg，空白对照组注射等体积的PBS。小鼠分别在注射后的2周和5周时，眼角取血，采用Uric Acid Assay Kit(sigma，MAK077)检测血中尿酸含量，具体方法参见实施例3，检测结果见图4和图5所示，不同时间点血尿酸的含量比较结果见图6所示。
从图4、图5和图6的结果可以看出，注射rAAV-shRNA#1的给药组中的血尿酸水平明显高于对照组(PBS组)中血尿酸水平，并且具有显著的统计学差异(P<0.01)，且高血尿酸水平在实验期间6周内保持持续的高水平。
另外，对不同组小鼠的存活率进行检测，结果见图7所示，显示给药组和对照组小鼠在实验期间6周内都可以正常存活，完全可以满足高尿酸和痛风药物的体内活性评价。
由上述实验可知，利用本发明的核酸序列和方法构建的高尿酸动物模型长期有效且效果稳定，可以用于后续的高尿酸和痛风药物的活性评价。
序列表
<110> 舒泰神（北京）生物制药股份有限公司
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Claims (22)
1.一种降低尿酸酶基因表达的RNA序列，其特征在于，所述RNA序列选自：
1)AUACCACAGCAUCAAAGAGGU(SEQ ID：1)；
2)AAGUGUACUGCAAGUGGCGCU(SEQ ID：2)；
3)CAGACACCAUCAAGAACACUG(SEQ ID：3)或
4)CAGACACCAUCAAGAACACAG(SEQ ID：4)。
2.权利要求1所述的RNA序列和与之反向互补的序列杂交形成的双链RNA序列。
3.权利要求1所述的RNA序列和与之反向互补的序列由中间的非互补连接序列共价连接后获得的可形成发夹样结构的RNA序列。
4.权利要求3所述的可形成发夹样结构的RNA序列和与之反向互补的序列杂交形成的双链RNA序列。
5.在权利要求1或2所述的RNA序列的5’端和/或3’端进行核苷酸修饰获得的序列。
6.一种与权利要求1所述的降低尿酸酶基因表达的RNA序列相对应的DNA序列，其特征在于，所述DNA序列选自：
1)ATACCACAGCATCAAAGAGGT(SEQ ID：5)；
2)AAGTGTACTGCAAGTGGCGCT(SEQ ID：6)；
3)CAGACACCATCAAGAACACTG(SEQ ID：7)或
4)CAGACACCATCAAGAACACAG(SEQ ID：8)。
7.权利要求6所述的DNA序列和与之反向互补的序列杂交形成的双链DNA序列。
8.权利要求6所述的DNA序列和与之反向互补的序列由中间的非互补连接序列共价连接后获得的可形成发夹样结构的DNA序列。
9.权利要求8所述的可形成发夹样结构的DNA序列和与之反向互补的序列杂交形成的双链DNA序列。
10.在权利要求6-9中任一项所述DNA序列的5’端和/或3’端进行核苷酸修饰获得的序列。
11.包含权利要求1-5中任一项所述的RNA序列或权利要求6-10中任一项所述的DNA序列的递送载体。
12.权利要求11所述的递送载体，其特征在于，所述递送载体选自病毒载体和非病毒载体。优选地，所述非病毒载体选自脂质体、质粒载体、噬菌体载体组成的组。优选地，所述病毒载体选自腺病毒载体、腺相关病毒载体、慢病毒载体和杂合病毒载体组成的组。更优选地，所述病毒载体选自腺相关病毒载体。
13.一种构建高尿酸血症模型动物的方法，包括如下步骤：利用权利要求1-10中任一项所述的序列或权利要求11-12中任一项所述的递送载体降低目的动物中尿酸酶基因的表达，得到的所述尿酸酶表达降低的动物即为高尿酸血症模型动物。
14.一种特异性降低尿酸酶基因表达的shRNA，其特征在于其碱基序列如SEQ ID NO：13所示。
15.根据权利要求14所述的一种特异性降低尿酸酶基因表达的shRNA，其特征在于其针对的靶序列为尿酸酶编码基因的第108-128位，如SEQ ID NO：5所示。
16.根据权利要求14所述的一种特异性降低尿酸酶基因表达的shRNA，其特征在于该shRNA通过如SEQ NO：21所示的DNA寡核苷酸链或包含如SEQ ID NO：21所示的DNA寡核苷酸链的质粒转录而成。
17.根据权利要求14所述的一种特异性降低尿酸酶基因表达的shRNA，其特征在于该shRNA可剪切形成含如SEQ ID NO：1和SEQ ID NO：9所示的siRNA正义链和反义链。
18.一种与权利要求14所述的shRNA相对应的DNA序列，其特征在于其碱基序列如SEQID NO：21所示。
19.权利要求18所述的DNA序列和与之反向互补的序列杂交形成的双链DNA序列。
20.一种包含权利要求14-17所述特异性降低尿酸酶基因表达的shRNA或权利要求18-19所述的DNA序列的载体。
21.一种如权利要求14-17所述的特异性降低尿酸酶基因表达的shRNA或权利要求18-19所述的DNA序列或权利要求20所述包含shRNA或DNA序列的载体在制备治疗高尿酸血症模型动物中的应用。
22.一种构建高尿酸血症模型动物的方法，包括利用权利要求14-17所述的特异性降低尿酸酶基因表达的shRNA或权利要求18-19所述的DNA序列或权利要求20所述包含shRNA或DNA序列的载体降低目的动物中尿酸酶基因的表达，从而得到的所述尿酸酶表达降低的动物即为高尿酸血症模型动物。
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