CN108085331B - 用于环状rna过表达的dna框架及其应用 - Google Patents
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Abstract
本发明涉及一种用于环状RNA过表达的DNA框架及其应用,将这种人工合成DNA框架序列***真核表达载体、慢病毒载体、腺病毒载体、腺相关病毒载体上,可形成circRNA专用表达载体。本发明涉及的DNA框架序列及其相应表达载体能广泛的应用于各种circRNA的表达,为研究circRNA的功能和机制以及进一步开发基于circRNA的基因治疗药物提供了一个有利的工具。
Description
[技术领域]
本发明涉及用于环状RNA过表达的DNA框架及其应用。
[背景技术]
环状RNA(circular RNA,circRNA)是区别于传统线性RNA的一类新型RNA,其不具有传统线性RNA中的5'末端帽子和3'末端poly(A)尾巴,而以闭合环状结构存在。研究表明环状RNA主要通过非典型可变剪切加工产生,广泛存在于各种生物细胞中,具有结构稳定,难以被RNA酶降解、表达丰度高、物种间保守性好,表达具有组织及时空特异性等特征。对于circRNA与疾病的研究表明circRNA与肿瘤、神经发育、动脉粥样硬化、强直性肌营养不良等疾病的发生和进展相关。另外,研究发现在人唾液和血液中能够检测到稳定的circRNA的存在,这些特点使得环状RNA在新型疾病诊断与治疗方法的开发应用上具有广阔的前景。
目前对于circRNA的研究尚不深入,circRNA在生物功能和调控机制仍然不甚明了。虽然已有的研究认为,circRNA可以作为“sponge”海绵吸附细胞内的microRNA,阻断microRNA对其靶基因的抑制作用;circRNA还可以通过碱基互补配对直接调控其他RNA水平;此外,研究认为circRNA可能具有与蛋白质结合,调控蛋白的活性等功能。然而,circRNA的生物学活性及其在胞内发挥作用的机制需要深入的研究。
正如常规基因功能和机制研究一样,研究circRNA的生物学功能和机制的一个重要研究手段就是在胞内过表达circRNA,观察其对细胞功能的影响并阐释其参与调控细胞功能的分子机制。那么,要在胞内引入过表达的circRNA就必须有稳定可靠的circRNA过表达工具。目前过表达环状RNA的方法通常以基因组DNA为模板,扩增目的circRNA序列及其上游1000bp和下游200bp的序列,然后将上游800bp的反向互补序列通过PCR拼接的方式***到下游,然后将得到的完整序列通过酶切连接的方式***商业化的过表达载体如pCDNA3.1上表达目的circRNA。此种方法不适合用于人工设计的环状RNA的过表达。2014年Liang利用对hsa_circ_0001727的上下游序列研究,优化构建了一个circRNA表达框架,但是该表达框架有局限性,仅对部分内源性circRNA的过表达有效,且过表达效率较低。
[发明内容]
本发明的目的是在于提供一种适用于circRNA表达的DNA序列及其载体,该序列普遍适用于各种circRNA的表达,表达效率高效、稳定;该序列能整合到各种类型的表达载体,能应用于普通真核表达、慢病毒表达、腺病毒表达、腺相关病毒表达、逆转录病毒表达等各种表达体系中;应用含有该序列的载体表达circRNA操作简单易行,易于推广。
为了实现上述目的,发明一种式I序列的用于环状RNA过表达的DNA框架,
US-[N]n-DS
I
其中,
US为上游序列,如SEQ ID NO.1所示,
DS为下游序列,如SEQ ID NO.2所示,
[N]n为中间序列,N代表A、T、G、C四种脱氧核糖核苷酸中任意一种,n个相同或者不同的N依次连接形成中间序列。
上述用于环状RNA过表达的DNA框架还具有如下优化结构:
所述的[N]n优选所需表达的circRNA的线性DNA序列。进一步优选内源性的circRNA的线性DNA序列,更进一步包括hsa_circ_0001727、hsa_circ_0001756、hsa_circ_0000284、hsa_circ_0000711或hsa_circ_0000268的线性DNA序列。
n的最佳范围为150≤n≤3000。
本发明还包括一种用于环状RNA过表达的DNA框架,其DNA序列如SEQ ID NO.4所示。
本发明还包括一种表达载体,通过上述用于环状RNA过表达的DNA框架序列***真核表达载体、慢病毒载体、腺病毒载体、腺相关病毒载体上形成。
本发明的用于环状RNA过表达的DNA框架可以用作circRNA的过表达。
该circRNA过表达DNA框架序列能整合到各种类型的表达载体,能应用于普通真核表达、慢病毒表达、腺病毒表达、腺相关病毒、逆转录病毒表达等各种表达体系中。
本发明的实验过程中设计了多种DNA序列,经过可靠的实验结果证实本发明涉及的circRNA过表达DNA框架序列设计简单,表达载体的选择多样,载体构建操作简便、circRNA过表达稳定高效(circRNA过表达倍数达数百倍以上)。该序列及相应的载体能广泛应用于各种circRNA的表达,为circRNA的功能和机制研究提供了一个有力的工具。
[附图说明]
图1为实施例中环状RNA通用过表达框架示意图。
图2为本发明构建的多个circRNA过表达质粒转染293T细胞后相应circRNA的过表达效果图。
[具体实施方式]
以下结合实施例来进一步解释本发明,但实施例并不对本发明做任何形式的限定。
本实施例利用真核生物基因剪接的GT-AG法则,在hsa_circ_0001727的上下游侧翼序列的基础上,分别对侧翼反向重复序列进行简化和优化,并在5'和3'剪切位点两侧同时进行优化设计,获得通用型的circRNA过表达框架(上游序列+中间序列+下游序列)。根据此通用的过表达框架序列设计原则,设计多条circRNA过表达框架DNA序列并利用化学合成的方式进行合成,然后利用酶切链接的方式将人工环状RNA过表达框架序列构建至商业化的表达载体(真核表达载体、慢病毒载体、腺病毒载体等)上。然后将上述构建好的circRNA过表达载体转染至293T细胞中,验证circRNA过表达框架的过表达效率。
本发明所涉及的技术为基因合成、载体构建、细胞培养、质粒转染、qPCR等常规技术手段,其中涉及的酶、引物、试剂、细胞培养反应条件、细胞转染条件在未作说明的情况下均可根据本领域技术人员的经验进行合理选择,其中涉及试剂耗材属于市售的普通产品,细胞为ATCC来源。其中涉及的检测手段以及仪器也均为本领域技术人员所熟知并熟练掌握。
下面通过实施例和试验例对本发明的技术方案做进一步的说明,但不应理解为对本发明的限制。
实施例一:has_circ_0001727过表达载体构建
1.hsa_circ_0001727过表达框架序列设计:
从circBase数据库查询获取编码hsa_circ_0001727环状RNA的线性DNA序列。序列如SEQ ID NO.3所示。
根据上述通用circRNA过表达DNA框架序列设计原则,将查询到的hsa_circ_0001727的DNA序列分别替换式I序列中的[N]n,得到hsa_circ_0001727的专用过表达框架DNA序列,序列如SEQ ID NO.4所示。
而进一步的,在其他实施例中,如图1所示,n还可以采用2、3、4、5、10、20、30、40、50、100、200、300、400、500、1000、2000、3000甚至更多的倍数,其可以是相同的N或者不同的N。
2.将hsa_circ_0001727的专用过表达框架DNA序列交由南京金斯瑞公司进行序列合成。
3.引物设计合成
用Primer5软件进行引物设计:
Circ-F:如SEQ ID NO.5所示。
Circ-R:如SEQ ID NO.6所示。
引物序列由上海华大基因公司进行合成。
4.PCR扩增hsa_circ_0001727的专用过表达框架DNA序列
以全基因合成得到的hsa_circ_0001727的专用过表达框架DNA序列为模板,PCR扩增目的片段,扩增体系如下:
10×Buffer | 10ul |
MgSO<sub>4</sub>(50mM) | 1ul |
dNTP(10mM) | 1.5ul |
transStart Fastpfu DNA polymerase(5U/ul) | 0.5ul |
Circ-F(10uM) | 2ul |
Circ-R(10uM) | 2ul |
模板DNA(50ng/ul) | 1ul |
加ddH<sub>2</sub>O至总体积 | 50ul |
PCR循环程序如下:
5.PCR产物回收
琼脂糖凝胶电泳PCR产物,并利用胶回收试剂盒(Axygen,AP-GX-50),按照试剂盒说明书的详细操作步骤回收PCR产物。
6.PCR回收产物Nhe I和HindⅢ双酶切以及酶切产物回收
将PCR回收产物用Asc I和Pme I进行双酶切,酶切体系如下:
10×Buffer | 3ul |
PCR产物/目的质粒 | 1ug |
限制性内切酶Nhe I | 0.5ul |
限制性内切酶HindⅢ | 0.5ul |
补ddH<sub>2</sub>0至总体积 | 30ul |
37℃酶切4-5小时后,电泳分离酶切片段,切胶回收目的片段。
7.pCDNA3.1质粒Nhe I和HindⅢ双酶切以及酶切产物回收
将真核表达质粒pCDNA3.1用Nhe I和HindⅢ进行双酶切,酶切体系如下:
10×Buffer | 3ul |
PCR产物/目的质粒 | 1ug |
限制性内切酶Nhe I | 0.5ul |
限制性内切酶HindⅢ | 0.5ul |
补ddH<sub>2</sub>0至总体积 | 30ul |
37℃酶切4-5小时后,电泳分离酶切片段,切胶回收线性化质粒。
8.目的片段与线性化pCDNA3.1质粒链接
利用T4 DNA ligase连接上述酶切后的PCR产物及慢病毒载体,连接体系如下:
室温连接1h。同时做阴性对照,以水代替基因与载体连接。
9.连接产物的转化
1)冰浴中将连接产物分别加入至50μl Tran5α感受态细胞中。轻轻旋转混匀,冰浴30min。
2)42℃水浴热激90s。
3)快速将管转移至冰浴中,冰浴2min。
4)分别加入500μl LB培养基,混匀,37℃、150g振荡培养40min。
5)将150ul菌液涂布于含氨苄青霉素(Amp)(100μg/ml)的LB平板表面,室温下放置,至液体吸收。倒置平板,转移入37℃生化培养箱过夜培养。
10.阳性克隆PCR鉴定
转化后次日,挑取单菌落进行菌落PCR,PCR扩增体系及循环程序如下:扩增体系如下:
10×Reaction Buffer | 1.5ul |
MgCl<sub>2</sub>(25mM) | 1.5ul |
dNTPs(10mM) | 0.5ul |
Circ F(10mM) | 0.5ul |
Circ R(10mM) | 0.5ul |
Taq(5U/ul) | 0.1ul |
菌落悬液 | 1ul |
补ddH<sub>2</sub>0至总体积 | 15ul |
循环条件如下:
11.阳性克隆摇菌和质粒抽提
将PCR鉴定呈阳性的克隆在含有相应抗生素的1ml LB液体培养基中培养,37℃摇菌过夜,次日利用质粒小量提取试剂盒(Axygen,AP-MN-P-50),按照说明书的详细步骤抽提质粒。
12.将抽提好的质粒送至华大基因进行测序,对测序结果进行序列比对。得到构建完成的hsa_circ_0001727的专用过表达质粒。
实施例二:qPCR检测hsa_circ_0001727过表达质粒转染后293T细胞中hsa_circ_0001727过表达效果
1,qPCR引物设计和合成
divergent primer-F:CAGGTCAAGTTCATGGACCTG
divergent primer-R:ATTCAGACTTACCTGAAGTA
引物序列由上海华大基因公司进行合成。
2,转染前24h,用胰酶消化对数生长期的293T细胞,传代到接种至六孔板,37℃、5%CO2培养箱内培养。24h待细胞密度达70%~80%时即可用于转染。
3,转染前将细胞培养基更换为无血清培养基。
4,向一灭菌离心管中加入所制备的hsa_circ_0001727过表达质粒DNA溶液25ug,与相应体积的Opti-MEM混合均匀,调整总体积为1.5ml。
5,将Lipofectamine 2000试剂轻柔摇匀,取60μl Lipofectamine 2000试剂在另一管中与1.5ml Opti-MEM混合,在室温下温育5分钟。
6,把稀释后的DNA与稀释后的Lipofectamine 2000进行混合,轻轻地颠倒混匀,不要振荡。
7,将DNA与Lipofectamine 2000混合液转移至293T细胞的培养液中,混匀,于37℃,5%CO2细胞培养箱中培养。
8,培养6小时后吸去含有转染混和物的培养基,每瓶细胞中加入含10%血清的细胞培养基10ml,于37℃、5%CO2培养箱内继续培养48小时。
9,6孔板每孔加入1ml Trizol,用1ml枪头反复吹打10次,收集到EP管内;12000g离心15分钟,取上清。
10,向上清中加入200ul氯仿,上下用力颠倒混匀半分钟,静置3分钟。
11,4℃,12000g离心15分钟,此时可见裂解液分三层:上层为水相的RNA;中层为DNA,脂类等;下层为细胞残渣,蛋白,多糖等。
12,取上清500ul到新的EP管内,167ul吸三次;加入等体积的异丙醇,混匀,静置10分钟后,4℃,12000g离心10分钟。
13,小心去掉上清,注意不要丢失RNA沉淀,加入1ml 75%乙醇,上下颠倒,使沉淀块重悬起。
14,4℃,12000g离心10分钟,小心去掉上清,尽量吸干管壁的液体,注意不要丢失RNA沉淀,若沉淀松动可再次离心。晾干约15分钟,至管壁无液体。
15,加入适量体积(20-30ul)的DEPC水溶解RNA,58℃水浴10分钟。
16,取出2ul定量,测量buffer:10mM TrisCl(pH7.8),根据定量结果进行逆转录。(1A260=40μg/ml,A260/A280=1.8~2.1)
17,RNA反转录
根据操作说明书进行反转录,体系如下:
在RNase-Free的PCR管中加入(总体系20ul)
RNA | 3μg |
DEPC·H 2O | 补足至11.0 |
混匀,65℃孵育10min,立刻冰浴,然后加入
2.5U/μl Poly A Polymerase | 1μl |
RTase Mix | 1μl |
5×PAP/RT Buffer | 5μl |
dd H2O(RNase/Dnase free) | 8μl |
37℃孵育60min,85℃,5min;cDNA冻存于-20℃或即刻PCR。
18,qPCR检测
1)在RT-PCR预实验摸索出最佳的引物退火温度和模板量的前提下,使用2×SYBRGreen Mix配制PCR Mix,根据需要上机的样品数和重复数,计算并配制PCR Mix,体系如下:
2×SYBR Green Mix | 10μl |
qPCR引物Mix | 1μl |
模板 | 5μl |
超纯水 | 4μl |
总体积 | 20μl |
2)分装至PCR8连管,微型离心机瞬时离心混匀PCR体系。
3)将上述样品放入IQ5荧光定量PCR仪,SYBR Green法荧光定量PCR以分析各基因的表达,PCR程序设置如下:
PCR反应可设成3步法:(退火温度结合引物的Tm值及RT-PCR预实验的结果自行设定,融解曲线可设60-95℃)
预变性Cycle 1:(1X)
Step 1:95.0℃for 02:00.
PCR循环Cycle 2:(40X)
Step 1:95.0℃for 00:15.
Step 2:60.0℃for 00:20.
Step 3:72.0℃for 00:20.
Data collection and real-time analysis enabled.
溶解曲线Cycle 3:(71X)
Step 1:60.0℃-95.0℃for 00:30.
Increase set point temperature after cycle 2 by 0.5℃
Melt curve data collection and analysis enabled.
19,qPCR相对定量结果分析
目标基因的相对表达量计算公式为:Ct目的为目标基因Ct值,Ct内参为管家基因Ct值。△Ct=Ct目的-Ct管家,表示各样本目的基因相对管家基因的相对Ct值,△△Ct=(△Ct)Test-(△Ct)Control,表示处理组相对对照组进行归一化,表示处理组相对对照组的相对表达量,表示目的基因的相对表达倍数。
采用上述相同的方法构建has_circ_0001756、has_circ_0000284、has_circ_0000711或has_circ_0000268的过表达载体,并通过qPCR检测这些过表达质粒转染后293T细胞中的表达效果。效果图如图1所示。
序列表
<110> 上海锐赛生物技术有限公司
<120> 用于环状RNA过表达的DNA框架及其应用
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 89
<212> DNA
<213> artificial sequence
<400> 1
tgagattaca ggtgtgagcc accacccccg gcctcacttt ttgtaaaggt acgtactaat 60
gacttttttt ttatacttca ggtaagtct 89
<210> 2
<211> 70
<212> DNA
<213> artificial sequence
<400> 2
tctctctctc ttcaggtaag tagcaaggaa aagagttagg cccggcacgg tagctcacac 60
ctgtaatccc 70
<210> 3
<211> 889
<212> DNA
<213> artificial sequence
<400> 3
gaatagtaaa gaaacacatc ataaaacctc ccaggacata aaggtgagca cagaccctgt 60
ttggatcaag tcagttcctg gagcctgaat gatgactgct gaatcacggg aagccacggg 120
tctgtcccca caggctgcac aggagaagga tggtatcgta atagtgaagg tggaagagga 180
agatgaggaa gaccacatgt gggggcagga ttccacccta caggacacgc ctcctccaga 240
cccagagata ttccgccaac gcttcaggcg cttctgttac cagaacactt ttgggccccg 300
agaggctctc agtcggctga aggaactttg tcatcagtgg ctgcggccag aaataaacac 360
caaggaacag atcctggagc ttctggtgct agagcagttt ctttccatcc tgcccaagga 420
gctccaggtc tggctgcagg aataccgccc cgatagtgga gaggaggccg tgacccttct 480
agaagacttg gagcttgatt tatcaggaca acaggtaaaa agaggtgaaa cctattatgt 540
gtgagcaggg cacagacgtt gaaactggag ccaggagaag tattggcagg ctttaggtta 600
ttaggtggtt actctgtctt aaaaatgttc tggctttctt cctgcatcca ctggcatact 660
catggtctgt ttttaaatat tttaattccc atttacaaag tgatttaccc acaagcccaa 720
cctgtctgtc ttcaggtccc aggtcaagtt catggacctg agatgctcgc aagggggatg 780
gtgcctctgg atccagttca ggagtcctcg agctttgacc ttcatcacga ggccacccag 840
tcccacttca aacattcgtc tcggaaaccc cgcctcttac agtcacgag 889
<210> 4
<211> 1048
<212> DNA
<213> artificial sequence
<400> 4
tgagattaca ggtgtgagcc accacccccg gcctcacttt ttgtaaaggt acgtactaat 60
gacttttttt ttatacttca ggtaagtctg aatagtaaag aaacacatca taaaacctcc 120
caggacataa aggtgagcac agaccctgtt tggatcaagt cagttcctgg agcctgaatg 180
atgactgctg aatcacggga agccacgggt ctgtccccac aggctgcaca ggagaaggat 240
ggtatcgtaa tagtgaaggt ggaagaggaa gatgaggaag accacatgtg ggggcaggat 300
tccaccctac aggacacgcc tcctccagac ccagagatat tccgccaacg cttcaggcgc 360
ttctgttacc agaacacttt tgggccccga gaggctctca gtcggctgaa ggaactttgt 420
catcagtggc tgcggccaga aataaacacc aaggaacaga tcctggagct tctggtgcta 480
gagcagtttc tttccatcct gcccaaggag ctccaggtct ggctgcagga ataccgcccc 540
gatagtggag aggaggccgt gacccttcta gaagacttgg agcttgattt atcaggacaa 600
caggtaaaaa gaggtgaaac ctattatgtg tgagcagggc acagacgttg aaactggagc 660
caggagaagt attggcaggc tttaggttat taggtggtta ctctgtctta aaaatgttct 720
ggctttcttc ctgcatccac tggcatactc atggtctgtt tttaaatatt ttaattccca 780
tttacaaagt gatttaccca caagcccaac ctgtctgtct tcaggtccca ggtcaagttc 840
atggacctga gatgctcgca agggggatgg tgcctctgga tccagttcag gagtcctcga 900
gctttgacct tcatcacgag gccacccagt cccacttcaa acattcgtct cggaaacccc 960
gcctcttaca gtcacgagtc tctctctctt caggtaagta gcaaggaaaa gagttaggcc 1020
cggcacggta gctcacacct gtaatccc 1048
<210> 5
<211> 29
<212> DNA
<213> artificial sequence
<400> 5
ttagctagct gagattacag gtgtgagcc 29
<210> 6
<211> 30
<212> DNA
<213> artificial sequence
<400> 6
gctaagcttg ggattacagg tgtgagctac 30
<210> 7
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 7
caggtcaagt tcatggacct g 21
<210> 8
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 8
attcagactt acctgaagta 20
Claims (7)
1.一种序列如式I所示的用于环状RNA过表达的DNA框架,
US-[N]n-DS
I
其中,
US为上游序列,其核苷酸序列如SEQ ID NO.1所示,
DS为下游序列,其核苷酸序列如SEQ ID NO.2所示,
[N]n为中间序列,N代表A、T、G、C四种脱氧核糖核苷酸中任意一种,n个相同或者不同的N依次连接形成中间序列,所述的[N]n是circRNA的序列。
2.如权利要求1所述的用于环状RNA过表达的DNA框架,其特征在于所述的[N]n是内源性circRNA的序列。
3.如权利要求2所述的用于环状RNA过表达的DNA框架,其特征在于所述内源性circRNA的序列为hsa_circ_0001727、hsa_circ_0001756、hsa_circ_0000284、hsa_circ_0000711或hsa_circ_0000268。
4.如权利要求1所述的用于环状RNA过表达的DNA框架,其特征在于150≤n≤3000。
5.一种用于环状RNA过表达的DNA框架,其特征在于其DNA序列如SEQ ID NO.4所示。
6.一种表达载体,所述表达载体为真核表达载体、慢病毒载体、腺病毒载体或腺相关病毒载体,其特征在于其携带如权利要求1~5任一所述的用于环状RNA过表达的DNA框架序列。
7.一种权利要求1~5任一所述用于环状RNA过表达的DNA框架的用途,其特征在于用作体外过表达circRNA。
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