CN107365770B - 针对自噬相关基因Beclin1编码区174-194位点的siRNA序列及其应用 - Google Patents
针对自噬相关基因Beclin1编码区174-194位点的siRNA序列及其应用 Download PDFInfo
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Abstract
本发明提供一种针对自噬相关基因Beclin1编码区174‐194位点的siRNA序列，其核苷酸序列是：5’‐GGAGGAAGAGACTAACTCAGG‐3’。本发明根据siRNA设计原则，选择的siRNA的cDNA序列GC含量为52.4％，对应Beclin1基因编码区的第174‐194位核苷酸。构建针对Beclin1的siRNA真核表达载体，转化大肠杆菌后提取质粒，转染AD293细胞，能有效抑制Beclin1基因的mRNA和蛋白表达水平，可作为人源细胞自噬水平调控的一个有效靶点。应用于人类细胞自噬水平的调控。
Description
技术领域
本发明属生物技术，涉及分子生物学和基因工程技术领域，具体涉及针对自噬相关基因Beclin1编码区174-194位点的siRNA序列的设计、筛选及其在调控人类细胞自噬水平中的应用。
背景技术
自噬(autophagy)是一种程序化的细胞内降解过程，细胞通过包裹降解物形成自噬体运送至溶酶体进行消化，从而满足代谢需要、细胞器更新及维持细胞稳态。根据降解物与溶酶体结合途径的不同，自噬可分为巨自噬、微自噬和分子伴侣介导的自噬三类。其中，对巨自噬的研究最为深入，在此过程中很多自噬相关基因(autophagy related gene,Atg)编码的蛋白定位到自噬前体结构，即双层扁平杯状分隔膜形成的自噬泡。自噬泡伸展变形吞食包裹细胞内老化或损伤的细胞器/蛋白质，形成闭合的双层膜结构，称为自噬体。在细胞骨架驱动下，自噬体的外膜与溶酶体膜融合，内膜及包裹物质进入溶酶体，溶酶体脱颗粒并释放蛋白水解酶降解自噬体，部分降解产物可被细胞再利用。
自噬是广泛存在于真核细胞中的生命现象，贯穿于正常细胞的生长发育和生理病理过程。自噬不仅能清除细胞内老化或损伤的细胞器和蛋白质，在维持蛋白质代谢平衡和细胞内环境稳定中起重要作用，而且也与病理性损伤的保护过程相关，对神经退行性病变、肿瘤、心肌病、病原微生物感染等疾病的预防有积极作用。自噬具有高度的进化保守性，其发生、发展受多种Atg基因的调控，至今已鉴定出30多种自噬特异性基因和50多种相关基因。
Beclin1基因是酵母自噬基因Atg6在哺乳类动物中的同源基因，是第一个被发现参与自噬过程的基因。其编码的Beclin1蛋白由450个氨基酸组成，包含BH3、卷曲螺旋结构域和进化保守结构域等主要结构域。Beclin1蛋白可调控自噬前体的形成，引导自噬相关蛋白定位于自噬体膜，是整个自噬过程中必不可少的关键分子。Beclin1及其上下游信号调节蛋白组成了重要的自噬调节通路，通常以Beclin1/PI3K3C复合体的形式与各种蛋白相互作用，从而达到调节自噬的目的。近年研究发现Beclin1通过对自噬的调节，在肿瘤的发生、发展中起着重要作用，是一个重要的抑癌基因。
RNA干扰(RNA interference,RNAi)是序列特异的双链RNA使细胞内同源mRNA降解，从而产生特异性基因表达沉默的过程。RNAi的作用主要由长约21～23nt的小干扰RNA(siRNA)介导，由于RNAi是siRNA靶向作用mRNA引起的特异性降解，因此要产生有效RNAi的关键是选择合适的siRNA作用靶点。RNAi靶点的选择原则主要包括：①从靶基因起始密码子下游50～100nt开始查找；②选择以AA或NA(N代表任意碱基)开始的序列；③选择GC含量在30％-60％左右的mRNA区域；④避免连续的单一碱基和反向重复序列；⑤保证靶序列与其他基因没有同源性。目前，制备siRNA的方法主要包括化学合成法、体外转录法、长片段dsRNA经RNaseⅢ降解法、siRNA表达载体转录法及PCR制备的siRNA表达框法等5种。
发明内容
本发明的目的是提供一种针对自噬相关基因Beclin1编码区174-194位点的siRNA的cDNA序列，其核苷酸序列是：5’-GGAGGAAGAGACTAACTCAGG-3’。
本发明的另一个目的是提供该cDNA序列在人类细胞自噬水平调控中的应用。
本发明根据siRNA设计原则，选择的siRNA的cDNA序列GC含量为52.4％，对应Beclin1基因编码区的第174-194位核苷酸。构建针对该siRNA的真核表达载体，转化大肠杆菌后提取质粒，转染AD293细胞，能有效抑制Beclin1基因的mRNA水平和蛋白水平，因此可应用于人类细胞自噬水平的调控。
本发明的有益之处是：提供的siRNA序列针对自噬相关基因Beclin1的编码区，以此序列为基础的真核表达载体在细胞中能持续、有效抑制Beclin1基因的mRNA水平和蛋白水平，故可作为人源细胞自噬水平调控的一个有效靶点。
具体实施方式
本发明通过实施例作进一步说明。应该理解，这些实施例仅用于说明目的，而不用于限制本发明的范围。
实施例1siRNA真核表达载体体外抑制Beclin1-EGFP融合蛋白的表达
1.siRNA的设计：根据siRNA设计原则，从Beclin1基因编码区起始密码子ATG下游50nt处搜索NA序列，其3’端相邻21nt序列作为候选靶点，从中选择GC含量在30～60％的siRNA序列，并通过GenBank数据库的Blast功能与人类基因组序列进行比对，确保无同源性。最终选择的siRNA其cDNA序列为5’-GGAGGAAGAGACTAACTCAGG-3’，GC含量为52.4％，对应Beclin1基因编码区的第174-194位核苷酸。
2.表达siRNA所需shDNA的合成与制备：表达siRNA所需shDNA的正义链序列为5’-GATCCGGAGGAAGAGACTAACTCAGGTTCGCCTGAGTTAGTCTCTTCCTCCTTTTTA-3’，反义链序列为5’-AGCTTAAAAAGGAGGAAGAGACTAACTCAGGCGAACCTGAGTTAGTCTCTTCCTCCG-3’，委托生物公司合成，将正、反义链分别退火形成双链。
3.siRNA真核表达载体的构建：具有U6启动子的真核表达质粒pSilencer 2.0-U6用BamHI和HindⅢ双酶切，与退火后的shDNA双链连接，转化大肠杆菌感受态细胞DH5α，37℃培养过夜，挑取克隆抽提质粒送生物公司进行DNA测序鉴定，选取测序结果正确的质粒扩增、保存。
4.siRNA表达质粒体外抑制Beclin1-EGFP融合蛋白表达的实验
(1)融合蛋白质粒pBeclin1-EGFP：为表达绿色荧光蛋白(EGFP)和Beclin1融合蛋白的质粒，由Beclin1基因cDNA***pEGFP-N1质粒的HindⅢ和EcoR I位点构建而成，由于EGFP位于Beclin1的下游，且共用一个启动子，故EGFP的表达情况可间接反映Beclin1的表达。
(2)siRNA表达质粒与融合蛋白质粒共转染AD293细胞：人胚肾细胞AD293接种12孔细胞培养板后，待细胞达到80％～90％融合率，用Invitrogen公司Lipofectamine 2000试剂将siRNA表达质粒与融合蛋白质粒pBeclin1-EGFP共转染细胞，操作方法参照试剂说明书，同时设转染空质粒pSilencer 2.0-U6的阴性对照组和不转染质粒的空白对照组，5小时后换培养液(含10％新生牛血清的DMEM)继续培养。
(3)荧光定量RT-PCR检测AD293细胞中Beclin1基因mRNA的表达：质粒转染后48h，收集AD293细胞，Trizol法抽提细胞总RNA，采用TAKARA公司的SYBR PrimeScript RT-RCPKit检测Beclin1基因的mRNA水平，实验方法参照试剂盒说明书。Beclin1基因PCR引物序列为：上游5’-CTGGGGACCTTTTTGACATC-3’，下游5’-TTGCGGTTCTTTTCCACGTC-3’。内参照采用GAPDH，PCR引物序列为：上游5’-GAAGGTGAAGGTCGGAGTC-3’，下游5’-GAAGATGGTGATGGGATTTC-3’。结果显示，与阴性对照组相比，siRNA表达质粒对Beclin1基因mRNA表达的抑制率为81.7％。
(4)流式细胞仪检测AD293细胞中Beclin1蛋白表达情况：质粒转染后72h，收集每孔内细胞，用PBS缓冲液洗涤2次后重悬于PBS中。用流式细胞仪在488nm激发波长下检测每孔细胞平均荧光强度(mean fluorescence intensity,MFI)及荧光阳性细胞比率α，计算每孔细胞的总荧光强度(total fluorescence intensity,TFI)＝MFI×α。结果显示，与阴性对照组相比，siRNA表达质粒对Beclin1蛋白表达的抑制率为73.4％。
本发明是结合最佳实施例进行描述的，然而在阅读了本发明的上述内容后，本领域技术人员可对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限度的范围。
<110> 浙江大学
<120> 针对自噬相关基因Beclin1编码区174-194位点的siRNA序列及其应用
<160> 7
<210> 1
<211> 21
<212> DNA
<213> 人工序列
<220>
<223> 根据自噬相关基因Beclin1设计的siRNA的cDNA序列
<400> 1
GGAGGAAGAG ACTAACTCAG G 21
<210> 2
<211> 57
<212> DNA
<213> 人工序列
<220>
<223> 制备Beclin1基因siRNA所需shDNA正义链的序列
<400> 2
GATCCGGAGG AAGAGACTAA CTCAGGTTCG CCTGAGTTAG TCTCTTCCTC CTTTTTA 57
<210> 3
<211> 57
<212> DNA
<213> 人工序列
<220>
<223> 制备Beclin1基因siRNA所需shDNA反义链的序列
<400> 3
AGCTTAAAAA GGAGGAAGAG ACTAACTCAG GCGAACCTGA GTTAGTCTCT TCCTCCG 57
<210> 4
<211> 20
<212> DNA
<213> 人工序列
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<223> Beclin1基因PCR扩增的上游引物序列
<400> 4
CTGGGGACCT TTTTGACATC 20
<210> 5
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> Beclin1基因PCR扩增的下游引物序列
<400> 5
TTGCGGTTCT TTTCCACGTC 20
<210> 6
<211> 19
<212> DNA
<213> 人工序列
<220>
<223> GAPDH基因PCR扩增的上游引物序列
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GAAGGTGAAG GTCGGAGTC 19
<210> 7
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<212> DNA
<213> 人工序列
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<223> GAPDH基因PCR扩增的下游引物序列
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GAAGATGGTG ATGGGATTTC 20
Claims (3)
1.一种针对自噬相关基因Beclin1的siRNA的靶序列核酸，其特征在于，所述靶序列核酸的序列是：5’-GGAGGAAGAGACTAACTCAGG-3’。
2.权利要求1所述的靶序列核酸在制备shDNA中的应用。
3.一种针对自噬相关基因Beclin1的shDNA，其特征在于，正义链序列为5’-GATCCGGAG GAAGAGACTAACTCAGGTTCGCCTGAGTTAGTCTCTTCCTCCTTTTTA-3’，反义链序列为5’-AGCTTAAAAAGGAGGAAGAGACTAACTCAGGCGAACCTGAGTTAGTCTCTTCCTCCG-3’。
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| CN104258394A (zh) * | 2014-09-28 | 2015-01-07 | 中国科学技术大学 | 纳米银联合自噬抑制剂杀伤肿瘤 |
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Non-Patent Citations (3)
| Title |
|---|
| Beclin1基因siRNA慢病毒载体构建和鉴定;王文玉等;《生物医学工程学杂志》;20130225;第30卷(第1期);摘要，第131页左栏第1.2节,引言 * |
| Electro-Magnetic Nano-Particle Bound Beclin1 siRNA Crosses the Blood-Brain Barrier to Attenuate the Inflammatory Effects of HIV-1 Infection in Vitro;Rodriguez M等;《J Neuroimmune Pharmacol》;20160610;第12卷(第1期);120-132 * |
| 自噬相关基因Beclin 1 沉默对人肺癌A549 细胞顺铂耐药性的影响;任爽;《中国老年学杂志》;20140531;第34卷;材料与方法1.2,1.4，结果2.1，讨论3 * |
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