CN101333539A - A DNA plasmid capable of expressing small-molecule interfering RNA and its construction method - Google Patents

Abstract

Translated fromChinese

本发明公开了一种能表达小分子干扰RNA的DNA质粒及其构建方法，以载体BS/U6为骨架，在载体BS/U6的Apa I和EcoR I的酶切位点上，插入同源酶切位点的长度为21nt的DNA靶序列构建获得能表达小分子干扰RNA的DNA质粒；使DNA质粒在细胞内或体内能稳定、高效地表达siRNA，从而达到较高的传染率和RNAi作用，能充分抑制靶蛋白，最高抑制率可以达到75％，而且本发明是以全新广谱的构建载体为骨架所构建的表达载体系统，在该构建载体BS/U6上可以构建各种目的基因的表达小分子干扰RNA的DNA质粒；且本发明的DNA质粒在细胞内介导RNAi具有表达稳定、持续时间长和抑制效果明显等优点。

The invention discloses a DNA plasmid capable of expressing small-molecule interfering RNA and a construction method thereof. The carrier BS/U6 is used as a backbone, and homologous enzymes are inserted into the restriction sites of Apa I and EcoR I of the carrier BS/U6 The length of the cutting site is 21nt to construct a DNA plasmid capable of expressing small interfering RNA; the DNA plasmid can stably and efficiently express siRNA in cells or in vivo, thereby achieving a higher infection rate and RNAi effect. It can fully inhibit the target protein, and the highest inhibition rate can reach 75%. Moreover, the present invention is an expression vector system constructed with a new broad-spectrum construction vector as the backbone. The expression of various target genes can be constructed on the construction vector BS/U6 The DNA plasmid of the small molecular interference RNA; and the DNA plasmid of the present invention mediates RNAi in cells and has the advantages of stable expression, long duration and obvious inhibitory effect.

Description

Translated fromChinese

一种能表达小分子干扰RNA的DNA质粒及其构建方法A DNA plasmid capable of expressing small-molecule interfering RNA and its construction method

技术领域technical field

本发明涉及基因工程技术，具体涉及一种能表达小分子干扰RNA的DNA质粒及其构建方法。The invention relates to genetic engineering technology, in particular to a DNA plasmid capable of expressing small molecule interference RNA and a construction method thereof.

背景技术Background technique

RNA干扰(RNA interference，RNAi)现象是在1998年被Fire等人发现于秀丽隐杆线虫(Caenorhabditis elegans)中，后来发现在植物、真菌、果蝇及哺乳动物细胞内均存在这种现象；RNAi自发现之日起便成为了研究热点，2001年被成功用于哺乳动物细胞。RNAi是生命最古老的基因免疫监视机制，依靠RNAi可以实现维持生命健康、抵御病毒侵入、抗肿瘤发生以及维持遗传稳定等生命机体的基本活动，因此，对RNAi的研究及应用受到了越来越多的科学家的青睐。The phenomenon of RNA interference (RNA interference, RNAi) was discovered in Caenorhabditis elegans by Fire et al. in 1998, and later found that this phenomenon exists in plants, fungi, fruit flies and mammalian cells; RNAi It has become a research hotspot since its discovery, and was successfully used in mammalian cells in 2001. RNAi is the oldest genetic immune surveillance mechanism in life. Relying on RNAi, it can realize the basic activities of living organisms such as maintaining life and health, resisting virus invasion, anti-tumor occurrence, and maintaining genetic stability. Therefore, the research and application of RNAi has received more and more attention. favored by many scientists.

RNAi技术是利用21nt或29nt的小分子干扰RNA(small interference RNA，siRNA)对基因进行封闭，达到干扰或封闭目的基因的作用，同时使生物体出现相应的表型缺失现象。由于siRNA作用的高效性及特异性，目前已经成为功能基因组研究的主要工具。已有的实验证实人工化学合成的siRNA能在体内产生RNAi现象，但它不能长期介导RNAi作用，用于基因治疗时，体内应用siRNA易被核糖核酸酶(RNase)降解，且合成siRNA的量难以满足动物或人体试验的大量需求，因此这在一定程度上限制了其适用范围；另外，RNAi还存在衰减现象，在较高级的哺乳动物或人类细胞中，细胞内的RNAi现象在数天之后逐渐消失。这些问题的存在，限制了RNAi的众多功能的发挥。目前制备siRNA的方法除了直接化学合成外，还有利用体外转录、Dicer(核酸水解酶)酶切双链RNA获得，以及制备siRNA表达框利用质粒或病毒表达载体在细胞内转录成siRNA；如中国专利号为ZL200510024242.5，名称为“低氧诱导因子小干扰RNA质粒”的发明专利，就公开了针对HIF-1α设计的抑制性siRNA插入到_pSilence U6-1.0载体中构建而成，而抑制性siRNA具有其SRQ ID NO：1或SRQ ID NO：2所表示的基因结构；该发明的骨架载体为_pSilence U6-1.0，该发明的siRNA质粒具有在细胞内能稳定高效地表达，使转染效率较高，抑制效果显著的优点。因此利用RNAi的表达载体siRNA质粒或为DNA质粒，在细胞内介导RNAi具有表达稳定、持续时间长和抑制效果明显等特征。RNAi technology is to use 21nt or 29nt small molecule interference RNA (small interference RNA, siRNA) to block the gene, so as to interfere or block the target gene, and at the same time cause the corresponding phenotype deletion phenomenon in the organism. Due to the high efficiency and specificity of siRNA, it has become the main tool for functional genomics research. Existing experiments have confirmed that artificial chemically synthesized siRNA can produce RNAi in vivo, but it cannot mediate RNAi for a long time. When used in gene therapy, siRNA in vivo is easily degraded by ribonuclease (RNase), and the amount of synthetic siRNA It is difficult to meet the large demand for animal or human experiments, so this limits its scope of application to a certain extent; in addition, RNAi also has a decay phenomenon, and in higher mammalian or human cells, the RNAi phenomenon in cells will not last for several days disappear slowly. The existence of these problems limits the many functions of RNAi. In addition to direct chemical synthesis, the current methods of preparing siRNA include in vitro transcription, Dicer (nucleic acid hydrolase) digestion of double-stranded RNA, and preparation of siRNA expression cassettes, which are transcribed into siRNA in cells using plasmids or viral expression vectors; such as China The patent number is ZL200510024242.5, and the invention patent named "hypoxia-inducible factor small interfering RNA plasmid" discloses that the inhibitory siRNA designed for HIF-1α is inserted into the_p Silence U6-1.0 vector to construct, and inhibits Sexual siRNA has the gene structure represented by its SRQ ID NO: 1 or SRQ ID NO: 2; the backbone vector of the invention is_p Silence U6-1.0, and the siRNA plasmid of the invention can be expressed stably and efficiently in cells, making the transgenic It has the advantages of high dyeing efficiency and remarkable inhibitory effect. Therefore, the use of RNAi expression vector siRNA plasmid or DNA plasmid to mediate RNAi in cells has the characteristics of stable expression, long duration and obvious inhibitory effect.

发明内容Contents of the invention

本发明所要解决的技术问题是提供一种能稳定高效表达小分子干扰RNA的DNA质粒，是用全新广谱的构建载体为骨架所构建的表达载体系统，该DNA质粒在细胞内介导RNAi具有表达稳定、持续时间长和抑制效果明显等特征。同时本发明还提供了该DNA质粒的构建方法。The technical problem to be solved by the present invention is to provide a DNA plasmid capable of stably and efficiently expressing small-molecule interfering RNA, which is an expression vector system constructed with a new broad-spectrum construction vector as the backbone, and the DNA plasmid mediates RNAi in cells. It has the characteristics of stable expression, long duration and obvious inhibitory effect. At the same time, the invention also provides a method for constructing the DNA plasmid.

本发明解决上述技术问题所采用的技术方案为：一种能表达小分子干扰RNA的DNA质粒，以载体pBlueScript(BS)/U6为骨架，在所述载体BS/U6的Apa I和EcoRI的酶切位点上，插入同源酶切位点的长度为21nt的DNA靶序列构建而成。The technical solution adopted by the present invention to solve the above technical problems is: a DNA plasmid capable of expressing small molecule interfering RNA, with the carrier pBlueScript (BS)/U6 as the backbone, the enzymes of Apa I and EcoRI in the carrier BS/U6 At the cleavage site, a DNA target sequence with a length of 21 nt inserted into the homologous cleavage site is constructed.

所述载体BS/U6为在BS的Kpn I和Apa I的酶切位点上，插入同源酶切位点的U6启动子构建而成。The vector BS/U6 is constructed by inserting a U6 promoter with a homologous restriction site at the Kpn I and Apa I restriction sites of BS.

所述DNA靶序列为报告基因(即绿色荧光蛋白基因gfp)的编码区的第101～121位片段；gfp的编号为Genebank no.U19281。The DNA target sequence is the 101st to 121st fragment of the coding region of the reporter gene (ie, the green fluorescent protein gene gfp); the number of gfp is Genebank no.U19281.

所述DNA靶序列为肿瘤细胞内源基因周期蛋白依赖性激酶2基因(cdk-2)的编码区的第66～86位片段；cdk-2的编号为Genebank no.NM001798。The DNA target sequence is the 66th to 86th fragment of the coding region of the tumor cell endogenous gene cyclin-dependent kinase 2 gene (cdk-2); the number of cdk-2 is Genebank no.NM001798.

所述DNA靶序列为DNA甲基转移酶1基因(dnmt-1)的编码区的第85～105位片段；dnmt-1的编号为Genebank no.NM001379。The DNA target sequence is the 85th to 105th fragment of the coding region of theDNA methyltransferase 1 gene (dnmt-1); the number of dnmt-1 is Genebank no.NM001379.

一种能表达小分子干扰RNA的DNA质粒的构建方法，包括下述步骤：A method for constructing a DNA plasmid capable of expressing small-molecule interfering RNA, comprising the steps of:

1)以pmU6质粒为模板，通过PCR扩增反应获得在左侧含有Kpn I酶切位点和在右侧含有Apa I酶切位点的U6启动子序列；1) Using the pmU6 plasmid as a template, the U6 promoter sequence containing the Kpn I restriction site on the left and the Apa I restriction site on the right is obtained by PCR amplification reaction;

2)将上述U6启动子序列插入到同样含有Kpn I和Apa I的酶切位点的BS上，然后转入感受态细胞E.coli TG1中，经过筛选、扩增和抽提获得载体BS/U6；2) Insert the above-mentioned U6 promoter sequence into the BS that also contains the restriction sites of Kpn I and Apa I, and then transfer it into the competent cell E.coli TG1, and obtain the vector BS/ U6;

3)应用siRNA target finder软件分析筛选出含有Apa I和EcoR I的酶切位点的长度为21nt的DNA靶序列，体外人工合成该DNA靶序列；3) The siRNA target finder software was used to analyze and screen out a DNA target sequence with a length of 21 nt containing restriction sites of Apa I and EcoR I, and artificially synthesize the DNA target sequence in vitro;

4)将上述DNA靶序列插入到所述载体BS/U6的Apa I和EcoR I的酶切位点上，利用质粒抽提试剂盒抽提得到能表达小分子干扰RNA的DNA质粒。4) The above-mentioned DNA target sequence is inserted into the enzyme cutting sites of Apa I and EcoR I of the vector BS/U6, and a DNA plasmid capable of expressing small interfering RNA is obtained by extracting with a plasmid extraction kit.

上述BS、pmU6和质粒抽提试剂盒都可以市售得到。The above-mentioned BS, pmU6 and plasmid extraction kits are all commercially available.

与现有技术相比，本发明的优点在于以载体BS/U6为骨架，在载体BS/U6的Apa I和EcoR I的酶切位点上，插入同源酶切位点的长度为21nt的DNA靶序列构建获得能表达小分子干扰RNA的DNA质粒；使DNA质粒在细胞内或体内能稳定、高效地表达siRNA，从而达到较高的传染率和RNAi作用，能充分抑制靶蛋白，最高抑制率可以达到75％，而且本发明是以全新广谱的构建载体为骨架所构建的表达载体系统，在该构建载体BS/U6上可以构建各种目的基因的表达小分子干扰RNA的DNA质粒；且本发明的DNA质粒在细胞内介导RNAi具有表达稳定、持续时间长和抑制效果明显等优点。Compared with the prior art, the present invention has the advantage of using the carrier BS/U6 as the backbone, inserting a homologous restriction site with a length of 21 nt at the Apa I and EcoR I restriction sites of the carrier BS/U6 The DNA target sequence is constructed to obtain a DNA plasmid capable of expressing small interfering RNA; the DNA plasmid can stably and efficiently express siRNA in the cell or in vivo, thereby achieving a high infection rate and RNAi effect, and can fully inhibit the target protein, the highest inhibition The rate can reach 75%, and the present invention is an expression vector system constructed with a new broad-spectrum construction vector as the backbone, on which construction vector BS/U6 can construct DNA plasmids expressing small molecule interference RNA of various target genes; Moreover, the DNA plasmid of the present invention mediates RNAi in cells and has the advantages of stable expression, long duration, obvious inhibitory effect, and the like.

附图说明Description of drawings

图1、为载体BS/U6结构示意图；其中U6表示启动子，Kpn I、Apa I、EcoR I分别表示酶切位点，LacZ表示β半乳糖苷酶基因启动子，f1 origin和ColE1 origin分别表示质粒复制起始区序列，Ampicillin表示氨苄西林抗生素筛选标记；Figure 1 is a schematic diagram of the structure of the vector BS/U6; where U6 represents the promoter, Kpn I, Apa I, and EcoR I represent the enzyme cleavage sites, LacZ represents the promoter of the β-galactosidase gene, and f1 origin and ColE1 origin represent respectively Plasmid replication initiation region sequence, Ampicillin means ampicillin antibiotic selection marker;

图2、为DNA质粒的模型示意图；其中U6promoter表示U6启动子，21nt coding seq表示21个核苷酸的DNA靶序列，6nt space表示6个核苷酸的间隔序列，TTTTT表示转录终止识别信号；Figure 2 is a schematic diagram of a DNA plasmid model; where U6promoter represents the U6 promoter, 21nt coding seq represents a DNA target sequence of 21 nucleotides, 6nt space represents a spacer sequence of 6 nucleotides, and TTTTT represents a transcription termination recognition signal;

图3、为BS/U6质粒的琼脂糖电泳图；箭头所指即为超螺旋质粒DNA的位置。Figure 3 is the agarose electrophoresis image of the BS/U6 plasmid; the arrow points to the position of the supercoiled plasmid DNA.

具体实施方式Detailed ways

以下结合附图实施例对本发明作进一步详细描述。The present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments.

实施例1Example 1

一种能表达小分子干扰RNA的DNA质粒，以载体BS/U6为骨架，在载体BS/U6的Apa I和EcoR I的酶切位点上，插入同源酶切位点的长度为21nt的gfp基因的编码区的第101～121位片段的DNA靶序列构建而成，得到BS/U6/gfp质粒，该质粒能在体内转录为siRNA，从而起到RNAi的作用。A DNA plasmid capable of expressing small interfering RNA, with the carrier BS/U6 as the backbone, inserting a homologous restriction site with a length of 21 nt at the Apa I and EcoR I restriction sites of the carrier BS/U6 The DNA target sequence of the 101st-121st fragment of the coding region of the gfp gene was constructed to obtain a BS/U6/gfp plasmid, which can be transcribed into siRNA in vivo to play the role of RNAi.

实施例2Example 2

一种能表达小分子干扰RNA的DNA质粒，与实施例1基本相同，所不同的只是插入的DNA靶序列为cdk-2基因的编码区的第66～86位片段，得到BS/U6/cdk-2质粒。A DNA plasmid capable of expressing small interfering RNA is basically the same as in Example 1, except that the inserted DNA target sequence is the 66th to 86th fragment of the coding region of the cdk-2 gene to obtain BS/U6/cdk -2 plasmid.

实施例3Example 3

一种能表达小分子干扰RNA的DNA质粒，与实施例1基本相同，所不同的只是插入的DNA靶序列为dnmt-1基因的编码区的第85～105位片段，得到BS/U6/dnmt-1质粒。A DNA plasmid capable of expressing small interfering RNA is basically the same as in Example 1, except that the inserted DNA target sequence is the 85th to 105th fragment of the coding region of the dnmt-1 gene to obtain BS/U6/dnmt -1 plasmid.

根据各种目的要求，本发明的DNA质粒中DNA靶序列也可以为其他21nt的DNA片段，在此不一一列举，只是它们的骨架载体都为BS/U6。According to the requirements of various purposes, the DNA target sequence in the DNA plasmid of the present invention can also be other 21nt DNA fragments, which are not listed here, but their backbone vectors are all BS/U6.

实施例4Example 4

一种能表达小分子干扰RNA的DNA质粒的构建方法，包括下述步骤：A method for constructing a DNA plasmid capable of expressing small-molecule interfering RNA, comprising the steps of:

1)以pmU6质粒为模板，通过PCR扩增反应获得在左侧含有Kpn I的酶切位点和在右侧含有Apa I的酶切位点的U6启动子序列，即形成含有U6启动子的Kpn I-U6-ApaI的基因片段；1) Using the pmU6 plasmid as a template, the U6 promoter sequence containing the restriction site of Kpn I on the left side and the restriction site of Apa I on the right side is obtained by PCR amplification reaction, that is, the U6 promoter sequence containing the U6 promoter is formed. The gene fragment of KpnI-U6-ApaI;

2)将上述U6启动子序列插入到同样含有Kpn I和Apa I的酶切位点的BS上，然后转入感受态细胞E.coli TGl中，经过氨苄西林抗生素筛选、在E.coli TG1中大量扩增，用质粒抽提试剂盒抽提获得载体BS/U6，得到的载体BS/U6的示意结构如图1所示，琼脂糖凝胶电泳的BS/U6产物如图3所示；2) Insert the above-mentioned U6 promoter sequence into the BS that also contains Kpn I and Apa I restriction sites, and then transfer it into the competent cell E.coli TG1, after ampicillin antibiotic selection, in E.coli TG1 A large amount of amplification was carried out, and the carrier BS/U6 was obtained by extraction with a plasmid extraction kit. The schematic structure of the obtained carrier BS/U6 is shown in Figure 1, and the BS/U6 product of agarose gel electrophoresis is shown in Figure 3;

3)应用siRNAtarget finder软件分析筛选出含有Apa I和EcoR I的酶切位点的长度为21nt的DNA靶序列：gfp基因的编码区的第101～121位片段，用BLAST序列分析获得的：gfp片段与其它基因无同源性，体外人工合成：gfp片段(101～121)；3) The siRNAtarget finder software was used to analyze and screen out a DNA target sequence with a length of 21 nt containing restriction sites of Apa I and EcoR I: the 101st to 121st fragment of the coding region of the gfp gene, which was obtained by BLAST sequence analysis: gfp The fragment has no homology with other genes, artificially synthesized in vitro: gfp fragment (101-121);

4)将上述gfp片段插入到载体BS/U6的Apa I和EcoR I酶切位点上，利用质粒抽提试剂盒抽提得到如图2模型所示的能表达小分子干扰RNA的DNA质粒：BS/U6/gfp。4) Insert the above-mentioned gfp fragment into the Apa I and EcoR I restriction sites of the carrier BS/U6, and use the plasmid extraction kit to extract the DNA plasmid capable of expressing small interfering RNA as shown in the model in Figure 2: BS/U6/gfp.

用同样的方法可以构建BS/U6/cdk-2和BS/U6/dnmt-1等质粒。Plasmids such as BS/U6/cdk-2 and BS/U6/dnmt-1 can be constructed in the same way.

上述实施例中所用的BS和pmU6购于Invitrogen公司；质粒抽提试剂盒购于TaKaRa公司；E.coli TG1感受态细胞为实验室制备。The BS and pmU6 used in the above examples were purchased from Invitrogen Company; the plasmid extraction kit was purchased from TaKaRa Company; E. coli TG1 competent cells were prepared in the laboratory.

在上述实施例的具体步骤中所用到的各种分子生物学方法：如质粒的提取、琼脂糖凝胶电泳、分离回收DNA片段、感受态细胞制备、质粒转化感受态细胞等均按《分子克隆实验指南》中的方法进行操作，具体操作细节在此不作详述。Various molecular biological methods used in the specific steps of the above-mentioned examples: extraction of plasmids, agarose gel electrophoresis, separation and recovery of DNA fragments, preparation of competent cells, transformation of competent cells with plasmids, etc. are all according to "Molecular Cloning" The method in the "Experiment Guide" was operated, and the specific operation details were not described in detail here.

实施例5Example 5

细胞培养与质粒转染Cell culture and plasmid transfection

实验室分别培养人宫颈癌细胞(Hela)、人胚肾细胞(HEK293)、人结肠癌细胞(HT29)、人肺腺癌细胞(SPCAl)，用购于Invitrogen公司的脂质体转染试剂(Lipofectamine)将本发明的BS/U6/gfp、BS/U6/cdk-2和BS/U6/dnmt-1质粒各自分别转染到Hela、HEK293、H1299、SPCAl细胞中，在6孔培养板中继续培养48小时，操作按照产品说明书操作规程完成。The laboratory cultured human cervical cancer cells (Hela), human embryonic kidney cells (HEK293), human colon cancer cells (HT29), and human lung adenocarcinoma cells (SPCA1) respectively, using liposome transfection reagents purchased from Invitrogen ( Lipofectamine) BS/U6/gfp, BS/U6/cdk-2 and BS/U6/dnmt-1 plasmids of the present invention are respectively transfected in Hela, HEK293, H1299, SPCA1 cells, continue in 6-well culture plate Cultivate for 48 hours, and the operation is completed according to the operating procedures in the product manual.

实施例6Example 6

表达siRNA对靶蛋白抑制作用分析Analysis of the inhibitory effect of expressed siRNA on target protein

收集实施例5中的各培养细胞，用倒置荧光显微镜分别观察各自的绿色荧光蛋白(gfp、cdk-2、dnmt-1)的表达情况，数字摄像后经过流式细胞仪(FACS calibur，BD公司)分别检测gfp、cdk-2、dnmt-1在不同的培养细胞表达情况并计算对靶基因的抑制效率，得到附表1的结果。从表中可以看出本发明的各种DNA质粒都能在各特征细胞内稳定高效表达出各自siRNA，如转染48小时后对gfp的抑制率为75％，而内源靶蛋白cdk-2和dnmt-1的最高抑制率分别61％和58％；也可以看出不同的小分子干扰RNA的DNA质粒对不同的细胞的RNAi的作用不同，因此可以进一步筛选出针对性的DNA质粒用于诊断、预防和治疗人类的各种病变，为基因治疗打下了基础。Collect each cultured cell inembodiment 5, observe the expression situation of respective green fluorescent protein (gfp, cdk-2, dnmt-1) respectively with inverted fluorescent microscope, after digital camera, pass through flow cytometer (FACS calibur, BD company) ) to detect the expression of gfp, cdk-2, and dnmt-1 in different cultured cells and calculate the inhibition efficiency of the target gene, and obtain the results in Table 1. As can be seen from the table, various DNA plasmids of the present invention can stably and efficiently express respective siRNAs in each characteristic cell, such as the inhibition rate to gfp after 48 hours of transfection, and the endogenous target protein cdk-2 and dnmt-1 have the highest inhibition rates of 61% and 58% respectively; it can also be seen that the DNA plasmids of different small interfering RNAs have different effects on the RNAi of different cells, so the targeted DNA plasmids can be further screened for use in Diagnosis, prevention and treatment of various human diseases have laid the foundation for gene therapy.

表1：不同RNA干扰质粒对在不同细胞株相应靶蛋白的抑制率Table 1: Inhibition rate of different RNA interference plasmids on corresponding target proteins in different cell lines

Claims (6)

Translated fromChinese

1、一种能表达小分子干扰RNA的DNA质粒，其特征在于以载体pBlueScript/U6为骨架，在所述载体pBlueScript/U6的Apa I和EcoR I的酶切位点上，插入同源酶切位点的长度为21nt的DNA靶序列构建而成。1. A DNA plasmid capable of expressing small-molecule interfering RNA is characterized in that the carrier pBlueScript/U6 is used as a backbone, and a homologous restriction enzyme is inserted into the restriction sites of Apa I and EcoR I of the carrier pBlueScript/U6 The length of the site is constructed from the DNA target sequence of 21 nt.2、如权利要求1所述的一种能表达小分子干扰RNA的DNA质粒，其特征在于所述载体pBlueScript/U6为在pBlueScript的Kpn I和Apa I的酶切位点上，插入同源酶切位点的U6启动子构建而成。2. A DNA plasmid capable of expressing small-molecule interfering RNA as claimed in claim 1, characterized in that said carrier pBlueScript/U6 inserts a homologous enzyme at the restriction sites of Kpn I and Apa I of pBlueScript The U6 promoter at the cleavage site was constructed.3、如权利要求1所述的一种能表达小分子干扰RNA的DNA质粒，其特征在于所述DNA靶序列为报告基因的编码区的第101～121位片段。3. A DNA plasmid capable of expressing small interfering RNA according to claim 1, characterized in that said DNA target sequence is the 101-121 fragment of the coding region of the reporter gene.4、如权利要求1所述的一种能表达小分子干扰RNA的DNA质粒，其特征在于所述DNA靶序列为肿瘤细胞内源基因周期蛋白依赖性激酶2基因的编码区的第66～86位片段。4. A DNA plasmid capable of expressing small interfering RNA according to claim 1, characterized in that the DNA target sequence is the 66th to 86th part of the coding region of the tumor cell endogenous gene cyclin-dependent kinase 2 gene. bit fragments.5、如权利要求1所述的一种能表达小分子干扰RNA的DNA质粒，其特征在于所述DNA靶序列为DNA甲基转移酶1基因的编码区的第85～105位片段。5. A DNA plasmid capable of expressing small interfering RNA according to claim 1, characterized in that said DNA target sequence is the 85th to 105th fragment of the coding region of DNA methyltransferase 1 gene.6、一种权利要求1所述的能表达小分子干扰RNA的DNA质粒的构建方法，其特征在于包括下述步骤：6. A method for constructing a DNA plasmid capable of expressing small interfering RNA according to claim 1, characterized in that it comprises the following steps:1)以pmU6质粒为模板，通过PCR扩增反应获得在左侧含有Kpn I酶切位点和在右侧含有Apa I酶切位点的U6启动子序列；1) Using the pmU6 plasmid as a template, the U6 promoter sequence containing the Kpn I restriction site on the left and the Apa I restriction site on the right is obtained by PCR amplification reaction;2)将上述U6启动子序列插入到同样含有Kpn I和Apa I的酶切位点的pBlueScript上，然后转入感受态细胞E.coli TGl中，经过筛选、扩增和抽提获得载体pBlueScript/U6；2) Insert the above-mentioned U6 promoter sequence into pBlueScript that also contains the restriction sites of Kpn I and Apa I, and then transfer it into the competent cell E.coli TG1, and obtain the vector pBlueScript/ U6;3)应用siRNA target finder软件分析筛选出含有Apa I和EcoR I的酶切位点的长度为21nt的DNA靶序列，体外人工合成该DNA靶序列；3) siRNA target finder software was used to analyze and screen out a DNA target sequence with a length of 21 nt containing restriction sites of Apa I and EcoR I, and artificially synthesize the DNA target sequence in vitro;4)将上述DNA靶序列插入到所述载体pBlueScript/U6的Apa I和EcoR I的酶切位点上，利用质粒抽提试剂盒抽提得到能表达小分子干扰RNA的DNA质粒。4) The above-mentioned DNA target sequence is inserted into the restriction sites of Apa I and EcoR I of the vector pBlueScript/U6, and a DNA plasmid capable of expressing small interfering RNA is obtained by extraction of a plasmid extraction kit.

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