WO2017219172A1 - 敲低 miRNA-140 和 miR-152 表达的慢病毒载体的构建及其应用 - Google Patents
敲低 miRNA-140 和 miR-152 表达的慢病毒载体的构建及其应用 Download PDFInfo
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- the present invention relates to the field of gene editing and epigenetics, and in particular to the construction and application of a lentiviral vector which knocks down the expression of miRNA-1 40 and miR-152.
- MicroRNAs are a class of endogenous, non-coding RNAs found in eukaryotes, typically between 22 and 25 nt in size. miRNAs are widely distributed in plants, animals, and multicellular organisms, and can Play an important regulatory role, and in the study of human miRNAs, it is found that the expression of miRNA in normal tissues and tumor tissues is significantly different, some miRNAs are lowly expressed in tumor tissues, and some are highly expressed in tumor tissues. This suggests that miRNAs play a crucial role in tumorigenesis.
- miR-140 is closely related to the development of various diseases, such as bone and joint diseases, liver diseases, pituitary adenomas, testicular development, head and neck tumors, ovarian and breast diseases. miR-140 can inhibit the proliferation and invasion and metastasis of hepatocellular carcinoma by targeting TG FBR1 and other gene expression. miR-140 is highly expressed in articular cartilage and plays a crucial role in the pathogenesis of osteoarthritis. miR-152 is a multifunctional miRNA. It was found that miR-152 is related to methylation. For example, it is related to methyltransferase DNMT1 content and enzyme activity. miR-152 can be methylated by endometrial cancer DNA.
- a tumor suppressor microRNA which is associated with many diseases such as pre-eclampsia, trophoblastic tumor, bladder cancer, gastrointestinal cancer, ovarian cancer and the like.
- miR-140 and miR-152 By controlling the expression of miR-140 and miR-152, peers work synergistically with other drugs to provide new epigenetic ideas for the treatment of cancer.
- miRNA silencing is the presentation of synthetic oligonucleotides into cells, with endogenous miRNAs.
- a heteroduplex causes the miRNA to reduce the inhibition of the target gene to achieve regulation of gene function.
- Silencing of miRNAs is currently difficult to achieve.
- Commonly used methods for silencing miRNA are anti- miR, antagomiR, miRNA sponge, etc., wherein anti-miR and antagomiR are With the transient transfection technology, the interference effect can not be maintained stably, and the miRNA sponge effect is far from optimal. At present, there is no report on the optimization of miR-140 and miR-152 interference.
- Tough Decoy RNA is a novel miRNA-inhibiting miRNA that inhibits miRNA by introducing double-stranded RNA to target miRNAs. Because the inserted RNA is double-stranded and has a secondary structure of stem-loops, it is resistant to intracellular nuclease degradation and inhibits miRNAs in a long-term, stable, and efficient manner.
- the object of the present invention is to provide a lentiviral vector for constructing homologous interference miR-140 and miR-152 and construct a lentiviral vector capable of stably maintaining interference effects, aiming at the deficiencies in the prior art. Applied to the field of gene editing.
- the gene interference sequence of the corresponding TuD RNA against miR-140 and miR-152 was designed and synthesized, and the nucleotide sequence thereof is shown in SEQ ID NO.: 1.
- This sequence was ligated to the lentiviral vector pLKO.l-puro to obtain a lentiviral vector pLKO-Tud-140-152 lentiviral vector capable of stably maintaining the interference effect, and the nucleotide sequence thereof is shown in SEQ ID NO.: 2.
- the present invention constructs and synthesizes the gene interference sequence of the corresponding TuD RNA of miR-140 and miR-152, and is ligated to the lentiviral vector pLKO.l-puro, and the resulting vector has the same interference with miR-140.
- the specific integration steps are as follows:
- the sequence information of miR-140 and miR-152 provided in miRBase was designed to design the same TuD RNA oligonucleotide sequence for mi R-140 and miR-152, and its sequence ⁇ ij is SEQ ID NO.: As shown in Fig. 1, Shanghai Shengong was commissioned to synthesize the sequence by means of primers.
- the synthesized sequence is two complementary single-stranded DNAs.
- the two single-stranded DNAs were dissolved in ddH 2 0, mixed in an equimolar ratio, treated at 95 ° C for 5 min, and then allowed to cool to room temperature at room temperature.
- RNA sequence was ligated into the vector pLKO.l-puro to form the recombinant vector pLKO-TuD-140-152, and finally the ligation product was transformed into competent E. coli ToplO and plated onto a plate containing ampicillin LB medium. Incubate at 37 °C for 14 h. Five single colonies were picked from the plates and added to 5 tubes containing ampicillin in liquid LB medium for 8 hours at 37 °C. The bacteria were sent to Shanghai Biotech for sequencing. Sequencing was performed in the correct strain and extracted with a small amount of endotoxin-free extraction kit. The extracted plasmid was the plasmid for miR-140 and miR-152 which were required for the present invention.
- the homologous interference miR-140 and miR-152 TuD RNA sequences designed by the present invention have a stem-loop structure and are not easily degraded, and the double-stranded Tud RNA has higher binding efficiency than the currently used single-stranded miRNA sponge. And the same target for two targets, can better achieve the interference of two miRNAs, improve the efficiency of miRNA function research.
- FIG. 1 is a schematic view showing the structure of a pLKO-TuD-140-152 lentiviral expression vector according to an embodiment
- FIG. 2 is a flow diagram showing the steps required to transform the lentiviral expression vector shown in FIG. 1 into the lentiviral vector of the present invention
- Example 3 is a miRNA expression level of 16HBE cells and TuD-140-152 cells in Example 6, wherein
- the lentiviral plasmid pLKO.l-puro vector used in the present invention was purchased from Addgene; the human bronchial epithelial cells (16HBE cell strain) used in the present invention were purchased from the United States ATCC; S-Poly(T) hsa-miR- 140 qPCR-assay primer set and S-Poly(T) hsa-miR-152 qPCR-assay primer set
- the miRNA reverse transcription and fluorescence quantification kit was purchased from Shenzhen Anran Biotechnology Co., Ltd.
- TuD RNA design sequence Based on the TuD RNA design sequence and the sequence information of miR-140 and miR-152 provided in miRBase, a TuD RNA oligonucleotide sequence targeting miR-140 and miR-152 was designed, and its sequence is SEQ ID. ⁇ .:1, commissioned by Shanghai Biotech to synthesize by means of gene synthesis.
- the synthesized sequence is two complementary single-stranded DNAs.
- the two single-stranded DNAs were dissolved in ddH 2 0, mixed in an equimolar ratio, treated at 95 ° C for 5 min, and allowed to stand at room temperature to allow them to naturally cool to room temperature.
- the vector pLK0.1-puro was extracted and digested with Age I and Eco RI for 16 h, and the digested vector was recovered with MinElute Reaction Cleanup Kit, and then T4 DNA was used.
- RNA sequence was ligated into the vector pLKO.l-puro to form the recombinant vector pLKO-Tud-140-152, and finally the ligation product was transformed into competent E. coli ToplO and plated onto a plate containing ampicillin LB medium. Incubate at 37 °C for 14 h. Five single colonies were picked from the plate and added to 5 test tubes of liquid LB medium containing ampicillin for 8 h at 37 °C. The bacteria were sent to Shanghai Biotech for sequencing. The sequencing results were completely correct. This is the pLKO-Tud-140-152 lentiviral recombinant vector.
- the dilution ratio of the recombinant lentivirus solution is 1, 10, 100, 1000, 10000, 10,000, 1000000, 10000000, and 100000000, and the solution gradient of the recombinant lentivirus is used in the medium. Diluted, then 100 gradient dilutions of the recombinant lentivirus solution and 10 (L perforated plate)
- the cell culture medium was mixed and transfected in different wells of the multi-well plate. 24 h after the start of transfection, the medium was aspirated and replaced with 50 (L containing 5 U DNasel in fresh medium, and cultured at 37 ° C for 30 min to remove possible adhesion. Remaining plasmid DNA on the cell surface, then changing the medium to 1 mL of normal medium, and continuing to culture for 48 hours;
- the medium in each well of the multiwell plate was aspirated, 50 (L-trypsin-EDTA solution was added to digest the cells, reacted at 37 ° C for 1 minute, and then the medium was added to terminate the digestion reaction. After the cells are purged, the cells of each well are collected by centrifugation, the total RNA of each well is extracted, and then the total cDNA of each well is reverse-transcribed; and the total cDNA of each of the obtained cells is separately fluorescent.
- Quantitative PCR was performed to obtain the ct value of each well of the cells, and the experimental group with the smallest difference from the ⁇ value of the control group but exceeding 2 was selected to obtain the dilution factor, and the lentivirus titer was calculated according to the following formula:
- T 20000 x R, where T is the lentivirus titer, T is in units of TU/mL, and R is the dilution factor.
- the lentivirus titer of the package is greater than 10000000 TU/mL, indicating that the packaging of the lentivirus is successful.
- 16HBE cells were seeded in 6-well plates, 1000000 cells per well, and the cell density was about 50% after 12 hours.
- the virus solution was taken separately, and the virus was diluted 10 times with DMEM complete medium, and then polyglycolamine was added.
- the homologous interference miR-140 and miR-152 TuD RNA sequences designed by the invention have a stem-loop structure and are not easily degraded, and the double-stranded Tud RNA has higher binding efficiency than the commonly used single-stranded miRNA sponge, and the same ⁇ Targeting two targets can better achieve the interference of two miRNAs and improve the efficiency of miRNA function research.
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Abstract
敲低miRNA-140和miR-152表达的慢病毒载体的构建及其应用,包括pLKO.1-puro表达载体的基本序列、抗性基因序列、多克隆位点序列、启动子序列和靶向miR-140和miR-152的寡核苷酸序列。所述多克隆位点包括Age I酶切位点和EcoR I酶切位点;所述靶向miR-140和miR-152的寡核苷酸序列正向***所述多克隆位点中。构建得到的pLKO-Tud-140-152慢病毒表达载体具有转染效率高,用量少,可持续、高效、稳定、特异地抑制人miRNA-140-152表达的优点,可作为有力工具应用于制备治疗miRNA-140-152表达异常相关疾病的药物。
Description
敲低 miRNA-140和 miR-152表达的慢病毒载体的构建及其应用
技术领域
[0001] 本发明涉及基因编辑领域和表观遗传领域研究, 具体地涉及一种敲低 miRNA-1 40和 miR- 152表达的慢病毒载体的构建及其应用。
背景技术
[0002] MicroRNA (miRNA) 是在真核生物中发现的一类内源性的非编码 RNA, 大小 一般在 22-25 nt之间, miRNA广泛分布于植物、 动物和多细胞生物中, 并且能发 挥重要的调节作用, 而在人类 miRNA的研究中, 发现 miRNA在正常组织和肿瘤 组织中的表达有着显著差异, 有些 miRNA会在肿瘤组织中有低表达, 有些则在 肿瘤组织中有高表达, 这说明 miRNA在肿瘤发生过程中起了至关重要的作用。
[0003] miR-140与多种疾病的发生发展密切相关, 如骨、 关节疾病, 肝脏疾病, 垂体 腺瘤, 睾丸发育, 头颈部肿瘤, 卵巢与乳腺疾病等。 miR-140能通过靶向调节 TG FBR1等基因表达抑制肝细胞癌增殖和侵袭转移, miR-140特异性高表达于关节软 骨中, 并在骨关节炎的发病机制中发挥至关重要的作用; miR-152是一种具有多 功能的 miRNA, 研究发现 miR-152与甲基化相关, 如与甲基转移酶 DNMT1含量 和酶活性相关, miR-152可被子宫内膜癌 DNA甲基化变为沉默基因, 并且其与多 种癌症的发生发展相关, 它是一种肿瘤抑制 microRNA, 与子痫前期、 滋养细胞 肿瘤、 膀胱癌、 胃肠癌、 卵巢癌等诸多疾病相关。 通过控制 miR- 140和 miR- 152 的表达, 同吋与其他药物协同作用, 能为治疗癌症提供新的表观遗传思路。 技术问题
[0004] 目前 miRNA功能研究一般通过 miRNA过表达和沉默来实现, miRNA沉默是把 人工合成的寡核苷酸小分子提呈到细胞内, 与内源性 miRNA
形成异源双链, 使 miRNA降低对靶基因的抑制作用, 以实现对基因功能的调控 。 miRNA沉默, 特别是长期稳定沉默目前较难以实现。 常用的沉默 miRNA的方 法主要有 anti- miR, antagomiR, miRNA sponge等, 其中 anti- miR和 antagomiR为
瞬吋转染技术, 其干扰效果不能稳定保持, 而 miRNA sponge效果远未达到最优 , 目前也未出现同吋针对 miR-140和 miR-152干扰进行优化提升其效果的报道。
[0005] Tough Decoy RNA (Tud RNA) 是一种新幵发出的 miRNA抑制手段, 其通过引 入双链 RNA对目标 miRNA进行吸附, 达到抑制 miRNA的目的。 由于弓 |入的 RNA 为双链并且带有茎环的二级结构, 因此其够抵抗胞内核酸酶的降解, 能长期、 稳定和高效地抑制 miRNA。
问题的解决方案
技术解决方案
[0006] 本发明的目的是针对现有技术中的不足, 提供一种用于构建同吋干扰 miR-140 和 miR-152的 TuD RNA, 并构建能稳定保持干扰效果的慢病毒载体, 将其应用到 基因编辑领域。
[0007] 为解决上述技术问题, 本发明采用的技术方案如下:
[0008] 设计并合成同吋针对 miR-140和 miR-152的相应 TuD RNA的基因干扰序列, 其 核苷酸序列如 SEQ ID NO.: 1所示。 将该序列与慢病毒载体 pLKO.l-puro连接, 获 得能稳定保持干扰效果的慢病毒载体 pLKO-Tud-140-152慢病毒载体, 其核苷酸 序列如 SEQ ID NO.: 2所示。
[0009] 本发明通过设计并合成同吋针对 miR-140和 miR-152的相应 TuD RNA的基因干 扰序列, 与慢病毒载体 pLKO.l-puro连接, 形成的载体具有能同吋干扰 miR-140 和 miR-152的作用, 具体整合的步骤如下:
[0010] S10、 同吋针对 miR-140和 miR-152d的 TuD的设计与合成: 根据 TuD
设计序列和 miRBase中提供的 miR- 140和 miR- 152的序列信息, 设计出同吋针对 mi R-140和 miR-152的 TuD RNA寡核苷酸序列, 其序歹 ij如 SEQ ID NO.: 1所示, 委托 上海生工以引物的方式合成该序列。
[0011] S20、 合成好的序列是两条互补的单链 DNA。 将两条单链 DNA溶解于 ddH 20中 , 按照等摩尔比混合后, 95°C处理 5 min, 再将其置于室温使其自然冷却至室温
[0012] S30、 提取载体 pLKO.l-puro, 使用 Age I和 Eco RI酶双酶切处理 16
h后, 用 MinElute Reaction Cleanup Kit回收酶切后的载体, 再用 T4 DNA连接酶将
上一步得到的 TuD
RNA序列连接到载体 pLKO.l-puro中, 形成重组载体 pLKO-TuD-140-152, 最后将 连接产物转化到感受态大肠杆菌 ToplO中, 并涂布到含氨苄青霉素 LB培养基的平 板上, 37 °C培养 14 h。 从平板中挑取 5个单菌落, 分别加入到 5支含氨苄青霉素的 液体 LB培养基的试管中 37 °C振荡培养 8 h后, 将菌液送至上海生工测序。 取测序 正确的菌株里并用无内毒素质粒小量提取试剂盒提取, 提取的质粒为本发明所 需的同吋干扰 miR- 140和 miR- 152的质粒。
发明的有益效果
有益效果
[0013] 本发明设计的同吋干扰 miR-140和 miR-152 TuD RNA序列带有茎环结构, 不容 易降解, 双链的 Tud RNA相对目前常用的单链的 miRNA sponge, 其结合效率更 高, 并且同吋针对两个靶点, 能较好地实现两个 miRNA的干扰, 提高 miRNA功 能研究的效率。
对附图的简要说明
附图说明
[0014] 图 1为一实施方式所述 pLKO-TuD-140-152慢病毒表达载体的结构示意图;
[0015] 图 2为将图 1所示的慢病毒表达载体改造为本发明所述慢病毒载体所需步骤的流 程图;
[0016] 图 3为实施例六中 16HBE细胞与 TuD-140-152细胞的 miRNA表达水平情况, 其中
, a. miR-140的表达情况, b. miR- 152的表达情况。
实施该发明的最佳实施例
本发明的最佳实施方式
[0017] 根据下述实施例, 可以更好地理解本发明。 然而, 本领域的技术人员容易理解 , 实施例所描述的具体的物料配比、 工艺条件及其结果仅用于说明本发明, 而 不应当也不会限制权利要求书中所详细描述的本发明。 下述实施例中所用的方 法如无特别说明均为常规方法; 所述试剂如无特殊说明, 均为市售产品。 具体 步骤可参见: 《Molecular Cloning: A Laboratory Manual》 (Sambrook, J., Russell,
David W., Molecular Cloning: A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring Harbor) °
[0018] 本发明所使用的慢病毒质粒 pLKO.l-puro载体购自 Addgene; 本发明所使用的人 支气管上皮细胞 (16HBE细胞株) 购自美国 ATCC; S-Poly(T) hsa-miR-140 qPCR-assay primer set和 S-Poly(T) hsa-miR-152 qPCR-assay primer set miRNA逆转 录和荧光定量试剂盒购自深圳市盎然生物科技有限公司。
[0019] 实施例一同吋针对 miR-140和 miR-152a的 TuD RNA的设计与合成
[0020] 根据 TuD RNA设计序列和 miRBase中提供的 miR-140和 miR-152的序列信息, 设 计出同吋针对 miR- 140和 miR- 152的 TuD RNA寡核苷酸序列, 其序列如 SEQ ID ΝΟ.:1所示, 委托上海生工以基因合成的方式合成。
[0021] 实施例二序列的退火
[0022] 合成好的序列是两条互补的单链 DNA。 将两条单链 DNA溶解于 ddH 20中, 按 照等摩尔比混合后, 95°C处理 5 min, 再将其置于室温使其自然冷却至室温。
[0023] 实施例三重组 pLKO-Tud-140慢病毒重组载体的构建
[0024] 提取载体 pLK0.1-puro, 使用 Age I和 Eco RI酶双酶切处理 16 h后, 用 MinElute Reaction Cleanup Kit回收酶切后的载体, 再用 T4 DNA
连接酶将上一步得到的 TuD
RNA序列连接到载体 pLKO.l-puro中, 形成重组载体 pLKO-Tud-140-152, 最后将 连接产物转化到感受态大肠杆菌 ToplO中, 并涂布到含氨苄青霉素 LB培养基的平 板上, 37 °C培养 14 h。 从平板中挑取 5个单菌落, 分别加入到 5支含氨苄青霉素的 液体 LB培养基的试管中 37 °C振荡培养 8 h后, 将菌液送至上海生工测序, 测序结 果完全正确的即为 pLKO-Tud-140-152慢病毒重组载体。
[0025] 实施例四慢病毒滴度测定
[0026] 第一天, 将 293FT细胞接种到多孔板中, 每个孔接种 200000个细胞, 每个孔加 入 500 培养基, 37°C、 5<¾C0 2培养过夜;
[0027] 第二天, 按所述重组慢病毒的溶液的稀释比例为 1、 10、 100、 1000、 10000、 1 00000、 1000000、 10000000和 100000000, 用培养基将所述重组慢病毒的溶液梯 度稀释, 接着分别将 100 梯度稀释的所述重组慢病毒的溶液与 10( L多孔板中
的细胞培养液在多孔板的不同孔中混合转染, 转染幵始后 24h, 吸去培养基并换 成 50( L含 5U DNasel的新鲜培养基, 37°C下培养 30min以去除可能附着于细胞表 面的残余质粒 DNA, 然后将培养基换成 l mL正常培养基, 继续培养 48h;
[0028] 第四天, 吸去所述多孔板的每个孔中的培养基, 加入 50( L胰酶 -EDTA溶液消 化细胞, 在 37°C反应 1分钟, 接着加入培养基终止消化反应并将细胞吹洗下, 离 心收集每个孔的细胞, 抽提每孔细胞的总 RNA, 接着逆转录得到每孔细胞的总 c DNA; 以及分别对得到的所述每孔细胞的总 cDNA进行荧光定量 PCR, 得到每孔 细胞的 ct值, 选择与对照组 α值差异最小但超过 2的实验组, 得到其稀释倍数, 按照以下公式计算慢病毒滴度:
[0029] T=20000xR, 其中, T为慢病毒滴度, T的单位为 TU/mL, R为稀释倍数。
[0030] 经计算, 本次包装的慢病毒滴度大于 10000000 TU/mL, 表明此次慢病毒的包装 是成功的。
[0031] 实施例五慢病毒转导 16HBE细胞
[0032] 接种 16HBE细胞于 6孔板中, 每孔 1000000个细胞, 12h后细胞密度约为 50% , 分别取病毒液, 用 DMEM完全培养基 10倍稀释病毒, 再加入聚凝胺
(polybrene)至终浓度为 8 g/mL。 去除 6孔板中的培养基, 加入含病毒的 DMEM 完全培养基(含 10%胎牛血清), 24h后弃去含病毒的 DMEM完全培养基, 更换新 鲜的 DMEM完全培养基, 24h后用 0.5 g/ml浓度的嘌呤霉素筛选细胞。 筛选 10d, 每隔 3d更换培养基一次, 并不断的增加嘌呤霉素的浓度至 1.0 g/ml。 筛 选获得的细胞株命名为 TuD-140-152细胞株。 [0034]实施例六荧光定量 PCR检 测 miR- 140的表达水平变化
[0033] 分别接种正常 16HBE细胞、 TuD-140-152细胞至 6孔板 (每孔约 300000个) , 培 养细胞约 24 h后至融合度 80%。 用 miRcute miRNA提取分离试剂盒提取这些细胞 的 miRNA, 然后用 S-Poly(T) hsa-miR-140 qPCR-assay primer set和 S-Poly(T) hsa-miR-152 qPCR-assay primer set试剂盒对 miRNA进行逆转录和加尾, 得到相应 的 cDNA。 取 2种细胞的 cDNA各 2
为模板, 荧光定量 PCR检测 miR-140和 miR-152表达水平的变化, 实验重复 3次 , 每孔设置 3个平行样,以 snord
44作为内参。 结果如图 3所示, 可以看到与 TuD-140-152细胞的 miR-140的表达水 平比 16HBE细胞低 57%, miR-152的表达水平比 16HBE细胞低 45%, 差异有统计 学意义 (/?<0.01) , 说明 TuD-140-152细胞株构建成功
工业实用性
本发明设计的同吋干扰 miR-140和 miR-152 TuD RNA序列带有茎环结构, 不容 易降解, 双链的 Tud RNA相对目前常用的单链的 miRNA sponge, 其结合效率更 高, 并且同吋针对两个靶点, 能较好地实现两个 miRNA的干扰, 提高 miRNA功 能研究的效率。
Claims
权利要求书
[权利要求 1] 一种敲低 miRNA-140和 miR-152表达的慢病毒载体, 其特征在于包括 pLKO.l-puro表达载体的基本序列、 抗性基因序列、 多克隆位点序列 、 启动子序列和靶向 miR-140和 miR-152的寡核苷酸序列。 所述多克 隆位点包括 Age I酶切位点和 EcoR
I酶切位点; 所述靶向 miR-140和 miR-152的寡核苷酸序列由 Age I酶切 位点 +颈 I序列 +靶核苷酸序列 +颈 II序列 +环 +颈 II序列互补序列 + 靶核苷酸序列互补序列 +颈 I序列互补序列 +终止位点序列 +EcoR I 酶切位点组成。
[权利要求 2] 根据权利要求 1所述的一种敲低 miRNA-140和 miR-152表达的慢病毒载 体, 其特征在于所述靶向 miR-140和 miRNA-152的寡核苷酸序列正向 ***所述多克隆位点中, 其核苷酸序列为 5'- A
TTTTTGAATTC -3' (SEQ ID NO.: l) 。
[权利要求 3] 根据权利要求 1-2所述的一种敲低 miRNA-140和 miR-152表达的慢病毒 载体, 其特征在于将所述靶向 miR-140和 miR-152的寡核苷酸序列插 入到 pLKO.l-puro表达载体中的步骤如下:
S1.同吋针对 miR-140和 miR-152的 TuD的设计与合成: 根据 TuD设计 序列和 miRBase中提供的 miR-140和 miR-152的序列信息, 设计出同吋 针对 miR-140和 miR-152的 TuD RNA寡核苷酸序列, 其序列如 SEQ ID
ΝΟ.: 1所示, 委托上海生工以引物的方式合成该序列; 合成好的序列是两条互补的单链 DNA。 将两条单链 DNA溶解于 ddH 2 0中, 按照等摩尔比混合后, 95°C处理 5 min, 再将其置于室温使其自 然冷却至室温。
S3.提取载体 pLK0.1-puro, 使用 Age I和 Eco RI酶双酶切处理 16 h后, 用 MinElute Reaction Cleanup Kit回收酶切后的载体, 再用 T4 DNA连 接酶将上一步得到的 TuD RNA序列连接到载体 pLKO.1-puro中, 形成 重组载体 pLKO-TuD-140-152, 最后将连接产物转化到感受态大肠杆 菌 ToplO中, 并涂布到含氨苄青霉素 LB培养基的平板上, 37
°C培养 14 h。 从平板中挑取 5个单菌落, 分别加入到 5支含氨苄青霉素 的液体 LB培养基的试管中 37 °di荡培养 8 h后, 将菌液送至上海生工 测序。 取测序正确的菌株里并用无内毒素质粒小量提取试剂盒提取, 提取的质粒为本发明所需的同吋干扰 miR-140和 miR-152的质粒。
[权利要求 4] 根据权利要求 1所述的一种敲低 miRNA-140和 miR-152表达的慢病毒载 体的应用方法, 其特征在于包含以下步骤:
51.将含有 pLKO-TuD-140-152载体的 ToplO大肠杆菌置于 10 mL LB培 养基中, 恒温空气摇床上 37°C, 300 rpm培养 12-16 h至 OD 600=0.6-0.8
, 将得到的菌液置于离心机中, 10000 rpm离心 1 min, 弃上清, 获得 所需菌体, 并用 E.Z.N.A. Endo-free Plasmid DNA Mini Kit I提取其中的 质粒。
52.培养 293FT细胞。 取生长状态良好的 293FT细胞接种到 10 cm培养 皿中, 每个皿接种 5000000个细胞, 加入 DMEM无双抗培养基培养细 胞约 18 h后至融合度达到 80-90%后, 取上一步得到的所需慢病毒载体 10 g、 pMDLg/pRRE、 pRSV-Rev和 pMD-G载体各 5
, 用 Lipofectamine 2000转染至 293FT细胞中, 转染后 4-6
h更换成 DMEM完全培养基, 继续培养 48h后收集含病毒的上清培养 基, 6000 g离心 lO min后, 取上清液再用 0.45μηι过滤头进行过滤, 获 得慢病毒液。
53.培养 293FT细胞, 取生长状态良好的 293FT细胞接种到 24孔板中, 每个孔接种 200000个细胞, 加入 500 培养基, 3TC, 5<¾CO 2培养 过夜。 第二天, 按病毒原液: 培养基的稀释比例为 10-10000000。 分 别制备病慢毒梯度稀释液各 100 μL! 然后吸取各孔原培养基各 100 μL
, 再加入慢病毒稀释液各 100 幵始转染。 转染幵始后 24 h, 吸出含 慢病毒的培养基, 换成 500 μί含 5 U DNaseI的新鲜培养基, 37°C下培 养 30 min以去除可能附着于细胞表面的残余质粒 DNA。 然后将培养基 换成 l mL正常培养基, 继续培养 48 h。
S4.小心吸走每个孔的全部培养基, 加入 500 胰酶 -EDTA溶液消化 细胞, 在 37°C反应 1分钟。 接着加入培养基终止消化反应并将细胞吹 洗下, 离心收集每个孔的细胞。 抽提每孔细胞的总 RNA, 接着逆转 录为 cDNA。 以分别对得到的所述每孔细胞的总 cDNA进行荧光定量 P CR, 得到每孔细胞的 Ct值, 选择与对照组 Ct值差异最小但超过 2的实 验组, 得到其稀释倍数, 按照以下公式计算慢病毒滴度:
T=20000xR , 其中, T为慢病毒滴度, T的单位为 TU/mL, R为稀释倍 数。
只要慢病毒滴度达到 10000000 TU/mL以上, 即认为成功获得所 需慢病毒液。
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