WO2018170653A1 - 一种降低miRNA-29a、miR-140和miR-424表达水平的Tud RNA及其应用 - Google Patents

一种降低miRNA-29a、miR-140和miR-424表达水平的Tud RNA及其应用 Download PDF

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WO2018170653A1
WO2018170653A1 PCT/CN2017/077208 CN2017077208W WO2018170653A1 WO 2018170653 A1 WO2018170653 A1 WO 2018170653A1 CN 2017077208 W CN2017077208 W CN 2017077208W WO 2018170653 A1 WO2018170653 A1 WO 2018170653A1
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mir
tud
rna
mirna
vector
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PCT/CN2017/077208
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毛吉炎
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深圳市博奥康生物科技有限公司
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  • the present invention relates to the field of gene editing and epigenetics, and in particular to a Tud RNA which reduces the expression levels of miRNA-29a, miR-140 and miR-424 and uses thereof.
  • 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-29a is a small RNA closely related to cell proliferation. It is involved in many diseases and can act as a tumor suppressor gene in a variety of tumors. It has the ability to grow and invade cells such as human gastric cancer and bladder cancer. Related, its expression level is an important reference for evaluating the benign and malignant glioma, and it is also associated with diseases such as atherosclerotic liver fibrosis, and has important potential application value for the treatment of various tumors; miR-140 and various diseases The occurrence and development are closely related, 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 TGFBR1 and other gene expression.
  • miR-140 is highly expressed in articular cartilage and plays a vital role in the pathogenesis of osteoarthritis. Under various mechanisms, miR-140 plays an oncogene role in some tumors, and plays a tumor suppressor role in other tumors, and is associated with multiple tumor chemotherapy resistance; miR-424 is a miRNA discovered in recent years. It acts on target genes and participates in the signal pathway of target gene regulation in a variety of tumors, thereby affecting the biological effects and development of tumor cells, playing a role similar to oncogenes, tumor suppressor genes, or promoting or inhibiting tumors. Invasion and transfer.
  • miR-424 is a multifunctional miRNA, which is associated with cell invasion and metastasis of cervical cancer, pancreatic cancer, etc.; it is associated with the expression of inflammatory factors such as IL-6 and TNF- ⁇ ; since the miR-424 promoter region has CpG islands It is also associated with methylation-induced gene silencing.
  • MiRNA functional studies often require miRNA silencing techniques, including anti-miR, antago miR, miRNA
  • 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.
  • Lentiviral vector is currently used to construct a stable cell line. Compared with expression vectors such as retrovirus and adenovirus, it can simultaneously infect dividing cells and non-dividing cells, and has high transfection efficiency. And high stability, can make the experimental target gene long-term stable expression
  • the primary object of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide a Tud RNA which can completely reduce the expression levels of miR NA-29a, miR-140 and miR-424.
  • Another object of the present invention is to provide the use of the Tud RNA which reduces the expression levels of miRNA-29a, miR-140 and miR-424.
  • a further object of the present invention is to provide a recombinant vector containing the TudRNA.
  • RNA is applied to inhibit the expression of miRNA-29a, miR-140 and miR-424, preferably the Tud RNA is constructed on a lentiviral vector, and the recombinant plasmid consisting of the Tud RNA and the lentiviral vector is applied to the WRL-68 Cells, for the purpose of inhibiting the expression of miRNA-29a, miR-140 and miR-424;
  • the lentiviral vector is preferably a CS-RfA-EG lentiviral vector
  • the construction of the Tud RNA on a CS-RfA-EG lentiviral vector comprises the following steps: (1) designing a DNA sequence containing Tud RNA which inhibits human miRNA-29a, miR-140 and miR-424, as shown in SEQ ID NO: 1; in order to clone it into pENTR/U6 vector, the following DNA was synthesized. sequence
  • the double-stranded DNA fragment on U6-Tud-29a-140-424 was transferred to the lentiviral vector CS-Rf A-EG to obtain CS
  • the TuD RNA sequence designed to inhibit human miR-29a, miR-140 and miR-424 has a stem-loop structure and is not easily degraded, and the double-stranded Tud RNA is relatively common with the currently used single-stranded miRNA sponge.
  • the binding efficiency is higher, and the same target can better achieve the interference of the three miRNAs for the three targets, and improve the efficiency of the miRNA function research.
  • FIG. 1 is a schematic diagram showing the structure of a CS-RfA-EG lentiviral vector
  • FIG. 2 is a WRL of a WRL-68 cell transfected with CS-RfA-EG-Tud-29a-140-424 lentivirus in one embodiment.
  • miRNA expression levels of 68 cells wherein a. expression of miR-29a, b. expression of miR-140, c. expression of miR-424. Best embodiment for carrying out the invention
  • ddH20 is supplemented to 20 ⁇ 1;
  • reaction was carried out at 25 ° C for 6 hours, and the reaction was carried out at 37 ° C for 10 minutes to remove excess LR.
  • Clonase II enzyme Then, the ⁇ competent Escherichia coli Stbl3 (purchased from Quanjin Company) was cultured at 30 ° C for 22 to 24 hours in LB solid medium containing 10 (Vg/ml Ampicillin), and the clone that grew was a positive clone. Positive clones were picked and cultured in LB liquid medium containing 10 (Vg/ml Ampicillin, cultured at 30 ° C for 20 hours, and the plasmid was purified and sent for sequencing. The correct sequencing result was obtained by correct sequencing.
  • This recombinant plasmid was defined as: CS-RfA-EG-Tud-29a-140-424, a Tud RNA lentiviral plasmid that reduced the expression levels of miRN A-29a, miR-140 and miR-424.
  • the recombinant plasmid was extracted from a box (purchased from Tiangen Biochemical).
  • CS-RfA-EG-Tud-29a-140-424, pCMV-VSV-G-RSV-Rev and pCAG-HIVgp plasmid were introduced into human embryonic kidney 293T cells in a ratio of 5:2:2 with Lipofectamine 2000. The supernatant of the cell culture was collected, and filtered with a 0.45 ⁇ filter to obtain a virus containing the CS-RfA-EG-Tud-29a-140-424 plasmid.
  • WRL-68 cells were infected with a virus containing the CS-RfA-EG-Tud-29a-140-424 plasmid.
  • the infection steps are as follows: 50,000 WRL-68 cells and 100,000 TU virus solution were suspended in DMEM medium and prepared according to the amount of ⁇ mixture/well. Each ⁇ mixture also contained 10% FBS and 8 g/ml.
  • the condensed amine was mixed, placed in a 96-well plate, and reacted at 37 ° C for 24 h.
  • the culture solution was replaced with fresh medium (DMEM + 10% FBS), and culture was continued for 24 h, subculture.
  • the miRNA extraction and isolation kit was used to extract miRNA from normal WRL-68 cells and WRL-68 cells transfected with CS-RfA-EG-Tud-29a-140-42 4 lentivirus. After reverse transcription and tailing, the corresponding cDNA was obtained. The cDNA of each of the two cells was used as a template, and the expression levels of miR-29a, miR-140 and miR-424 were detected by real-time PCR. The experiment was repeated 3 times, and 3 parallel samples were set per well, with snord 44 as the internal reference. . The results are shown in Figure 3.
  • the expression level of miR-29a in TuD-29a-140-424 cells is 49% lower than that in WRL-68 cells, and the expression level of miR-140 is 53% lower than that in WRL-68 cells.
  • the expression level of miR-424 was 76% lower than that of WR L-68 cells. The difference was statistically significant (/? ⁇ 0.01), indicating that the TuD-29a-140-424 cell line was successfully constructed.
  • the TuD RNA sequence designed to inhibit human miR-29a, miR-140 and miR-424 has a stem-loop structure and is not easily degraded, and the double-stranded Tud RNA is relatively common with the currently used single-stranded miRNA sponge.
  • the binding efficiency is higher, and the same target can better achieve the interference of the three miRNAs for the three targets, and improve the efficiency of the miRNA function research.

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Abstract

一种降低miRNA-29a、miR-140和miR-424表达水平的Tud RNA及其应用。所述降低miRNA-29a、miR-140和miR-424表达水平的Tud RNA的DNA序列如SEQ ID NO 1所示。包含所述Tud RNA的重组质粒及其制备方法,包括先设计含有抑制人miRNA-29a、miR-140和miR-424的Tud RNA的DNA序列,将此DNA序列克隆至pENTR/U6载体上,再通过Gateway技术将位于重组载体pENTR/U6-U6-Tud-29a-140-424上的此DNA序列转移至慢病毒载体CS-RfA-EG上,得到CS-RfA-EG-Tud-29a-140-424重组质粒,该重组质粒能完全抑制人miRNA-29a、miR-140和miR-424表达。

Description

发明名称:一种降 fi;miRNA-29a、 miR-140和 miR-424表达水平的
Tud RNA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, 具体地涉一种降低 miRNA-29a 、 miR- 140和 miR-424表达水平的 Tud RNA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-29a是一个与细胞增殖紧密相关的小分子 RNA, 参与多种疾病, 可在多种 肿瘤中起到抑癌基因作用, 它与人胃癌和膀胱癌等细胞的生长和侵袭能力相关 , 其表达水平是评价胶质瘤良恶性的重要参考指标, 还与动脉粥样硬化肝纤维 化等疾病相关, 对多种肿瘤的治疗具有重要的潜在应用价值; 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 TGFBR1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, 在多种机制调控下, miR-140在一些肿 瘤中发挥癌基因作用, 而在另一些肿瘤中发挥抑癌作用, 并且与多种肿瘤化疗 耐药性有关; miR-424是近年来发现的一个 miRNA, 其在多种肿瘤中通过作用于 靶基因, 参与靶基因调控的信号通路, 从而影响肿瘤细胞生物学效应和发生发 展, 发挥类似于癌基因、 抑癌基因的作用, 或促进、 抑制肿瘤的侵袭转移。 有 研究表明 miR-424是多功能 miRNA, 它与宫颈癌, 胰腺癌等细胞侵袭转移相关; 与炎性因子如 IL-6、 TNF-α的表达相关; 由于 miR-424启动子区域具有 CpG岛, 它与甲基化诱导的基因沉默也相关。 通过控制 miR-29a、 miR- 140和 miR-424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] MiRNA的功能研究通常需要用到 miRNA沉默技术, 主要包括 anti-miR, antago miR, miRNA
sponge等, 这些技术均属于瞬吋转染技术, 无法保持对目的 miRNA的长期稳定 沉默, 沉默效果远未达到最优。
[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。
[0006] 慢病毒载体是目前采用较多的构建稳定细胞株的载体, 与逆转录病毒和腺病毒 等表达载体相比, 它能同吋感染分裂细胞和非分裂细胞, 转染效率较高, 并且 稳定性高, 能使实验目的基因长期稳定表达
问题的解决方案
技术解决方案
[0007] 本发明的首要目的在于克服现有技术的缺点与不足, 提供一种能完全降低 miR NA-29a、 miR- 140和 miR-424表达水平的 Tud RNA。
[0008] 本发明的另一目的在于提供所述降低 miRNA-29a、 miR- 140和 miR-424表达水平 的 Tud RNA的应用。
[0009] 本发明的再一目的在于提供一种含有所述 TudRNA的重组载体。
[0010] 本发明的目的通过下述技术方案实现: 一种降低 miRNA-29a、 miR-140和 miR-4 24表达水平的 Tud RNA, 其 DNA序歹 ij如 SEQ ID NO 1所示;
[0011] 所述降低 miRNA-29a、 miR- 140和 miR-424表达水平的 Tud
RNA应用于抑制 miRNA-29a、 miR-140和 miR-424的表达, 优选将所述 Tud RNA 构建于慢病毒载体上, 再将由所述 Tud RNA和慢病毒载体组成的重组质粒作用 于 WRL-68细胞, 达到抑制 miRNA-29a、 miR- 140和 miR-424表达的目的;
[0012] 所述的慢病毒载体优选为 CS-RfA-EG慢病毒载体;
[0013] 将所述的 Tud RNA构建于 CS-RfA-EG慢病毒载体上, 包括以下步骤: [0014] (1) 设计含有抑制人 miRNA-29a、 miR-140和 miR-424的 Tud RNA的 DNA序列 , 如 SEQ ID NO l所示; 为了将其克隆至 pENTR/U6载体上, 合成以下 DNA序列
[0015] F: 5,-CACC
Figure imgf000004_0001
ATCAAGATGATCCTAGCGCCACCTTTTT -3'
R: 5'-AAAA
Figure imgf000004_0002
TGAGTGCAAAACGTTGATGATCCTAGCGCC -3';
[0017] (2) 将 DNA序列 FP和 RP按等摩尔量进行混合, 于 95°C处理 5 min, 然后按 0.1
°C/s的速度降温至室温, 沉淀, 得到双链 DNA片段;
[0018] (3) 将 pENTR/U6载体与步骤 (2) 得到的双链 DNA片段进行连接, 得到重组 载体 pENTR/U6-Tud-29a- 140-424;
[0019] (4) 通过 Gateway技术将位于重组载体 pENTR/
U6-Tud-29a- 140-424上的双链 DNA片段转移至慢病毒载体 CS-Rf A-EG上, 得到 CS
-RfA-EG-Tud-29a- 140-424重组质粒。
发明的有益效果
有益效果
[0020] 本发明设计的抑制人 miR-29a、 miR-140和 miR-424的 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, 并且同吋针对三个靶点, 能较好地实现三个 miRNA的干扰, 提高 m iRNA功能研究的效率。 对附图的简要说明
附图说明
[0021] 图 1为 CS-RfA-EG慢病毒载体的结构示意图; 图 2为一实施例中 WRL-68细胞与 转染 CS-RfA-EG-Tud-29a- 140-424慢病毒的 WRL-68细胞的 miRNA表达水平情况 , 其中, a. miR-29a的表达情况, b. miR-140的表达情况, c. miR-424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
[0022] 下面结合实施例及附图对本发明作进一步详细的描述, 但本发明的实施方式不 限于此。
[0023] 实施例一
[0024] (1) 设计降低 miRNA-29a、 miR-140和 miR-424表达水平的 Tud RNA的 DNA序 歹 |J, 如 SEQ ID NO l所示; 为了将其克隆至 pENTR/U6载体上, 合成以下 DNA序 列:
[0025] F: 5,-CACC
Figure imgf000005_0001
ATCAAGATGATCCTAGCGCCACCTTTTT -3'
R: 5'-AAAA
Figure imgf000005_0002
TGAGTGCAAAACGTTGATGATCCTAGCGCC -3';
(2) 将 DNA序列 FP和 RP按等摩尔量进行混合, 于 95°C处理 5 min, 然后按 0.1 °C/s的速度降温至室温, 2倍冰冻的无水乙醇 (加入 0.1倍 pH 5.6的 3 mol/L NaAc) 沉淀, 得到双链 DNA片段;
[0028] (3) 将 pENTR/U6载体 (购自 Thermo
Fisher公司) 与步骤 (2) 得到的双链 DNA片段进行连接, 得到重组载体 pENTR/
U6-Tud-29a- 140-424。 连接反应体系如下:
[0029] 双链 DNA片段 100 ng
[0030] pENTR/U6载体 20 ng
[0031] T4 DNA连接酶 (购自 NEB公司) Ιμΐ
[0032] Τ4 DNA连接酶 Buffer 2 μL
[0033] ddH20补至 20μ1;
[0034] 16°C连接 5 h, 接着取 5μ1连接产物转化感受态大肠杆菌 Stbl3 (购自全式金公 司) , 培养于含 5(Vg/ml的 Kanamycin的 LB固体培养基。 生长出来的克隆即为阳 性克隆, 挑取阳性克隆培养于依据分子克隆配方配制的含 5(Vg/ml的 Kanamycin 的 LB液体培养基中, 进行测序, 确定得到含有抑制人 miR-29a、 miR-140和 miR-4 24的 TuD RNA的重组载体 pENTR/U6-Tud-29a- 140-424。
[0035] (4) 通过 Gateway技术将位于重组载体 pENTR/U6-Tud-29a- 140-424上的双链 DNA片段转移至慢病毒载体 CS-RfA-EG上, CS-RfA-EG-Tud-29a- 140-424重组质 粒, 具体操作过程如下:
[0036] pENTR/U6-Tud-29a- 140-424 100 ng
[0037] CS-RfA-EG载体 150ng
[0038] LR clonase II enzyme mix (购自 Invitrogen公司 ) Ιμΐ
[0039] TE共计 9μ1
[0040] 25°C下反应 6小吋, 力 Β ΐμΐ蛋白酶 K, 37°C反应 10分钟以清除多余的 LR
clonase II enzyme。 接着取 Ιμΐ感受态大肠杆菌 Stbl3 (购自全式金公司) , 于 30°C 在含有 10(Vg/ml Ampicillin的 LB固体培养基中培养 22〜24小吋, 生长出来的克隆 即为阳性克隆。 挑取阳性克隆, 培养于含有 10(Vg/ml Ampicillin的 LB液体培养 基中, 30°C下培养 20小吋, 纯化质粒, 并送测序。 测序结果正确即为获得了正确 的重组质粒, 将此重组质粒定义为: CS-RfA-EG-Tud-29a- 140-424, 即降低 miRN A-29a、 miR-140和 miR-424表达水平的 Tud RNA慢病毒质粒。 用无内毒素质粒提 取盒 (购自天根生化) 提取该重组质粒。
[0041] (5) 慢病毒的包装
[0042] 用 Lipofectamine 2000将 CS-RfA-EG-Tud-29a- 140-424、 pCMV-VSV-G-RSV-Rev 以及 pCAG-HIVgp质粒按照 5:2:2的比例导入到人胚肾 293T细胞中; 收集细胞培 养的上清液, 0.45 μηι滤头过滤后即得到含有 CS-RfA-EG-Tud-29a-140-424质粒的 病毒。
[0043] (6) 病毒转染 WRL-68细胞
[0044] 用含有 CS-RfA-EG-Tud-29a-140-424质粒的病毒感染 WRL-68细胞。 感染步骤如 下: 将 5万个 WRL-68细胞与 10万 TU病毒液混悬于 DMEM培养基中, 按 ΙΟΟμΙ混合 液 /孔的用量配制, 每 ΙΟΟμΙ混合液还包含 10%FBS和 8 g/ml的凝聚胺, 混匀后放 于 96孔板中, 37°C下反应 24 h, 将培养液换成新鲜的培养液 (DMEM+10%FBS ) , 继续培养 24 h, 传代培养。
[0045] (7) 定量 PCR检测 miRNA的表达水平变化
[0046] 以正常 WRL-68细胞未对照, 分别用 miRcute
miRNA提取分离试剂盒提取正常 WRL-68细胞和转染 CS-RfA-EG-Tud-29a-140-42 4慢病毒的 WRL-68细胞的 miRNA, 逆转录和加尾后, 得到相应的 cDNA。 取 2种 细胞的 cDNA各 2 μί为模板, 荧光定量 PCR检测 miR-29a、 miR- 140和 miR-424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-29a-140-424细胞的 miR-29a的表达水平比 WRL-68细 胞低 49%, miR- 140的表达水平比 WRL-68细胞低 53%, miR-424的表达水平比 WR L-68细胞低 76%。 差异有统计学意义 (/?<0.01) , 说明 TuD-29a-140-424细胞株 构建成功。
工业实用性
[0047] 本发明设计的抑制人 miR-29a、 miR- 140和 miR-424的 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, 并且同吋针对三个靶点, 能较好地实现三个 miRNA的干扰, 提高 m iRNA功能研究的效率。

Claims

权利要求书
[权利要求 1] 一种降低 miRNA-29a、 miR-140和 miR-424表达水平的 Tud RNA, 其特 征在于: 编码所述 Tud RNA的 DNA序列如 SEQ ID NO 1所示。
[权利要求 2] 权利要求 1所述抑制人 miRNA-29a、 miR-140和 miR-424表达的 TudRN
A的应用, 其特征在于: 所述 Tud
RNA应用于抑制人源 miRNA-29a、 miR-140和 miR-424的表达。
[权利要求 3] 根据权利要求 2所述的应用, 其特征在于: 先将所述 Tud RNA构建于 慢病毒载体上, 再将由所述 Tud RNA和慢病毒载体组成的重组质粒应 用于抑制 miRNA-29a、 miR-140和 miR-424表达。
[权利要求 4] 根据权利要求 3所述的应用, 其特征在于: 所述的慢病毒载体为 CS-Rf
A-EG慢病毒载体。
[权利要求 5] 根据权利要求 4所述的应用, 其特征在于: 将所述的 shRNA构建于 CS-
RfA-EG慢病毒载体上, 包括以下步骤:
(1) 设计含有抑制人 miRNA-29a、 miR-140和 miR-424的 Tud
RNA的 DNA序歹 ij, 如 SEQ ID NO 1所示; 为了将其克隆至 pENTR/U6 载体上, 合成以下 DNA序列:
F: 5'-CACC
Figure imgf000008_0001
AGCGCCACCTTTTT -3'
R: 5'-AAAA GATGATCCTAGCGCC -3';
(2) 将 DNA序列 FP和 RP按等摩尔量进行混合, 于 95°C处理 5 min, 然后按 O.rC/s的速度降温至室温, 沉淀, 得到双链 DNA片段;
(3) 将 pENTR/U6载体与步骤 (2) 得到的双链 DNA片段进行连接, 得到重组载体 pENTR/U6-Tud-29a-140-424;
(4) 通过 Gateway技术将位于重组载体 pENTR/ U6-Tud-29a- 140-424 上的双链 DNA片段转移至慢病毒载体 CS-Rf A-EG上, 得到 CS-Rf A-EG -Tud-29a- 140-424重组质粒。
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