WO2020000455A1 - 靶向人APRF基因的新型RNAi干扰片段、RNAi载体及其制备方法和应用 - Google Patents

靶向人APRF基因的新型RNAi干扰片段、RNAi载体及其制备方法和应用 Download PDF

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WO2020000455A1
WO2020000455A1 PCT/CN2018/093865 CN2018093865W WO2020000455A1 WO 2020000455 A1 WO2020000455 A1 WO 2020000455A1 CN 2018093865 W CN2018093865 W CN 2018093865W WO 2020000455 A1 WO2020000455 A1 WO 2020000455A1
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aprf
rnai interference
vector
gene
fragment
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PCT/CN2018/093865
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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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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host

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  • the invention relates to the field of biotechnology, in particular to a novel RNAi interference fragment targeting a human APRF gene, an RNAi vector, a preparation method and an application thereof.
  • STAT transcriptional activators
  • APRF is an important member of the STAT family and is closely related to cell proliferation, apoptosis and differentiation. Under normal conditions, the activation of APRF by extracellular signals is strictly regulated. The process of APRF activation is short and rapid, which can maintain the biological function of normal cells. Deletion of APRF causes death of early embryonic cells.
  • APRF is continuously activated.
  • Continuous activation of APRF can mediate tumor immune escape, promote tumor neovascularization, inhibit tumor cell apoptosis, promote tumor cell proliferation and other mechanisms, leading to malignant transformation of tumor cells. Therefore, APRF is an ideal target for potential tumor therapy.
  • RNAi technology has been used to knock down the APRF gene and its function has been reported, due to the poor specificity of this technology, potential off-target risks Larger, which has caused certain obstacles to the progress of related research.
  • RNA-targeted CRISPR enzyme Cas13a
  • CRISPR / Cas9 which cuts DNA
  • CRISPR / Cas13a can be used to cut specific RNA sequences in bacterial cells.
  • the present invention provides a novel RNAi interference fragment targeting human APRF gene, RNAi vector, preparation method and application thereof.
  • the main purpose is to improve the RNAi specificity of APRF gene, reduce or eliminate off-target effects, and promote APRF. Study of gene function.
  • the present invention mainly provides the following technical solutions:
  • RNAi interference fragment for interfering with human APRF gene, its sequence is as SEQ ID NO.1.
  • RNAi interference vector is used to interfere with the human APRF gene.
  • the novel RNAi interference vector clone carries a novel RNAi interference fragment, and the sequence of the novel RNAi interference fragment is as shown in SEQ ID. NO.1.
  • novel RNAi interference vector includes a pRNAT-LwCas13a-Neo vector, and the pRNAT-LwCas13a-Neo vector is connected to the novel RNAi interference fragment.
  • a method for preparing a novel RNAi interference vector includes the following steps:
  • novel RNAi interference fragment and the pRNAT-LwCas13a-Neo after digestion
  • the vector is ligated to obtain a ligated product; wherein the sequence of the novel RNAi interference fragment is as shown in SEQ ID NO.1;
  • the novel RNAi interference fragment and RNAi vector provided by the present invention can efficiently and specifically knock down the expression of the human APRF gene, and the application cost is low, which can promote the research of the function of the human APRF gene.
  • Figure 1 is a map of pRNAT-LwCas13a-Neo plasmid
  • FIG. 2 shows the results of the quantitative detection of APRF gene in the experimental and control cells.
  • RNAi interference fragment targeting human APRF gene was designed according to the crRNA design rules of Cas13a, and BamHI and AflII restriction sites were added to its 5 'and 3' ends, respectively, and its sequence is shown in SEQ ID NO.1.
  • pRNAT-LwCas13a-Neo plasmid (plasmid map shown in Figure 1) and the novel RNAi interfering fragment were digested with BamHI and AflII endonucleases respectively, and the target fragment was recovered by agarose gel electrophoresis and ligated with T4 DNA ligase Transformation of competent E. coli Stbl3 and sequencing.
  • the correct sequence is the new RNAi interference vector targeting the human APRF gene, named pRNAT-LwCas13a-APRF.
  • the correct strain was sequenced and identified in Example 1, and placed in an LB liquid medium having an ampicillin concentration of 100 ⁇ g / ml, and cultured with shaking at 250 rpm and 37 ° C. for 12-16 h.
  • the bacterial solution was collected by centrifugation at 4 ° C and 10,000 rpm, the supernatant was discarded, the bacterial cells were collected, and then the plasmid was extracted according to the instructions of the Endo-Free Plasmid Mini Kit kit to obtain the endotoxin-free pRNAT-LwCas13a-APRF vector.
  • Untreated HepG2 cells were used as the control group, and the cells selected in Example 3 were used as the experimental group. After total RNA was extracted and reverse transcription was performed, the expression level of APRF gene was detected by fluorescent quantitative PCR. . It can be seen that the expression level of APRF gene in the cells of the experimental group is significantly lower than that in the control group, indicating that the novel RNAi interference sequences and vectors targeting the human APRF gene can achieve RNA interference to the APRF gene.
  • the novel RNAi interference fragment and RNAi vector provided by the present invention can efficiently and specifically knock down the expression of the human APRF gene, and the application cost is low, which can promote the research of the function of the human APRF gene.

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Abstract

本发明提供一种靶向人APRF基因的新型RNAi干扰片段、RNAi载体及其制备方法和其应用，涉及生物技术领域。其中，一种靶向人APRF基因的新型RNAi干扰片段，其特征在于，所述新型RNAi干扰片段序列为SEQ ID NO.1所示。本发明提供的新型RNAi干扰片段可引导LwCas13a对人APRF基因转录形成的mRNA进行精准的识别与切割，实现对APRF基因的高效和特异性表达干扰。

Description

靶向人APRF基因的新型RNAi干扰片段、RNAi载体及其制备方法和应用 技术领域
本发明涉及生物技术领域，具体涉及一种靶向人APRF基因的新型RNAi干扰片段、RNAi载体及其制备方法和其应用。
 背景技术
信号转导子和转录激活子（STAT）是细胞内重要的DNA结合蛋白，这些蛋白具有双重作用，是细胞质信号转导子和细胞核的转录因子。APRF是STAT家族中重要一员，与细胞的增殖、凋亡和分化密切相关。在正常条件下，细胞外信号激活APRF是被严格调控的，APRF被激活的过程短暂而迅速，可以维持正常细胞的生物学功能。APRF的缺失会导致早期胚胎细胞的死亡。
 技术问题
在许多肿瘤中，例如白血病、乳腺癌、头颈部肿瘤、恶性黑色素瘤、前列腺癌和胰腺癌，APRF处于持续激活的状态。研究表明APRF持续活化有助于肿瘤的形成。APRF持续性激活，可以通过介导肿瘤免疫逃逸，促进肿瘤新生血管的形成，抑制肿瘤细胞凋亡，促进肿瘤细胞增殖等机制，导致肿瘤细胞的恶性转化。因此APRF是一个理想的潜在肿瘤治疗靶点，但现有技术中虽然已有通过传统RNAi技术敲低APRF基因对其功能进行研究的报道，但由于这一技术特异性较差，潜在的脱靶风险较大，对相关研究的进展造成了一定的阻碍。
 技术解决方案
2016首次报道了一种RNA靶向的CRISPR酶——Cas13a。与CRISPR/Cas9切割DNA的活性不同，CRISPR/Cas13a能够用于切割细菌细胞中特定的RNA序列。研究表明，来自Leptotrichia wadei的LwCas13a能够以比现有RNAi工具更强的特异性在目标RNA上切割特定的位点。
有鉴于此，本发明提供一种靶向人APRF基因的新型RNAi干扰片段、RNAi载体及其制备方法和其应用，主要目的是提升针对APRF基因的RNAi特异性，减少或消除脱靶效应，促进APRF基因功能的研究。
为达到上述目的，本发明主要提供如下技术方案：
一种新型RNAi干扰片段，用于干扰人APRF基因，其序列如SEQ ID NO.1所示。
另一方面，一种新型RNAi干扰载体，用于干扰人APRF基因，所述新型RNAi干扰载体克隆携带新型RNAi干扰片段，所述新型RNAi干扰片段的序列如SEQ ID NO.1所示。
进一步地，所述新型RNAi干扰载体包括pRNAT-LwCas13a-Neo载体，所述pRNAT-LwCas13a-Neo载体与所述新型RNAi干扰片段连接。
一种新型RNAi干扰载体的制备方法，包括如下步骤 ：
a. 酶切pRNAT-LwCas13a-Neo载体，所述酶切位点为BamHI和AflII；
b. 将所述新型RNAi干扰片段与酶切后的所述pRNAT-LwCas13a-Neo 载体连接，得到连接产物；其中，所述新型RNAi干扰片段序列如SEQ ID NO.1所示；
c. 将所述连接产物转化至感受态大肠杆菌Stbl3中，筛选并将得到的菌液进行测序鉴定，并将测序结果与所述新型RNAi干扰片段序列完全一致的菌液进行扩增；
d. 从所述扩增的菌液中提取新型RNAi干扰载体。
 有益效果
本发明提供的一种靶向人APRF基因的新型RNAi干扰片段、RNAi载体可高效、特异地敲低人APRF基因的表达，而且应用成本低廉，可很好地推动人APRF基因功能的研究。
 附图说明
图1为pRNAT-LwCas13a-Neo质粒的图谱；
图2为实验组和对照组细胞的APRF基因荧光定量PCR检测结果。
 本发明的实施方式
实施例一
根据Cas13a的crRNA设计规则设计靶向人APRF基因的新型RNAi干扰片段，并在其5’端和3’端分别加上BamHI和AflII酶切位点，其序列如SEQ ID NO.1所示。
用BamHI和AflII内切酶分别对pRNAT-LwCas13a-Neo质粒（质粒图谱如图1所示）和所述新型RNAi干扰片段进行酶切，琼脂糖凝胶电泳回收目的片段并用T4 DNA连接酶进行连接、转化感受态大肠杆菌Stbl3及测序。测序正确的即为所述靶向人APRF基因的新型RNAi干扰载体，命名为pRNAT-LwCas13a-APRF。
实施例二
取实施例一中测序鉴定正确的菌株，置于氨苄青霉素浓度为100 μg/ml的LB液体培养基中，250 rpm、37℃振荡培养12-16 h。4℃，10000 rpm离心收集菌液，弃上清，收集菌体，然后按照Endo-Free Plasmid Mini Kit试剂盒说明书操作步骤提取质粒，得无内毒素的pRNAT-LwCas13a-APRF载体。
实施例三
培养HepG2细胞，待HepG2细胞的融合率达到50％～60％，接种后12～18h为最佳转染时间；转染前更换新鲜培养液，60 mm培养皿中加入3 ml培养基；转染时按照Lipofectamine 2000试剂盒说明书导入4μg的pRNAT-LwCas13a-APRF质粒，转染后48 h，加入800 μg/mL G418筛选10 d。筛选完成后，将嘌呤霉素的浓度降为200 μg/ml继续扩大培养细胞。。
实施例四
以未经任何处理的HepG2细胞作为对照组，实施例三中筛选出的细胞为实验组，提取总RNA并进行逆转录后，荧光定量PCR检测APRF基因的表达水平，其结果如图2所示。可以看到，实验组细胞的APRF基因表达水平显著低于对照组细胞，说明所述靶向人APRF基因的新型RNAi干扰序列及载体可以实现对APRF基因的RNA干扰。
 工业实用性
本发明提供的一种靶向人APRF基因的新型RNAi干扰片段、RNAi载体可高效、特异地敲低人APRF基因的表达，而且应用成本低廉，可很好地推动人APRF基因功能的研究。

Claims (4)

  1. 一种新型RNAi干扰片段，用于干扰人APRF基因，其特征在于，所述RNAi干扰片段序列如SEQ ID NO.1所示。
  2. 一种新型RNAi干扰载体，用于干扰人APRF基因，其特征在于，所述新型RNAi干扰载体克隆携带新型RNAi干扰片段，所述新型RNAi干扰片段的序列如SEQ ID NO.1所示。
  3. 根据权利要求2所述的新型RNAi干扰载体，其特征在于，所述新型RNAi干扰载体包括pRNAT-LwCas13a-Neo载体，所述pRNAT-LwCas13a-Neo载体与所述新型RNAi干扰片段连接。
  4. 一种新型RNAi干扰载体的制备方法，其特征在于，包括如下步骤 ：
    a. 酶切pRNAT-LwCas13a-Neo载体，所述酶切位点为BamHI和AflII；
    b. 将所述新型RNAi干扰片段与酶切后的所述pRNAT-LwCas13a-Neo 载体连接，得到连接产物；其中，所述新型RNAi干扰片段序列如SEQ ID NO.1所示；
    c. 将所述连接产物转化至感受态大肠杆菌Stbl3中，筛选并将得到的菌液进行测序鉴定，并将测序结果与所述新型RNAi干扰片段序列完全一致的菌液进行扩增；
    d. 从所述扩增的菌液中提取新型RNAi干扰载体。
PCT/CN2018/093865 2018-06-29 2018-06-29 靶向人APRF基因的新型RNAi干扰片段、RNAi载体及其制备方法和应用 WO2020000455A1 (zh)

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Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ABUDAYYEH, O. O. ET AL.: "RNA Targeting with CRISPR-Cas13", NATURE, vol. 550, 12 October 2017 (2017-10-12), pages 280 - 284, XP055529736, ISSN: 0028-0836, DOI: 10.1038/nature24049 *
DATABASE NCBI 26 March 2018 (2018-03-26), Database accession no. XM_011525145.3 *

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