WO2020000450A1 - 靶向人CD272基因的新型RNAi干扰片段、RNAi载体及其制备方法和其应用 - Google Patents

靶向人CD272基因的新型RNAi干扰片段、RNAi载体及其制备方法和其应用 Download PDF

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WO2020000450A1
WO2020000450A1 PCT/CN2018/093860 CN2018093860W WO2020000450A1 WO 2020000450 A1 WO2020000450 A1 WO 2020000450A1 CN 2018093860 W CN2018093860 W CN 2018093860W WO 2020000450 A1 WO2020000450 A1 WO 2020000450A1
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rnai interference
gene
vector
novel
fragment
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PCT/CN2018/093860
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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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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 CD272 gene, an RNAi vector, a preparation method and an application thereof.
  • CD272 is the third inhibitory molecule found in the CD28 family. It is mainly expressed on the surface of B cells, T cells, and macrophages. It has a similar structure to CTLA-4 and PD-1, but their expression characteristics are different. CTLA-4 and PD-1 are not expressed on resting T cells, and the expression gradually increases after activation, while CD272 is constitutively expressed on resting T cells, and continues to be expressed after activation. CD272 signal can inhibit the proliferation of CD3 activated T cells and the secretion of IL-2 and IFN- ⁇ .
  • CD272 plays an important role in maintaining peripheral immune tolerance and transplantation immunity, and has good prospects for clinical transformation. Further research is needed, but in the prior art, the function of CD272 has been knocked down by traditional RNAi technology. Research reports, but due to the poor specificity of this technology, the potential off-target risk is greater, which has caused some 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 targeted to the human CD272 gene, an RNAi vector, a preparation method and application thereof.
  • the main purpose is to improve the RNAi specificity of the CD272 gene, reduce or eliminate off-target effects and promote CD272 Study of gene function.
  • the present invention mainly provides the following technical solutions:
  • RNAi interference fragment used to interfere with human CD272 gene, the sequence of which is as SEQ ID NO.1.
  • RNAi interference vector is used to interfere with the human CD272 gene.
  • the novel RNAi interference vector clone carries a novel RNAi interference fragment, and the sequence of the novel RNAi interference fragment is as 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 CD272 gene, and the application cost is low, which can well promote the research of the function of the human CD272 gene.
  • Figure 1 is a map of pRNAT-LwCas13a-Neo plasmid
  • Figure 2 shows the results of CD272 gene fluorescence quantitative PCR detection in experimental and control cells.
  • RNAi interference fragment targeting the human CD272 gene was designed according to the crRNA design rule 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 CD272 gene, which is named pRNAT-LwCas13a-CD272.
  • 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. Collect the bacterial solution by centrifugation at 10,000 rpm at 4 ° C, discard the supernatant, collect the bacterial cells, and then extract the plasmid according to the instructions of the Endo-Free Plasmid Mini Kit kit to obtain the endotoxin-free pRNAT-LwCas13a-CD272 vector.
  • the novel RNAi interference fragment and RNAi vector provided by the present invention can efficiently and specifically knock down the expression of the human CD272 gene, and the application cost is low, which can well promote the research of the function of the human CD272 gene.

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Abstract

本发明提供一种靶向人CD272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CD272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CD272基因转录形成的mRNA进行精准的识别与切割，实现对CD272基因的高效和特异性表达干扰。

Description

靶向人CD272基因的新型RNAi干扰片段、RNAi载体及其制备方法和其应用 技术领域
本发明涉及生物技术领域，具体涉及一种靶向人CD272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
CD272是CD28家族发现的第3个抑制性分子，主要表达在B细胞、T细胞、巨噬细胞等细胞表面，具有和CTLA-4、PD-1相似的结构，但它们的表达特点有所不同，CTLA-4和PD-1在静止的T细胞上不表达，活化后表达逐步升高，而CD272在静止T细胞上组成性表达，活化后继续表达。CD272信号能够抑制CD3活化的T细胞增殖和IL-2、IFN-γ的分泌。
 技术问题
研究表明，CD272在维持外周免疫耐受和移植免疫中具有重要作用，具有很好的临床转化前景，需进一步研究，但现有技术中虽然已有通过传统RNAi技术敲低CD272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CD272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CD272基因的RNAi特异性，减少或消除脱靶效应，促进CD272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CD272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CD272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CD272基因的新型RNAi干扰片段、RNAi载体可高效、特异地敲低人CD272基因的表达，而且应用成本低廉，可很好地推动人CD272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CD272基因荧光定量PCR检测结果。
 本发明的实施方式
为更进一步阐述本发明为达成预定发明目的所采用的技术手段及功效，结合较佳实施例详细说明如下。
实施例一
根据Cas13a的crRNA设计规则设计靶向人CD272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CD272基因的新型RNAi干扰载体，命名为pRNAT-LwCas13a-CD272。
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-CD272载体。
实施例三
培养Jurkat细胞，待Jurkat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-CD272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100 μg/ml继续扩大培养细胞。。
实施例四
以未经任何处理的Jurkat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CD272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CD272基因表达水平显著低于对照组细胞，说明所述靶向人CD272基因的新型RNAi干扰序列及载体可以实现对CD272基因的RNA干扰。
 工业实用性
本发明提供的一种靶向人CD272基因的新型RNAi干扰片段、RNAi载体可高效、特异地敲低人CD272基因的表达，而且应用成本低廉，可很好地推动人CD272基因功能的研究。

Claims (4)

  1. 一种新型RNAi干扰片段，用于干扰人CD272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CD272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/CN2018/093860 2018-06-29 2018-06-29 靶向人CD272基因的新型RNAi干扰片段、RNAi载体及其制备方法和其应用 WO2020000450A1 (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-Casl3", NATURE, vol. 550, 12 October 2017 (2017-10-12), pages 280 - 284, XP055529736, DOI: 10.1038/nature24049 *
DATABASE NCBI 20 April 2007 (2007-04-20), STSUKI, N. ET AL., Database accession no. NM_181780.3 *

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