WO2020000452A1 - 一种靶向人NGL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

一种靶向人NGL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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WO2020000452A1
WO2020000452A1 PCT/CN2018/093862 CN2018093862W WO2020000452A1 WO 2020000452 A1 WO2020000452 A1 WO 2020000452A1 CN 2018093862 W CN2018093862 W CN 2018093862W WO 2020000452 A1 WO2020000452 A1 WO 2020000452A1
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rnai interference
vector
novel
gene
ngl
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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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    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • 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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells

Definitions

  • the present invention relates to the field of biotechnology, and in particular to a novel RNAi interference fragment targeting a human NGL gene, an RNAi vector, a preparation method and an application thereof.
  • NGL is a member of the epidermal growth factor receptor (EGFR) family of proto-oncogenes, and is involved in the regulation of growth and development of normal tissues. It can promote cell division and secretion of proteolytic enzymes, and enhance cell mobility. In this state, the expression of NGL increases, which in turn promotes tumor invasion and metastasis.
  • EGFR epidermal growth factor receptor
  • NGL is involved in the regulation of growth and development of normal tissues, can promote cell division and secretion of proteolytic enzymes, and enhance the ability of cells to move, but under pathological conditions, the expression of NGL increases, thereby promoting tumor invasion and metastasis.
  • NGL has intracellular tyrosine kinase-like activity, plays a role in embryonic development and differentiation, is widely expressed in human epithelial cells, and is closely related to the survival and evolution of a variety of tumors. Therefore, the research on NGL can greatly promote the development of the field of tumor prevention and treatment.
  • RNAi technology due to the poor specificity of this technology, the potential The risk of off-target is great, 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 NGL gene, an RNAi vector, a preparation method and application thereof.
  • the main purpose is to improve the specificity of RNAi against NGL gene, reduce or eliminate off-target effects, and promote NGL Study of gene function.
  • the present invention mainly provides the following technical solutions:
  • RNAi interference fragment for interfering with human NGL gene the sequence of which is as SEQ ID NO.1.
  • RNAi interference vector is used to interfere with the human NGL gene.
  • the clone of the novel RNAi interference vector 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:
  • the novel RNAi interference fragment and RNAi vector provided by the present invention can efficiently and specifically knock down the expression of the human NGL gene, and the application cost is low, which can promote the research of the function of the human NGL gene.
  • Figure 1 is a map of pRNAT-LwCas13a-Neo plasmid
  • Figure 2 shows the results of NGL gene fluorescence quantitative PCR detection in experimental and control cells.
  • RNAi interference fragment targeting human NGL gene was designed, and the 5 'and 3' ends were added with BamHI and AflII restriction sites, respectively, and the 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 NGL gene, which is named pRNAT-LwCas13a-NGL.
  • 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-NGL 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 NGL gene, and the application cost is low, which can promote the research of the function of the human NGL gene.

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Abstract

一种靶向人NGL基因的RNAi干扰片段、包含该RNAi的载体及其制备方法和其应用。该RNAi干扰片段如SEQ ID NO.1所示，可引导LwCas13a对人NGL基因转录形成的mRNA进行精准的识别与切割，实现对NGL基因的高效和特异性表达干扰。

Description

一种靶向人NGL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NGL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
NGL是原癌基因为表皮生长因子受体(EGFR)家族成员之一，参与了正常组织的生长与发育调节，可以促进细胞分裂和蛋白水解酶的分泌，并增强细胞的运动能力，但在病理状态下，NGL表达增多，进而促进肿瘤的侵袭和转移。
 技术问题
NGL参与了正常组织的生长与发育调节，可以促进细胞分裂和蛋白水解酶的分泌，并增强细胞的运动能力，但在病理状态下，NGL表达增多，进而促进肿瘤的侵袭和转移。NGL具有细胞内酪氨酸激酶样活性，在胚胎发育形成及分化中有一定的作用，在人类组织的上皮细胞中有广泛表达，并且与多种肿瘤的生存和演进有密切的关系。因此对NGL的研究可以大大促进肿瘤防治领域的发展，但现有技术中虽然已有通过传统RNAi技术敲低NGL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NGL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NGL基因的RNAi特异性，减少或消除脱靶效应，促进NGL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NGL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NGL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NGL基因的新型RNAi干扰片段、RNAi载体可高效、特异地敲低人NGL基因的表达，而且应用成本低廉，可很好地推动人NGL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NGL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NGL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NGL基因的新型RNAi干扰载体，命名为pRNAT-LwCas13a-NGL。
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-NGL载体。
实施例三
培养Raji细胞，待Raji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-NGL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Raji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NGL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NGL基因表达水平显著低于对照组细胞，说明所述靶向人NGL基因的新型RNAi干扰序列及载体可以实现对NGL基因的RNA干扰。
 工业实用性
本发明提供的一种靶向人NGL基因的新型RNAi干扰片段、RNAi载体可高效、特异地敲低人NGL基因的表达，而且应用成本低廉，可很好地推动人NGL基因功能的研究。

Claims (4)

  1. 一种新型RNAi干扰片段，用于干扰人NGL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NGL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/CN2018/093862 2018-06-29 2018-06-29 一种靶向人NGL基因的新型RNAi干扰片段、RNAi载体及其制备方法和其应用 WO2020000452A1 (zh)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104232573A (zh) * 2014-09-11 2014-12-24 安沂华 一种生长培养基及其用途和培养脐带间充质干细胞的方法
CN104531760A (zh) * 2014-12-26 2015-04-22 中南大学 Dp71蛋白的短发夹RNA干扰质粒及其应用方法
CN107446045A (zh) * 2016-07-22 2017-12-08 北京天广实生物技术股份有限公司 一种抗her2的抗体、其药物组合物及用途
CN107557455A (zh) * 2017-09-15 2018-01-09 国家纳米科学中心 一种基于CRISPR‑Cas13a的特异性核酸片段的检测方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104232573A (zh) * 2014-09-11 2014-12-24 安沂华 一种生长培养基及其用途和培养脐带间充质干细胞的方法
CN104531760A (zh) * 2014-12-26 2015-04-22 中南大学 Dp71蛋白的短发夹RNA干扰质粒及其应用方法
CN107446045A (zh) * 2016-07-22 2017-12-08 北京天广实生物技术股份有限公司 一种抗her2的抗体、其药物组合物及用途
CN107557455A (zh) * 2017-09-15 2018-01-09 国家纳米科学中心 一种基于CRISPR‑Cas13a的特异性核酸片段的检测方法

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