CN105925578A - 靶向沉默Neogenin的shRNA - Google Patents

靶向沉默Neogenin的shRNA Download PDF

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CN105925578A
CN105925578A CN201610367273.9A CN201610367273A CN105925578A CN 105925578 A CN105925578 A CN 105925578A CN 201610367273 A CN201610367273 A CN 201610367273A CN 105925578 A CN105925578 A CN 105925578A
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neogenin
silence
shrna
targeted
sequence
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俞华莉
陈晶滢
马驰
朱筱娟
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Northeastern University China
Northeast Normal University
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Northeast Normal University
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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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid

Abstract

本发明属于生物工程中的DNA重组技术领域，具体涉及特异性靶向沉默Neogenin的shRNA。首先是根据人源的neogenin的CDS区序列设计出19bp的目的片段，合成的目的片段两端带有合适的酶切位点，将目的片段连接到PLL3.7载体上，连接转化，挑取阳性克隆鉴定。鉴定正确后，将提取的质粒送去进行测序，测序比对正确之后转染细胞并进行neogenin沉默效率的检测。本发明设计合成了能够靶向人源neogenin的19bp的靶向沉默Neogenin的shRNA，序列为GCATCACCTTTATGGAGTGTA。

Description

靶向沉默Neogenin的shRNA
技术领域
本发明属于生物工程中的DNA重组技术领域，具体涉及特异性靶向沉默Neogenin的shRNA。
背景技术
Neogenin最早是从鸡胚中分离出来的一个蛋白，是DCC的同系物，属于免疫球蛋白超家族，结构与DCC及其相似，膜外是由4个免疫球蛋白和6个III型纤维连接蛋白功能区组成，一个跨膜区域和胞内段结构域组成。
Neogenin作为依赖性受体家族的一个成员，能够与其配体结合介导多种生理功能，包括轴突导向、血管生成、细胞迁移、信号转导以及细胞凋亡功能，它的结构与DCC高度相似，它作为DCC的同系物在神经系统中广泛表达，所以最初的研究都集中在其在神经系统中的功能。近些年来，依赖性受体的功能在肿瘤中被广泛阐述，Neogenin作为其中的一员，研究表明其对体内多种肿瘤的发生发展都有促进作用，但是目前神经系统的肿瘤中研究表明由于其启动子区域的甲基化作用，Neogenin在胶质瘤中表达下调，但是其机制有待更进一步的阐明。以上表明在肿瘤中减少Neogenin的表达能加速肿瘤的进程，所以构建能特异性沉默Neogenin的载体对于了解它的功能至关重要。
发明内容
本发明的目的是合成靶向沉默Neogenin的shRNA，，为更加深入的研究neogenin以及neogenin在选择、制备治疗肿瘤药物中的作用提供了有效的手段。
本发明中所需要构建的主要是neogenin的沉默表达载体。核心沉默表达载体构建分为两个大的步骤，首先是根据人源的neogenin的CDS区序列设计出19bp的目的片段，合成的目的片段两端带有合适的酶切位点，将目的片段连接到PLL3.7载体上，连接转化，挑取阳性克隆鉴定。鉴定正确后，将提取的质粒送去进行测序，测序比对正确之后转染细胞并进行neogenin沉默效率的检测。
本发明中设计合成了能够靶向人源neogenin的19bp的靶向沉默Neogenin的shRNA，序列为GCATCACCTTTATGGAGTGTA
能够特异性沉默neogenin的表达载体的构建包括以下步骤：
第一步，序列设计
根据人源neogenin基因序列特征设计特异性识别UNC5A的沉默序列。
第二步，对靶序列进行退火成双链以及对PLL3.7载体进行酶切
将核心载体PLL3.7进行双酶切后, 利用胶回收试剂盒回收线性化载体，为下一步的实验准备。
将合成的靶序列利用退火buffer溶解之后，利用PCR仪进行退火，95℃/4min，72℃/10min，之后降温到4℃，获得的样品拿去测浓度之后放在-20℃备用。
第三步，将合成的靶序列以及核心载体进行连接
将上一步中的两个产物在T4连接酶的作用下进行连接，经转化、提取等步骤获得沉默载体。
第四步，沉默靶蛋白效率的检测
经转染细胞后提取蛋白，利用 western blot检测沉默效率。
附图说明：
图1为neogeninshRNA菌液PCR结果凝胶电泳图，框中为阳性结果；
图2为neogeninshRNA酶切验证凝胶电泳图，框中为阳性结果；
图3为检测neogeninshRNA沉默效率图。
本发明中设计合成的19bp的目的片段，能够特异性的沉默neogenin，同时也为构建neogenin-shRNA提供关键的序列片段。Neogenin属于依赖性受体，该家族成员在神经发生以及肿瘤中都有很重要的作用。经研究表明，neogenin在肿瘤的进程中发挥一定的作用，构建能够特异性沉默neogenin的载体，能也异性的降低neogenin蛋白的表达，这能为研究neogenin在肿瘤中的作用提供一个好的手段，所以neogenin沉默载体的应用能为更好的研究其在肿瘤中的作用机制以及为提供制备抗肿瘤药物提供理论基础。
具体实施方式
实施例一: 特异性沉默靶标蛋白neogenin的表达载体构建
1、特异性沉默靶蛋白neogenin的序列设计
（1）根据人源neogenin基因序列在Invitrogen公司主页上用相应的软件设计，设计的原则：19bp的特异性结合neogenin的序列的GC含量为45%-55%，退火温度45℃-65℃，同时按照载体的要求，第一个碱基必须为G。将选取的片段进行BLAST人基因组比对，选择特异性识别人源Neogenin的序列。
（2）HpaⅠ- GN18-TT-loop- 81NC-XhoⅠ形式合成一段59bp序列，81NC为NG18的反向互补，5’端为HpaⅠ酶切位点，3’端为XhoⅠ酶切位点。设计合成的特异性识别UNC5A的序列片段为 GCATCACCTTTATGGAGTGTA。
2、特异性沉默靶蛋白neogenin的序列的合成与储存
（1）将合成的两段Oligo利用退火buffer溶解至50 µM，各取100µl混合。
（2）各取10µl上述储存液，放在PCR管中，混匀放入PCR仪中，95 ℃变性4min，72 ℃退火10min，之后可以放在4℃保存。
3、neogenin沉默载体的构建
（1）取1µg PLL3.7载体，利用HpaⅠ和XhoⅠ进行双酶切，电泳，回收线性PLL3.7大片段。
（2）连接和转化。将线性PLL3.7浓度稀释至100ng/µl，将退火形成的DNA片段稀释成合适的浓度，进行连接反应（Promega公司的T4 DNA 连接酶）
16 ℃过夜连接，将连接产物转化Stb13感受态细胞，涂布在具有氨苄青霉素的LB平板上。
（3）阳性克隆鉴定。在转化平板上挑取单克隆摇菌，试剂盒提取质粒，通过PCR和双酶切鉴定阳性克隆【图1】【图2】。鉴定引物为pLL3.7 test-F：5’-GCACAGACTTGTGGGAGAAG-3’，pLL3.7 test-R:5’CCTGCTGGAATCTCGTGAA-3’。以U6启动子为测序引物，送北京金唯智进行测序。
实施例二:
沉默靶蛋白效率的检测
（1）将ScrabmbleshRNA和neogeninshRNA共同转染进入胶质瘤细胞
（2）提取细胞总蛋白，利用Western blot免疫印迹检测沉默效率，结果显示，转入neogenin沉默载体后，neogenin表达量显著降低，GAPDH为内参【图3】。
序列表
<110>东北师范大学
<120>特异性沉默靶标蛋白neogenin的表达载体构建及沉默效率的检测
<141> 2016-3-30
<160>19
<210> 1
<211> 19
<212> DNA
<213>人工序列
<220>
<221>misc_RNA
<222> (1)...(19)
<223>
<400> 1
GCATCACCTTTATGGAGTGTA。

Claims (1)

1.靶向沉默Neogenin的shRNA，其特征是序列为GCATCACCTTTATGGAGTGTA。
CN201610367273.9A 2016-05-30 2016-05-30 靶向沉默Neogenin的shRNA Pending CN105925578A (zh)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11008388B2 (en) 2015-04-28 2021-05-18 Mitsubishi Tanabe Pharma Corporation RGMa binding protein and use thereof

Citations (2)

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Publication number Priority date Publication date Assignee Title
WO2007099133A1 (en) * 2006-02-28 2007-09-07 Centre National De La Recherche Scientifique (Cnrs) Screening for anti-cancer compounds using netrin-1 activity
WO2012065143A1 (en) * 2010-11-12 2012-05-18 The General Hospital Corporation Polycomb-associated non-coding rnas

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007099133A1 (en) * 2006-02-28 2007-09-07 Centre National De La Recherche Scientifique (Cnrs) Screening for anti-cancer compounds using netrin-1 activity
WO2012065143A1 (en) * 2010-11-12 2012-05-18 The General Hospital Corporation Polycomb-associated non-coding rnas

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
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CAROLINE A. ENNS ET AL.: "Neogenin interacts with matriptase 2 to facilitate hemojuvelin cleavage", 《THE JOURNAL OF BIOLOGICAL CHEMISTRY》 *
JAE HO LEE等: "Neogenin as a Receptor for Early Cell Fate Determination in Preimplantation Mouse Embryos", 《PLOS ONE》 *
未知: "PREDICTED:HOMO sapiens neogenin 1（NEO1），transcript variant X7，mRNA", 《GENBANK》 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11008388B2 (en) 2015-04-28 2021-05-18 Mitsubishi Tanabe Pharma Corporation RGMa binding protein and use thereof

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Application publication date: 20160907