WO2020000469A1 - 促进 at2r 蛋白过表达的重组载体及其构建方法 - Google Patents

促进 at2r 蛋白过表达的重组载体及其构建方法 Download PDF

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WO2020000469A1
WO2020000469A1 PCT/CN2018/093879 CN2018093879W WO2020000469A1 WO 2020000469 A1 WO2020000469 A1 WO 2020000469A1 CN 2018093879 W CN2018093879 W CN 2018093879W WO 2020000469 A1 WO2020000469 A1 WO 2020000469A1
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at2r
vector
overexpression
recombinant vector
pegfp
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PCT/CN2018/093879
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English (en)
French (fr)
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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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Publication of WO2020000469A1 publication Critical patent/WO2020000469A1/zh

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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
    • C12N15/63—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
    • 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

Definitions

  • the invention belongs to the field of biotechnology, and particularly relates to a recombinant vector for promoting AT2R protein overexpression and a construction method thereof.
  • liver cancer is a clinically common malignant tumor. It has an insidious onset, rapid progress, and high mortality. It is known as the "King of Cancer". China is a country with a high incidence of liver cancer, accounting for 55% of the global incidence. It kills about 110,000 people each year, accounting for 45% of the world ’s liver cancer deaths. Its death rate is second only to lung cancer, ranking second, and a serious threat. The people's lives are healthy.
  • the recombinant vector for promoting AT2R protein overexpression provided by the present invention has the advantages of high transfection efficiency, promoting AT2R protein overexpression, and continuous and stable expression, and the construction method is simple.

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Abstract

一种促进AT2R蛋白过表达的重组载体，属于生物技术领域，包括AT2R编码序列和pEGFP-C1载体；所述pEGFP-C1载体包括XhoI和EcoRI酶切位点，所述AT2R序列正向插入所述酶切位点。所述促进AT2R蛋白过表达的重组载体具有转染效率高，促进AT2R蛋白过表达且表达持续稳定的优点，构建方法简单。

Description

促进AT2R蛋白过表达的重组载体及其构建方法 技术领域
本发明属于生物技术领域，尤其涉及一种促进AT2R蛋白过表达的重组载体及其构建方法。
 背景技术
原发性肝癌是临床上常见的恶性肿瘤，其发病隐匿，进展快，死亡率高，被称为“癌中之王”。我国是肝癌高发国家，发病人数占全球发病人数的55%，每年死于肝癌约11万人，占全世界肝癌死亡人数的45%，其死亡率仅次于肺癌，居第2位，严重威胁着人民生命健康。
肝癌的发病机制很复杂，目前仍不明确，总体来说，肝癌组织中细胞增生和调亡的异常失衡对于肝癌的发生发展是关键环节。凋亡抑制因子可加速肿瘤的增长，而凋亡诱导因子能诱导肝癌细胞凋亡而抵消肿瘤细胞生长。当这些因子之间失衡时，细胞凋亡与增殖之间的平衡发生障碍，肝癌由此发生并发展。
 技术问题
A1TR和A2TR分别是血管紧张素II（AngII）作用的靶细胞表面特异性的G蛋白偶联1型和2型受体。AngII是通过上述两种受体参与调节血管舒缩、水盐平衡、细胞增殖、炎性反应、细胞凋亡等生物学功能。A1TR和A2TR都是蛋白偶联受体，具有七次跨膜结构域，但两者只有的34%同源性。现有研究表明，AT2R在肝癌的发展过程中发挥了一定的作用，但其作用机制仍不明确，现有技术中也缺乏促进AT2R蛋白过表达的重组载体。
 技术解决方案
为解决现有技术中存在的问题，本发明提供了一种促进AT2R蛋白过表达的重组载体，包括AT2R编码序列和pEGFP-C1载体；所述pEGFP-C1载体包括XhoI和EcoRI酶切位点，所述AT2R序列正向插入所述酶切位点。
采用上述技术方案，获得的重组载体具有转染效率高，促进AT2R蛋白过表达且表达持续稳定的优点。
优选地，所述AT2R编码序列包含GenBank基因编号为NM_000686.4 的序列，且其5’端含有XhoI酶切位点，3’端含有EcoRI酶切位点。
 有益效果
本发明提供的促进AT2R蛋白过表达的重组载体具有转染效率高，促进AT2R蛋白过表达且表达持续稳定的优点，构建方法简单。
 附图说明
图1为pEGFP-C1载体图谱；
图2为对照组和实验组HepG2细胞的AT2R表达水平。
 本发明的实施方式
下面结合附图与具体实施例对本发明做进一步的说明。
实施例一 ： AT2R 蛋白过表达的重组载体的构建
委托上海生工采用基因合成的方式直接合成所述AT2R编码序列。使用XhoI和EcoRI酶分别对含有合成序列的质粒和pEGFP-C1载体进行双酶切，然后进行回收纯化。回收后的合成序列与pEGFP-C1载体按1:10混匀后，T4 DNA连接酶进行连接。
连接产物转化感受态大肠杆菌JM107，扩大培养后测序，筛选出测序结果与预期完全相符的菌。再扩大培养，并应用无内毒素质粒提取试剂盒提取大肠杆菌中的重组质粒，命名为pEGFP-AT2R。
实施例二： pEGFP-AT2R 转染 HepG2 细胞
接种HepG2细胞于六孔板中，每孔1000000个细胞，12h后细胞密度约为50%，，用Lipofectamine 2000将pEGFP-AT2R质粒分别转染至HepG2细胞，每孔1 μg，并对细胞进行继续培养。
实施例三：荧光定量 PCR 检测 AT2R 基因 表达水平
分别接种正常HepG2细胞（对照组）、实施例二中获得经转染的HepG2细胞（实验组）至六孔板。细胞密度达到80%-90%时，提取各组细胞的总RNA，并将mRNA逆转录为cDNA，-20℃保存。
取各组细胞的cDNA 1 μL为模板，以β-actin为内参，实时荧光定量PCR检测AT2R基因的相对表达水平，设置反应条件：95℃ 30s，1循环；60℃ 30s 40循环；95℃ 5s，60℃ 1min，95℃ 15s。结果如图1所示，可以看到，实验组细胞的AT2R基因表达水平较对照组细胞有92.4倍的升高，说明本发明提供的促进AT2R蛋白过表达的重组载体能特异、高效地促进AT2R基因过表达。
 工业实用性
本发明提供的促进AT2R蛋白过表达的重组载体具有转染效率高，促进AT2R蛋白过表达且表达持续稳定的优点，构建方法简单。

Claims (3)

  1. 一种促进AT2R蛋白过表达的重组载体，其特征在于，包括AT2R编码序列和pEGFP-C1载体；所述pEGFP-C1载体包括XhoI和EcoRI酶切位点，所述AT2R序列正向插入所述酶切位点。
  2. 根据权利要求1所述的促进AT2R蛋白过表达的重组载体，其特征在于：所述AT2R编码序列包含GenBank基因编号为NM_000686.4 的序列，且其5’端含有XhoI酶切位点，3’端含有EcoRI酶切位点。
  3. 根据权利要求1所述的促进AT2R蛋白过表达的重组载体的构建方法，其特征在于，该载体制备过程包含如下步骤：
    S1、将所述AT2R编码序列和pEGFP-C1载体分别用XhoI和EcoRI酶进行双酶切，琼脂糖凝胶电泳后进行胶回收；
    S2、将所述AT2R编码序列与所述pEGFP-C1载体在T4 DNA连接酶的作用下连接，转化大肠杆菌挑单克隆提取质粒，测序验证正确，得到促进AT2R蛋白过表达的重组载体。
PCT/CN2018/093879 2018-06-29 2018-06-29 促进 at2r 蛋白过表达的重组载体及其构建方法 WO2020000469A1 (zh)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102943063A (zh) * 2012-09-26 2013-02-27 浙江大学 提高骨髓单个核细胞心肌细胞保护及迁移能力的处理方法
WO2013091883A2 (en) * 2011-12-23 2013-06-27 Medical Research Council Selective gpcr ligands
WO2016081733A2 (en) * 2014-11-19 2016-05-26 Novopyxis Inc. Compositions and methods for modulating at2r activity

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013091883A2 (en) * 2011-12-23 2013-06-27 Medical Research Council Selective gpcr ligands
CN102943063A (zh) * 2012-09-26 2013-02-27 浙江大学 提高骨髓单个核细胞心肌细胞保护及迁移能力的处理方法
WO2016081733A2 (en) * 2014-11-19 2016-05-26 Novopyxis Inc. Compositions and methods for modulating at2r activity

Non-Patent Citations (3)

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Title
QU, XIAOLING ET AL.: "Electroporation-mediated in Vivo AT2R Gene Transfection Inhibitis Neointimal Hyperplasia after Balloon Angioplasty in Rat Common Carotid Arteries", ACTA ACADEMIAE MEDICINAE MILITARIS TERTIAE, vol. 33, no. 6, 30 March 2011 (2011-03-30), pages 562 - 565 *
YU, YIYAN ET AL.: "Construction and Identification of AT1R and AT2R Expression Plasmids from Human Atrial Tissue", JOURNAL OF SOUTHWEST MEDICAL UNIVERSITY, vol. 40, no. 1, 31 December 2017 (2017-12-31), pages 31 - 34 *
ZUO, YUMEI ET AL.: "Construction of Eukaryotic Expression Vector pEGFP-N2/AT2R and Its Expression in Primary Vascular Smooth Muscle Cells", ACTA ACADEMIAE MEDICINAE MILITARIS TERTIAE, vol. 33, no. 9, 15 May 2011 (2011-05-15), pages 940 - 943, XP055670393 *

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