WO2020000470A1 - 促进cdc42蛋白过表达的重组载体及其构建方法 - Google Patents

促进cdc42蛋白过表达的重组载体及其构建方法 Download PDF

Info

Publication number
WO2020000470A1
WO2020000470A1 PCT/CN2018/093880 CN2018093880W WO2020000470A1 WO 2020000470 A1 WO2020000470 A1 WO 2020000470A1 CN 2018093880 W CN2018093880 W CN 2018093880W WO 2020000470 A1 WO2020000470 A1 WO 2020000470A1
Authority
WO
WIPO (PCT)
Prior art keywords
cdc42
vector
promoting
recombinant vector
pegfp
Prior art date
Application number
PCT/CN2018/093880
Other languages
English (en)
French (fr)
Inventor
毛吉炎
Original Assignee
深圳市博奥康生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 深圳市博奥康生物科技有限公司 filed Critical 深圳市博奥康生物科技有限公司
Priority to PCT/CN2018/093880 priority Critical patent/WO2020000470A1/zh
Publication of WO2020000470A1 publication Critical patent/WO2020000470A1/zh

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
    • 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 invention belongs to the field of biotechnology, and particularly relates to a recombinant vector and a method for constructing the same, which promote the overexpression of CDC42 protein.
  • Cholangiocarcinoma is one of the relatively common malignant tumors of the digestive tract system. With the development of medical science, the diagnosis and discovery rate of this tumor has been increasing in recent years, especially the incidence of extrahepatic cholangiocarcinoma. As a solid malignant tumor of the digestive tract, its typical biological manifestations are mainly in situ swelling and invasive growth. Invasion and metastasis are its main cause of death.
  • CDC42 is a member of the Rho family of proteins.
  • the members of Rho's family mainly include Cde42, RhoA, RhoB, RhoC, Rac1, Rac2, Rac4 and other molecules.
  • the members of this family are important signaling intermediate molecules in the cell.
  • CDC42 is a small G protein. In the cytoplasm, one form is GDP (inactive state) and the other form is GTP (active state). Depending on the changes in the cell's internal and external environment and the stimulation of foreign factors, a transition can be made between active and inactive forms.
  • the increased expression of CDC42 in malignant tumors has various effects on the biological behavior of tumor cells.
  • the increased expression of CDC42 in breast cancer mainly promotes the migration and metastasis of cancer cells; while in leiomyosarcoma, it can affect the cell cycle and promote cell growth and colony formation. In colorectal cancer, it can affect downstream active molecules and participate in tumor adhesion.
  • Previous studies have shown that the expression of CDC42 in cholangiocarcinoma cells is related to the invasion ability of cholangiocarcinoma cells, but its role in the pathogenesis of cholangiocarcinoma and its related mechanisms are not clear. Recombinant vectors that promote CDC42 protein overexpression are also lacking in the prior art.
  • the present invention provides a recombinant vector that promotes the overexpression of CDC42 protein, including a CDC42 coding sequence and a pEGFP-C1 vector; the pEGFP-C1 vector includes XhoI and EcoRI restriction sites, The CDC42 sequence is inserted into the restriction site in the forward direction.
  • the obtained recombinant vector has the advantages of high transfection efficiency, promotion of CDC42 protein overexpression and sustained and stable expression.
  • the CDC42 coding sequence comprises a sequence of GenBank gene number NM_001039802.1, and its 5 'end contains an XhoI restriction site and the 3' end contains an EcoRI restriction site.
  • the recombinant vector for promoting the overexpression of CDC42 protein provided by the present invention has the advantages of high transfection efficiency, promoting overexpression of CDC42 protein and sustained and stable expression, and has a simple construction method.
  • Figure 1 shows the pEGFP-C1 vector map
  • Figure 2 shows the CDC42 expression levels of SGC996 cells in the control and experimental groups.
  • Example one CDC42 Construction of recombinant vector for protein overexpression
  • the ligation product was transformed into competent E. coli Top 10, followed by expansion and sequencing, and screening of bacteria that completely matched the expected results. Then expand the culture and use the endotoxin-free plasmid extraction kit to extract the recombinant plasmid in E. coli and name it pEGFP-CDC42.
  • Embodiment two pEGFP-CDC42 Transfection SGC996 cell
  • SGC996 cells were seeded in a six-well plate with 1,000,000 cells per well, and the cell density was about 50% after 12 hours.
  • the pEGFP-CDC42 plasmid was transfected into SGC996 cells with Lipofectamine 2000, 1 ⁇ g per well, and the cells were continued. to cultivate.
  • the normal SGC996 cells (control group) and the transfected SGC996 cells (experimental group) obtained in Example 2 were respectively inoculated into a six-well plate. When the cell density reached 80% -90%, the total RNA of each group of cells was extracted, and the mRNA was reverse-transcribed into cDNA, and stored at -20 ° C.
  • the recombinant vector for promoting the overexpression of CDC42 protein provided by the present invention has the advantages of high transfection efficiency, promoting overexpression of CDC42 protein and sustained and stable expression, and has a simple construction method.

Landscapes

  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicinal Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

提供一种促进CDC42蛋白过表达的重组载体，包括CDC42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CDC42序列正向插入所述酶切位点。所述促进CDC42蛋白过表达的重组载体具有转染效率高，促进BNP蛋白过表达且表达持续稳定的优点，构建方法简单。

Description

促进CDC42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CDC42蛋白过表达的重组载体及其构建方法。
 背景技术
胆管细胞癌是相对比较常见的消化道系统恶性肿瘤之一，随着医学科学的发展，近年来该肿瘤诊断、发现率不断提高，特别是肝外胆管细胞癌发生率最为明显。作为消化道的实体性恶性肿瘤，其典型的生物学表现主要是原位膨胀及浸润生长，侵袭转移是其主要的致死原因。
 技术问题
CDC42是Rho家族蛋白中的成员之一。Rho其家族成员主要包括有Cde42、RhoA、RhoB、RhoC、Rac1、Rac2、Rac4等多个分子，该族成员分子为细胞内重要的信号中间参与分子，在细胞的粘附和细胞的移动过程中，需要肌动蛋白关联细胞骨架结构的变化，而这些变化需要一个重要的信号分子-CDC42。CDC42属于小G蛋白，在细胞浆内，一种形式为GDP（非活性态），另外一种形式为GTP（活性态）。根据细胞的内外界环境的变化以及外来因子的刺激，活性和非活性形式之间可以进行转变。
恶性肿瘤中CDC42表达增高对肿瘤细胞生物学行为影响是多样的。乳腺癌中CDC42表达增高主要促进了癌细胞移动和转移；而平滑肌肉瘤中可影响细胞周期，促进细胞增长和集落形成。在大肠癌中，可影响下游的活性分子，参与肿瘤的粘附。前期研究表明，胆管癌细胞中CDC42的表达与胆管癌细胞侵袭能力相关，但其在胆管癌发病过程中的所起作用及相关机制尚不清晰。现有技术中也缺乏促进CDC42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CDC42蛋白过表达的重组载体，包括CDC42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CDC42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CDC42蛋白过表达且表达持续稳定的优点。
优选地，所述CDC42编码序列包含GenBank基因编号为NM_001039802.1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CDC42蛋白过表达的重组载体具有转染效率高，促进CDC42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SGC996细胞的CDC42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 ： CDC42 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CDC42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Top 10，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-CDC42。
实施例二： pEGFP-CDC42 转染 SGC996 细胞
接种SGC996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-CDC42质粒分别转染至SGC996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 CDC42 基因 表达水平
分别接种正常SGC996细胞（对照组）、实施例二中获得经转染的SGC996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CDC42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CDC42基因表达水平较对照组细胞有206.4倍的升高，说明本发明提供的促进CDC42蛋白过表达的重组载体能特异、高效地促进CDC42基因过表达。
 工业实用性
本发明提供的促进CDC42蛋白过表达的重组载体具有转染效率高，促进CDC42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CDC42蛋白过表达的重组载体，其特征在于，包括CDC42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CDC42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CDC42蛋白过表达的重组载体，其特征在于：所述CDC42编码序列包含GenBank基因编号为NM_001039802.1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CDC42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CDC42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CDC42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CDC42蛋白过表达的重组载体。
PCT/CN2018/093880 2018-06-29 2018-06-29 促进cdc42蛋白过表达的重组载体及其构建方法 WO2020000470A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2018/093880 WO2020000470A1 (zh) 2018-06-29 2018-06-29 促进cdc42蛋白过表达的重组载体及其构建方法

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2018/093880 WO2020000470A1 (zh) 2018-06-29 2018-06-29 促进cdc42蛋白过表达的重组载体及其构建方法

Publications (1)

Publication Number Publication Date
WO2020000470A1 true WO2020000470A1 (zh) 2020-01-02

Family

ID=68985359

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2018/093880 WO2020000470A1 (zh) 2018-06-29 2018-06-29 促进cdc42蛋白过表达的重组载体及其构建方法

Country Status (1)

Country Link
WO (1) WO2020000470A1 (zh)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050112748A1 (en) * 2001-10-03 2005-05-26 Takeshi Endo Low molecular weight gtpase rhot
WO2005079847A1 (ja) * 2004-02-25 2005-09-01 Kowa Company, Ltd. Cdc42タンパク質の核内移行促進剤及びそのスクリーニング方法
CN102209522A (zh) * 2008-09-10 2011-10-05 布拉德福德大学 调节皮肤色素沉着的组合物和方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050112748A1 (en) * 2001-10-03 2005-05-26 Takeshi Endo Low molecular weight gtpase rhot
WO2005079847A1 (ja) * 2004-02-25 2005-09-01 Kowa Company, Ltd. Cdc42タンパク質の核内移行促進剤及びそのスクリーニング方法
CN102209522A (zh) * 2008-09-10 2011-10-05 布拉德福德大学 调节皮肤色素沉着的组合物和方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LIU L., ET. AL.: "Construction of cdc42 gene and promoter cpg island of hazak esophageal ancer in Eukaryotic Expression Vector", JOURNAL OF XINJIANG MEDICAL UNIVERSTIY, vol. 33, no. 7, 31 July 2010 (2010-07-31), pages 735 - 738 *
ZHANG Q. ,ET. AL.: "Construction of FRET-based single-olecule probes for monitoring spatial and temporal imaging of induced activation of Rac,cdc42 in living cells", ACTA LASER BIOLOGY SINICA, vol. 17, no. 5, 15 October 2008 (2008-10-15), pages 684 - 688 *

Similar Documents

Publication Publication Date Title
CN108102940B (zh) 一株利用CRISPR/Cas9系统敲除XKS1基因的工业酿酒酵母菌株及构建方法
EP2714913B1 (en) Transcription terminator sequences
WO2024002149A1 (zh) 一种重组iii型胶原蛋白及其制备方法
CN106978407B (zh) 一种β-葡萄糖醛酸苷酶及其基因与应用
WO2023045682A1 (zh) 一种提高多肽可溶性表达产量的方法
CN117645996A (zh) 核酸5’utr分子及其用途
WO2012126265A1 (zh) 用于口服的表面展示有人血清白蛋白重组芽孢及其制备方法
US10508279B2 (en) Recombinant Escherichia coli for high efficiency production of fructosylated chondroitin and method for making thereof
CN112608933B (zh) 一种重组蓝铜肽前体-寡肽的高纯度制备方法
WO2020000470A1 (zh) 促进cdc42蛋白过表达的重组载体及其构建方法
JP2001046078A (ja) タンパク質の高発現システム
WO2020000471A1 (zh) 促进pdss2蛋白过表达的重组载体及其构建方法
CN109022442B (zh) 一种可有效抑制骨肉瘤生长的生物重组型miR124-3p
CN102399810B (zh) 一种携带attB高效定向整合假attP位点的载体及其转基因方法
WO2020000474A1 (zh) 促进pou5f1蛋白过表达的重组载体及其构建方法
WO2020000469A1 (zh) 促进 at2r 蛋白过表达的重组载体及其构建方法
WO2020000472A1 (zh) 促进pink1蛋白过表达的重组载体及其构建方法
WO2017214940A1 (zh) 特异促进Cplx2基因高表达的慢病毒表达载体及其应用
WO2020000466A1 (zh) 一种lncRNA AFAP1-AS1过表达载体及其制备方法
KR102365768B1 (ko) 개선된 슬리핑 뷰티 트랜스포존 시스템 및 이를 이용한 유전자 전위 방법
WO2020000467A1 (zh) 一种LncRNA DB327252过表达载体及其制备方法
WO2020000468A1 (zh) 一种LncRNA DLX6-AS1过表达载体及其制备方法
CN110156877B (zh) 抑制肿瘤的多肽类似物UBE2Z1-330aa及其编码核酸、引物对、表达载体和应用
WO2023184539A1 (zh) 一种甲醇诱导启动子及其在制备丙谷二肽中的应用
WO2024131232A1 (zh) 环状rna分离和纯化方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18924132

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18924132

Country of ref document: EP

Kind code of ref document: A1