[go: up one dir, main page]

WO2019036870A1 - Procédé de construction d'un vecteur d'expression élevée d'un gène arntl humain, et applications - Google Patents

Procédé de construction d'un vecteur d'expression élevée d'un gène arntl humain, et applications Download PDF

Info

Publication number
WO2019036870A1
WO2019036870A1 PCT/CN2017/098368 CN2017098368W WO2019036870A1 WO 2019036870 A1 WO2019036870 A1 WO 2019036870A1 CN 2017098368 W CN2017098368 W CN 2017098368W WO 2019036870 A1 WO2019036870 A1 WO 2019036870A1
Authority
WO
WIPO (PCT)
Prior art keywords
arntl
gene
vector
primers
expression vector
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/CN2017/098368
Other languages
English (en)
Chinese (zh)
Inventor
毛吉炎
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shenzhen Biocan Technologies Co Ltd
Original Assignee
Shenzhen Biocan Technologies Co Ltd
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 Shenzhen Biocan Technologies Co Ltd filed Critical Shenzhen Biocan Technologies Co Ltd
Priority to PCT/CN2017/098368 priority Critical patent/WO2019036870A1/fr
Publication of WO2019036870A1 publication Critical patent/WO2019036870A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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

Definitions

  • the present invention relates to a method and application for constructing a human ARNTL gene high expression vector.
  • Biological rhythm refers to the cyclical changes in the process of long-term biological evolution, in order to adapt to the cycle of nature.
  • the living organisms from single cells to higher animals and plants and human life all form a regular life.
  • Activity phenomenon The ARNTL gene is an important circadian clock gene involved in the regulation of the biological rhythm of the organism and is closely related to the occurrence and development of tumors.
  • the object of the present invention is to provide a method for constructing a human ARNTL gene high expression vector, and to explore ARNT.
  • the present invention discloses a method for constructing a human ARNTL gene high expression vector.
  • pCDNA3.1 as an intermediate vector, designing a pair of primers, introducing Xba l and EcoR into the primers
  • ARNTL-F 5,- GTCTAGAATGGCAGACCAGAGAATGGAC -3' (SEQ ID No: 1
  • ARNTL-R 5, - GGAATTCTTACAGCGGCCATGGCAAGTC -3' (SEQ ID No: 2
  • the amplified product obtained in the step 2) is ligated with the double-digested pCDNA3.1 vector obtained in the step 3), and then cultured and cloned to obtain the human ARNTL gene high expression vector pCDNA. 3.1-ARNTL.
  • the human ARNTL gene high expression vector provided by the invention has the advantages of high transfection efficiency, low dosage, and can promote the high expression of ARNTL gene specifically and efficiently, and can be used as a powerful tool for drug research related to ARNTL and ⁇ Hair.
  • FIG. 1 is a schematic diagram showing the results of ARFTL expression level detection by transcytometry of pCDNA3.1-ARNTL vector.
  • the ARNTL gene coding sequence it was analyzed using 01igo7 to find upstream primers and downstream primers (requiring as little primer-free dimer as possible and the annealing temperature difference is small), and then at the 5' end of the upstream primer and the downstream primer. Adding protective bases and restriction sites Xba I and EcoR, respectively I, The designed primer sequences are shown in SEQ ID No: 1 and SEQ ID No: 2.
  • the coding sequence of the ARNTL gene was amplified by Premix PrimeSTAR HS enzyme, and Xba I and EcoR were used after electrophoresis recovery.
  • the enzyme I is digested, and the double-cut product is recovered by electrophoresis.
  • the pcDNA3.1 vector was electrophoresed and recovered by Xba l and EcoR I enzyme.
  • the double-digested PCR product and the pcDNA3.1 vector were ligated with T4 DNA ligase, transformed into competent E. coli DH50C, uniformly coated onto an ampicillin-containing LB medium plate, and cultured at 37 ° C. After h, pick a single colony culture and send it to Shanghai Yingjun for sequencing.
  • the recombinant E. coli was sequenced and the recombinant plasmid pCDNA3.1-ARNTL was extracted with Endo-Free Plasmid Mini Kit II.
  • A375 cells were seeded into six-well plates, cultured 18
  • pCDNA3.1-ARNTL was transduced into A375 cells by PEI and cultured for 48 h.
  • Example 4 Quantitative PCR was used to detect the expression level of ARNTL gene.
  • A375 cells and ARNTL high expressing cells were inoculated separately into 6-well plates. Cell density reached 80 ⁇ 3 ⁇ 4-90 ⁇ 3 ⁇ 4 ⁇ , total RNA was extracted from each group using RNeasy Mini Kit, using PrimeScrip RT reagent
  • ARNTL gene expression level of ARNTL high expression cells is more than 160 times higher than that of normal A375 cells, indicating that the ARNTL gene cDNA sequence provided by the present invention is successfully inserted into the PCDNA3.1 expression vector, which can be specific, sustained and efficient. Promote high expression of ARNTL gene.
  • the human ARNTL gene high expression vector provided by the invention has the advantages of high transfection efficiency, low dosage, and can promote the high expression of ARNTL gene specifically and efficiently, and can be used as a powerful tool for drug research and development related to ARNTL.

Landscapes

  • Genetics & Genomics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé de construction d'un vecteur d'expression élevée d'un gène ARNTL humain, et des applications. Le procédé comprend les étapes consistant : premièrement, à amplifier et à purifier un gène cible : à concevoir une amorce supérieure et une amorce inférieure, à extraire un ARN cellulaire, et à réaliser une amplification par PCR au moyen d'un ADNc transcrit en sens inverse en tant que matrice, et après la découpe d'une bande cible, à réaliser un recyclage et une purification à l'aide d'un kit de réactifs; et deuxièmement, à construire et à vérifier un plasmide recombiné pCDNA3.1-ARNTL : après la réalisation d'une double digestion enzymatique sur le produit de PCR purifié et sur un vecteur pCDNA3.1 au moyen de Xba I et de EcoR I, à recycler et à purifier respectivement les produits de digestion enzymatique à l'aide de kits de réactifs, à relier des segments, à réaliser un criblage, une culture, un clonage et un séquençage, et à détecter l'état d'expression génique à l'aide d'une QPCR.
PCT/CN2017/098368 2017-08-21 2017-08-21 Procédé de construction d'un vecteur d'expression élevée d'un gène arntl humain, et applications Ceased WO2019036870A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2017/098368 WO2019036870A1 (fr) 2017-08-21 2017-08-21 Procédé de construction d'un vecteur d'expression élevée d'un gène arntl humain, et applications

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2017/098368 WO2019036870A1 (fr) 2017-08-21 2017-08-21 Procédé de construction d'un vecteur d'expression élevée d'un gène arntl humain, et applications

Publications (1)

Publication Number Publication Date
WO2019036870A1 true WO2019036870A1 (fr) 2019-02-28

Family

ID=65438289

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2017/098368 Ceased WO2019036870A1 (fr) 2017-08-21 2017-08-21 Procédé de construction d'un vecteur d'expression élevée d'un gène arntl humain, et applications

Country Status (1)

Country Link
WO (1) WO2019036870A1 (fr)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003064647A1 (fr) * 2002-01-31 2003-08-07 Sato Honma Procede de regulation du rythme circadien
CN101358199A (zh) * 2008-02-22 2009-02-04 东南大学 一种表达人cdc25 b3质粒及其构建方法
CN101591669A (zh) * 2009-05-15 2009-12-02 西安交通大学 一种野生型egfr高表达的重组hek293细胞
US20130237716A1 (en) * 2012-03-07 2013-09-12 Shiseido Company, Ltd. Expression Modulator For Clock Gene

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003064647A1 (fr) * 2002-01-31 2003-08-07 Sato Honma Procede de regulation du rythme circadien
CN101358199A (zh) * 2008-02-22 2009-02-04 东南大学 一种表达人cdc25 b3质粒及其构建方法
CN101591669A (zh) * 2009-05-15 2009-12-02 西安交通大学 一种野生型egfr高表达的重组hek293细胞
US20130237716A1 (en) * 2012-03-07 2013-09-12 Shiseido Company, Ltd. Expression Modulator For Clock Gene

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
V. MATAGNE ET AL.: "Thyroid transcription factor 1, a homeodomain containing transcription factor, contributes to regulating periodic oscillations in GnRH gene expression", JOURNAL OF NEUROENDOCRINOLOGY, vol. 24, no. 6, 30 June 2012 (2012-06-30), pages 916 - 929, XP055579341, ISSN: 1365-2826, DOI: 10.1111/j.1365-2826.2012.02302.x *

Similar Documents

Publication Publication Date Title
US12049623B2 (en) Compositions and methods for identifying polynucleotides of interest
CN105087570B (zh) 一种环状rna人工过表达框架及其表达载体及构建方法
CN115960802B (zh) 高产重组蛋白的需钠弧菌工程菌及其应用
CN106947766A (zh) 一种枯草芽孢杆菌具有启动子功能的dna片段及其应用
CN112226460B (zh) 稳定表达猪源宿主限制因子a3z2分子的细胞系的筛选、培育方法及其应用
WO2019036870A1 (fr) Procédé de construction d'un vecteur d'expression élevée d'un gène arntl humain, et applications
CN106086025A (zh) 一种具有启动子功能的dna片段及其应用
CN114561411A (zh) Dicer1基因及其dsRNA在害虫防治中的应用
CN115896143B (zh) 一种通过构建核酸支架提升色氨酸合成酶活性的方法
WO2019037133A1 (fr) Arnsh ciblant app silencieux
WO2019037054A1 (fr) Procédé de construction de vecteur à expression élevée du gène cvid1 humain et applications
WO2019037132A1 (fr) Procédé de construction d'un vecteur d'expression élevée du gène ebln1 humain et application correspondante
CN101250510A (zh) 丙肝病毒特异性核酶M1GS-hcv/C20的构建方法和应用
WO2018170712A1 (fr) Procédé de construction d'un vecteur d'expression élevée du gène du ligand 2 de mort programmée humaine et application associée
WO2017214940A1 (fr) Vecteur d'expression lentiviral pour favoriser spécifiquement l'expression élevée du gène cplx2, et ses applications
WO2019037048A1 (fr) Vecteur lentiviral à haute expression de gène de l'indoléamine 2,3-dioxygénase, lentivirus, et sa méthode de construction
CN103525854A (zh) 高基因敲除效率的谢瓦氏曲霉间型变种工程菌株的构建方法
CN118109472A (zh) 一种可翻译蛋白的环形rna表达载体及构建方法和表达方法
WO2019000145A1 (fr) Procédé de construction de cellule cho recombinée exprimant fortement l'ampar
Wang et al. Monitoring promoter activity by RNA editing based reporter
CN118222638B (zh) 一种干扰蚯蚓目的基因表达的方法
CN111944849B (zh) 应用hpv18间隔序列构建双顺反子表达质粒的方法
WO2018170710A1 (fr) Vecteur d'expression élevée du gène imd16 humain et son application
WO2019037052A1 (fr) Arnsh pour le silençage ciblé de wucam
WO2018170711A1 (fr) Vecteur d'expression élevée du gène gp34 humain et son application

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: 17922784

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: 17922784

Country of ref document: EP

Kind code of ref document: A1