WO2019037054A1 - Procédé de construction de vecteur à expression élevée du gène cvid1 humain et applications - Google Patents

Procédé de construction de vecteur à expression élevée du gène cvid1 humain et applications Download PDF

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Publication number
WO2019037054A1
WO2019037054A1 PCT/CN2017/098911 CN2017098911W WO2019037054A1 WO 2019037054 A1 WO2019037054 A1 WO 2019037054A1 CN 2017098911 W CN2017098911 W CN 2017098911W WO 2019037054 A1 WO2019037054 A1 WO 2019037054A1
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WIPO (PCT)
Prior art keywords
cvid1
gene
vector
expression vector
human
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PCT/CN2017/098911
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English (en)
Chinese (zh)
Inventor
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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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/CN2017/098911 priority Critical patent/WO2019037054A1/fr
Publication of WO2019037054A1 publication Critical patent/WO2019037054A1/fr

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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
    • 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 CVID1 gene high expression vector.
  • CVID1 is expressed on activated T cells.
  • the interaction of CVID1 with its ligand CVID1L plays an important biological role in the regulation of immune response in the body.
  • This signal has been shown to significantly up-regulate the expression of cytokines such as IL-4, IL-5, IL-10 and IL-13.
  • cytokines such as IL-4, IL-5, IL-10 and IL-13.
  • the significant up-regulation of IL-4 and IL-10 suggests that the signal is extremely important for the immune response of T h2 cells.
  • the various cytokines produced by T cells can be regulated by CVID1.
  • CVID1 plays a key role in determining the polarization direction of T cells, plays an important role in the immunotherapy of tumors, and requires a lot of research to achieve clinical transformation, but the lack of coding expression CVID1 in the prior art The carrier of the gene has caused certain obstacles to the progress of related research.
  • the object of the present invention is to provide a method for constructing a human CVID1 gene high expression vector, and to investigate the effect of CVID1 on cell proliferation and apoptosis.
  • the present invention discloses a method for constructing a human CVID1 gene high expression vector, which is characterized in that: pCDNA3.1 is used as an intermediate vector, a pair of primers are designed, and Xba is introduced into the primer.
  • a pair of primers are designed as follows:
  • CVID1-F 5'- GTCTAGAATGAAGTCAGGCCTCTGGTAT -3' (SEQ ID No: 1
  • CVID1 -R 5' - GGAATTCTTTATAGGGTCACATCTGTGAG -3' (SEQ ID No:
  • the amplified product obtained in the step 2) is ligated to the double-digested pCDNA3.1 vector obtained in the step 3), and then cultured and cloned to obtain the human CVID1 gene high expression vector pCDNA3. .1-CVID1.
  • the human CVID1 gene high expression vector provided by the invention has the advantages of high transfection efficiency, low dosage, and can promote the high expression of CVID1 gene specifically and efficiently, and can be used as a powerful tool for drug research related to CVID1 and ⁇ Hair.
  • FIG. 1 is a schematic diagram showing the results of CRFID1 expression level detection by transcytometry of pCDNA3.1-CVID1 vector.
  • CVID1 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
  • Example 2 Construction of human CVID1 gene high expression vector [0019] After diluting the synthetic antibody, the coding sequence of the CVID1 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 on 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-CVIDl was extracted with Endo-Free Plasmid Mini Kit II.
  • pCDNA3.1-CVID1 was transduced into HepG2 cells and cultured for 48 h.
  • Example 4 Quantitative PCR was used to detect the expression level of CVID1 gene.
  • HepG2 cells and CVID1 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 with RNeasy Mini Kit, and mRNA was reverse transcribed into cDNA using PrimeScrip RT reagent Kit.
  • Reverse transcription conditions 37 ° C, 15 min; 85 ° C, 5s; 4°C, ⁇ . After the end of the reverse transcription, 9 (L of RNase Free dH 20 diluted cDNA was added and stored at -20 ° C for later detection.
  • CVID1 was detected by real-time fluorescent quantitative PCR.
  • the reaction conditions were set: 95. C 30s, 1 cycle, 54°C 30s 40 cycles, 95. C 5s, 60 ° C lmin, 95. C 15s, the results are shown in Figure 1. It can be seen that the CVID1 gene expression level of CVID1 high expression cells is 270-fold higher than that of normal HepG 2 cells, indicating that the CVID1 gene cDNA sequence provided by the present invention is successfully inserted into the PCDNA3.1 expression vector, which can be specific, sustained, and Efficiently promote high expression of CVID1 gene.
  • the human CVID1 gene high expression vector provided by the invention has the advantages of high transfection efficiency, low dosage, and can promote the high expression of CVID1 gene specifically and efficiently, and can be used as a powerful tool for drug research related to CVID1. Hair.

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  • 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 à expression élevée d'un gène CVID1 humain, et des applications. Le procédé comprend les étapes consistant à : premièrement, amplifier et purifier un gène cible : concevoir une amorce supérieure et une amorce inférieure, extraire un ARN cellulaire, et réaliser une amplification par PCR au moyen d'un ADNc transcrit par transcription inverse en tant que modèle, et après la coupure d'une bande cible, réaliser un recyclage et une purification au moyen d'un kit de réactifs ; et deuxièmement, construire et vérifier un plasmide recombiné pCDNA3.1-CVID1 : après la mise en œuvre de la double digestion enzymatique sur le produit de PCR purifié et un vecteur pCDNA3.1 au moyen de Xba I et EcoR I, recycler et purifier respectivement les produits de digestion enzymatique au moyen des kits de réactifs, relier les segments, réaliser un criblage, une culture, un clonage et un séquençage, et détecter l'état d'expression génique au moyen d'une QPCR.
PCT/CN2017/098911 2017-08-24 2017-08-24 Procédé de construction de vecteur à expression élevée du gène cvid1 humain et applications WO2019037054A1 (fr)

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Application Number Priority Date Filing Date Title
PCT/CN2017/098911 WO2019037054A1 (fr) 2017-08-24 2017-08-24 Procédé de construction de vecteur à expression élevée du gène cvid1 humain et applications

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2017/098911 WO2019037054A1 (fr) 2017-08-24 2017-08-24 Procédé de construction de vecteur à expression élevée du gène cvid1 humain et applications

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
US20100303811A1 (en) * 2008-06-16 2010-12-02 Atsuo Ochi Recombinant multiple domain fusion protein mitogens and use thereof for inducing enhancement or repression of antigen-specific immunity.
WO2012037116A2 (fr) * 2010-09-13 2012-03-22 The Children's Hospital Of Philadelphia Variations génétiques communes et rares associées à une immunodéficience commune variable (icv) et procédés d'utilisation de celles-ci pour le traitement et le diagnostic de cet état
CN103509812A (zh) * 2013-08-07 2014-01-15 吴珍芳 唾液腺组织特异性的转基因载体及其构建方法
CN104531761A (zh) * 2015-01-07 2015-04-22 天津市第一中心医院 脂肪间充质干细胞的构建方法与应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100303811A1 (en) * 2008-06-16 2010-12-02 Atsuo Ochi Recombinant multiple domain fusion protein mitogens and use thereof for inducing enhancement or repression of antigen-specific immunity.
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
WO2012037116A2 (fr) * 2010-09-13 2012-03-22 The Children's Hospital Of Philadelphia Variations génétiques communes et rares associées à une immunodéficience commune variable (icv) et procédés d'utilisation de celles-ci pour le traitement et le diagnostic de cet état
CN103509812A (zh) * 2013-08-07 2014-01-15 吴珍芳 唾液腺组织特异性的转基因载体及其构建方法
CN104531761A (zh) * 2015-01-07 2015-04-22 天津市第一中心医院 脂肪间充质干细胞的构建方法与应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ZHAO ET AL: "Construction and expression of eukaryotic expression vector pcDNA3-ICOSIg", CLINICAL REHABILITATIVE TISSUE ENGINEERING RESEARCH, vol. 17, no. 37, 10 September 2013 (2013-09-10), pages 6642 - 6643 *

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