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WO2018170712A1 - Procédé de construction d'un vecteur d'expression élevée du gène du ligand 2 de mort programmée humaine et application associée - Google Patents

Procédé de construction d'un vecteur d'expression élevée du gène du ligand 2 de mort programmée humaine et application associée Download PDF

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Publication number
WO2018170712A1
WO2018170712A1 PCT/CN2017/077399 CN2017077399W WO2018170712A1 WO 2018170712 A1 WO2018170712 A1 WO 2018170712A1 CN 2017077399 W CN2017077399 W CN 2017077399W WO 2018170712 A1 WO2018170712 A1 WO 2018170712A1
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WO
WIPO (PCT)
Prior art keywords
gene
vector
expression vector
primers
kit
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/CN2017/077399
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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/CN2017/077399 priority Critical patent/WO2018170712A1/fr
Publication of WO2018170712A1 publication Critical patent/WO2018170712A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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  • the present invention belongs to the technical field of molecular biology, and relates to a method and application for constructing a human PD-L2 gene high expression vector.
  • Non-small cell lung cancer is a common malignant tumor in the world, and its incidence is increasing year by year. Studying NSCLC tumor recurrence factors and tumor immune escape mechanisms has important theoretical significance and clinical application value. T lymphocytes are important effector cells that mediate tumor immune responses, and T cell activation requires TCR-mediated antigen-specific signaling and costimulatory molecule-mediated costimulatory signals.
  • PD-L2 Programmed death ligand 2
  • B7-H1 is a negative T cell costimulatory molecule in the B7 family
  • PD-L2 inhibits proliferation of CD4 and CD8 T cells by binding to its receptor PD-1
  • activation negative regulation of the body's immune response process, thereby mediating tumor immune escape, promoting tumor growth
  • the study of the role of PD-L2 in tumor escape plays an important role in the prevention and treatment of tumors, but the lack of targets in the prior art
  • Vectors that promote the expression of programmed death ligand 2 genes have hampered the progress of related research.
  • the object of the present invention is to provide a method for constructing a human PD-L2 gene high expression vector, and to investigate the effect of PD-L2 on cell proliferation and apoptosis.
  • the present invention discloses a method for constructing a human PD-L2 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:
  • PD-L2-F 5'- GTCTAGAATGATCTTCCTCCTGCTAATG -3' (SEQ ID No: l)
  • PD-L2-R 5, - GGAATTCTCAGATAGCACTGTTCACTTC -3' (SEQ ID No: 2
  • 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 high expression of the human PD-L2 gene.
  • the human PD-L2 gene high expression vector provided by the invention has the advantages of high transfection efficiency, low dosage, and can promote the high expression of PD-L2 gene specifically and efficiently, and can be used as a powerful tool for PD-L2. Related drug research and development.
  • FIG. 1 is a schematic diagram showing the results of detecting the PD-L2 expression level by transcytometry pCDNA3.1-PD-L2 vector.
  • the coding sequence of the PD-L2 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-PD-L2 was extracted with Endo-Free Plasmid Mini Kit II.
  • A375 cells were seeded into six-well plates, cultured 18
  • pCDNA3.1-PD-L2 was transduced into A375 cells and cultured for 48 h.
  • the Primer 5 was designed using the primer design software Premier Primer 5.
  • A375 cells and PD-L2 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
  • ⁇ was used as a template, and GAPDH was used as an internal reference.
  • the relative expression of PD-L2 was detected by real-time fluorescent quantitative PCR, and 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 result is shown in Figure 1. It can be seen that the PD-L2 gene expression level of PD-L2 high expression cells is 190-fold higher than that of normal A375 cells, indicating that the PD-L2 gene cDNA sequence provided by the present invention is successfully inserted into the pCD NA3.1 expression vector. It can promote the high expression of PD-L2 gene specifically, continuously and efficiently.
  • the human PD-L2 gene high expression vector provided by the invention has the advantages of high transfection efficiency, low dosage, and can promote the high expression of PD-L2 gene specifically and efficiently, and can be applied as a powerful tool to PD-L2. Related drug research and development.

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Abstract

L'invention concerne un procédé pour construire un vecteur d'expression élevée du gène du ligand 2 de mort programmée humaine (PD-L2) et une application associée. Le procédé de construction comprend les étapes consistant : 1) à amplifier et à purifier un gène cible : concevoir une amorce supérieure et une amorce inférieure, extraire un ARN cellulaire, utiliser un ADNc soumis à une transcription inverse comme modèle pour effectuer une amplification par PCR, couper une bande cible et utiliser un kit pour la récupération et la purification ; et 2) à construire et à identifier un plasmide recombinant pCDNA3.1-PD-L2 : effectuer une digestion enzymatique double sur le produit de PCR purifié et un vecteur pCDNA3.1 avec Xba I et EcoR I, récupérer et purifier un produit digéré par une enzyme avec le kit séparément, ligaturer des fragments et cribler, mettre en culture, cloner et séquencer, et détecter des expressions géniques par QPCR.
PCT/CN2017/077399 2017-03-20 2017-03-20 Procédé de construction d'un vecteur d'expression élevée du gène du ligand 2 de mort programmée humaine et application associée Ceased WO2018170712A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2017/077399 WO2018170712A1 (fr) 2017-03-20 2017-03-20 Procédé de construction d'un vecteur d'expression élevée du gène du ligand 2 de mort programmée humaine et application associée

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2017/077399 WO2018170712A1 (fr) 2017-03-20 2017-03-20 Procédé de construction d'un vecteur d'expression élevée du gène du ligand 2 de mort programmée humaine et application associée

Publications (1)

Publication Number Publication Date
WO2018170712A1 true WO2018170712A1 (fr) 2018-09-27

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Application Number Title Priority Date Filing Date
PCT/CN2017/077399 Ceased WO2018170712A1 (fr) 2017-03-20 2017-03-20 Procédé de construction d'un vecteur d'expression élevée du gène du ligand 2 de mort programmée humaine et application associée

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WO (1) WO2018170712A1 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020164600A1 (en) * 2000-06-28 2002-11-07 Gordon Freeman PD-L2 molecules: novel PD-1 ligands and uses therefor
CN103820481A (zh) * 2014-01-26 2014-05-28 新乡学院 鸡外周血单核淋巴细胞pd-l2重组质粒的构建、基因丰度实时检测方法及其应用

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020164600A1 (en) * 2000-06-28 2002-11-07 Gordon Freeman PD-L2 molecules: novel PD-1 ligands and uses therefor
CN103820481A (zh) * 2014-01-26 2014-05-28 新乡学院 鸡外周血单核淋巴细胞pd-l2重组质粒的构建、基因丰度实时检测方法及其应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
HE, YUFEI ET AL.,: "Eukaryotic Expression and Functional Characterization of PD-1 Extracellular Domain", CHINESE JOURNAL OF BIOTECHNOLOGY, vol. 20, no. 5, 30 September 2014 (2014-09-30), pages 699 - 703 *

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