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WO2018170710A1 - Vecteur d'expression élevée du gène imd16 humain et son application - Google Patents

Vecteur d'expression élevée du gène imd16 humain et son application Download PDF

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Publication number
WO2018170710A1
WO2018170710A1 PCT/CN2017/077397 CN2017077397W WO2018170710A1 WO 2018170710 A1 WO2018170710 A1 WO 2018170710A1 CN 2017077397 W CN2017077397 W CN 2017077397W WO 2018170710 A1 WO2018170710 A1 WO 2018170710A1
Authority
WO
WIPO (PCT)
Prior art keywords
imd16
pegfp
expression vector
gene
pcr
Prior art date
Application number
PCT/CN2017/077397
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English (en)
Chinese (zh)
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/CN2017/077397 priority Critical patent/WO2018170710A1/fr
Publication of WO2018170710A1 publication Critical patent/WO2018170710A1/fr

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Definitions

  • the present invention belongs to the field of biotechnology, and relates to a method for constructing a human IMD16 gene high expression vector and an application thereof.
  • IMD16 is a member of the TNF receptor superfamily and is a type I transmembrane glycoprotein.
  • the expression profile of IMD16 is restricted to the surface of activated CD4+ and CD8+ T cells, and is CD4+
  • IMD16L/CD 134L Human IMD16 ligand contained 183 amino acids in 1991 (extracellular 139 amino acids, transmembrane 21 amino acids, intracellular 23 amino acids), belonging to the TNF family, and is a scorpion-type transmembrane glycoprotein.
  • IMD16/GP34 is an important co-stimulatory molecule that plays an important role in the body's immune response and various diseases. Its interaction can promote the activation, proliferation, migration, prolongation of life and promote hair growth of CD+4 T cells. The formation of the center and the differentiation and maturation of DC.
  • the object of the present invention is to overcome the deficiencies in the prior art and to provide a method for constructing a human IMD16 gene expression vector.
  • the overexpression vector pEGFP-Cl/IMD16 was constructed to lay a foundation for the subsequent study of human IMD16 gene function.
  • a method for constructing a human IMD16 gene overexpression vector comprising the steps of:
  • the purified PCR product and the eukaryotic expression vector pEGFP-Cl were digested with restriction enzymes Xho I and EcoR I, identified and recovered by 1% agarose gel electrophoresis;
  • the target gene fragment was ligated with the same double-digested expression vector pEGFP-Cl; the reaction system was: 10XT4 DNA ligase Buffer 1 ⁇ , linearized pEGFP-Cl vector 1 ⁇ , PCR product 3 ⁇ , T4 DNA ligase 1 ⁇ , dH20 4 L, 4 °C ligation overnight; the ligation product was transformed into E.
  • coli DH5ot competent cells ice bath for 30 min, heat shock at 42 °C for 90 s, immediately transferred to ice for 2 min, then added to 500 37
  • the pre-warmed non-resistant LB medium was incubated at 37 °C for 1 h.
  • the bacterial solution was uniformly coated on LB solid medium containing kanamycin, and placed in a 37 ° C incubator for overnight culture. Positive colonies were screened; 3 monoclonal colonies were randomly picked. After the colony PCR was positive, the bacterial liquid was sent to Shanghai Biotech for sequencing.
  • the obtained recombinant eukaryotic expression plasmid was named pEGFP-Cl/IMD16.
  • the present invention constructs the overexpression vector pEGFP-Cl/IMD16 of the IMD16 gene. Subsequent development of overexpression I MD16 gene will play an important role in IMD16-related drug research and development.
  • Jurkat cells and 293T cells were purchased from ATCC, Premix PrimeSTAR
  • HS enzyme was purchased from Takara, RNeasy Mini Kit was purchased from QIAGEN, Endo-Free Plasmid
  • Mini Kit II was purchased from Omega bio-tek.
  • Example 1 Cloning of the IMD16 gene [0016] Total RNA of Jurkat cells was extracted, reverse-transcribed into cDNA, primers (IMD16-F, IMD16-R) were designed, cDNA was used as a template, and primers and PrimeStar high-fidelity DNA polymerase were used for PCR amplification according to a conventional method. The reaction conditions were: 98 ° C 2 min; 98. C 10 s, 58. C 10 s, 72 ° C 50 s, 30 cycles; 72 ° C 5 min. The PCR amplification products were identified by agarose gel electrophoresis. The nucleotide sequence of IMD16-F is shown in SEQ ID No: 1, and the nucleotide sequence of IMD16-R is shown in SEQ ID No: 2.
  • the purified PCR product and the eukaryotic expression vector pEGFP-Cl were digested with restriction enzymes Xho I and EcoR I, identified and recovered by 1% agarose gel electrophoresis;
  • the target gene fragment was ligated with the same double-digested expression vector pEGFP-Cl; the reaction system was: 10XT4 DNA ligase Buffer 1 ⁇ , linearized pEGFP-Cl vector 1 ⁇ , PCR product 3 ⁇ , T4 DNA ligase 1 ⁇ , dH20 4 L, 4 °C ligation overnight; the ligation product was transformed into E.
  • coli DH5ot competent cells ice bath for 30 min, heat shock at 42 °C for 90 s, immediately transferred to ice for 2 min, then added to 500 37
  • the pre-warmed non-resistant LB medium was incubated at 37 °C for 1 h.
  • the bacterial solution was uniformly coated on LB solid medium containing kanamycin, and placed in a 37 ° C incubator for overnight culture. Positive colonies were screened; 3 monoclonal colonies were randomly picked. After the colony PCR was positive, the bacterial liquid was sent to Shanghai Biotech for sequencing.
  • the obtained recombinant eukaryotic expression plasmid was named pEGFP-Cl/IMD16.
  • Escherichia coli containing the PEGFP-C1/IMD16 plasmid was cultured in large amounts, and the pEGFP-Cl/IMD16 plasmid was extracted from Endo-Free Plasmid Mini Kit II. 293T cells with good growth were inoculated into six wells with 1000 000 cells per well. After 18 h of culture, Lipofectamine was used.
  • the pEGFP-Cl/IMD16 plasmid was transduced into 293T cells in 2000 and cultured for 48 h.
  • the primer design software Oligo 7.0 was used to design the bow.
  • the RT reagent kit reverse-transcribes mRNA into cDNA and stores it at -20 °C.
  • Kit detected the relative expression of IMD16 gene in each group, and the results are shown in Figure 1. It can be seen that the expression level of IMD 16 gene in 293T cells transfected with pEG FP-C1/IMD 16 plasmid is more than 160-fold higher than that of normal 293T cells, indicating that the present invention provides PEGFP-C1/IMD16 plasmid specificity, Continuous, efficient, and stable promotion of high expression of IMD16 gene.
  • the present invention constructs the overexpression vector pEGFP-Cl/IMD16 of the IMD16 gene. Subsequent development of overexpression I MD16 gene will play an important role in IMD16-related drug research and development.

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  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne un procédé de construction d'un vecteur d'expression du gène IMD16 humain, qui comprend les étapes consistant à : (1) cloner un gène IMD16 ; et (2) construire un vecteur de surexpression pEGFP-C1/IMD16.
PCT/CN2017/077397 2017-03-20 2017-03-20 Vecteur d'expression élevée du gène imd16 humain et son application WO2018170710A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2017/077397 WO2018170710A1 (fr) 2017-03-20 2017-03-20 Vecteur d'expression élevée du gène imd16 humain et son application

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2017/077397 WO2018170710A1 (fr) 2017-03-20 2017-03-20 Vecteur d'expression élevée du gène imd16 humain et son application

Publications (1)

Publication Number Publication Date
WO2018170710A1 true WO2018170710A1 (fr) 2018-09-27

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Family Applications (1)

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WO (1) WO2018170710A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2718868A1 (fr) * 2008-03-18 2009-09-24 Centre National De La Recherche Scientifique Polynucleotides et polypeptides chimeriques permettant la secretion d'un polypeptide d'interet en association avec des exosomes et leur utilisation pour la production de compositions immunogenes
CN102061310A (zh) * 2010-11-24 2011-05-18 中国人民解放军第四军医大学 人fhl1c真核表达载体的构建及其应用
CN102337297A (zh) * 2011-10-21 2012-02-01 南京医科大学 一种mbr-FPGS高效表达载体及其构建方法和应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2718868A1 (fr) * 2008-03-18 2009-09-24 Centre National De La Recherche Scientifique Polynucleotides et polypeptides chimeriques permettant la secretion d'un polypeptide d'interet en association avec des exosomes et leur utilisation pour la production de compositions immunogenes
CN102061310A (zh) * 2010-11-24 2011-05-18 中国人民解放军第四军医大学 人fhl1c真核表达载体的构建及其应用
CN102337297A (zh) * 2011-10-21 2012-02-01 南京医科大学 一种mbr-FPGS高效表达载体及其构建方法和应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LIU, HONGXIANG ET AL.,: "Advances in Research on TNF-TNFR Superfamily T Cell Costimulatory Molecules and Organ Transplantation", SURGERY FOREIGN MEDICAL SCIENCES, vol. 32, no. 6, 30 November 2005 (2005-11-30), pages 401 - 404 *

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