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WO2018170709A1 - Vecteur d'expression du gène tnlg5a humain et son application - Google Patents

Vecteur d'expression du gène tnlg5a humain et son application Download PDF

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Publication number
WO2018170709A1
WO2018170709A1 PCT/CN2017/077396 CN2017077396W WO2018170709A1 WO 2018170709 A1 WO2018170709 A1 WO 2018170709A1 CN 2017077396 W CN2017077396 W CN 2017077396W WO 2018170709 A1 WO2018170709 A1 WO 2018170709A1
Authority
WO
WIPO (PCT)
Prior art keywords
tnlg5a
pegfp
gene
vector
cells
Prior art date
Application number
PCT/CN2017/077396
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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/CN2017/077396 priority Critical patent/WO2018170709A1/fr
Publication of WO2018170709A1 publication Critical patent/WO2018170709A1/fr

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  • the present invention belongs to the field of biotechnology, and relates to a method for constructing a human TNLG5A gene expression vector and an application thereof.
  • ILA belongs to the TNFR superfamily and is mainly expressed in activated T cells and is an inducible T cell surface receptor;
  • TNLG5A belongs to the TNF superfamily and is mainly expressed in concentrated antigen presenting cells (APC).
  • APC concentrated antigen presenting cells
  • IL A/TNLG5A is another important costimulatory molecule other than CD28/B7, which may or may not be dependent on CD28/
  • the B7 pathway mediates the production of costimulatory signals that induce T cell activation, proliferation, and cytokine secretion.
  • ILA and its ligand system have two-way signal transduction, which can transmit cells to T cells through TNLG5A, and can transmit signals to cells expressing ligands, which plays an important role in tumor immunotherapy, and needs to be done.
  • a large number of studies can achieve clinical transformation, but the lack of recombinant vectors with high expression of TNLG5A gene in the prior art has hindered the progress of related research.
  • the object of the present invention is to overcome the deficiencies in the prior art and to provide a method for constructing a human TNLG5A gene expression vector.
  • the expression vector pEGFP-Cl/TNLG5A was constructed, which laid a foundation for the subsequent study of human TNLG5A gene function.
  • a method for constructing a human TNLG5A gene overexpression vector comprising the steps of:
  • RNA of Jurkat cells was extracted and reverse transcribed into cDNA, and primers TNLG5A-F and TNLG5A-R were designed.
  • the 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 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/TNLG5A.
  • the present invention constructs the overexpression vector pEGFP-Cl/TNLG5A of the TNLG5A gene. Subsequent development of the TNLG5A gene will play an important role in TNLG5A-related drug research and development.
  • 1 is the relative level of the TNLG5 A gene of 293T cells transfected with pEGFP-C 1/TNLG5 A vector.
  • 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.
  • 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/TNLG5A.
  • the primer design software Oligo 7.0 was used to design bows [0023] , CR production of the country
  • GGGTCGG GA@CTTTGeCCeG
  • Kit extracts total RNA from each group of cells, using PrimeScrip RT reagent
  • Kit reverse transcribes mRNA into cDNA and stores at -20 °C.
  • TNLG5A As a template, GAPDH was used as an internal reference, and the relative expression of TNLG5A was detected by real-time PCR. The reaction conditions were set: 95 ° C for 30 s, 1 cycle, 95 ° C for 10 s, and 54 ° C for 30 s for 40 cycles. The relative expression of TNLG5A gene in each group was detected by SYBR Primescript RT-PCR Kit. The results are shown in Figure 1. It can be seen that the expression level of TNLG5A gene in 293T cells transfected with pEGFP-Cl/TNLG5A plasmid is 170-fold higher than that of normal 293T cells, indicating that the pEGFP-Cl/TNLG5A plasmid can be specific, sustained and efficient. , stably promote high expression of TNLG5A gene.
  • the present invention constructs the overexpression vector pEGFP-Cl/TNLG5A of the TNLG5A gene. Subsequent development of the TNLG5A gene will play an important role in TNLG5A-related drug research and development.

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  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne une méthode de construction d'un vecteur d'expression du gène TNLG5A humain, comprenant les étapes suivantes : (1) clonage d'un gène TNLG5A ; et (2) construction d'un vecteur de surexpression pEGFP-Cl/TNLG5A.
PCT/CN2017/077396 2017-03-20 2017-03-20 Vecteur d'expression du gène tnlg5a humain et son application WO2018170709A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2017/077396 WO2018170709A1 (fr) 2017-03-20 2017-03-20 Vecteur d'expression du gène tnlg5a humain et son application

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2017/077396 WO2018170709A1 (fr) 2017-03-20 2017-03-20 Vecteur d'expression du gène tnlg5a humain et son application

Publications (1)

Publication Number Publication Date
WO2018170709A1 true WO2018170709A1 (fr) 2018-09-27

Family

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2017/077396 WO2018170709A1 (fr) 2017-03-20 2017-03-20 Vecteur d'expression du gène tnlg5a humain et son application

Country Status (1)

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WO (1) WO2018170709A1 (fr)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101528779A (zh) * 2006-10-16 2009-09-09 斯克利普斯研究院 炎症性疾病中的4-1bb配体

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101528779A (zh) * 2006-10-16 2009-09-09 斯克利普斯研究院 炎症性疾病中的4-1bb配体

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE Nucleotide 1 September 2016 (2016-09-01), GOODWIN RG: "Homo sapiens tumor necrosis factor superfamily member 9 (TNFSF9), mRNA", XP055608407, retrieved from NCBI Database accession no. NM_003811 *
SUN, SHUNTAO ET AL.,: "Construction of Expression Vector with Human Costimulatory Molecules 4-1BBL and its Stable Expression in Tca8113 Cells", CHINA JOURNAL OF ORAL AND MAXILLOFACIAL SURGERY, vol. 6, no. 3, 31 May 2008 (2008-05-31), pages 188 - 193 *

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