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WO2018170711A1 - Vecteur d'expression élevée du gène gp34 humain et son application - Google Patents

Vecteur d'expression élevée du gène gp34 humain et son application Download PDF

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Publication number
WO2018170711A1
WO2018170711A1 PCT/CN2017/077398 CN2017077398W WO2018170711A1 WO 2018170711 A1 WO2018170711 A1 WO 2018170711A1 CN 2017077398 W CN2017077398 W CN 2017077398W WO 2018170711 A1 WO2018170711 A1 WO 2018170711A1
Authority
WO
WIPO (PCT)
Prior art keywords
pegfp
expression vector
gene
pcr
cells
Prior art date
Application number
PCT/CN2017/077398
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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/CN2017/077398 priority Critical patent/WO2018170711A1/fr
Publication of WO2018170711A1 publication Critical patent/WO2018170711A1/fr

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Definitions

  • the present invention belongs to the field of biotechnology, and relates to a method for constructing a human GP34 gene high expression vector and an application thereof.
  • GP34 is a member of the TNF receptor superfamily and is a type I transmembrane glycoprotein.
  • the expression profile of GP34 is restricted to the surface of activated CD4+ and CD8+ T cells, and is predominantly CD4+ T cells.
  • the human GP34 ligand (GP34/CD 134L) 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.
  • GP34 can synergistically stimulate the activation of T cells, promote the production of high titer antibodies and class switching of B cells, mediate the infiltration of I MD16+ T cells into the inflammatory reaction site, and play an important role in the immunotherapy of tumors.
  • the clinical transformation value is very large, and it requires a solid research before it can be put into practical use.
  • the lack of the plasmid of the recombinant GP34 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 GP34 gene expression vector.
  • the over-expression vector pEGFP-Cl/GP34 was constructed, which laid a foundation for the subsequent study of human GP34 gene function.
  • a method for constructing a human GP34 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/GP34.
  • the present invention constructs the overexpression vector pEGFP-Cl/GP34 of the GP34 gene. Subsequent development of overexpression GP3
  • the 4 gene will play an important role in GP34-related drug research and development.
  • 1 is the relative level of GP34 gene of 293T cells transfected with pEGFP-C 1/GP34 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
  • RNA of Jurkat cells was extracted, reverse-transcribed into cDNA, primers (GP34-F, GP34-R) were designed, and PCR was carried out according to a conventional method using c-DNA as a template, using primers and PrimeStar high-fidelity DNA polymerase.
  • 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
  • the PCR amplification products were identified by agarose gel electrophoresis.
  • the nucleotide sequence of GP34-F is shown in SEQ ID No: 1
  • the nucleotide sequence of GP34-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/GP34.
  • the primer design software Oligo 7.0 was used to design the bow.
  • the present invention constructs the overexpression vector pEGFP-Cl/GP34 of the GP34 gene. Subsequent development of overexpression GP3
  • the 4 gene will play an important role in GP34-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 GP34 humain, qui comprend les étapes consistant à : (1) cloner un gène GP34 ; et (2) construire un vecteur de surexpression pEGFP-C1/GP34.
PCT/CN2017/077398 2017-03-20 2017-03-20 Vecteur d'expression élevée du gène gp34 humain et son application WO2018170711A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2017/077398 WO2018170711A1 (fr) 2017-03-20 2017-03-20 Vecteur d'expression élevée du gène gp34 humain et son application

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2017/077398 WO2018170711A1 (fr) 2017-03-20 2017-03-20 Vecteur d'expression élevée du gène gp34 humain et son application

Publications (1)

Publication Number Publication Date
WO2018170711A1 true WO2018170711A1 (fr) 2018-09-27

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Application Number Title Priority Date Filing Date
PCT/CN2017/077398 WO2018170711A1 (fr) 2017-03-20 2017-03-20 Vecteur d'expression élevée du gène gp34 humain et son application

Country Status (1)

Country Link
WO (1) WO2018170711A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
US20110268750A1 (en) * 2008-03-18 2011-11-03 Universite Montpellier 2 Sciences Et Techniques Chimeric polynucleotides and polypeptides enabling secretion of a polypeptide of interest in association with exosomes and use thereof for the production of immunogenic compositions
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
US20110268750A1 (en) * 2008-03-18 2011-11-03 Universite Montpellier 2 Sciences Et Techniques Chimeric polynucleotides and polypeptides enabling secretion of a polypeptide of interest in association with exosomes and use thereof for the production of immunogenic compositions
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, JIAN ET AL.,: "OX40/OX40L in Autoimmune Diseases", MODERN PREVENTIVE MEDICINE, 31 December 2013 (2013-12-31), pages 956 - 959 *

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