WO2019033247A1 - Production d'une lignée cellulaire à expression stable et élevée du gène sisp1 et son utilisation - Google Patents

Production d'une lignée cellulaire à expression stable et élevée du gène sisp1 et son utilisation Download PDF

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Publication number
WO2019033247A1
WO2019033247A1 PCT/CN2017/097430 CN2017097430W WO2019033247A1 WO 2019033247 A1 WO2019033247 A1 WO 2019033247A1 CN 2017097430 W CN2017097430 W CN 2017097430W WO 2019033247 A1 WO2019033247 A1 WO 2019033247A1
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WIPO (PCT)
Prior art keywords
sisp1
gene
cell line
expression vector
cell
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PCT/CN2017/097430
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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/CN2017/097430 priority Critical patent/WO2019033247A1/fr
Publication of WO2019033247A1 publication Critical patent/WO2019033247A1/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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention belongs to the fields of bioengineering and medical technology, and particularly relates to a SISP1 gene stable high expression cell line and its application in drug screening.
  • the SISP1 protein is a novel member of the PD-L1 protein family and is a novel and structurally different Ig superfamily inhibitory ligand whose extracellular domain has homology to the B7 family ligand PD-L1.
  • SISP1 protein plays an important role in various cancers and autoimmune diseases, allergies, infections and inflammatory diseases such as multiple sclerosis and cellular immunity of joint disorders, and requires extensive transformation studies for clinical application.
  • the lack of cell lines that promote the expression of SISP1 gene in the prior art has hindered the progress of related research.
  • a method for establishing a stable high expression cell line of SISP1 gene comprises the following steps:
  • the recombinant expression vector obtained in the step 1 is introduced into a host cell, and a cell line stably expressing the SISP1 gene is selected.
  • the eukaryotic expression vector of the present invention is pIRES2-EGFP, and its map is shown in FIG.
  • the host cell of the present invention is HeLa cell, HepG2 cell or MCF-7 cell.
  • the method of the present invention wherein the prepared SISP1 gene stably stabilizes a cell line.
  • the SISP1 gene stably expressing cell line according to the present invention is characterized in that the SISP1 gene stable expression cell line is a HepG2 cell line stably expressing the SISP1 gene.
  • the present invention encodes a SISP1 gene protein coding frame cDNA sequence, and the primers are as follows:
  • SISP1 -sense 5,- GGAATTCATGGGCGTCCCCACGGCC -3
  • SISPl -antisense 5'-GCCCGGGCTAGATGACCTCAAAGTTTG -3'.
  • the SISP1 gene stable high expression cell line of the present invention provides an experimental technical platform for further exploration of the role of the SISP1 gene, and can be used in drug research and development related to abnormal expression of SISP1.
  • 1 is a map of the pIRES2-EGFP vector.
  • FIG. 2 is a schematic diagram showing the results of SISP1 gene expression levels of cells transduced with pIRES2-EGFP-SISP1 vector.
  • PCR primers were designed as follows: Forward primer: 5'-GGAATTCATGGGCGTCCCCACGGCC-3'; Reverse primer: 5'-GCCCGGGCTAGATGACCTCAAAGTTTG-3'.
  • Jurkat cells were taken and total RNA was extracted, reverse-transcribed into cDNA and used as a template, and PCR amplification was carried out using the above primers.
  • the PCR amplification product was purified and recovered by agarose gel electrophoresis.
  • the purified product was digested with restriction endonucleases Ec oR I and Xma l, and the digested product was subjected to agarose gel electrophoresis purification and recovery. , the cDNA sequence of the SISP1 protein coding frame was obtained.
  • the eukaryotic expression vector pIRES 2-EGFP was also digested with EcoR I and Xma I, and the digested product was purified and recovered by agarose gel electrophoresis.
  • the cDNA sequence of the SISP1 protein coding frame amplified by the above amplification and the product of the eukaryotic expression vector PIRES2-EGFP were ligated with DNA ligase, and the ligated product was transformed into competent E. coli JM107 and coated with kanamycin. On the LB plate, it was sent to Shanghai Biotech for sequencing after cultivation. The sequencing results showed that the cDNA sequence of the inserted SISP1 protein coding frame was identical to the sequence recorded on GenBank.
  • the resulting recombinant expression vector was named pIRES2-EGFP-SISP1.
  • the recombinant Escherichia coli obtained in the above step 1 was extracted with a Plasmid Plus SV Miniprep (purchased from Promega) to obtain a high-purity recombinant expression vector pIRES2-EGFP-SISP1, and the recombinant expression vector pIRES2-EGFP-SISP1 was utilized Lipofectamine2000 ( Transfected HepG2 cells from Invitrogen and continued to culture the transfected HepG2 cells for more than 24 hours. With neomycin (concentration 1
  • HepG2 cells and HepG2 cells transduced with pIRES2-EGFP-SISP1 vector were separately inoculated into 6-well plates. Cell density reaches ⁇ , with RNeasy Mini
  • Kit extracts total RNA from each group of cells, using PrimeScrip RT reagent
  • Kit reverse transcribes mRNA into cDNA and stores at -20 °C. Take cDNA 1 from each group of cells
  • SISP1 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 2. It can be seen that the expression level of SISP1 gene in HepG2 cells transfected with pIRES2-EGFP-SISP1 vector is more than 150-fold higher than that of Jurkat cells, indicating that the SISP1 gene cDNA sequence provided by the present invention is successfully inserted into the PIRES2-EGFP expression vector. It can promote the high expression of SI SP1 gene specifically, continuously, efficiently and stably.
  • the SISP1 gene stable high expression cell line of the present invention provides an experimental technical platform for further exploration of the role of the SISP1 gene, and can be used in drug research and development related to abnormal expression of SISP1.

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  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Cell Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne un procédé de production d'une lignée cellulaire à expression stable et élevée du gène SISP1, le procédé comprenant : (1) l'insertion de la séquence codante du gène SISP1 dans des sites de clonage multiples d'un vecteur d'expression eucaryote afin d'obtenir un vecteur d'expression recombinant; et (2) l'introduction du vecteur d'expression recombinant obtenu à l'étape (1) dans une cellule hôte, et le criblage des cellules afin d'obtenir une lignée cellulaire exprimant le gène SISP1 de manière stable. La lignée cellulaire à expression stable et élevée du gène SISP1 peut être utilisée pour cribler des médicaments associés.
PCT/CN2017/097430 2017-08-14 2017-08-14 Production d'une lignée cellulaire à expression stable et élevée du gène sisp1 et son utilisation WO2019033247A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2017/097430 WO2019033247A1 (fr) 2017-08-14 2017-08-14 Production d'une lignée cellulaire à expression stable et élevée du gène sisp1 et son utilisation

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Application Number Priority Date Filing Date Title
PCT/CN2017/097430 WO2019033247A1 (fr) 2017-08-14 2017-08-14 Production d'une lignée cellulaire à expression stable et élevée du gène sisp1 et son utilisation

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WO2019033247A1 true WO2019033247A1 (fr) 2019-02-21

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030083482A1 (en) * 1999-09-30 2003-05-01 Maureen Murphy Compositions and methods for p53-mediated repression of gene expression
WO2007100211A1 (fr) * 2006-02-28 2007-09-07 Curonix Co., Ltd Sisp-1, un nouveau gène cible p53 et son utilisation
CN103614414A (zh) * 2013-11-25 2014-03-05 河南省华隆生物技术有限公司 Afp和il-2双基因共表达重组载体及其制备方法和应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030083482A1 (en) * 1999-09-30 2003-05-01 Maureen Murphy Compositions and methods for p53-mediated repression of gene expression
WO2007100211A1 (fr) * 2006-02-28 2007-09-07 Curonix Co., Ltd Sisp-1, un nouveau gène cible p53 et son utilisation
CN103614414A (zh) * 2013-11-25 2014-03-05 河南省华隆生物技术有限公司 Afp和il-2双基因共表达重组载体及其制备方法和应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DONG-WOOK KIM: "Anti-proliferative effect of honokiol in oral squamous cancer through the regulation of specificity protein 1", INTERNATIONAL JOURNAL OF ONCOLOGY, vol. 43, no. 4, October 2013 (2013-10-01), pages 1103 - 1110, XP055576415, ISSN: 1019-6439, DOI: 10.3892/ijo.2013.2028 *
YE JIANXIN ET AL.: "Construction and Identification of the Recombinant Vaccione of attenuated Salmonella Typhimurium Containing pIRES2-EGFP-4-IBBL vector.", CHINESE JOURNAL OF IMMUNOLOGY, vol. 25, no. 4, 31 December 2009 (2009-12-31), pages 336 - 340 *

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