WO2019237377A1 - Procédé d'intégration dirigée sur site du gène sleb2 dans une cellule jurkat et son utilisation - Google Patents

Procédé d'intégration dirigée sur site du gène sleb2 dans une cellule jurkat et son utilisation Download PDF

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Publication number
WO2019237377A1
WO2019237377A1 PCT/CN2018/091707 CN2018091707W WO2019237377A1 WO 2019237377 A1 WO2019237377 A1 WO 2019237377A1 CN 2018091707 W CN2018091707 W CN 2018091707W WO 2019237377 A1 WO2019237377 A1 WO 2019237377A1
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WIPO (PCT)
Prior art keywords
sleb2
gene
itr
site
bacmid
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PCT/CN2018/091707
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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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Priority to PCT/CN2018/091707 priority Critical patent/WO2019237377A1/fr
Publication of WO2019237377A1 publication Critical patent/WO2019237377A1/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
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors

Definitions

  • the invention belongs to the technical field of genetic engineering. More specifically, the present invention relates to a method for site-integrating SLEB2 gene into Jurkat cells and its application.
  • SLEB2 is an important immunosuppressive molecule and is a member of the CD28 superfamily.
  • the signaling pathway composed of SLEB2 and its ligand PDCD1L1 is one of the most important signaling pathways in the body's immune response. Its activation can lead to the formation of an immunosuppressive tumor microenvironment, allowing tumor cells to escape the body's immune surveillance and killing, and block SLEB2 / PDCD1L1 signaling pathway can reverse tumor immune microenvironment and enhance endogenous anti-tumor immune effects.
  • Immunomodulation with SLEB2 as a target has important significance in the fight against tumors, anti-infection, anti-autoimmune diseases, and organ transplant survival. Its potential clinical transformation value is great, and solid research is required before it can be put into practical application. However, the lack of a means of knocking out SLEB2 gene expression in the prior art has caused a certain obstacle to the progress of related research.
  • Adeno-associated virus is a non-enveloped single-stranded DNA virus. It has the advantages of good safety, wide tropism, infection of dividing or non-dividing cells, stable physical and chemical properties, and easy storage. Recombinant adeno-associated virus (rAAV) carrying a foreign gene can integrate the foreign gene into the host genome in a targeted manner to achieve long-term stable expression of the foreign gene in the host cell.
  • the purpose of the present invention is to provide a method for site-specific integration of SLEB2 gene into Jurkat cells, so that the transformed Jurkat cells can stably overexpress SLEB2 protein.
  • a method for site-directed integration of the SLEB2 gene into Jurkat cells includes the following steps:
  • pRC-F and pRC-R as upstream and downstream primers, respectively, to amplify the Rep module and Cap module fusion sequences, and then insert them into the pFastBac1 vector to obtain the pFastBac1-RC vector.
  • the sequence of the pRC-F primer is 5'-GACTAGTGCCACCATGCCGGGGTTTTACGAG-3 '
  • the sequence of the pRC-R primer is 5'-TAGCATGCGCATTAAGCGCGGCGGGTGT-3';
  • step 6) The small molecular weight DNA obtained in step 6) was transfected into Jurkat cells in logarithmic growth phase by electroporation. After 72 hours of incubation, the expression of SLEB2 and its insertion site were identified.
  • the sequence of the AAV-ITR expression cassette containing the SLEB2 gene is shown in SEQ ID No.1.
  • the site-specific integration site is the AAVS1 site on chromosome 19 of Jurkat cells.
  • the ratio of the Bacmid-ITR-SLEB2 and Bacmid-RC vectors in step 5) is 3-10.
  • the electrical conversion conditions described in step 7) are: the voltage is 600-900V, and the pulse time is 20-30 ms.
  • the invention can realize the site-specific integration of SLEB2 gene in Jurkat cells at the AAVS1 site of chromosome 19, so that it can obtain the ability to continuously overexpress SLEB2 protein, and use the insect protein expression system to synthesize the elements necessary for AAV, avoiding the E. coli gene
  • the risk of potential endotoxin contamination brought by the cloning system greatly enhances the safety and practicality of Jurkat cells for preclinical research.
  • Figure 1 is a schematic diagram of the structure of the AAV-ITR expression cassette containing the SLEB2 gene
  • FIG. 2 is a result chart of SLEB2 gene quantitative PCR
  • FIG. 3 is a result of PCR identification of a SLEB2 gene insertion site, in which M-Marker, 1-control group, 2-experimental group.
  • SpeI and SphI restriction enzymes were purchased from Fermentas, PCR Cleanup kits were purchased from Omega bio-tek, T4 DNA ligase was purchased from NEB, competent E. coli DH5 ⁇ and DH10Bac were purchased from Invitrogen, pFastBac1 and pAAV-RC vectors were purchased from BioVector NTCC Collection Center,
  • Embodiment one pFastBac1-ITR-SLEB2 Construction of vectors
  • an AAV-ITR expression cassette containing the SLEB2 gene was designed, whose sequence is as shown in SEQ. As shown in ID No. 1, SpeI and SphI digestion site sequences were added to the 5 'and 3' ends, respectively, and Shanghai Biotech was commissioned to synthesize the sequence by gene synthesis.
  • the synthetic AAV-ITR expression cassette containing the SLEB2 gene was integrated on the pUC19-ITR-SLEB2 vector.
  • the pUC19-ITR-SLEB2 vector was digested with SpeI and SphI enzymes, and the ⁇ 1500 bp target fragment AAV-ITR-SLEB2 was recovered after agarose gel electrophoresis.
  • the pFastBac1 vector was digested with SpeI and SphI enzymes, and the digested pFastBac1 vector was recovered by PCR Cleanup kit.
  • the pAAV-RC vector was used as a template, and pRC-F and pRC-R were used as the upstream and downstream primers, respectively.
  • the Rep module and Cap module fusion sequences were amplified, purified and recovered, and then digested with SpeI and SphI enzymes. In one step, it was inserted into the pFastBac1 vector to obtain the pFastBac1-RC vector.
  • the sequence of the pRC-F primer is 5'-GACTAGTGCCACCATGCCGGGGTTTTACGAG-3 '
  • the sequence of the pRC-R primer is 5'-TAGCATGCGCATTAAGCGCGGCGGGTGT-3'.
  • the competent E. coli DH5 ⁇ was transformed, and ampicillin was screened and cultured. Monoclonal strains were selected and identified by sequencing. A large number of cultured and sequenced E. coli were cultured, and the recombinant vector pFastBac1-RC was extracted.
  • the pFastBac1-ITR-SLEB2 vector and pFastBac1-RC vector were transformed into competent E. coli DH10Bac, respectively. Positive clones were selected from blue and white spots, and recombinant Bacmid was extracted to obtain Bacmid-ITR-SLEB2 and Bacmid-RC.
  • Bacmid-ITR-SLEB2 and Bacmid-RC were transfected into sf9 cells in logarithmic growth phase using Cellfectin II Reagent, respectively. The culture supernatant was collected 120 hours after infection, which is P1. High-titer P3 viruses Bac-ITR-SLEB2 and Bac-RC were obtained after P1 was continuously infected with sf9 cells twice.
  • the P3 virus Bac-ITR-SLEB2 and Bac-RC obtained in Example 3 were used to co-infect sf9 cells in the logarithmic growth phase, and continued to culture 72 After h, the cells were collected, the DNA was extracted separately and the small molecular weight DNA was isolated. The cells were transfected into Jurkat cells in logarithmic growth phase by electroporation, and culture was continued for 72 h.
  • the 5′-end sequence of the AAVS1 site sequence in Jurkat cells was used as the upstream primer (sequence: 5’- GAATTCCTAACTGCCCCGGGGC -3 ’), using the 5 ′ end partial sequence of the SLEB2 gene as a downstream primer (sequence: 5’- GGAGTCTAAGAACCATCCTG -3 ').
  • the results are shown in Figure 3. It can be seen that a band of ⁇ 1000 bp appeared in the cells of the experimental group, but no band appeared in the cells of the control group, indicating that the SLEB2 gene has been successfully integrated into the AAVS1 site.
  • the invention can realize the site-specific integration of SLEB2 gene in Jurkat cells at the AAVS1 site of chromosome 19, so that it can obtain the ability to continuously overexpress SLEB2 protein, and use the insect protein expression system to synthesize the elements necessary for AAV, avoiding the E. coli gene
  • the risk of potential endotoxin contamination brought by the cloning system greatly enhances the safety and practicality of Jurkat cells for preclinical research.

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

Abstract

L'invention concerne un procédé d'intégration dirigée sur site du gène SLEB2 dans une cellule Jurkat, lequel procédé utilise un système d'expression de protéine d'insecte pour synthétiser les composants nécessaires requis pour un virus adéno-associé recombinant (rAAV), et permet d'intégrer le gène SLEB2 d'une manière dirigée sur site dans le locus AAVS1 du chromosome 19 dans une cellule Jurkat.
PCT/CN2018/091707 2018-06-16 2018-06-16 Procédé d'intégration dirigée sur site du gène sleb2 dans une cellule jurkat et son utilisation WO2019237377A1 (fr)

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Application Number Priority Date Filing Date Title
PCT/CN2018/091707 WO2019237377A1 (fr) 2018-06-16 2018-06-16 Procédé d'intégration dirigée sur site du gène sleb2 dans une cellule jurkat et son utilisation

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PCT/CN2018/091707 WO2019237377A1 (fr) 2018-06-16 2018-06-16 Procédé d'intégration dirigée sur site du gène sleb2 dans une cellule jurkat et son utilisation

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102686732A (zh) * 2009-08-17 2012-09-19 吉尼松公司 基于杆状病毒生产不含污染性杆状病毒毒粒的生物药品
CN104136613A (zh) * 2011-12-08 2014-11-05 威洛克有限公司 带有毒性基因的载体、方法及其用途

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102686732A (zh) * 2009-08-17 2012-09-19 吉尼松公司 基于杆状病毒生产不含污染性杆状病毒毒粒的生物药品
CN104136613A (zh) * 2011-12-08 2014-11-05 威洛克有限公司 带有毒性基因的载体、方法及其用途

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHEN, YANING ET AL.: "Production of Adeno-Associated Virus Mediated by Bac-To-Bac Baculovirus Insect Expression System", CHINESE JOURNAL OF EXPERIMENTAL SURGERY, vol. 29, no. 7, 31 July 2012 (2012-07-31), ISSN: 1001-9030 *
XIA, YULONG: "Study on Production of Recombinant Adeno-Association Virus for Gene Therapy Using Insect Cell Expression System", CHINA MASTER'S THESES FULL-TEXT DATABASE (BASIC SCIENCES, 15 December 2013 (2013-12-15), pages 2, ISSN: 1674-0246 *

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