WO2019237376A1 - Procédé d'intégration dirigée sur site d'un gène txgp1 dans une cellule raji et son utilisation - Google Patents

Procédé d'intégration dirigée sur site d'un gène txgp1 dans une cellule raji et son utilisation Download PDF

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Publication number
WO2019237376A1
WO2019237376A1 PCT/CN2018/091706 CN2018091706W WO2019237376A1 WO 2019237376 A1 WO2019237376 A1 WO 2019237376A1 CN 2018091706 W CN2018091706 W CN 2018091706W WO 2019237376 A1 WO2019237376 A1 WO 2019237376A1
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WIPO (PCT)
Prior art keywords
txgp1
gene
itr
site
bacmid
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Ceased
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PCT/CN2018/091706
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English (en)
Chinese (zh)
Inventor
毛吉炎
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Shenzhen Biocan Technologies Co Ltd
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Shenzhen Biocan Technologies Co Ltd
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Priority to PCT/CN2018/091706 priority Critical patent/WO2019237376A1/fr
Publication of WO2019237376A1 publication Critical patent/WO2019237376A1/fr
Anticipated expiration legal-status Critical
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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-integrated TXGP1 gene into Raji cells and its application.
  • TXGP1 is a member of the TNF receptor superfamily and is a type I transmembrane glycoprotein. TXGP1 expression profile is limited to the surface of activated CD4 + and CD8 + T cells, and is predominantly CD4 + T cells. Human TXGP1 ligand belongs to the TNF family and is a type II transmembrane glycoprotein. IMD16 / TXGP1 is an important pair of co-stimulatory molecules that play an important role in the body's immune response and various diseases. Their interactions can promote the activation, proliferation, and migration of CD + 4 T cells, extend their life span, and promote germination. The formation of centers and the differentiation of DCs mature.
  • TXGP1 can synergistically stimulate the activation of T cells, promote the production of high titer antibodies and class conversion by B cells, and mediate the infiltration of IMD16 + T cells into the inflammatory response site, which plays an important role in tumor immunotherapy and its potential clinical transformation value It is very large and requires solid research before it can be put into practical use. However, the lack of a means of knocking out TXGP1 gene expression in the prior art has caused certain obstacles 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 TXGP1 gene into Raji cells, so that the transformed Raji cells stably overexpress TXGP1 protein.
  • a method for site-directed integration of TXGP1 gene into Raji cells which includes the following steps:
  • the AAV-ITR expression cassette containing the TXGP1 gene is inserted into a pFastBac1 vector to construct a plasmid pFastBac1-ITR-TXGP1;
  • 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) Transfection of small molecular weight DNA obtained in step 6) into Raji cells in logarithmic growth phase by electroporation. After 72 hours of incubation, the expression of TXGP1 and its insertion site were identified.
  • the sequence of the AAV-ITR expression cassette containing the TXGP1 gene is shown in SEQ ID No.1.
  • the site-specific integration site is the AAVS1 site of chromosome 19 of Raji cells.
  • the ratio of the Bacmid-ITR-TXGP1 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 TXGP1 gene in Raji cells at the AAVS1 site of chromosome 19, so that it can obtain the ability to continuously overexpress TXGP1 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 has greatly enhanced the safety and practicality of Raji cells for preclinical research.
  • Figure 1 is a schematic diagram of the structure of the AAV-ITR expression cassette containing the TXGP1 gene
  • FIG. 2 is a result chart of TXGP1 gene fluorescent quantitative PCR
  • FIG. 3 is a result of PCR identification of a TXGP1 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-TXGP1 Construction of vectors
  • an AAV-ITR expression cassette containing the TXGP1 gene was designed, and its 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 TXGP1 gene was integrated on the pUC19-ITR-TXGP1 vector.
  • the pUC19-ITR-TXGP1 vector was digested with SpeI and SphI enzymes, and the ⁇ 1500 bp target fragment AAV-ITR-TXGP1 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.
  • Example three High titer baculovirus Bac-ITR-TXGP1 with Bac -RC Preparation
  • the pFastBac1-ITR-TXGP1 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-TXGP1 and Bacmid-RC.
  • Cellfectin II Reagent was used to transfect Bacmid-ITR-TXGP1 and Bacmid-RC into sf9 cells in logarithmic growth phase. The culture supernatant was collected 120 hours after infection, which is P1. High-titer P3 virus Bac-ITR-TXGP1 and Bac-RC were obtained after P1 was continuously infected with sf9 cells twice.
  • the P3 virus Bac-ITR-TXGP1 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, DNA was extracted separately and the small molecular weight DNA was isolated, and it was transfected into Raji cells in the logarithmic growth phase by electroporation, and culture was continued for 72 h.
  • TXGP1 gene expression levels of transfected Raji cells (experimental group) and normal Raji cells (control group) were detected by real-time quantitative PCR. The results are shown in Figure 2. It can be seen that the TXGP1 gene expression level of the experimental group cells was significantly higher. In control cells, the TXGP1 gene sequence was successfully integrated into Raji cells.
  • the 5′-end sequence of the AAVS1 site of Raji cells was used as the upstream primer (sequence: 5’- GAATTCCTAACTGCCCCGGGGC -3 ’), using the 5’ end of the TXGP1 gene as a downstream primer (sequence: 5’- GAAACCTTTCTCCTTCTTATA -3 ') PCR, 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 TXGP1 gene has been successfully integrated into the AAVS1 site.
  • the invention can realize the site-specific integration of TXGP1 gene in Raji cells at the AAVS1 site of chromosome 19, so that it can obtain the ability to continuously overexpress TXGP1 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 has greatly enhanced the safety and practicality of Raji cells for preclinical research.

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
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  • 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)
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  • Virology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne un procédé d'intégration dirigée sur site du gène TXGP1 dans une cellule Raji, ledit procédé utilisant un système d'expression de protéine d'insecte pour synthétiser les composants nécessaires requis pour un virus adéno-associé recombinant (rAAV), et parvenant à intégrer le gène TXGP1 de manière ciblée dans le locus AAVS1 du chromosome 19 dans une cellule Raji.
PCT/CN2018/091706 2018-06-16 2018-06-16 Procédé d'intégration dirigée sur site d'un gène txgp1 dans une cellule raji et son utilisation Ceased WO2019237376A1 (fr)

Priority Applications (1)

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PCT/CN2018/091706 WO2019237376A1 (fr) 2018-06-16 2018-06-16 Procédé d'intégration dirigée sur site d'un gène txgp1 dans une cellule raji et son utilisation

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PCT/CN2018/091706 WO2019237376A1 (fr) 2018-06-16 2018-06-16 Procédé d'intégration dirigée sur site d'un gène txgp1 dans une cellule raji 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.: "Bac-to-Bacffl (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), pages 1363 - 1366, ISSN: 1001-9030 *
XIA, YULONG: "Production of Recombinant Adeno-Associated Virus for Gene Therapy by Insect Cell Expression System", BASIC SCIENCES, CHINA MASTER'S THESES FULL-TEXT DATABASE, 15 December 2013 (2013-12-15), ISSN: 1674-0246 *

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