WO2019237374A1 - Procédé d'intégration dirigée sur un site du gène mtabc3 dans une cellule a375 et son utilisation - Google Patents

Procédé d'intégration dirigée sur un site du gène mtabc3 dans une cellule a375 et son utilisation Download PDF

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Publication number
WO2019237374A1
WO2019237374A1 PCT/CN2018/091704 CN2018091704W WO2019237374A1 WO 2019237374 A1 WO2019237374 A1 WO 2019237374A1 CN 2018091704 W CN2018091704 W CN 2018091704W WO 2019237374 A1 WO2019237374 A1 WO 2019237374A1
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WO
WIPO (PCT)
Prior art keywords
mtabc3
gene
itr
site
cells
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PCT/CN2018/091704
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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/CN2018/091704 priority Critical patent/WO2019237374A1/fr
Publication of WO2019237374A1 publication Critical patent/WO2019237374A1/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-directed integration of the MTABC3 gene into A375 cells and its application.
  • MTABC is a large class of transmembrane proteins. It uses the energy of hydrolyzing ATP to transmembrane a variety of endogenous and exogenous biomolecules.
  • the substrates for translocation include: sugar, amino acid, metal ion, peptide, protein, cell Metabolites and drugs.
  • MTABC transporters are widely found in eukaryotes and prokaryotes, and 49 members of the MTABC transporter superfamily have been identified in the human genome to date.
  • MTABC tumor multidrug resistance
  • MTABC transporter MDR family MTABC transporter MDR family.
  • MTABC3 is not only related to MDR, but also to the pathogenesis of many tumors.
  • the function and role of MTABC3 in it have not been clarified so far, and further research is needed, but the existing technology lacks a means to knock out the expression of MTABC3 gene. This has hindered 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 the MTABC3 gene into A375 cells, so that the modified A375 cells stably overexpress the MTABC3 protein.
  • a method for site-directed integration of the MTABC3 gene into A375 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 pRC-F primer is 5’- GACTAGTGCCACCATGCCGGGGTTTTACGAG-3 '
  • the sequence of the pRC-R primer is 5'-TAGCATGCGCATTAAGCGCGGCGGGTGT-3';
  • Bacmid-ITR-MTABC3 and Bacmid-RC were transfected into sf9 cells in logarithmic growth phase with Cellfectin II Reagent. The culture supernatant was collected 120 hours after infection, which is P1. High-titer P3 virus Bac-ITR-MTABC3 and Bac-RC were obtained after P1 was continuously infected with sf9 cells twice.
  • step 6) The small molecular weight DNA obtained in step 6) was transfected into A375 cells in logarithmic growth phase by electroporation. After 72 hours of incubation, the expression of MTABC3 and its insertion site were identified.
  • the sequence of the AAV-ITR expression cassette containing the MTABC3 gene is shown in SEQ ID No.1.
  • the site-specific integration site is AAVS1 site of chromosome 19 of A375 cells.
  • the ratio of the Bacmid-ITR-MTABC3 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 the MTABC3 gene in A375 cells at the AAVS1 site of chromosome 19, so that it can obtain the ability to continuously overexpress the MTABC3 protein, and use the insect protein expression system to synthesize the elements necessary for AAV to avoid the E. coli gene
  • the risk of potential endotoxin contamination brought by the cloning system greatly improves the safety and practicability of A375 cells for preclinical research.
  • Figure 1 is a schematic diagram of the structure of the AAV-ITR expression cassette containing the MTABC3 gene
  • FIG. 2 is a result chart of MTABC3 gene quantitative PCR
  • FIG. 3 is a result of PCR for identifying the insertion site of the MTABC3 gene, in which M-Marker, 1-control group, 2-experiment 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 Deposit center,
  • Embodiment one pFastBac1-ITR-MTABC3 Construction of vectors
  • an AAV-ITR expression cassette containing the MTABC3 gene was designed. Its sequence is shown in SEQ ID No. 1. SpeI and SphI were added to the 5 'and 3' ends of the cassette, respectively. Site sequence, commissioned Shanghai Biotech to synthesize the sequence by gene synthesis.
  • the synthetic AAV-ITR expression cassette containing the MTABC3 gene was integrated on the pUC19-ITR-MTABC3 vector.
  • the pUC19-ITR-MTABC3 vector was digested with SpeI and SphI enzymes, and the ⁇ 1500 bp target fragment AAV-ITR-MTABC3 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.
  • Embodiment two pFastBac1-RC Construction of vectors
  • 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 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 3 High titer baculovirus Bac-ITR-MTABC3 with Bac-RC Preparation
  • the pFastBac1-ITR-MTABC3 vector and pFastBac1-RC vector were transformed into competent E. coli DH10Bac, respectively. Positive clones were selected by blue and white spots, and recombinant Bacmid was extracted to obtain Bacmid-ITR-MTABC3 and Bacmid-RC.
  • Cellfectin II Reagent was used to transfect Bacmid-ITR-MTABC3 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 viruses Bac-ITR-MTABC3 and Bac-RC were obtained after P1 was continuously infected with sf9 cells twice.
  • Embodiment 4 Fixed-point insertion MTABC3 genetic A375 Construction and identification of cells
  • the sf9 cells in the logarithmic growth phase were co-infected with the P3 virus Bac-ITR-MTABC3 and Bac-RC obtained in Example 3. After the culture continued for 72 h, the cells were collected, DNA was extracted and small molecular weight DNA was isolated, and It was transfected into A375 cells in logarithmic growth phase by electrotransfection, and culture was continued for 72 h.
  • the insertion site of the MTABC3 gene was identified.
  • the upstream primer sequence: 5’- GAATTCCTAACTGCCCCGGGGC -3 '
  • using the 5' end partial sequence of the MTABC3 gene as a downstream primer sequence: 5'- CTGGCTCCGCTCCACGACAAGC -3 '
  • PCR was performed, and the results are shown in FIG. 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 MTABC3 gene has been successfully integrated into the AAVS1 site.
  • the invention can realize the site-specific integration of the MTABC3 gene in A375 cells at the AAVS1 site of chromosome 19, so that it can obtain the ability to continuously overexpress the MTABC3 protein, and use the insect protein expression system to synthesize the elements necessary for AAV to avoid the E. coli gene
  • the risk of potential endotoxin contamination brought by the cloning system greatly improves the safety and practicability of A375 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)

Abstract

L'invention concerne un procédé pour l'intégration dirigée sur un site du gène MTABC3 dans une cellule A375, lequel procédé utilise un système d'expression de protéines d'insecte pour synthétiser les composants nécessaires requis pour un virus adénoassocié recombinant (rAAV), et atteint l'objectif consistant à intégrer le gène MTABC3 de manière dirigée sur un site dans le locus AAVS1 du chromosome 19 dans une cellule A375.
PCT/CN2018/091704 2018-06-16 2018-06-16 Procédé d'intégration dirigée sur un site du gène mtabc3 dans une cellule a375 et son utilisation WO2019237374A1 (fr)

Priority Applications (1)

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PCT/CN2018/091704 WO2019237374A1 (fr) 2018-06-16 2018-06-16 Procédé d'intégration dirigée sur un site du gène mtabc3 dans une cellule a375 et son utilisation

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PCT/CN2018/091704 WO2019237374A1 (fr) 2018-06-16 2018-06-16 Procédé d'intégration dirigée sur un site du gène mtabc3 dans une cellule a375 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, YA'NING ET AL.: "Bac-to-Bac (Production of Adeno-associated Virus Mediated by Bac-to-Bac Baculovirus Insect Expression System", CHINESE JOURNAL OF EXPERIMENTED SURGERY, vol. 29, no. 7, 31 July 2012 (2012-07-31), ISSN: 1001-9030 *
XIA, YULONG: "Study on Production of Recombinant Adeno-associated Virus for Gene Therapy Using 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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