WO2019237375A1 - Procédé d'intégration à site fixe de gène hdmx dans des cellules mcf-7, et son application - Google Patents

Procédé d'intégration à site fixe de gène hdmx dans des cellules mcf-7, et son application Download PDF

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Publication number
WO2019237375A1
WO2019237375A1 PCT/CN2018/091705 CN2018091705W WO2019237375A1 WO 2019237375 A1 WO2019237375 A1 WO 2019237375A1 CN 2018091705 W CN2018091705 W CN 2018091705W WO 2019237375 A1 WO2019237375 A1 WO 2019237375A1
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hdmx
cells
gene
mcf
itr
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PCT/CN2018/091705
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Chinese (zh)
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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/CN2018/091705 priority Critical patent/WO2019237375A1/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
    • C12N15/866—Baculoviral vectors

Definitions

  • the invention belongs to the technical field of genetic engineering. More specifically, the present invention relates to a method for site-integrating the HDMX gene into MCF-7 cells and its application.
  • the p53 tumor suppressor gene is located on the short arm of human chromosome 17 and encodes a protein containing 393 amino acids, namely the p53 protein.
  • Wild-type p53 protein can be used as a cell cycle regulator protein to monitor damage and induce apoptosis, inhibit excessive cell proliferation, and have anti-tumor cell proliferation functions.
  • a variety of target genes of the p53 gene mainly include: P21, HDMX, bax, etc. It is these boot genes regulated by the p53 gene and form a network to achieve the functions jointly. Among them, HDMX-p53-p21 signaling pathway is one of the important pathways in the gene pathway.
  • the human HDMX gene encodes a protein with a molecular weight of kD.
  • P1 is upstream of the coding gene
  • P2 is in the first intron, which is controlled by p53 through two nearby p53 binding sites.
  • HDMX-encoded protein is divided into 4 functional regions: about 10 amino acid residues at the N-terminus, which can be combined with p53. This region also has a nuclear localization sequence and a highly acidic region of nuclear export signals; a zinc finger structure that can bind to genes and activate genes To make cells from G1 phase to S phase; ring finger structure, which can mediate protein-protein interactions, participate in cell regulation and promote cell proliferation.
  • HDMX negatively regulates p53 through multiple pathways.
  • HDMX protein can directly bind to p53 to inhibit its activity, resulting in p53 being degraded by the ubiquitin system.
  • Different splicing forms of HDMX also participate in regulatory activity.
  • the abnormal amplification of HDMX or the increase in protein expression levels lead to the inactivation of p53 function, so it is considered to be a newly discovered important proto-oncogene.
  • HDMX protein has been confirmed to be closely related to the degree of tumor metastasis. In tumor metastasis and recurrence, high amplification of HDMX gene or high expression of HDMX protein were observed. Therefore, HDMX is an ideal target for potential tumor treatment.
  • the lack of a method for site-integrating the HDMX gene into MCF-7 cells 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-integrating the HDMX gene into MCF-7 cells, so that the transformed MCF-7 cells can stably overexpress HDMX protein.
  • a method for site-integrating the HDMX gene into MCF-7 cells which 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) Transfection of small molecular weight DNA obtained in step 6) into MCF-7 cells in logarithmic growth phase by electroporation, and continue to culture 72 After h, the expression of HDMX and its insertion site were identified.
  • the sequence of the AAV-ITR expression cassette containing the HDMX gene is shown in SEQ ID No.1.
  • the site-specific integration site is the AAVS1 site of chromosome 19 of MCF-7 cells.
  • the ratio of the Bacmid-ITR-HDMX 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 fixed-point integration of HDMX gene in MCF-7 cells at the AAVS1 site of chromosome 19, so that it can obtain the ability to continuously overexpress HDMX protein, and use the insect protein expression system to synthesize the elements necessary for AAV to avoid the large intestine
  • the potential risk of endotoxin contamination brought by the Bacillus gene cloning system has greatly enhanced the safety and practicability of MCF-7 cells for preclinical research.
  • Figure 1 is a schematic diagram of the structure of the AAV-ITR expression cassette containing the HDMX gene
  • FIG. 2 is a result chart of HDMX gene quantitative PCR
  • Figure 3 is a result of PCR identification of HDMX gene insertion sites, wherein 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-HDMX Construction of vectors
  • an AAV-ITR expression cassette containing the HDMX 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 HDMX gene was integrated on the pUC19-ITR-HDMX vector.
  • the pUC19-ITR-HDMX vector was digested with SpeI and SphI enzymes, and the ⁇ 1500 bp target fragment AAV-ITR-HDMX 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-HDMX 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-HDMX and Bacmid-RC.
  • Cellfectin II Reagent was used to transfect Bacmid-ITR-HDMX 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-HDMX and Bac-RC were obtained after P1 was continuously infected with sf9 cells twice.
  • the P3 virus Bac-ITR-HDMX 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 and the small molecular weight DNA was isolated, and it was transfected into MCF-7 cells in logarithmic growth phase by electroporation, and culture was continued for 72 h.
  • HDMX gene in MCF-7 cells (experimental group) and normal MCF-7 cells (control group) was detected by real-time quantitative PCR. The results are shown in Figure 2. It can be seen that the HDMX gene in the experimental group of cells The expression level was significantly higher than the control cells, indicating that the HDMX gene sequence was successfully integrated into MCF-7 cells.
  • the 5 ′ end sequence of the AAVS1 site of MCF-7 cells was used as the upstream primer (sequence: 5’- GAATTCCTAACTGCCCCGGGGC -3 ’), using the 5’ end of the HDMX gene as a downstream primer (sequence: 5’- CAGGAAGCCTGTCTTGGGCATG -3 '), 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 HDMX gene has been successfully integrated into the AAVS1 site.
  • the invention can realize the fixed-point integration of HDMX gene in MCF-7 cells at the AAVS1 site of chromosome 19, so that it can obtain the ability to continuously overexpress HDMX protein, and use the insect protein expression system to synthesize the elements necessary for AAV, avoiding the large intestine
  • the potential risk of endotoxin contamination brought by the Bacillus gene cloning system has greatly enhanced the safety and practicability of MCF-7 cells for preclinical research.

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  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
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  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
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  • General Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Virology (AREA)
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  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
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Abstract

L'invention concerne un procédé d'intégration à site fixe d'un gène HDMX dans des cellules MCF-7, au moyen d'un système d'expression de protéine d'insecte pour synthétiser les éléments nécessaires requis pour le virus adéno-associé recombinant (rAAV), le procédé permettant de réaliser une intégration à site fixe d'un gène HDMX dans le site AAVS1 du chromosome 19 des cellules MCF-7. Les cellules MCF-7 transformées surexpriment de manière stable la protéine HDMX, et les éléments nécessaires pour le rAAV synthétisés à partir d'un système d'expression de protéine d'insecte évitent le risque de contamination d'endotoxine potentielle par un système de clonage de gène E. coli, ce qui améliore la sécurité et l'utilisation des cellules MCF-7 dans des recherches précliniques.
PCT/CN2018/091705 2018-06-16 2018-06-16 Procédé d'intégration à site fixe de gène hdmx dans des cellules mcf-7, et son application Ceased WO2019237375A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008024998A2 (fr) * 2006-08-24 2008-02-28 Virovek, Inc. Expression dans des cellules d'insecte de gènes ayant des cadres ouverts de lecture chevauchants, procédés et compositions pour cela
CN105524943A (zh) * 2016-01-13 2016-04-27 中国科学院苏州生物医学工程技术研究所 基于双链微载体将car基因定点整合至t细胞aavs1位点的方法
CN106544325A (zh) * 2015-09-23 2017-03-29 中国科学院武汉物理与数学研究所 一种重组杆状病毒及其应用
CN106916793A (zh) * 2015-12-24 2017-07-04 中国科学院武汉物理与数学研究所 一种重组腺相关病毒的制备方法及重组杆状病毒
CN106987603A (zh) * 2016-01-20 2017-07-28 中国科学院武汉物理与数学研究所 一种重组腺相关病毒的制备方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008024998A2 (fr) * 2006-08-24 2008-02-28 Virovek, Inc. Expression dans des cellules d'insecte de gènes ayant des cadres ouverts de lecture chevauchants, procédés et compositions pour cela
CN106544325A (zh) * 2015-09-23 2017-03-29 中国科学院武汉物理与数学研究所 一种重组杆状病毒及其应用
CN106916793A (zh) * 2015-12-24 2017-07-04 中国科学院武汉物理与数学研究所 一种重组腺相关病毒的制备方法及重组杆状病毒
CN105524943A (zh) * 2016-01-13 2016-04-27 中国科学院苏州生物医学工程技术研究所 基于双链微载体将car基因定点整合至t细胞aavs1位点的方法
CN106987603A (zh) * 2016-01-20 2017-07-28 中国科学院武汉物理与数学研究所 一种重组腺相关病毒的制备方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LI, TAIMING ET AL.: "Preparation of a Novel AAV-ITR Gene Expression Mini Vector in Sf9 Insect Cells via Baculovirus", CHINESE JOURNAL OF BIOTECHNOLOGY, vol. 31, no. 8, 14 January 2015 (2015-01-14), pages 1232 - 1236 *
SHIGEHIRA, S: "Overexpression of MDM2 in MCF-7 promotes both growth advantage and p53 accumulation in response to estradiol", JPN. J. CANCER RES., vol. 90, no. 2, 28 February 1999 (1999-02-28), pages 210 - 218, XP055669029 *

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