WO2017214833A1 - Vecteur lentiviral pour favoriser spécifiquement l'expression élevée du gène erbb2, et ses applications - Google Patents

Vecteur lentiviral pour favoriser spécifiquement l'expression élevée du gène erbb2, et ses applications Download PDF

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Publication number
WO2017214833A1
WO2017214833A1 PCT/CN2016/085638 CN2016085638W WO2017214833A1 WO 2017214833 A1 WO2017214833 A1 WO 2017214833A1 CN 2016085638 W CN2016085638 W CN 2016085638W WO 2017214833 A1 WO2017214833 A1 WO 2017214833A1
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erbb2 gene
sequence
erbb2
cdna sequence
expression vector
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PCT/CN2016/085638
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Chinese (zh)
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石庆学
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石庆学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • 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
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • 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
    • 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
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • 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
    • 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
    • C12N15/867—Retroviral vectors

Definitions

  • the present invention belongs to the field of genetic engineering technology, and particularly relates to a lentiviral vector which specifically promotes high expression of the ERBB2 gene, and a construction method and application thereof.
  • ERBB2 is a 185 kDa cell membrane receptor encoded by the proto-oncogene ERBB2 and is a member of the epidermal growth factor receptor (EGFR) family.
  • the family includes four members, ERBB1 (also known as EGFR, HER1), ERBB2 (also known as HER2), ERBB3 (HER3), and ERBB4 (HER4).
  • ERBB2 is involved in the regulation of growth and development of normal tissues, promotes cell division and secretion of proteolytic enzymes, and enhances cell motility, but under pathological conditions, ERBB2 expression increases, thereby promoting tumor invasion and metastasis.
  • ERBB2 has intracellular tyrosine kinase-like activity and plays a role in embryonic development and differentiation. It is widely expressed in epithelial cells of human tissues and is closely related to the survival and progression of various tumors. Therefore, the research on ERBB2 can greatly promote the development of tumor prevention and control, but the existing technology still lacks the lentiviral expression vector which specifically promotes the high expression of ERBB2 gene, which hinders the development of related fields.
  • the ERBB2 gene cDNA sequence of the I cleavage site was inserted into the multiple cloning site of the pLVX-IRES-Puro expression vector to construct a lentiviral expression vector which specifically promotes the high expression of the ERBB2 gene, thereby completing the present invention.
  • the present invention provides a lentiviral expression vector which specifically promotes high expression of the ERBB2 gene, including the basic sequence of the pLVX-IR ES-puro expression vector, the resistance gene sequence, the multiple cloning site sequence, the promoter sequence and the E RBB2 a cDNA sequence of the gene; the multiple cloning site includes a Xho I cleavage site and a Spe I cleavage site.
  • the ERBB2 gene cDNA sequence includes an Xho I restriction site, an ERBB2 gene coding sequence, and a Spe I restriction site, and the ERBB2 gene cDNA sequence is inserted into the multiple cloning site sequence.
  • the lentiviral expression vector constructed by inserting the ERBB2 gene cDNA sequence into the pLVX-IRES-Puro expression vector has high transfection efficiency and low dosage, and can stably, efficiently and stably increase ERBB2.
  • the advantages of gene expression can be used as a powerful tool in the preparation and treatment of ERBB2 gene expression for diseases such as Alzheimer's disease.
  • the ERBB2 gene coding sequence is obtained by PCR amplification
  • the PCR primer comprises an upstream primer and a downstream primer
  • the sequence of the upstream primer is: 5'-GCTCGAGATGAAGCTGCGGCTCCCTGC-3, ie, SEQ ID NO: 1
  • the sequence of the downstream primer is: 5, - GACTAGTTCACACTGGCACGTCCAGACC -3', ie SEQ ID NO: 2.
  • the ERBB2 gene coding sequence can be amplified by PCR, and can be successfully inserted into the pLVX-IRES-Puro expression vector to continuously express the ERBB2 gene, which reduces the cost of sequence synthesis and lowers the cost.
  • the present invention also provides a method for constructing a lentiviral expression vector which specifically promotes high expression of the ERBB2 gene, comprising the following steps:
  • A) ERBB2 gene primer design According to the ERBB2 gene coding sequence, use Oligo
  • B) obtaining the ERBB2 gene cDNA sequence PCR amplification using the upstream primer and the downstream primer to obtain a large number of ERBB2 gene coding sequences, and then adding the A tail reaction to the sequence, using T4 DNA ligase
  • the ligation product is obtained by ligation to the pGM-T vector, and the ligation product is transformed into competent E. coli ToplO, uniformly coated on an ampicillin-containing LB medium plate, and the positive monoclonal colony culture preservation liquid is picked and subjected to PCR.
  • Preliminary identification the preliminary identification results indicate that the ERBB2 gene cDNA sequence was inserted into the successful bacterial solution for sequencing and identification; the correct E.
  • coli was identified by liquid LB medium culture sequencing, and the pGM-T vector carrying the ERBB2 gene cDNA sequence was extracted and used. Restriction enzyme Xho I enzyme and Spe I enzyme double digestion, electrophoresis, gelatinization recovery of about 3500 bp fragment, this fragment is ERBB2 base Due to the cDNA sequence;
  • the present invention utilizes genetic engineering technology to construct a lentiviral expression vector which specifically promotes high expression of ERBB2 gene, and after being successfully constructed, it is packaged into a virus and introduced into Jurkat cells, and after puromycin is used to screen cells, real-time quantitative PCR is used.
  • the Western Blot technique verifies the change of ERBB2 gene expression from mRNA and protein levels, respectively.
  • the experimental results show that the ERBB2 gene cDNA sequence provided by the present invention is successfully inserted into the pLV X-IRES-Puro expression vector, and can be specifically, continuously, efficiently and stably promoted. High expression of ERBB2 gene
  • the present invention also provides the use of a lentiviral expression vector which specifically promotes high expression of the ERBB2 gene for the preparation of a medicament for treating a disease associated with abnormal expression of the ERBB2 gene.
  • the lentiviral expression vector which specifically promotes the high expression of ERBB2 gene provided by the invention has the advantages of high transfection efficiency, low dosage, specific, sustained, high efficiency and stable promotion of high expression of ERBB2 gene, and can be used as a powerful tool.
  • the present invention also provides a method for constructing a lentiviral expression vector which specifically promotes high expression of the E RBB2 gene, which has a good operation effect, reduces the cost of sequence synthesis, and has a low cost.
  • DRAWINGS 1 is a plasmid map of the pLVX-IRES-Puro expression vector.
  • FIG. 2 is a schematic diagram showing the results of fluorescent quantitative PCR detection after puromycin screening of cells.
  • Jurkat cells were purchased from the Cell Resource Center of the Shanghai Institute of Biological Sciences. 293FT cells were purchased from Thermo Fisher, Premix PrimeSTAR HS enzyme, lentiviral expression vector pLVX-IRES-Puro, virus packaging auxiliary kit, and Lenti-X GoStix kit. Purchased from Takara, RNeasy Mini
  • Kit was purchased from QIAGEN, pGM-T was purchased from Tiangen, and Endo-Free Plasmid Mini Kit II was purchased from Omega bio-tek.
  • ERBB2 gene coding sequence GenBank NM_001005862.2
  • the designed PCR primers were synthesized by Shanghai Shenggong Bioengineering Technology Service Co., Ltd.
  • Example 2 Construction of a lentiviral vector that specifically promotes high expression of the ERBB2 gene
  • the coding sequence of the ERBB2 gene was amplified by Premix PrimeSTAR HS enzyme, and then electrophoresed and then subjected to A-tail reaction, and then T4 was used.
  • DNA ligase is ligated to the pGM-T vector to obtain a ligation product (ERBB2-T vector), which is produced by the ligation
  • ERBB2-T vector a ligation product
  • the material was transformed into competent E. coli ToplO, uniformly coated on ampicillin-containing LB medium plate, cultured at 37 ° C for 12 h, and the negative control group 1 was set up (the competent cells were uniformly coated in the absence of ampicillin).
  • the negative control group 2 the competent cells were uniformly coated on the plate containing 100 g/ml ampicillin
  • the positive control group 1 the connection product of the double enzyme-cut empty vector was uniformly coated in the 100 g/ml ampicillin on the plate
  • positive control group 2 the empty carrier was evenly spread on a plate containing 100 g/mL ampicillin.
  • the experimental group grew a single colony, and the negative control group 1 grew colonies; the negative control group 2, the positive control group 1, and the positive control group 2 did not grow colonies.
  • the bacteria with the correct sequencing result were cultured in liquid LB medium for 14 h, and then the recombinant T vector containing the ERBB2 gene sequence was extracted, and the pLVX-IRES-Puro vector was separately used with Xho I enzyme and Spe I, respectively.
  • the enzyme was double-digested, electrophoresed and recovered, and the product was recovered by T4 DNA ligase, and then transformed into competent E. coli ToplO, uniformly coated on ampicillin-containing LB medium plate, and cultured at 37 ° C for 12 h.
  • the negative control group 1 was set up (the competent cells were uniformly coated on the ampicillin-free plate), and the negative control group 2 (the competent cells were uniformly coated on the plate containing 100 g/ml ampicillin), Positive control group 1 (the ligation product of the double enzyme-cut empty vector was uniformly coated on a plate containing 100 g/ml ampicillin), positive control group 2 (the empty vector was uniformly coated on 100 g/mL ampicillin) On the tablet).
  • the experimental group grew a single colony, and the negative control group 1 grew colonies; the negative control group 2, the positive control group 1, and the positive control group 2 did not grow colonies.
  • 293FT cells were cultured, and cells with good growth state were inoculated into six wells, 1,000,000 cells per well, and the extracted recombinant plasmid pLVX-ERBB2 2 ⁇ ⁇ was transfected into 293FT cells using a lentiviral packaging auxiliary kit. After 48 h, the virus-containing supernatant medium was collected, and the virus solution was filtered through a sieve of 0.45 ⁇ m for infecting Jur kat cells, and the Lenti-X GoStix kit was used to detect a virus titer of 5,000,000 to 5,000,000 IFU.
  • Jurkat cells were inoculated into 6-well plates at 1000000 cells per well. After 12 hours, the cell density was about 50 ⁇ 3 ⁇ 4, and the virus solution was taken separately. The virus was diluted 10 times with DMEM complete medium, and polybrene was added. ) The final concentration is 8 g/mL. The medium in the 6-well plate was added to the virus-containing DMEM complete medium (containing 10% fetal bovine serum). After 24 hours, the virus-containing DMEM complete medium was discarded, and the fresh DMEM complete medium was replaced. After 24 hours, 0.5 was used. The cells were screened at a g/ml concentration of puromycin. After 10 days of screening, the medium was changed once every 3 days, and the concentration of puromycin was continuously increased to 1.00 g/ml.
  • Example 5 Fluorescence quantitative PCR was used to detect the expression level of ERBB2 gene.
  • the primer design software Oligo 7.0 was used to design the bow.
  • Jurkat cells, pLVX empty vector control Jurkat cell group, and pLVX-ERBB2 high-expressing cells were inoculated into 6-well plates, respectively.
  • 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, reverse transcription conditions: 37 ° C, 15 min; 85 ° C, 5 s; 4 ° C, ⁇ . After the reverse transcription, add 9 (L RNase Free dH20 diluted cDNA, stored at -20 ° C, for later detection. Take the cDNA of each group of cells
  • ERBB2 gene cDNA sequence provided by the present invention is successfully inserted into the pLVX-IRES-Puro expression vector, and can specifically, stably, efficiently and stably promote the high expression of the ERBB2 gene.
  • the lentiviral expression vector which specifically promotes the high expression of ERBB2 gene provided by the invention has the advantages of high transfection efficiency, low dosage, specific, sustained, high efficiency and stable promotion of high expression of ERBB2 gene, and can be used as a powerful tool.
  • the present invention also provides a method for constructing a lentiviral expression vector which specifically promotes high expression of the E RBB2 gene, which has a good operation effect, reduces the cost of sequence synthesis, and has a low cost.

Abstract

L'invention concerne un vecteur d'expression lentiviral destiné à favoriser spécifiquement l'expression élevée d'un gène ERBB2, ledit vecteur comprenant une séquence fondamentale, une séquence de gène de résistance, une séquence de sites multiples de clonage, une séquence de promoteur et une séquence d'ADNc du gène ERBB2 d'un vecteur d'expression de pLVX-IRES-puro. Les sites multiples de clonage comprennent un site de découpe d'enzyme Xho I et un site de découpe d'enzyme Spe I, la séquence d'ADNc du gène ERBB2 comprend un site de découpe d'enzyme Xho I, une séquence de codage du gène ERBB2 et un site de découpe de l'enzyme Spe I, et la séquence d'ADNc du gène ERBB2 est insérée vers l'avant dans la séquence de sites multiples de clonage. Le vecteur d'expression lentiviral présente les avantages d'une efficacité de transfection élevée et d'une faible quantité requise, et il est capable d'exprimer, de manière spécifique, en continu, avec efficacité et stabilité le gène ERBB2, et peut servir d'outil puissant pour la recherche et le développement de médicaments associés à ERBB2.
PCT/CN2016/085638 2016-06-14 2016-06-14 Vecteur lentiviral pour favoriser spécifiquement l'expression élevée du gène erbb2, et ses applications WO2017214833A1 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105254767A (zh) * 2015-11-17 2016-01-20 重庆科润生物医药研发有限公司 一种Protein D与HER2融合蛋白及其制备方法和应用

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105254767A (zh) * 2015-11-17 2016-01-20 重庆科润生物医药研发有限公司 一种Protein D与HER2融合蛋白及其制备方法和应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LETO, S.M. ET AL.: "Sustained Inhibition of HER3 and EGFR Is Necessary to Induce Regression of HER2-Amplified Gastrointestinal Carcinomas", CLINICAL CANCER RESEARCH, vol. 21, no. 24, 15 December 2015 (2015-12-15), pages 5519 - 5531, XP055448957 *
PAKRAVAN, N. ET AL.: "N-terminally Fusion of Her2/neu to HSP70 Decreases Efficiency of Her2/neu DNA Vaccine", CELL STRESS AND CHAPERONES., vol. 15, 12 March 2010 (2010-03-12), pages 631 - 638, XP055448958 *

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