WO2017214832A1 - Vecteur lentiviral pour favoriser spécifiquement l'expression élevée du gène foxp3, et ses applications - Google Patents

Vecteur lentiviral pour favoriser spécifiquement l'expression élevée du gène foxp3, et ses applications Download PDF

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Publication number
WO2017214832A1
WO2017214832A1 PCT/CN2016/085637 CN2016085637W WO2017214832A1 WO 2017214832 A1 WO2017214832 A1 WO 2017214832A1 CN 2016085637 W CN2016085637 W CN 2016085637W WO 2017214832 A1 WO2017214832 A1 WO 2017214832A1
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Prior art keywords
foxp3 gene
sequence
foxp3
cdna sequence
vector
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PCT/CN2016/085637
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English (en)
Chinese (zh)
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石庆学
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石庆学
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Priority to PCT/CN2016/085637 priority Critical patent/WO2017214832A1/fr
Publication of WO2017214832A1 publication Critical patent/WO2017214832A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • 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
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
    • 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 lentivirus expression which specifically promotes high expression of Foxp3 gene, and a construction method and application thereof.
  • Foxp3 is a member of the transcription factor fork ead/winged-helix (fork-like/winged helix) family, FOX
  • Foxp3 is a generic term for the vertebrate fork ead transcription factor. Different subclasses are represented by the following English letters and the last digits. Foxp3 comprises at least three distinct domains: the N-terminal C2H2 zinc finger structure, the leucine zipper motif, and the C-terminal fork ead DNA binding region (FKH).
  • the FKH domain is a common feature of FOX family proteins and plays a key role in nuclear localization and DNA binding.
  • the Foxp3 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 Foxp3 gene, thereby completing the present invention.
  • the present invention provides a lentiviral expression vector which specifically promotes high expression of the Foxp3 gene, including the basic sequence of the pLVX-IRE S-puro expression vector, a resistance gene sequence, a multiple cloning site sequence, a promoter sequence, and Fo xp3 a gene cDNA sequence;
  • the multiple cloning site comprises an EcoR I cleavage site and a Spe I cleavage site
  • the Foxp3 gene cDNA sequence comprises an EcoR I cleavage site, a Foxp3 gene coding sequence, and a Spe I cleavage site
  • the Foxp3 gene cDNA sequence is positively inserted into the multiple cloning site sequence.
  • the lentiviral expression vector constructed by inserting the Foxp3 gene cDNA sequence into the pLVX-IRES-Puro expression vector has high transfection efficiency and low dosage, and can stably, efficiently and stably increase Foxp3.
  • the advantages of gene expression can be used as a powerful tool in the preparation and treatment of drugs for the treatment of Foxp3 gene expression in diseases such as Alzheimer's disease.
  • the Foxp3 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'-GCGAATTCATGCCCAACCCCAGGCCTGG-3, ie, SEQ ID NO: 1
  • the sequence of the downstream primer is: 5'- GACTAGTTCAGGGGCCAGGTGTAGGGTTG -3', gPSEQ ID NO: 2.
  • the Foxp3 gene coding sequence can be amplified by PCR, and can be successfully inserted into the pLVX-IRES-Puro expression vector to continuously express the Foxp3 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 Foxp3 gene, and comprises the following steps:
  • A) Foxp3 gene primer design According to the Foxp3 gene coding sequence, using 01igo 7 analysis, select 5, -GCGAATTCATGCCCAACCCCAGGCCTGG-3, ie SEQ ID NO: 1 as the upstream primer, select 5,- GACTAGTTCAGGGGCCAGGTGTAGGGTTG -3', ie SEQ ID NO:
  • NO: 2 is used as a downstream primer, and then the upstream primer and the downstream primer are synthesized; the upstream primer and the downstream primer have no primer dimer, and the annealing temperature difference is small;
  • B) obtaining the Foxp3 gene cDNA sequence PCR amplification using the upstream primer and the downstream primer to obtain a large number of Foxp3 gene coding sequences, and then adding the A tail reaction to the sequence, using T4 DNA ligase
  • the ligation product was obtained by ligating to the pGM-T vector, and the ligation product was transformed into competent E. coli DH50C, uniformly coated on an ampicillin-containing LB medium plate, and the positive monoclonal colony culture preservation liquid was picked and subjected to PCR.
  • a lentiviral vector that specifically promotes high expression of Foxp3 gene The plasmid pLVX-IRES-P uro was extracted, digested with restriction endonuclease EcoR I enzyme and Spe l enzyme, electrophoresis, gel-removed recovery vector, and then used. T4 DNA ligase ligated the Foxp3 gene cDNA sequence into the pLVX-IRES-Puro expression vector to obtain a ligation product, which was transformed into competent E. coli DH50C and uniformly coated onto an ampicillin-containing LB medium plate. The positive monoclonal colonies were picked and cultured to preserve the bacterial liquid, and the preliminary identification of PCR was carried out. The preliminary identification results indicated that the Foxp3 gene cDNA sequence was inserted into the successful bacterial solution for sequencing and identification;
  • the present invention utilizes genetic engineering technology to construct a lentiviral expression vector which specifically promotes high expression of Foxp3 gene. After successful identification, it is packaged into a virus and introduced into Jurkat cells. After puromycin is used to screen cells, real-time quantitative PCR is used. Western Blot technology verified the change of Foxp3 gene expression from mRNA and protein levels respectively. The experimental results confirmed that the Foxp3 gene cDNA sequence provided by the present invention was successfully inserted into the pLVX-I RES-Puro expression vector, which can promote specifically, continuously, efficiently and stably. The Foxp3 gene is highly expressed.
  • the present invention also provides the use of a lentiviral expression vector which specifically promotes high expression of the Foxp3 gene for the preparation of a medicament for treating a disease associated with abnormal expression of Foxp3 gene.
  • the lentiviral expression vector which specifically promotes the high expression of Foxp3 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 Foxp3 gene, and can be used as a powerful tool.
  • the present invention also provides specific promotion of Foxp
  • the method for constructing a lentiviral expression vector with high expression of 3 gene has good operation effect and reduced sequence synthesis cost
  • 1 is a plasmid map of the pLVX-IRES-Puro expression vector.
  • 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.
  • Example 2 Construction of a lentiviral vector that specifically promotes high expression of Foxp3 gene
  • the coding sequence of Foxp3 gene was amplified by Premix PrimeSTAR HS enzyme, and then electrophoresed and then subjected to A tail reaction, and then ligated to pGM-T with T4 DNA ligase.
  • the ligation product (Foxp3-T vector) was obtained on the vector, and the ligation product was transformed into competent E. coli DH 5 ⁇ , uniformly coated on an ampicillin-containing LB medium plate, and cultured at 37 ° C for 12 h.
  • Negative control group 1 consistentt cells were uniformly coated on ampicillin-free plates
  • negative control group 2 peripheral cells were uniformly coated in 100
  • positive control group 1 the connection product of the double enzyme-cut empty vector was evenly coated in 100
  • 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, the negative control group 1 grew colonies; the negative control group 2, the positive control group 1, 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 Foxp3 gene sequence was extracted, and the pLVX-IRES-Puro vector was firstly used with EcoR 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 DH50C, uniformly coated on an 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 grown in good condition were inoculated into six wells, 1,000,000 cells per well, and the extracted recombinant plasmid pLVX-Foxp3 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 Jurka t cells, and the Lenti-X GoStix kit was used to detect a virus titer of 5,000,000 to 50,000,000 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 Foxp3 gene.
  • the primer design software Oligo 7.0 was used to design the bow.
  • Jurkat cells, pLVX empty vector control Jurkat cell group, and P LVX-Foxp3 high expressing cells were inoculated into 6-well plates, respectively.
  • the cell density reached 80 ⁇ 3 ⁇ 4-90 ⁇ 3 ⁇ 4 ⁇ , and the total RNA of each group was extracted with RNeasy Mini Kit, and PrimeScrip RT reagent was used for He 1 J. Kit reverse transcribes mRNA into cDNA, reverse transcription conditions: 37 ° C, 15 min; 85 ° C, 5 s; 4 ° C, ⁇ . After the end of reverse transcription, add 9 (L of RNase Free dH 2 0 diluted cDNA, and store at -20 ° C for later detection. Take the cDNA of each group of cells.
  • ⁇ was used as a template, and GAPDH was used as an internal reference.
  • Real-time quantitative PCR (QPCR) was used to detect the relative expression of Foxp3, and the reaction conditions were set: 95. C 30s, 1 cycle, 54°C 30s 40 cycles, 95. C 5s, 60. C
  • the lentiviral expression vector which specifically promotes the high expression of Foxp3 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 Foxp3 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 Foxp 3 gene, which has good operation effect, reduces sequence synthesis cost, and has low cost.

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Abstract

L'invention concerne un vecteur d'expression lentiviral destiné à favoriser spécifiquement l'expression élevée d'un gène Foxp3, 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 Foxp3 d'un vecteur d'expression de pLVX-IRES-puro. Les sites multiples de clonage comprennent un site de découpe d'enzyme EcoR I et un site de découpe d'enzyme Spe I, la séquence d'ADNc du gène Foxp3 comprend un site de découpe d'enzyme EcoR I, une séquence de codage du gène Foxp3 et un site de découpe de l'enzyme Spe I, et la séquence d'ADNc du gène Foxp3 est insérée vers l'avant dans la séquence de sites multiples de clonage. Le vecteur d'expression lentiviral peut servir pour la préparation de médicaments associés à Foxp3.
PCT/CN2016/085637 2016-06-14 2016-06-14 Vecteur lentiviral pour favoriser spécifiquement l'expression élevée du gène foxp3, et ses applications WO2017214832A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101575604A (zh) * 2009-05-15 2009-11-11 苏州大学 一种慢病毒载体及其制备和应用
CN105452546A (zh) * 2012-08-31 2016-03-30 斯克利普斯研究院 与真核细胞调节剂相关的方法和组合物

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101575604A (zh) * 2009-05-15 2009-11-11 苏州大学 一种慢病毒载体及其制备和应用
CN105452546A (zh) * 2012-08-31 2016-03-30 斯克利普斯研究院 与真核细胞调节剂相关的方法和组合物

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CHEN, CHUN: "XIE2DAI4 YOU3 Foxp3 JI1YIN1 MAN4BING4DU2 ZAI3TI3 DEI GOU4JIAN4", SHANDONG MEDICAL JOURNAL, vol. 51, no. 26, 31 December 2011 (2011-12-31) *
KWON, H. S. ET AL.: "Three Novel Acetylation Sites in the Foxp3 Transcription Factor Regulate the Suppressive Activity of Regulatory T Cells", THE JOURNAL OF IMMUNOLOGY, vol. 188, no. 6, 6 February 2012 (2012-02-06), pages 2712 - 2721, XP055447718 *
MCINNES, N. ET AL.: "FOXP3 and FOXP3-regulated microRNAs suppress SATB1 in breast cancer cells", ONCOGENE, vol. 31, no. 8, 11 July 2011 (2011-07-11), pages 1045 - 1054, XP055447725 *
XIAO, JIANGWEI ET AL.: "xie2dai4 you3 foxp3 jilyinl man4bing4du2 zai3ti3 del gou4jian4 ji2qil chuan2ran3 da4shu3 t xi4baol del shi2yan4yan2jiu3", MODERN PREVENTIVE MEDICINE, vol. 37, no. 9, 31 December 2010 (2010-12-31) *

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