WO2017214831A1 - Vecteur lentiviral pour favoriser spécifiquement l'expression élevée du gène nrg1, et ses applications - Google Patents

Vecteur lentiviral pour favoriser spécifiquement l'expression élevée du gène nrg1, et ses applications Download PDF

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Publication number
WO2017214831A1
WO2017214831A1 PCT/CN2016/085636 CN2016085636W WO2017214831A1 WO 2017214831 A1 WO2017214831 A1 WO 2017214831A1 CN 2016085636 W CN2016085636 W CN 2016085636W WO 2017214831 A1 WO2017214831 A1 WO 2017214831A1
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Prior art keywords
nrg1 gene
sequence
nrg1
cdna sequence
gene
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PCT/CN2016/085636
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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/CN2016/085636 priority Critical patent/WO2017214831A1/fr
Publication of WO2017214831A1 publication Critical patent/WO2017214831A1/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 lentiviral vector which specifically promotes high expression of the NRG1 gene, and a construction method and application thereof.
  • NRG1 neuregulin l
  • ERBB receptor ERBB
  • All NRG1 share a common EGF-like domain located in the extracellular domain near the transmembrane segment.
  • the EGF-like domain is an essential structure for activating the NRGI receptor ERBB, and only the EGF-like domain is sufficient to activate ErbB tyrosine kinase activity.
  • NRG1 is associated with schizophrenia, and thus it is expected that a certain intervention effect on schizophrenia may be exerted by regulating the expression level of NRG1, but the relationship between the expression level of NRG1 and schizophrenia remains to be studied, and in the prior art, Lack of a lentiviral expression vector that specifically promotes high expression of the NRG1 gene is not conducive to the development of related research.
  • the NRG 1 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 NRG1 gene, thereby completing the present invention.
  • the present invention provides a lentiviral expression vector that specifically promotes high expression of the NRG1 gene, including pLVX-IRE
  • the NRG1 gene cDNA sequence comprises an EcoR I restriction site, a NRG1 gene coding sequence and a Not I restriction site, and the NRG1 gene cDNA sequence is inserted into the multiple cloning site sequence.
  • the lentiviral expression vector constructed by inserting the cDNA sequence of the NRG1 gene into the pLVX-IRES-Puro expression vector has high transfection efficiency and low dosage, and can stably, efficiently and stably increase NRG1.
  • the advantages of gene expression can be used as a powerful tool in the preparation and treatment of NRG1 gene expression for diseases such as Alzheimer's disease.
  • the NRG1 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'-GGAATTCATGGGGAAAGGACGCGCGGGC-3, ie, SEQ ID NO: 1
  • the sequence of the downstream primer is: 5, - AGCGGCCGCTTATACAGCAATAGGGTCTTGG -3, ie SEQ ID NO: 2.
  • the NRG1 gene coding sequence can be amplified by PCR, and can be successfully inserted into the pLVX-IRES-Puro expression vector to continuously express the NRG1 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 NRG1 gene, comprising the following steps:
  • NRG1 gene primer design According to the NRG1 gene coding sequence, using Oligo 7 analysis, select 5'-GGAATTCATGGGGAAAGGACGCGCGGGC-3, ie, SEQ ID NO: 1 as the upstream primer, select 5, - AGCGGCCGCTTATACAGCAATAGGGTCTTGG-3, gPSEQ ID NO
  • the upstream primer and the downstream primer have no primer dimer, and the annealing temperature difference is small;
  • 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 NRG1 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.
  • the ligation product is obtained, 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 preliminary identification by PCR, and preliminary identification is performed.
  • the present invention utilizes genetic engineering technology to construct a lentiviral expression vector which specifically promotes high expression of NRG1 gene, and after successful identification, is packaged into a virus and transduced into HEB cells, and after puromycin is used to screen cells, real-time quantitative PCR is used.
  • the Western Blot technique verified the change of NRG1 gene expression from mRNA and protein levels, respectively.
  • the experimental results confirmed that the NRG1 gene cDNA sequence provided by the present invention was successfully inserted into the pLVX-I RES-Puro expression vector, and can be specifically, continuously, efficiently and stably promoted.
  • the NRG1 gene is highly expressed.
  • the present invention also provides the use of a lentiviral expression vector which specifically promotes high expression of the NRG1 gene for the preparation of a medicament for treating a disease associated with abnormal expression of a NRG1 gene.
  • the lentiviral expression vector which specifically promotes the high expression of the NRG1 gene provided by the invention has the advantages of high transfection efficiency, low dosage, specific, sustained, efficient and stable promotion of high expression of the NRG1 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 NRG1 gene, which has a good operation effect, reduces the cost of sequence synthesis, and has a low cost.
  • 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.
  • HEB cells were purchased from the Cell Resource Center of Shanghai Institute of Biological Sciences, and 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.
  • NRG1 gene coding sequence GenBank NM_001159995.2
  • the designed PCR primers were synthesized by Shanghai Shenggong Bioengineering Technology Service Co., Ltd.
  • the coding sequence of the NRG1 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 (NRG1-T vector) was obtained on the vector, and the ligation product was transformed into competent E. coli To plO, uniformly coated on an ampicillin-containing LB medium plate, and cultured at 37 ° C for 12 h. Negative control group 1 (consistent 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 NRG1 gene sequence was extracted, and the pLVX-IRES-Puro vector was firstly used with EcoR I enzyme and Not 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-NRG1 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 0.45 ⁇ filter for infection of sputum cells, and the Lenti-X GoStix kit was used to detect the virus titer of 5,000,000 to 50000000 IFU.
  • HEB 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 NRG1 gene.
  • the primer design software Oligo 7.0 was used to design the bow.
  • HEB cells HEB cells, pLVX empty vector control HEB cells, and pLVX-NRG1 high expressing cells were inoculated separately. 6-well plate. Cell density reached 80 ⁇ 3 ⁇ 4-90 ⁇ 3 ⁇ 4 ⁇ , total RNA was extracted from each group 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 the reverse transcription, add 9 (L RNase Free dH20 diluted cDNA, and store at -20 °C for later detection. Take the cDNA of each group of cells.
  • NRG1 gene is more than 210-fold higher than that of HEB cells, whether it is just after screening or after 20 generations of pLVX-NRG1 cells, and the expression of NRG1 gene in pLVX empty vector cells.
  • NRG 1 gene cDNA sequence provided by the present invention is successfully inserted into the pLVX-IRES-Puro expression vector, and can promote the high expression of NRG1 gene specifically, continuously, efficiently and stably.
  • the lentiviral expression vector which specifically promotes the high expression of the NRG1 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 the NRG1 gene, and can be used as a powerful tool.
  • the present invention also provides specific promotion of NRG
  • the method for constructing a lentiviral expression vector with high expression of 1 gene has good operation effect, reduces the cost of sequence synthesis, 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 NRG1, 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 NRG1 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 Not I, la séquence d'ADNc du gène NRG1 comprend un site de découpe d'enzyme EcoR I, une séquence de codage du gène NRG1 et un site de découpe de l'enzyme Not I, et la séquence d'ADNc du gène NRG1 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 NRG1.
PCT/CN2016/085636 2016-06-14 2016-06-14 Vecteur lentiviral pour favoriser spécifiquement l'expression élevée du gène nrg1, et ses applications WO2017214831A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090291893A1 (en) * 2008-05-23 2009-11-26 Morehouse School Of Medicine Compositions for the prevention and treatment of neuroinjury and methods of use thereof
CN104922153A (zh) * 2015-04-02 2015-09-23 连祺周 Nrg1-erbb4复合体在制备治疗心肌缺血的药物中的应用
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 (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090291893A1 (en) * 2008-05-23 2009-11-26 Morehouse School Of Medicine Compositions for the prevention and treatment of neuroinjury and methods of use thereof
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
CN104922153A (zh) * 2015-04-02 2015-09-23 连祺周 Nrg1-erbb4复合体在制备治疗心肌缺血的药物中的应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
J IANG, H. ET AL.: "Expression of peroxiredoxin 1 and 4 promotes human lung cancer malignancy", AM. J. CANCER RES., vol. 4, no. 5, 6 September 2014 (2014-09-06), pages 445 - 460, XP055447826 *
LI, B. G. ET AL.: "Gene transfer of human neuregulin-1 attenuates ventricular remodeling in diabetic cardiomyopathy rats", EXPERIMENTAL AND THERAPEUTIC MEDICINE, vol. 6, 31 December 2013 (2013-12-31), XP055447825 *
WILSON, T. R. ET AL.: "Neuregulin-l-Mediated Autocrine Signaling Underlies Sensitivity to HER2 Kinase Inhibitors in a Subset of Human Cancers", CANCER CELL, vol. 20, 16 August 2011 (2011-08-16), pages 158 - 172, XP028263270 *

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