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WO2017214945A1 - Vecteur d'expression lentiviral pour améliorer le taux d'expression du gène de l'hepcidine, et ses applications - Google Patents

Vecteur d'expression lentiviral pour améliorer le taux d'expression du gène de l'hepcidine, et ses applications Download PDF

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Publication number
WO2017214945A1
WO2017214945A1 PCT/CN2016/086069 CN2016086069W WO2017214945A1 WO 2017214945 A1 WO2017214945 A1 WO 2017214945A1 CN 2016086069 W CN2016086069 W CN 2016086069W WO 2017214945 A1 WO2017214945 A1 WO 2017214945A1
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WIPO (PCT)
Prior art keywords
hepcidin gene
sequence
hepcidin
vector
expression vector
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PCT/CN2016/086069
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English (en)
Chinese (zh)
Inventor
毛侃琅
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Priority to PCT/CN2016/086069 priority Critical patent/WO2017214945A1/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 expression vector for increasing the expression level of Hepcidin gene and application thereof.
  • Prostate cancer is a common malignant tumor in men and ranks second among male cancer deaths.
  • New research suggests that cellular iron metabolism plays an important role in prostate cancer growth, angiogenesis and metastasis. Iron accumulation is involved in the growth and metabolism of the body and is a necessary trace element for human growth.
  • Iron is considered to be the basic substance for maintaining the growth and development of tumor cells. Iron metabolism regulating proteins affect tumor changes, and reducing iron metabolism can be considered as an anti-tumor research direction. . It has been suggested that proteins that regulate iron metabolism can affect tumor growth and reduce the utilization of intracellular iron as a pathway for anti-tumor therapy.
  • Hepcidin may be a potential tumor therapeutic target with good bursting potential, but the lack of lentiviral expression vectors in the prior art that specifically promote the high expression of Hepcidin gene does not facilitate the research in related fields.
  • the present invention provides a lentiviral expression vector which specifically promotes high expression of the Hepcidin gene, including the basic sequence of the pLVX-I RES-puro expression vector, the resistance gene sequence, the multiple cloning site sequence, the promoter sequence and the Hepcidin gene.
  • a cDNA sequence including an EcoR I cleavage site and a Spe I cleavage site
  • the Hepcidin gene cDNA sequence includes EcoR
  • the I cleavage site, the Hepcidin gene coding sequence and the Spe I cleavage site, and the Hepcidin gene cDNA sequence is inserted into the multiple cloning site sequence.
  • the lentiviral expression vector constructed by inserting the cDNA sequence of the Hepcidin gene into the pLVX-IRES-Pur o expression vector has the advantages of high transfection efficiency, low dosage, sustainable, high efficiency and stable improvement.
  • the advantages of Hepcidin gene expression can be used as a powerful tool in the preparation and treatment of Hepcidi n gene expression in the treatment of diseases such as Alzheimer's disease.
  • the Hepcidin gene coding sequence is obtained by PCR amplification
  • the PC R primer comprises an upstream primer and a downstream primer
  • the sequence of the upstream primer is: 5'-GGAATTCATGGCACTGAGCTCCCAGATCTG -3, ie SEQ ID NO: 1
  • the sequence of the downstream primer is: 5, - GACTAGTCTACGTCTTGCAGCACATCCC -3, ie SEQ ID NO: 2.
  • the Hepcidin gene coding sequence can be amplified by PCR, and can be successfully inserted into the pLVX-IRES-Puro expression vector to continuously express the Hepcidin 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 Hepcidin gene, comprising the following steps:
  • A) Hepcidin gene primer design According to the Hepcidin gene coding sequence, using 01igo 7 analysis, select 5, - GGAATTCATGGCACTGAGCTCCCAGATCTG -3, ie SEQ ID NO: 1 as the upstream bow, select 5,- GACTAGTCTACGTCTTGCAGCACATCCC -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 Hepcidin gene cDNA sequence PCR amplification using the upstream primer and the downstream primer to obtain a large number of Hepcidin gene coding sequences, and then adding the A tail reaction to the sequence, using T4 DNA ligase Attached to the pGM-T vector to obtain the ligation product, the ligation product was transformed into competent E. coli ToplO, uniformly coated onto the ampicillin-containing LB medium plate, and the positive monoclonal bacteria were picked. The culture supernatant was cultured and the PCR was initially identified. The preliminary identification results indicated that the Hepcidin gene cDNA sequence was inserted into the successful bacterial solution for sequencing. The correct E.
  • coli was identified by liquid LB medium, and the Hepcidin gene cDNA was extracted.
  • the sequence of pGM-T vector was digested with restriction endonuclease Eco RI enzyme and Spe l enzyme, and the fragment of about 250 bp was recovered by electrophoresis and gel-cutting. This fragment is the Hepcidin gene cDNA sequence;
  • the present invention utilizes genetic engineering technology to construct a lentiviral expression vector which specifically promotes high expression of Hepcidin gene, and after being successfully constructed, it is packaged into a virus and introduced into RWPE-2 cells, and the cells are selected by puromycin, and then quantified by real-time fluorescence.
  • the PCR and Western Blot techniques were used to verify the expression of Hepcidin gene from mRNA and protein levels.
  • the experimental results showed that the Hepcidin gene cDNA sequence provided by the present invention was successfully inserted into the pLVX-IRES-Puro expression vector, which was specific, sustained, efficient and stable. Promote high expression of Hepcidin gene.
  • the present invention also provides the use of a lentiviral expression vector which specifically promotes high expression of the Hepcidin gene for the preparation of a medicament for treating a disease associated with abnormal expression of Hepci din gene.
  • the lentiviral expression vector which specifically promotes high expression of Hepcidin gene has the advantages of high transfection efficiency, low dosage, specific, sustained, efficient and stable promotion of high expression of Hepcidin gene, and can be used as a powerful tool.
  • the present invention also provides specific promotion
  • the construction method of the lentiviral expression vector with high expression of Hepcidin gene has good operation effect, reduces the cost of sequence synthesis, and has 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.
  • RWPE-2 cells were purchased from the Cell Resource Center of 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, Lenti-X GoStix reagent Boxes are 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.
  • the coding sequence of the Hepcidin gene was amplified by Premix PrimeSTAR HS enzyme, and then electrophoresed and then subjected to A tail reaction, and then T4 was used.
  • the DNA ligase was ligated to the pGM-T vector to obtain the ligation product (Hepcidin-T vector), and the ligation product was transformed into competent E. coli ToplO and uniformly coated onto ampicillin-containing LB medium plate at 37 ° C.
  • 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 with 100 g/ml ampicillin).
  • positive control group 1 the connection product of the double enzyme-cut empty vector was uniformly coated on the plate containing 100 g/ml ampicillin
  • positive control group 2 the empty carrier was uniformly coated in 100 g/ mL of ampicillin on the plate).
  • 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 Hepcidin 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 ToplO, 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-Hepcidin 2 g was transfected into 293FT cells using a lentiviral packaging assistant kit. After 48 hours, the virus-containing supernatant medium was collected, and the virus solution was filtered through a sieve of 0.45 ⁇ m for infection of RWPE-2 cells, and the titer of the virus detected by Lenti-X GoStix kit was 5000000 to 50000000 IFU.
  • the virus-containing DMEM complete medium was discarded, and fresh DME M complete medium was replaced. After 24 hours, the cells were selected with 0.5 g/ml concentration of puromycin. After 10 days of screening, the medium was changed 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 Hepcidin gene.
  • the primer design software Oligo 7.0 was used to design bows.
  • RWPE-2 cells, pLVX empty vector control RWPE-2 cell group, and pLVX-Hepcidin high expression cells were inoculated into 6-well plates, respectively.
  • Cell density reached 80 ⁇ 3 ⁇ 4-90 ⁇ 3 ⁇ 4 ⁇
  • total RNA was extracted from each group with RNeasy Mini Kit, and mRNA was reverse transcribed into cDNA using PrimeScrip RT reagent Kit.
  • Reverse transcription conditions 37 ° C, 15 min ; 85 ° C, 5s; 4°C, ⁇ . After the reverse transcription was completed, the cDNA was diluted with 90 ⁇ M of RNase Free dH 20 and stored at -20 ° C for later detection.
  • the cDNA of each group of cells was used as a template, GAPDH was used as an internal reference, and the relative expression of Hepcidin was detected by real-time quantitative PCR (QPCR).
  • the reaction conditions were set: 95. C 30s, 1 cycle, 54°C 30s 40 cycles, 95. C 5s, 60 ° C lmin, 95. C 15s , using S YBR Primescript RT-PCR
  • Kit detects the relative expression of Hepcidin gene in each group of cells. After continuous culture of pLVX-Hepcidin cells for 20 passages, the above experiment was repeated. The summarized results are shown in Figure 2. It can be seen that whether it is just after screening or after 20 generations of pLVX-Hepcidin cells, the expression of Hepcidin gene is about 240 times higher than that of RWPE-2 cells, and the Hepccidin gene of pLVX empty vector cells. The expression level of the Hepcidin gene was successfully inserted into the p LVX-IRES-Puro expression vector, which promoted the high expression of Hepcidin gene specifically, continuously, efficiently and stably.
  • the lentiviral expression vector which specifically promotes high expression of Hepcidin gene provided by the invention has high transfection efficiency
  • the utility model has the advantages of low dosage, specific, sustained, high efficiency and stable promotion of high expression of the Hepcidin gene, and can be used as a powerful tool in drug research and development related to Hepcidin; the invention also provides specific promotion of high expression of Hepcidin gene.
  • the construction method of the lentiviral expression vector has good operation effect, reduces the cost of sequence synthesis, and has low cost.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
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Abstract

L'invention concerne un vecteur d'expression lentiviral destiné à favoriser spécifiquement l'expression élevée du gène de l'hepcidine, 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 de l'hepcidine d'un vecteur d'expression de pLVX-IRES-puro. Le sites multiples de clonage comprend un site de découpe d'enzyme EcoR I et un site de découpe d'enzyme Spe I, la séquence d'ADNc du gène de l'hepcidine comprend le site de découpe d'enzyme EcoR I, une séquence de codage du gène de l'hepcidine et le site de découpe de l'enzyme Spe I, la séquence d'ADNc du gène de l'hepcidine étant 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, il est capable d'exprimer, de manière spécifique, en continu, avec efficacité et stabilité le gène de l'hepcidine, et peut servir d'outil puissant pour la recherche et le développement de médicaments associés à l'hepcidine.
PCT/CN2016/086069 2016-06-16 2016-06-16 Vecteur d'expression lentiviral pour améliorer le taux d'expression du gène de l'hepcidine, et ses applications Ceased WO2017214945A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102766654A (zh) * 2012-06-20 2012-11-07 深圳市疾病预防控制中心 特异促进肝细胞cyp1a2基因高表达的慢病毒表达载体及其构建方法与应用
CN102876716A (zh) * 2012-09-28 2013-01-16 深圳市疾病预防控制中心 特异促进肝细胞cyp2e1基因高表达的慢病毒表达载体及其构建方法与应用
WO2013075066A2 (fr) * 2011-11-18 2013-05-23 Eleven Biotherapeutics, Inc. Protéines ayant une demi-vie et d'autres propriétés améliorées
CN105331635A (zh) * 2015-11-05 2016-02-17 深圳大学 一种诱导型慢病毒表达体系及其构建方法和应用
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 (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013075066A2 (fr) * 2011-11-18 2013-05-23 Eleven Biotherapeutics, Inc. Protéines ayant une demi-vie et d'autres propriétés améliorées
CN102766654A (zh) * 2012-06-20 2012-11-07 深圳市疾病预防控制中心 特异促进肝细胞cyp1a2基因高表达的慢病毒表达载体及其构建方法与应用
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
CN102876716A (zh) * 2012-09-28 2013-01-16 深圳市疾病预防控制中心 特异促进肝细胞cyp2e1基因高表达的慢病毒表达载体及其构建方法与应用
CN105331635A (zh) * 2015-11-05 2016-02-17 深圳大学 一种诱导型慢病毒表达体系及其构建方法和应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
FU, LIJUAN ET AL.: "Iron metabolism and iron-regulatory hormone, hepcidin", PROGRESS IN PHYSIOLOGICAL SCIENCES, vol. 36, no. 03, 25 July 2005 (2005-07-25), pages 233 - 236 *
WANG, YUANZHONG ET AL.: "HEPC1 and HEPC2 gene transfection increases iron retention in RAW264.7 cells", ACTA ACADEMIAE MEDICINAE MILITARIS TERTIAE., vol. 28, no. 17, 15 September 2006 (2006-09-15), pages 1772 - 1774 *
ZHANG, ZIYAN ET AL.: "Hepcidin yan2jiul jin4zhan3", FOREIGN MEDICAL SCIENCES(SECTION OF INTERNAL MEDICINE, vol. 33, no. 10, 28 October 2006 (2006-10-28), pages 453 and 454 *

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