WO2017214939A1 - Vecteur d'expression lentiviral pour améliorer le taux d'expression du gène ccr7, et ses applications - Google Patents

Vecteur d'expression lentiviral pour améliorer le taux d'expression du gène ccr7, et ses applications Download PDF

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Publication number
WO2017214939A1
WO2017214939A1 PCT/CN2016/086063 CN2016086063W WO2017214939A1 WO 2017214939 A1 WO2017214939 A1 WO 2017214939A1 CN 2016086063 W CN2016086063 W CN 2016086063W WO 2017214939 A1 WO2017214939 A1 WO 2017214939A1
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ccr7 gene
sequence
ccr7
gene
expression vector
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PCT/CN2016/086063
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English (en)
Chinese (zh)
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毛侃琅
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毛侃琅
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Priority to PCT/CN2016/086063 priority Critical patent/WO2017214939A1/fr
Publication of WO2017214939A1 publication Critical patent/WO2017214939A1/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
    • C12N15/66—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
    • 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 chronic virus expression vector for increasing the expression level of a CCR7 gene and an application thereof.
  • Chemokine is a class of low molecular weight (8-12 kda) cytokines secreted by different cell types that are chemotactic to immune cells and capable of chemotaxis. Directed migration, hematopoietic cells, development and differentiation of immune cells, inflammation, angiogenesis, and tumorigenesis play different roles. The function of chemokines is mediated by chemokine receptors, and the interaction of chemokines with their receptors controls the directed migration of various immune cells between the circulatory system and tissues. Chemokines can be divided into four subfamilies: CXC, CC, C and CX3C. Their corresponding receptors are called CC receptors (CCR), CXC receptors (CXCR), C and CX3C receptors (CR, CX3CR). .
  • CCR7 is a member of the CC chemokine receptor and contains 378 amino acids.
  • CCR7 was originally discovered as a result of B cell infection with Epstein-Barr virus and plays an important role in the maturation of dendritic cells (DC) and the homing of T cells to secondary lymphoid organs such as lymph nodes and spleen.
  • DC dendritic cells
  • CC chemokine receptor There are many studies on it, but the prior art lacks a lentiviral expression vector for specifically promoting the high expression of the CCR7 gene, which hinders the progress of research in related fields.
  • the present invention provides a lentiviral expression vector which specifically promotes high expression of a CCR7 gene, including a basic sequence of a pLVX-IRE S-puro expression vector, a resistance gene sequence, a multiple cloning site sequence, a promoter sequence and C CR7 gene cDNA sequence; the multiple cloning site includes EcoR I cleavage site and Spe l cleavage site, and the CCR7 gene cDNA sequence includes EcoR I cleavage site, CCR7 gene coding sequence and Spe I cleavage site The CCR7 gene cDNA sequence is inserted into the multiple cloning site sequence.
  • the lentiviral expression vector constructed by inserting the cDNA sequence of the CCR7 gene into the pLVX-IRES-Puro expression vector has high transfection efficiency and low dosage, and can stably, efficiently and stably increase CCR7.
  • the advantages of gene expression can be used as a powerful tool in the preparation and treatment of drugs for the treatment of CCR7 gene expression in diseases such as Alzheimer's disease.
  • the CCR7 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'-CAGAATTCATGTACTCCATCATTTGTTTCG-3 ', ie SEQ ID NO: 1
  • the sequence ⁇ 'J of the downstream bow is: 5'- GTACTAGTCTATGGGGAGAAGGTGGTGGTG -3', ie SEQ ID NO: 2.
  • the CCR7 gene coding sequence can be amplified by PCR, and can be successfully inserted into the pLVX-IRES-Puro expression vector to continuously express the CCR7 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 a CCR7 gene, comprising the following steps:
  • A) CCR7 gene primer design According to the CCR7 gene coding sequence, using Oligo 7 analysis, select 5'-CAGAATTCATGTACTCCATCATTTGTTTCG-3, ie SEQ ID NO: 1 as the upstream primer, select 5, - GTACTAGTCTATGGGGAGAAGGTGGTGGTG -3, ie SEQ ID NO:
  • the upstream primer and the downstream primer have no primer dimer, and the annealing temperature difference is small;
  • B) obtaining the CCR7 gene cDNA sequence PCR amplification using the upstream primer and the downstream primer to obtain a large number of CCR7 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.
  • Preliminary identification the preliminary identification results indicate that the CCR7 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 CCR7 gene cDNA sequence was extracted and used.
  • the present invention utilizes genetic engineering technology to construct a lentiviral expression vector which specifically promotes high expression of CCR7 gene. After successful identification, it is packaged into a virus and introduced into Jurkat cells. After screening cells with puromycin, real-time quantitative PCR and The Western Blot technique verified the change of CCR7 gene expression from mRNA and protein levels, respectively. The experimental results showed that the CCR7 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 CCR7 gene is highly expressed.
  • the present invention also provides the use of a lentiviral expression vector which specifically promotes high expression of a CCR7 gene for the preparation of a medicament for treating a disease associated with abnormal expression of a CCR7 gene.
  • the lentiviral expression vector which specifically promotes the high expression of CCR7 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 CCR7 gene, and can be used as a powerful tool.
  • the present invention also provides specific promotion of CCR
  • a method for constructing a lentiviral expression vector with high expression of 7 gene which has a good operation effect and reduces the cost of sequence synthesis
  • 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 ATCC, 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 were purchased from Takara.
  • the RNeasy Mini Kit was purchased from QIAGEN, the pGM-T carrier was purchased from Tiangen, and the 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 the CCR7 gene
  • the coding sequence of the CCR7 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 (CCR7-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 CCR7 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-CCR7 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 CCR7 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-CCR7 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.
  • CCR7 gene is 80-fold higher than that of Jurkat cells, whether it is just after screening or after 20 generations of pLVX-CCR7 cells, and the expression of CCR7 gene in pLVX empty vector cells.
  • the CCR7 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 CCR7 gene.
  • the lentiviral expression vector which specifically promotes the high expression of CCR7 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 CCR7 gene, and can be used as a powerful tool.
  • the present invention also provides specific promotion of CCR
  • the construction method of the lentiviral expression vector with high expression of 7 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 CCR7, 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 CCR7 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 CCR7 comprend un site de découpe d'enzyme EcoR I, une séquence de codage du gène CCR7 et un site de découpe de l'enzyme Spe I, et la séquence d'ADNc du gène CCR7 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, il est capable d'exprimer, de manière spécifique, en continu, avec efficacité et stabilité le gène CCR7, et peut servir d'outil puissant pour la recherche et le développement de médicaments associés à CCR7.
PCT/CN2016/086063 2016-06-16 2016-06-16 Vecteur d'expression lentiviral pour améliorer le taux d'expression du gène ccr7, et ses applications WO2017214939A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1480465A (zh) * 2002-09-02 2004-03-10 中国科学院遗传与发育生物学研究所 重组人外周淋巴组织趋化性细胞因子及其制备方法和用途
WO2008085564A2 (fr) * 2006-09-20 2008-07-17 The Board Of Regents Of The University Of Texas System Composés et procédés impliquant des récepteurs de recombinaison tronqués couplés à la protéine g(7)
CN101400785A (zh) * 2005-09-30 2009-04-01 宝生物工程株式会社 T细胞群的制备方法
CN102031244A (zh) * 2010-11-08 2011-04-27 中国人民解放军军事医学科学院基础医学研究所 重组间充质干细胞及其制备方法与应用
CN103619351A (zh) * 2010-12-14 2014-03-05 詹姆斯·W·利拉德 用于预防和治疗癌症和癌细胞迁移的抗ccl25抗体和抗ccr9抗体
CN104800243A (zh) * 2014-01-28 2015-07-29 中国人民解放军军事医学科学院基础医学研究所 一种重组间充质干细胞在制备免疫抑制剂中的应用

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1480465A (zh) * 2002-09-02 2004-03-10 中国科学院遗传与发育生物学研究所 重组人外周淋巴组织趋化性细胞因子及其制备方法和用途
CN101400785A (zh) * 2005-09-30 2009-04-01 宝生物工程株式会社 T细胞群的制备方法
WO2008085564A2 (fr) * 2006-09-20 2008-07-17 The Board Of Regents Of The University Of Texas System Composés et procédés impliquant des récepteurs de recombinaison tronqués couplés à la protéine g(7)
CN102031244A (zh) * 2010-11-08 2011-04-27 中国人民解放军军事医学科学院基础医学研究所 重组间充质干细胞及其制备方法与应用
CN103619351A (zh) * 2010-12-14 2014-03-05 詹姆斯·W·利拉德 用于预防和治疗癌症和癌细胞迁移的抗ccl25抗体和抗ccr9抗体
CN104800243A (zh) * 2014-01-28 2015-07-29 中国人民解放军军事医学科学院基础医学研究所 一种重组间充质干细胞在制备免疫抑制剂中的应用

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