WO2019237383A1 - Vecteur modifié utilisé pour l'édition du gène tnfsf18 humain, son procédé de préparation et son application - Google Patents

Vecteur modifié utilisé pour l'édition du gène tnfsf18 humain, son procédé de préparation et son application Download PDF

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Publication number
WO2019237383A1
WO2019237383A1 PCT/CN2018/091713 CN2018091713W WO2019237383A1 WO 2019237383 A1 WO2019237383 A1 WO 2019237383A1 CN 2018091713 W CN2018091713 W CN 2018091713W WO 2019237383 A1 WO2019237383 A1 WO 2019237383A1
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WO
WIPO (PCT)
Prior art keywords
tnfsf18
gene
human
vector
editing
Prior art date
Application number
PCT/CN2018/091713
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English (en)
Chinese (zh)
Inventor
毛吉炎
Original Assignee
深圳市博奥康生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 深圳市博奥康生物科技有限公司 filed Critical 深圳市博奥康生物科技有限公司
Priority to PCT/CN2018/091713 priority Critical patent/WO2019237383A1/fr
Publication of WO2019237383A1 publication Critical patent/WO2019237383A1/fr

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Classifications

    • 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
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • 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
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses

Definitions

  • the invention relates to the technical field of genetic engineering, in particular to a modified vector for human TNFSF18 gene editing, a preparation method and application thereof.
  • TNFSF18 is a ligand for the surface molecule CD357 on CD4 + CD25 + Treg cells of thymus origin. Studies have shown that CD357 / TNFSF18 has many important biological activities, including cell proliferation, differentiation, and survival.
  • the CD357 / TNFSF18 system participates in the role of Treg cells in immune regulation, plays an important role in tumor immunotherapy, and has good clinical transformation prospects, but the prior art lacks a means to target the knockout of TNFSF18 gene expression. Related research Progress has caused certain obstacles.
  • a first object of the present invention is to provide a modified vector for human TNFSF18 gene editing.
  • a second object of the present invention is to provide a method for preparing a modified vector for human TNFSF18 gene editing.
  • the present invention provides a targeted sgRNA for human TNFSF18 gene editing, and its sequence is 5′- AATTTCTACTCTTGTAGATCTGGAGAAATGAGAGCTGTGGAA -3 ’.
  • the invention also provides a method for preparing the modified vector for editing human TNFSF18 gene, which includes the following steps:
  • step (3) ligating the sgRNA obtained in step (1) to a linearized core vector with T4 DNA ligase;
  • the ligation product is transformed into competent E. coli Stbl3. After a large amount of culture, the recombinant vector is extracted and commissioned for sequencing. The correct sequencing result is the modified vector for human TNFSF18 gene editing.
  • the core vector is pLVX-ascpf1-puro.
  • the modified vector for human TNFSF18 gene editing provided by the present invention has strong specificity, can edit human TNFSF18 gene through the CRISPR / Cpf1 system very efficiently, and can be used in research and development of drugs related to abnormal expression of TNFSF18 gene.
  • Figure 1 is a vector map of pLVX-AsCpf1-puro
  • FIG. 1 T7 Endonuclease I test results of Jurkat cells in the control group and the experimental group, wherein: 1-experimental group, 2-control group.
  • Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System provides crRNA design rules and gRNA sequences that target TNFSF18 gene, design crRNA that targets TNFSF18 gene, and according to the actual situation of pLVX-AsCpf1-puro vector in its 5 ' The sticky ends of BamHI and EcoRI digestion sites were added at the 3 'and 3' ends, respectively.
  • the forward sequence was 5'-GATCCT AATTTCTACTCTTGTAGATCTGGAGAAATGAGAGCTGTGGAAG -3 ', and the reverse sequence was 5'-GTTCCACAGCTCTCATTTCTCCAGATCTACAAGAGTAGAAATTC-3'.
  • 5 ⁇ L of each was mixed, heated at 95 ° C for 5 minutes, and then naturally cooled to room temperature to form a double-stranded DNA with sticky ends of BamHI and EcoRI.
  • T4 DNA ligase was used to ligate a and b products.
  • the ligated product was then transformed into E. coli Stbl3 and identified by sequencing.
  • the recombinant vector contained in the correct strain was the pLVX-AsCpf1-puro-TNFSF18 vector.
  • the correct strain was sequenced and identified in Example 1 and placed in an LB liquid medium having an ampicillin concentration of 100 ⁇ g / ml, and cultured with shaking at 250 rpm and 37 ° C. for 12-16 h. Collect the bacterial solution by centrifugation at 10,000 rpm at 4 ° C, discard the supernatant, collect the bacterial cells, and then extract the plasmid according to the instructions of the Endo-Free Plasmid Mini Kit kit to obtain the endotoxin-free pLVX-AsCpf1-puro-TNFSF18 plasmid.
  • 293T cells were cultured and transfected after 2 passages of growth and culture: pLVX-AsCpf1-puro-TNFSF18 vector was taken and transfected with the packaging plasmid and transfection reagent provided by the integrase-deficient Lenti-X HTX lentivirus packaging system Stained in 293T cells. 48 hours before transfection, inoculate cells into a well plate or petri dish for lentivirus production. During transfection, the confluence of cells is about 70% -80% is the best infection state, and the viability is ⁇ 95%. The staining time was the starting point. The supernatants were harvested after 48 h and 72 h, filtered through a 0.45 ⁇ m filter and stored at -80 ° C.
  • Untreated Jurkat cells (control group) and lentivirus-treated Jurkat cells (experimental group) were seeded into six-well plates. After the cells were full, genomic DNA was extracted, and then the high-fidelity PCR enzyme PrimeSTAR HS was used to expand The gene editing site is expected to be amplified, and the PCR product is recovered by electrophoresis.
  • the modified vector for human TNFSF18 gene editing provided by the present invention has strong specificity, can edit human TNFSF18 gene through the CRISPR / Cpf1 system very efficiently, and can be used in research and development of drugs related to abnormal expression of TNFSF18 gene.

Abstract

La présente invention concerne un vecteur modifié utilisé pour l'édition du gène tnfsf18 humain, son procédé de préparation et son application. Le vecteur modifié utilisé pour l'édition du gène TNFSF18 a une forte spécificité et peut être utilisé pour modifier extrêmement efficacement le gène TNFSF18 humain au niveau cellulaire au moyen d'un système CRISPR/Cpf1, étant utilisé pour obtenir des cellules positives d'édition de gène TNFSF18, réunissant ainsi les conditions propices pour la recherche associée au gène TNFSF18.
PCT/CN2018/091713 2018-06-16 2018-06-16 Vecteur modifié utilisé pour l'édition du gène tnfsf18 humain, son procédé de préparation et son application WO2019237383A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2018/091713 WO2019237383A1 (fr) 2018-06-16 2018-06-16 Vecteur modifié utilisé pour l'édition du gène tnfsf18 humain, son procédé de préparation et son application

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2018/091713 WO2019237383A1 (fr) 2018-06-16 2018-06-16 Vecteur modifié utilisé pour l'édition du gène tnfsf18 humain, son procédé de préparation et son application

Publications (1)

Publication Number Publication Date
WO2019237383A1 true WO2019237383A1 (fr) 2019-12-19

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100240732A1 (en) * 2007-10-01 2010-09-23 University Of Miami Aptamer-targeted sirna to prevent attenuation or suppression of a t cell function
CN105907785A (zh) * 2016-05-05 2016-08-31 苏州吉玛基因股份有限公司 化学合成的crRNA用于CRISPR/Cpf1***在基因编辑中的应用
CN106978428A (zh) * 2017-03-15 2017-07-25 上海吐露港生物科技有限公司 一种Cas蛋白特异结合靶标DNA、调控靶标基因转录的方法及试剂盒
WO2017152149A1 (fr) * 2016-03-03 2017-09-08 University Of Massachusetts Adn double hélice linéaire à extrémité fermée pour transfert de gène non viral
CN107312761A (zh) * 2017-07-18 2017-11-03 江苏溥博生物科技有限公司 一种AsCpf1突变体蛋白、编码基因、重组表达载体及其制备方法与应用
CN107406878A (zh) * 2015-02-03 2017-11-28 昂科梅德制药有限公司 肿瘤坏死因子受体超家族(tnfrsf)结合剂类及其用途
CN107488649A (zh) * 2017-08-25 2017-12-19 南方医科大学 一种Cpf1和p300核心结构域的融合蛋白、相应的DNA靶向激活***和应用

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100240732A1 (en) * 2007-10-01 2010-09-23 University Of Miami Aptamer-targeted sirna to prevent attenuation or suppression of a t cell function
CN107406878A (zh) * 2015-02-03 2017-11-28 昂科梅德制药有限公司 肿瘤坏死因子受体超家族(tnfrsf)结合剂类及其用途
WO2017152149A1 (fr) * 2016-03-03 2017-09-08 University Of Massachusetts Adn double hélice linéaire à extrémité fermée pour transfert de gène non viral
CN105907785A (zh) * 2016-05-05 2016-08-31 苏州吉玛基因股份有限公司 化学合成的crRNA用于CRISPR/Cpf1***在基因编辑中的应用
CN106978428A (zh) * 2017-03-15 2017-07-25 上海吐露港生物科技有限公司 一种Cas蛋白特异结合靶标DNA、调控靶标基因转录的方法及试剂盒
CN107312761A (zh) * 2017-07-18 2017-11-03 江苏溥博生物科技有限公司 一种AsCpf1突变体蛋白、编码基因、重组表达载体及其制备方法与应用
CN107488649A (zh) * 2017-08-25 2017-12-19 南方医科大学 一种Cpf1和p300核心结构域的融合蛋白、相应的DNA靶向激活***和应用

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