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WO2019037057A1 - ARNSH CIBLANT DD1α SILENCIEUX - Google Patents

ARNSH CIBLANT DD1α SILENCIEUX Download PDF

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Publication number
WO2019037057A1
WO2019037057A1 PCT/CN2017/098914 CN2017098914W WO2019037057A1 WO 2019037057 A1 WO2019037057 A1 WO 2019037057A1 CN 2017098914 W CN2017098914 W CN 2017098914W WO 2019037057 A1 WO2019037057 A1 WO 2019037057A1
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WO
WIPO (PCT)
Prior art keywords
vector
shrna
dd1α
fragment
ddla
Prior art date
Application number
PCT/CN2017/098914
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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/CN2017/098914 priority Critical patent/WO2019037057A1/fr
Publication of WO2019037057A1 publication Critical patent/WO2019037057A1/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
    • C12N15/11—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

Definitions

  • the present invention belongs to the field of DNA recombination technology in bioengineering, and specifically relates to shRNA which specifically silences the target protein D Dla.
  • DDla protein is a new member of the PD-L1 protein family and is a novel and structurally different Ig superfamily inhibitory ligand whose extracellular domain has homology to the B7 family ligand PD-L1.
  • DDla protein plays an important role in various cancers and autoimmune diseases, allergies, infections and inflammatory diseases such as multiple sclerosis and cellular immunity of joint disorders, and requires extensive transformation studies for clinical application.
  • lentiviral vectors that specifically inhibit the expression of DDla gene in the prior art has made the related studies not well developed.
  • the object of the present invention is to synthesize a specific base sequence capable of targeting silencing DDla, and provides an effective means for further research on the action of DDla.
  • the main construct required in the present invention is a silencing vector of DDla.
  • the construction of the core silencing vector is divided into two major steps. The first step is to design a 19 bp shRNA core fragment based on the human DDla sequence. The synthesized fragment has appropriate restriction sites at both ends, and the target fragment is ligated to the pSUPER vector to form The core silencing vector, after transformation, pick positive clones for PCR identification. The correct vector was identified, DNA sequencing was performed, and silencing efficiency was subsequently detected by immunoblotting.
  • a 19 bp shRNA core fragment capable of targeting human DDla was designed and synthesized, and its sequence was 5,-GGTGCAGACAGGCAAAGAT-3, (SEQ ID No: 1).
  • Construction of an expression vector that specifically silences DDla includes the following steps:
  • the sequence of DDla is specifically identified based on the human DDla gene sequence using the corresponding software design.
  • the second step the artificial synthesis of double-stranded shRNA fragments
  • the linearized vector is recovered by using a recovery kit, and the recovered fragment is ligated with the shRNA fragment obtained by annealing, transformed, and the positive clone is identified, and the successfully constructed silencing vector is obtained by sequencing.
  • the plasmid was extracted, and the total RNA was extracted after transfecting the cells. After reverse transcription into cDNA, the silencing efficiency was detected by quantitative PCR.
  • the shRNA fragment designed in the present invention can specifically silence DDloc.
  • the experimental results show that the successful construction of the vector can specifically silence DDlot and reduce the expression of DDlot in cells. Therefore, the application of DDloc silencing vector can study the function of DDloc more elaborately, providing a theoretical basis for explaining related mechanisms.
  • FIG. 1 is a schematic diagram showing the results of quantitative PCR detection of DDla gene expression of Jurkat cells after transfection of pSUPER-DDla vector.
  • Jurkat cells were purchased from the Cell Resource Center of the Shanghai Institute of Biological Sciences, and the RNeasy Mini Kit was purchased from Qiagen.
  • the complete medium described below is a cell culture medium supplemented with 10% fetal bovine serum.
  • Example 1 Design of shRNA Oligonucleotide Sequences
  • the full sequence of DDla mRNA was found in GenBank, and the specificity was confirmed by BLAST homology alignment.
  • the secondary structure of the target mRNA sequence was evaluated by RNAstmcture 4.4 software, and finally the target nucleotide sequence, such as SEQ ID, was obtained. NO: 1 is shown.
  • the 59 bp sequence was designed in the form of Bgl II-GN18-TT-loop-81NC-Hind III, 81NC is the reverse complement of NG18, the 5' end is the Bgl II restriction site, and the 3' end is the Hind III cleavage site. Point, the obtained sequences are respectively SEQ ID NO:
  • the synthesized shRNA oligonucleotides are mixed in equal amounts in a single strand and annealed to form a double-stranded shRNA. Take 2 g
  • the pSUPER vector, Bgl II and Hind III were digested, electrophoresed, and the linear pSUPER large fragment was recovered. Dilute the linearized pSUPER concentration to 100
  • Ng/ ⁇ , and the double-stranded shRNA obtained by annealing were ligated overnight at 4 °C with T4 DNA ligase (NEB).
  • the ligation product was transformed into ToplO competent cells and plated on LB plates with Kan resistance. Monoclonal shakes were picked on the transformation plates and cultured overnight before being sent to Shanghai Biotech for sequencing. The correct bacteria were sequenced for the extraction of endotoxin-free plasmids, and the pSUPER-DDla vector was obtained.
  • Example 3 The pSUPER-DDla vector transduced Jurkat cells.
  • Jurkat cells were cultured, and cells grown in good condition were inoculated into a six-well plate at 10 6 cells per well, and the cell density was about 60% after 18 h.
  • the extracted pSUPER-DDla vector was transferred to 3 g using Lipofectamin 2000. Guide Jurkat cells. After 6 h, the medium was changed to fresh complete medium and cultured for 48 h.
  • the shRNA fragment synthesized in the present invention is designed to specifically silence DDla.
  • the experimental results show that the successful construction of the vector can specifically silence DDlot and reduce the expression of DDla in cells. Therefore, the application of DDla silencing vector can more closely study the function of DDla, providing a theoretical basis for explaining related mechanisms.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne un ARNsh spécifique pour la protéine DD1α cible silencieuse. Un vecteur de silençage pour DD1α est construit. Tout d'abord, un fragment de noyau d'ARNsh de 19 pb est conçu selon la séquence d'un gène DD1α humain, deux extrémités du fragment synthétisé ayant des sites de coupure de l'enzyme de restriction appropriés ; le fragment cible est raccordé à un vecteur pSUPER pour former un vecteur de silençage de noyau ; après transformation, un clone positif est sélectionné pour une vérification par PCR ; un vecteur correct est vérifié, un séquençage d'ADN est effectué, et ensuite l'efficacité de silençage est mesurée au moyen d'une expérience d'immunotransfert. Un fragment de noyau d'ARNsh de 19 pb capable de cibler DD1α humain est synthétisé et conçu, et la séquence du fragment de noyau d'ARNsh est 5'-GGTGCAGACAGGCAAAGAT-3'.
PCT/CN2017/098914 2017-08-24 2017-08-24 ARNSH CIBLANT DD1α SILENCIEUX WO2019037057A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2017/098914 WO2019037057A1 (fr) 2017-08-24 2017-08-24 ARNSH CIBLANT DD1α SILENCIEUX

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2017/098914 WO2019037057A1 (fr) 2017-08-24 2017-08-24 ARNSH CIBLANT DD1α SILENCIEUX

Publications (1)

Publication Number Publication Date
WO2019037057A1 true WO2019037057A1 (fr) 2019-02-28

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105586320A (zh) * 2016-02-01 2016-05-18 厚朴生物科技（苏州）有限公司 一种重组腺相关病毒及其构建方法和应用
WO2017112741A1 (fr) * 2015-12-22 2017-06-29 Novartis Ag Récepteur d'antigène chimérique (car) contre la mésothéline et anticorps contre l'inhibiteur de pd-l1 pour une utilisation combinée dans une thérapie anticancéreuse
CN106955354A (zh) * 2016-01-11 2017-07-18 中国科学院上海生命科学研究院 用于肿瘤免疫治疗的联合用药物组合

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017112741A1 (fr) * 2015-12-22 2017-06-29 Novartis Ag Récepteur d'antigène chimérique (car) contre la mésothéline et anticorps contre l'inhibiteur de pd-l1 pour une utilisation combinée dans une thérapie anticancéreuse
CN106955354A (zh) * 2016-01-11 2017-07-18 中国科学院上海生命科学研究院 用于肿瘤免疫治疗的联合用药物组合
CN105586320A (zh) * 2016-02-01 2016-05-18 厚朴生物科技（苏州）有限公司 一种重组腺相关病毒及其构建方法和应用

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