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WO2018170764A1 - Vecteur pour arni et son application - Google Patents

Vecteur pour arni et son application Download PDF

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Publication number
WO2018170764A1
WO2018170764A1 PCT/CN2017/077606 CN2017077606W WO2018170764A1 WO 2018170764 A1 WO2018170764 A1 WO 2018170764A1 CN 2017077606 W CN2017077606 W CN 2017077606W WO 2018170764 A1 WO2018170764 A1 WO 2018170764A1
Authority
WO
WIPO (PCT)
Prior art keywords
sequence
vector
gene
rnai
rna interference
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/CN2017/077606
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English (en)
Chinese (zh)
Inventor
毛吉炎
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shenzhen Biocan Technologies Co Ltd
Original Assignee
Shenzhen Biocan Technologies Co Ltd
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 Shenzhen Biocan Technologies Co Ltd filed Critical Shenzhen Biocan Technologies Co Ltd
Priority to PCT/CN2017/077606 priority Critical patent/WO2018170764A1/fr
Publication of WO2018170764A1 publication Critical patent/WO2018170764A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention belongs to the field of genetic engineering, and relates to a vector for RNAi and application thereof, and particularly to an RNA interference vector of GP34 gene and application thereof.
  • GP34 is a member of the TNF receptor superfamily and is a type I transmembrane glycoprotein. The expression profile of GP34 is restricted to the surface of activated CD4+ and CD8+ T cells, and is predominantly CD4+ T cells. OX40/GP34 is an important co-stimulatory molecule that plays an important role in the body's immune response and various diseases, and its interaction can promote CD+4.
  • Activation, proliferation, and migration of T cells prolong their life span and promote the formation of germinal centers and the differentiation and maturation of DCs.
  • GP34 can synergistically stimulate the activation of T cells, promote the production of high-valent antibodies and class switching of B cells, mediate the infiltration of OX40+ T cells into the inflammatory reaction site, and play an important role in the immunotherapy of tumors.
  • the research can achieve clinical transformation, but the lack of vectors specifically inhibiting the expression of GP34 gene in the prior art makes the related research not well developed.
  • the object of the present invention is an RNA interference vector of GP34 gene and a construction method and application thereof.
  • an isolated polynucleotide is provided, the nucleotide sequence of which is set forth in SEQ ID NO: 1.
  • a recombinant vector comprising the nucleotide sequence set forth in SEQ ID NO: 2, wherein the recombinant vector is a pLVX-shRNA1 vector as a backbone vector, in a polyclonal enzyme GP34- consisting of Bam HI restriction site + target nucleotide sequence + stem loop structure sequence + target nucleotide sequence complementary sequence + termination site sequence + EcoR I restriction site at the cleavage site RNAi sequence.
  • RNA interference vector of the aforementioned GP34 gene including the following steps:
  • RNA interference vector of GP34 gene The synthesized GP34-RNAi sequence and the extracted plasmid pLVX-shRNA1 were digested with restriction endonucleases Bam HI and EcoR I, electrophoresis and gelatinization. The vector was recovered, and the GP34-RNAi sequence was ligated into the SJpLVX-shRNA1 expression vector by T4 DNA Ligase to obtain a ligation product; the ligation product was transformed into competent E. coli Stbl3 and uniformly coated onto an ampicillin-containing LB medium plate. The positive monoclonal colonies were picked and cultured and identified by PCR. The preliminary identification results indicated that the GP34-RNAi sequence was inserted into the successful bacterial solution for sequencing and identification;
  • RNA interference vector of GP34 gene Extraction of RNA interference vector of GP34 gene: The GP34-RNAi sequence was confirmed to be inserted into a successful bacterial cell expansion culture, and a large amount of recombinant plasmid was extracted to obtain an RNA interference vector of GP34 gene.
  • the present invention utilizes RNAi technology to construct an RNA interference vector targeting GP34 gene expression, and after successful identification, electrotransformation of DC2.4 cells, using the real-time fluorescent quantitative PCR technique to verify the inhibitory effect of GP34 gene expression from the mRNA level, the experiment As a result, it was confirmed that the GP34-RNAi sequence provided by the present invention was successfully inserted into the pLVX-shRNA1 expression vector, and the inhibitory effect on the expression of the GP34 gene was remarkable.
  • polynucleotide of the polynucleotide represented by 1 or the RNA interference vector of the aforementioned GP34 gene for the drug of the GP34 gene expression abnormality-related disease.
  • RNA interference vector of the GP34 gene provided by the invention has the advantages of high transfection efficiency, low dosage, high and specific inhibition of GP34 gene expression in DC2.4 cells, and can be used as a powerful tool for preparing and treating the G P34 gene.
  • a drug that expresses an abnormally related disease A drug that expresses an abnormally related disease.
  • FIG. 1 is a schematic diagram showing the results of real-time quantitative PCR detection of RNA interference vector cells transfected with GP34 gene.
  • DC2.4 cells were purchased from Wuxi Yingnuui Biomedical Technology Co., Ltd., RNAi vector pLVX-shRNAl was purchased from Clontech, RNeasy MiniKit was purchased from Qiagen, and endotoxin free plasmid extraction kit was purchased from Omega bio-tek.
  • the complete medium described below is a cell culture medium supplemented with 10% fetal bovine serum.
  • GP34 mRNA The complete sequence of GP34 mRNA was found in GenBank. According to the full sequence of GP34 mRNA, the interference fragment was designed according to the design principle of RNAi fragment, NCBI BLSAT was used for homology comparison, and the target nucleotide sequence was obtained after confirming specificity.
  • the GP34-RNAi sequence was designed as follows: Bam
  • the designed GP34-RNAi sequence chain was synthesized by Shanghai Biotech Engineering Services Co., Ltd.
  • the synthesized GP34-RNAi sequence and the extracted plasmid pLVX-shRNA1 were digested with restriction endonucleases Bam HI and EcoR I, electrophoresed, and the gel was recovered, and the GP34-RNAi sequence was further amplified by T4 DNA Ligase.
  • An RNA interference vector that forms the GP34 gene is ligated into the vector pLVX-shRNA1.
  • the ligation product was transformed into competent E. coli Stbl3, plated on a plate containing ampicillin LB medium, and cultured at 37 ° C for 14 h. Pick up the grown colonies and send them to Shanghai Biotech for sequencing. The sequencing results are fully consistent with the designed sequence and can be used in subsequent experiments.
  • DC2.4 cells were cultured, and 35,000 cells in good growth state were taken, and the cells were collected by centrifugation, and then resuspended in 500 L of PBS with 20 g.
  • RNA interference vector of GP34 gene was mixed and added to the electric shock cup, and the electric rotation was performed by BTX ECM830 electro-rotation instrument.
  • the electroporation procedure was: 2.1 KV, 25 ⁇ , pulse electric shock once; the cells were transferred to 5 mL DMEM. In a 6 cm dish of complete medium, the cells were gently shaken to mix the cells, and the expression of GP34 gene was detected 48 h later.
  • DC2.4 cells of normal DC2.4 cells and RNA interference vector of GP34 gene were cultured for 48 h, respectively, 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 reverse transcription was completed, the cDNA was diluted with 90 ⁇ l of RNase-Free dH20 and stored at -20 ° C for later detection. Take the cDNA of each group of cells
  • ⁇ is the template, GAPDH is used as the internal reference, and the relative expression of GP34 is detected by real-time quantitative PCR (QPCR).
  • the reaction conditions are set: 95 ° C for 10 s, 1 cycle; 95 ° C 5 s, 54 ° C
  • the NP34 gene RNA interference vector provided by the invention has the advantages of high transfection efficiency, low dosage, high and specific inhibition of GP34 gene expression in DC2.4 cells, and can be used as a powerful tool for preparing and treating G P34 gene.
  • a drug that expresses an abnormally related disease is a drug that expresses an abnormally related disease.

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  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne : une séquence GP34-ARNi et un vecteur recombinant contenant la séquence, la séquence GP34-ARNi étant telle que représentée dans la SEQ ID NO:1 ; un vecteur recombinant contenant une séquence telle que représentée dans la SEQ ID NO:2 ; et un vecteur d'ARN interférent du gène GP34 et sa méthode de préparation. La méthode de préparation comprend : l'utilisation de pLVX-shRNA1 en tant que vecteur squelette, et l'insertion dans un site recombinant d'une séquence d'oligonucléotides d'ARNsh consistant en un site de coupe de l'enzyme Bam HI + une séquence nucléotidique cible + une séquence de structure tige-boucle + une séquence complémentaire de séquence nucléotidique cible + une séquence de site de terminaison + un site de coupe de l'enzyme EcoR I en connexion séquentielle.
PCT/CN2017/077606 2017-03-22 2017-03-22 Vecteur pour arni et son application Ceased WO2018170764A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2017/077606 WO2018170764A1 (fr) 2017-03-22 2017-03-22 Vecteur pour arni et son application

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2017/077606 WO2018170764A1 (fr) 2017-03-22 2017-03-22 Vecteur pour arni et son application

Publications (1)

Publication Number Publication Date
WO2018170764A1 true WO2018170764A1 (fr) 2018-09-27

Family

ID=63586177

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2017/077606 Ceased WO2018170764A1 (fr) 2017-03-22 2017-03-22 Vecteur pour arni et son application

Country Status (1)

Country Link
WO (1) WO2018170764A1 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101948859A (zh) * 2010-08-17 2011-01-19 深圳市疾病预防控制中心 特异性抑制SET蛋白表达的shRNA表达载体及其构建方法和应用
WO2016022468A1 (fr) * 2014-08-04 2016-02-11 Baylor Research Institute Anticorps anti-ox40l antagonistes et leurs procédés d'utilisation

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101948859A (zh) * 2010-08-17 2011-01-19 深圳市疾病预防控制中心 特异性抑制SET蛋白表达的shRNA表达载体及其构建方法和应用
WO2016022468A1 (fr) * 2014-08-04 2016-02-11 Baylor Research Institute Anticorps anti-ox40l antagonistes et leurs procédés d'utilisation

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