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Specific STING expression isomer, cell line, preparation method and application

An isoform and cell line technology, applied in the field of biomedicine, can solve problems such as obstacles, lack of pmSTING molecular mechanism cell model, and no report on STING isoform technology, and achieve the effect of promoting expression

Pending Publication Date: 2022-03-01
哈尔滨医科大学附属肿瘤医院
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on the technology of preparing and expressing specific STING isoforms in the prior art, which creates obstacles for the study of STING mechanism and the development of new small molecule immunotherapeutic drugs targeting STING
[0005] Through the above analysis, the existing problems and defects of the prior art are: there is no report on the preparation and expression of specific STING isomers in the prior art, and there is a lack of cell models for pmSTING molecular mechanism research and drug development

Method used

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  • Specific STING expression isomer, cell line, preparation method and application
  • Specific STING expression isomer, cell line, preparation method and application
  • Specific STING expression isomer, cell line, preparation method and application

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preparation example Construction

[0062] like figure 1 As shown, the preparation method of the cell line expressing a specific STING isoform provided by the embodiments of the present invention includes:

[0063] S101, the culture of mouse splenocytes and the isolation and culture of human peripheral blood mononuclear cells PBMCs;

[0064] S102, using PBMCs cDNA as a template to synthesize the open reading frame ORF of human pmSTING and erSTING; using mouse splenocyte cDNA as a template to synthesize the ORF of mouse pmSTING and erSTING;

[0065] S103, respectively cloning the ORFs of the synthesized alternatively spliced ​​isoforms into the pcDNA3.1-GFP vector to construct pmSTING-GFP and erSTING-GFP fusion gene plasmids;

[0066] S104, transfect the constructed fusion gene plasmid into pre-cultured B16 Tmem173- / - cells and HEK293T cells, the B16 cell line expressing only erSTING and only pmSTING and the HEK293T cell line expressing only erSTING and expressing pmSTING can be obtained.

[0067] The culture ...

Embodiment 1

[0083] 1. C57BL / 6 Tmem173wt / wt Mice were purchased from the Animal Center of the Second Affiliated Hospital of Harbin Medical University, C57BL6 Tmem173gt / gt Mice were purchased from Jackson Laboratory. Take C57BL / 6 Tmem173wt / wt and C57BL6 Tmem173gt / gt The fresh spleen of the mouse was gently crushed with a syringe plunger in sterile PBS, and the splenocytes suspended in PBS were filtered through a filter (100 μm), placed in a centrifuge, and centrifuged at 1500g for 5 minutes. Remove the supernatant, resuspend the cell pellet in 1 mL of red blood cell lysis buffer, and lyse at room temperature for 3 minutes to remove red blood cells. Place in a centrifuge and centrifuge at 1500g for 5 minutes. Remove the supernatant, resuspend the cell pellet with PBS, and centrifuge at 1500g for 5 minutes to successfully separate the mouse splenocytes.

[0084] 2. Collect blood from healthy volunteers into 5mL EDTA anticoagulant tubes and dilute with 6mL sterile PBS. Then the diluted b...

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Abstract

The invention belongs to the technical field of biomedicine, and discloses an isomer for expressing specific STING, a cell line, a preparation method and application, the STING isomer comprises erSTING and pmSTING, and Pre-mRNA transcribed by an STING gene has two transcripts with different splicing modes; the longer transcript is translated and processed into classical erSTING to be positioned on the endoplasmic reticulum; the short transcript lacks a transmembrane region due to exon jumping, and is translated and processed into pmSTING to be positioned on a cytoplasmic membrane. According to the invention, mouse and human cell lines expressing specific STING isomers are constructed for the first time, namely, a B16 cell line only expressing erSTING and only expressing pmSTING and an HEK293T cell line only expressing erSTING and only expressing pmSTING, and a foundation is laid for research and development of novel micromolecular immunotherapy drugs taking STING as a target spot.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a method for expressing a specific STING isomer, a cell line, a preparation method and an application. Background technique [0002] At present, Stimulator of interferon genes (STING) is a new protein molecule that plays an important role in the natural immune response process, and is widely expressed in immune cells and tumor cells. [0003] As the main regulator of the anti-tumor immune cycle, STING regulates all links in the whole cancer immune cycle. The type I IFN produced by STING activation can promote the activation of various immune cells, increase the ability of antigen presentation, and activate the anti-tumor immune response. Activating STING has been proven to be effective in a variety of cancers. There have been more than 20 clinical trials related to STING agonists. A series of high-level studies on STING in tumors, compelling in vivo and in vitro d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/47C12N15/12C12N5/10C12N15/85C12N15/62C12R1/91
CPCC07K14/4702C12N5/0686C12N5/0693C12N5/0626C12N15/85C07K2319/60C12N2510/00
Inventor 郑桐森李晓波时佳琪刘偲奇聂建华李顺李雪寒
Owner 哈尔滨医科大学附属肿瘤医院
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