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Nucleotide molecule for in-vitro transcription of mRNA, presenting cell and application

An in vitro transcription and nucleotide technology, applied in the field of abnormal protein expression of disease antigens and applications, can solve the problems of not meeting the needs of disease treatment, unable to produce stable antigen presentation ability, poor mRNA stability, etc.

Active Publication Date: 2020-04-17
北京立康生命科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, to obtain a single tumor antigen by this method, patients with high expression of AKR1B10 protein are required as test samples, and the selection range of test subjects is narrow; since the full-length protein is used, it is impossible to determine which part of the vaccine is activated by the presentation of the epitope, and the expression of mRNA Poor stability
[0006] In summary, the prior art using TAA to design tumor antigen mRNA cannot meet the current treatment needs of diseases caused by abnormal protein expression, and the existing mRNA preparation samples cannot produce stable and continuous antigen presentation ability

Method used

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  • Nucleotide molecule for in-vitro transcription of mRNA, presenting cell and application
  • Nucleotide molecule for in-vitro transcription of mRNA, presenting cell and application
  • Nucleotide molecule for in-vitro transcription of mRNA, presenting cell and application

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Experimental program
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Embodiment 1

[0075] The acquisition of the nucleotide molecule of embodiment 1 in vitro transcription mRNA

[0076] 1. Codon optimization

[0077] Use the codon optimization software and website to optimize the codons of the N-terminal signal peptide of wild-type human MHC class I, the coding region of the human class I MHC transport signal sequence, and the coding region of the CEF epitope to obtain the N-terminal of MHC class I. Terminal signal peptide, nucleotide sequence of human MHC class I transit signal sequence, CEF epitope.

[0078]It should be noted that, in one embodiment of the present invention, the CEF epitope is only an exemplary epitope, and those skilled in the art can use other epitopes with tandem lymphocytes as nucleotide analysis tandem epitope elements to replace CEF epitope in this example.

[0079] The CEF epitopes used in the present invention are positive epitopes of Cytomegalovirus, Epstein-Barr Virus and InfluenzaVirus Control Peptide, each epitope is 8-12 ami...

Embodiment 2

[0104] Example 2 Antigen Presenting Cells

[0105] 1. Induction and culture of DC in vitro

[0106] 1.1 Isolation of high-purity monocytes

[0107] In order to obtain higher purity mononuclear cells, the mononuclear cells in the peripheral blood of healthy volunteers were firstly separated and purified by a mononuclear cell collection machine, and then the peripheral blood mononuclear cells were separated by lymphocyte separation medium in an ultra-clean bench. Select the kit to purify monocytes to obtain high-purity monocytes.

[0108] 1.2 Culture of DC

[0109] Adding monocytes to ImmunoCult TM Differentiation medium (ImmunoCult TM Add ImmunoCult to the medium TM DC Differentiation Medium), placed at 37°C, 5% CO 2 Cultured in the incubator for 6 days, half of the medium was changed every 3 days.

[0110] Perform maturation culture of DC on day 6 and replace with ImmunoCult TM In maturation medium (ImmunoCult TM Add ImmunoCult to the medium TM DC Maturation Medium),...

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Abstract

The invention provides a nucleotide molecule for in-vitro transcription of mRNA, a presenting cell and application, and relates to the technical field of biology. The nucleotide molecule provided by the invention at least has a nucleic acid sequence shown as SEQ ID NO.3, is used for transcribing at least mRNA with an amino acid sequence shown as SEQ ID NO.1 or / and an amino acid sequence shown as SEQ ID NO.2; the presenting cell is a dendritic cell loaded with the nucleotide molecule for in-vitro transcription of mRNA. The nucleotide molecule for in-vitro transcription of mRNA can be used for preparing an mRNA screening or / and evaluation model, or directly for screening or / and evaluating antigen epitope or mRNA. The nucleotide molecule for in-vitro transcription of mRNA provided by the invention has the advantages of higher stability of mRNA expressed by the nucleotide molecule for in-vitro transcription of mRNA, prolonged continuous expression time, low cost and wide universality; andthe technical problem that the existing tumor antigen vaccine can only present a single antigen is solved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a protein abnormal expression disease antigen and its application. Background technique [0002] With the advancement of biomedical research, more and more diseases related to abnormal protein expression have been discovered. Among them, the antigens abnormally produced by tumor proteins mainly include tumor-associated antigen (Tumor associated Antigen, TAA) and tumor neoantigen (Neo-antigen). At present, antibodies achieve their therapeutic effects by binding to target proteins located on the cell surface or outside the cells. However, the number of extracellular target proteins is very limited. Due to this limitation, the target proteins recognized by antibody drugs in the market are highly concentrated. On the one hand, this greatly limits the scope of indications of antibody drugs, and many diseases lack suitable antibody drugs; on the other hand On the one hand, limited target...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C12N5/10C12N15/38
CPCC07K14/005C07K14/4717C12N5/0639C07K2319/02C12N2710/16222C12N2760/16122C12N2760/16222C12N2760/16322C12N2510/00
Inventor 陈立贾明明黎小珠俞洋洋
Owner 北京立康生命科技有限公司
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