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Method for improving stability of in-vitro synthetic mRNA

An in vitro synthesis and stability technology, applied in fermentation and other directions, can solve problems such as poor stability, achieve the effect of prolonging expression time and improving translation efficiency

Pending Publication Date: 2019-09-10
湖北爱济莱斯生物科技有限公司
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Problems solved by technology

In vitro synthesis of mRNA has broad clinical application prospects in the preparation of nucleic acid vaccines, gene therapy and other fields, but its stability is poor

Method used

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  • Method for improving stability of in-vitro synthetic mRNA
  • Method for improving stability of in-vitro synthetic mRNA
  • Method for improving stability of in-vitro synthetic mRNA

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Embodiment 1

[0023] A method for improving the stability of mRNA synthesized in vitro, comprising the steps of:

[0024] (1) Construction of a luciferase mRNA synthesis template plasmid: the plasmid EF1-GFP-T2A-LuciferaseMinicircle DNA was used as a template to amplify the sequence encoding luciferase by PCR, and the primers for amplifying the sequence encoding luciferase were: forward primer, 5'-cgcggatccgccaccatggaagatgccaaaaacattaag-3 'Reverse primer, 5'-ccgctcgagttacacgg cgatcttgccgccc-3'; PCR reaction conditions: 95°C for 5 minutes, 95°C for 10 seconds, 56°C for 30 seconds, 72°C for 1 minute, and 72°C for 10 minutes. and pcDNA3.4 plasmids were digested with BamHI and XhoI respectively, recovered from the gel, ligated with T4 ligase, transformed into Escherichia coli DH5α and screened for recombinants, and the recombinant plasmids were identified by double digestion, PCR and sequencing to obtain luciferase mRNA synthesis template plasmids ;

[0025] (2) Use the luciferase mRNA synthes...

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Abstract

The invention discloses a method for improving the stability of in-vitro synthetic mRNA. After PCR tailing, cap structure analogue modification and base analogue modification are carried out respectively with a luciferase mRNA synthesis template plasmid as a template, the mRNA is synthesized by using an MEGAscript T7 kit according to an instruction book under the action of T7 RNA polymerase. Through researches about the length of polyA tails and the translation efficiency and stability of cap structure analogues and base analogues on the in-vitro synthetic mRNA, it is found that through a series of modification, the translation efficiency of the in-vitro synthetic mRNA in cells can be improved, and the expression time can be prolonged.

Description

technical field [0001] The invention relates to the technical field of mRNA synthesis, in particular to a method for improving the stability of mRNA synthesized in vitro. Background technique [0002] The mRNA synthesized in vitro is mainly obtained by in vitro reverse transcription using cDNA as a template, and the most commonly used one is synthesized by RNA polymerase transcription using plasmid DNA as a template. At present, the RNA in vitro transcription kit containing RNA polymerase is mainly used to obtain in vitro synthesized mRNA, which uses linearized plasmid DNA as a template under the condition of RNA polymerase (T7, T3 or SP6) and NTP as the template. The substrate synthesizes mRNA complementary to one strand of the template DNA from the downstream of the promoter, and obtains a large number of mRNA molecules simply and quickly. [0003] The cap structure is the basic component that mature mRNA should have. The cap structure of in vitro synthesized mRNA can be ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/34
CPCC12P19/34
Inventor 王小莉李东升
Owner 湖北爱济莱斯生物科技有限公司
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