Chimeric enzyme, application thereof in-vitro one-step reaction synthesis of Cap0 mRNA and method

Abstract

The invention relates to the field of molecular biology, and particularly relates to a chimeric enzyme, application thereof in-vitro one-step reaction synthesis of Cap0mRNA and a method. The method comprises the steps that the chimeric enzyme and an in-vitro transcription template are mixed and then subjected to a co-reaction, and a capped and tailed mRNA product is obtained through a one-step reaction, wherein the chimeric enzyme comprises an RNA polymerase structural domain and a capping enzyme catalytic structural domain, and the chimeric enzyme has the dual functions of in-vitro transcription and capping through chimeric design. The method for in-vitro one-step reaction synthesis of the mRNA is developed, and the synthesis efficiency and the mRNA yield are improved.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a chimeric enzyme and its use and method for synthesizing Cap0 mRNA by one-step reaction in vitro. Background technique [0002] In recent years, respiratory-based infectious diseases have seriously threatened public health. In this context, mRNA-based in vitro transcription technology only needs to provide the gene sequence of the antigen in the research and development design, and does not need to isolate and cultivate highly pathogenic pathogens. , has obvious advantages in dealing with viruses with high transmission risks, and the mRNA vaccine production process is simple, the synthesis is fast, the cost is low, and natural antigenic epitopes are provided, so it has become a new type of vaccine with great advantages. [0003] The working principle of the mRNA in vitro transcription system is to use DNA as a template, and under the action of a series of transcription factors, ...

AI Technical Summary

Problems solved by technology

[0004] At present, in vitro transcription kits on the market are mainly developed for the above two methods. The first method relies on cap analogs to generate a cap structure during the transcription process, which is not efficient and has a low yield. High immunogenicity, causing difficulties in the application of subsequent transcription product mRNA

The second one uses capping enzymes to add a cap structure to the 5' end of the RNA. This type of product is also based on the in vitro transcribed RNA, and the product is capped. This method involves many times from transcription, capping to tailing The enzyme addition operation takes a long time and the procedure is cumbersome. The process of circulation will also cause the degradation and loss of intermediate products, and the overall efficiency and yield need to be improved.

[0005] Aiming at the technical problems existing in the above-mentioned market products, especially the problem of how to synchronize transcription and capping while producing high-quality transcription products, many professionals have carried out relevant transformation and exploration, such as using relevant transcription tools in prokaryotic systems Enzymes prepare mRNA, and select RNA polymerase and capping enzyme after lysing prokaryotic cells, but the cleavage products of this method are all enzymes in prokaryotic cells, including other impurities such as nucleases or nucleic acids endogenous to the large intestine, which will cause The specificity of the enzyme system is not high, which reduces the efficiency, quality and yield of subsequent mRNA production

Even directly degrade RNA, unable to effectively transcribe the target fragment

[0006] Therefore, there is currently a lack of large-scale mass-produced mRNA production technology with simple process and short process. Establishing such a set of easy-to-operate, stable, and efficient mRNA preparation methods will promote the mRNA vaccine platform and facilitate the future vaccine research and development process.

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