Method for preparing recombinant eukaryotic mRNA by using prokaryotic transcription system and application thereof

A transcription system and RNA polymerase technology, applied in the field of preparing recombinant eukaryotic mRNA molecules, can solve the problem of lack of recombinant mRNA preparation technology and the like

Pending Publication Date: 2020-11-06
WUHAN DANGKANG XING ZHONG BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, there is a lack of recombinant mRNA preparation technology suitable for industrialized large-scale production

Method used

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  • Method for preparing recombinant eukaryotic mRNA by using prokaryotic transcription system and application thereof
  • Method for preparing recombinant eukaryotic mRNA by using prokaryotic transcription system and application thereof
  • Method for preparing recombinant eukaryotic mRNA by using prokaryotic transcription system and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089] The design of embodiment 1 capping enzyme plasmid

[0090] Preferably, the present invention selects the capping enzyme system of vaccinia virus for the capping of recombinant mRNA. The vaccinia virus capping enzyme consists of two subunits (D1 and D12) with three enzymatic activities (D1 subunit has RNA triphosphatase and guanyl transferase activities; D12 subunit has guanine methyltransferase activity) , all of these activities are required to add a complete cap structure, m7Gppp(5')N.

[0091] Preferably, the present invention selects T7 RNA polymerase gene for transcription of recombinant mRNA. T7 RNA polymerase is highly promoter-specific and will only transcribe DNA downstream of the T7 promoter. Therefore, it is suitable for transcription of recombinant target mRNA. Some prokaryotic cells, such as Escherichia coli BL21(DE3), contain T7 RNA polymerase gene in their genome. Therefore, it is very convenient for subsequent operations. If the selected prokaryote ...

Embodiment 2

[0099] Example 2 Preparation of Cell Lysate Containing Recombinant RNA Polymerase and Capping Enzyme

[0100] 1) Construction of recombinant expression capping enzyme strain

[0101] Transfer the p4helpers described in Example 1 into the E. coli expression strain BL21(DE3), and then obtain a strain expressing the capping enzyme recombinantly, which is named CAP4.

[0102] 2) Preparation of strain lysis buffer

[0103] The lysis buffer was prepared (all final concentrations): 10mM Tris-Ac pH 7.4, 14mM manganese acetate, 60mM potassium acetate, 1mM DTT, 0.5ml / L 2-mercaptoethanol.

[0104] 3) Induced expression of recombinant capping enzyme and preparation of cell lysate

[0105] CAP4 was inoculated in liquid LB containing kanamycin at a ratio of 1:1000 and cultured overnight. Inoculate 1 L of fresh liquid LB medium containing kanamycin at a ratio of 1:100 to 1 L of fresh liquid LB medium containing kanamycin, and cultivate for 3 hours at 37° C. on a shaker at 200 rpm, so that...

Embodiment 3

[0108] Embodiment 3 The design of recombinant mRNA transcription template plasmid

[0109] Preferably, the present invention uses linearized plasmid DNA as a transcription template for recombinant mRNA. The plasmid DNA can meet the requirements of cloning and transcribing various mRNAs. At its multiple cloning site, the DNA sequence corresponding to the target mRNA can be directly inserted into the gene expression frame in the form of enzyme cutting-ligation. The linearized transcription template DNA can then be directly obtained by hydrolyzing the reserved plasmid linearization site on the plasmid.

[0110] In order to improve the stability of the recombinant mRNA and the efficiency of subsequent translation, the present invention has designed the plasmid DNA sequence as follows:

[0111] Preferably, the present invention uses the T7 promoter to initiate the transcription of the recombinant mRNA, and its corresponding DNA sequence is: 5'-TCTCGATCCCGCGAAATTAATACGACTCACTATAGG-...

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Abstract

The invention provides a method for preparing a recombinant eukaryotic mRNA molecule by using a prokaryotic transcription system. The method comprises the following steps: 1) expressing an enzyme system required for the transcription and capping process of the recombinant eukaryotic mRNA, and obtaining a prokaryotic in-vitro transcription-capping enzyme system; 2) preparing a linear transcriptiontemplate DNA; and 3) transcribing, capping and purifying the recombinant mRNA. The recombinant mRNA molecule prepared by the method provided by the invention has all mRNA characteristics required by eukaryotic organisms in the protein translation process, so that the recombinant mRNA molecule can be further identified and utilized by a eukaryotic translation system. The method provided by the invention overcomes the defects in the field of recombinant mRNA at present, can produce the recombinant eukaryotic mRNA with stable genetic characteristics and no carrier inhibition effect in a simple process and at low cost, and meets the large-scale industrial production demand.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular, the invention relates to the preparation of recombinant eukaryotic mRNA molecules by using a prokaryotic transcription system. Background technique [0002] Gene expression refers to the process in which cells convert genetic information stored in DNA sequences into biologically active protein molecules through transcription and translation during the life process. It includes two stages: transcription of genes and translation of genes. Gene transcription refers to the process of synthesizing messenger RNA (mRNA) using a strand of DNA as a template and according to the principle of complementary base pairing. Gene translation refers to the process in which mRNA synthesizes the corresponding amino acid sequence according to its sequence order according to the triplet code. It can be seen that mRNA plays an intermediate role in the flow of genetic information. Like DNA and protein, mR...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/12
CPCC12N15/70C07K14/43595C12N2830/55
Inventor 陈博李冉王儒恺
Owner WUHAN DANGKANG XING ZHONG BIOTECHNOLOGY CO LTD
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