Preparation method of RNA polymerase using RNA as formwork and application of polymerase

A technology of RNA polymerase and polymerase, which is applied in the field of RNA polymerase preparation, can solve the problems of inability to efficiently synthesize RNA from RNA templates and the inability to apply industrial production, and achieve the effects of improving RNA synthesis efficiency, reducing frequent movement, and reducing difficulty

Inactive Publication Date: 2019-08-20
通用生物(安徽)股份有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In the prior art, RNA cannot be synthesized efficiently through RNA templates, so this technology cannot be applied to industrial production. In order to solve this problem, the present invention provides the following technical solutions

Method used

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  • Preparation method of RNA polymerase using RNA as formwork and application of polymerase
  • Preparation method of RNA polymerase using RNA as formwork and application of polymerase
  • Preparation method of RNA polymerase using RNA as formwork and application of polymerase

Examples

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Effect test

Embodiment 1

[0037] A method for preparing an RNA polymerase using RNA as a template, comprising the steps of:

[0038] a, select tobacco necrosis virus Ac as raw material;

[0039] b. Use truncation or mutation to remove the transmembrane region of the N-terminus of the viral RNA to remove the cytotoxicity of the viral protein and enhance the solubility of the recombinant protein;

[0040] c. Intracellular or extracellular synthesis of the RNA polymerase by prokaryotic expression or eukaryotic expression.

[0041] The specific method of removing the N-terminal transmembrane region by mutation in the step b is deletion mutation;

[0042] When the target RNA is prepared extracellularly, the RNA polymerase is firstly expressed in prokaryotic or eukaryotic and then purified by the primary extraction device to become an enzyme product. In the extracellular buffer environment, the target template RNA is used to synthesize complementary strand RNA, buffer Composed of 100mM KCl, 5mM MgCl2 and 5...

Embodiment 2

[0053] A method for preparing an RNA polymerase using RNA as a template, comprising the steps of:

[0054] a, select tobacco necrosis virus Ac as raw material;

[0055] b. Use truncation or mutation to remove the transmembrane region of the N-terminus of the viral RNA to remove the cytotoxicity of the viral protein and enhance the solubility of the recombinant protein;

[0056] c. Intracellular or extracellular synthesis of the RNA polymerase by prokaryotic expression or eukaryotic expression.

[0057] The specific method of removing the N-terminal transmembrane region by mutation in the step b is point mutation;

[0058] When preparing the target RNA in the cell, the polymerase template RNA is constructed in a eukaryotic expression vector, and after expression in the eukaryotic cell, the target template RNA in the cell is used to synthesize a complementary strand RNA; the eukaryotic expression vector is a mammalian Expression vector;

[0059] By designing the 3' end of the...

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Abstract

The invention discloses a preparation method of RNA polymerase using RNA as formwork and an application of the polymerase. The preparation method comprises the following steps of using alpha necrosisviruses as a raw material, preparing recombinant protein based on virus replication protein containing a polymerase structural domain and free from N-terminal transmembrane domain or transmembrane domain mutation, and the RNA polymerase having RNA dependence is obtained. The preparation method specially comprises the steps of firstly, selecting alpha necrosis viruses of tomato bushy stunt virusesas the raw material; then removing the N-terminal transmembrane domain of the virus RNA through cutting short or mutation to obtain the formwork RNA; and finally, performing synthesis on the RNA polymerase in cells or outside cells through prokaryotic expression or eukaryotic expression. According to the recombinant protein obtained by the method, the RNA yield is greater than 1 milligram/milliliter, and the preparation method is suitable for synthesis and production of industrial RNA.

Description

technical field [0001] The invention belongs to the technical field of enzyme production, and in particular relates to a method for preparing RNA polymerase using RNA as a template and the application of the polymerase. Background technique [0002] RNA is an important component in eukaryotic cells and prokaryotic cells. It participates in many important functions such as transcription and translation. Transcription is the transfer of genome information. Genome information refers to DNA information, which is transferred from the nucleus to the cytoplasm , translation refers to the translation of transcriptional information into amino acids or proteins, and RNA polymerase uses a DNA chain or RNA as a template, ribonucleoside triphosphate as a substrate, and an enzyme that synthesizes RNA that polymerizes through phosphodiester bonds, because In the cell, it is related to the transcription of the genetic information of gene DNA into RNA, so it is also called transcriptase; ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12C12M1/42C12M1/33C12M1/00
CPCC12N9/127C12M21/14C12M47/06C12Y207/07048
Inventor 雍金贵许凯
Owner 通用生物(安徽)股份有限公司
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