Replication system and application thereof in gene expression

A gene and replicase technology, applied in bacteria, fermentation, etc., can solve the problems of RNA replication speed and replicon stability, unfavorable long-term retention of RNA replicon, etc.

Active Publication Date: 2019-06-28
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In traditional research, RNA replicons are mainly used as the only carrier of target genes, but a good balance cannot be achieved between the speed of RNA replication and the stability of replicons, which is not conducive to the long-term persistence of RNA replicons
At present, there is no research that explicitly proposes the RNA replication process as a stable carrier for the study of target gene expression, and there is no research on the use of RNA replication-related tools in prokaryotic systems

Method used

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  • Replication system and application thereof in gene expression
  • Replication system and application thereof in gene expression
  • Replication system and application thereof in gene expression

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0102] Example 1 Verification of replication efficiency when replicase and replication sequences are expressed separately

[0103] The design of the replication system for expressing the replicase and replicating sequences separately is as follows: figure 1 As shown in the schematic diagram of A, the replication sequence and replicase are expressed separately, the replication system expresses the replication sequence, but not the replicase, and the replicase is expressed in another replicase plasmid. The replication sequence contained in the replication system includes the promoter sequence, the 5' linker sequence, the target gene sequence (that is, the "target protein region" in the figure), and the inactive replicase sequence (that is, the target protein region) from the 5' end to the 3' end. "phage sequence (replicase inactive)") and 3' linker sequence, such as figure 1 Shown in "Replication System" in A. Since the replicase in the replication system is inactivated, the...

Embodiment 2

[0107] Example 2 Construction of preferred RNA replication system and detection of its working effect

[0108] (1) construct the plasmid of preferred RNA replication system

[0109] A schematic diagram of the preferred replication system is shown in figure 2 As shown in "preferred replication system" in A, the replicase sequence is placed in the replication system and co-expressed with the target gene. The preferred RNA molecule of the replication system comprises a promoter, a 5' linker sequence, an objective gene sequence (i.e. the "target protein region" in the figure), a replicase sequence (i.e. the "Qβ replicase" in the figure), a 3' linker sequence and HDV ribozyme sequence, wherein the HDV ribozyme sequence is used for self-cleavage after expression.

[0110] In this embodiment, the blue fluorescent protein BFP is used as the target gene, and the T7M5 promoter is used to transcribe the RNA molecule of the replication system. The schematic diagram of the replication...

Embodiment 3

[0133] Embodiment 3 cuts the upstream and downstream of the replication system

[0134] In this embodiment, HDV ribozyme and tRNA were used to cut the 3' end and 5' end of the replication system, respectively, to test the impact of removing the remaining sequences outside the 3' end and 5' end on the performance of the replication system.

[0135] (1) Use HDV ribozyme to cut the 3' end of the replication system, the schematic diagram is as follows Figure 4 As shown in A, the replication system RRS-T7M5-BFP constructed in Example 2 and the control replication system RRS mutation-T7M5-BFP were used. In addition, the replication system RRS-T7M5-BFP and the control replication system RRS mutation-T7M5-BFP not containing the HDV ribozyme sequence after the 3' linker sequence were constructed.

[0136] Using the same plasmid construction method as in Example 2 and the attB / P integration method of HK022, the plasmids expressing these replication systems were integrated into the g...

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Abstract

The invention provides a method for obtaining a target gene expression product, which comprises expressing a recombinant RNA molecule in a host bacterium and culturing the host bacterium in the presence of a phage RNA replicase and under conditions suitable for expression of the target gene; wherein the recombinant RNA molecule comprises a 5' replication recognition sequence and a 3' replication recognition sequence which are recognizable by a phage RNA replicase and perform an RNA replication process, and a target gene sequence arranged between the 5' replication recognition sequence and the3' replication recognition sequence. The invention also provides s recombinant bacterium capable of expressing the recombinant RNA molecule. The method can modify engineering bacteria from the level of directly increasing the RNA expression quantity so as to promote the expression of the target gene.

Description

technical field [0001] The present invention relates to the field of biotechnology, and more specifically, relates to the use of a replicable RNA sequence to enhance the content of target gene messenger RNA and protein under the action of replicase. Background technique [0002] Microbial fermentation is an important way to prepare food and medicine in modern industrial production, which relies on artificially modified industrial microorganisms. For many metabolites that natural microorganisms cannot produce or produce poorly, metabolic engineering can be used to introduce new metabolic pathways in chassis strains (Steen, E.J., Kang, Y., Bokinsky, G., Hu, Z., Schiremer, A. .,McClure,A.,Del Cardayre,S.B.,and Keasling,J.D.(2010).Microbialproduction of fatty-acid-derived fuels and chemicals from plant biomass.Nature,463,559.) and regulation of existing metabolic pathways (Varma,A. , and Palsson, B.O. (1994). MetabolicFlux Balancing: Basic Concepts, Scientific and Practical Use...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P21/02
Inventor 吴琼姚绎张文慧张敏金守红
Owner TSINGHUA UNIV
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