Application of tRNA subjected to anticodon editing as molecular switch in precise control of protein expression and virus replication

An anti-codon, protein expression technology, applied in the direction of DNA / RNA fragment, virus / phage, recombinant DNA technology, etc., to achieve good application prospects and improve the effect of safety

Pending Publication Date: 2021-08-27
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Application Information

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Problems solved by technology

[0005] In summary, there are still various challenges in the application of codon expansion technology to the selection of orthogonal pairs, the selection of mutation sites and the selection of substrates[1]

Method used

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  • Application of tRNA subjected to anticodon editing as molecular switch in precise control of protein expression and virus replication
  • Application of tRNA subjected to anticodon editing as molecular switch in precise control of protein expression and virus replication
  • Application of tRNA subjected to anticodon editing as molecular switch in precise control of protein expression and virus replication

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Embodiment 1

[0057] method

[0058] 1. Materials and methods

[0059] 1.1 Samples, cells and virus strains

[0060] Human embryonic kidney cells (HEK293T) were preserved by our laboratory. The pDC-315-GFP plasmid (using the pEGFP-N1 plasmid as a template, PCR amplified EGFP gene, and cloned it into the pDC-315 vector to obtain pDC-315-GFP) was constructed and preserved by our laboratory. Lentiviral packaging system plasmids PLVX-IRES-ZSGreen1, HIV-1-Luc (use pGL3-Basic vector as template, PCR amplify luciferase gene, clone it into pFUGW vector to obtain HIV-1-Luc), psPAX2 and pMD2.G Plasmids are kept by our laboratory. The pUC57 plasmid was purchased from Beijing Qingke Biotechnology Co., Ltd. DH5α competent cells used for plasmid transformation were purchased from Beijing Qingke Biotechnology Co., Ltd. (Beijing, China). Three anticodon-edited tRNAs——Arg UGA tRNA, Gly UGA tRNA and Trp UGA The tRNAs are respectively obtained by transcribing the nucleotide sequences shown in SEQ ID N...

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Abstract

The invention discloses application of tRNA subjected to anticodon editing as a molecular switch in precise control of protein expression and virus replication. The tRNA subjected to anticodon editing carries a normal amino acid, can identify the early termination codon and inserts the carried amino acid into a corresponding position, so that the protein and the replication-deficient virus can complete expression and replication in the presence of the tRNA. A method does not depend on special aminoacyl tRNA synthetase and UAA. Only the anticodon coding sequence of the tRNA is subjected to mutation modification, the tRNA carrying normal amino acid can recognize a termination codon, and the carried amino acid is inserted into a corresponding position, so that protein expression and replication-deficient virus are precisely regulated and controlled, and the safety of the vaccine is improved. The invention provides a novel method for accurately controlling protein or virus. The tRNA subjected to anticodon editing particularly has a good application prospect in the production of novel replication-deficient virus vaccines.

Description

technical field [0001] The present invention relates to a new application of anticodon-edited tRNA, in particular to the application of anticodon-edited tRNA as a molecular switch in precise control of protein expression and replication-deficient virus replication, and the invention belongs to the technical field of biomedicine. Background technique [0002] Diseases caused by pathogenic microorganisms have had a huge impact on human and animal health, and making safe and effective vaccines is a priority for controlling pathogenic microorganism infections. However, it is difficult to balance the safety and effectiveness of vaccines. If a "molecular switch" that precisely regulates its replication is installed on pathogenic microorganisms, it can control its replication in vitro and cannot replicate in vivo, but it can activate both cellular and humoral immune responses at the same time, thus making the vaccine safer. This form of vaccine represents a fantastic intermediate ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/85C12P21/00C12N7/00
CPCC12N15/11C12N15/85C12P21/00C12N7/00
Inventor 汤艳东蔡雪辉
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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