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Fidelity mutant of swine fever virus ns5b protein and its application

A technology of swine fever virus and mutants, applied in the direction of virus/bacteriophage, virus, virus peptide, etc., can solve the problems of low conservation of vaccine virus genome and low broad-spectrum protection effect, and achieve the effect of solving vaccine failure

Active Publication Date: 2021-07-13
WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to overcome the deficiencies in the prior art, provide a fidelity mutant of CSFV NS5B protein and its application, to solve the problem that the attenuating site of the existing vaccine is mainly located in the relatively low conservation site of the virus genome. Regional, technical issues with low broad-spectrum protection

Method used

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  • Fidelity mutant of swine fever virus ns5b protein and its application
  • Fidelity mutant of swine fever virus ns5b protein and its application
  • Fidelity mutant of swine fever virus ns5b protein and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Analysis of NS5B protein of swine fever virus stone plants, crystallization and structural analysis

[0026] Construction of the expression plasmid of NS5B of swine fever virus stone plants:

[0027] The plasmid PMAL-C2X-CSFV_NS5B of the swine fever virus NS5B (stone door strain) is subjected to a DNA coded fragment of the 1-694AA of the amino acid of the CSFV NS5B shown in SEQ ID NO to SEQ ID NOS1 to the prokaryotic expression vector PET26b. The prokaryotic expression plasmid PET26B-CSFV_NS5B (1-694) of CSFV NS5B was constructed.

[0028]The genome of the amino acid 1-694 for amplifying the stone strain NS5B is designed, and the primer sequence is as follows:

[0029] F1: 5'-attctorcgcggggggggtagtaattggtgatgcaag-3 '

[0030] R1: 5'-gtgatggctcgctccccctgtacctgtctgcccctTG-3 '

[0031] F2: 5'-attcgggggggggtagtaattgggtgatgcaag-3 '

[0032] R2: 5'-atatgaattcttagtgatgtgatgtgatgctcgagctcccatg-3 '

[0033] The first round of PCR is performed by F1 and R1 as a top and dow...

Embodiment 2

[0048] Example 2: Design and Get of NS5B protein mutant in the swine fever virus

[0049] According to the above-described unique NTD-RDRP molecular interaction interface, for the Y471 and E472 design series of mutations, at the same time, the amino acid E481 (NS5B protein) is not involved in the surface of the NTD-RDRP interface (NS5B protein) is selected near the NTD-RDRP interface. Aproductone) and mutated to alanine and aspartic acid as a control.

[0050] The design of the mutant and control group is as follows:

[0051] structure Note structure Note WT Wild-type (non-mutant) E472D E472 mutation is D Y471A Y471 mutation is a E472Q E472 mutation is Q Y471H Y471 mutation is h Y471A-E472A [AA] Y471 mutation is A, E472 mutation is A Y471F Y471 mutation is f E481A E481 mutation is a Y471W Y471 mutation is W E481D E481 mutation is D E472A E472 mutation is A -- --

[0052] Using the QuickChange method (Vandeyar, MA, Weiner, M...

Embodiment 3

[0073] Example 3: Detecting Fire Demonity from Head Synthesis RNA Experiment

[0074] As shown in FIG. 2, NS5B protein fidelity was detected using an experiment from head synthesis RNA, wherein: Template RNAT30 sequence is: 5'-gggaugaaaaucuccaacgauuauauaucc-3 ', the template RNA T30 and dikacino primer GG (P2) final concentration of 1: 1.25 molar concentration ratio after mixing, 45 ℃ incubated for 3min, then slowly cooled at room temperature. T30 / p2 (P2 concentration of 5 μm) (P2 concentration of 5 μm), 15 μm p2, 6 μm Csfv NS5B, 300 μmatp, 300 μm Utp, 20 mM NaCl, 50 mM Tris (pH 7.0), 5 mm mgCl were added to a 20 μl system. 2 5 mM DTT, 45 mination was incubated for 45 minutes (Stop Solution: 95% [V / V] formamide, 20 mM EDTA [pH 8.0], 0.02% [w / v] bromophenol blue, 0.02% [W / v] Ttolin Blue) Terminate the reaction, and then heated the sample at 100 ° C for 1 min, and the polyacrylamide gel is stained with Stains-ALL (Sigma-Aldrich), and the treatment method is the same as JeV N...

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Abstract

The invention discloses a fidelity mutant of the NS5B protein of swine fever virus and its application, and belongs to the field of biotechnology. Specifically, two proteins closely related to the fidelity of RdRP of the swine fever virus are confirmed through crystal structure analysis and in vitro biochemical experiments. The key amino acid sites Y471 and E472, the mutation of these two sites can clearly regulate the fidelity of RdRP synthesis, and can prepare an attenuated vaccine of any strain of CSFV to solve the problem caused by the mutation of CSFV. The problem of vaccine failure.

Description

Technical field [0001] The present invention relates to the field of biological technology, and more particularly to a swine fever virus NS5B protein firming mutant and its application thereof. Background technique [0002] Pig fever is a class of infectious diseases listed in the World Animal Health Organization. Classical Swinefever Virus, CSFV, there is currently no special medicine to control it, mainly using rabbit disinfection vaccines for prevention (Paton, DJ & Greiser-wilke, I.Classical Swine Fever - An Update.Research Inveterinary Science Science 75, 169-178 (2003)). The swine fever virus is a single positive chain RNA virus, which is affiliated to the Braviosis of the yellow strain. Similar to most other RNA viruses, the virus variability of the swine fever virus genome is high, resulting in differences in the genome of the population plant in different regions, and affects the protection effect of the vaccine strain. The cause of the rabbit vaccine strain is mainly lo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/18A61K39/187A61P31/14
CPCA61K39/12A61K2039/5254A61P31/14C07K14/005C12N2770/24322C12N2770/24334
Inventor 龚鹏刘伟驰石晓玲潘兹书
Owner WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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