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Hog cholera virus NS5B protein fidelity mutant and application thereof

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: 2018-10-26
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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  • Hog cholera virus NS5B protein fidelity mutant and application thereof
  • Hog cholera virus NS5B protein fidelity mutant and application thereof
  • Hog cholera virus NS5B protein fidelity mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Obtainment, crystallization and structural analysis of NS5B protein of CSFV Shimen strain

[0026] Construction of NS5B expression plasmid of classical swine fever virus Shimen strain:

[0027] Using the plasmid pMal-c2X-CSFV_NS5B of CSFV NS5B (Shimen strain) as material, the DNA coding fragment corresponding to 1-694aa of the amino acids of CSFV NS5B shown in SEQ ID NO.1 was cloned onto the prokaryotic expression vector pET26b, The prokaryotic expression plasmid pET26b-CSFV_NS5B(1-694) of CSFV NS5B was constructed.

[0028]Primers were designed to amplify the genome of amino acid 1-694 of Shimen strain NS5B, and the primer sequences were as follows:

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

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

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

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

[0033] Use F1 and R1 as upstream and downstream primers for the first roun...

Embodiment 2

[0048] Embodiment 2: Design and acquisition of NS5B protein mutant of classical swine fever virus Shimen strain

[0049] According to the above-mentioned unique NTD-RdRP intramolecular interaction interface, a series of mutations were designed for Y471 and E472. At the same time, an amino acid E481 (the 481st valley of the NS5B protein) that was not involved in the formation of the interface was selected near the NTD-RdRP interface. amino acid), and mutated it to alanine and aspartic acid as controls.

[0050] The design of mutants and controls is as follows:

[0051] structure

Remark

structure

Remark

WT

wild type (not mutant)

E472D

E472 mutation to D

Y471A

Y471 mutation to A

E472Q

E472 mutated to Q

Y471H

Y471 mutation to H

Y471A-E472A[AA]

Y471 is mutated to A, E472 is mutated to A

Y471F

Y471 mutation to F

E481A

E481 is mutated to A

Y471W

Y471 mutation to W

E481D

...

Embodiment 3

[0073] Example 3: Experimental detection of de novo synthetic RNA fidelity

[0074] As shown in Figure 2, the fidelity of NS5B protein was detected by de novo synthesis of RNA experiment, wherein: the template RNAT30 sequence is: 5'-GGGAGAUGAAAAUCUCCAACGAUUAUAUCC-3', template RNA T30 and dinucleotide primer GG (P2) according to After mixing at a molar ratio of final concentration 1:1.25, incubate at 45°C for 3 minutes, and then slowly cool down at room temperature. Add T30 / P2 with a final concentration of 4 μM (P2 concentration is 5 μM), 15 μM P2, 6 μM CSFV NS5B, 300 μM ATP, 300 μM UTP, 20 mM NaCl, 50 mM Tris (pH 7.0), 5 mM MgCl in 20 μL of the system 2 , 5mM DTT, incubated at 45°C for 45min, then added stop solution (Stop Solution: 95% [v / v] formamide, 20mM EDTA [pH 8.0], 0.02% [w / v] bromophenol blue, 0.02% [w / v] xylene blue) to terminate the reaction, and then the samples were heated at 100°C for 1 min, and stained with Stains-All (Sigma-Aldrich) after polyacrylamide gel e...

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Abstract

The invention discloses a hog cholera virus NS5B protein fidelity mutant and application thereof, and belongs to the technical field of biology. Through crystallographic structural analysis and an in-vitro biochemical test, it is verified that two key amino acid sites of Y471 and E72 are closely related with the fidelity of hog cholera virus RdRP, the mutation of the two sites can definitely regulate and control the fidelity of RdRP synthesis, and according to the mutant, any attenuated vaccine of the hog cholera virus can be prepared to solve the problem that the variation of the hog choleravirus causes vaccine failure.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a fidelity mutant of the NS5B protein of swine fever virus and its application. Background technique [0002] Classical swine fever is a class A infectious disease listed by the World Organization for Animal Health. It is caused by classical swinefever virus (CSFV). Currently, there is no specific drug to control it. The main method is rabbitized attenuated vaccine (Paton, D.J. & Greiser-Wilke, I. Classical swine fever--an update. Research in Veterinary Science 75, 169-178 (2003)). Classical swine fever virus is a single-stranded positive-sense RNA virus belonging to the genus Pestivirus in the family Flaviviridae. Similar to most other RNA viruses, the genome replication and mutation rate of CSFV is high, which leads to differences in the genomes of epidemic strains in different regions and in different periods, thus affecting the protective effect of vaccine strains. The reason i...

Claims

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Application Information

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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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