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A kind of goose source mitochondrial antiviral signal protein and its application

A mitochondrial and anti-viral technology, applied in the field of gene and protein engineering, can solve the problems of different animals, innate immune molecular function differences, etc., and achieve the effect of inhibiting early replication

Active Publication Date: 2021-08-03
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it did not elaborate on the impact of different sources of MAVS on different animals. Compared with mammals, the total amount of immune genes in birds is less, which means that the innate immunity of birds has its own uniqueness. The innate immune mechanisms of different species are also different. For example, chickens lack the important RNA pattern recognition receptor RIG-I, but ducks and geese have RIG-I, indicating that there are large differences in the molecular functions of innate immunity among different species.

Method used

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  • A kind of goose source mitochondrial antiviral signal protein and its application
  • A kind of goose source mitochondrial antiviral signal protein and its application
  • A kind of goose source mitochondrial antiviral signal protein and its application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] The determination of embodiment 1 goose source MAVS gene sequence

[0059] According to the existing goose genome on GenBank and the predicted MAVS sequence of goose MAVS, design overlapPCR amplification primers P1, P2, P3, and P4 to amplify the goose MAVS gene by fusion PCR, where P1 and P2 are the first round The upstream and downstream primers of PCR, P3 and P4 are the upstream and downstream primers of the second round of PCR. After the two rounds of PCR products are mixed, P1 and P4 are used for the third round of PCR to obtain the full-length sequence, which is connected to the T vector for sequencing. Primers P5 and P6 are MAVS The fluorescent quantitative PCR detection sequence of the sequence is specifically shown in Table 1 below:

[0060] Table 1

[0061]

[0062] Prepare the PCR amplification system as shown in Table 2, and perform overlapping PCR amplification in two rounds, as follows:

[0063] Table 2

[0064]

[0065]

[0066] Concrete reacti...

Embodiment 2

[0074] Embodiment 2 constructs goose source MAVS eukaryotic expression plasmid

[0075] In order to construct the full-length expression plasmid of goose MAVS connected with p3XFLAG-CMV-14, the full-length goose MAVS connected with T vector was used as a template, and P7 and P8 were used as primers to amplify, and EcoR I / Xba I (purchased from takara company) enzyme After cutting, connect into the p3XFLAG-CMV-14 expression vector, as shown in Table 4, wherein P7 and P8 are primers for constructing wild-type goose MAVS expression plasmid, and P9 and P10 are primers for constructing goose MAVS expression plasmid with deletion of PRR domain:

[0076] Table 4

[0077]

[0078] The specific PCR system is shown in Table 5 below, specifically as follows:

[0079] table 5

[0080]

[0081]

[0082] The specific PCR conditions are shown in Table 6 below, specifically as follows:

[0083] Table 6

[0084]

[0085] The first round of amplification: use T-goMAVS as a templa...

Embodiment 3

[0088] Example 3 Exogenous transfection of goose MAVS can induce the expression of high-level interferon-stimulated genes in goose cells

[0089] In addition to constructing eukaryotic expression plasmids of goose-derived MAVS, the researchers also constructed human-derived and chicken-derived MAVS, and overexpressed goose-derived, chicken-derived, and human-derived MAVS in goose primary fibroblasts (GEF). The specific steps As follows: Use an endotoxin-free plasmid extraction kit (purchased from Tiangen Biochemical Technology Co., Ltd.) to extract p3XFLAG-CMV-14 empty vector, goose MAVS, chicken MAVS and human MAVS recombinant plasmids, and pass through liposomes after enzyme digestion and identification (purchased from American Life Company) Transfection method Transiently transfect GEF cells, collect cell samples 24 hours after transfection, extract RNA, and perform reverse transcription with promega M-MLV reverse transcription kit.

[0090] The obtained goose source, chick...

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Abstract

The invention relates to a goose-derived mitochondrial anti-virus signal protein and its application, in particular to a goose-derived mitochondrial anti-virus signal protein with anti-virus and immune activation functions and its application. The signal protein includes an active region, a proline Any one or a combination of at least two of the enrichment region, domain A, domain B or transmembrane region. The goose-derived mitochondrial antiviral signal protein can induce a high level of innate antiviral response in primary goose embryo fibroblasts, and can inhibit the early replication of Newcastle disease virus.

Description

technical field [0001] The invention relates to the field of gene and protein engineering, in particular to a goose-derived mitochondrial anti-virus signal protein and its application, in particular to a goose-derived mitochondrial anti-virus signal protein with anti-virus and immune activation functions and its application. Background technique [0002] It is the most important type of innate antiviral mechanism in mammals to recognize the pathogenic pattern molecules of viruses through pattern recognition receptors (PRR), induce the production of interferon (IFN) and inflammatory factors to inhibit virus replication. Among all conserved pattern recognition receptors, retinoic acid-inducible gene protein I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are the most important cytoplasmic pattern recognition receptors that professionally recognize viral RNA. RIG-I and MDA5 are two similar receptors that recognize different types of viral RNA. They share the sam...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/465C12N15/12C12N1/21A61K39/39A61K39/00A61P31/14C12R1/19
CPCA61K39/0005A61K39/39A61K2039/55516A61P31/14C07K14/465
Inventor 丁铲孙英杰谭磊孟春春仇旭升廖瑛宋翠萍于圣青郑航
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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