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ChIFNGR1 gene and its coding protein and application

A technology of protein and gene, applied in the direction of peptide/protein composition, application, genetic engineering, etc.

Inactive Publication Date: 2008-06-04
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the chicken IFNGR1 gene has not been publicly reported at home and abroad, and only a computer analysis and prediction sequence XM 419722 can be found on GenBank. The correctness of this sequence needs further experimental proof

Method used

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  • ChIFNGR1 gene and its coding protein and application
  • ChIFNGR1 gene and its coding protein and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1. Obtaining of Chicken IFNγ Receptor Alpha Chain (chIFNGR1)

[0020] 1. The cDNA sequence of chicken chIFNGR1 was obtained by rapid amplification of cDNA ends (RACE) method

[0021] The total RNA of chicken peripheral blood lymphocytes was extracted, and the total RNA was used with 100 U MMLV-RT (promega company product), 100 ng of upstream and downstream primers 5'-AACATATGCGGCCGCATTATGGCCGGG-3' and 5'-GATCTTCCACGCGTCGAC(T)30MN-3'(M =A, G, C; N=A, G, C, T) Reverse transcription was performed at 42°C. The obtained cDNA was subjected to 3'RACE, and RACE primers were designed according to the conserved region of the red jungle fowl IFNGR1 computer predicted sequence XM_419722 registered on GenBank, and the primers for the first round of PCR were specific upstream primers GSP1 (5'-CAGAAGCAAAAGGCATTTGTGCCGTC-3') and Universal downstream primer UP (5'-GATCTTCCACGCGTCGACT-3'), primers for the second round of PCR are nested upstream primers GSP2 (5'-TGTGAGTGTCCCATTGA...

Embodiment 2

[0027] Embodiment 2, expression of chicken IFNGR1 extracellular domain (EC) recombinant protein

[0028] 1. Construction of chicken IFNGR1EC recombinant expression vector

[0029] The extracellular domain of chicken IFNGR1 was amplified by conventional PCR, and the primers used were chIFNGR1EC-F (5'-TAGGATCCGAGCGTCTTCCCGCAGT-3) and chIFNGR1EC-R (5'-GCTCTAGATCAAGCCTGCGTGATAGGAACC-3'), and the PCR reaction conditions were: 94°C for 5 minutes, 94°C for 1 minute, 60°C for 1 minute, 72°C for 1 minute, 30 cycles; 72°C for 10 minutes. After the reaction, the amplified PCR product was double-digested with BamHI and Xba I, and inserted into the pMAL-p2x (product of New England Biolabs) vector, and the ligated product was transformed into Escherichia coli competent cell DH5α and sequenced. A recombinant plasmid containing IFNGR1EC was obtained and named pMAL-p2x / IFNGR1EC.

[0030] 2. Transform Escherichia coli TB1 competent cells with the recombinant plasmid in step 1, and shake the b...

Embodiment 3

[0031] Embodiment 3, the determination of antiviral activity of chicken IFNGR1 ectodomain (EC) recombinant protein

[0032] After the concentration of the purified rIFNGR1EC protein was determined, the lesion-inhibiting effect test was carried out. The rIFNGR1EC tagged with maltose binding protein (MBP) was serially diluted (1:4), inoculated on the monolayer of chicken CEF cells, and after culturing for 24 hours, 100 TCID was added to each well 50 Equine vesicular stomatitis virus) (VSV), when the virus control wells were completely diseased, the antiviral activity of rIFNGR1EC was calculated using the calculation method of interferon titer. rIFNGR1EC has high antiviral activity and can effectively inhibit the destructive effect of VSV on fibroblasts. The antiviral activity of rIFNGR1EC was 4.58×10 3 U / mg.

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Abstract

The invention provides a chick IFN-gamma receptor alpha chain (IFNGR1) gene which comprises a nucleotide sequence shown in SEQ ID No.1 or a nucleotide sequence which is obtained after changing, adding and / or deleting one or a plurality of nucleotides in the prior nucleotide sequence and has the same functions. The invention also provides a protein chick IFNGR1 coded by the gene, and proteins have functions of ligand synthesis and transmission of IFN-gamma stimulus cue. Chick IFNGR1 extracellular fused proteins have antivirus activity and are a potential antivirus biological agent.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to chicken interferon-gamma receptor alpha chain (chIFNGR1) gene and encoded amino acid, and also relates to the antiviral activity of its ectodomain recombinant protein. Background technique [0002] Interferon-γ (IFN-γ) is mainly produced by activated T cells and NK cells. It is a cytokine with various biological activities such as anti-virus, regulating immunity and promoting apoptosis. Its functional pleiotropy is achieved by binding to high-affinity receptors expressed on almost all cell surfaces in a strictly species-specific manner, and inducing cells to express a variety of proteins. [0003] Many biological functions related to IFN-γ have been known for a long time, but the understanding of the structure and function of IFN-γ cell surface receptors has only made meaningful progress in the past ten years. The IFN-γ receptor consists of a ligand binding chain (IFN-γ receptor α c...

Claims

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Application Information

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IPC IPC(8): C12N15/12C07K14/465A61K38/17A61P31/12
Inventor 汪明韩雪
Owner CHINA AGRI UNIV
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