Domestic pig ISG15 recombination protein and encoding gene, recombination plasmid, recombination strain and application thereof

A technology of recombinant protein and recombinant plasmid, which is applied in the field of genetic engineering to achieve the effects of controlling acute infection, reducing the amount of antigen, and controlling viral infection

CN106496320AInactive Publication Date: 2017-03-15HENAN AGRICULTURAL UNIVERSITY
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Patent Information

Authority / Receiving Office
CN · China
Patent Type
Applications(China)
Current Assignee / Owner
Publication Date
2017-03-15
Estimated Expiration
Not applicable · inactive patent

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Abstract

The invention belongs to the technical field of gene engineering, and particularly discloses a domestic pig ISG15 recombination protein and an encoding gene, a recombination plasmid, a recombination strain and application thereof. An amino acid sequence of the domestic pig ISG15 reconstruction protein is shown in SEQ ID No.2. A gene of the domestic pig ISG15 recombination protein, and the recombination plasmid and the recombination strain containing the gene are encoded. One of the domestic pig ISG15 recombination protein, the gene, the recombination plasmid and the recombination strain is applied to prepare a medicine for treating PR (pseudorabies). The domestic pig ISG15 recombination protein has the advantages that the antigen amount of the PRV (pseudorabies virus) is effectively reduced, the acute infection of the PRV is effectively controlled, and a new path is provided for controlling the virus infection.
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Description

technical field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a domestic pig ISG15 recombinant protein and its coding gene, recombinant plasmid, recombinant bacterial strain and application. Background technique

[0002] ISG15 is an interferon-stimulated gene 15 (isg15), and its encoded protein is ISG15 protein. The stimulation of virus or type I interferon can induce the high expression of ISG15 protein, and its molecular mass is about 17ku. ISG15 has the functions of cytokine activity, regulating interferon signaling pathway, anti-virus and anti-bacterial infection, etc., and plays an important role in innate immunity. ISG15 protein has certain differences among different organisms. At present, the reports on ISG15 mainly focus on human and mouse, and they are all researches on anti-virus and other aspects. At present, there is no report on the function of this protein in domestic pigs.

[0003] Porcine pseudor...

Examples

Embodiment 1

[0024] Example 1 Preparation of domestic pig ISG15 recombinant protein

[0025] 1 Materials and methods

[0026] 1.1 Test animals

[0027] 5 nursery pigs were sent for inspection from the pig disease clinic of Henan Agricultural University.

[0028] 1.2 Strains, cells and vectors

[0029] Prokaryotic expression vector pET28a, Escherichia coli DH5α competent cells were purchased from Dalian Bao Biological Engineering Co., Ltd.; PK15 cells were purchased from China Center for Type Culture Collection.

[0030] 1.3 Reagents and media

[0031] 1.3.1 Reagents

[0032] T4 DNA ligase, pMD18-T vector, DL2000 DNA Marker, DL15000 DNA Marker, low molecular weight protein marker (14.3 KDa ~ 97.2 KDa), restriction endonuclease, etc. were purchased from Takara Company; SanPrep column plasmid DNA mini-extraction The kit and SanPrep Column DNA Gel Recovery Kit were purchased from Sangon Bioengineering (Shanghai) Co., Ltd.; Trizol reagent was purchased from Invitrogen; DEPC was purchased f...

Embodiment 2

[0061] Example 2 Effect of domestic pig ISG15 protein on porcine virus

[0062] The present invention constructs a PiggyBac-ISG15 eukaryotic expression vector, establishes a PK15 cell line stably expressing porcine ISG15, and explores the effect of ISG15 on porcine-derived viruses.

[0063] 2.1 Cells, strains, plasmids and bacterial strains

[0064] PK15 cells were purchased from the China Center for Type Culture Collection in DMEM containing 10% fetal bovine serum at 37°C, 5% CO 2 nourish. Pseudorabies virus (PRV) Fa strain was purchased from China Veterinary Drug Control Institute. PiggyBac transposon system (Novagen) was purchased from Shanghai Beinuo Biotechnology Co., Ltd. TOP10 competent cells were purchased from Beijing Dingguo Biotechnology Co., Ltd.

[0065] 2.2 Construction and identification of PiggyBac-ISG15 eukaryotic expression vector

[0066] The upstream and downstream primers were designed according to the porcine ISG15 gene sequence, and inserted into th...

Embodiment 3

[0076] Example 3 Application of domestic pig ISG15 recombinant protein

[0077] The present invention establishes a porcine pseudorabies model by artificially infecting domestic pigs with pseudorabies virus, uses the ISG15 recombinant protein obtained in Example 1 to carry out treatment experiments, and simultaneously measures the relative expression changes of several cytokines during the experiment, in order to explore ISG15 Gene lays a theoretical foundation for the disease resistance mechanism of porcine pseudorabies.

[0078] 3.1 Main reagents and instruments

[0079]Pseudorabies virus (PRV) Fa strain was purchased from China Veterinary Drug Administration; TRIzol was purchased from Invitrogen; SYBRGreen I Mix was purchased from Dalian Bao Biological Engineering (Takara); lymphocyte separation fluid was purchased from Haoyang Biological Products Company.

[0080] 3.2 Preparation of pig ISG15 recombinant protein (prepared by the method in Example 1)

[0081] 3.3 Grouping...