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Recombinant porcine alpha interferon and application thereof in preparing medicines for treating Porcine cytomegalovirus (PCMV)

A technology of alpha interferon and interferon, applied in the field of recombinant porcine alpha interferon and its application in the preparation of drugs for the treatment of porcine cytomegalovirus disease, can solve the problem of prevention and treatment of specific drugs that have not yet been developed, and have not yet been developed. PCMV vaccine and other issues

Inactive Publication Date: 2012-11-28
CHENGDU QIANKUN VETERINARY PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] So far, no effective PCMV vaccine has been developed, and no specific drug has been developed to prevent and treat this disease among chemical therapeutic drugs.

Method used

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  • Recombinant porcine alpha interferon and application thereof in preparing medicines for treating Porcine cytomegalovirus (PCMV)
  • Recombinant porcine alpha interferon and application thereof in preparing medicines for treating Porcine cytomegalovirus (PCMV)
  • Recombinant porcine alpha interferon and application thereof in preparing medicines for treating Porcine cytomegalovirus (PCMV)

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] The preparation method of embodiment 1 porcine alpha interferon

[0022] 1. Synthesis of artificially modified porcine interferon alpha gene

[0023] The 1-69 nucleotides of the signal peptide sequence in the pig natural IFNα nucleotide sequence are removed, and the XhoI and XbaI restriction sites and protective bases are added.

[0024] The specific operation is as follows: the 1-69 nucleotides of the signal peptide sequence in the natural IFNα nucleotide sequence are removed, and the XhoI and XbaI restriction site sequences and protective bases are added. The third base of the triplet codon of the 24th amino acid in the gene is subjected to a point mutation, that is, TGC is mutated into TGT; the second base of the 29th amino acid is subjected to a point mutation, that is, ACC is mutated into ATC; the 48th amino acid Carry out a point mutation at the third base of the amino acid at position 101, that is, mutate TCC to TCT; perform a point mutation at the first base of...

Embodiment 2

[0043] Embodiment two porcine alpha interferon biological activity assay

[0044] 1. Material method

[0045] Virus source: vesicular stomatitis virus VSV (preservation number: GDV-011, purchased from China Center for Type Culture Collection).

[0046] Cells: bovine kidney cells MDBK (NBL-1, Shanghai Biochemical Reagent Co., Ltd.).

[0047] Sample to be tested (porcine alpha interferon): prepared by genetic engineering methods.

[0048] Detection method: cytopathic inhibition method, the titer unit is calculated according to 50% pathological changes. The cells used in the determination are bovine kidney cells MDBK, and the challenge virus is VSV.

[0049] Biological activity calculation method:

[0050]

[0051] Pr is the biological activity of the standard, IU / ml;

[0052] Ds is the pre-dilution multiple of the test product;

[0053] Dr is the pre-dilution multiple of the standard;

[0054] Es is the dilution factor that the test product is equivalent to the half-ef...

Embodiment 3

[0060] Embodiment 3 Porcine alpha interferon clinical treatment of porcine cytomegalovirus disease

[0061] Select 120 diseased pigs as pig interferon treatment objects. According to the injection of 15,000 units per kilogram of body weight, once a day, a course of treatment for 3 days, after treatment, the clinical symptoms of most pigs were relieved.

[0062] Observation of Clinical Symptoms of Experimental Pigs

[0063]

[0064] Cured: 107; invalid: 13; the cure rate was 89.17%, and the ineffective rate was 10.83%.

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Abstract

The invention discloses a recombinant porcine alpha interferon, prepared by the following methods: A1, synthesizing an artificially modified porcine alpha interferon; A2, constructing a recombinant eukaryotic expression vector pGAPZ alpha-IFN alpha; and A3, highly expressing IFN alpha by an eukaryotic cell yeast expression system. The invention is characterized by taking a supernatant to purifying the porcine alpha interferon which is cultured by a lot of engineering bacteria and expressed with a chromatography column, then collecting a target protein eluate, filtering through a 0.22 mum microfiltration membrane, then determining the activity by cytopathic inhibition, and calculating the potency unit according to 50% pathology; wherein the cell used for determination is Madin-Darby bovine kidney (MDBK), and vesicular stomatitis virus (VSV) is used for attacking the virus. According to the invention, the recombinant porcine alpha interferon obtained by purification is applied in controlling PCMV, and experiments prove that the recombinant porcine alpha interferon has obvious protection effect on PCMV infected cells. The route of administration of the porcine alpha interferon is injection or mucous membrane administration, and the dosage form comprises injection or nasal drops.

Description

technical field [0001] The invention relates to a new application of recombinant porcine alpha interferon, in particular to recombinant pig alpha interferon and its application in preparing medicine for treating porcine macrophage virus disease. Background technique [0002] Interferon (Interferon, IFN) is a kind of secreted glycoprotein with broad-spectrum anti-virus, anti-proliferation and immunomodulatory effects, and is an important part of the body's defense system. According to the sensitivity of interferon to acid, it is usually divided into two types: type I interferon (acid-sensitive type) and type II interferon (acid-resistant type), and almost all vertebrates can produce these two types of interferon. Type I interferon mainly plays an antiviral and antitumor role. So far, INF-α, β, ω, κ, τ, δ and interferon-like cytokines limitin, IL28, IL29, etc. have been discovered; type II is also called immune interferon, Only one type of INF-γ has been found so far. Interf...

Claims

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Application Information

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IPC IPC(8): C12N15/81A61K38/21A61P31/22C12R1/84
Inventor 李犹平刘宗秀王建军房春林陈元坤
Owner CHENGDU QIANKUN VETERINARY PHARMA
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