Preparation method and application of cytokine medicine

A technology of cytokines and drugs, applied in the field of genetic engineering, can solve problems such as increased economic costs, risks of drug dispersal, and unknown therapeutic effects, and achieve the effect of rapid scab formation and inhibition of effects

Inactive Publication Date: 2019-07-16
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

After the onset, farmers often use symptomatic treatment to reduce losses, but the effect of treatment is unknown, and it takes a long time for the sheep to survive the disease, which increases the economic cost and the risk of poison shedding during the treatment process

Method used

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  • Preparation method and application of cytokine medicine
  • Preparation method and application of cytokine medicine
  • Preparation method and application of cytokine medicine

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Induced expression of embodiment 1 sheep interferon protein

[0051] (1) Amplification of sheep interferon gene

[0052] The sheep interferon α gene was amplified by using the cDNA of sheep peripheral blood lymphocytes as a template, connected into the T vector, and sequenced to verify the correctness of the gene sequence. Wherein, the nucleotide sequence of the sheep interferon alpha gene is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2.

[0053] (2) Construction of expression vector

[0054] The sequence of the sheep interferon alpha gene sequence correctly sequenced was connected into the pET-43.1a expression vector by the double enzyme digestion method (BamHI and HindIII), and the vector pET-43.1a-IFN-α was obtained. The vector map is as follows: figure 1 shown; the correct construction of the expression vector was verified by double enzyme digestion and sequencing, and the results were as follows figure 2 shown.

[0055] (3) Screeni...

Embodiment 2

[0069] Embodiment 2 antiviral effect analysis

[0070] For checking whether recombinant interferon has antiviral activity, test has used porcine pseudorabies virus (PRV, DNA virus) as research object (the present invention uses pseudorabies virus as research object, mainly because pseudorabies virus is easy to cultivate, and cell test operation It is convenient, but there is no available passage cell line for sheep oral ulcer virus at present, and the cell anti-virus experiment is not easy to operate, and interferon is a general anti-virus mechanism evolved by animals, which can exert similar anti-virus effects on different animals or different animal cells. viral effects).

[0071] The porcine pseudorabies virus CRB-1 isolate was inoculated into BHK-21 cells (containing 2ml culture fluid) that had been cultivated for 24 hours in a 24-well culture plate according to the amount inoculated at 1 MOI, and the cell density was about 80%. After 1 hour of infection, the cells 2 μl, ...

Embodiment 3

[0074] Embodiment 3 determines storage conditions

[0075] The recombinant protein was added to different concentrations (0%, 5%, 10%, 20%, 40% and 50%) of glycerol and stored at different temperatures (-20°C, -80°C) for 6 months, then SDS-PAGE was carried out , the result is as Figure 9-12 shown.

[0076] The results showed that, when stored at -20°C, 50% glycerol had the best preservation effect, and the degradation rate of interferon was 33%. 37%; therefore, the final storage concentration is determined to be 50% glycerol, and the storage condition is -20°C.

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Abstract

The invention discloses a preparation method and application of a cytokine medicine. Escherichia coli is used for expressing recombinant interferon of sheep, and is used for preventing and treating infectious pustule of the sheep, and infected local blisters of infected sheep can quickly scab and heal; and the recombinant interferon has significant effects on restraining proliferation of infectious pustule viruses of the sheep. The recombinant interferon disclosed by the invention is used for preparing medicines for treating infectious pustule of the sheep, the present situation that no special medicines for treating the infectious pustule of the sheep exist at present is overcome, and technical guarantee is provided for quick development of the sheep husbandry of Shanxi province.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, and more specifically relates to a preparation method of cytokine medicine and its application in treating sheep contagious pustule. Background technique [0002] Sheep contagious pustules seriously endanger the production of sheep farming. At present, vaccine immunization can be used to prevent and control sheep contagious pustules, but because of the irregular incidence of the disease, farmers are generally reluctant to use immunization methods to control the disease. After the onset of the disease, farmers often adopt symptomatic treatment to reduce losses, but the treatment effect is unknown, and it takes a long time for the sheep to survive the disease. The treatment process increases the economic cost and the risk of dispersing the poison. [0003] Interferon (Interferon, IFN) is a class of highly active multifunctional glycoprotein produced by interferon-inducing agents in bio...

Claims

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Application Information

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IPC IPC(8): A61K38/21A61K47/10A61P31/20C07K14/56C12N15/70
CPCA61K38/212A61K47/10A61P31/20C12N15/70C07K14/56
Inventor 王兴龙仇相书吕长杰张学志万文静刘蕴蝶杨增岐
Owner NORTHWEST A & F UNIV
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