Composition for treating and/or preventing influenza, method and application thereof

A technology for influenza virus and influenza A, which is applied in the field of protein engineering to achieve the effect of high safety, resistance to resistant strains, and inhibition of virus infection

Active Publication Date: 2019-04-26
SHANXI JINBO BIO PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These drugs have a certain inhibitory effect on H5N1 and H7N9 viruses, but whether they can inhibit future new mutant influenza viruses has not yet been confirmed

Method used

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  • Composition for treating and/or preventing influenza, method and application thereof
  • Composition for treating and/or preventing influenza, method and application thereof
  • Composition for treating and/or preventing influenza, method and application thereof

Examples

Experimental program
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Effect test

preparation example Construction

[0063] The invention provides a method for preparing an acid-anhydride-treated protein that has preventive and control effects on various subtypes of seasonal influenza viruses and highly pathogenic influenza viruses. The steps are as follows:

[0064] Dissolve 3-hydroxy-phthalic anhydride powder in dimethyl sulfoxide to make a 1M anhydride solution; use disodium hydrogen phosphate dodecahydrate powder to prepare a 0.1M phosphate solution with a pH of 8.5, and add 400 mg of β-lactoglobulin (β-lactoglobulin, β-LG) powder was dissolved in 20 mL of phosphate solution; 250 μL of 1 M acid anhydride solution was added to the β-LG solution, repeatedly blown and mixed, and the pH was maintained at 9.0, Incubate for 20 minutes, repeat 6 times in total, place at 25°C for 1-2 hours for the last time, so that the protein is completely modified; dialyze the anhydrided protein with pH 7.4 phosphate buffer saline (PBS), change the medium twice during the period, dialyze for 60 Remove the imp...

Embodiment 1

[0072] Example 1. Preparation of bovine β-lactoglobulin treated with acid anhydride

[0073] (1) 3-hydroxy-phthalic anhydride (3-hydroxy phthalic anhydride, HP) powder (purchased from Sigma, product number 320064) was dissolved in dimethyl sulfoxide to prepare a 1M anhydride solution;

[0074] (2) Prepare a 0.1 M phosphate solution with pH 8.5 with disodium hydrogen phosphate dodecahydrate powder, and dissolve 400 mg of bovine β-lactoglobulin (β-lactoglobulin, β-LG) powder (purchased from Sigma, product number L8005) In 20mL phosphate solution;

[0075] (3) Add 250 μL of 1M acid anhydride solution into the β-LG solution (in step (2)), repeatedly blow and mix, adjust the pH to 8.5 with 1M NaOH solution, let stand at 25°C for 20 minutes, repeat 6 times , the last time at 25 ° C for 1-2 hours, so that the protein is completely modified;

[0076] (4) Dialyze the acid-anhydrided protein with phosphate buffered saline (PBS) at pH 7.4, and use a 3.5KD dialysis bag for dialysis, and...

Embodiment 2

[0080] Embodiment 2. detect the concentration of acid anhydride protein with BCA method

[0081] (1) Dilute the anhydrided protein prepared in Example 1 with PBS at a ratio of 10 times, 50 times, and 100 times; and dilute the 2 mg / mL protein standard sample (BSA) with PBS solution to keep the protein 0, 0.1, 0.2, 0.4, 1, 2 mg / mL concentration;

[0082] (2) Add 10 μL of the diluted sample and different concentrations of standard substances into a 96-well plate, and then add 15 μL of PBS to each well to set up 3 duplicate wells;

[0083] (3) Mix liquid A and liquid B in the BCA detection kit (purchased from Takara, product number T9300A) at a ratio of 50:1, add 200 μL in each well to the sample to be tested and the standard, and repeatedly blow and mix, Incubate at 37°C in the dark for 0.5 hours;

[0084] (4) Detect the absorbance of each sample at 562nm with a microplate reader;

[0085] (5) Use GraphPad Prism 5.0 software to draw a scatter diagram of standard substance conc...

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Abstract

The present invention provides a composition for treating and / or preventing influenza, a method and an application thereof. The composition contains protein treated with acid anhydride and another drug for treating and / or preventing influenza. The acid anhydride-treated protein prepared by the invention is acid anhydride-treated lactoglobulin. An in vitro experiment shows that the protein can broadly inhibit influenza virus infected cells of various subtypes. In animal in vivo experiments, the protein has a significant prevention and treatment effect on influenza virus infection. Influenza viruses have many subtypes and high mutation rates, so that the development of broad-spectrum anti-influenza preparations is very important. The acid anhydride-treated protein can not only inhibits the infection of many seasonal influenza viruses, but also significantly inhibits the highly pathogenic avian influenza virus H7N9, and the influenza virus H1N1 (2009) which caused a pandemic, suggesting that the protein can be developed as a highly effective and broad-spectrum biological preparation for the prevention and treatment of influenza viruses.

Description

technical field [0001] The invention belongs to the field of protein engineering, and in particular relates to a method and application of an acid anhydride-treated protein for inhibiting influenza virus. Background technique [0002] Influenza Viruses and Influenza Epidemics [0003] Influenza (Influenza) is caused by influenza virus (Influenza viruses, IVs) infection, which can cause acute respiratory tract infection, mainly discomfort in the mouth, pharynx, bronchi and even lungs, characterized by sudden high fever, sore throat, cough , runny nose, muscle and joint pain, etc., will lead to death in severe cases. Statistics from the World Health Organization at the end of 2018 show that respiratory diseases caused by seasonal influenza alone cause 290,000 to 650,000 deaths worldwide each year. The four outbreaks of large-scale influenza in history and the outbreak and prevalence of seasonal influenza and new influenza each year indicate that influenza viruses pose a cont...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K45/06A61K38/17A61K38/38A61K38/46A61P31/16
CPCA61K38/1722A61K38/385A61K38/465A61K45/06A61P31/16A61K2300/00
Inventor 杨霞陆路姜世勃付玉红李佩玉徐巍
Owner SHANXI JINBO BIO PHARMA CO LTD
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