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Preparation method and application of thermal denaturation lysozyme with anti-influenza A virus activity

A technology of influenza A virus and heat denaturation, applied in the field of biomedicine, can solve the problems such as the research report on anti-influenza A virus of heat denatured lysozyme, etc., and achieve the effect of clinical transformation or market promotion with less toxic and side effects

Active Publication Date: 2021-09-07
金橄榄科技(上海)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] There is no relevant research report on the anti-influenza A virus of heat-denatured lysozyme

Method used

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  • Preparation method and application of thermal denaturation lysozyme with anti-influenza A virus activity
  • Preparation method and application of thermal denaturation lysozyme with anti-influenza A virus activity
  • Preparation method and application of thermal denaturation lysozyme with anti-influenza A virus activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1: preparation of heat-denatured lysozyme

[0035] 1. Source of lysozyme

[0036] Natural egg white lysozyme was purchased from Suo Laibao Biotechnology Co., Ltd., item number: L8120.

[0037] 2. Preparation of denatured lysozyme

[0038] A, solution configuration: configuration mass volume ratio is 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 4%, 5% heat-denatured lysozyme, solvent is water;

[0039] B. pH adjustment: use NaOH, KOH, HCl or H 3 PO 4 Wait for the inorganic acid to adjust the pH value of the lysozyme solution to the desired value, usually 4 to 8. When the pH value is greater than 8, the egg white lysozyme will be difficult to dissolve in the solvent and precipitate;

[0040] C. Heat denaturation: after sterilizing the lysozyme solution through a 0.22 μm filter membrane, place it in a water bath or a metal bath, heat it at 75-100°C (preferably 90-100°C) for 2-3 hours, and immediately Take it out and place it on ice to cool down, and store it in a sealed c...

Embodiment 2

[0041] Embodiment 2: Hydrophobicity test of heat-denatured lysozyme (HDL)

[0042] 1. Materials

[0043] Orifice plate: 96-well non-removable microtiter plate (Corning, 3590)

[0044] Fluorescent agent: 8-aniline-1-naphthalenesulfonic acid (ANS, Solarbio, A9470)

[0045] Buffer: 0.1M PB (pH7.0)

[0046] 0.2M PB (pH7.0)

[0047] Microplate reader: Tecan infinite 200Pro

[0048] 2. Specific steps:

[0049] (1) Fluorescent agent ANS is dissolved in 0.1M PB, so that the final concentration is 8mM. ANS needs to be stored in the dark, and it is prepared and used immediately;

[0050] (2) The heat-denatured lysozyme was diluted with 0.2M PB to a concentration of 0.05%, that is, 0.5mg / mL;

[0051] (3) Add 10 μL of ANS solution to every 2 mL of diluted heat-denatured lysozyme solution, and use 0.2M PB solution as a control group, add 10 μL of ANS solution to the same 2 mL solution, mix well and react at room temperature for 30 minutes;

[0052] (4) Add 200uL of the mixed solutio...

Embodiment 3

[0055] Embodiment 3: in vitro anti-influenza virus plaque-loving experiment

[0056] 1. Materials:

[0057] DMEM basic medium (gibco, C11995500BT);

[0058] Fetal bovine serum (FBS gibco, 10270-106);

[0059] TPCK-trypsin (sigma, T1426-100MG);

[0060] PBS (Corning, 21-040-CV);

[0061] Penicillin / Streptomycin (p / s);

[0062] Orifice plate: 24-well plate (Nest, 702001);

[0063] Target cells: MDCK (Madin-Darby canine kidney) cell line;

[0064] Influenza virus: H1N1;

[0065] 2. Virus spotting test method:

[0066] (1) The genotype of the H1N1 virus is Influenza A / WSN / 33 virus (H1N1).

[0067] (2) 37°C, 5% CO 2 Cultivate MDCK cells, the medium is DMEM medium containing 10% fetal bovine serum (FBS), 1% penicillin / streptomycin (P / S), inoculated in the cell culture microwell plate one day before the experiment, it is required to be exactly plated A monolayer of cells in a well-filled plate.

[0068] (3) Dilute the virus infection titer used to 10 4～8 In between, mix t...

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Abstract

The invention relates to the technical field of medicines, and provides a preparation method and application of thermal denaturation lysozyme with anti-influenza A virus activity. The preparation method of the thermal denaturation lysozyme comprises the following steps of: A, preparing a solution, namely preparing a lysozyme solution of which the mass volume ratio is at least 2%, and adjusting the pH value to be within the range of 4-8; and B, heating for denaturation, namely filtering the lysozyme solution for sterilization, heating for 2-3 hours at 75-100 DEG C, immediately taking out, cooling on ice, and sealing and storing at low temperature. Furthermore, the invention provides application of the thermal denaturation lysozyme in preparation of an anti-influenza A virus medicinal composition, and the anti-influenza A virus medicinal composition with the thermal denaturation lysozyme as an active component. Results show that the lysozyme has the anti-influenza A virus activity after being subjected to thermal denaturation according to the method.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a preparation method and application of heat-denatured lysozyme with anti-influenza A virus activity. Background technique [0002] Lysozyme (LZ), also known as N-acetylmuramide glycanohydrase or Muramidase, was first discovered in human secretions by British bacteriologist Fleming in 1922. (Fleming, A., Lysozyme: President's Address. Proc R Soc Med, 1932.26 (2): p.71-84.), it acts on the N-acetylmuramic acid and N-acetylamino of the peptidoglycan layer of the bacterial cell wall The β-1,4-glucosidic bond between glucose causes the bacterial cell wall to rupture, eventually leading to bacterial lysis and death (Phillips, D.C., The three-dimensional structure of an enzyme molecule. Sci Am, 1966.215(5): p.78 -90.). [0003] The antiviral activity of lysozyme has been reported in the literature (Lee-Huang, S., et al., Lysozyme and RNases as anti-HIV components in beta-core p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/36A61K38/47A61K45/06A61P31/16
CPCC12N9/2462A61K45/06A61P31/16C12Y302/01017A61K38/00
Inventor 范国荣
Owner 金橄榄科技(上海)有限公司
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