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Production and use of high expression, high purity, high activity gene recombinant human lysozyme

A technology of human lysozyme and genetic recombination, which is applied in genetic engineering, plant genetic improvement, recombinant DNA technology, etc., to achieve high purity and high activity

Inactive Publication Date: 2003-04-16
长春奇龙生物技术研究所 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Methicillin-resistant Staphylococcus aureus (MRSA) has no cure other than vancomycin

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0010] Embodiment 1: the production method of the human lysozyme expressed by genetic engineering;

[0011] Materials: The expression system of genetically engineered human lysozyme is characterized by using methanolotrophic yeast as the host strain "Pichia genus strain". In the phenotype of methanol utilization, both fast methanol utilization (mut+) and slow methanol utilization (muts or mut-) were used. The smd1165(his4.prb1), smd1163(his.pep4.prb1), Gs115 and km71 strains of Pichia pastoris were mainly used. The plasmid of the genetically engineered human lysozyme of the present invention is mainly integrated into the chromosome of the above bacterial strain by using the plasmid of the pPIC9k series.

[0012] Steps: 1. Shake flask seed preparation: based on the preparation of 200 milliliters of culture medium, use H 3 PO 4 (phosphoric acid) 6 ml, MgSO 4 (magnesium sulfate) 3 grams, K 2 SO 4 (potassium sulfate) 4 grams, KOH (potassium hydroxide) 1 gram, CaSO 4 2H 2 O...

Embodiment 2

[0014] Embodiment 2: the identification of human lysozyme bacteriolytic activity (test tube method)

[0015] Material

[0016] 1. Buffer pH7.2 10mM Tris-Cl buffer

[0017] 2. Strains Staphylococcus aureus, Escherichia coli

[0018] 3. 10% SDS

[0019] 4. LB medium

[0020] method:

[0021] Staphylococcus aureus with pH 7.2 10mM Tris-Cl buffer

[0022] (or E. coli) to make 10 7 -10 8 Bacterial solution with a concentration of about CFU / mL

[0023] ↓

[0024] Add to different test tubes respectively, 2mL in each tube

[0025] ↓

[0026] Add different doses of human lysozyme (100μg-1mg) to different test tubes

[0027] 37C° overnight Take 20 μL from each tube and dilute with 10-fold dilution method Add 100 μL 10% SDS to each tube (final concentration 0.5%)

[0028] ↓ ↓ Shake well, take 100μL and spread it on LB medium, observe the change of turbidit...

Embodiment 3

[0032] Example 3: The bactericidal effect of human lysozyme on drug-resistant bacteria, as shown in Figures 6 and 7, with Bacillus abalone and Staphylococcus aureus as examples.

[0033] Drug-resistant strains: 30 strains of different drug-resistant bacteria came from the Laboratory Department of Xijing Hospital, Xi'an

[0034] method:

[0035] Prepare 1% agarose with pH7.2 10mM Tris-Cl buffer

[0036] After autoclaving, cool to 40 °C and add bacteria to 10 6 dump flat

[0037] ↓

[0038] Punch holes in the plate with a hole punch

[0039] ↓

[0040] Add different concentrations of lysozyme solution (0-100mg / ml) to the wells

[0041] ↓

[0042] Incubate in a 37°C incubator for 4-6 hours

[0043] ↓

[0044] After LB nutrient agar is autoclaved, cool to 40°C and cover on the above agarose plate

...

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Abstract

A high-expression, high-purity and high-activity gene-recombinant human lysozyme, its preparing process and the medical composition of antibiotic and the said human lysozyme for treating bacterial and fungus infection, drug-resistant bacteria infection, and the sequela caused by gram-negative bacteria infection are disclosed.

Description

Technical field: [0001] The invention relates to a method and application of gene recombinant human lysozyme, in particular to the production and application of a gene recombinant human lysozyme with high expression, high purity and high activity, belonging to the technical field of bioengineering. Background technique: [0002] Lysozyme was discovered by Fleming in 1922 (Proc. R. Soc. Lond. B. Biol. Sci. 1922). It is an effective antibacterial agent, its full name is: 1,4-β-N-lysozyme or: mucopeptide N-acetylmuramoyl hydrolase. It can cut off the β-1,4 glycosidic linkage between N-acetylglucosamine and N-acetylmuramic acid in the peptidoglycan of the bacterial cell wall, and destroy the peptidoglycan scaffold. Swells and cracks, causing bacteria to lyse. Human and animal cells have no cell wall structure and no peptidoglycan, so lysozyme has no toxic effect on human cells. Lysozyme widely exists in animals, plants and microorganisms in nature. At present, people mainly ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/47A61P31/00A61P31/04C12N1/19C12N9/36C12N15/56C12N15/81
Inventor 安米张华李元安献禄
Owner 长春奇龙生物技术研究所
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