Penicillium chrysogenum antifungal protein Pc-Arctin and preparation method thereof

A technology of antifungal protein and Penicillium chrysogenum, applied in peptide preparation methods, biochemical equipment and methods, fungi, etc., can solve unseen food preservation and clinical treatment problems

Active Publication Date: 2012-02-01
THIRD INST OF OCEANOGRAPHY STATE OCEANIC ADMINISTATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, although antifungal proteins exist widely in nature and have some excellent properties, there are basically no direct applications of such antifungal proteins in food preservation and clinical treatment of related fungal diseases
This is due to the fact that the use of these antimicrobial peptides (proteins) in clinical medicine still faces some challenges

Method used

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  • Penicillium chrysogenum antifungal protein Pc-Arctin and preparation method thereof
  • Penicillium chrysogenum antifungal protein Pc-Arctin and preparation method thereof
  • Penicillium chrysogenum antifungal protein Pc-Arctin and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1: Chromatographic separation of Penicillium chrysogenum A096 protein components on DEAE-sepharose Fast Flow column

[0048] Equilibrate the DEAE-sepharose Fast Flow column with the starting buffer (PH8.10, 20mM Tris). After loading the crude protein fraction of Penicillium chrysogenum A096 on the DEAE-sepharose Fast Flow column, continue to use two column volumes of starting Buffer Wash the column with the initial buffer (PH8.10, 20mM Tris), elute to obtain non-adsorbed component A on the DEAE-sepharose Fast Flow column under the condition of low salt concentration, and then use the washing buffer (PH8.10, 20mM Tris, 1MNaCl) to continue washing the DEAE-sepharose FastFlow column to obtain the adsorption component B ( figure 1 ).

Embodiment 2

[0049] Embodiment 2: Non-adsorbed component A is separated by CM-sepharose Fast Flow column chromatography

[0050] Also use the starting buffer (PH8.10, 20mM Tris) to equilibrate the CM-sepharose Fast Flow column, load the non-adsorbed component A on the DEAE column on the CM-sepharose Fast Flow column, and use the starting buffer of two column volumes The initial buffer (pH8.10, 20mM Tris) was used to wash the column to obtain non-adsorbed component C; then the initial buffer (pH8.10, 20mM Tris) was completely switched to the washing buffer (pH8. 10, 20mM Tris, 1M NaCl), and obtained the adsorption fraction D on the CM-sepharose Fast Flow column ( figure 2 ).

Embodiment 3

[0051] Embodiment 3: SDS-PAGE electrophoresis pattern of purified product Pc-Arctin

[0052] The purified component D ran 15% SDS-PAGE electrophoresis under normal temperature conditions at a voltage of 180V. The purity of component D was detected by rapid silver nitrate staining. The electrophoretic staining pattern showed that the purified component D had only one line under silver staining conditions. A single band was developed, and the protein was named Pc-Arctin.

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Abstract

The invention discloses Penicillium chrysogenum antifungal protein Pc-Arctin and a preparation method thereof, and relates to microbial protein. The preparation method comprises the following steps of: picking Penicillium chrysogenum A096 from a flat, inoculating into an SGY liquid medium for culture, taking supernate, adding saturated ammonium sulfate to obtain a mixed solution, centrifuging, re-dissolving a precipitate in water to obtain a crude protein solution, dialyzing and centrifuging; separating and purifying the obtained crude protein solution on a diethyl amino ethyl anion exchange chromatographic column, collecting various eluted ingredients, performing centrifugal ultrafiltration and condensation, and tracking active ingredients; continuously separating and purifying concentrated active non-adsorption ingredients on a carboxymethyl anion exchange chromatographic column, collecting various eluted ingredients, performing centrifugal ultrafiltration and condensation on the various ingredients until a required volume is reached, tracking antifungal active ingredients by using Paecilomyces varioti as sensitive testee indicator bacteria, and judging the purity of the proteinfor the determined active ingredients; and cutting a single band from a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) map and performing matrix-assisted laser desorption ionization-time of flight-time of flight (MALDI-TOF-TOF) identification.

Description

technical field [0001] The invention relates to a microbial source protein, in particular to a Penicillium chrysogenum antifungal protein Pc-Arctin and a preparation method thereof. Background technique [0002] The distribution of fungi on the earth is extremely wide, and the species are also very rich and diverse. Among them, there are about 200 kinds of fungi that have pathogenic effects on humans and other mammals, plus some fungi that can cause allergic symptoms, and there are about 300 kinds of pathogenic fungi that affect human health. Aspergillus fumigatus, Cryptococcus neoformans and Candida albicans are considered the main fungi responsible for human disease. Compared with the infections caused by bacteria, viruses and protozoa, the cases of fungal infectious diseases are much less, but fungal diseases have become a serious international problem, and many patients have no effective drug treatment for fungal infections And die. [0003] Fungi are not only an impo...

Claims

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Application Information

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IPC IPC(8): C12N1/14C07K14/385C12P21/02C07K1/36C07K1/34C07K1/30C07K1/18C12R1/82
Inventor 陈新华陈志腾敖敬群
Owner THIRD INST OF OCEANOGRAPHY STATE OCEANIC ADMINISTATION
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