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Method for extracting small-molecular alpha-defensin polypeptide from sputum of patient

A technology for small molecule polypeptides and defensins, applied in the biological field, can solve the problems of low yield, less activity than natural proteins, and high production cost, and achieve the effects of high yield, improved sensitivity and accuracy, and low cost

Inactive Publication Date: 2015-02-04
王烁
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The α-defensins currently on the market mainly rely on chemical synthesis or bacterial expression of recombinant proteins. Although they have the amino acid sequence of natural polypeptides, their activity is not as good as natural proteins due to the lack of proper intracellular expression and post-translational protein folding and modification. Moreover, the production cost is high and the output is low

Method used

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  • Method for extracting small-molecular alpha-defensin polypeptide from sputum of patient
  • Method for extracting small-molecular alpha-defensin polypeptide from sputum of patient
  • Method for extracting small-molecular alpha-defensin polypeptide from sputum of patient

Examples

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Embodiment 1

[0041] A method for extracting small molecule α-defensin polypeptides from patient sputum, comprising the following steps:

[0042] Step 1: Collect 15mL of the patient's sputum, put it into a dry ice box quickly to keep the sputum in the ice-water mixture state at low temperature, and store it in a -80°C refrigerator until it is extracted and used, and mark it 135#.

[0043] Step 2: Add 15 mL of 10% (V / V) acetic acid solution to the 15 mL of sputum in the previous step, and homogenize with a homogenizer for 5 min. Collect about 30 mL of sputum homogenate, and put it in a 4°C refrigerator for overnight rotation.

[0044] Step 3: Take out the sputum homogenate obtained by stirring and leaching with acetic acid in the previous step, centrifuge at 4° C., 14,000 rpm for 30 minutes, remove the precipitate, and keep the supernatant as the crude extract.

[0045] Step 4: Add 0.2mM DTT to the crude supernatant extract at a ratio of 1:10 by volume (take 1M DTT stored in a -20°C refrige...

Embodiment 2

[0053] A method for extracting small molecule α-defensin polypeptides from patient sputum, comprising the following steps:

[0054] Step 1: Collect 20mL of sputum from the patient, quickly put it into a dry ice box to keep the sputum in the ice-water mixture state at low temperature, and store it in a -80°C refrigerator until it is extracted and used, and mark it 136#.

[0055] Step 2: Add 20 mL of 10% (V / V) acetic acid solution to 20 mL of sputum in the previous step, and homogenize for 5 minutes with a homogenizer. Collect about 40 mL of sputum homogenate, and put it in a 4°C refrigerator for overnight rotation.

[0056] Step 3: Take out the sputum homogenate obtained by stirring and leaching with acetic acid in the previous step, centrifuge at 4° C., 14,000 rpm for 30 minutes, remove the precipitate, and keep the supernatant as the crude extract.

[0057] Step 4: Add 0.2mM DTT to the crude supernatant extract at a ratio of 1:10 by volume (take 1M DTT stored in a -20°C refr...

Embodiment 3

[0065] A method for extracting small molecule α-defensin polypeptides from patient sputum, comprising the following steps:

[0066] Step 1: Collect 25mL of the patient's sputum, put it into a dry ice box quickly to keep the sputum in the ice-water mixture state at low temperature, and store it in a -80°C refrigerator until it is extracted and used, and mark it 137#.

[0067] Step 2: Add 25 mL of 10% (V / V) acetic acid solution to 25 mL of sputum in the previous step, and homogenize for 5 minutes with a homogenizer. Collect about 50 mL of sputum homogenate, and put it in a 4°C refrigerator for overnight rotation.

[0068] Step 3: Take out the sputum homogenate obtained by stirring and leaching with acetic acid in the previous step, centrifuge at 4° C., 14,000 rpm for 30 minutes, remove the precipitate, and keep the supernatant as the crude extract.

[0069] Step 4: Add 0.2mM DTT to the crude supernatant extract at a ratio of 1:10 by volume (take 1M DTT stored in a -20°C refrige...

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Abstract

The invention relates to a method for extracting small-molecular alpha-defensin polypeptide from sputum of a patient, which comprises the following steps: 1. collecting and preserving the sputum of the patient; 2. adding acetic acid into the sputum specimen, preparing a homogenate, stirring and leaching, centrifugating the homogenate, and taking the supernatant to prepare the crude extract; 3. adding DTT into the crude extract, and evenly mixing to obtain a mixture; 4. loading the mixture to a Pullulan gel column, eluting and purifying with acetic acid at normal temperature, preparing an elution curve, monitoring the collection of the eluate, sampling the extract, and carrying out mass spectrometric detection; 5. carrying out ultrafiltration concentration, carrying out vacuum freeze-drying, and storing; and 6. carrying out protein amino acid sequencing on the extract, and detecting the antimicrobial activity of the extract for Escherichia coli by using an agarose dispersion process. The method has the advantages of simple preparation process, low cost and high yield; the obtained product is natural alpha-defensin with high purity and high antimicrobial bioactivity; and the method provided by the invention is obviously better than the common chemical synthesis and recombinant protein method.

Description

technical field [0001] The invention relates to a method for extracting a small molecular peptide alpha defensin with anti-pathogenic primary immune biological activity from patient's sputum, which belongs to the field of biotechnology. Background technique [0002] Defensins are antimicrobial peptides widely present in a variety of organisms including plants, invertebrates and vertebrates. Human neutrophil peptide (HNP) is the most important antimicrobial polypeptide of human beings, mainly expressed in neutrophils (PMN), accounting for 5% of total cell protein, and more than 50% of intracellular azurophilin granulin. HNP is a kind of active peptide, which has broad-spectrum antibacterial effect on Gram-positive bacteria and Gram-negative bacteria, and also has anti-virus and anti-specific cytotoxic substances. There are six known α-defensins in the human body, namely human neutrophil defensins 1-4 and human defensins 5-6. Human neutrophil defensin 1 mainly exists in neut...

Claims

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Application Information

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IPC IPC(8): C07K14/47C07K1/16
CPCC07K14/4723
Inventor 韩兵王烁万君花
Owner 王烁
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