阅读说明：本技术 一种尘螨致敏蛋白Der p 1的分离纯化方法 (Separation and purification method of dust mite allergenic protein Der p 1 ) 是由 陈晴 何韶衡 张杨 张丹丹 张晓楠 于 2021-07-29 设计创作，主要内容包括：本发明公开了一种尘螨致敏蛋白Der p 1的分离纯化方法,包括以下步骤：1)尘螨水溶蛋白粗提液的制备：取包含尘螨的灰尘样品,加入PBS-T溶液,在4℃下搅拌过夜,5000×g离心,取上清液；再向上清液中加入醋酸钠缓冲液,在4℃下透析3-4天,4℃保存待用；2)阳离子交换层析：将上述步骤1)所制备的尘螨水溶蛋白粗提液上样,使用结合缓冲液进行冲洗,随后固定结合缓冲液并利用洗脱缓冲液进行梯度洗脱,收集洗脱峰,获得目的产物。本方法具有快速、高效、生产稳定,成本低等特点；所制备的提取液或纯化蛋白可用于患者血清样品中主要致敏蛋白的检测,同时还可进一步制备重组过敏原,设计低过敏衍生物以及预防性疫苗方面具有广泛应用。(The invention discloses a separation and purification method of dust mite allergenic protein Der p 1, which comprises the following steps: 1) preparing a coarse extract of dust mite water-soluble protein: adding PBS-T solution into dust sample containing dust mite, stirring overnight at 4 deg.C, centrifuging at 5000 × g, and collecting supernatant; adding sodium acetate buffer solution into the supernatant, dialyzing at 4 deg.C for 3-4 days, and storing at 4 deg.C; 2) cation exchange chromatography: loading the crude extract of the dust mite water soluble protein prepared in the step 1), washing by using a binding buffer solution, fixing the binding buffer solution, performing gradient elution by using an elution buffer solution, and collecting an elution peak to obtain a target product. The method has the characteristics of high speed, high efficiency, stable production, low cost and the like; the prepared extracting solution or purified protein can be used for detecting main allergic protein in a serum sample of a patient, and meanwhile, the recombinant allergen can be further prepared, and the method has wide application in the aspects of designing hypoallergenic derivatives and preventing vaccines.)

1. A separation and purification method of dust mite sensitization protein Der p 1 is characterized by comprising the following steps:

1) preparing a coarse extract of dust mite water-soluble protein: adding PBS-T solution into dust sample containing dust mite, stirring overnight at 4 deg.C, centrifuging at 5000 × g, and collecting supernatant; adding sodium acetate buffer solution into the supernatant, dialyzing at 4 deg.C for 3-4 days, and storing at 4 deg.C;

2) cation exchange chromatography: loading the crude extract of the dust mite water soluble protein prepared in the step 1), washing by using a binding buffer solution, fixing the binding buffer solution, performing gradient elution by using an elution buffer solution, and collecting an elution peak to obtain a target product.

2. The method for separating and purifying a dust mite sensitizing protein Der p 1 as claimed in claim 1, further comprising:

3) freeze-drying: freeze-drying the target product obtained in the step 2) to obtain a pure product of the dust mite allergenic protein Der p 1.

3. The method for separating and purifying a dust mite-sensitized protein Der p 1 according to claim 1, wherein in the step 1), the solid-to-liquid ratio of the dust mite-containing dust sample to the PBS-T solution is 1 (100-200).

4. The method for separating and purifying a dust mite sensitizing protein Der p 1 as claimed in claim 1, wherein the binding buffer in the step 2) is a sodium acetate buffer solution with a pH of 10 to 100mM and 4.5.

5. The method for separating and purifying a dust mite sensitized protein Der p 1 according to claim 1, wherein the elution buffer solution in the step 2) is 100 to 1000mM NaCl solution.

6. The method for separating and purifying the dust mite sensitizing protein Der p 1 according to claim 1, wherein the step 2) is specifically as follows:

21) preparing a binding buffer solution and an elution buffer solution, filtering and storing at room temperature;

22) loading, eluting and collecting: washing the system pump with 20% ethanol, connecting the column to the chromatography system instrument, washing the sample pump with binding buffer, and equilibrating the column with 2 column volumes of binding buffer; sample is loaded through a sample pump, the sample injection volume is 1mL, after sample injection is carried out for 5min, the column is washed by 1 column volume binding buffer, then gradient elution is carried out on the column by using 0-30% elution buffer, 10 column volumes are washed, and after the elution is finished, the column is balanced by using the binding buffer, and 5 column volumes are balanced.

7. The method for separating and purifying the dust mite sensitized protein Der p 1 according to claim 1, wherein the binding buffer solution is a 10-100mM sodium acetate buffer solution with pH 4.5, the solution is prepared and filtered by 0.2 μm filter paper, and the solution is stored at room temperature for later use; the elution buffer solution is 100-1000 mM NaCl solution, and the solution is prepared, filtered by 0.2 mu m filter paper and stored at room temperature for later use.

8. The method for separating and purifying a dust mite sensitizing protein Der p 1 as claimed in claim 1, further comprising identifying the Der p 1 protein by SDS-PAGE from the elution peaks collected in the step 2).

9. A dust mite sensitizing protein Der p 1 prepared by the method for separating and purifying the dust mite sensitizing protein Der p 1 according to any one of claims 1 to 8.

10. An application of the dust mite sensitizing protein Der p 1 prepared by the method for separating and purifying the dust mite sensitizing protein Der p 1 according to any one of claims 1-8 in preparing recombinant allergens, hypoallergenic derivatives and preventive vaccines.

Technical Field

The invention relates to the technical field of separation and purification of proteins, in particular to a separation and purification method of dust mite sensitizing protein Der p 1.

Background

Allergic diseases comprise allergic asthma, allergic rhinitis, food allergy and the like, and are clinically common diseases and frequently encountered diseases. Allergies are caused by the immune response of the body to large molecular (e.g. proteinaceous) substances that are normally harmless. Dust mites are one of the leading causes of allergic diseases worldwide. Dust mites cause allergic diseases of the respiratory system such as allergic asthma and rhinitis, and in 2004, the world health organization estimates about 3 billion patients with asthma and rhinitis worldwide and is on the rise (Chen K W, Hypoallengenic Der p 1/Der p 2combination vaccines for immunological therapy of house dust mite allergy, 2012). The content of Der p 1 in house dust mite is 0.02-14.30 μ g/g, the amino acid number of Der p 1 is 320, it is glycoprotein with molecular weight of 25kDa, and isoelectric point is 4.7-7.4. In the secondary structure of Der p 1, 43.75% is alpha helical structure, and as a papain family member, the papain family member contains active sites such as histidine, cysteine, aspartic acid, magnesium binding site and the like, and plays a key role in the process of generating anaphylactic reaction with later stage. However, the special extraction processes developed for dust mites, including long extraction processes, may lead to degradation and aggregation of glycoproteins.

Therefore, in order to reduce the risk of introducing any possible artefacts at the level of the major allergens, the present application has developed a rapid extraction method, resulting in a major allergen enrichment and formulated a separation and purification method aimed at obtaining a suitable amount of the 25kDa allergen.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a method for separating and purifying dust mite sensitizing protein Der p 1, which solves the problems in the prior art.

The technical problem to be solved by the invention is realized by the following technical scheme:

a method for separating and purifying dust mite sensitizing protein Der p 1 comprises the following steps:

1) preparing a coarse extract of dust mite water-soluble protein: adding PBS-T solution into dust sample containing dust mite, stirring overnight at 4 deg.C, centrifuging at 5000 × g, and collecting supernatant; adding sodium acetate buffer solution into the supernatant, dialyzing at 4 deg.C for 3-4 days, and storing at 4 deg.C;

2) cation exchange chromatography: loading the crude extract of the dust mite water soluble protein prepared in the step 1), washing by using a binding buffer solution, fixing the binding buffer solution, performing gradient elution by using an elution buffer solution, and collecting an elution peak to obtain a target product.

Preferably, the method further comprises the following steps:

3) freeze-drying: and (3) carrying out freeze-drying treatment on the target product in the step 2) to obtain a pure product of the dust mite allergenic protein Der p 1.

Preferably, in the step 1), the solid-to-liquid ratio of the dust sample containing dust mites to the PBS-T solution is 1: (100-.

Preferably, the binding buffer in the step 2) is 10-100mM sodium acetate buffer with pH 4.5.

Preferably, the elution buffer solution in the step 2) is 100-1000 mM NaCl solution.

Preferably, step 2) is specifically:

21) preparing a binding buffer solution and an elution buffer solution, filtering and storing at room temperature;

22) loading, eluting and collecting: washing the system pump with 20% ethanol, connecting the column to the chromatography system, washing the sample pump with binding buffer, and balancing the column with 2 column volumes of binding buffer; sample is loaded through a sample pump, the sample injection volume is 1mL, after sample injection is carried out for 5min, the column is washed by 1 column volume binding buffer, then gradient elution is carried out on the column by using 0-30% elution buffer, 10 column volumes are washed, and after the elution is finished, the column is balanced by using the binding buffer, and 5 column volumes are balanced.

Preferably, the binding buffer solution is 10-100mM sodium acetate buffer solution with pH 4.5, the solution is filtered by 0.2 μm filter paper after being prepared, and is stored at room temperature for later use; the elution buffer solution is 100-1000 mM NaCl solution, and the solution is prepared, filtered by 0.2 mu m filter paper and stored at room temperature for later use.

Preferably, the method further comprises identifying the Der p 1 protein by SDS-PAGE from the elution peaks collected in step 2).

A dust mite sensitizing protein Der p 1 prepared by the method for separating and purifying the dust mite sensitizing protein Der p 1.

An application of the dust mite sensitizing protein Der p 1 prepared by the method for separating and purifying the dust mite sensitizing protein Der p 1 in preparing recombinant allergens, hypoallergenic derivatives and preventive vaccines.

The technical scheme of the invention has the following beneficial effects:

(1) according to the separation and purification method of the main allergic protein, the content of the main allergic protein Der p 1 in the dust mite crude extract can be obviously improved by improving the components of the dust mite protein extract, and then the target protein in the dust mite crude extract is separated and purified through cation exchange chromatography, so that 25kDa allergen with high purity and proper quantity is obtained.

(2) The method for detecting the allergen in vitro, namely ImonoCAP, used in clinic at present does not contain Der p 1, and the invention is beneficial to applying crude extract or purified protein of the protein to preparation of recombinant allergen, design of hypoallergenic derivatives and preventive vaccination and is expected to develop a new generation of candidate drugs for desensitization treatment.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description, serve to explain the principles of the invention.

FIG. 1 shows the distribution of major proteins in the crude extract of dust mites of this application. Wherein, Lane 1: protein marker; lane 2: dust mite extract.

FIG. 2 is the results of cation exchange chromatography in accordance with the present application. Arrows indicate the collection peak containing the protein of interest.

FIG. 3 shows the results of cation exchange chromatography SDS-PAGE according to the present application. Wherein, M: protein marker; lane 1 is collection I; lane 2 is collection II; wherein Lane 3 is a 25kDa protein band (i.e., the target protein) visible on SDS-PAGE of the collection III; lane 4 is collection IV; lane 5 is pool V.

Detailed Description

Various exemplary embodiments of the present invention will now be described in detail with reference to the accompanying drawings. It should be noted that: the relative arrangement of the components and steps, the numerical expressions and numerical values set forth in these embodiments do not limit the scope of the present invention unless specifically stated otherwise.

Example 1

Dust mite raw materials: dust samples containing dust mites were provided by the university of medical, california.

The operation method comprises the following steps: the conventional operation method adopted by the application is detailed in Experimental anaphylaxis, Yangshao Heng, 2010.

(1) Preparation of a crude extract: a dust mite-containing dust sample (0.10 g) was weighed, added to 20mL of PBS-T solution, stirred overnight at 4 ℃, and the supernatant was collected by centrifugation at 5000 Xg for 20min, to which 20mL of 10-100mM sodium acetate buffer solution (pH 4.5) was added, dialyzed, and dialyzed at 4 ℃ for 3-4 days. After dialysis, the cells were stored at 4 ℃. The distribution of main proteins in the coarse extract of dust mites extracted by the method is shown in figure 1.

The allergen protein in the dust mite is subjected to crude extraction by adopting a PBS-T solution, and is dialyzed by using a 10-100mM sodium acetate buffer solution with pH 4.5, so that the target allergen protein Der p 1 in the dust mite crude extract has the highest content (25.0kDa) and other proteins have lower contents.

(2) Cation exchange chromatography:

21) solution preparation:

binding buffer: 10-100mM sodium acetate buffer solution with pH 4.5, filtering the solution with 0.2 μm filter paper, and storing at room temperature.

Elution buffer: filtering the solution with 100-1000 mM NaCl and 0.2 μm filter paper, and storing at room temperature.

22) Loading, eluting and collecting: the system pump was first washed with 20% ethanol, then a HiTrap Capto MMC (1ml, GE Healthcare) column was attached to the AKTA pure instrument, the sample pump was washed with binding buffer, and the column was equilibrated with 2 column volumes of binding buffer. Sample loading is carried out through a sample pump, the sample injection volume is 1mL, after sample injection is carried out for 5min, the column is washed by 1 column volume binding buffer, then gradient elution is carried out on the column by using 30% elution buffer, 10 column volumes are washed, after the elution is finished, the column is balanced by using the binding buffer, and 5 column volumes are balanced. Collecting the target eluent to obtain the target product.

The results of cation exchange chromatography are shown in FIG. 2, in which the arrow indicates the peak containing the target protein, and the peak III pool is selected.

(3) Different eluents were selected according to the collected peaks for SDS-PAGE electrophoresis to analyze the protein separation effect.

The peak III pools were selected for SDS-PAGE analysis, as shown in FIG. 3, wherein a distinct 25kDa protein band (i.e., the protein of interest) was observed on SDS-PAGE in the peak III pools.

Example 2

Dust mite raw materials: dust samples containing dust mites were provided by the university of medical, california.

The operation method comprises the following steps: the conventional operation method adopted by the application is detailed in Experimental anaphylaxis, Yangshao Heng, 2010.

(1) Preparation of a crude extract: a dust mite-containing dust sample (0.10 g) was weighed, added to 10mL of PBS-T solution, stirred overnight at 4 ℃, and the supernatant was collected by centrifugation at 5000 Xg for 20min, to which 20mL of 10-100mM sodium acetate buffer solution (pH 4.5) was added, dialyzed, and dialyzed at 4 ℃ for 3-4 days. After dialysis, the cells were stored at 4 ℃.

(2) Cation exchange chromatography:

21) solution preparation:

binding buffer: 10-100mM sodium acetate buffer solution with pH 4.5, filtering the solution with 0.2 μm filter paper, and storing at room temperature.

Elution buffer: filtering the solution with 100-1000 mM NaCl and 0.2 μm filter paper, and storing at room temperature.

22) Loading, eluting and collecting: the system pump was first washed with 20% ethanol, then a HiTrap Capto MMC (1ml, GE Healthcare) column was attached to the AKTA pure instrument, the sample pump was washed with binding buffer, and the column was equilibrated with 2 column volumes of binding buffer. Sample loading is carried out through a sample pump, the sample injection volume is 1mL, after sample injection is carried out for 5min, the column is washed by 1 column volume binding buffer, then gradient elution is carried out on the column by using 30% elution buffer, 10 column volumes are washed, after the elution is finished, the column is balanced by using the binding buffer, and 5 column volumes are balanced. Collecting the target eluent to obtain the target product.

(3) Different eluents were selected according to the collected peaks for SDS-PAGE electrophoresis to analyze the protein separation effect.

Example 3

Dust mite raw materials: dust samples containing dust mites were provided by the university of medical, california.

The operation method comprises the following steps: the conventional operation method adopted by the application is detailed in Experimental anaphylaxis, Yangshao Heng, 2010.

(1) Preparation of a crude extract: a dust mite-containing dust sample (0.10 g) was weighed, added to 15mL of PBS-T solution, stirred overnight at 4 ℃, and the supernatant was collected by centrifugation at 5000 Xg for 20min, to which 20mL of 10-100mM sodium acetate buffer solution (pH 4.5) was added, dialyzed, and dialyzed at 4 ℃ for 3-4 days. After dialysis, the cells were stored at 4 ℃.

(2) Cation exchange chromatography:

21) solution preparation:

binding buffer: 10-100mM sodium acetate buffer solution with pH 4.5, filtering the solution with 0.2 μm filter paper, and storing at room temperature.

Elution buffer: filtering the solution with 100-1000 mM NaCl and 0.2 μm filter paper, and storing at room temperature.

22) Loading, eluting and collecting: the system pump was first washed with 20% ethanol, then a HiTrap Capto MMC (1ml, GE Healthcare) column was attached to the AKTA pure instrument, the sample pump was washed with binding buffer, and the column was equilibrated with 2 column volumes of binding buffer. The sample is loaded through a sample pump, the sample injection volume is 1mL, after the sample injection is carried out for 5min, the column is washed by 1 column volume binding buffer, then the column is subjected to gradient elution by using 0-30% elution buffer, 10 column volumes are washed, and after the elution is finished, the column is balanced by using the binding buffer, and 5 column volumes are balanced. Collecting the target eluent to obtain the target product.

(3) Different eluents were selected according to the collected peaks for SDS-PAGE electrophoresis to analyze the protein separation effect.

And (4) analyzing results:

(1) preparation of a crude extract: by adopting PBS-T solution to carry out rough extraction on the allergen protein in the dust mites and dialyzing the dust mites by using 10-100mM sodium acetate buffer solution with pH 4.5, the content of the target allergen protein Der p 1 in the dust mite rough extract is the highest (25.0kDa), while the content of other proteins is less, the operation is green, mild, simple and easy to implement, and the protein is not easy to degrade, wherein the target allergen protein is the main protein component of the extract. The distribution of main proteins in the coarse extract of dust mites extracted by the method is shown in figure 1.

(2) Cation exchange chromatography: the results of cation exchange chromatography are shown in FIG. 2, in which the arrow indicates the peak containing the target protein, and the peak III pool is selected.

(3) SDS-PAGE electrophoresis: the peak III pools were selected for SDS-PAGE analysis, as shown in FIG. 3, wherein a distinct 25kDa protein band (i.e., the protein of interest) was observed on SDS-PAGE in the peak III pools.

The dust mite sensitizing protein Der p 1 obtained by the separation and purification method according to the above embodiment can be applied in various fields, such as the preparation of recombinant allergens, hypoallergenic derivatives and prophylactic vaccines. This will be a major direction for future exploration.

Fig. 1, 2 and 3 are graphs showing the results of example 1. The results of the embodiments 2 and 3 are similar to those of the embodiment 1, and are not repeated herein.

Therefore, the invention provides a method for separating and purifying the main allergic protein Der p 1 of dust mites. The separation and purification method comprises the steps of preparing a dust mite water soluble protein crude extract, further separating and purifying by cation exchange chromatography HiTrap Capto obtain a purified main allergenic protein Der p 1 by using a pH 4.5 sodium acetate buffer solution as a binding solution (10-100 mM) and a 30% NaCl solution (100-1000 mM NaCl) as an eluent. The method has the characteristics of high speed, high efficiency, stable production, low cost and the like. The prepared extracting solution or purified protein can be used for detecting main allergic protein in a serum sample of a patient, and meanwhile, the recombinant allergen can be further prepared, and the method has wide application in the aspects of designing hypoallergenic derivatives and preventing vaccines.

Although the present invention has been described with reference to the above embodiments, it should be understood that the present invention is not limited thereto, and various changes and modifications may be made by those skilled in the art without departing from the spirit and scope of the present invention.