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Enrichment method of low abundance protein in bee venom

A protein, low abundance technology, applied in the field of protein processing, can solve the problems of protein loss, poor method reproducibility, cumbersome separation process, etc., and achieve high specificity, simple operation, and good repeatability.

Inactive Publication Date: 2013-07-17
BEE RES INST CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The biggest disadvantage of these two methods as traditional protein separation and purification methods is that the separation process is cumbersome, and many proteins are lost during the extraction and purification process, resulting in protein loss, so that some proteins with specific biological functions have not been discovered and studied; in addition , the cumbersome test process leads to poor reproducibility of the method, and the method established in the laboratory is difficult to apply to actual production
These are technical difficulties that are difficult to overcome by traditional separation and enrichment methods

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0032] Example 1 Enrichment of low-abundance proteins in the venom of Apis mellifera

[0033] 1. Soak 500 mg of hexapeptide ligand microbeads bonded to porous polyhydroxymethacrylate particles and 2.0 mL of pure water in a syringe for 5 min, then mix on a shaker for 1 min, centrifuge for 2 min, discard the effluent, and repeat the above Operate 1 time.

[0034] 2. Add 2.0mL PBS buffer solution and place it in the above syringe, shake for 1 min, centrifuge for 2 min, discard the effluent, and repeat the above operation once.

[0035] 3. Accurately weigh 1.0g of the bee venom sample of Apis mellifera and place it in a 2.5mL centrifuge tube, add 1.0mL of pure water, vortex to dissolve it completely, then centrifuge at 4°C and 10000rpm for 5min at high speed, and take the supernatant .

[0036]4. Soak the sample supernatant in the above-mentioned syringe containing the hexapeptide ligand microbeads and mix for 30 minutes, mix in the shaker for 1 minute every 5 minutes, make the ...

Embodiment 2

[0046] 1. Soak 300 mg of hexapeptide ligand microbeads bonded to porous polyhydroxymethacrylate particles and 1.0 mL of pure water in a syringe for 5 minutes, then mix on a shaker for 1 minute, centrifuge for 2 minutes, discard the effluent, and repeat the above Operate 1 time.

[0047] 2. Add 1.0mL PBS buffer solution and place it in the above syringe, shake for 1 min, centrifuge for 2 min, discard the effluent, and repeat the above operation once.

[0048] 3. Accurately weigh 1.0g of the bee venom sample of Apis mellifera and place it in a 2.5mL centrifuge tube, add 1.0mL of pure water, vortex to dissolve it completely, then centrifuge at 4°C and 10,000rpm for 5min at high speed, and take the supernatant .

[0049] 4. Soak the sample supernatant in the above-mentioned syringe containing the hexapeptide ligand microbeads and mix for 25 minutes, mix in the shaker for 1 minute every 5 minutes, make the sample supernatant fully contact with the microbeads, centrifuge for 2 minu...

Embodiment 3

[0055] 1. Soak 500 mg of hexapeptide ligand microbeads bonded to porous polyhydroxymethacrylate particles and 2.5 mL of pure water in a syringe for 5 minutes, then mix on a shaker for 3 minutes, centrifuge for 5 minutes, discard the effluent, and repeat the above Operate 1 time.

[0056] 2. Add 2.0mL PBS buffer solution and place it in the above syringe, shake for 3 minutes, centrifuge for 5 minutes, discard the effluent, and repeat the above operation once.

[0057] 3. Accurately weigh 1.0g of the bee venom sample of Apis mellifera and place it in a 2.5mL centrifuge tube, add 2.0mL of pure water, vortex to dissolve it completely, then centrifuge at 4°C and 10000rpm for 5min at high speed, and take the supernatant .

[0058] 4. Soak the sample supernatant in the above-mentioned syringe containing the hexapeptide ligand microbeads and mix for 40 minutes, mix in the shaker for 1 minute every 5 minutes, make the sample supernatant fully contact with the microbeads, centrifuge fo...

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Abstract

The invention relates to an enrichment method of low abundance protein in bee venom. The method includes: using hexapeptide ligand microspheres bonded with porous poly(2-hydroxyethyl methacrylate) granules as absorbent base, mixing the absorbent base with bee venom to absorb the protein in the bee venom, washing with PBS (phosphate buffered saline) buffer solution to remove excess high-abundance protein, and eluting the protein absorbed to the absorbent base with eluent to obtain the eluent with the enriched low-abundance protein. The enrichment method of low abundance protein is efficient, stable, well repeatable and highly specific and has great significance to enhancing the study on low-abundance protein, the search on information of more allergens in bee venom, the development of traditional Chinese medicines, the study on disease diagnosis markers and occurrence mechanisms of disease, and the like.

Description

technical field [0001] The invention relates to the field of protein treatment, in particular to a method for enriching low-abundance proteins in bee venom. Background technique [0002] The main bee species raised in our country is the Italian honeybee with strong production capacity, which has strong disease resistance and cold resistance. Bee venom is a liquid secreted by the venom glands and accessory glands of worker bees and stored in the poison sac. Italian bee venom has a variety of pharmacological and biological activities, mainly including anti-inflammatory, antihypertensive, analgesic, antiviral, etc., especially for the treatment of rheumatism, rheumatoid arthritis, frozen shoulder, and cardiovascular diseases , tumors and multiple sclerosis and other diseases have a good therapeutic effect (Jilin Science and Technology Press, 2000, 1-121). In order to fully understand the relevant information of all low-abundance proteins in bee venom, all proteins should be e...

Claims

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Application Information

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IPC IPC(8): C07K1/22
Inventor 周金慧李熠吴黎明赵静薛晓锋
Owner BEE RES INST CHINESE ACAD OF AGRI SCI
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