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Method of extracting phytohemagglutinin

A plant lectin and extraction solution technology, which is applied in the preparation method of peptides, plant peptides, chemical instruments and methods, etc., can solve the problems of limited amount of PHA, high cost, precipitation, etc., and achieve good growth, good effect, and purity high effect

Inactive Publication Date: 2019-09-27
河南赛诺特生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In these methods, the use of ethanol gradient precipitation or ammonium sulfate gradient precipitation will result in the prepared PHA containing a large amount of foreign proteins, which will cause serious precipitation or coagulation in the subsequent application of lymphocyte culture.
[0004] Moreover, other extraction methods existing in the prior art, such as molecular exclusion chromatography, ion chromatography, etc., cannot obtain high-purity PHA, and there will also be subsequent problems similar to ethanol gradient precipitation or ammonium sulfate gradient precipitation.
Although at present, it is also reported that relatively high-purity PHA protein can be obtained by using an affinity chromatography method (Con A-Sepharose, etc.), but the cost of this preparation method is too high, and the price of the affinity filler itself is very high , and if the affinity filler is used or not cleaned well, it will cause damage to the affinity filler, and the amount of PHA purified by the affinity filler is limited, which cannot be suitable for a large amount of PHA required by industrial production

Method used

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  • Method of extracting phytohemagglutinin
  • Method of extracting phytohemagglutinin
  • Method of extracting phytohemagglutinin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] The method for extracting lectins in this embodiment comprises the following steps:

[0028] 1) Pulverize the purchased beans into fine powder (Xinjiang red kidney beans);

[0029] 2) Soak soybean powder in 10mM PBS buffer solution with pH7.4, wherein the mass / volume ratio of soybean powder to PBS buffer solution is 1:10 (that is, 1g soybean powder is added to 10mL PBS buffer solution), and put in a refrigerator at 5°C Soak for 2 days, during which you can take it out and shake it 1-2 times a day;

[0030] 3) After soaking for 2 days, the extract was centrifuged at 10,000 rpm for 15 minutes to obtain supernatant I;

[0031] 4) With 1M (i.e. 1N, equivalent concentration) dilute hydrochloric acid, the pH value of the supernatant I was adjusted to 4.8, and a large amount of white precipitates would appear at this moment;

[0032] 5) Centrifugation: rotate at 10000rpm, centrifuge for 15min; get supernatant II;

[0033] 6) Add 1 volume of sodium chloride solution with a m...

Embodiment 2

[0037] In the method for extracting lectins in this embodiment, the beans used are Yunnan white kidney beans.

[0038] Including the following steps:

[0039] 1) Pulverize the purchased beans into fine powder;

[0040] 2) Soak soybean powder in 10mM PBS buffer solution with pH7.4, wherein the mass / volume ratio of soybean powder to PBS buffer solution is 1:10 (that is, 1g soybean powder is added to 10mL PBS buffer solution), and put in a refrigerator at 6°C Soak for 2 days, during which you can take it out and shake it 1-2 times a day;

[0041] 3) After soaking for 2 days, the extract was centrifuged at 10,000 rpm for 15 minutes to obtain supernatant I;

[0042] 4) With 1M (i.e. 1N, normal concentration) dilute hydrochloric acid, the pH value of the supernatant I is adjusted to 4.8, and a large amount of white precipitates will appear at this moment;

[0043] 5) Centrifugation: rotate at 10000rpm, centrifuge for 15min; get supernatant II;

[0044] 6) Add 1 volume of sodium ...

Embodiment 3

[0048] In the method for extracting lectins in this embodiment, the beans used are Shandong red kidney beans.

[0049] Including the following steps:

[0050] 1) Pulverize the purchased beans into fine powder;

[0051] 2) Soak soybean powder in 10mM PBS buffer solution with pH7.4, wherein the mass / volume ratio of soybean powder to PBS buffer solution is 1:10 (that is, 1g soybean powder is added to 10mL PBS buffer solution), and put in a refrigerator at 5°C Soak for 2 days, during which you can take it out and shake it 1-2 times a day;

[0052] 3) After soaking for 2 days, the extract was centrifuged at 10,000 rpm for 15 minutes to obtain supernatant I;

[0053] 4) With 1M (i.e. 1N, equivalent concentration) dilute hydrochloric acid, the pH value of the supernatant I was adjusted to 4.8, and a large amount of white precipitates would appear at this moment;

[0054] 5) Centrifugation: rotate at 10000rpm, centrifuge for 15min; get supernatant II;

[0055] 6) Add 1 volume of s...

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Abstract

The invention relates to a method of extracting phytohemagglutinin and belongs to the technical field of phytohemagglutinin extraction. The phytohemagglutinin can be extracted from beans by soaking in a buffer solution, acid conditioning to settle protein and adding 0.1-0.4% of sodium chloride solution to settle the protein; the extraction method is simple and convenient; and a reagent used in an extraction process is cheap, easy to obtain and harmless to a human body. Extracted PHA (phytohemagglutinin) protein is high in purity and good in activity; and when the extracted PHA protein is applied to a lymphocyte culture medium, blood coagulation or hemolysis can be avoided, and a good effect of stimulating lymphocyte growth is achieved. The extraction method is suitable for large-scale production of the PHA protein.

Description

technical field [0001] The invention relates to a method for extracting plant lectin, belonging to the technical field of plant lectin extraction. Background technique [0002] Phytohemagglutinin (Phytohemagglutinin PHA) is a sugar-containing protein extracted from the seeds of legumes. PHA is further divided into E-type and L-type, among which PHA-E type has erythrocyte agglutination; PHA-L type can stimulate lymphocyte transformation and is a stimulator of human lymphocyte mitosis, that is, under the action of PHA-L, it was originally in G0 Lymphocytes transform into lymphoblasts and undergo mitosis. [0003] Currently, various methods for extracting lectins from leguminous plants have been disclosed. For example, the Chinese invention patent application with the publication number CN101508730 A discloses a method for extracting bean lectin, specifically: using a 40-60°C water extraction method and assisting the alternate application of ultrasound and microwaves to extra...

Claims

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Application Information

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IPC IPC(8): C07K14/42C07K1/30
CPCC07K14/42
Inventor 梁永波陈玲李莉刘玲玲齐华马锦华耿伟杨晓华
Owner 河南赛诺特生物技术有限公司
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