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Fast united purification method of thyroglobulin and red kidney bean vegetable hemagglutinin

A technology of thyroglobulin and hemagglutination, applied in the field of bioengineering, can solve the problems of inability to extract plant hemagglutinin, unfavorable large-scale production, high production cost, etc., and achieve the effect of high economic value of the product, low production cost and low cost

Inactive Publication Date: 2012-05-02
CHENGDU MEDICAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Separation, Purification and Characteristic Research of Red Kidney Bean Hemagglutinin Monomer, Journal of Chengdu Medical College, June 2008, which discloses the extraction method of hemagglutinin monomer. The specific method is: prepare the initial plant lectin extract, and then Phytohemagglutinin monomers were prepared by GM-Sepharose chromatography, DEAE-Sepharose chromatography, and Sephadex G 100 chromatography; however, these methods cannot directly extract phytohemagglutinin from raw materials, and because multi-step chromatography takes a long time, High production cost, not conducive to mass production

Method used

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Examples

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Comparison scheme
Effect test

Embodiment 1

[0019] Example 1 The separation and purification method of red kidney bean plant hemagglutinin and thyroglobulin of the present invention

[0020] 1. Preparation of phytohemagglutinin crude extract: Crush red kidney beans, weigh 50g of red kidney bean powder, soak in 500ml of acetic acid-sodium acetate buffer solution (pH 4.0, 35mmol / L, 0.14mol / L NaCl), soak overnight, and use Filter through four layers of gauze, collect the filtrate, centrifuge at 10,000rpm for 15min at 4°C, take the supernatant, add ammonium sulfate solid powder to the supernatant to make the final concentration 26% (mass volume ratio, m / v), in Centrifuge at 10,000 rpm for 15 minutes at 4°C, and take the supernatant to obtain the crude phytohemagglutinin extract.

[0021] 2. Crude extraction of thyroglobulin Take 50g of thyroid gland and remove connective tissue, add 450ml of acetic acid-sodium acetate buffer solution (pH 4.0, 35mmol / L, 0.14mol / L NaCl) to pulverize, soak overnight, filter with four layers of...

Embodiment 2

[0030] Example 2 The separation and purification method of red kidney bean plant hemagglutinin and thyroglobulin of the present invention

[0031] 1. Preparation of phytohemagglutinin crude extract Weigh 100g of red kidney bean powder, soak in 1000ml of acetic acid-sodium acetate buffer solution (pH 4.5, 30mmol / L, 0.14mol / L NaCl), and then filter with four layers of gauze. Collect the filtrate, centrifuge at 10,000rpm at 4°C for 15min, take the supernatant, add ammonium sulfate solid powder to the supernatant to make the final concentration 30% (mass volume ratio, m / v), and centrifuge at 10,000rpm at 4°C Centrifuge for 15 minutes, and take the supernatant to obtain the crude phytohemagglutinin extract.

[0032] 2. Crude extraction of thyroglobulin Take 100g of thyroid connective tissue and add 800ml of acetic acid-sodium acetate buffer solution (pH 4.5, 30mmol / L, 0.14mol / L NaCl) to pulverize, soak overnight, filter with four layers of gauze, and collect the filtrate , centrif...

Embodiment 3

[0036] Example 3 The separation and purification method of red kidney bean plant hemagglutinin and thyroglobulin of the present invention

[0037] 1. Preparation of plant hemagglutinin crude extract Weigh 150g of red kidney bean powder, soak in 1200ml of acetic acid-sodium acetate buffer solution (pH5, 25mmol / L, 0.14mol / L NaCl), soak overnight, filter with four layers of gauze, and collect The filtrate was centrifuged at 10,000rpm at 4°C for 15min, the supernatant was taken, and ammonium sulfate solid powder was added to the supernatant to make the final concentration 25% (mass volume ratio, m / v), centrifuged at 10,000rpm at 4°C After 15 minutes, the supernatant was taken to obtain the crude phytohemagglutinin extract.

[0038] 2. Crude extraction of thyroglobulin Take 150g of thyroid gland and remove connective tissue, add 1000ml of acetic acid-sodium acetate buffer (pH5, 25mmol / L, 0.14mol / L NaCl) to pulverize, soak overnight, filter with four layers of gauze, and collect the...

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Abstract

The invention provides a method for separating and purifying red kidney bean vegetable hemagglutinin and thyroglobulin. The method mixes crude extract of red kidney bean vegetable hemagglutinin in redkidney bean with crude extract of thyroglobulin; the mixture is then extracted by coprecipitation method, purified and separated to respectively obtain hemagglutinin monomer and thyroglobulin. The method directly extracts vegetable agglutinin from red kidney bean and is applicable to obtaining of vegetable hemagglutinin without extraction of vegetable hemagglutinin after extracting of vegetable agglutinin, thereby simplifying purification process. Material used in the method is low in cost and wide in sources, and the raw materials are easy to obtain. The method has low production cost, simple operation and high yield which are applicable to industrial mass production. The vegetable hemagglutinin extracted by the method has not only high activity and high yield, but also low cost and short cycle. The method obtains hemagglutinin monomer together with thyroglobulin that the method has high product economic value, excellent generalizability, and relatively high economical and utility value.

Description

technical field [0001] The invention relates to a new technology for extracting plant hemagglutinin monomer from red kidney beans by co-precipitation method and simultaneously purifying thyroglobulin, which belongs to the technical field of bioengineering. Background technique [0002] Thyroglobulin is a glycoprotein found primarily in mammalian thyroid tissue. Because it can reversibly bind to lectins or glycoproteins, thyroglobulin is often used as a ligand in the field of biotechnology coupled to Sepharose 4B and other carriers for affinity chromatography separation and purification of glycoproteins. Thyroglobulin has a huge molecular weight (660kD), and the current literature reports are mainly purified by molecular sieve methods. Such as: Nubuo U etc. (Nubuo U.and Osamu T., J Biochem.1961,50 (6): 508-518) and Zeng Zhongkui Wu Qiaqing etc. (Zeng Zhongkui Wu Qiaqing, Sichuan University Journal (Natural Science Edition) 1993) use Ammonium sulfate and molecular sieves wer...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/42C07K14/47C07K1/14
Inventor 张涛何涛杨雨晗刘桦
Owner CHENGDU MEDICAL COLLEGE
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