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Amaranth seed ribosome inactivating protein

A ribosome inactivation and amaranth seed technology is applied to the ribosome inactivation protein-amaranth seed protein and the fields of its preparation and application, and achieves the effects of vigorous growth and high yield

Inactive Publication Date: 2004-04-28
FUJIAN INST OF RES ON THE STRUCTURE OF MATTER CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Other ribosome inactivating proteins in this genus have not been reported

Method used

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  • Amaranth seed ribosome inactivating protein
  • Amaranth seed ribosome inactivating protein
  • Amaranth seed ribosome inactivating protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Embodiment 1. Isolation and purification of amaranth seed protein (Amaramangin) from amaranth seed

[0017] Take 200g of amaranth seeds, add appropriate amount of 20mMol / L acetic acid buffer (pH4.5) to homogenate, centrifuge or filter to collect the extract, adjust the pH to 4.0 with 50% acetic acid, let stand to separate layers, centrifuge or filter to collect the supernatant , put the supernatant on the SP-agarose fast flow gel ion exchange column, wash the column with 20mMol / L pH4.5 acetate buffer, then wash away the impurity proteins with 20mMol / L pH6.0 phosphate buffer, and finally wash with 0~1M NaCl (in 20 mM pH 6.0 phosphate buffer) gradient elution. See Figure 1 for the elution profile.

[0018] Combine 9-15 tubes of collected liquid, adjust the pH to 4.0 with 50% acetic acid, add it to the SP-agarose fast-flow ion exchange column again, wash the column with 20mMol / L pH4.5 acetate buffer, and then wash the column with 10mMol / LpH7 .2 phosphate buffer to wash a...

Embodiment 2

[0019] Embodiment 2.SDS-polyacrylamide gel electrophoresis (SDS-PAGE)

[0020] Ribosome inactivating protein-maranth seed protein, carried out SDS-PAGE on the micro vertical plate electrophoresis instrument of Bio-Rad company, and SDS-PAGE is carried out by the method for Leammli, and separating gel concentration is 12%, and stacking gel concentration is 4%, After electrophoresis, according to Guo Yaojun's method (1991, Advances in Biochemistry and Biophysics, 18: 32-37), stain with Coomassie Brilliant Blue, and use trichosanthin (MW ~ 27kDa) and lysozyme (MW ~ 14kDa) as molecular weight standards to control, showing The molecular weight of amaranth seed protein is 29kDa. The SDS-PAGE results are shown in Figure 3.

Embodiment 3

[0021] Example 3. Alcian blue staining after SDS-PAGE

[0022] The amaranth seed protein was subjected to SDS-PAGE according to the method in Example 2, and the film after electrophoresis was stained with Alcian blue (Sun Chu, "Lectin", p21). The gel after SDS-PAGE was fixed in 12.5% ​​trichloroacetic acid for 30 minutes, then oxidized in a periodic acid solution containing 7% acetic acid for one hour, and then placed in 0.5% sodium metabisulfite solution and fully shaken for 30 minutes. Finally stained with 0.5% Alcian blue (dissolved in 7% acetic acid) for 4 hours. The staining results showed that the amaranth protein band was blue-green, which proved that the amaranth protein was a glycoprotein.

[0023] The gel after glycoprotein staining was re-stained with Coomassie Brilliant Blue according to the method in Example 2, and it was observed that the position of the original staining band was the band of amaranth seed protein.

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Abstract

The present invention relates to amaranth seed ribosome inactivating protein, which is separated form the seed of Amaranthus mangostanus as one kind of vegetable, has molecular weight of 29 KDa and isoelectric point greater than 9. By means of mild extraction method, and through twice fast SP-agarose gel ion exchange chromatography, high purity of protein is obtained. The protein may be used as medicine material applied in preparing antitumor preparation, antiviral preparation and anti-HIV preparation, and may used as RNAN-glycoside for molecular biological and immulogical research and as toxin material for immunotoxin.

Description

technical field [0001] The invention relates to the field of organic chemistry, in particular to a kind of ribosome inactivation protein-maranth seed protein in biochemistry and its preparation and application. Background technique [0002] In this context, a ribosome-inactivating protein can be defined as a protein having at least one of the following activities: RNA N-glycosidase activity, polynucleotide: adenosylglucosidase activity, antitumor activity, antiviral activity, especially anti- HIV activity, inducer protein activity. The protein is an oligomeric, single-peptide chain simple protein or glycoprotein. [0003] Ribosome-inactivating proteins extracted from higher plants can be divided into two types according to their molecular structure. Type I is a single-peptide chain protein, for example, trichosanthin from roots and seeds of Cucurbitaceae plants, bitter melon seed protein (Momorcharin) and pokeweed antiviral protein (PAP) from p...

Claims

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Application Information

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IPC IPC(8): A61K38/16A61P31/12A61P31/18A61P35/00C07K1/14C07K14/415
Inventor 陈明晃王艳芹王梓壬石小莉叶晓明
Owner FUJIAN INST OF RES ON THE STRUCTURE OF MATTER CHINESE ACAD OF SCI
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