Protein as well as separating method thereof and application thereof

A separation method and protein technology, applied in the field of vegetable protein separation, can solve the problems of low protein content, lack of scientific quantitative judgment, limited target plants, etc., to achieve the effect of ensuring purity

Inactive Publication Date: 2017-05-24
HUAZHONG AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Although there have been related studies on plants and their extracts that can easily cause the human body to get angry in the prior art, the purity of the extracts obtained by these extraction methods is not high, and people cannot use these extracts to obtain accurate specific protein sequences; On the one hand, the plants that these extraction methods can target are relatively limited. Taking Rutaceae plants as an example, the fruit of Rutaceae plants is one of the most commonly consumed fruits in China. A large number of secondary metabolites, sugar, protease and other substances, the relative content of protein is low
These unfavorable factors will lead to low protein extraction efficiency
In addition, because different Rutaceae plants (such as tangerine, tangerine, pomelo, orange, lemon, etc.) have different effects on causing inflammation, there is currently a lack of scientific and quantitative means to judge their inflammatory effects in addition to qualitative judgments based on experience.

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  • Protein as well as separating method thereof and application thereof
  • Protein as well as separating method thereof and application thereof
  • Protein as well as separating method thereof and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Separation and purification of substances that induce "get angry" in Wenzhou satsuma

[0039] 1. Extraction of total protein in Satsuma citrus pulp

[0040] Such as figure 1 As shown, pick fresh Wenzhou satsuma citrus fruit, manually peel off the white cortex outside the pulp. The homogenizer extracts the juice from the pulp, filters it with 4 layers of gauze to remove fat-soluble parts such as juice cells and other impurities, collects the filtrate and centrifuges (5000rpm, 15min, 4°C) to remove the residual filter residue. The collected supernatant is placed in a freeze dryer, freeze-dried to obtain a solid powder, and stored in an ultra-low temperature refrigerator for a long time.

[0041] Weigh 5g powder sample, add 5mL~7mL protein extraction buffer (containing 250mM sucrose, 20mM Tris, 10mM EGTA, 1mM PMSF, 1mM DTT, 1% TritonX-100 and Tris-HCl at pH=7.5) on ice Shake well for 10 minutes, fully shake for 20 minutes to make it evenly mixed, and the whole...

Embodiment 2

[0048] Example 2 Determining whether the isolated substance triggers an inflammatory phenomenon

[0049] After dialysis to remove inorganic salts, the four separation solutions were concentrated in a freeze dryer to obtain solid powder. The powder was dissolved in serum-free DMEM medium (Hyclone) to treat the macrophage cell line RAW264.7. The cells were subcultured on a 24-well plate, and after 1 day of adherent growth, different treatments were added. These treatments were the blank control group, that is, the serum-containing medium DMEM; the positive control, that is, the 1 μg / mL LPS treatment group; and then is 10 mg / mL of C2.1, C2.2, C2.3 and C2.4. After maintaining the growth for about 18 hours, the RNA of the treated cells was extracted with Trizol reagent. The up-regulated expression of the inflammatory factor cyclooxygenase-2 was detected by real-time PCR method, and the lipopolysaccharide LPS could significantly induce the expression of COX-2. C2.1, C2.2, C2.3 an...

Embodiment 3

[0056] Example 3 Determining Protein Peptides Using Mass Spectrometry

[0057] Collect C2.3 for identification. C2.3 was first collected and tested for purity by HPLC. Parameter conditions: The mobile phase is prepared by acetonitrile solution of 0.05% trifluoroacetic acid and aqueous solution of 0.1% trifluoroacetic acid at a volume ratio of 10:90; the temperature of the column oven is 30°C; the model of the C18 column is COSMOSILPACKED COLUMN 5C18-MS SIZE 10I D×250mm; instrument: dionex Ultimate3000. The DAD detector (ie photodiode array detector) scans the full wavelength to detect the purity. Finally, after in-gel enzymatic hydrolysis of Trypsin for 20 hours, peptide extraction, and Ziptip desalting treatment, the separated substances that can cause "inflammation" were detected by MALDI-TOF / TOF mass spectrometry, and their peptides were determined.

[0058] Use MALDI-TOF / TOF to detect and identify the peptides identified in the isolated substances as follows: 1)

[005...

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Abstract

The invention discloses a protein as well as a separating method thereof and application thereof. The protein is selected from: i, a protein consisting of amino acid sequences as shown in SEQ ID No.1 or SEQ ID No.2; ii, a protein homologous with i-type protein; and iii, a derived protein of the i-type protein. The separating method for the protein comprises the following steps of: (i), extracting total proteins of rutaceae plant fruits; (2) re-extracting the total proteins; and (3) performing crude extraction on target proteins. According to the protein disclosed by the invention, proteins which can cause inflammations such as a human heat syndrome are firstly separated out; the final fine extract obtained by the separating method is high in purity, and the fine extract can be utilized to accurately obtain amino acid sequences of proteins which cause inflammations; besides, the fine extract or intermediate crude extract obtained by the separating method can be used for performing quantitative judgment about whether the rutaceae plant fruits have effects of causing inflammations or not, and therefore, the practicability is good.

Description

technical field [0001] The invention belongs to the technical field of plant protein separation, and more specifically relates to a protein and its separation method and application. The protein (namely, Citsh1 protein) is isolated and extracted from Wenzhou citrus, and is a protein capable of inducing inflammatory reactions such as "getting angry" in the human body . The present invention uses Sephadex gel and reverse chromatographic separation method to separate the total protein in Satsuma citrus pulp, and determines the transcription level of the final inflammatory factor cyclooxygenase 2 by treating the macrophage cell line RAW 264.7 with different extracts Changes, so as to screen and functionally verify the isolated product. Background technique [0002] Wenzhou citrus (C.unshiu Marcov.satsuma) is native to Wenzhou, Zhejiang, China. It has the advantages of "bright color, beautiful shape, large fruit, thin skin, seedless, and juicy". It is an excellent variety for bo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C07K1/20C07K1/16C07K1/14
CPCC07K14/415
Inventor 马兆成闫慧清王小丽邓秀新
Owner HUAZHONG AGRI UNIV
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