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A kind of camel velvet transfer protein and its preparation method and application

A technology of transferring protein and camel canopy, applied in the direction of peptide preparation method, botanical equipment and method, application, etc., to achieve the effect of inhibiting HIV-1 reverse transcriptase activity

Inactive Publication Date: 2011-12-21
XINJIANG UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The present invention isolates and purifies a lipid transfer protein PHP with a molecular weight of 16kDa from the seeds of Camelina for the first time, which has antifungal and antitumor activities and the effect of inhibiting HIV-1 reverse transcriptase activity, which has not been reported in the existing literature

Method used

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  • A kind of camel velvet transfer protein and its preparation method and application
  • A kind of camel velvet transfer protein and its preparation method and application
  • A kind of camel velvet transfer protein and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1: Preparation of camel velvet transfer protein PHP

[0036] (1) Crude product preparation: use camel cape seeds as materials, crush them into powder, add 0.02mol / L PBS phosphate buffer solution with pH 7.2, stir and extract overnight at 4°C, collect supernatant after low-temperature centrifugation, and use Slowly add ammonium sulfate to the supernatant to adjust the saturation to 50%, let it stand at 4°C for 4 hours, collect the supernatant by centrifugation, and discard the precipitate. Continue to slowly add ammonium sulfate to the supernatant to adjust the saturation to 70%. After standing at 4°C for 4 hours, the precipitate is collected by centrifugation, dissolved in a small amount of phosphate buffer, dialyzed to remove salt, and freeze-dried to obtain crude protein;

[0037] (2) Separation and purification by cation exchange chromatography: Use Resource S cation exchange column to perform chromatography on the crude protein obtained in the first step,...

Embodiment 2

[0040] Example 2: Research on some physicochemical properties of camel velvet transfer protein

[0041] (1) Purity and molecular weight detection

[0042] The PHP protein obtained in Example 1 is measured by Tricine-SDS-PAGE electrophoresis (gel concentration 12%), and the experimental results are as attached figure 1 As shown in panel A, the molecular weight of the subunit of the PHP protein is about 8 kDa. Adopting model is that C18ZORBAX ODS reversed-phase liquid chromatography column (Agilent) carries out high performance liquid chromatography (HPLC) identification protein PHP purity, and sample purity result shows that PHP sample purity is higher than 95% (attachment figure 1 Middle B panel). The molecular weight of PHP protein in camel camel was detected by high-performance liquid chromatography (HPLC) with model TSK-G4000 PWxl. The column temperature was 25°C, the detection wavelength was 280nm, the mobile phase was 0.05mol / LPBS (pH7.2), the solvent was 0.05mol / L PBS...

Embodiment 3

[0051] Example 3: Inhibitory effect of camel velvet transfer protein PHP on fungi

[0052] Inoculate five plant pathogenic fungi that can cause fruit rot and grain mildew, such as Rhizopus stuolonifer, Penicillium italicum, Alternaria alternata, Magnaporthe grisea and Penicillium degitatum On potato culture medium, cultivate for 48 hours at 28°C for activation to obtain activated new strains. Take 1 mL of sterile distilled water to wash the surface of the culture medium to make the fungal spores fall off and make a bacterial suspension. Preparation of bacteria-containing plates: Pour 20 mL of medium that has been autoclaved and cooled to 50°C-60°C into a plate with a diameter of 10 cm. After cooling, absorb 200 μL of bacterial suspension and drop it onto the center of the plate to make a plate containing bacteria. . When the diameter of the round surface of the mycelia spreading outward is about 2-3cm, place a sterilized filter paper sheet with a diameter of 6mm at a distanc...

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Abstract

The invention discloses a peganum harmala lipid transfer protein (PHP) and relates to the preparation and use of new protein with antifungal, anticancer and antivirus activities. The method uses seeds of a peganum harmala plant as raw materials and comprises the following steps: preparing a coarse product, performing cationic exchange chromatographic separation and purifying by using a molecular sieve; and gradually separating and collecting a purified component having antibacterial activity, dialyzing, desalting, freeze-drying and thus, obtaining purified PHP. In the invention, a filter paper disc agar diffusion method is adopted, and researches prove the protein has growth inhibiting effect on five plant pathogenic fungi, namely alternaria alternate, penicillium degitatum, rhizopus stuolonifer, magnaporthe grisea and penicillium italicum. An 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method proves that the protein has inhibiting effect on proliferation of human esophageal carcinoma cell Eca109, human cervical cancer cells HeLa, human stomach cancer cells MGC-7, mouse melanoma cells B16 and the like. The protein can inhibit the activity of human immunodeficiency virus 1 (HIV-1) reverse transcriptase. The protein can be used for developing medicines for resisting agricultural pathogenic fungi, tumors and viruses.

Description

Technical field: [0001] The invention belongs to the field of biotechnology, and relates to a lipid transfer protein PHP with a molecular weight of 16 kDa isolated and purified from camel camel seeds. Transcriptase activity camel bales transfer protein, and a method for preparing the protein by using camel bales seeds as a raw material, performing crude product preparation, cation exchange chromatography separation and molecular sieve purification, and the application of the protein. Background technique: [0002] Plant growth is often attacked by various fungi. During the long-term evolution process, plants have formed a set of natural defense systems to fight against foreign invaders. Plant antifungal protein (antifungal protein, AFP) is one of them, they can inhibit the growth of fungi or kill fungi. According to the mechanism of action, structure and sequence characteristics of plant antifungal proteins, they can be roughly divided into the following categories: pathoge...

Claims

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Application Information

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IPC IPC(8): C07K14/415C07K1/36C07K1/30C07K1/18C07K1/16A23L3/3472A01N43/36A01P3/00A61K38/16A61P35/00A61P31/18
Inventor 孙素荣马晓瑾刘东亮吕慧李阳张富春
Owner XINJIANG UNIVERSITY
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