Protein extracted from perinereis aibuhitensis, its preparation method and use

A bidentate clamworm and protein technology, applied in the field of protein, can solve the problems that the research and development of active protein has not received due attention, and achieve the effect of meticulous and rigorous preparation method, easy material acquisition, and inhibition of proliferation

Inactive Publication Date: 2004-08-18
INST OF ZOOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the research and development of antibacterial and anticancer active proteins prepared from clam worms has not received due attention, and domestic and foreign research reports and related patents on this aspect are also rare.

Method used

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  • Protein extracted from perinereis aibuhitensis, its preparation method and use
  • Protein extracted from perinereis aibuhitensis, its preparation method and use
  • Protein extracted from perinereis aibuhitensis, its preparation method and use

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Effect test

Embodiment 1

[0034] Using the preparation method described in the present invention to extract protein from P. bidentate Nereis, the specific steps of preparation are as follows:

[0035] 1. Put the artificially cultured fresh Nereis NEREIDAE Perinereis Kinberg Perinereis aibuhitensis Grube into the artificial seawater prepared by sea crystal for 24 hours to let it spit out the contents;

[0036] 2. After washing the nereis, blot it dry quickly on filter paper, shock the body wall of the nereis with 5V DC, and collect the body fluid secreted by the nereis;

[0037] 3. Put the clam worm body fluid into an ultracentrifuge, centrifuge at 108,000×g for 30 minutes at 4°C, collect the supernatant, and filter and sterilize it with a 0.20 μm filter.

[0038] 4. Apply the sterilized supernatant to Sep-Pak C 18 After the cartridge reversed-phase column was loaded, it was washed with 0.1% (v / v) trifluoroacetic acid, then eluted with acetonitrile solution containing 80% (v / v) trifluoroacetic acid, a...

Embodiment 2

[0043] Use MTS / PMS (nitro blue tetrazolium salt / phenazine methyl sulfate) method to detect the antibacterial activity of protein of the present invention to multiple pathogenic bacteria such as Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus etc. ; The specific detection steps are as follows:

[0044] (1) After the protein prepared by the method of the present invention is resuspended with Dulbecco's PBS (pH7.2), it is determined by the Bradford method, and then diluted to 60 μg / ml with RPMI 1640 medium;

[0045] (2) Escherichia coli (CGMCC 1.1543), Pseudomonas aeruginosa (CGMCC 1.50), Staphylococcus aureus (CGMCC 1.879) and Arthrobacter sp. (CGMCC 1.8) were used as materials , add 150 μl culture medium and 50 μl bacterial suspension (titer: 10 4 cells / ml) for 24 hours;

[0046] (3) Select 48 experimental groups on the culture plate, add 30 μl active protein (concentration: 60 μg / ml) to each experimental group, and add 30 μl phosphate buffer saline PBS to the...

Embodiment 3

[0052] Detect the impact of protein of the present invention on the proliferation of human liver cancer cell line HepG2 with MTS / PMS (nitroblue tetrazolium salt / phenazine methyl sulfate), and the specific detection steps are as follows:

[0053] (1) After the protein prepared by the method of the present invention is resuspended with Dulbecco's PBS (pH7.2), it is determined by the Bradford method, and then diluted to 60 μg / ml with RPMI 1640 medium;

[0054] (2) Using human liver cancer cell line HepG2 as material, according to MTS standard curve and HepG2 cell growth curve, cancer cells were divided into 2×10 4 Inoculate cells / ml in 96-well culture plate, add 80 μl cancer cells to each well and culture for 24 hours;

[0055] (3) Select 48 experimental groups on the culture plate, add 20 μl of the protein prepared in step (1) to each experimental group, add 20 μl phosphate buffer saline PBS to the other 48 control groups, and continue culturing for 24 hours after treatment like...

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Abstract

The present invention is one kind of protein extracted from Perinereis aibuhiteris and its preparation process and use. The protein is alkali protein with molecular weight 65000 Da and isoelectric point of 8.3. The protein preparing process includes raising Perinereis aibuhiteris in artificial sea water for 24-48 hr to make it become clean, electrically striking Perinereis aibuhiteris with 5-10 V DC to obtain body fluid, centrifugally filtering body fluid, reverse concentration, gel filtering, and chromatographic elution in anionic exchange column to collect active protein. The protein is used in preparing antibiotic medicine resisting escherichia coli, verdigris pseudomonad, staphylococcus aureus and arthrobacter and anticancer medicine for suppressing human liver cancer cell strain proliferation and producing alpha-fetoglobulin, and has high antibiotic activity and obvious anticancer effect.

Description

technical field [0001] The present invention relates to a kind of protein extracted from P. didentate nereis, and the preparation method and application of the protein. Background technique [0002] Antibacterial protein (abbreviated as ABP) is a kind of defensive substance produced by the animal immune system to resist the pathogenic effect of foreign pathogens, and has many characteristics superior to the currently used antibiotics. First of all, it has a wide antibacterial spectrum, and it has a certain killing or inhibiting effect on Gram-positive and Gram-negative bacteria, fungi, and viruses; secondly, continuous use of antibacterial proteins rarely produces drug resistance, and will not Cross-resistance with other drugs; thirdly, antibacterial proteins have a wide range of sources in nature, and are easy to extract and low cost; finally, ABP has high antibacterial activity, and the lethal concentration for bacteria is in umol.L -1 It also has selective killing effect...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/02A61K38/16A61P31/04A61P35/00C07K1/14C07K1/36C07K2/00C07K14/435
CPCY02A50/30
Inventor 潘卫东戈峰
Owner INST OF ZOOLOGY CHINESE ACAD OF SCI
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