CN1521188A - Protein extracted from perinereis aibuhitensis, its preparation method and use - Google Patents
Protein extracted from perinereis aibuhitensis, its preparation method and use Download PDFInfo
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- CN1521188A CN1521188A CNA03102033XA CN03102033A CN1521188A CN 1521188 A CN1521188 A CN 1521188A CN A03102033X A CNA03102033X A CN A03102033XA CN 03102033 A CN03102033 A CN 03102033A CN 1521188 A CN1521188 A CN 1521188A
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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
The present invention relates to a kind of protein that from perinereis aibuhitensis, extracts, and this proteinic preparation method and purposes.
Background technology
Antibacterial protein (antibacterial protein abbreviates ABP as) is the defensive material of the class opposing exotic disease substance pathogenic effects of animal immune system generation, has many antibiotic characteristics of use at present that are better than.At first, its antimicrobial spectrum is extensive, and Gram-positive, Gram-negative bacteria, fungi and virus etc. are all had certain killing or restraining effect; Secondly, use antibacterial protein seldom to produce resistance continuously, also can not produce crossing drug resistant with other drug; Once more, antibacterial protein is at the occurring in nature wide material sources, and easily extract, cost is low; At last, the anti-microbial activity of ABP is very high, to the lethal concentration of bacterium all at umol.L
-1Level is to the also selective lethal effect of tumour cell.
Marine organisms are because kind diversity and its natural pharmaceutical use of areal distribution popularity more and more come into one's own, and " drugs from the sea " now become the hot topic of China's marine industries research tackling key problem.Clam worm is a very important section in the Annelita Polychaeta; its ecological distribution all has distribution for 5023 meters from top, tideland to bathymetric fascia; studies show that; except clam worm antithrombotic plasmin-Nereid kinase of comparatively paying close attention to; also contain various active and medicinal ingredientss such as β-guanidine radicals isopropylformic acid, hematin, phosphotaurocyamine, arenicochrome in the clam worm body, its application and development prospect is very extensive.But, be that the research and development of the antibiotic and anticancer active protein of feedstock production do not obtain due attention with the clam worm, both at home and abroad about research report is also rarely seen with relevant patented technology in this respect.
Summary of the invention
The objective of the invention is to: filling up with the clam worm is the research and development blank of the antibiotic and anticancer active protein of feedstock production, thereby a kind of protein that extracts from perinereis aibuhitensis and its production and use is provided.
The object of the present invention is achieved like this: a kind of protein that from perinereis aibuhitensis, extracts of the present invention, it is characterized in that this protein is a basic protein, and its molecular weight is 65,000Da, iso-electric point is 8.3, as shown in Figure 1.
A kind of proteinic preparation method who extracts from perinereis aibuhitensis of the present invention is characterized in that this method comprises following steps:
(1) the fresh perinereis aibuhitensis that will propagate artificially was put into artificial seawater foster 24-48 hour, made it tell clean inclusion;
(2) after the perinereis aibuhitensis that will tell clean inclusion is cleaned, be placed on the filter paper and blot fast,, and collect perinereis aibuhitensis excretory body fluid again with 5-10V direct current electric shock perinereis aibuhitensis body wall;
(3) body fluid with the perinereis aibuhitensis collected places ultracentrifuge centrifugal, collects supernatant liquor, and filtration sterilization;
(4) supernatant liquor after the filtration sterilization is carried out anti-phase concentrating, collect concentrated solution;
(5) concentrated solution is carried out chromatography with gel-filtration column, collect the active ingredient that obtains through chromatography;
(6) active ingredient of collecting is carried out gradient elution after, obtain activated protein, again this activated protein is carried out desalination, concentrates and freeze-drying is handled.
Centrifugal in the described step (3) is under 4-10 ℃ condition, clam worm body fluid put in the ultracentrifuge with 108 000-120, the centrifugal 20-30 of the rotating speed of 000 * g minute.
Anti-phase concentrate in the described step (4), be with supernatant liquor to sample on the reversed-phase column after with the trifluoroacetic acid flushing, concentrate component with the acetonitrile solution wash-out that contains trifluoroacetic acid again.
Gel filtration chromatography in the described step (5) is after concentrated solution is suspended again, last gel-filtration column wash-out.
Gradient elution in the described step (6) is that anion-exchange column on the active ingredient of gel permeation chromatography preparation is carried out gradient elution.
A kind of proteinic application of extracting from perinereis aibuhitensis of the present invention is characterized in that, this is applied as the application in preparing antibiotic or cancer therapy drug.
Describedly be meant in preparation that in the application of preparation in the antibacterials colon bacillus, Pseudomonas aeruginosa, streptococcus aureus or Arthrobacter are had application in the inhibiting antibacterials.
Described application in the preparation cancer therapy drug is meant the application in the cancer therapy drug of preparation inhibition human hepatoma cell strain propagation.
Described application in the preparation cancer therapy drug is meant the application for preparing in the cancer therapy drug that suppresses human hepatoma cell strain generation alpha-fetoprotein.
The invention has the advantages that: a kind of protein that extracts from perinereis aibuhitensis of the present invention and its production and use is raw material with the perinereis aibuhitensis of propagating artificially, and with the method acquisition perinereis aibuhitensis body fluid of electric shock, it is easy, easy and simple to handle to draw materials; The preparation method is careful rigorous, and repeatability is high; After testing, prepared protein anti-microbial activity height, and can suppress human hepatoma cell strain HepG2 propagation, can suppress the secretory volume of human hepatoma cell strain HepG2 characteristic protein AFP (being alpha-fetoprotein).
Purpose of the present invention, feature and advantage will be illustrated in conjunction with the accompanying drawings by preferred embodiment.
The drawing explanation
Fig. 1 is the proteinic SDS-PAGE electrophoretogram that the present invention extracts from perinereis aibuhitensis
Fig. 2 is to use the OD value comparison diagram of the protein handler hepatoma cell strain HepG2 that the present invention makes
Fig. 3 is to use the MTS canonical plotting of the OD value of the protein handler hepatoma cell strain HepG2 that the present invention makes
Fig. 4 is the effect curves figure of the protein that extracts from perinereis aibuhitensis of the present invention of different concns to cancer cells
Fig. 5 is to use the AFP value comparison diagram of the protein handler hepatoma cell strain HepG2 that the present invention makes
Fig. 6 is to use the canonical plotting of the AFP value of the protein handler hepatoma cell strain HepG2 that the present invention makes
Fig. 7 is that the human hepatoma cell strain HepG2 of normal growth differs sem observation figure
Fig. 8 is that effect differs sem observation figure to the protein that extracts from perinereis aibuhitensis of the present invention of lower concentration to human hepatoma cell strain HepG2
Fig. 9 is that effect differs sem observation figure to the protein that extracts from perinereis aibuhitensis of the present invention of high density to human hepatoma cell strain HepG2
Embodiment
Embodiment 1
Extract protein with preparation method of the present invention from perinereis aibuhitensis, the concrete steps of preparation are as follows:
1, the fresh Nereidae NEREIDAE that will propagate artificially encloses Nereis PerinereisKinberg perinereis aibuhitensis Perinereis aibuhitensis Grube, puts into the artificial seawater of sea crystal preparation and supports 24 hours, makes it tell clean inclusion;
2, clam worm is cleaned after, on filter paper, blot fast, with 5V direct current shock clam worm body wall, collect clam worm excretory body fluid;
3, clam worm body fluid is put in the ultracentrifuge, in the time of 4 ℃, with 108,000 * g centrifugal 30 minutes, collected supernatant liquor, with 0.20 μ m filter filtration sterilization.
4, with the supernatant liquor after the degerming to Sep-Pak C
18On the cartridge reversed-phase column behind the sample, with the trifluoroacetic acid flushing of 0.1% (v/v), again with contain trifluoroacetic acid 80% (v/v) the acetonitrile solution wash-out, collect elution fraction.
5, above-mentioned elution fraction is suspended again with Dulbecco ' s PBS (pH7.2) after, last gel-filtration column Sephadex G-75 (1.6 * 90cm is with Dulbecco ' s PBS (pH7.2) balance) chromatography is collected the chromatography component.
6, above-mentioned chromatography component is carried out gradient elution 0-1.0M NaCl with anion-exchange column DEAE-52 (2.5 * 30cm is with 0.02M Tris-HCl (pH 8.0) balance), collect activated protein, desalination, concentrated back freeze-drying.
The protein that is extracted from perinereis aibuhitensis is done the SDS-PAGE electrophoretogram, and the result as shown in Figure 1.
Detect the anti-microbial activity of protein of the present invention with MTS/PMS (nitrogen blue tetrazole salt/phenazine methosulfate) method to various pathogens such as large intestine dust west Salmonella, Pseudomonas aeruginosa, streptococcus aureus etc.; Concrete detection step is as follows:
(1) will suspend again with Dulbecco ' s PBS (pH7.2) by the protein of the inventive method preparation after, with Bradford method mensuration, be diluted to 60 μ g/ml with RPMI 1640 substratum again;
(2) be material with colon bacillus Escherichia coli (CGMCC 1.1543), Pseudomonas aeruginosa Pseudomonas aeruginosa (CGMCC 1.50), streptococcus aureus Staphylococcus aureus (CGMCC 1.879) and Arthrobacter Arthrobacter sp. (CGMCC 1.8), every hole adds 150 μ l substratum on the flat enzyme plate in 96 holes, (titre is 10 to 50 μ l bacterial suspensions
4Cells/ml) cultivated 24 hours;
(3) in selected 48 experimental group of culture plate, each experimental group respectively adds 30 μ l activated proteins (concentration is 60 μ g/ml), and other 48 control groups add 30 μ l phosphate buffered saline buffer PBS, after handling like this, puts 96 orifice plates and continues incubation 24 hours in 37 ℃;
(4) in each hole, add MTS/PMS solution 0.02ml again, send culture plate back to CO
2Incubator is cultivated after 1 hour and is taken out, and slight shaking culture plate 1 minute is measured the OD value with microplate reader with the 490nm wavelength, and detected result is as shown in table 1:
Table 1
| Bacterial strain | The relative OD value of antibacterial protein (%) | The antibacterial protein vigor (%) that compares | Student-T checks (P value) | Antibacterial protein minimum inhibitory concentration (μ g/ml) |
| Streptococcus aureus | 14.22±2.12 | ?302.30±45.23 | ?0.001 | ??16 |
| Arthrobacter | 26.41±4.40 | ?84.47±14.07 | ?0.016 | ??25 |
| Pseudomonas aeruginosa | 32.17±7.03 | ?185.66±29.01 | ?0.032 | ??50 |
| Colon bacillus | 19.83±2.75 | ?55.86±8.73 | ?0.045 | ??18 |
As shown in Table 1, the prepared protein of the present invention has anti-microbial activity to various pathogens, its to minimum inhibitory concentration of each pathogenic bacterium all≤50 μ g/ml, particularly to causing that clinically some cause death or the inhibiting rate of the arch-criminal-Pseudomonas aeruginosa of chronic disease such as microbemia, urinary tract infection (UTI) and chronic pneumonia reaches 68.83%, and the bacteriostasis rate of traditional anti-pseudomonas medicine quinlone class only has 63.2-67.0%, has shown good prospects for application.
Employed JEG-3 is in following examples: human hepatoma cell strain HepG2 (ATCCHB8065).This human hepatoma cell strain be with MEM nutrient solution (containing penicillin 100,000 units, Streptomycin sulphate 0.1g and foetal calf serum 100ml in the 1000ml nutrient solution) under 37 ℃, the CO with 5%
2Constant temperature culture was changed a nutrient solution in per 2 days or 3 days, detected used human hepatoma cell strain in the preparation cost inventive embodiments.
Embodiment 3
Detect the influence of protein of the present invention to human hepatoma cell strain HepG2 propagation with MTS/PMS (nitrogen blue tetrazole salt/phenazine methosulfate) method, it is as follows specifically to detect step:
(1) will suspend again with Dulbecco ' s PBS (pH7.2) by the protein of the inventive method preparation after, with Bradford method mensuration, be diluted to 60 μ g/ml with RPMI 1640 substratum again;
(2) be material with human hepatoma cell strain HepG2,, cancer cells pressed 2 * 10 according to MTS typical curve and HepG2 cell growth curve
4Individual/ml is inoculated in 96 well culture plates, and every hole adds 80 μ l cancer cells and cultivated 24 hours;
(3) in selected 48 experimental group of culture plate, each experimental group respectively adds the protein that 20 μ l make through step (1), and other 48 control groups add 20 μ l phosphate buffered saline buffer PBS, handles the back like this and continues to cultivate 24 hours;
(4) in each hole, add MTS/PMS solution 0.02ml again, send culture plate back to CO
2Incubator is cultivated after 4 hours and is taken out, and slight shaking culture plate 1 minute is measured the OD value with microplate reader with the 490nm wavelength, and measurement result as shown in Figure 2.
Detect the influence of protein of the present invention to human hepatoma cell strain HepG2 propagation with MTS/PMS (nitrogen blue tetrazole salt/phenazine methosulfate) method, it is as follows specifically to detect step:
(1) protein is suspended again with Dulbecco ' s PBS (pH7.2) after, measure protein content, be diluted to 60 μ g/ml with RPMI 1640 substratum;
(2) be material with human hepatoma cell strain HepG2,, cancer cells pressed 2 * 10 according to MTS typical curve and HepG2 cell growth curve
4Individual/ml is inoculated in 96 well culture plates, and every hole adds 80 μ l cancer cells, cultivates 24 hours;
(3) in selected 48 experimental group of culture plate, each experimental group respectively adds the protein that 20 μ l make through step (1), and other 48 control groups add 20 μ l phosphate buffered saline buffer PBS, handles the back like this and continues to cultivate 24 hours;
(4) again with culture plate with the centrifugal 5min of 3000rpm, after supernatant liquor 50 μ l are got in every hole, respectively 50 μ l alpha-fetoprotein AFP standard substance, protein of the present invention are added in the respective aperture, add 150 μ lAFP enzyme labelling things more respectively; After jog made abundant mixing, at 37 ℃ of following incubations 1 hour, reaction solution in the hole of inclining, with washings washing 3 times, each washing all must be got rid of the globule in the most hole, and culture plate is patted dry;
(5) add 200 μ lTMB substrate solutions again in each hole, at room temperature the lucifuge color development is 15 minutes; At last, the 2M H that in each hole, adds 50 μ l
2SO
4Termination reaction, the jog mixing guarantees that blueness is transformed into yellow fully, measures the OD value with microplate reader with the 450nm wavelength.
Embodiment 5
Detect the influence of the protein of different concns to liver cancer cell AFP (alpha-fetoprotein) secretory volume with the ELISA method, concrete detection step is as follows:
(1) after the active ingredient that obtains by anion exchange chromatography suspends again with Dulbecco ' s PBS (pH7.2), measures protein content, be diluted to 60 μ g/ml with RPMI 1640 substratum with the Bradford method;
(2) be material with human hepatoma cell strain HepG2,, cancer cells pressed 2 * 10 according to MTS typical curve and HepG2 cell growth curve
4Individual/ml is inoculated in 96 well culture plates, and every hole adds 80 μ l cancer cells and cultivated 24 hours;
(3) in selected 48 experimental group of culture plate, each experimental group respectively adds 20 μ l protein, and other 48 control groups add 20 μ l phosphate buffered saline buffer PBS, handles the back like this and continues to cultivate 24 hours;
(4) again with culture plate with the centrifugal 5min of 3000rpm, supernatant liquor 50 μ l are got in every hole, with ELISA-AFP (alpha-fetoprotein) kit measurement alpha-fetoprotein content.
In a word, data measured among the embodiment 3-5 show, are the protein of feedstock production with the clam worm with the inventive method, can obviously suppress human hepatoma cell strain HepG2 propagation, and inhibiting rate reaches 82.3%, as shown in Figure 2, handle back cancer cells number and have only 2.21 * 10
4Individual/ml as shown in Figure 3; Can also obviously suppress human hepatoma cell strain HepG2 and produce alpha-fetoprotein AFP, inhibiting rate reaches 60.6%, and as shown in Figure 5, the content of handling the alpha-fetoprotein AFP of back cancer cells has only 81.28ng/ml, as shown in Figure 6.
In addition, the protein of different concns becomes positive correlation with the degree that suppresses human hepatoma cell strain HepG2 propagation, as shown in Figure 4; The protein of different concns becomes positive correlation with the degree of the alpha-fetoprotein AFP that suppresses human hepatoma cell strain HepG2 generation, sees the following form 2:
Table 2
| Concentration (μ g/ml) | AFP content (ng/ml) (X ± SE) |
| ????20 | ????81.28±1.72 |
| ????10 | ????107.15±2.49 |
| ????5 | ????164.44±2.19 |
| ????2.5 | ????186.37±2.37 |
| Contrast | ????206.54±4.25 |
As shown in Table 2, the protein of different concns acts on human hepatoma cell strain HepG2, cause that cytolemma destroys, entocyte releases, last cancer cells crumbles and dead to such as Fig. 7, Fig. 8 and Fig. 9 as can be known, and the effect that the protein that concentration is high more suppresses human hepatoma cell strain HepG2 is good more.
Claims (10)
1, a kind of protein that extracts from perinereis aibuhitensis is characterized in that, this protein is a basic protein, and its molecular weight is 65,000Da, and iso-electric point is 8.3.
2, the described proteinic preparation method who extracts from perinereis aibuhitensis of a kind of claim 1 is characterized in that this method comprises following steps:
(1) the fresh perinereis aibuhitensis that will propagate artificially was put into artificial seawater foster 24-48 hour, made it tell clean inclusion;
(2) after the perinereis aibuhitensis that will tell clean inclusion is cleaned, be placed on the filter paper and blot fast,, and collect perinereis aibuhitensis excretory body fluid again with 5-10V direct current electric shock perinereis aibuhitensis body wall;
(3) body fluid with the perinereis aibuhitensis collected places ultracentrifuge centrifugal, collects supernatant liquor, and filtration sterilization;
(4) supernatant liquor after the filtration sterilization is carried out anti-phase concentrating, collect concentrated solution;
(5) concentrated solution is carried out chromatography with gel-filtration column, collect the active ingredient that obtains through chromatography;
(6) active ingredient of collecting is carried out gradient elution after, obtain activated protein, again this activated protein is carried out desalination, concentrates and freeze-drying is handled.
3, by the described proteinic preparation method who extracts from perinereis aibuhitensis of claim 2, its feature also is, centrifugal in the described step (3), be under 4-10 ℃ condition, clam worm body fluid is put in the ultracentrifuge with 108 000-120, the centrifugal 20-30 of the rotating speed of 000 * g minute.
4, by the described proteinic preparation method who from perinereis aibuhitensis, extracts of claim 2, its feature also is, anti-phase concentrate in the described step (4), be with supernatant liquor to sample on the reversed-phase column after with the trifluoroacetic acid flushing, concentrate component with the acetonitrile solution wash-out that contains trifluoroacetic acid again.
5, by the described proteinic preparation method who extracts from perinereis aibuhitensis of claim 2, its feature also is, the gel filtration chromatography in the described step (5) is after concentrated solution is suspended again, last gel-filtration column wash-out.
6, by the described proteinic preparation method who extracts from perinereis aibuhitensis of claim 2, its feature also is, the gradient elution in the described step (6) is that anion-exchange column on the active ingredient of gel permeation chromatography preparation is carried out gradient elution.
7, the described proteinic application of extracting from perinereis aibuhitensis of a kind of claim 1 is characterized in that, this is applied as the application in preparing antibiotic or cancer therapy drug.
8, by the described proteinic application of from perinereis aibuhitensis, extracting of claim 7, its feature also is, described application in the preparation antibacterials is meant in preparation has application in the inhibiting antibacterials to colon bacillus, Pseudomonas aeruginosa, streptococcus aureus or Arthrobacter.
9, by the described proteinic application of extracting from perinereis aibuhitensis of claim 7, its feature is that also described application in the preparation cancer therapy drug is meant the application in the cancer therapy drug of preparation inhibition human hepatoma cell strain propagation.
10, by the described proteinic application of extracting from perinereis aibuhitensis of claim 7, its feature is that also described application in the preparation cancer therapy drug is meant the application for preparing in the cancer therapy drug that suppresses human hepatoma cell strain generation alpha-fetoprotein.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106632634A (en) * | 2016-11-28 | 2017-05-10 | 浙江海洋大学 | Neanthes succinea anti-lung cancer peptide and application thereof |
| CN106755230A (en) * | 2016-11-28 | 2017-05-31 | 浙江海洋大学 | A kind of preparation method of perinereis aibihitensis Grube anti-lung cancer polypeptide |
| CN107259293A (en) * | 2017-06-20 | 2017-10-20 | 中国海洋大学 | A kind of method that natural is prepared from clam worm |
| KR101807080B1 (en) * | 2016-04-21 | 2017-12-08 | 전북대학교 산학협력단 | Antioxidant or antiinflammatory composition comprising clamworm |
| CN109954003A (en) * | 2019-05-05 | 2019-07-02 | 北京颢美细胞基因生物技术有限公司 | Clam worm sperm plasmin and the preparation method and application thereof |
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- 2003-01-28 CN CNB03102033XA patent/CN1226305C/en not_active Expired - Fee Related
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR101807080B1 (en) * | 2016-04-21 | 2017-12-08 | 전북대학교 산학협력단 | Antioxidant or antiinflammatory composition comprising clamworm |
| CN106632634A (en) * | 2016-11-28 | 2017-05-10 | 浙江海洋大学 | Neanthes succinea anti-lung cancer peptide and application thereof |
| CN106755230A (en) * | 2016-11-28 | 2017-05-31 | 浙江海洋大学 | A kind of preparation method of perinereis aibihitensis Grube anti-lung cancer polypeptide |
| CN106632634B (en) * | 2016-11-28 | 2020-03-20 | 浙江海洋大学 | Perinereis aibuhitensis lung cancer resisting polypeptide and application thereof |
| CN106755230B (en) * | 2016-11-28 | 2020-03-20 | 浙江海洋大学 | Preparation method of perinereis aibuhitensis anti-lung cancer polypeptide |
| CN107259293A (en) * | 2017-06-20 | 2017-10-20 | 中国海洋大学 | A kind of method that natural is prepared from clam worm |
| CN109954003A (en) * | 2019-05-05 | 2019-07-02 | 北京颢美细胞基因生物技术有限公司 | Clam worm sperm plasmin and the preparation method and application thereof |
| CN109954003B (en) * | 2019-05-05 | 2022-08-16 | 北京颢美细胞基因生物技术有限公司 | Nereid semen cytoplasm essence and preparation method and application thereof |
| CN111647094A (en) * | 2020-06-11 | 2020-09-11 | 福建省水产研究所（福建水产病害防治中心） | Method for extracting nereis polysaccharide with auxiliary protection effect on chemical liver injury |
| CN111647094B (en) * | 2020-06-11 | 2021-11-23 | 福建省水产研究所（福建水产病害防治中心） | Method for extracting nereis polysaccharide with auxiliary protection effect on chemical liver injury |
| CN114250192A (en) * | 2021-11-12 | 2022-03-29 | 宁波大学 | Method for extracting antibacterial active substance of phascolosoma esculenta coelomic fluid cells |
| CN114250192B (en) * | 2021-11-12 | 2023-11-03 | 宁波大学 | Method for extracting antibacterial active substances from delicious euglena coelomica cells |
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| Publication number | Publication date |
|---|---|
| CN1226305C (en) | 2005-11-09 |
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