CN104740599A - Compound housefly antimicrobial peptide preparation - Google Patents
Compound housefly antimicrobial peptide preparation Download PDFInfo
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- CN104740599A CN104740599A CN201510094256.8A CN201510094256A CN104740599A CN 104740599 A CN104740599 A CN 104740599A CN 201510094256 A CN201510094256 A CN 201510094256A CN 104740599 A CN104740599 A CN 104740599A
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- 239000003910 polypeptide antibiotic agent Substances 0.000 title claims abstract description 51
- 241000257159 Musca domestica Species 0.000 title claims abstract description 20
- 102000044503 Antimicrobial Peptides Human genes 0.000 title abstract description 17
- 108700042778 Antimicrobial Peptides Proteins 0.000 title abstract description 17
- 238000002360 preparation method Methods 0.000 title abstract description 9
- 150000001875 compounds Chemical class 0.000 title abstract 7
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims abstract description 19
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims abstract description 19
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims abstract description 19
- 239000012528 membrane Substances 0.000 claims abstract description 8
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 8
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 8
- 238000011978 dissolution method Methods 0.000 claims abstract description 3
- 238000010438 heat treatment Methods 0.000 claims abstract 2
- 239000002131 composite material Substances 0.000 claims description 22
- 239000000203 mixture Substances 0.000 claims description 19
- 238000009472 formulation Methods 0.000 claims description 15
- 239000006228 supernatant Substances 0.000 claims description 9
- 238000000108 ultra-filtration Methods 0.000 claims description 9
- 239000000706 filtrate Substances 0.000 claims description 7
- 238000004108 freeze drying Methods 0.000 claims description 5
- 238000001556 precipitation Methods 0.000 claims description 5
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 claims description 4
- 238000001914 filtration Methods 0.000 claims description 4
- 230000014759 maintenance of location Effects 0.000 claims description 4
- 239000000284 extract Substances 0.000 claims description 3
- 238000000034 method Methods 0.000 abstract description 17
- 230000000844 anti-bacterial effect Effects 0.000 abstract description 15
- 239000007788 liquid Substances 0.000 abstract description 8
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 abstract description 6
- 238000002156 mixing Methods 0.000 abstract 1
- 230000001376 precipitating effect Effects 0.000 abstract 1
- 238000000967 suction filtration Methods 0.000 abstract 1
- 238000009777 vacuum freeze-drying Methods 0.000 abstract 1
- 230000001408 fungistatic effect Effects 0.000 description 7
- 241000588724 Escherichia coli Species 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 5
- 230000003115 biocidal effect Effects 0.000 description 5
- 230000003385 bacteriostatic effect Effects 0.000 description 4
- 108090000765 processed proteins & peptides Proteins 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 244000063299 Bacillus subtilis Species 0.000 description 3
- 235000014469 Bacillus subtilis Nutrition 0.000 description 3
- 241000607142 Salmonella Species 0.000 description 3
- 241000607768 Shigella Species 0.000 description 3
- 241000191967 Staphylococcus aureus Species 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 238000009295 crossflow filtration Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000012466 permeate Substances 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 108010001394 Disaccharidases Proteins 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 235000002918 Fraxinus excelsior Nutrition 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 241000191940 Staphylococcus Species 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000000843 anti-fungal effect Effects 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 229940121375 antifungal agent Drugs 0.000 description 1
- 239000002956 ash Substances 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 238000007906 compression Methods 0.000 description 1
- 230000006835 compression Effects 0.000 description 1
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
- 239000012531 culture fluid Substances 0.000 description 1
- 230000012969 defense response to bacterium Effects 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 230000015788 innate immune response Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000003223 protective agent Substances 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000008719 thickening Effects 0.000 description 1
- 238000003828 vacuum filtration Methods 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
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- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention discloses a compound housefly antimicrobial peptide preparation. A method for preparing the compound housefly antimicrobial peptide preparation comprises the steps of extracting protein in housefly bodies by utilizing a conventional acetic acid dissolution method, centrifuging an extracting solution to obtain supernate, heating the supernate at 100 DEG C for 10min, precipitating thermal-instability protein, centrifuging, performing suction filtration on the supernate through a filter membrane, performing twice molecule cut-off on a filter liquid by utilizing10Kda and 5Kda films, thereby obtaining a compound antimicrobial peptide solution with the molecular weight of 5Kda to 10Kda; adding trehalose to the compound antimicrobial peptide solution to ensure that the final concentration of the trehalose in the solution is 0.1-0.2mol/L, uniformly mixing, and performing vacuum freeze drying to obtain the compound housefly antimicrobial peptide preparation. The compound housefly antimicrobial peptide preparation has broad-spectrum bactericidal ability and long shelf life.
Description
Technical field
The invention belongs to biological technical field, be specifically related to provide a kind of housefly composite antibacterial peptide formulations.
Background technology
Antibiotic medically plays an important role always, is the core drug that western medical treatment catches.But along with antibiotic extensive and life-time service, and the situation of abuse of antibiotics is day by day serious, causes the mutant strain of many tool drug resistance to occur, makes existing antibiotic lose efficacy.
Antibacterial peptide is a kind of micromolecule polypeptide with antibacterial functions produced in organism, in low grade and higher organism system, all demonstrate innate immunity function.Research shows, containing abundant antibacterial peptide in housefly body, this kind of active polypeptide has molecular weight little (most of antibacterial peptide is all at below 20Kda), heat stability is strong, good water solubility, has a broad antifungal spectrum, disinfection vitality are high and the advantage such as abundant raw material source, particularly it can kill Resistant strain, and bactericidal mechanism makes pathogen not easily produce resistance mutation, become the ideal substitute of conventional antibiotic, cause the extensive attention of biological educational circles and the world of medicine.
At present about the report that house fly antibiotic peptide extracts, basic skills is the protein extracted by the method for acetate dissolution in housefly body, again by 100 DEG C of thermal precipitation thermally labile albumen, be adsorbed on trehalose by the remaining heat-stable protein be dissolved in acetic acid further by absorption method, by carrying out eluting to the albumen of absorption, concentrated (lyophilizing or the sedimentation method) obtain recombinant antimicrobial peptide afterwards.But the cost of trehalose is high, can not reuse, affect the industrial applications of the method.The people such as Hou also obtain concentrated recombinant antimicrobial peptide by the method for direct lyophilizing acid extraction liquid, but because a large amount of ashes cannot be removed after lyophilizing, and then the dissolubility of recombinant antimicrobial peptide can be reduced, affect the further abstraction and purification of recombinant antimicrobial peptide.
Cross-flow ultrafiltration technology refers to liquid flow direction and filtering direction filtered version in vertical direction, and during tangential flow filtration, the flow direction of liquid to be filtered is parallel with the direction of filter membrane plane, and liquid will perpendicular to film surface through fenestra.During ultrafiltration, filter fairly large feed liquid just to need to adopt tangential flow filtration mode, its meeting turbulization (Secondary Flow), owing to there being turbulent flow, liquid flow produces shearing force at filter media surface (i.e. ultrafiltration membrane surface), reduce cake layer or the accumulation of gel layer on film surface, make precipitation from film sur-face peeling, reduce fouling membrane, ensure the stable rate of filtration, the difference of ultrafilter membrane molecular cut off, can make cross-flow ultrafiltration technology be applied to the albumen being separated different molecular weight, in large-scale protein compression and extensive use in being separated.
Antibacterial peptide has higher stability at normal temperatures, is not easy inactivation, but also exists along with the holding time extends, the problem that antibacterial activity goes down.The stable chemical nature of trehalose, has the unique property being different from other disaccharidase, has no research to the protective effect of antibacterial little peptide.
Summary of the invention
The invention provides a kind of housefly composite antibacterial peptide formulations, said preparation extracts the protein in housefly body by conventional acetate dissolution method, extracting solution centrifuging and taking supernatant, supernatant is placed in 100 DEG C of thermal precipitation thermally labile albumen, centrifugal, supernatant filter membrane sucking filtration, filtrate adopts 10Kda and 5Kda film twice molecular retention, acquisition molecular weight is the composite antibacterial peptide solution between 5Kda ~ 10Kda, trehalose is added in composite antibacterial peptide solution, trehalose final concentration in solution is made to be 0.1mol/L ~ 0.2mol/L, the lyophilization of mix homogeneously final vacuum, obtained housefly composite antibacterial peptide formulations.
Described filtrate adopts 10Kda and 5Kda film bag cross-flow ultrafiltration method to carry out molecular retention.
The advantage of the inventive method and technique effect:
The recombinant antimicrobial peptide be made up of multiple antibacterial peptide of invention formulation to be molecular weight be continuous distribution; it not only has fungistatic effect to bacillus subtilis; also there is antibacterial action to staphylococcus aureus, escherichia coli, Salmonella, shigella; its antimicrobial spectrum can not only suppress gram-positive bacterium; also can suppress gram negative bacteria, add trehalose can also improve antibacterial peptide activity and stability as protective agent.
In view of cross-flow ultrafiltration method once can process large volume antibacterial peptide acid extraction liquid, thickening efficiency is high, and can reuse, and do not need expensive equipment, for suitability for industrialized production antibacterial peptide, this method is more reliably convenient.
Accompanying drawing explanation
Fig. 1 is the fungistatic effect figure of composite antibacterial peptide solution, and wherein 1 is staphylococcus aureus S.
aureusaTCC6538; 2 is bacillus subtilis B.
subtiliscMCC (B) 63501; 3 is escherichia coli E.
colirosetta; 4 is escherichia coli E.
coli0157NOTC12900; 5 is Salmonella S.
enteritidiscMCC (B) 50335; 6 is shigella S.
dysenteriaea7CC10231);
Fig. 2 is the recombinant antimicrobial peptide formulation against S staphylococcus (S. containing variable concentrations trehalose
aureusaTCC6538) antibacterial result schematic diagram;
Fig. 3 is that composite antibacterial peptide formulations containing variable concentrations trehalose is to escherichia coli (E.
coli0157NOTC12900) antibacterial result schematic diagram.
Detailed description of the invention
Below by embodiment, the inventive method is described in further detail, but the protection domain of the inventive method is not limited to described content.
Embodiment 1: this housefly recombinant antimicrobial peptide preparation, is prepared as follows:
(1) 400g fly larvae powder (20 order) is dissolved in the acetum of 2L 0.5mol/L, extraction of spending the night at 4 DEG C;
(2) extracting solution collects supernatant with the centrifugal 15min of 7000rpm, supernatant in 100 DEG C water-bath 10min to precipitate macromole thermal instability albumen, the centrifugal 15min of 7000rpm removes thermal instability albumen precipitation, and supernatant utilizes the filter membrane of 0.22 μm to carry out vacuum filtration, collects filtrate;
(3) filtrate step (2) obtained is that 10Kda film bag tangential flow systems carries out ultrafiltration and concentration by molecular cut off, collect permeate, be that 5Kda film bag tangential flow systems carries out ultrafiltration and concentration by above-mentioned permeate by molecular cut off again, collect concentrated solution 50ml, obtain the antibacterial peptide solution that slipstream concentration method is concentrated.
(4) after filtrate lyophilizing step (2) obtained, then use the acetate dissolution of 50ml 0.1mol/L, obtain the antibacterial peptide solution that lyophilization is concentrated.
(5) utilize Coomassie Brilliant Blue to measure the concentration of the antibacterial peptide solution soluble albumen that different concentration method obtains, the results are shown in Table 1.
Table 1:
Adopt the first slipstream method for concentration to obtain dark brown antibacterial peptide concentrated solution, soluble protein content is 13.7mg/ml; Adopt lyophilization method for concentration to have also been obtained dark brown antibacterial peptide concentrated solution equally, soluble protein content is 1.5mg/ml, and the former response rate is almost 10 times of the latter.
Embodiment 2: the bacteriostatic experiment of antibacterial peptide solution obtained in embodiment 1
(1) testing bacterium is staphylococcus aureus S.
aureusaTCC6538, bacillus subtilis B.
subtiliscMCC (B) 63501, escherichia coli E.
colirosetta, escherichia coli E.
coli0157NOTC12900, Salmonella S.
enteritidiscMCC (B) 50335, shigella S.
dysenteriaea7CC10231, cultivate and all adopt LB culture medium, its composition is (often liter): yeast extract 5g; Peptone 10g; NaCl 10g; Adjust ph is 7.0, if preparation solid medium, then adds 15 grams, agar;
(2) above bacterial strain is inoculated in the LB fluid medium of 5ml respectively, on 37 DEG C of shaking tables with 150rpm constant temperature culture to OD
600≈ 0.8;
(3) respectively above-mentioned test bacteria culture fluid 500 μ l is mixed homogeneously with the LB solid medium of 20ml preheating, be then down flat plate;
(4) after the culture medium solidifying in flat board, punch in culture medium with the card punch of sterilizing, diameter is 0.4cm;
(5) in hole, add the concentrated antibacterial peptide solution 40 μ l prepared, leave standstill dull and stereotyped 15min and composite antibacterial Toplink is fully spread in agar;
(6) be finally inverted into by flat board in 37 DEG C of incubators and measure antibacterial circle diameter (antibacterial circle diameter=transparent circle diameter-bore dia) after constant temperature culture 10h, 10h, different test the antibacterial of bacterium the results are shown in Table 2.
Table 2:
Experimental result shows: this antibacterial peptide solution has broad-spectrum antibacterial effect.
Embodiment 3: the preparation of composite antibacterial peptide formulations
Be add trehalose in the composite antibacterial peptide solution between 5Kda ~ 10Kda at the molecular weight that embodiment 1 is obtained, trehalose final concentration in solution is made to be 0.05 mol/L, 0.1 mol/L, 0.2mol/L, the lyophilization of mix homogeneously final vacuum, obtained housefly composite antibacterial peptide formulations.
Measure the fungistatic effect of composite antibacterial peptide formulations: be dissolved in 1 ml sterile water 14 milligrams of composite antibacterial peptide formulations as composite antibacterial solution, carry out bacteriostatic experiment as a comparison with the composite antibacterial solution not adding trehalose, bacteriostatic experiment method is with embodiment 2 simultaneously.
Measured its fungistatic effect every 10 days, Fig. 2 and Fig. 3 is the bacteriostatic activity result of the composite antibacterial peptide formulations containing variable concentrations trehalose under two kinds of different test strain conditions;
As can be seen from the figure: within 60 day shelf-life; when there is no trehalose; recombinant antimicrobial peptide fungistatic effect disappears obviously; after adding certain density trehalose; the fungistatic effect of recombinant antimicrobial peptide disappears and weakens; wherein 0.1mol/L-0.2mol/L trehalose concentration has best protection effect, and at this concentration, recombinant antimicrobial peptide is good fungistatic effect still.
Claims (2)
1. a housefly composite antibacterial peptide formulations, it is characterized in that: extract the protein in housefly body by conventional acetate dissolution method, extracting solution centrifuging and taking supernatant, supernatant is placed in 100 DEG C of heating 10min precipitation heat labile proteins, centrifugal, supernatant filter membrane sucking filtration, filtrate adopts 10Kda and 5Kda film twice molecular retention, acquisition molecular weight is the composite antibacterial peptide solution between 5Kda ~ 10Kda, trehalose is added in composite antibacterial peptide solution, trehalose final concentration in solution is made to be 0.1mol/L ~ 0.2mol/L, the lyophilization of mix homogeneously final vacuum, obtained housefly composite antibacterial peptide formulations.
2. housefly composite antibacterial peptide formulations according to claim 1, is characterized in that: filtrate adopts 10Kda and 5Kda film bag cross-flow ultrafiltration method to carry out molecular retention.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510094256.8A CN104740599A (en) | 2015-03-03 | 2015-03-03 | Compound housefly antimicrobial peptide preparation |
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|---|---|---|---|
| CN201510094256.8A CN104740599A (en) | 2015-03-03 | 2015-03-03 | Compound housefly antimicrobial peptide preparation |
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ID=53581219
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105085610A (en) * | 2015-08-17 | 2015-11-25 | 张卫民 | Graded secondary ultrafiltration purification method for antibacterial peptide |
| CN106581699A (en) * | 2016-12-30 | 2017-04-26 | 江西宜信堂医疗科技有限公司 | Bactericidal aseptic solid medical ultrasonic coupling patch and preparation method thereof |
| US10039801B2 (en) | 2016-02-16 | 2018-08-07 | Strongbridge Ireland Limited | Pharmaceutical compositions of water soluble peptides with poor solubility in isotonic conditions and methods for their use |
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| CN101054408A (en) * | 2007-04-02 | 2007-10-17 | 贵阳医学院 | Method for separating housefly secretion type antibacterial peptide, product and application thereof |
| CN101392020A (en) * | 2008-11-06 | 2009-03-25 | 中山大学 | Musca domestic pupae natural antimicrobial peptide products and preparation method and use thereof |
| CN102321164A (en) * | 2011-05-25 | 2012-01-18 | 绍兴市曙光科技开发有限公司 | Preparation method and application of insect natural antibacterial peptide products |
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2015
- 2015-03-03 CN CN201510094256.8A patent/CN104740599A/en active Pending
Patent Citations (3)
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| CN101054408A (en) * | 2007-04-02 | 2007-10-17 | 贵阳医学院 | Method for separating housefly secretion type antibacterial peptide, product and application thereof |
| CN101392020A (en) * | 2008-11-06 | 2009-03-25 | 中山大学 | Musca domestic pupae natural antimicrobial peptide products and preparation method and use thereof |
| CN102321164A (en) * | 2011-05-25 | 2012-01-18 | 绍兴市曙光科技开发有限公司 | Preparation method and application of insect natural antibacterial peptide products |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105085610A (en) * | 2015-08-17 | 2015-11-25 | 张卫民 | Graded secondary ultrafiltration purification method for antibacterial peptide |
| US10039801B2 (en) | 2016-02-16 | 2018-08-07 | Strongbridge Ireland Limited | Pharmaceutical compositions of water soluble peptides with poor solubility in isotonic conditions and methods for their use |
| US10398751B2 (en) | 2016-02-16 | 2019-09-03 | Strongbridge Dublin Limited | Pharmaceutical compositions of water soluble peptides with poor solubility in isotonic conditions and methods for their use |
| US10987402B2 (en) | 2016-02-16 | 2021-04-27 | Strongbridge Dublin Limited | Pharmaceutical compositions of water soluble peptides with poor solubility in isotonic conditions and methods for their use |
| CN106581699A (en) * | 2016-12-30 | 2017-04-26 | 江西宜信堂医疗科技有限公司 | Bactericidal aseptic solid medical ultrasonic coupling patch and preparation method thereof |
| CN106581699B (en) * | 2016-12-30 | 2018-04-17 | 江西宜信堂医疗科技有限公司 | A kind of antibacterial type sterile solid medical supersonic coupled patch and preparation method thereof |
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