CN102757490A - Chemical synthesis method of hippocampus antimicrobial peptide hkplp and application thereof in breeding industry - Google Patents
Chemical synthesis method of hippocampus antimicrobial peptide hkplp and application thereof in breeding industry Download PDFInfo
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Abstract
The invention relates to a chemical synthesis method of a natural antimicrobial peptide hkplp derived from a hippocampus. The chemical synthesis method comprises the following main steps: with amino resin as a carrier, firstly synthesizing hkplp-amino resin according to a solid-phase synthesis method; after TFA treatment, obtaining a crude peptide; and performing G10 processing and drying on the crude peptide to obtain a finished product hkplp. The invention further relates to application of the antimicrobial peptide finished product in the breeding industry. The problems in detecting the antibacterial spectrum and the minimum inhibitory concentration of the synthetic antimicrobial peptide hkplp and determining an antibacterial activity unit are mainly solved; in addition, experiments of adding the synthetic antimicrobial peptide finished product into feed, with shrimps and chickens as experimental subjects preferably, show that the synthetic hkplp can prevent gastrointestinal diseases instead of antibiotics, promote growth and increase body weight and can be used as livestock and aquaculture feed additives.
Description
Technical field: the present invention relates to the chemical synthesis process of a kind of natural antibiotics hippocampus antibacterial peptide hkplp, particularly solid phase synthesis hippocampus antibacterial peptide; The invention still further relates to the application of synthetic hippocampus antibacterial peptide in aquaculture.
Background technology: in recent years along with antibiotic abuse, anti-medicine bacterium is more and more, and resistance is serious day by day, not only shows clinically, also involves aquaculture simultaneously.According to data; The golden Portugal bacterium that is separated to clinically in the U.S. has 95% pair of penicillium mould resistance; More than 50% to methicillin resistance (Frindkin SK; Gaynes RP.Antimicrobial resistance in intensive care units.Clin Chest Med.1999,20 (2): 303-316); Since the eighties in 20th century; The drug resistance of vancomycin bacterial strain also has rising tendency; Comprise vancomycin resistance streptococcus aureus and faecalis (The centers for Disease Control and Prevention.Staphylococus aureus with reduced sueceptibility to vancomycin-United States 1997.JAMA; 1997,278 (11): 891-892).Along with people's is to the enhancing of environmental consciousness and the improve of food safety rank consciousness, and traditional antibiotic abuse causes the appearance of " superbacteria ", and the microbiotic of new generation that presses for environmental protection, safe overriding resistance replaces.
Antibacterial peptide is the important component part of immune defense system in the animal body; It is the small molecule peptide; Be called as free of contamination natural antibiotics, have broad-spectrum antimicrobial, have no drug resistance, advantage such as thermally-stabilised, good water solubility, nontoxic residue-free, meet the idea of modern environmental protection.According to statistics, China still has 20 kinds of microbiotic that people and animals are shared or livestock and poultry are special-purpose of reaching to make fodder additives at present, and along with China joined WTO, microbiotic is made fodder additives will have a strong impact on the competitive power of our tame livestock product in the world market.Green feed additive with the replacement of antibacterial peptide product development Cheng Xin tradition Antibiotic Additive; To inject a pin cardiotonic drug to the feed additive industry of China; Integral body is promoted the level of feedstuff industry; Make the feeds product of China more competitive, promote China's feedstuff industry and constantly advance in world market.
Two antibacterial peptide gene hkplp and hkabf that the experimental result in we shows from hippocampus that the clone obtains in early stage have the excellent antibiotic activity.With HKABF is example, and HKABF is to G in reorganization
+Bacterium is relatively responsive, especially to streptococcus aureus, can suppress dilution at the protein concentration of 0.5 μ g/ml and be about 5 * 10
6The streptococcus aureus of CFU/ml concentration.In the microbial bacteria of test; Secondly responsive is Staphylococcus albus; MIC is 2.5 μ g/ml; Be Warner staphylococcus (MIC is 5 μ g/ml) and the isolated streptococcus aureus III of Guangzhou Disease Control and Prevention Center (MIC is 5 μ g/ml) once more, the streptococcus aureus II that the attached Second Academy of Zhongshan University isolation identification goes out (MIC is 15 μ g/ml) is for morganella morganii (MIC is 30 μ g/ml) and these two kinds of G of Vibrio vulnificus (MIC is 120 μ g/ml)
-Bacterium also can suppress its growth under low peptide concentration.For other G
+And G
-What bacterium even fungi (Candida albicans, yeast, Oidium tropicale) had under than higher protein concentration also can demonstrate different fungistatic effects.Two antibacterial peptide gene hkplp and hkabf are simultaneously recombinant expressed through pichia spp KM71; Detect the anti-microbial activity that its anti-microbial activity shows hkplp and will obviously be superior to HKABF, so we have selected the development object of antibacterial peptide gene hkplp synthetic as the antibacterial peptide fodder additives.
Hippocampus antibacterial peptide hkplp sequence: FLGLIFHGLVHAGKLIHGLIHRNR-NH
2Molecular formula: C
128H
205N
41O
25, molecular weight MW
[AV]: 2718.32, MW
[mon]: 2716.6; Do not contain rare amino acid and exogenous chemical components; In animal digestive tract, has satisfactory stability property; Have simultaneously that immunogenicity is little, good water solubility, broad-spectrum sterilization in addition can fungicidal, protozoon and do not produce advantages such as endurance strain, can tolerate the degraded of proteolytic enzyme and peptase in the gi tract.Particularly under acidic conditions; Therefore heating or even high temperature high pressure process not only can tolerate the violent condition of HTHP in the feed course of processing to the not influence of its anti-microbial activity, and can in feed preservation process, continue its function of performance; Keep feed quality, prolong the shelf-life.Hkplp can be used as the green feed additive of a new generation, is good traditional microbiotic substitute, can effectively must solve the residual and chemical sproof problem of pathogenic bacteria of humans and animals medicine.
Summary of the invention: the object of the present invention is to provide the chemical synthesis process of a kind of hippocampus antibacterial peptide hkplp,, solve the unstable difficult big technical problem of producing of fermentation process through solid-phase synthesis synthetic hkplp; Another object of the present invention provides the application of synthetic hippocampus antibacterial peptide in fodder additives.
Method of the present invention comprises the steps:
(1) with aminoresin is starting raw material, connects amino acid successively with blocking group according to the method for solid phase synthesis.Wherein Fmoc-AA, condensing agent ratio were thrown in 1: 1 in molar ratio; Fmoc-AA doubly measures adding by the 2-5 of condensation amount; Obtain protection 24 peptide resins, during remove the Fmoc blocking group successively, each step amino acid condensation all adopts Kaise Test detection reaction, and whether condensation is complete; Like coupling reaction this amino acid of repetition condensation that is positive, the sequence condensation directly removes the Fmoc blocking group at last.Amino acid condensation reaction one time 2-4 hour is taken off Fmoc reaction branch and is carried out for 2 times, is respectively 5min, 15min.
(2) use TFA: Tis: 95: 2.5: 2.5 by volume mixing solutions of Phenol was handled peptide resin 3-4 hour, scaled off and excise Side chain protective group to peptide from resin, concentrated or blew to doing the back and separate out solid with the ice ether, and whiz gets bullion.
(3) handle the hkplp bullion with 1% acetum, concentrate drying gets finished product behind the G10 post excessively.
(4) mensuration of synthetic antibacterial peptide hkplp antimicrobial spectrum.
(5) survey OD with the doubling dilution culture method
630Value is confirmed synthetic hkplp minimal inhibitory concentration (MIC).
(6) synthetic hkplp anti-microbial activity unit confirms
(7) add the hkplp finished product in the feed application in aquaculture.
Aminoresin comprises all Fmoc-Rink amino resins in the above-mentioned steps (1), is preferably Fmoc-Rink Amide Resin here; The condensing agent system is DIC+A or A+B+C, and wherein A is HOBt or HOAt, and B is HBTU, HATU, PyBOP or TBTU, and C is DIPEA or TMP, can adopt the mode of permutation and combination to make up each other, is preferably DIC+HOBt; Fmoc-AA is respectively: Fmoc-Phe-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-His (Trt)-OH, Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Lys (Boc)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Asn (Trt)-OH; Solvent is preferably DMF.
Wherein the aminoresin substitution degree is elected 0.2mmol-1.0mmol/g as, the Fmoc-Rink Amide resin of selecting for use according to the invention.The coupling agent system is DIC+A or A+B+C; Wherein A is HOBt or HOAt; B is HBTU, HATU, PyBOP or TBTU; C is DIPEA or TMP, and this programme technician can make up modes such as comprising DIC/HOBt, DIC/HOAt, HBTU/HOBt, HATU/HOAt, PyBOP/HOBt, PyBOP/HOAt, TBTU/HOBt TBTU/HOAt each other according to the mode of permutation and combination, and the organic bases C of employing is DIPEA or TMP.
Some abbreviations commonly used have following implication among the present invention:
Fmoc-Rink Amide Resin: fluorenylmethyloxycarbonyl-aminoresin
Fmoc: fluorenylmethyloxycarbonyl
Fmoc-AA:N-fluorenylmethyloxycarbonyl amino acid
Fmoc-Phe-OH:N-fluorenylmethyloxycarbonyl-phenylalanine(Phe)
Fmoc-Leu-OH:N-fluorenylmethyloxycarbonyl-leucine
Fmoc-Gly-OH:N-fluorenylmethyloxycarbonyl-glycocoll
Fmoc-Ile-OH:N-fluorenylmethyloxycarbonyl-Isoleucine
Fmoc-His (Trt)-OH:N-fluorenylmethyloxycarbonyl-Histidine (triphenyl)
Fmoc-Val-OH:N-fluorenylmethyloxycarbonyl-Xie Ansuan
Fmoc-Ala-OH:N-fluorenylmethyloxycarbonyl-L-Ala
Fmoc-Lys (Boc)-OH:N-fluorenylmethyloxycarbonyl-Methionin (tertbutyloxycarbonyl)
Fmoc-Arg (Pbf)-OH:N-fluorenylmethyloxycarbonyl-l-arginine (alkylsulfonyl)
Fmoc-Asn (Trt)-OH:N-fluorenylmethyloxycarbonyl-l-asparagine (triphenyl)
TFA: trifluoroacetic acid
Tis: tri isopropyl silane
Phenol: phenol
DIC: DIC
The HOBt:1-hydroxybenzotriazole
HOAt:1-hydroxyl-7-azo benzotriazole
HBTU:O-benzotriazole-N, N, N ', N '-tetramethyl-urea hexafluorophosphate
TBTU:O-(benzotriazole-1-oxygen)-N, N, N ', N '-tetramethyl-urea hexafluoro borate
HATU:O-(7-azo benzotriazole-1-oxygen)-N, N, N ', N '-tetramethyl-urea hexafluorophosphate
PyBOP: (benzotriazole-1-oxygen) tripyrrole alkyl phosphorus hexafluorophosphate
DIPEA: diisopropylethylamine
TMP:2,4, the 6-trimethylpyridine
DMF:N, dinethylformamide
DCM: methylene dichloride
MeOH: methyl alcohol
Synthetic route is following:
The invention has the beneficial effects as follows that method is simple through artificial solid phase synthesis antibacterial peptide hkplp, productive rate is higher can to obtain stable product, has avoided fermented bacterium structure, the low unsettled shortcoming of productive rate.
Step (4) the present invention adopts plating method that synthetic antibacterial peptide hkplp antimicrobial spectrum is measured, and shows streptococcus aureus, intestinal bacteria K
12D
31, bacillus coli DH 5 alpha, Salmonellas, animal pathogenic escherichia coli and the isolating escherichia coli of hospital clinical case, urine enterococcus, MRSH antibacterial experiment as shown in Figure 2, this antibacterial peptide all has good antibacterial activity to above each bacterium.
Adopt the hkplp solution of the method preparation different concns of doubling dilution in the step (5), join in the culture bacteria liquid and survey OD behind the cultivation 16h
630Value is confirmed the minimal inhibitory concentration MIC of synthetic hkplp to different strains.
To add in the feed mainly be the application to cultivated animals to synthetic hkplp finished product in the step (7), does comparative study with conventional feed, and preferred prawn of the present invention and chicken are as experimental subjects.
Description of drawings:
Fig. 1 antibacterial peptide hkplp finished product preparation figure.
Fig. 2 synthetic antibacterial peptide hkplp is to streptococcus aureus, intestinal bacteria K
12D
31, bacillus coli DH 5 alpha, Salmonellas, animal pathogenic escherichia coli and the isolating escherichia coli of hospital clinical case, urine enterococcus, MRSH the antibacterial experiment result, among the figure: the anti-microbial activity of 1 expression penbritin; 2 negative contrast ddH
2O; 3,4,5,6,7 is the anti-microbial activity of artificial synthetic antimicrobial peptide.
Embodiment:
Below provide embodiment the present invention is specifically described, but be pointed out that and the invention is not restricted to specific embodiment, embodiment only is used for further specifying of the present invention, can not be interpreted as limitation of the scope of the invention.
The specific embodiment explanation:
The preparation of embodiment 1:Fmoc-Arg (pbf)-aminoresin
With aminoresin Fmoc-RinkAmide 10.1g, substitution degree 0.84mmol/g joins in the solid phase reactor; Add DMF and soak, make its abundant swelling 30min, drain; Add 20% piperidines/DMF solution and take off Fmoc 5min, 15min, with DMF, DCM, MeOH washes clean.Get 11.05g Fmoc-Arg (Pbf)-OH; 2.32gHOBt 6.54gHBTU joins balance 5min in the reactor drum after dissolving with DMF, adds 6.0ml DIPEA room temperature reaction 1 hour; Drain; Successively respectively wash 3 times with DMF, DCM, MeOH, drying 17.06g Fmoc-Arg (the pbf)-Rink Amide resin of weighing, the detection substitution value is 0.63mmol/g.
The preparation of embodiment 2:hkplp-aminoresin
Above-mentioned resin is put in connects in the peptide bottle, add 20% piperidines/DMF solution and take off Fmoc 5min, 15min, with DMF, DCM, MeOH washes clean.Then by sequence Phe-Leu-Gly-Leu-Ile-Phe-His-Gly-Leu-Val-His-Ala-Gly-Lys-Leu-Ile-His-Gly-Leu-Ile-His-Arg-Asn-Arg-NH
2Order connect the amino acid of Fmoc protection successively, the reaction system mol ratio is that Fmoc-AA: DIC: HOBt threw in by 1: 1: 1, Fmoc-AA extraordinarily goes into by 3 of amount of resin in this example, each DIC adds about 4.07g, HOBt adds about 4.24g.20% piperidines/DMF removes the Fmoc blocking group 2 times successively during this time, and first set reaction 5min reacts 15min for the second time; Each step amino acid condensation reaction one time 2-4 hour, whether condensation is complete all to adopt Kaise Test detection reaction, and like coupling reaction this amino acid of repetition condensation that is positive, the sequence condensation directly removes the Fmoc blocking group at last, and DMF, DCM, MeOH wash several times.
The preparation of embodiment 3:hkplp bullion
Hkplp-aminoresin is used TFA: Tis: 95: 2.5: 2.5 by volume mixing solutions of Phenol was handled peptide resin 3-4 hour; Scale off and excise Side chain protective group to peptide from resin; Be concentrated into dried back and separate out solid with the ice ether, whiz gets bullion 28.42g.
The preparation of embodiment 4:hkplp finished product
After the Hkplp bullion dissolved with 1% acetum, the centrifuging and taking supernatant was crossed the G10 post, collected antibacterial peptide hkplp peak concentrate drying and got finished product 15.68g.Purity is greater than 60%, and total recovery reaches 54%.
Embodiment 5: the mensuration of synthetic antibacterial peptide hkplp antimicrobial spectrum
Picking PCR is accredited as male list colony inoculation in the 100mlBMGY substratum from the YPD flat board, and 30 ℃ of shaking culture are to OD
6002～6, remove supernatant, add the resuspended thalline of 20mlBMMY substratum, 30 ℃ of shaking culture 96 hours were whenever got the 1ml sample in the Eppendorff pipe at a distance from 24 hours, got supernatant and were used for the anti-microbial activity detection.
With nutrient agar heat fused in water-bath, be cooled to about 50 ℃, draw giving birth to of 60 μ l with aseptic method and survey bacterium (OD600=0.3); Add in the 20ml nutrient agar; Rapid mixing, pouring into diameter is that horizontal positioned is waited to solidify in the aseptic plane ware of 9cm.On agar, beat the circular hole that diameter is 2.7mm, in the hole, add synthetic hkplp respectively, sterilized water and 100mg/ml Amplicin with the phosphate buffered saline buffer preparation.Plate is inverted in 37 ℃ of overnight cultures, next day observations.
The antibacterial peptide hkplp of synthetic is to streptococcus aureus, intestinal bacteria K
12D
31, bacillus coli DH 5 alpha, Salmonellas, animal pathogenic escherichia coli and the isolating escherichia coli of hospital clinical case, urine enterococcus, MRSH antibacterial experiment as shown in Figure 2, this antibacterial peptide all has good antibacterial activity to above each bacterium.
Embodiment 6: the mensuration of synthetic hkplp minimal inhibitory concentration (MIC)
MIC: the bacterium liquid dilution that test strain is cultured to logarithmic phase is about 5 * 10
6CFU/ml adds in 96 well culture plates, and the every hole of test hole adds bacterium liquid 90 μ l, adds the synthetic antimicrobial peptide solution of the different concns of doubling dilution then, 10 μ l/ holes.Positive control is the bacterium liquid in 100 μ l/ holes, and negative control is corresponding 100 μ l substratum.Slowly shake in 37 ℃ then and cultivate about 16h, survey OD with ELIASA
630The minimum concentration of bacteria growing inhibiting is MIC, and the result sees table 1.
The MIC of table 1 recombinant antibacterial peptide HKPLP
Embodiment 7: artificial synthetic antimicrobial peptide hkplp anti-microbial activity unit confirms
The antibiotic vigor that we are not less than 1mg purity 95% solid state chemistry synthetic antibacterial peptide hkplp is defined as 1000U.Therefore the antibiotic vigor of the antibacterial peptide hkplp of synthetic is not less than 600U/mg.
Embodiment 8: artificial synthetic antimicrobial peptide hkplp promotes the application of prawn growth
Synthetic antibacterial peptide finished product among the present invention is processed feed as fodder additives raise prawn; Three groups of experiment components are low, the high antibacterial peptide group of neutralization, and adding final concentration respectively to is 200ppm, 500ppm and 1000ppm; Divide spring and autumn to carry out two groups of parallel laboratory tests; Each experimental group is established three processing, and each handles the first mantissa of prawn is 30, controls the identical water quality of each experimental group, temperature and dissolved oxygen; Respectively each surviving rate of handling of record, the whole story single tail counterpoise and single tail food ration, its result is like following table 2, shown in 3.
Surviving rate average and the single tail rate of body weight gain that can know the experimental group of adding the antibacterial peptide fodder additives by following two table results are apparently higher than control group, and The mean feed is a little less than control group; Especially the group that has added antibacterial peptide hkplp autumn and vernal aspect be not than there being too big difference, and the control group surviving rate, and the equal rate of body weight gain of single tail all has decline, and feed coefficient has in a small amount and increases.This antimicrobial peptide products have dosage little, have no side effect, efficient, quick-acting, long-acting, drug residue free, have no drug resistance and the characteristics of double cross infection, in the prawn culturing whole process, can preventing disease take place, promote growth, improve quality.
Table 2 antibacterial peptide fodder additives is raised prawn test-results in spring
Table 3 antibacterial peptide fodder additives is raised prawn test-results in autumn
Embodiment 9 artificial synthetic antimicrobial peptide hkplp promote the application of chicken growth
Select 1000 of commodity AA fryer that just go out shell for use, consistent control group, the test group of being divided into of public female ratio, every group is 500.Control group is selected the complete diet pellet that uses 4% fryer Preblend preparation for use, adds microbiotic by recommended amounts.Test group is not added microbiotic, and feed uses the complete diet pellet of 4% fryer Preblend preparation, adds synthetic antibacterial peptide hkplp finished product among 0.04% the present invention, and trial period was for 6 weeks.It is identical that each organizes the raising condition, and feeding and management is undertaken by the conventional feeding and management system of chicken house.
Claim during on-test respectively to organize the chicken body weight, the 21st age in days, the 42nd age in days claim that on an empty stomach chicken body weight and material weigh morning after on-test, and record feed intake, changes of weight and death toll are calculated feed conversion rate, average daily gain and surviving rate.Observe chicken healthy state, loose and watery stool is thick and situation such as smell.
Experimental result is following:
(1) the overall observation: compare with adding antibiotic control group, the experimental group fryer mental status is good, honey stomach; The speed of growth is very fast, good evenness, and general healthy body is strong; Suffer from respiratory tract disease and intestinal tract disease and obviously reduce, control group then is prone to ill, and especially the white scour of chicken takes place more.Experimental group is high than the control group surviving rate, and feather is clean and tidy, and ight soil is comparatively dry, and stink is little, and hen house ammonia flavor and stink reduce.
(2) artificial synthetic antimicrobial peptide hkplp is to the influence of meat chicken production performance: as shown in table 4, the experimental group average daily gain all is significantly higher than control group with full phase average daily gain.
Table 4 different days is respectively organized mean body weight and stage weightening finish (g)
(3) full phase feed conversion rate and surviving rate such as following table 5:
Table 5 feed conversion rate and surviving rate
In daily ration of broiler, add an amount of synthetic antibacterial peptide hkplp, through the feeding experiment in 6 weeks, the material anharmonic ratio has reduced by 15.54%, has verified that this antibacterial peptide hkplp has significantly improvement effect to meat chicken production performance.Simultaneously, compare with adding antibiotic control group, the experimental group cost is lower, and benefit is higher.
Above content is to combine concrete preferred implementation to further explain of the present invention, can not assert that practical implementation of the present invention is confined to these explanations.Under the prerequisite that does not break away from the present invention's design, person of ordinary skill in the field of the present invention can make some deduction or replace, all should be regarded as protection scope of the present invention.
Claims (15)
1. the chemical synthesis process of a hippocampus antibacterial peptide hkplp and the application in aquaculture thereof mainly may further comprise the steps:
1) with aminoresin be carrier, connect amino acid successively, obtain hkplp-aminoresin with blocking group according to the method for solid phase synthesis, during remove the Fmoc blocking group successively;
2) downcut peptide from resin with the trifluoroacetic acid mixing solutions, handle the dry bullion that gets;
3) bullion is crossed the G10 column purification, and concentrate drying obtains finished product;
4) antimicrobial spectrum of the antibacterial peptide hkplp of mensuration synthetic;
5) minimal inhibitory concentration (MIC) of the antibacterial peptide hkplp of mensuration synthetic;
6) the antibacterial peptide hkplp with synthetic adds the application in aquaculture in the feed to.
2. according to the said method of claim 1, it is characterized in that: said aminoresin is Fmoc-aminoresin.
3. according to the said method of claim 1; It is characterized in that: solid phase synthesis connects in the amino acid process; The Fmoc blocking group removes agent with 20% piperidines/DMF; Fmoc-AA doubly measures adding by the 2-5 of condensation amount, and wherein Fmoc-AA, condensing agent ratio were thrown in the each 2-4 of condensation reaction hour in 1: 1 in molar ratio.
4. according to claim 1 and 3 said methods, it is characterized in that: in the amino acid connection procedure, each step amino acid condensation all adopts Kaise Test detection reaction, and whether condensation is complete, like coupling reaction this amino acid of repetition condensation that is positive.
5. according to claim 1 and 3 said methods, it is characterized in that: the condensing agent system adopts DIC+A or A+B+C, and wherein A is HOBt or HOAt, and B is HBTU, HATU, PyBOP or TBTU, and C is DIPEA or TMP, can adopt the mode of permutation and combination to make up each other.
6. according in the claim 1 2) said method, it is characterized in that: the trifluoroacetic acid mixing solutions is TFA: Tis: Phenol 95: 2.5: 2.5 by volume, and the reaction times is 3-4 hour.
7. according in the claim 1 2) said method, it is characterized in that: handle drying and be meant that cutting liquid concentrates or blows to doing the back and separate out solid with the ice ether, whiz.
8. according in the claim 1 3) said method, it is characterized in that: the acetum with 1% is handled the hkplp bullion, and the centrifuging and taking supernatant is crossed the G10 post.
9. according in the claim 1 3) said method, it is characterized in that: the hkplp of collection crosses post liquid through concentrating after drying.
10. according in the claim 1 4) said content, it is characterized in that: the hkplp that shows synthetic with plating method is to streptococcus aureus, intestinal bacteria K
12D
31, bacillus coli DH 5 alpha, Salmonellas, animal pathogenic escherichia coli and the isolating escherichia coli of hospital clinical case, urine enterococcus, MRSH all have good antibacterial activity.
11. according in the claim 1 5) said content, it is characterized in that: adopt the hkplp solution of the method preparation different concns of doubling dilution, join and survey OD after cultivating 16h in the culture bacteria liquid
630Value is confirmed the minimal inhibitory concentration MIC of synthetic hkplp to different strains.
12. according in the claim 1 6) said content, it is characterized in that: the application in the aquaculture is meant the application to cultivated animals, comprises that beast is herded, aquatic products and poultry etc.
13. according in the claim 1 6) said content, it is characterized in that: artificial synthetic antimicrobial peptide hkplp is added in the feed influences to the prawn growth in two seasons in spring and autumn with 200ppm, 500ppm, 1000ppm different concns.
14. according in the claim 1 6) said content, it is characterized in that: artificial synthetic antimicrobial peptide hkplp adds in the feed with conventional feed and does relatively the influence to chicken.
15. according in the claim 1 6) said content, it is characterized in that: the application of artificial synthetic antimicrobial peptide hkplp is meant as fodder additives to be added in the feed.
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Cited By (5)
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| CN103305577A (en) * | 2013-05-22 | 2013-09-18 | 宁波大学 | Preparation method of hippocampus polypeptide enzyme solution |
| CN105985442A (en) * | 2015-02-04 | 2016-10-05 | 广东中大南海海洋生物技术工程中心有限公司 | Hippocampus kuda-like antimicrobial peptide hkplpl-1 |
| CN105985407A (en) * | 2015-02-04 | 2016-10-05 | 广东中大南海海洋生物技术工程中心有限公司 | Hippocampus kuda-like antimicrobial peptide hkplpl-2 |
| CN108059682A (en) * | 2015-02-04 | 2018-05-22 | 广东中大南海海洋生物技术工程中心有限公司 | One species striped perch antibacterial peptide sb-M1-4 |
| CN111518167A (en) * | 2020-03-30 | 2020-08-11 | 东北农业大学 | Antibacterial peptide with antibacterial activity in acidic environment, and preparation method and application thereof |
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| CN103305577A (en) * | 2013-05-22 | 2013-09-18 | 宁波大学 | Preparation method of hippocampus polypeptide enzyme solution |
| CN103305577B (en) * | 2013-05-22 | 2016-01-27 | 宁波大学 | A kind of preparation method of hippocampus polypeptide enzymolysis solution |
| CN105985442A (en) * | 2015-02-04 | 2016-10-05 | 广东中大南海海洋生物技术工程中心有限公司 | Hippocampus kuda-like antimicrobial peptide hkplpl-1 |
| CN105985407A (en) * | 2015-02-04 | 2016-10-05 | 广东中大南海海洋生物技术工程中心有限公司 | Hippocampus kuda-like antimicrobial peptide hkplpl-2 |
| CN108059682A (en) * | 2015-02-04 | 2018-05-22 | 广东中大南海海洋生物技术工程中心有限公司 | One species striped perch antibacterial peptide sb-M1-4 |
| CN111518167A (en) * | 2020-03-30 | 2020-08-11 | 东北农业大学 | Antibacterial peptide with antibacterial activity in acidic environment, and preparation method and application thereof |
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