CN102295694B - Antimicrobial polypeptide, and preparation method and application thereof - Google Patents
Antimicrobial polypeptide, and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to an antimicrobial polypeptide, and a preparation method and application thereof, belonging to the field of biomedicine. The amino acid sequence of the antimicrobial polypeptide provided by the invention is disclosed as SEQ ID NO.2. The preparation method comprises the following steps: after linearizing the coding sequence of the antimicrobial polypeptide with restriction endonuclease, transforming into Pichia pastoris GS115 by an electric shock method, recombining onto the Pichia pastoris chromosome by homology, screening high-expression strains with G418, and inducing the expression with methanol, thereby obtaining the antimicrobial polypeptide. The test proves that the antimicrobial polypeptide has the functions of inhibiting and killing multiple bacteria, viruses, tumor cells, carcinomatous solid tumors, fungi and protozoa, can not easily generate resistance, and thus, can be used as an ideal antibiotic substitute, biological medicine for human and livestock, feed additive, preservative, bactericide or the like.
Description
Technical field
The invention belongs to bioengineering field, relate to preparation method and the purposes of a kind of antimicrobial polypeptide and this antimicrobial polypeptide.
Background technology
Microbiotic is for controlling, preventing and treat various infectious diseases and brought into play vital role.As time goes on and antibiotic abuse due to antibiotic use, to the eighties in 20th century, the mankind almost can conquer all infection class diseases, yet, Resistant strain is more and more many.Have monitoring to find, in the middle of the most representative Resistant strain, the staphylococcus of resistance has risen to 40%; The coagulase negative staphylococcus of resistance surpasses 77%.Now, all antibiotic resistant organisms occurred resisting, clearly, the serious threat of antibiotics resistance problem is to the mankind's health.
Removing resistant organism has been a suitable urgent problems to the day by day serious threat of the mankind, so the discovery of new class antimicrobial material and development have become the focus of research in the world.Polypeptide class antimicrobial substance is compared with now widely used traditional class microbiotic, has lot of advantages: as when the least action concentration, fast and the killing microorganisms of wide spectrum (comprising clinical anti-medicine bacterium at present), fungi also there is restraining effect, the resistant strain generative nature is little, and is all effective to local infection and systemic infection.At present, extracted multiple antibacterial peptide from different biologies.
Human alpha-defensin belongs to antibacterial peptide family, it is the one group of homeopeptide class that is present in people's M7, scavenger cell, small intestine Paneth cell, it is the cationic polypeptide that is rich in arginine residues, molecular weight is 4～6kDa, contain 6-8 Cys residue in molecule, and form 3～4 pairs of intramolecular disulfide bonds, have stable molecular structure and broad spectrum antibiotic activity.In vivo and in vitro various bacteria, virus, tumour cell, cancer solid tumor, fungi and protozoon are all had inhibitory or killing effect, and microorganism is difficult for it is produced resistance.Therefore human alpha-defensin is expected to become desirable Substitutes For Antibiotic, has huge using value.But because antibacterial peptide content in organism is not high, and extract difficulty, cost is high, therefore seek the source easily, cost is low, the high antibacterial peptide of tiring is paid attention to just widely.
Summary of the invention
First purpose of the present invention is to provide a kind of antimicrobial polypeptide;
Second purpose of the present invention is to provide the gene of this antimicrobial polypeptide of coding;
The 3rd purpose of the present invention is to provide the preparation method of this antimicrobial polypeptide;
A further object of the present invention is to provide the purposes of this antimicrobial polypeptide.
The present invention improves and optimizates on the basis to the sequence of human alpha-defensin 4, structural analysis, compares with human alpha-defensin 4, except having anti-microbial effect, also has antivirus action, and its aminoacid sequence is as shown in SEQ ID No.2.
Be to be understood that, those skilled in the art can be according to aminoacid sequence disclosed by the invention, do not affecting under its active prerequisite, replace, lack and/or increase one or several amino acid, obtain the mutant nucleotide sequence of described albumen, for example, the Leu of the 6th is replaced with Ile, perhaps at three amino acid of its terminal deletion Thr Arg Val.Therefore, antimicrobial polypeptide of the present invention comprises that also shown in SEQ ID No.2, aminoacid sequence is substituted, replaces and/or increases one or several amino acid, and what have same function derives by SEQ ID No.2 the polypeptide that obtains.For convenience of statement, with this polypeptide called after ABP-j1.Gene of the present invention comprises the nucleotide sequence of coding said polypeptide, and preferably its nucleotide sequence is as shown in SEQ ID No.1.
In addition, should be understood that the degeneracy of considering codon and the preferences of different plant species codon, those skilled in the art can use as required and be fit to the codon that specific species are expressed.
Gene of the present invention can be operably connected with expression vector, obtain to express the recombinant expression vector of polypeptide of the present invention, and then can by such as transgenic methods such as electrotransformations, described expression vector be imported host cell, obtain turning the transformant of ABP-j1 gene, for example genetic engineering bacterium.
Therefore, the present invention also comprises and contains the expression vector of above-mentioned antimicrobial polypeptide of encoding, and the host cell that has transformed this expression vector.
The present invention also provides a kind of method for preparing aforementioned polypeptides, and it is that above-mentioned expression vector is transformed Host Strains, screening positive clone, the described antimicrobial polypeptide of abduction delivering claim 1, and separation and purification.
In one embodiment of the invention, the gene (its nucleotide sequence is as shown in SEQ ID No.1) of coding aforementioned polypeptides is cloned into Expression vector pPIC9K, obtain recombinant expression vector pPIC9K-ABP-j1, and then be transformed in pichia spp (Pichia pastoris) GS115 by electric shocking method; Be incorporated on yeast chromosomal by homologous recombination; With G418 screening high expression level bacterial strain; Methanol induction is expressed.
Particularly, the present invention's method of preparing aforementioned polypeptides comprises the steps:
1, the restructured Pichia pastoris in expression Vector construction of this antimicrobial polypeptide: according to the restriction endonuclease sites characteristic on ABP-j1 nucleotide sequence and yeast expression vector pPIC9K, increase respectively EcoRI and NotI site in ABP-j1 nucleotide sequence upstream and downstream, with EcoRI and NotI double digestion, be connected with the pPIC9K that the NotI double digestion is crossed with EcoRI again, transform bacillus coli DH 5 alpha, build the yeast expression vector pPIC9K-ABP-j1 of this antimicrobial polypeptide.Extract plasmid, identify through EcoRI and NotI double digestion, order-checking.Confirm that this antimicrobial polypeptide yeast expression vector pPIC9K-ABP-j1 successfully constructs.
2, the conversion of pichia spp and screening: use restriction enzyme such as SacI, Bg1II with the recombinant plasmid linearizing, getting approximately, 5 μ l linear DNA 1 μ g mix with 200 μ l methanol yeast GS115 competent cells, then inject the 0.2cm electric shock cup of precooling, rap the electric shock cup, make mixture fall within electric shock cup bottom, setting voltage is 1500V on electric shock instrument, electric capacity is 50 μ F, electric current 25mA, electric power 25W, resistance 200 Ω carry out the electroporation operation.The 1M D-glucitol that adds immediately the 1ml precooling after electroporation, after mixing therefrom sucking-off 200 μ l coat immediately the MD D-glucitol that contains 2.0mg/ml G418 dull and stereotyped (1.34%YNB, 4 * 10
-5The % vitamin H, 2%D-glucose, 1M D-glucitol), then be inverted flat board in 28～30 ℃ of cultivations, after 2～4 days, transformant appears, filter out the high expression level bacterial strain.
The yeast colony that random picking grows is cultivated in YPD, extract Yeast genome according to the described method of Promega company's Yeast genome extraction test kit, and carry out pcr amplification ABP-j1 gene take it as template, agarose gel electrophoresis, the obvious band of visible approximately 110bp size under UV-light, with ABP-j1 nucleotide sequence in the same size, illustrate that the yeast efficient expression strain is successfully established.
3, abduction delivering: picking positive colony, be inoculated in respectively in the 50mlBMGY substratum, 30 ℃, the 250rpm shaking culture is taken out when OD600 is between 4～6, the centrifugal 5min of 1500g collects yeast cell, then is resuspended in to continue in the BMMY substratum triangular flask that 1/5 volume is housed to cultivate, after 24 hours, adding methyl alcohol to final concentration every day is 0.5～1%.Continue to cultivate 6～10 days the centrifuging and taking supernatant.
Directly get ABP-j1 yeast expression supernatant and make SDS-PAGE, electrophoresis showed has target protein to express, and protein content can reach the 500mg/L left and right after measured.Western-blotting detects visible ABP-j1 band of expression.
With standard intestinal bacteria, the mixed bacterium plate identified activity of staphylococcus aureus strains, and show that by mensuration antimicrobial polypeptide of the present invention all has stronger restraining effect to virus, common bacteria, animal acute toxicity test, systemic allergy test and long term toxicity test show that this antimicrobial polypeptide is safe, but the efficient Substitutes For Antibiotic of Using such method development of new.Antimicrobial polypeptide of the present invention can be for the preparation of antibacterials, antiviral, fodder additives, sanitas or elimination agent.And then the present invention also provides a kind of antibacterials, antiviral, fodder additives, sanitas or elimination agent that contains the described antimicrobial polypeptide of effective dose.
The inventive method can prepare this antimicrobial polypeptide in a large number, efficiently, and the Substitutes For Antibiotic that makes of method has the biological characteristics of broad-spectrum antimicrobial, antiviral, anti-tumor activity thus, and is difficult for producing resistance.
Description of drawings
Fig. 1: the structure schema of recombinant expression vector pPC9K-ABP-j1.
Fig. 2: ABP-j1 antibacterial peptide SDS-PAGE electrophorogram, wherein, 1: protein molecular weight standard; 2: induce rear supernatant; 3: induce front supernatant.
Fig. 3: ABP-j1 antibacterial peptide Western blotting detects figure, and wherein 1: the target protein band; M: protein molecular weight standard.
Embodiment
Following examples further illustrate content of the present invention, but should not be construed as limitation of the present invention.Without departing from the spirit and substance of the case in the present invention, modification or replacement to the inventive method, step or condition are done all belong to scope of the present invention.
If do not specialize, the conventional means that in embodiment, technique means used is well known to those skilled in the art.
The preparation of embodiment 1 antimicrobial polypeptide
Bacillus coli DH 5 alpha is available from precious biotechnology (Dalian) company limited, methanol yeast (Pichia pastoris) GS115, and Expression vector pPIC9K is available from Invitrogen company.
With the single colony inoculation of GS115 in 5mlYPD (1% yeast extract, 2% Tryptones, 2%D-glucose) in liquid nutrient medium, 28～30 ℃ of incubated overnight, get 100 μ l and be transferred to continuation cultivation in 50ml YPD substratum, stop cultivating when its OD600 is between 1.1～1.3, centrifugal 5000rpm 5min, collect thalline, then wash 2 times with ice-cold deionized water and D-glucitol respectively, the 1M D-glucitol that adds at last the 1ml precooling is distributed into standby methanol yeast GS115 competent cell by every pipe 200 μ l, uses immediately or be stored in-70 ℃.
According to the restriction endonuclease sites characteristic on ABP-j1 nucleotide sequence (SEQ ID No.1) and yeast expression vector pPIC9K, pass through synthetic, increase respectively EcoRI and NotI site in ABP-j1 nucleotide sequence upstream and downstream, with EcoRI and NotI double digestion, be connected with the pPIC9K that the NotI double digestion is crossed with EcoRI again (seeing accompanying drawing 1), transform bacillus coli DH 5 alpha, build the yeast expression vector pPIC9K-ABP-j1 of this antimicrobial polypeptide.Extract plasmid, identify through EcoRI and NotI double digestion, order-checking.Confirm that this antimicrobial polypeptide pichia spp recombinant expression vector pPIC9K-ABP-j1 successfully constructs.
Use respectively restriction enzyme BGIII, SacI etc. with the recombinant plasmid linearizing, line taking DNA1 μ g (about 5 μ l) (aseptic) mixes with 200 μ l yeast GS115 competent cells, then inject the 0.2cm electric shock cup of precooling, rap the electric shock cup, make mixture fall within electric shock cup bottom, setting voltage is 1500V on electric shock instrument, electric capacity is 50 μ F, electric current 25mA, electric power 25W, resistance 200 Ω carry out the electroporation operation.The 1M D-glucitol that adds immediately the 1ml precooling after electroporation, after mixing therefrom sucking-off 200 μ l coat immediately the MD D-glucitol that contains 2.0mg/mlG418 dull and stereotyped (1.34%YNB, 4 * 10
-5The % vitamin H, 2%D-glucose, 1M D-glucitol), then be inverted flat board in 28～30 ℃ of cultivations, after 2～4 days, transformant appears, filter out the high expression level bacterial strain.
The yeast colony YPD that random picking grows cultivates, extract Yeast genome according to the described method of Promega company's Yeast genome extraction test kit, and carry out pcr amplification ABP-j1 gene, upstream primer take it as template: 5 '-gtc tgc tct tgc aga tta gta-3 '; Downstream primer: 5 '-gac acg cgt gca gca gta tgt-3 ', observe under ultraviolet lamp, identify conversion results, show the purpose band and exist.
The different positive colonies of picking, be inoculated in respectively in 50ml BMGY substratum, 30 ℃, the 250rpm shaking culture is taken out when OD600 is between 4～6, the centrifugal 5min of 1500g collects yeast cell, then is resuspended in to continue in the BMMY substratum triangular flask that 1/5 volume is housed to cultivate, after 24 hours, adding methyl alcohol to final concentration every day is 0.5～1%.Continue to cultivate 6～10 days the centrifuging and taking supernatant.
Directly get ABP-j1 yeast expression supernatant and make SDS-PAGE (seeing accompanying drawing 2), electrophoresis showed has target protein to express, and protein content can reach the 500mg/L left and right after measured.Western-blotting detects visible ABP-j1 band of expression (seeing accompanying drawing 3).Sequencing shows, this peptide sequence is consistent with prediction.
The heat stability test of embodiment 2 antimicrobial polypeptides
Get each 1ml of yeast expression supernatant be placed in respectively process respectively 10,20 under 40,60,80 and 100 ℃ of water bath condition, 30min, with untreated yeast expression supernatant in contrast, take the standard gold staphylococcus aureus as indicator, measure the fungistatic effect of processing the front and back sample with the antibacterial circle diameter method.Experiment repeats 3 times, and getting its mean value is measurement result.
The antibacterial circle diameter of yeast expression supernatant to the standard gold staphylococcus aureus after table 1 heat treated
Annotate: contrast and be the antibacterial circle diameter without heat treated yeast expression supernatant.
The result of table 1 shows, is different through the processing of different heating temperature and time on the impact of yeast expression supernatant fungistatic effect, and when Heating temperature was no more than 60 ℃, along with the prolongation of heat-up time, its antibacterial circle diameter not have variation substantially; When Heating temperature is increased to 80～100 ℃, its antibacterial circle diameter increases with temperature and time lengthening is slightly dwindled, but still shows certain fungistatic effect.
The protest test of embodiment 3 antimicrobial polypeptides
Strain is intestinal bacteria K88.
The mensuration of medium lethal dose (LD50) is inoculated in intestinal bacteria K88 and contains in 0.5% glucose nutrient broth, cultivates 24h for 37 ℃, and the OD650 value is 0.765.Respectively with 0.05,0.10,0.15,0.20,0.25mL dosage abdominal injection small white mouse, death condition is observed and recorded to 5 of every kind of dose inoculations.
The death condition of table 2 small white mouse inoculation various dose bacterium liquid
| Dosage (ml/ only) | 0.05 | 0.10 | 0.15 | 0.20 | 0.25 |
| Inoculation number (only) | 5 | 5 | 5 | 5 | 5 |
| Death toll (only) | 0 | 1 | 3 | 5 | 4 |
With the increase of bacterium liquid inoculum size, the mice dying number is linear to be increased.Calculate with Reed and Meunch method, this bacterium liquid is 0.142mL to the medium lethal dose (LD50) of small white mouse.
30 of small white mouses are divided into 4 groups, be respectively blank group, strain control group, empty carrier yeast supernatant control group, antibacterial peptide yeast expression supernatant group, small white mouse is adopted the mode that gavages, empty carrier, antibacterial peptide yeast supernatant group only gavage yeast supernatant 0.5mL/ at every turn, control group only gavages physiological saline 0.5mL/ at every turn, attack front 1 week of administration of poison, every day 2 times.With the strain nutrient solution (the OD650 value is 0.765) of surveying LD50 the same generation, with the dosage intraperitoneal inoculation small white mouse of a LD50, observed and recorded clinical manifestation and death condition.
Table 3 antimicrobial polypeptide attack malicious provide protection
| Group | Number of animals (only) | Death toll (only) | Survival number (only) | Protection ratio (%) |
| The blank group | 10 | 0 | 10 | 100 |
| The strain control group | 10 | 8 | 2 | 20 |
| Empty carrier yeast supernatant control group | 10 | 8 | 2 | 20 |
| Yeast expression supernatant group | 10 | 2 | 8 | 80 |
As can be seen from Table 3, than strain and empty carrier yeast supernatant control group (20%) height, difference is (P＜0.01) extremely significantly, illustrates that antimicrobial polypeptide has the stronger malicious provide protection of attacking at yeast expression supernatant group protection ratio 80%.
The restraining effect of embodiment 4 antimicrobial polypeptides to common bacteria
Will be through (160 ℃ of dry sterilizations, filter paper 2h), put into respectively the abundant 24h of immersion of cleer and peaceful antibacterial peptide yeast expression supernatant liquor on the empty carrier yeast controls, dip for examination bacterium liquid with aseptic spreader, the surface that is coated in equably substratum is made and is contained the bacterium flat board, takes out filter paper through fully soaking and is attached to and contains on the bacterium flat board, and each culture dish pastes 1, the constant incubator that is placed in 37 ℃ is cultivated 24h, observes and measure the diameter of inhibition zone.
The antibacterial circle diameter of table 4 yeast expression supernatant to each common bacteria
| Bacteria name | Empty carrier yeast controls supernatant antibacterial circle diameter (mm) | Yeast expression supernatant antibacterial circle diameter (mm) |
| Streptococcus aureus (CVCC laboratory preservation) | 0.0 | 9.0 |
| Streptococcus aureus (CICC 10001) | 0.0 | 18.4 |
| Streptococcus aureus (CVCC 1885) | 0.0 | 19.2 |
| Sarcina (CVCC laboratory preservation) | 0.0 | 9.8 |
| Intestinal bacteria (CVCC 195) | 0.0 | 17.2 |
| Intestinal bacteria (CVCC 222) | 0.0 | 17.7 |
| Intestinal bacteria (CICC 1.907) | 0.0 | 16.1 |
| Clostridium (CICC 20464) | 0.0 | 18.3 |
| Yeast (CICC 2.120) | 0.0 | 14.7 |
| Yeast (this laboratory separating and preserving) | 0.0 | 12.3 |
| Ocean rhodotorula (CICC) | 0.0 | 18.9 |
| Yeast (laboratory separating and preserving NM6) | 0.0 | 16.6 |
| Aspergillus oryzae (CICC 3.482) | 0.0 | 11.1 |
| Bacillus cereus (CICC 10024) | 0.0 | 19.7 |
| Bacillus cereus (CICC 10041) | 0.0 | 18.6 |
| Bacstearothermophilus (CICC 10091) | 0.0 | 17.1 |
| Gall's chain coccus (CICC 1.2496) | 0.0 | 19.4 |
| Thermophilus streptococcus (CICC 1.2471) | 0.0 | 18.9 |
| Streptococcus bovis (CICC 1.1624) | 0.0 | 18.4 |
| Anthrax bacillus (CVCC laboratory preservation) | 0.0 | 9.9 |
| Bacillus subtilus (CVCC laboratory preservation) | 0.0 | 8.0 |
| Corynebacterium diphtheriae (CVCC laboratory preservation) | 0.0 | 12.5 |
| Bacillus typhi murium (CVCC laboratory preservation) | 0.0 | 13.9 |
| Sonne bacillus (CVCC laboratory preservation) | 0.0 | 11.0 |
| Vibrio cholerae (CVCC laboratory preservation) | 0.0 | 12.9 |
| Pneumobacillus (CVCC laboratory preservation) | 0.0 | 11.5 |
As seen from Table 4, antimicrobial polypeptide all has in various degree fungistatic effect to each bacterial strain.
The restraining effect of embodiment 5 antimicrobial polypeptides to virus
1, drug cell poison experiment (LD0 mensuration)
The 2 times of dilutions respectively of empty carrier yeast controls supernatant and antibacterial peptide yeast expression supernatant liquor, totally 8 extent of dilution, namely 1/2,1/4,1/8,1/16,1/32,1/64,1/128 and 1/256.Every extent of dilution liquid 0.2ml adds (every extent of dilution 3 pipes) in the cell pipe, cultivates 7 days the observation of cell pathology for 37 ℃.Result: pathology does not appear in antibacterial peptide yeast expression supernatant group the 2nd pipe (1/4) to the 8th pipe (1/256), therefore LD0=1/4.
2, virus infection minim fixed (TCIDS0 mensuration)
(I) 10 times of dilutions of influenza virus, totally 3 extent of dilution, namely 10
-1, 10
-2, 10
-3Every extent of dilution virus liquid 0.2ml adds (every extent of dilution 5 pipes) in the cell pipe, cultivates 7 days the observation of cell pathology for 37 ℃.Press Reed-Muench method calculation result: TCID=1
-3.2..
(II) 10 times of dilutions of simplexvirus, totally 3 extent of dilution, namely 10
-1, 10
-2, 10
-3Every extent of dilution virus liquid 0.2ml adds in the cell pipe (every extent of dilution 5 pipes), and 37 ℃ of 1h are hatched, after suck virus liquid, hand \ S liquid is washed 3 times, adds maintenance medium, cultivates 7 days the observation of cell pathology for 37 ℃.Press Reed-Muench method calculation result: TCID50=1
-3.2
(III) sample is to virus function
Mix with LD0 sample and each 1ml of 100TCID50 virus liquid, put 37 ℃ and cultivated 7 days, the observation of cell pathology.Do simultaneously virus control, cell contrast, empty carrier yeast supernatant sample and the contrast of antibacterial peptide yeast supernatant sample, influenza virus mdck cell, simplexvirus HEP-2 cell.
The result demonstration, cytopathy all appears in each virus and empty carrier yeast supernatant control tube 24h; The cell control tube all occurred without pathology in 7 days; Medicine control tube 7 days is acellular pathology all; The influenza virus developmental tube began to occur cytopathy on the 5th day, therefore the sample infected by influenza has certain restraining effect; The simplexvirus developmental tube began to occur cytopathy on the 6th day, therefore sample has certain restraining effect to simplexvirus.
Show by high temperature resistant experiment, protest test, bacteriostatic experiment, antiviral experiment etc., antimicrobial polypeptide is a kind of stable broad-spectrum antimicrobial peptide, and gram-positive microorganism is had stronger lethality, and this may be to be determined by the kind of antibacterial peptide and characteristic.Simultaneously, although antimicrobial polypeptide has broad spectrum antibiotic activity, it may be variant on killing-efficiency for different bacterium bacterium even of the same race homophyletic not.
The acute toxicity test of embodiment 6 antimicrobial polypeptides
1, antimicrobial polypeptide mouse stomach acute toxicity test
Get 20 of healthy Kunming mouses, male female half and half, press 0.5ml/10g (but mouse maximum gavage amount) single gavage with the antimicrobial polypeptide of 50% (but antimicrobial polypeptide maximum gavage concentration) after fasting at night 12h.Result is, after medicine in 10 days tested mouse any toxic reaction does not all appear, none death of duration of test animal.But because being subjected to the restriction of drug level and medication volume, can not find the lethal dose of antimicrobial polypeptide mouse single gastric infusion, can't measure its LD50 value.
Separately get 40 Kunming mouses, be divided at random two dosage groups of A and B, every group of 20 mouse, male female half and half.But after fasting at night 12h with interval in peak concentration maximum dosage one day 5 hours gastric infusion 2 and 3 times respectively.Each gavage capacity is every 10g body weight 0.5ml.
A group: in interval 5 hours a day, gastric infusion is 2 times, and its day, total dosage was 25 * 2g/kg;
B group: in interval 5 hours a day, gastric infusion is 3 times, and its day, total dosage was 25 * 3g/kg.
The tight observation 10 after medicine.Observation item comprises proterties and the color of active situation, the mental status, breathing situation, fur situation, urine and ight soil, and nose, eye, oral cavity have or not the abnormal secretion thing, has or not the intoxicating phenomenons such as calmness, excitement, tic, cyanosis, has or not death.Record feed consumption every day of tested mouse.
Result is, any unusual phenomenon does not all appear in proterties and the color of duration of test A, B two groups of tested mouse activities, the mental status, breathing situation, fur, urine and ight soil, and nose, eye, oral cavity occur without dead without abnormal secretory product.Body weight and feed consumption there are no significant difference every day (table 5-6) of tested mouse is respectively organized in test.Test end dead mouse, anatomic observation have no the important organ such as the heart, liver, spleen, lung, kidney of tested mouse and occur any abnormal.
The impact of table 5 antimicrobial polypeptide gastric infusion on Mouse Weight
The impact of table 6 antimicrobial polypeptide gastric infusion on the mouse feed consumption
According to this test, antimicrobial polypeptide mouse single gastric infusion fails to cause that any toxic reaction appears in tested mouse, and none death of tested mouse is therefore can't measure the LD50 value.3 gastric infusions in interval 5 hours a day, in one day, total dosage is 75g/kg, any toxic reaction does not appear in tested mouse.
2, antimicrobial polypeptide general sensitivity test
36 of undercoat cavys are planted by Britain, and the male and female dual-purpose is divided into 4 groups at random, 9 every group.Each group is respectively by following method administration: the salt solution group: the next day abdominal injection (ip) physiological saline 0.5ml/ only, totally 3 times; The administration group: the next day abdominal injection (ip) antimicrobial polypeptide 0.5ml/ only, totally 5 times; Chymotrypsin group (positive controls): the next day ip Chymotrypsin (2000U/ml) 0.5ml/ only, totally 3 times.After the 1st sensitization the 14th, the 21st, the 28th, get respectively 3 for every group and only carry out challenge trial through the above-mentioned relative medicine 1ml/ of the quiet notes of cavy hind leg small saphenous vein.In 15min, observe cavy and anaphylaxis whether occurs after injection.Mark by table 7 pair cavy anaphylaxis.
Table 7 cavy anaphylaxis standards of grading
Table 8 antimicrobial polypeptide cavy systemic administration anaphylaxis test
Table 8 result shows, antimicrobial polypeptide is identical with the salt solution group, and the 14th day, the 21st day, the 28th day tested liquid of intravenous injection attacked after the 1st injection sensitization, has no in 15min any symptoms of allergic to occur, and cavy breathes freely, and is normally movable.After Chymotrypsin group cavy sensitization, only excite in the 14th day, the 21st day, the 28th day intravenous injection Chymotrypsin (2000U/ml) 1ml/, 9 animals expiratory dyspnea, spasm, tic all occur to severe allergic reactions such as death as a result.Result shows that antimicrobial polypeptide does not cause the cavy anaphylaxis under this test conditions.
Embodiment 7 antimicrobial polypeptide long term toxicity tests
Antimicrobial polypeptide is intended clinical daily dosage portion 0.25 μ g/ (kgd).Be about 1.5 μ g/ (kgd) according to the rat dose,equivalent of body surface area conversion; The basic, normal, high dosage group of antimicrobial polypeptide and control group are established in this test, and the dosage of basic, normal, high dosage group is respectively 0.01,0.03,0.10mg/kg.
120 rats on average are divided into 4 groups of Normal group, the basic, normal, high dosage groups of antimicrobial polypeptide at random, 30 every group, male female half and half.Rat begins test after the indoor normal feed of experiment raised for 2 weeks.
Every day entry food consumption quantity, and observe in detail tested rat and have or not abnormal response.Measure body weight per weekend one time.Duration of test is if any animal dead or be at death's door, and in time carries out anatomic observation, and carries out the system organization morphological examination.Put to death 1/3 surviving animals in the 91st day each group of administration; In the 181st day (drug withdrawal day) of medication, each being organized 1/2 of institute's survival rats puts to death; Remaining each treated animal all raises not contain the normal feed of antimicrobial polypeptide, all puts to death afterwards in 15 days.The execution program is abdominal injection (ip) urethane 1g/kg anesthesia, then processes in the following order rat:
1. hematological examination, blood biochemistry checking are carried out in blood sampling;
2. dissect and check each internal organs;
3. put to death rat, core, liver, spleen, lung, kidney, suprarenal gland, thymus gland, pancreas, testis or uterus, weigh, fixing, and carry out histological examination.
1, the impact of antimicrobial polypeptide on the general status of rat
Whole duration of test, the general state of the basic, normal, high dosage group of antimicrobial polypeptide rat is good, and spirit, behavioral activity, diet, urine, stool, hair color gloss, breathing etc. are showed no any abnormal; Each organizes none death of rat.Table 9 and table 10 result show, the body weight of antimicrobial polypeptide medication group and food consumption quantity and concurrent control group are than there are no significant difference.
Table 9 antimicrobial polypeptide ig is to rat body weight
Impact
Between each group, the same period is more all without statistical significant difference.
Table 10 antimicrobial polypeptide ig is on the impact in (g/ group/week) of rat food consumption quantity
Between each group, the same period is more all without statistical significant difference.
2, the impact of antimicrobial polypeptide on the rat blood index
The check result of each test group different time physiochemical indice sees Table 11 and table 12.Continuously ig antimicrobial polypeptide 90 days, 180 days low, in and the high dose group rat blood learn index and blood parameters and control group than there are no significant difference; After 2 weeks of drug withdrawal, between each group, hematology and blood biochemistry checking result also are showed no significant difference.
Table 11 (1) antimicrobial polypeptide ig is to the rat blood index
Impact
Table 11 (2) antimicrobial polypeptide ig is to the rat blood index
Impact
Between each group, the same period is more all without statistical significant difference.
Table 12 (1) antimicrobial polypeptide ig is to the rat blood biochemical indicator
Impact
Table 12 (2) antimicrobial polypeptide ig is to the rat blood biochemical indicator
Impact
Between each group, the same period is more all without statistical significant difference.
TP: blood plasma total protein, ALB: albumin, GLU: blood sugar, T-CHO blood total cholesterol, TBIL: total bilirubin, AST: aspartate amino transferase, ALT: alanine aminotransferase, ALP: alkaline phosphatase, CREA: creatinine, BUN: blood urea nitrogen.
3, each internal organs visual inspection and histological examination
The medication dissection finding of naked eye result of respectively organizing rat that was condemned to death in the 91st day and the 181st day is, antimicrobial polypeptide is low, in and high dose group animal thoracic cavity, abdominal cavity, pericardial cavity without hydrops, the internal organs colors such as brain stem, heart, liver, spleen, lungs, kidney, spleen, stomach and intestine, Tiroidina, suprarenal gland, testis, ovary, uterus are normal, without enlargement, hemostasis, the phenomenon such as hemorrhage, downright bad.Each each internal organs visual control of organizing rat the results are shown in Table 13.Table 14 result shows each organ coefficient (g/100g body weight) difference that there are no significant of respectively organizing rat.Histological examination result (table 15) is, antimicrobial polypeptide is low, in and heart, liver, spleen, lungs, kidney, thymus gland and the suprarenal gland of high dose group rat do not check out that all any specificity toxicity changes, the abnormal incidence of histology there are no significant difference between each group.
The visual inspection of each treated animal internal organs of table 13 antimicrobial polypeptide ig
Each the group between more all without statistical significant difference (X
2Check).
Table 14 (1) antimicrobial polypeptide ig to each organ coefficient of rat (the g/100g body weight,
) impact
Between each group, the same period is more all without statistical significant difference.
Each organ coefficient of table 14 (2) antimicrobial polypeptide ig rat
Impact
Between each group, the same period is more all without statistical significant difference.
Table 15 antimicrobial polypeptide ig is on the pathological impact of the important viscera tissue of rat
Each the group between more all without statistical significant difference (X
2Check).
4, the restorative experimental observation of toxic action
After drug withdrawal, until drug withdrawal the 15th day, each organizes general state, spirit, activity, food consumption quantity, the body weight of rat there are no significant difference; It is any abnormal that the every inspection of blood does not find that all antimicrobial polypeptide medication group has; Each is organized Rats Organs and Tissues visual inspection, organ coefficient and histological examination and also is showed no any significant difference (table 9-15).
There are no significant the impact of antimicrobial polypeptide 0.01,0.03,0.10mg/ (kgd) ig 180 days general status on tested rat, each index of hematology, each index of blood biochemical and each important organ tissue morphologies.Decubation, observations showed, antimicrobial polypeptide fails to cause any delayed toxicity reaction.Therefore this test prompting, the antimicrobial polypeptide long-term prescription is safe.
The application of embodiment 8 antimicrobial polypeptide of the present invention in the animal intestinal disease treatment
150 7 age in days broiler chicken are divided into three groups of A, B, C at random, 50 every group, every group of 5 repetitions, each repeats 10 chickens.Test early stage takes pathogenic white dysentery Salmonellas and attacks poison to mix, the 2d that feeds continuously, and the A group is control group, do not do any treatment in whole trial period and process, the B group is for adding antimicrobial polypeptide of the present invention, and duration of test is to add in feed at 25mg/kg.BW, early, late each 1 time, mix continuously and take 3d, the processing of normally feeding afterwards in 3 days, the C group is the antibiotic treatment group, 50mg/kg.BW gavages with aminobenzylpenicillin, early, evening each 1 time, continuous 3d, the processing of normally feeding afterwards in 3 days.The whole test period is 10d, the ill and death condition of observed and recorded chicken, and trial period, finishes laggard line number and analyzes according to statistics (seeing Table 16).
The impact of table 16 antimicrobial polypeptide on the broiler chicken intestinal tract disease
Test-results shows, antimicrobial polypeptide is better than microbiotic to the prevention effect of the microbial intestinal tract disease of causing a disease, and can effectively replace antibiotic therapeutic action, more is conducive to food safety, is antibiotic ideal substitute.
Claims (10)
1. an antimicrobial polypeptide, is characterized in that, the aminoacid sequence of described antimicrobial polypeptide is as shown in SEQ ID No.2.
2. the gene of coding claim 1 described antimicrobial polypeptide.
3. gene as claimed in claim 2, is characterized in that, the nucleotide sequence of this gene is as shown in SEQ ID No.1.
4. the expression vector that contains claim 2 or 3 described genes.
5. the host cell that is transformed by the described expression vector of claim 4.
6. host cell as claimed in claim 5, it is pichia spp.
7. method for preparing the described antimicrobial polypeptide of claim 1, it comprises the steps: expression vector claimed in claim 4 is transformed Host Strains, screening positive clone, the described antimicrobial polypeptide of abduction delivering claim 1, and separation and purification.
8. method as claimed in claim 7, is characterized in that, described Host Strains is Pichia pastoris GS115.
9. the application of the described antimicrobial polypeptide of claim 1 in preparation antibacterials, antiviral, fodder additives, sanitas or elimination agent.
10. the antibacterials, antiviral, fodder additives, sanitas or the elimination agent that contain the described antimicrobial polypeptide of claim 1.
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| CN107586329B (en) * | 2017-11-03 | 2020-08-28 | 宁波大学 | Polypeptide separated from pomfret |
| CN110526960B (en) * | 2018-05-24 | 2021-04-16 | 中国农业大学 | Antiviral polypeptide and preparation method and application thereof |
| CN109512692B (en) * | 2018-12-29 | 2021-09-07 | 浙江国能科技有限公司 | Antibacterial additive solution for wet tissue and preparation method thereof |
| CN112812159B (en) * | 2021-01-22 | 2022-11-08 | 杭州吾尾科技有限公司 | Yeast polypeptide derived from saccharomyces boulardii as well as preparation method and application thereof |
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| Title |
|---|
| AGNIESZKA SZYK et al.Crystal structures of human alpha-defensins HNP4, HD5, and HD6.《Protein Science》.2006, |
| Crystal structures of human alpha-defensins HNP4, HD5, and HD6;AGNIESZKA SZYK et al;《Protein Science》;20061231;2749–2760 * |
| 人防御素的研究进展及其应用前景;李春丽等;《中国生物制品学杂志》;20070228;第20卷(第2期);146-149 * |
| 李春丽等.人防御素的研究进展及其应用前景.《中国生物制品学杂志》.2007,第20卷(第2期), |
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