CN105001308B - A kind of antibacterial peptide PD22 and application thereof - Google Patents
A kind of antibacterial peptide PD22 and application thereof Download PDFInfo
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- CN105001308B CN105001308B CN201510519074.0A CN201510519074A CN105001308B CN 105001308 B CN105001308 B CN 105001308B CN 201510519074 A CN201510519074 A CN 201510519074A CN 105001308 B CN105001308 B CN 105001308B
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Abstract
The invention discloses a kind of antibacterial peptide PD22 and application thereof.The amino acid sequence of the antibacterial peptide PD22 such as SEQ ID NO:Shown in 1.Antibacterial peptide PD22 in the present invention have small molecular weight, artificial synthesized convenience and it is at low cost, strong to the bacteriums bactericidal effect such as Escherichia coli, salmonella and clostridieum welchii, cytotoxicity is low, and application can significantly reduce diarrhea of weaned piglets rate and improve the speed of growth of weanling pig in weanling pig feed the advantages of.
Description
Technical field
The present invention relates to genetic engineering fields more particularly to a kind of antibacterial peptide PD22 and application thereof.
Background technology
In order to improve sow reproductive efficiency and reduce the vertical transmission of Disease in Infants disease, China pig farm piglet is typically chosen
3-4 week old weans, and the process that weans typically occurs in 3-4 months in nature.Early weaning piglet causes huge to piglet
It is big stress, piglet and sow separate, source of nutrition becomes the variation of solid feed, mixed group and environment from the breast milk of liquid,
And early-weaned piglets digestive system and developing immune system and not perfect, it is easy to be infected disease by the attack of various pathogens
Disease, wherein disease of digestive tract are one of most common, most serious disease and lead to the even death of weanling pig growth inhibition
Major reason.For a long time, it is the main means of prevention and treatment diarrhea of weaned piglets using antibiotic, but antibiotic is big
Amount use causes pathogenic bacteria resistance to drugs constantly to enhance, antibiotic usage effect constantly weakens, and live pig produce in antibiotic length
Phase is largely had become using caused livestock products medicament residue and the enhancing of harmful bacteria drug resistance threatens the great of human security
Hidden danger.Therefore, find can substitute antibiotics solve the problems, such as that the product of weanling pig intestines problem is current Animal nutrition research
In hot subject.Antibacterial peptide is the micromolecule active polypeptide being widely present in nature biotechnology, has has a broad antifungal spectrum, thermostabilization
Property it is strong and the features such as be not likely to produce bacterial drug resistance, be substitute antibiotics preferably potential substance.However, natural antibacterial
Peptide mostly with molecular weight is big, antibacterial activity is not high, to host cell with toxic side effect or the defects of host's haemolysis can be caused,
Popularization and application of the antibacterial peptide in weanling pig cultivates are limited to a certain extent.It is to overcome day by MOLECULE DESIGN and transformation
So come derived antimicrobial peptide defect, the effective means that antibacterial peptide is promoted to be applied in weanling pig cultivates.
Invention content
The purpose of the present invention is to provide a kind of new antibacterial peptides.
To achieve the above object, the present invention provides a kind of antibacterial peptide PD22, it is characterised in that：The ammonia of the antibacterial peptide PD22
Base acid sequence such as SEQ ID NO:Shown in 1.Specially：KKIVKAALQAATQ, molecular weight 1369.67Da, isoelectric point are
10.30。
The present invention also protects the purposes of the antibacterial peptide PD22, which is characterized in that the purposes is the purposes of antibacterial.
Further, the antibacterial is anti-Escherichia coli, salmonella and/or clostridieum welchii.
Further, the purposes is the purposes for raise poultry.
Further, the poultry is pig, chicken, duck or goose.
Further, the pig is suckling piglet, weanling pig, grower pigs or boar.
Further, the chicken is broiler chicken or laying hen.
Further, the purposes refers to improving weanling pig daily gain and reduces feed-weight ratio.
Further, the purposes refers to reducing diarrhea of weaned piglets rate.
Further, the purposes refers to improving weanling pig function of intestinal canal.
The invention has the advantages that：Antibacterial peptide PD22 in the present invention have small molecular weight, artificial synthesized convenience and into
The advantages such as this is low, strong to the bacteriums bactericidal effect such as Escherichia coli, salmonella and clostridieum welchii, cytotoxicity is low, it is young in wean
Application can significantly reduce diarrhea of weaned piglets rate and improve the speed of growth of weanling pig in pannage.
The embodiment of the present invention is by measuring antibacterial peptide PD22 minimal inhibitory concentrations (minimal inhibitory
Concentration, MIC) reflect its antibacterial effect, minimal inhibitory concentration be can't detect bacterial growth minimum sample it is dense
Degree.The result shows that the present invention antibacterial peptide PD22 have to Escherichia coli, clostridieum welchii, salmonella and bacillus subtilis it is non-
The lethal effect of Chang Qiang.On cytotoxicity experiment, even if under the concentration of 300ug/mL, antibacterial peptide PD22 is to chitterlings epithelium
There is also the cellulotoxic side effects of very low (being less than 10%) by cell IPEC-1.Compared with negative control, antibacterial peptide PD22 can be notable
Reduce diarrhea of weaned piglets rate.Antibacterial peptide PD22 is remarkably improved weanling pig daily gain and reduces feed-weight ratio.It significantly improves disconnected
Milk Jejunum of Piglets and ileum height of naps and the ratio of height of naps and Crypt depth illustrate that antibacterial peptide PD22 can be significantly improved
Weanling pig function of intestinal canal.
Description of the drawings
Fig. 1 is antibacterial peptide PD22 cytotoxicity analysis figures；
Fig. 2 antibacterial peptides PD22 is to the influence figure of challenge with E.coli weanling pig (24 age in days) diarrhea；
Fig. 3 antibacterial peptides PD22 is to the influence figure of 24 age in days diarrhea of weaned piglets rates.
Specific embodiment
The embodiment of the present invention is described below in detail, the example of the embodiment is shown in the drawings, wherein from beginning to end
Same or similar label represents same or similar element or the element with same or like function.It is not specified in embodiment
Particular technique or condition person carry out according to the described technology of document in the art or condition or according to product description.
Reagents or instruments used without specified manufacturer, being can be with conventional products that are commercially available.
Embodiment 1：The preparation of antibacterial peptide PD22
The chemical synthesis process of antibacterial peptide PD22：The amino acid sequence according to invention content, is closed with full-automatic polypeptide
Cheng Yi (ABI433) synthesis PD22 complete sequences (Hunan Normal University's Life Science College synthesis), and pass through HPLC reversed phase column chromatographies
Desalting and purifying；
The antibacterial peptide PD22 molecular weight of purifying uses Matrix-Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF)
It measures, isoelectric point is measured using isoelectric focusing electrophoresis, and amino acid sequence is parsed using automatic determined amino acid sequence instrument.
Antibacterial peptide PD22, the antibacterial peptide PD22 are the active peptides of engineer's synthesis, residual comprising 13 amino acid
Base, molecular weight 1369.67Da, isoelectric point 10.30, amino acid sequence are KKIVKAALQAATQ (SEQ ID NO:1).
Embodiment 2：Antibacterial peptide PD22 bacteriostatic experiments
By measuring antibacterial peptide PD22 minimal inhibitory concentrations (minimal inhibitory concentration, MIC)
Reflect its antibacterial effect, minimal inhibitory concentration is that can't detect the minimum sample concentration of bacterial growth.Using doubling dilution,
Specific method is as follows：
Test bacteria is inoculated on LB solid mediums, and culture is then inverted in 37 DEG C of incubators.After bacterium colony is grown,
It is dropped down onto in LB fluid nutrient mediums with each test bacteria single bacterium of disposable ring picking, shake culture is to logarithm in 37 DEG C of incubators
Growth period.Bacterium solution OD600 is detected on ultraviolet specrophotometer, according to 1OD600=1 × 109CFU/mL is by tested bacterium bacterium solution liquid
Body LB culture mediums are diluted to 2 × 105CFU/mL.Then 100 μ L LB fluid nutrient mediums are added in sterile each hole of 96 orifice plate, so
Added in A1 holes afterwards 100 μ L be diluted to certain density antibacterial peptide PD22 samples (antibacterial peptide sample add in before with 0.22 μm it is micro-
Hole membrane filtration degerming), 100 μ L is taken after mixing to add in A2 holes, 100 μ L are taken to add in A3 holes after the mixing of A2 holes again, multiple proportions is dilute successively
It releases, 100 μ L finally, which are sucked out, from A12 holes discards, and so far bis- times of concentration gradient samples of antibacterial peptide PD22 are made.To being previously added antibacterial peptide
The 100 μ L mixings of bacterium solution diluted are sequentially added in each hole of PD22, in 37 DEG C of slow shake culture 16h, are then measured
Light absorption value at 600nm.As a result it calculates：Take antibacterial in the hole that can't detect bacterial growth and the hole for having bacterial growth adjacent thereto
The average value of the sum of peptide PD22 concentration is as antibacterial peptide PD22 minimal inhibitory concentrations.Test result is shown in Table 1.It can from table 1
To find out antibacterial peptide PD22 to Escherichia coli, clostridieum welchii, salmonella and bacillus subtilis (Chinese Academy of Sciences's subtropical zone agriculture
Industry ecological Studies are provided) there is very strong lethal effect.
Table 1：Antibacterial peptide PD22 is to the minimal inhibitory concentration table of each bacterium
| Tested strain | PD22MIC(ug/mL) |
| Escherichia coli 09 | 2.34 |
| Escherichia coli 064 | 9.83 |
| Escherichia coli 0141 | 4.7 |
| Salmonella | 4.7 |
| Clostridieum welchii | 9.83 |
Note：1) minimal inhibitory concentration of wherein PD22 MIC, that is, antibacterial peptide PD22, Escherichia coli 09,064 and of Escherichia coli
Escherichia coli 0141 represent the pathogenic escherichia coli that serotype is 09,064 and 0141 respectively.
2) result is five independent average values for repeating experimental result.
Embodiment 3：The cytotoxicity experiment of antibacterial peptide PD22
Chitterlings epithelial cell IPEC-1 (the Institute of Subtropical Agriculture, The Chinese Academy of Sciences's offer) is incubated at containing 5%
Too cow's serum, 200U/mL dual anti-(penicillin and each 100U/mL of streptomysin), the HAM ' S/ of 5ug/L epidermal growth factor (EGF)
Culture after cleaning three times with phosphate buffer (HyClone), adds in 0.25% (2.5g/L) to logarithmic phase in F12 culture mediums
Trypsin digestion to individual cells, 400g is centrifuged 4 minutes after adding in HAM ' S/F12 culture mediums, is abandoned after supernatant with fresh
HAM ' S/F12 culture medium suspension cells, it is 1 × 10 to adjust to cell density6A/mL, then toward 96 porocyte culture plates per hole
Middle addition 200uL, after cell is adherent add in various concentration antibacterial peptide PD22, be placed in carbon dioxide incubator (37 DEG C, 5%
CO2) after culture 24 hours, per 20uL CCK-8 (the green skies) CCK-8 solution is added in hole, continue culture 2 hours, then use enzyme
Mark the light absorption value of instrument detection 450nm wavelength.(wherein PD22-300 represents 300ug/mL antibacterial peptides to testing result as shown in Figure 1
PD22；PD22-200 represents 200ug/mL antibacterial peptides PD22；PD22-100 represents 100ug/mL antibacterial peptides PD22；PBS is phosphoric acid
Salt buffer (control group), average value of the experimental result for five independent experiment results, although from figure 1 it appears that
Antibacterial peptide PD22 cytotoxicity increases as it adds concentration, but the cell toxicant very little of antibacterial peptide PD22.Even if
Under the concentration of 300ug/mL, there is also the cell toxicants of very low (being less than 10%) to chitterlings epithelial cell IPEC-1 by antibacterial peptide PD22
Side effect.
Embodiment 4：Antibacterial peptide PD22 tests weanling pig (24 age in days) challenge with E.coli
Choose 24 age in days Durocs × length it is white × great Bai weanling pigs 240, designed using single factor experiment, by weight and
Gender assigns to 4 processing groups at random, and negative control group is basic daily ration, and PD22-50 groups are that addition 50mg/kg resists in basic daily ration
Bacterium peptide PD22；PD22-100 groups are that 100mg/kg antibacterial peptide PD22 are added in basic daily ration, and positive controls are in basic daily ration
100mg/kg aureomycin is added, every group of 6 repetitions each repeat 10 pigs, and experimental period is 14 days.Each processing group weaned piglet is worked as
It starts to feed different daily rations, starts to attack poison after 4 days, every piglet presses total Escherichia coli 1 × 109A cell point is given for 2 times sooner or later
Clothes culture solution (the shake culture acquisition in 48 hours in 37 DEG C of constant incubators by the LB culture solutions of inoculation Escherichia coli) twice.It is anti-
Fig. 3 is shown in influences of the bacterium peptide PD22 to challenge with E.coli diarrhea of weaned piglets rate.Wherein PD22-50 represents to add in basal diet
50mg/kg antibacterial peptides PD22；PD22-300 represents addition 100mg/kg antibacterial peptides PD22 in basal diet；Based on negative control
Daily ration；Positive control is to add 100mg/kg aureomycin in basic daily ration.It can be seen that from the result of the test of Fig. 2：It is right with feminine gender
Photograph ratio, antibacterial peptide PD22 can significantly reduce diarrhea of weaned piglets rate.
Embodiment 5：Influences of the antibacterial peptide PD22 to Growth Performance of Weaning Piglets and intestinal health
Choose 24 age in days Durocs × length it is white × great Bai weanling pigs 240, designed using single factor experiment, by weight and
Gender assigns to 3 processing groups at random, and control group, which does not add, adds antibacterial peptide PD22 in antibacterial peptide PD22, PD22-45 group daily ration
45mg/kg；Antibacterial peptide PD22 90mg/kg are added in PD22-90 group daily rations, every group of 8 repetitions each repeat 10 pigs, experiment
10 days phases.Basal diet formula is shown in Table 2.Antibacterial peptide PD22 is to Production Performance of Weaning Pigs, diarrhea rate and intestinal structure
Fig. 3 and table 3-4 are shown in influence.From the result of the test of table 2 can be seen that antibacterial peptide PD22 be remarkably improved weanling pig daily gain and
Reduce feed-weight ratio.It can be seen that antibacterial peptide PD22 can significantly reduce diarrhea of weaned piglets rate from the result of the test of Fig. 3.From table 3
Result of the test can be seen that antibacterial peptide PD22 can significantly improve weanling pig jejunum and ileum height of naps and height of naps with
The ratio of Crypt depth illustrates that antibacterial peptide PD22 can significantly improve weanling pig function of intestinal canal.
Table 2：Basal diet forms table
Composite vitamin formula：Vitamin A 4g, vitamin D 31g, vitamin E 16g, vitamin k 0.6g, vitamin
B1 0.8g, vitamin B2 1.7g, vitamin B6 1g, vitamin B12 1.3g, niacin 15g, D-VB5 calcium 5g, biotin
0.5g supplies zeolite powder to 1000g.
Composite mineral matter formula：Cupric sulfate pentahydrate 102g, ferrous sulfate 61g, monohydrate zinc sulphate 20g, manganese sulfate 30g contain
The sodium selenite 4g of selenium 1%, the calcium iodate 10g containing iodine 1%, supplies zeolite powder to 1000g.
Table 3：Antibacterial peptide PD22 is to the influence table of 24 age in days Growth Performance of Weaning Piglets
| Project | Control group | PD22-45 groups | PD22-90 groups |
| Daily gain (g/d) | 285.3 | 307.2 | 327.9 |
| Daily ingestion amount (g/d) | 373.7 | 374.8 | 383.6 |
| Feedstuff-meat ratio | 1.31 | 1.22 | 1.17 |
Table 4：Antibacterial peptide PD22 is to the influence table of 24 age in days weanling pig intestinal structures
Although the embodiments of the present invention has been shown and described above, it is to be understood that above-described embodiment is example
Property, it is impossible to limitation of the present invention is interpreted as, those of ordinary skill in the art are not departing from the principle of the present invention and objective
In the case of can make changes, modifications, substitutions and variations to the above described embodiments within the scope of the invention.
Claims (10)
1. antibacterial peptide PD22 is used to prepare the purposes of the drug with antibacterial functions, the amino acid sequence of the antibacterial peptide PD22 is such as
SEQ ID NO:Shown in 1.
2. antibacterial peptide PD22 is used to prepare the purposes of the drug with antibacterial functions described in claim 1, which is characterized in that described
Antibacterial is anti-Escherichia coli, salmonella and/or clostridieum welchii.
3. antibacterial peptide PD22 is used to prepare the purposes of the drug with antibacterial functions described in claim 1, which is characterized in that described
Drug is used for raise poultry.
4. antibacterial peptide PD22 is used to prepare the purposes of the drug with antibacterial functions described in claim 3, which is characterized in that described
Poultry is chicken, duck or goose.
5. antibacterial peptide PD22 is used to prepare the purposes of the drug with antibacterial functions described in claim 1, which is characterized in that described
Drug is used to raise pig.
6. antibacterial peptide PD22 is used to prepare the purposes of the drug with antibacterial functions described in claim 5, which is characterized in that described
Pig is suckling piglet, weanling pig, grower pigs or boar.
7. antibacterial peptide PD22 is used to prepare the purposes of the drug with antibacterial functions described in claim 4, which is characterized in that described
Chicken is broiler chicken or laying hen.
8. antibacterial peptide PD22 is used to prepare the purposes of the drug with antibacterial functions described in claim 6, which is characterized in that described
Drug is used to improve weanling pig daily gain and reduces feed-weight ratio.
9. antibacterial peptide PD22 is used to prepare the purposes of the drug with antibacterial functions described in claim 6, which is characterized in that described
Drug is for reducing diarrhea of weaned piglets rate.
10. antibacterial peptide PD22 is used to prepare the purposes of the drug with antibacterial functions described in claim 6, which is characterized in that institute
Drug is stated for improving weanling pig function of intestinal canal.
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Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106883289B (en) * | 2016-02-03 | 2020-02-04 | 亚太星原农牧科技海安有限公司 | Antibacterial peptide BV21 |
| CN106883288B (en) * | 2016-02-03 | 2020-08-14 | 辽宁傲农饲料有限公司 | Antibacterial peptide BV21 and application thereof |
| CN107722114A (en) * | 2016-07-07 | 2018-02-23 | 胡国田 | A kind of polypeptide and its application |
| CN106036185A (en) * | 2016-07-28 | 2016-10-26 | 四川昕泰科技有限公司 | Laying hen feed additive |
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