CN103539850A - Peptide separately separated from pig and use thereof - Google Patents
Peptide separately separated from pig and use thereof Download PDFInfo
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- CN103539850A CN103539850A CN201210245816.1A CN201210245816A CN103539850A CN 103539850 A CN103539850 A CN 103539850A CN 201210245816 A CN201210245816 A CN 201210245816A CN 103539850 A CN103539850 A CN 103539850A
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4723—Cationic antimicrobial peptides, e.g. defensins
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- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
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Abstract
The invention provides a peptide separately separated from a pig and use thereof. The peptide comprises an amino acid sequence shown in a sequence table SEQ ID NO:1, has bacteriostatic and bactericidal activities, and can be applied to treatment of infection caused by escherichia coli (Escherichia coli) or staphylococcus aureus (Staphylococcus aureus).
Description
Technical field
The present invention about a kind of from the independent separated peptide of pig, in detail, about a kind of peptide with antibacterial and fungicidal activity.
Background technology
For solving animal intestinal functional disorder, using microbiotic be current the most frequently used method, yet excessively uses that microbiotic can cause that bacterium develops immunity to drugs, the problem such as drug residue and environmental pollution.
Antibacterial peptide (antimicrobial peptide, AMPs) gradually coming into one's own in recent years, as far back as generation nineteen fifty, just there is scientist to find in the blood of different animals, existence has the material of anti-microbial activity, finds afterwards the chance that AMPs that insect scavenger cell can be secreted positively charged infects to reduce bacterium to extracellular.Nineteen eighty-three, Lehrer and Selsted are purified into two kinds and have the AMPs that three pairs of cysteine residues form disulfide linkage in rabbit lungs scavenger cell, and by its called after alexin (defensin).The people such as Zilbauer in 2005 find that enteron aisle is subject to bacterium initial infection, alexinic amount can promote to adjusted, in addition also there are many evidences to show, the running balance that the alexin of lower concentration or inactivation can affect in intestines causes enteron aisle chronic disease, as Crohn disease and ulcerative colitis (Wehkamp et al., 2005).
Antibacterial peptide is distributed in vertebrates, non-vertebrates, plant, bacterium and fungi, belongs to a wherein part for innate immunity reaction.Alexin is one of AMPs family, and tool is anti-microbial activity widely, and the chitling road alexin (Porcine β-defensin2, pBD2) of take is example, and many pig pathogenic bacterias have very high susceptibility to pBD2.
AMPs has the mechanism of action that physical property is destroyed bacterial cell membrane, therefore for antibiotic weapon in ancient body, there are great potentiality can assist antibiotic use people such as (, 2008) Linde, but with the somewhat expensive of chemosynthesis AMPs, therefore producing the strategy of AMPs, exploitation will have prospect.
Summary of the invention
For reaching above-mentioned and other object, the invention provides a kind of from the independent separated peptide of pig, this peptide has the aminoacid sequence shown in sequence table SEQ ID NO:1, and this aminoacid sequence contains the disulfide linkage that 6 halfcystines (cysteine) residue forms, it is characterized in that, this peptide is in order to antibacterial and sterilization.
According to the present invention, this peptide contains 36 amino-acid residues, and has approximately 5 molecular weight to 7kDa.
In concrete application, the present invention from pig separately separated peptide can with membrane interaction outside the phosphatide of intestinal bacteria (Escherichia coli) or streptococcus aureus (Staphylococcus aureus), make the unbalance apoptosis that causes of its osmotic pressure, therefore realize antibacterial and sterilizing use.
The present invention also provides the nucleotide fragments of the above-mentioned peptide of a kind of codified, and it has the nucleotide sequence shown in sequence table SEQ ID NO:2.
The present invention can express from the independent separated peptide of pig in pichia spp (Pichia pastoris), through example of the present invention, confirms, the peptide that the present invention produces in pichia spp has antibacterial effect, and this peptide can be further purified.
Preparation the present invention comprises the following steps: the nucleic acid fragment of this peptide fragment to construct in expression vector from the method for the independent separated peptide of pig; This expression vector is converted in pichia spp, to express this peptide fragment; And separated this peptide fragment of this pichia spp certainly.
The present invention's expression vector used can be (but being not limited to): pGAPZ α A and pPICZ α A, and expression vector can be converted into and in pichia spp, carry out recombinant peptide expression, through example of the present invention, confirm, this recombinant peptide has inhibition for intestinal bacteria and streptococcus aureus.
For realizing above-mentioned antibacterial and sterilization object, the present invention separately provides a kind of medical composition, the peptide and the pharmaceutically acceptable supporting agent that comprise medical significant quantity, it is characterized in that, this peptide is to have the aminoacid sequence shown in sequence table SEQ ID NO:1, and this bacterium is intestinal bacteria and streptococcus aureus.
The form of medical composition of the present invention can be (but being not limited to): embedding thing, soak solution, feed, feed additive, oral liquid, vaccinate, paster, pulvis, lozenge, injecting fluid, suspension, external use liquid, drops, liniment, paint, creme, ointment, ointment, paste, glue and gel.In one specific examples, this medical composition is creme, lozenge or pulvis.
Medical composition of the present invention can be selected suitable supporting agent, for example (but being not limited to): the matrix of vehicle, thinner, thickening material, weighting agent, bonding agent, disintegrating agent, lubricant, grease or non-grease, tensio-active agent, suspension agent, jelling agent, auxiliary, sanitas, antioxidant, stablizer, tinting material and or spices.
In the present invention, suitable lubricant is including (but not limited to) glycerine and oils, and this oils can be peanut oil or Viscotrol C; Suitable matrix is including (but not limited to) hydrocarbon polymer, and this hydrocarbon polymer is including (but not limited to) paraffinum durum, soft wax, paraffin oil, glycerine, beeswax, metallic soap, natural oil, lanolin and derivative thereof, lipid acid and composition thereof; Suitable natural oil is including (but not limited to) Prunus amygdalus oil, Semen Maydis oil, peanut oil, Viscotrol C and sweet oil; Suitable lipid acid is including (but not limited to) stearic acid and oleic acid; Suitable tensio-active agent comprises anion surfactant, cats product and nonionogenic tenside, and this tensio-active agent can be including (but not limited to): sorbitan ester, polyoxyethylene and derivative thereof, carboxyvinyl polymer and derivative thereof, and the derivative of this polyoxyethylene can be polyoxyethylene fatty acid ester, the derivative of this carboxyvinyl polymer can be carbomer (carbopol); Suitable suspension agent is including (but not limited to) natural resin, derivatived cellulose inorganic substance, polyoxyethylene glycol 540, PEG3350 and propylene glycol; Suitable jelling agent is including (but not limited to) derivatived cellulose, vinyl polymer, carboxyvinyl polymer derivative, pectin and glue class, this derivatived cellulose can be methylcellulose gum, hydroxy ethyl cellulose or carboxymethyl cellulose, and this glue class can be Sudan Gum-arabic, tragacanth gum, phycocolloid, agar or gelatin.
Can by medical composition of the present invention with oral, soak, inject, smear or the mode of paster is administered in human body or animal body, wherein, this animal can be livestock products animal or hydrocoles, for example (but being not limited to): pig, ox, sheep, chicken or cultivation fish.Preferably medical composition of the present invention can be administered in pig body with oral way, to suppress or to reduce intestinal bacteria in pig body or the illness of infection of staphylococcus aureus.
In order to treat by the animal of intestinal bacteria or infection of staphylococcus aureus, the present invention is applied to prepare immune inducing agent by the peptide with the aminoacid sequence shown in sequence table SEQ ID NO:1, in order to antibacterial and sterilization.Particularly, peptide of the present invention, to destroy the mechanism of action of the cytolemma of intestinal bacteria or streptococcus aureus, reaches antibacterial and biocidal efficacies, and then makes the infected animals of administration immune inducing agent increase immunizing power.
Peptide of the present invention separately can be applicable to the method for immune inducing agent that screening can increase animal immunizing power, comprise the following steps: by the immune inducing agent of wish screening mix with feed, to obtain mixture; This mixture is administered to animal; Detect the expression amount of the peptide fragment as shown in sequence table SEQ ID NO:1 in this animal body; And just judge that with the expression amount of this peptide fragment this immune inducing agent is on this animal sterilization and antibacterial impact, wherein, this bacterium comprises intestinal bacteria or streptococcus aureus.
Accompanying drawing explanation
Fig. 1 shows that the present invention is from the electrophorogram of the pBD2 gene fragment of the independent separated peptide of pig, with the primer of design, carry out the PCR of pBD2 gene, its gene fragment length is 108bp, the tack temperature scope of test is 67 ℃ to 59 ℃, result has the most obvious band at 59 ℃, represents the more pBD2 gene of amplification;
Fig. 2 is pPICZ α A/mPBD2 double digestion electrophoresis confirmation figure, it is characterized in that, M represents the DNA marker (marker) of 1kb, and band (lane) 1 and lane2 are respectively difference and turn and grow the plasmid purification of bacterial strain by the gene fragment of EcoR1 and Xba1 double digestion;
Fig. 3 shows the mpBD2 gene fragment of purifying and plasmid pPICZ α A concentration ratio, it is characterized in that, M represents the DNAmarker of 1kb, and lane1 and lane2 represent pPICZ α A plasmid, and lane3 represents the pBD2 gene fragment of purifying;
Fig. 4 show with Tricine-SDS-PAGE analyze different yeast turn grow bacterial strain inducing and express after extracellular fluid protein change situation, wherein, M represents protein labeling (protein marker), and lane1 to lane7 represents that different yeast turn the analytical results of the recombinant protein of growing bacterial strain expression separately;
Fig. 5 shows that with pPICZ α A/mPBD2, turning the Western Blot His taq that grows the supernatant liquor that bacterial strain inducing expresses demarcates and analyze, and wherein, M represents protein marker, and lane1 to the lane6 different yeast of respectively doing for oneself turn and grow the recombinant protein that bacterial strain is expressed; And
Fig. 6 A-6B shows that the present invention is from the pig anti-harmful intestinal tract bacteria activation analysis of separated peptide separately, Fig. 6 A is the anti-bacterial result to streptococcus aureus, Fig. 6 B is to colibacillary the anti-bacterial result, its longitudinal axis (log cfu/ml) represents to deposit viable count, wherein, CTRL represents not add the present invention from the control group of the independent separated peptide of pig, CTRL 1D represents that control group was with 37 ℃ of cultivations one day, mPBD21D represents that the peptide of control group interpolation 20ul 1mg/ml was with 37 ℃ of cultivations one day, Amp ' 1D represents that the positive control that control group adds the Ampicillin of 20ul1mg/ml cultivates one day with 37 ℃.
Embodiment
By particular specific embodiment, embodiments of the present invention are described below, these those skilled in the art can understand advantage of the present invention and effect easily by content disclosed in the present specification.The present invention also can be implemented or be applied by other different embodiment, and the every details in this specification sheets also can, based on different viewpoints and application, be given different modifications and change under not departing from disclosed spirit.
I. will construct in expression vector from the nucleic acid fragment of the independent separated peptide fragment of pig
With currently known methods, in pig tongue tissue, extract RNA, the reversed transcriptive enzyme of take is cDNA by the RNA reverse transcription of all extractions.
The partial sequence (108bp fragment) of corresponding chitling road alexin (Porcine β-defensin2, pBD2) of take is basis, uses by Sigma company on behalf of two synthetic primers, and the primer of design is as following table 1.
Table 1
Warp is with polymerase chain reaction (Polymerase Chain Reaction, PCR) amplify this pBD2 gene fragment, wherein, use PCR reactor (GeneAmp PCR System2400, Applied Biosystems), reaction conditions is that 94 ℃ of heating make DNA sex change for 10 minutes, then with 30 be cycled to repeat 94 ℃ 30 seconds, 67 ℃ are binded 30 seconds, and 72 ℃ of amplification 30 seconds, finally completes reaction for 10 minutes with 72 ℃.This PCR product is through sepharose (agarose gel) electrophoretic analysis, and as shown in Figure 1, pBD2 full length gene is 210bp to its result, and designed primer can amplification goes out the nucleic acid fragment of about 108bp.
Then, carrier for expression of eukaryon pGAPZ α A and pPICZ α A (being purchased from Promega) are mixed with the PCR product after above-mentioned purifying, add and engage damping fluid (ligation buffer) and T4DNA joining enzyme (being purchased from GeneMark), under room temperature, react and within 1 hour, carry out conjugation (Ligation).Afterwards, get and engage gained 10 μ l recombinant plasmid dnas conversions (Transformation) to the competent cell (Escherichia coli DH5 α) of intestinal bacteria, in 42 ℃ of dry (FIRSTEK DB200-2-110) ice baths 2 minutes at once after 45 seconds of bathing, be applied in surface and contain bleomycin (Zeiocin, 25 μ g/mL) less salt LB substratum (LB Agar), cultivates 16 to 24 hours for 37 ℃.
By the bacterium liquid after transforming with purification kit (Plasmid Miniprep Purification Kit, be purchased from GeneMark) carry out plasmid extraction, then the recombinant plasmid dna warp extracting is confirmed with EcoR1 (being purchased from GeneMark) and the cutting of Xba1 (being purchased from GeneMark) restriction enzyme, PCR evaluation and DNA sequencing.
(1) restriction enzyme cutting is confirmed
Use dry bath (FIRSTEK DB200-2-110) to carry out double digestion in 37 ℃ and react one hour 30 minutes, the formula using is as following table 2.After having reacted, with 2% sepharose, carry out electrophoretic analysis.Fig. 2 shows pPICZ α A/mPBD2 double digestion confirmation figure, wherein, band 1 (lane1) and lane2 are that difference turns and grows the plasmid purification of bacterial strain by the pBD2 gene fragment of EcoR1 and Xba1 double digestion, and result shows that mrna length is about 108bp, and plasmid size is 3.6kb.
Table 2
| Formula | Content |
| 10x reaction buffer | 1μl |
| Restriction enzyme EcoR1 | 0.3μl |
| Restriction enzyme Xba1 | 0.3μl |
| Recombinant plasmid dna | 3μl |
| Water | 5.4μl |
(2) PCR identifies
The upper single bacterium colony of culture plate (plate) is chosen to bacterium after 37 ℃ of cultivations, get machine on 1 μ l bacterium liquid, after having reacted, with 2% sepharose, carry out electrophoretic analysis.Fig. 3 shows the pBD2 gene fragment of purifying and plasmid pPICZ α A concentration ratio, and result shows that pBD2 gene fragment length is 108bp, and pPICZ α A plasmid size is 3.6kb.
(3) DNA sequencing is confirmed (DNA sequencing)
Bacterium liquid is applied in and contains bleomycin (Zeocin is purchased from Invitrogen) (25 μ g/ml) LB substratum, send to Taiwan Yuan Zisheng skill company and carry out sequencing, with the pBD2 gene of confirming that choosing is grown.The present invention after sequencing obtains the nucleotide sequence as shown in sequence table SEQ ID NO:2 from the independent separated peptide of pig.The present invention, through having 108 Nucleotide from the independent separated peptide of pig, obtains having 36 amino acid whose peptides after deduce (deduced), and its sequence is as shown in sequence table SEQ ID NO:1.
II. expression vector is converted into pichia spp
The pGAPZ α A-pBD2 constructing and pPICZ α A-pBD2 carrier are cut into linear rear purifying with restriction enzyme Avr2 and Sac1 (being purchased from GeneMark) respectively, carry out electroporation (Electroporation).
In centrifuge tube, add respectively the linear recombinant plasmid of 10 μ l, and and the competent cytomixis of 80 μ l, inserted in electroporation light suction pipe (cuvette) in standing 5 minutes on ice, use electroporation machine (Electroporator, Genepulser Xcell system, Bio-Red) at voltage, be 1500V, electric capacity is 25uF, and resistance is to carry out electroporation under the condition of 200 Ω, after energising, add immediately the 1M sorbyl alcohol that 1ml is ice-cold, in 30 ℃, cultivate one hour, be applied in the 100 μ g/ml YPDS dishes (plate) containing Zeocin (being purchased from Invitrogen), with 30 ℃, cultivate 3 to 7 days.The bacterium colony growing is provoked, and respectively to containing Zeocin500,1000,2000 μ g/ml YPDS plate, to select the yeast containing high tricks recombination, by the bacterium colony that can grow on high multiple YPDSZ plate, with Zeocin100 μ g/ml YPDS broth take 30 ℃ cultivate 1 day to OD600 numerical value as 0.6 to 1.The formula of YPDS dish is as shown in table 3 below.
Table 3
With 1500xg, within centrifugal 10 minutes, remove after supernatant liquor, nutrient solution (the buffered methanol-complex medium that adds 2ml to contain 100 μ g/mlZeocin, BMMY) Eddy diffusion bacterium piece, cultivate 30 ℃, 300rpm, and in every 24 hours, add 100 μ l methyl alcohol with successive induction expression of recombinant proteins.The formula of BMMY is as shown in table 4 below.
Table 4
Sampling respectively after 1 to 2 day after induction, by bacterium liquid with centrifugal 5 minutes postlyophilizations of 3000xg, and use a little aqua sterilisa back dissolving, carry out the analyses of laurylene sodium sulfate-polyacrylamide gel electrophoresis (Tricine-SDS PAGE) and west ink dot method (Western blot), to have judged whether that recombinant protein produces.Fig. 4 analyzes the protein electrophoresis result of the pBD2 of Pichia anomala expression with Tricine-SDS-PAGE; Fig. 5 turns with pPICZ α A/mPBD2 the west ink dot of growing the supernatant liquor that bacterial strain inducing expresses to remove Histidine label (Western Blot His taq) and demarcate analytical results, the recombinant protein of demarcating is approximately positioned at 20kDa, and expection recombinant protein size is about 5.7kDa.
Antibacterial and the bactericidal assay of embodiment 3
The nutrient solution of three days induction recombinant proteins with 3000xg after centrifugal 5 minutes, is got to supernatant liquor and is deposited in-20 ℃, after lyophilize powdered with appropriate sterilized water back dissolving, as preparing before antibacterial ring analysis sample.Intestinal bacteria (Escherichia coli) and streptococcus aureus (Staphylococcus aureus) two pathogen strain bacterium are activated to one day with 37 ℃ respectively, in 96 orifice plates, inject respectively the restructuring pBD2 (20 μ g) that 190 μ g bacterium liquid and concentration are 1mg/ml, and using the microbiotic Ampicillin of 1mg/ml as positive control, every processing repeats 3 times, cultivate 37 ℃ after 24 hours, with OD600, measure bacterial concentration.Fig. 6 A and Fig. 6 B show that respectively, to streptococcus aureus and colibacillary the anti-bacterial result, result all has inhibition (p<0.05).
According to above-mentioned test result, show, the present invention has antibacterial effect from the independent separated peptide of pig, is applicable to being applied to as medical composition or for the preparation of immune inducing agent.
The preparation of embodiment 4 medical compositions of the present invention
The formula of following table 5 listed materials (one) to formula (three) is mixed, press the method described in pharmaceutical purpose general rule, make the medical composition that is creme.
Table 5
The preparation of embodiment 5 medical compositions of the present invention
By the listed material mixing of following table 6, press the method described in pharmaceutical purpose general rule, and use bead machine to make lozenge, make the medical composition that is lozenge.
Table 6
The preparation of embodiment 6 medical compositions of the present invention
By the listed material mixing of following table 7, press the method described in the general rule of pharmaceutical purpose, make the medical composition that is pulvis.
Table 7
Above-described embodiment is illustrative principle of the present invention and effect thereof only, but not for limiting the present invention.Any these those skilled in the art all can, under spirit of the present invention and category, modify and change above-described embodiment.Therefore, these those skilled in the art, not departing from all equivalence modifications that complete under disclosed spirit and technological thought or changing, must be contained by claims scope such as.
Sequence table (SEQUENCE LISTING)
<110> Bai Ning biotech inc
<120> is from independent separated peptide of pig and uses thereof
<160>1
<170>PatentIn Version3.5
<210>1
<211>36
<212>PRT
<213> pig
<400>1
His Tyr Ile Cys Ala Lys Lys Gly Gly Thr Cys Asn Phe Ser Pro Cys Pro Leu
Phe Asn Arg Ile Glu Gly Thr Cys Thr Ser Gly Lys Ala Lys Cys Cys Ile Arg
<210>2
<211>108
<212>DNA
<213> pig
<400>2
cactacatat gtgccaagaa aggggggacc tgcaacttct ccccctgccc gctcttcaac 60
aggattgaag ggacctgtta cagtggcaag gccaagtgct gcatccgc 108
Claims (10)
1. from the independent separated peptide of pig, it has the aminoacid sequence shown in sequence table SEQ ID NO:1, and this aminoacid sequence contains the disulfide linkage that 6 cysteine residues form, and it is characterized in that, this peptide is for antibacterial and sterilization.
2. peptide as claimed in claim 1, is characterized in that, this bacterium comprises intestinal bacteria (Escherichia coli) and streptococcus aureus (Staphylococcus aureus).
3. from the independent separated Nucleotide of pig, it has the nucleotide sequence shown in sequence table SEQ ID NO:2, and the peptide as claimed in claim 1 of encoding.
4. a method of preparing peptide as claimed in claim 1, comprises the following steps:
The nucleic acid fragment of peptide fragment as claimed in claim 1 is constructed in expression vector;
This expression vector is converted in pichia spp (Pichia pastoris), to express this peptide fragment; And
From separated this peptide fragment of this pichia spp.
One kind in pichia spp for expressing the purposes of peptide as claimed in claim 1.
6. a medical composition, comprises peptide as claimed in claim 1 and the pharmaceutically acceptable supporting agent of medical significant quantity.
7. medical composition as claimed in claim 6, it is characterized in that, this supporting agent can be matrix, tensio-active agent, suspension agent, jelling agent, auxiliary, sanitas, antioxidant, stablizer, tinting material or the spices of vehicle, thinner, thickening material, weighting agent, bonding agent, disintegrating agent, lubricant, grease or non-grease.
8. medical composition as claimed in claim 6, with oral, soak, inject, smear or the mode of paster is administered in animal body.
9. a purposes for peptide as claimed in claim 1, for the preparation of immune inducing agent.
10. in order to screening, can increase a method for the immune inducing agent of animal immunizing power, comprise the following steps:
By the immune inducing agent of wish screening mix with feed, to obtain mixture;
This mixture is administered to animal;
Detect the expression amount of the peptide fragment as shown in sequence table SEQ ID NO:1 in this animal body; And
Expression amount with this peptide fragment just judges that this immune inducing agent is on this animal sterilization and antibacterial impact,
It is characterized in that, this bacterium comprises intestinal bacteria or streptococcus aureus.
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| CN201210245816.1A CN103539850A (en) | 2012-07-16 | 2012-07-16 | Peptide separately separated from pig and use thereof |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101250572A (en) * | 2007-10-29 | 2008-08-27 | 中国农业大学 | Method for extracting pig blood antibiotic peptide |
| CN101275118A (en) * | 2007-12-13 | 2008-10-01 | 河北农业大学 | Preparation for porcine defensin engineering yeast strain |
| CN102229934A (en) * | 2011-06-14 | 2011-11-02 | 华南农业大学 | Efficient production method and application of antimicrobial peptide PR-39 of pig small intestine in pichia pastoris |
-
2012
- 2012-07-16 CN CN201210245816.1A patent/CN103539850A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101250572A (en) * | 2007-10-29 | 2008-08-27 | 中国农业大学 | Method for extracting pig blood antibiotic peptide |
| CN101275118A (en) * | 2007-12-13 | 2008-10-01 | 河北农业大学 | Preparation for porcine defensin engineering yeast strain |
| CN102229934A (en) * | 2011-06-14 | 2011-11-02 | 华南农业大学 | Efficient production method and application of antimicrobial peptide PR-39 of pig small intestine in pichia pastoris |
Non-Patent Citations (2)
| Title |
|---|
| 李牧等: "猪源抗菌肽及其应用前景", 《饲料工业》 * |
| 韩军等: "猪源抗菌肽的研究进展", 《中国兽医杂志》 * |
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Application publication date: 20140129 |