CN101182360A - Fusion protein having antibiotic function and uses thereof - Google Patents
Fusion protein having antibiotic function and uses thereof Download PDFInfo
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- CN101182360A CN101182360A CNA2007101138678A CN200710113867A CN101182360A CN 101182360 A CN101182360 A CN 101182360A CN A2007101138678 A CNA2007101138678 A CN A2007101138678A CN 200710113867 A CN200710113867 A CN 200710113867A CN 101182360 A CN101182360 A CN 101182360A
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Abstract
The invention discloses a fusion protein with antibacterial function and an application thereof. The gene sequence compositions of the fusion protein are hepcidinl precursor antibacterial peptide gene which is obtained by cloning and synthetic magainin antibacterial peptide which are combined as a novel fusion protein cDNA and integrated into a yeast gene group by the gene engineering means; the fusion protein cDNA is induced to express at the yeast, and furthermore, the yeast ferments and cultivates to obtain a plurality of fusion protein. The fusion protein of the invention is detected having obvious antibacterial activity, is applied to prevent and remedy the bacterial disease of aquatic organisms and cultured animals, or the purified fusion protein is directly used as a drug to replace antibiotic to be applied to the treatment of the disease caused by bacteria and even virus.
Description
Technical field
The present invention relates to a kind of fusion rotein and application thereof, relate in particular to a kind of animal antibacterial peptide gene carrier of setting up with genetic engineering technique and the antibacterial peptide fusion protein that obtains by yeast expression system, and in the preparation antibacterials or have the hydrobiont of disease control function or culture application in the animal and fowl fodder product.
Background technology
Antibacterial peptide is that organism self produces, and pathogenic agent such as opposing external microbe, virus infect, and are used for the phylactic agent of human body nonspecific defense.It extensively is present in the animal and plant, has the disinfection vitality height, effect rapidly, does not produce characteristics such as resistance, is the effective means of multicellular organism immune defense, is bringing into play important effect in the existence of biology and evolutionary process.Antibacterial peptide is pathogenic microbe killing directly, and can activate the immunity system of organism, increases the ability of the extraneous pathogenic agent invasion of opposing.Up to now, had and comprised separated, the purifying of multiple antibacterial peptide to high Mammals and human origin from the comparatively low animal of waiting.For example: the cecropin in the silkworm, cecatotoxin, alexin in shark, shellfish, the tunicate (defensin), pleurocidin in seven eels, the flatfish, magainin of Amphibians and dermasepin, alexin in the birds, α-defensin in the Mammals, β-defensin, the thionin in the plant, plant alexin etc.Kind surplus the antibacterial peptide that has been found that outnumbers 800.
The Allopelagic sterilizing peptide is the important component part of body natural immune system, because the immunity system of most of marine animal is an innate immune system, thereby, the effect of marine animal is just seemed even more important as innate immune system member's antibacterial peptide than Mammals.Research to the Allopelagic sterilizing peptide mainly concentrates on some Crustaceans, as aspects such as king crab, crabs, in recent years also relevant for the research of shrimp, shellfish antibacterial peptide or antibacterial peptide similar substance report, show the Allopelagic sterilizing peptide and receiving more and more widely concern in the application prospect of culture fishery.But the research to fish antibacterial peptide is started late, and the antibacterial peptide kind that separation obtains is limited.
Defensin is the important antibacterial peptide family of a class, wherein hepcidin be in β-defensin protein family a member (Verga F, et al, Gene.2005,364:37-44).Hepcidin separates to obtain from people's blood plasma suction filtration liquid or urine, confirms that after testing it is a kind of antibacterial protein that is rich in halfcystine, and its anti-microbial activity has obtained confirmation (Krause A in many ways, et al, FEBS Lett, 2000,480:147-150; Park C H, et al, J Biol Chem, 2001,276:7806-7810).When it being carried out anti-microbial activity research, find again Hepcidin in iron metabolism, also bringing into play effect (Nicolas G, et al, Proc Natl Acad, 2001,98:8780-8785).Obtained the homology isomer gene of multiple hepcidin with rear clone.Shike etc. have obtained the hepcidin gene from hybridization lithosporic perch, be the hepcidin gene that from fish, obtains first (Shike H, et al, Eur J Biochem, 2002,269:2232-2237).Magainin separates to obtain from the skin of toad, and this class antibacterial peptide has αLuo Xuanjiegou, normally is made up of L type amphoteric amino acids, to gram-negative bacteria, positive bacteria, all lethal effect (Zasloff M arranged, Proc Natl Acad Sci USA, 1987,84:5449-5453; BarrellPJ, et al, Protein Expr Purif, 2004,33:153-159).
Although the antibacterial peptide antibiotic effect is remarkable, have a extensive future, the content of natural antibacterial peptide is very low, can't obtain in a large number in organism; And chemical synthesising peptide costs an arm and a leg, and has limited its widespread use.Because the aquiculture disease that seriously causes of environmental pollution frequently takes place, often cause bigger financial loss at present.Culture fishery extensively and a large amount of microbiotic such as paraxin that uses causes the microbiotic accumulation that exceeds standard in fishery products, bring significant damage to human consumer's health.European Union has forbidden the product water outlet European Union member countries that China's antibiotic exceeds standard at present.Therefore, utilize the antibacterial peptide with efficient sterilizing effect of genetic engineering technique producer gene engineering reorganization and be applied to culture fishery, and the raising that is used for various livestock and poultry animals, become the inevitable choice of China's culture fishery and livestock industry to substitute the antibiotic of being forbidden day by day.
Using yeast expression system expression eukaryotic gene is obtaining application more and more widely in the DNA recombinant technology.What use at first is the yeast saccharomyces cerevisiae system, up to after have a good characteristic the pichia pastoris phaff expression system more and more be subjected to the attention of science one industry member, and replace the yeast saccharomyces cerevisiae system gradually.Yeast expression system has the unique biological characteristic, has the proteic expression of strict regulation and control external source eukaryotic cell, and expression product is modified in processing, expression amount height, advantage such as nutritional requirement is low.Because antibacterial peptide directly acts on the prokaryote film, suppress its growth so that kill thalline, therefore adopt eukaryote zymic expression system almost to become the unique selection of producer gene engineering recombinant antibacterial peptide.In addition, pichia yeast expression system can utilize methyl alcohol as a large amount of synthetic proteins of sole carbon source, once is used for the production of plant-scale single cell protein.With Yeast engineering bacteria expressing gene engineering antibacterial peptide, can the mass production hydrobiont and vivarium animal autogene that be badly in need of, that can the substitute antibiotic antibacterial peptide of expressing, for market provides nontoxic, healthy green product.And the further development and utilization to antibacterial peptide will produce very high economic worth and huge social benefit.
Summary of the invention
The purpose of this invention is to provide a kind of animal antibacterial peptide gene carrier of setting up with genetic engineering technique and the antibacterial peptide fusion protein that obtains by yeast expression system, and in the preparation antibacterials or have the hydrobiont of disease control function or culture application in the animal and fowl fodder product.
Technical conceive of the present invention is that the antibacterial peptide of antibacterial peptide of fish and Amphibians is formed novel fusion rotein, uses engineered means to make up to contain the expression vector of fusion rotein cDNA sequence and be integrated into the yeast genes group and expresses.Its expression product can be applied in hydrobiont or the livestock and poultry animal breed; Also can pass through purifying or spissated method, obtain highly purified fusion rotein, directly be used for biological disease control, the treatment of bacillary even virus disease as antibiotic substitute.
Fusion rotein with antibacterial of the present invention is by forming through fish antibacterial peptide hepcidin and the Amphibians antibacterial peptide magainin that modifies, and it is characterized in that: described fusion rotein is the small peptide that contains aminoacid sequence shown in the SEQ ID NO:1.
The gene of above-mentioned fusion rotein is by the cDNA sequence in the cDNA sequence of the cDNA sequence of coding fish hepcidinl precursor mature peptide, coding Amphibians antibacterial peptide magainin mature peptide, restriction enzyme EcroR I site and meet eukaryote and express the gene order of preference and form, and it is characterized in that: the nucleotide sequence of described antigen-4 fusion protein gene is the nucleotide sequence shown in the SEQ ID NO:2.
Fusion rotein of the present invention is in the application of preparation in the preparation antibacterials.
Fusion rotein of the present invention has the hydrobiont of disease control function or the application in the breed animal and fowl fodder in preparation.
Wherein: described fusion rotein directly adds in hydrobiont or the animal and fowl fodder as immunity additive, or purified back is directly used or used as fodder additives as the disease control medicine.
The yeast expression system of fusion rotein of the present invention, it is characterized in that, contain the interior expression vector pAO815 of yeast cell of external source fusion rotein cDNA or the Yeast expression carrier of other type, and utilize carrier to make antigen-4 fusion protein gene in pichia spp, yeast saccharomyces cerevisiae, candiyeast or other edibility yeast, carry out in the cell or the extracellular expression.Its step is:
The structure of expression vector: the restriction enzyme site of the restricted property restriction endonuclease of design on the antibiotic fusion rotein cDNA sequence of synthetic, be linked at together after expression plasmid pAO815 enzyme is cut in the yeast cell of fusion rotein cDNA sequence and Invitrogen company, by the direction that PCR reaction detection antigen-4 fusion protein gene inserts, filter out the clone of the antigen-4 fusion protein gene that contains the forward insertion.
The structure of yeast expression system: use the digestion with restriction enzyme expression plasmid of yeast, make its linearizing, the method by electric shock transforms is incorporated into fusion rotein cDNA sequence in the zymic genome.Thalline suspension after transforming is coated on the MD flat board, and 30 ℃ of cultivations induce screening to occur until single bacterium colony.Picking list bacterium colony as template, is confirmed positive colony by the PCR reaction with it.
The cell of fusion rotein interior or extracellular abduction delivering and active the detection.
The detection method of expression product anti-microbial activity is as follows:
Select the positive colony bacterium colony,, collect thalline,, make the foreign gene can be by abduction delivering with the resuspended thalline of substratum that contains methyl alcohol in 30 ℃ of cultivations.As use the cell inner expression carrier, and collect thalline, obtain protein crude extract behind the smudge cells.As use the extracellular expression vector, by ammonium sulfate precipitation, ion exchange chromatography and gel chromatography, obtain electrophoretically pure fusion rotein behind the collecting cell nutrient solution by Polyacrylamide gel electrophoresis separation.
Also inoculate kind pathogenic bacterium such as intestinal bacteria, Vibrio anguillarum, vibrio alginolyticus simultaneously by coating fusion rotein crude extract on culture dish, carry out the inhibition zone experiment, or, detect the A600 light absorption value and detect its anti-microbial activity by in above-mentioned pathogenic bacterium nutrient solution, adding the fusion rotein crude extract.
Fusion rotein of the present invention has tangible anti-microbial activity after testing, can be used for hydrobiont, cultivated animals have chemical sproof bacteriosis to antibiosis control, perhaps with the fusion rotein of purifying directly as medicine, be used for bacterium even viral caused treatment of diseases.
The invention has the beneficial effects as follows:
(1) can the mass production novel hydrobiont of using gene engineering technique and the antibacterial peptide of cultivated animals.
(2) solve the problem that the accumulation of long-standing disease hazard of aquaculture and fishery products and livestock product microbiotic exceeds standard.
(3) drive the technical progress of industry of aquaculture and breeding bait industry with hi-tech.
(4) provide healthy green aquatic product and livestock product to society.
Description of drawings
Fig. 1. the clone of fish hepcidinl precursor gene.
Fig. 2. the carrier pAO815-antibac plasmid of expressed fusion protein cDNA.
The detection of Fig. 3 .pAO815-antibac positive colony.
Fig. 4. the antibiotic fusion rotein that pichia yeast expression system is expressed is to colibacillary fungistatic effect.
Fig. 5. the antibiotic fusion rotein that pichia yeast expression system is expressed is to the fungistatic effect of Vibrio anguillarum.
Fig. 6. the antibiotic fusion rotein that pichia yeast expression system is expressed is to the fungistatic effect of vibrio alginolyticus.
Wherein: in Fig. 4～6 1 *, 2 *, 3 * be the antibiotic expressing fusion protein systematic protein coarse body fluid of different concns, 0 is contrast (expressing the yeast expression system albumen coarse body fluid of β-Gal).
Embodiment
Also in conjunction with the accompanying drawings content of the present invention is described further with embodiment below:
1. the preparation of fish liver cDNA: after big dace flounder injection Vibrio anguillarum infects 24h, take out liver, be put in rapidly in the liquid nitrogen, hard tissue block is placed-80 ℃ of freezings.With reference to TRIZOL
The reagent operation instructions is got the about 100mg of tissue of freezing, joins in the 1ml TRIZOL reagent, puts under the room temperature after the homogenize 5 minutes, adds 200 μ l chloroform thermal agitations 15 seconds, incubation 3min under the room temperature.Under 4 ℃ of conditions with 12, the centrifugal 15min of 600rpm rotating speed.Obtain the interface supernatant liquid and add the high salt concentration solution that 250 μ l form with 0.8M sodium acetate and 1.2M sodium-chlor, add 250 μ l Virahol mixings behind the mixing again, under room temperature, precipitate 10min, again 4 ℃ with 12, the centrifugal 10min of 600rpm rotating speed.Precipitate with 1ml 70% ethanol rinsing after removing supernatant, with 11,4 ℃ of centrifugal 5min of 000rpm rotating speed.Behind 55 ℃ of dissolvings of the distilled water of handling with 20 μ lDEPC (no RNase) RNA precipitation 10min, get the total RNA of 2.0ug, add the random primer of 1 μ l50pM, be diluted to 17.1 μ l with DEPC water.70 ℃ of 5min destroy the quenching on ice of RNA secondary structure postposition.Add 5 μ l M-MLV 5 * transcribe damping fluid, 1.3 μ l 10mM dNTP, 24U rRNA enzyme inhibitors and 200UM-MLV reversed transcriptive enzyme successively.Behind 42 ℃ of reaction 1h, keep the 5min termination reaction, in 4 ℃ of preservations at 94 ℃.
2. the clone and the sequential analysis of fish antibacterial peptide hepcidinl precursor gene: with turbot liver cDNA is template, use primer WBHsp (5`-CAAACCCTCCTAAGATGAAG-3`), WBHap (5`-AATCCTCAGAACCTACAGCA-3`) carries out the PCR reaction.The reaction annealing temperature is 52 ℃, and 30 circulations obtain the PCR band (see figure 1) of about 300bp.The PCR product that obtains uses the gel electrophoresis test kit to reclaim, and confirms that through order-checking this sequence total length 293bp comprises complete coding region, the long 273bp of open reading frame wherein, and 90 amino acid of encoding see GenBank sequence number AM113708 for details.。Blastn server by NCBI carries out the comparison of homology similarity to nucleotide sequence.The higher sequence of homology similarity mostly is the gene order of Hepcidin family, wherein the homology similarity with lefteye flounder is 90%[Pseudosciaena crocea (DQ307050)], with the homology similarity of white bass be 84%[Morone chrysops (AF394246)], with the homology similarity of black porgy be 83%[Acanthopagrusschlegelii (AY669380)].Determine that this sequence is a turbot hepcidinl precursor gene.Use the signal peptide zone of signalP server prediction hepcidinl precursor protein sequence, 24 amino acid of C end are signal peptide in the hepcidinl precursor protein sequence.Use the secondary structure of software Omiga2.0 prediction hepcidinl precursor protein sequence, 21 amino acid of N end are the beta sheet structure in the protein sequence of hepcidinl precursor, and this part sequence comprises eight halfcystines.The notable attribute of Hepcidin protein family mature peptide is exactly the beta sheet structure that is formed by these eight halfcystines, and this meets the feature of hepcidin protein family.Compare perch, zebra fish Hepcidin mature peptide, thereby the mature peptide that draws turbot hepcidinl precursor have 21 amino acid.
Fusion rotein cDNA design and preparation: fusion rotein cDNA sequence is by the sequence in the gene order of the gene order of coding fish hepcidinlprecursor mature peptide, coding Amphibians antibacterial peptide Magainin mature peptide, restriction enzyme EcroR I site and meet eukaryote and express preference, and the gene order that meets the Kozak rule is simultaneously formed.The site of carrying out the genetically engineered modification is described below: (1) the 4th preference base is G; (2) alkali-free base T in the flanking sequence of the about 15bp scope of 5 ' of ATG end; (3)-3 ,-6 and-9 positions, G is the preference base; (4) remove-3 ,-6 and-9, in whole flanking sequence district, C is the preference base.So being modified through the gene engineering method optimization design by two sections mature peptide sequences of hepcidinl precursor and Magainin, the protein sequence of antigen-4 fusion protein gene sequence encoding forms.Use this fragment gene sequence of solid phase phosphorous acid acid amides method synthetic, the long 151bp of sequence (seeing the nucleotide sequence shown in the SEQ ID NO:2), 39 amino acid of encoding, the expressed albumen of the yeast expression system of subsequent builds is exactly that this contains 39 amino acid whose small peptides (seeing aminoacid sequence shown in the SEQ ID NO:1).
4. the structure of yeast expression system pAO815-antibac carrier: the restriction enzyme site of the restricted property restriction endonuclease EcroR I of design on the antibiotic antigen-4 fusion protein gene sequence of synthetic, expression plasmid carrier pAO815 has the single endonuclease digestion site of EcroR I equally in the yeast cell, has same sticky end behind antigen-4 fusion protein gene and the pAO815 plasmid enzyme restriction.Use EcroR I enzyme respectively cuts antigen-4 fusion protein gene and pAO815 plasmid, and use gel electrophoresis recovery test kit reclaims the product after enzyme is cut respectively.Use DNA Ligation Kit test kit to connect, get antigen-4 fusion protein gene 11 μ l and add pAO815 carrier 2 μ l connection damping fluid 2.5 μ l, ligase enzyme 4.5 μ l, 16 ℃ of connections are spent the night.It is linked at together, and leads the people in bacillus coli DH 5 alpha.Using primer HMsp (5`-GGACAGCCACATCTCCCTTT-3`) and HMap (5`-GTGCGAATTCTCAGAACTTC-3`), is template to transform back bacillus coli DH 5 alpha bacteria suspension, carries out the PCR reaction, and 53 ℃ of annealing temperatures are carried out 30 circulations.Connect product and detect the direction that antigen-4 fusion protein gene inserts.The PCR band that can occur about 130bp if the antigen-4 fusion protein gene forward is inserted among the plasmid pAO815 is if antigen-4 fusion protein gene oppositely inserts then the PCR band can not occur.Filter out the clone of the antigen-4 fusion protein gene that contains the forward insertion.With the expression vector called after pAO815-antibac plasmid (Fig. 2) that obtains.This plasmid is imported in the intestinal bacteria, make it form polyclone.Preparation pAO815-antibac plasmid uses restriction enzyme Sal I digested plasmid, makes its linearizing.With the linearizing pAO815-antibac plasmid solution of about 20 μ g and the GS115 competent cell mixing of 80 μ l, the electricity that goes to the precooling of 0.2ml ice transforms in the cup.To behind the electricity conversion cup ice bath 5min electricity consumption conversion instrument be transformed.Condition is: voltage 1.5kV, electric shock 10msec makes exogenous origin gene integrator in the genome of yeast GS115.After the conversion thalline is added the 1mol Sorbitol Solution USP mixing of 1ml ice precooling, go in the EP pipe of 1.5ml.Thalline suspension after the conversion is evenly coated on the MD flat board subsequently in 30 ℃ of cultivations, until single bacterium colony appearance.Picking list bacterium colony as template, uses primer 5`AOX (5`-GACTGGTTCCAATTGACAAGC-3`) and 3`AOX (5`-GCAAATGGCCATTCTGACATCC-3`) with it, has the positive colony of pAO815-antibac by PCR reaction screening.PCR band (Fig. 3) about 600bp appears in the positive colony of integration fusion rotein cDNA sequence.Select the bacterium colony that contains pAO815-antibac, place the bottle that shakes that 25ml BMGY substratum is housed, in 30 ℃ of OD that are cultured to bacterium liquid
600=2.0.Collect thalline,, make OD with the resuspended thalline of BMMY
600About=1.0.The bacterium liquid of gained places the bottle that shakes of 1L, and is positioned over 30 ℃ of cultivations, and every 24h adds anhydrous methanol to final concentration in substratum be 0.5% to be cultured to OD
600=3.0.Collect thalline, obtain protein crude extract behind the smudge cells, wherein be rich in fusion rotein.
Also protein crude extract can be used the gradient ammonium sulfate precipitation, will precipitate then collect be dissolved in phosphoric acid buffer after, handle through ion exchange chromatography and gel chromatography respectively, obtain electrophoretically pure fusion rotein.
Simultaneously, down cultivate with similarity condition, the yeast expression system albumen coarse body fluid that can express β-Gal in contrast.
5. bacteriostatic test: be coated with Yeast protein crude extract or the purified fusion rotein that drips different concns on the dull and stereotyped culture dish of intestinal bacteria, Vibrio anguillarum or vibrio alginolyticus.Culture dish as for 37 ℃ of incubated overnight, is observed and the record fungistatic effect next day.
Above-mentioned crude extract and the purified fusion rotein that is rich in fusion rotein, all performance has anti-microbial activity (the results are shown in Figure 4～6).
6. fusion rotein is in the application of preparation in the antibacterials: will contain the yeast of fusion rotein secreted expression carrier, and cultivate the back through routine and collect nutrient solution, again through routine precipitates, steps such as centrifugal, desalination, chromatography obtain the veterinary drug level fusion rotein; Or, use affinity chromatography to obtain more highly purified fusion rotein at labels such as fusion rotein sequence two ends interpolation Histidine cDNA sequences.
The fusion rotein that obtains can be applied to the control of hydrobiont or other breed diseases of bird and livestock directly as antibacterials with the routine administration amount.
7. fusion rotein has the hydrobiont of disease control function or the application in the breed animal and fowl fodder in preparation: because yeast contains rich in protein, VITAMIN and other nutritive substance, the yeast that therefore contains expression vector in the born of the same parents can be used as fodder additives.Add hydrobiont to or culture in the animal and fowl fodder containing in the born of the same parents yeast of expression vector according to preset proportion,, improve its disease defence capability for hydrobiont or when culturing livestock and poultry nutritive substance is provided.
As use aforesaid method, use the 100L fermentor tank, the pichia spp GS115 that in 30 ℃ of following enlarged culturing genomes, includes nucleotide sequence shown in the SEQ ID NO:2, by adding the expression of the antibiotic antigen-4 fusion protein gene of 0.5% methanol induction in the fermented liquid, cultivated through 3-5 days, after filtering, had the GS115 yeast of expressing the anti-microbial activity fusion rotein in a large number, described yeast count is no less than 5 * 10
4Cfu/g, water content≤10%.
Select Semen Maydis powder in a usual manner, chaff, wheat bran, soya-bean cake, fish meal, bone meal is the basal feed of hydrobiont or breed livestock and poultry, with the yeast of above-mentioned acquisition as fodder additives, directly make an addition to above-mentioned hydrobiont or culture in the basal feeds of livestock and poultry and mix, described fodder additives adding proportion amount is 20% consumption of other protein feed (can the corresponding minimizing fish meal or) of basal feed weight, make crude protein 〉=35% in the final nutritive ingredient of the basal feed that is mixed with additive, crude fat 〉=3%, robust fibre 〉=3%, coarse ash 〉=15%, calcium 〉=2%, phosphorus 〉=1%, Methionin 〉=1.8% makes the hydrobiont with disease control function or cultures animal and fowl fodder.
Perhaps, directly above-mentioned gained fermentation liquid is used for the nursing of livestock and poultry and aquaculture organisms with the adding proportion amount of basal feed weight 5%～25%.
Further, the yeast that will contain the fusion rotein secreted expression carrier with aforesaid method, cultivate the back through routine and collect nutrient solution, again through routine precipitates, steps such as centrifugal, desalination, chromatography obtain the veterinary drug level the fusion rotein that contains aminoacid sequence shown in the SEQ ID NO:1.
Select Semen Maydis powder in a usual manner, chaff, wheat bran, soya-bean cake, fish meal, bone meal is the basal feed of hydrobiont or breed livestock and poultry, with the fusion rotein of above-mentioned acquisition as fodder additives, directly make an addition to above-mentioned hydrobiont or culture in the basal feeds of livestock and poultry and mix, described fodder additives adding proportion amount is 10% of a basal feed weight, make crude protein 〉=35% in the final nutritive ingredient of the basal feed that is mixed with additive, crude fat 〉=3%, robust fibre 〉=3%, coarse ash 〉=15%, calcium 〉=2%, phosphorus 〉=1%, Methionin 〉=1.8% makes the hydrobiont with disease control function or cultures animal and fowl fodder.
Sequence table
＜110〉Oceanographic Inst. No.1 of State Bureau of Oceanography
＜120〉a kind of fusion rotein and application thereof with antibacterial
<141>2007-9-11
<160>2
<210>1
<211>39
<212>PRT
＜213〉artificial sequence
<400>1
Met?Asp?Ser?His?Ile?Ser?Leu?Cys?Arg?Trp?Cys?Cys?Asn?Cys?Cys
5 10 15
Lys?Ala?Tyr?Lys?Gly?Cys?Gly?Phe?Cys?Cys?Arg?Phe?Gly?Ile?Gly
20 25 30
Lys?Phe?Leu?His?Ser?Ala?Lys?Lys?Phe
35
<210>2
<211>151
<212>cDNA
＜213〉the cDNA Xu Lie ﹠amp of fish hepcidinl precursor mature peptide; The cDNA sequence of coding Amphibians antibacterial peptide magainin mature peptide
<400>2
ccctcgaatt?cgccgccacc?atggacagcc?acatctccct?ttgccgctgg?tgctgcaact?60
gctgcaaggc?ctacaagggc?tgtggcttct?gctgtaggtt?cggcattggc?aagttcctgc?120
actctgccaa?gaagttctga?gaattcgcac?c 151
Claims (5)
1. the fusion rotein with antibacterial is made up of fish antibacterial peptide hepcidin and Amphibians antibacterial peptide magainin through modification, and it is characterized in that: described fusion rotein is the small peptide that contains aminoacid sequence shown in the SEQ ID NO:1.
2. the gene of the described fusion rotein of claim 1, by the cDNA sequence in the cDNA sequence of the cDNA sequence of coding fish hepcidinl precursor mature peptide, coding Amphibians antibacterial peptide magainin mature peptide, restriction enzyme EcroR I site and meet eukaryote and express the gene order of preference and form, it is characterized in that: the nucleotide sequence of described antigen-4 fusion protein gene is the nucleotide sequence shown in the SEQ ID NO:2.
3. the application of the described fusion rotein of claim 1 in the preparation antibacterials.
4. the described fusion rotein of claim 1 has the hydrobiont of disease control function or the application in the breed animal and fowl fodder in preparation.
5. application as claimed in claim 4 is characterized in that: described fusion rotein directly adds in hydrobiont or the animal and fowl fodder as immunity additive, or purified back is directly used or used as fodder additives as antibacterials.
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| CN101906165A (en) * | 2010-07-09 | 2010-12-08 | 厦门大学 | Expression product in series of two fish antibacterial peptide genes and expression method thereof |
| CN101955525A (en) * | 2010-07-13 | 2011-01-26 | 中国农业科学院兰州畜牧与兽药研究所 | Artificial antimicrobial peptide, gene and preparation method thereof |
| CN102199215A (en) * | 2011-03-22 | 2011-09-28 | 成都市金之源生物技术有限公司 | MAPWA fusion antibacterial peptide, preparation method and application thereof |
| CN101481420B (en) * | 2009-01-22 | 2012-08-15 | 中山大学 | Heterozygous antibacterial peptide CA-MA and recombinant expression method thereof |
| CN104710523A (en) * | 2013-12-11 | 2015-06-17 | 华东理工大学 | Pseudomonas aeruginosa resistant Magainin peptide modifier and preparation method thereof |
| CN110693809A (en) * | 2019-11-21 | 2020-01-17 | 旌德县万方日用品有限公司 | Skin-care moisturizing and whitening beauty lotion and preparation method thereof |
| CN116003541A (en) * | 2022-07-27 | 2023-04-25 | 武汉大学 | Multifunctional fungus defensin modified peptide, preparation method and application thereof |
| CN116640203A (en) * | 2023-06-15 | 2023-08-25 | 自然资源部第一海洋研究所 | Antibacterial peptide Gigantin of deep sea Shendun snail and application thereof |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1158304C (en) * | 2001-05-10 | 2004-07-21 | 上海华谊生物技术有限公司 | Antibacterial frog skin peptide derivative |
| CN1778920A (en) * | 2005-09-27 | 2006-05-31 | 中国水产科学研究院黄海水产研究所 | Antibiotic peptide gene and its yeast expression carrier |
| US7579005B2 (en) * | 2005-11-28 | 2009-08-25 | E. I. Du Pont De Nemours And Company | Process for recombinant expression and purification of antimicrobial peptides using periplasmic targeting signals as precipitable hydrophobic tags |
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2007
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Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101481420B (en) * | 2009-01-22 | 2012-08-15 | 中山大学 | Heterozygous antibacterial peptide CA-MA and recombinant expression method thereof |
| CN101906165A (en) * | 2010-07-09 | 2010-12-08 | 厦门大学 | Expression product in series of two fish antibacterial peptide genes and expression method thereof |
| CN101906165B (en) * | 2010-07-09 | 2012-11-14 | 厦门大学 | Expression product in series of two fish antibacterial peptide genes and expression method thereof |
| CN101955525A (en) * | 2010-07-13 | 2011-01-26 | 中国农业科学院兰州畜牧与兽药研究所 | Artificial antimicrobial peptide, gene and preparation method thereof |
| CN102199215A (en) * | 2011-03-22 | 2011-09-28 | 成都市金之源生物技术有限公司 | MAPWA fusion antibacterial peptide, preparation method and application thereof |
| CN102199215B (en) * | 2011-03-22 | 2014-03-19 | 成都市金之源生物技术有限公司 | MAPWA fusion antibacterial peptide, preparation method and application thereof |
| CN104710523A (en) * | 2013-12-11 | 2015-06-17 | 华东理工大学 | Pseudomonas aeruginosa resistant Magainin peptide modifier and preparation method thereof |
| CN104710523B (en) * | 2013-12-11 | 2017-12-29 | 华东理工大学 | There is Magainin peptide trims of anti-microbial property and preparation method thereof to pseudomonas aeruginosa |
| CN110693809A (en) * | 2019-11-21 | 2020-01-17 | 旌德县万方日用品有限公司 | Skin-care moisturizing and whitening beauty lotion and preparation method thereof |
| CN116003541A (en) * | 2022-07-27 | 2023-04-25 | 武汉大学 | Multifunctional fungus defensin modified peptide, preparation method and application thereof |
| CN116003541B (en) * | 2022-07-27 | 2024-05-28 | 武汉大学 | Multifunctional fungus defensin modified peptide, preparation method and application thereof |
| CN116640203A (en) * | 2023-06-15 | 2023-08-25 | 自然资源部第一海洋研究所 | Antibacterial peptide Gigantin of deep sea Shendun snail and application thereof |
| CN116640203B (en) * | 2023-06-15 | 2023-12-29 | 自然资源部第一海洋研究所 | Antibacterial peptide Gigantin of deep sea Shendun snail and application thereof |
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