CN103665137A - Alligator mississrppinsis Cathelicidin-AM antibacterial peptide as well as coded sequence and application thereof - Google Patents
Alligator mississrppinsis Cathelicidin-AM antibacterial peptide as well as coded sequence and application thereof Download PDFInfo
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Abstract
The invention provides an alligator mississrppinsis Cathelicidin-AM antibacterial peptide, a polynucleotide for coding the antibacterial peptide and an application of the antibacterial peptide. The antibacterial peptide has an amino acid sequence as shown in SEQ ID NO: 2. The polynucleotide for coding the antibacterial peptide has a nucleotide sequence containing the coded amino acid sequence as shown in SEQ ID NO: 2 or a complementary sequence of the nucleotide sequence. The antibacterial peptide has a remarkable inhibiting effect on various bacteria and has the minimal inhibitory concentration (MIC) of 62.5mug/mL for Echerichia coli (CMCC44102), Escherichia coli (ATCC25922) and Salmonella typhi (CMCC50115). The antibacterial peptide can be used for preparing feed additives, preservatives, agricultural biocontrol agents, therapeutic drug compositions for controlling microorganism infection and the like.
Description
Technical field
The present invention relates to the antibacterial peptide of Cathelicidin antibacterial peptide family, relate in particular to a kind of America alligator Cathelicidin-AM antibacterial peptide.In addition, the invention still further relates to the coding nucleotide sequence of this antibacterial peptide and the purposes of this antibacterial peptide.
Background technology
Due to antibiotic, abuse and abuse, many bacteriums have produced Resistant strain, and the research and development speed of new antibiotic is fast not, so the contradiction between new antibiotic research and development and antibacterial demand.In addition, the residue problem of microbiotic in livestock product also more and more causes people's attention, and it has directly jeopardized the mankind's health.The solution that the Antibacterial Mechanism of antibacterial peptide uniqueness is this problem provide may, so the utmost point to be hopeful to be developed to be the novel high-efficiency antimicrobial medicine of a class.
Antibacterial peptide is to be present in the polypeptide that a class in animal body has broad spectrum antibiotic activity, is also integral part important in innate immune system.Up to the present, people have found hundreds of kind antibacterial peptide from Mammals, insect and Amphibians, and its structure, activity, the mechanism of action etc. have been done to a large amount of research, find that antibacterial peptide anti-microbial activity is high, has a broad antifungal spectrum, the mechanism of action and common microbiotic are very different by the synthetic feature that reaches sterilization effect of arrestin, and they are to carry out lethal bacterium by destroying the cytolemma of bacterium, are therefore difficult for producing resistance.Most antibacterial peptide all has following characteristics: molecular weight is little, and Heat stability is good with positive charge, and is amphipathic molecule.According to these features of antibacterial peptide, people are divided into two large class: defensins (alexin) and Cathelicidins by antibacterial peptide.
This noun of Cathelicidin is used to describe the molecule with Cathelin and two kinds of structural domains of C end antibacterial peptide since nineteen ninety-five.Because Cathelicidin family member is varied, its antibacterial mechanisms is not quite similar.They all have resistant function to bacterium, fungi, virus and parasitic infection, or direct pathogen kill, or by alleviate the various biological effects that cause because of cause pathogeny imcrobe infection in conjunction with intracellular toxin.Wherein one of topmost mechanism is that Cathelicidin can form cross-film ionic channel in the plasma membrane surfaces of pathogenic agent, thereby causes oozing out of entocyte to cause the death of bacterium.Cathelicidin has the ability of powerful combination bacteria lipopolysaccharide (LPS).With bacterium endochylema membrane phospholipid molecular adsorption after being combined on lipid film, Cathelicidin molecular aggregates is agglomerating, its hydrophobic side is inserted in plasma membrane by syndeton flexible in molecule, and water-wet side points to the inside of film to form cross-film ionic channel, can allow many kinds of substance to comprise that hydrophobic mixture, small molecules pass through.So the integrity of film is damaged, in born of the same parents, ion is lost in a large number, finally causes the death of bacterium.The test of plane phospholipid bilayer shows: the change of plasma membrane permeability may be that the formation due to passage causes.The Cathelicidin family peptides with αhelix, can penetrate into rapidly in the plasma membrane of sensitive organism, thereby change its permeability, and under bacteriocidal concentration, the destroy integrity of inner membrance, causes cellular respiration significantly to reduce, and bacterium is thereupon dead.Except anti-microbial activity, Cathelicidin also has other important biological functions, comprise promote trauma repair, as chemical inhibitor convene inflammatory cell arrive inflammation part, induction of vascular generate, in conjunction with intracellular toxin and inducing cell dissolving etc.These biological effects of Cathelicidin have strengthened host's defense mechanism, impel body from pathogenic infection, to recover.
Chinese medicine treasured book < < Compendium of Materia Medica > > thinks " Chinese alligator (being crocodile) first: cure mainly trusted subordinate's abdominal mass; Volt sons and daughters gather; Fever and chills; The blood five colors and the dead flesh of sore scabies under coupling pain, metrorrhagia in woman's underbelly the moon ... meat: cure mainly weak breath and inhale, foot is not on the spot; Do not record moisture perverse trend, all legendary venomous insects, abdominal mass, evil sore in abdomen.Fat: cure mainly and rub wind and dislike sore ... ".Above-mentioned " abdominal mass " and " gathering " mainly refer to ovarian cancer and the liver cancer in modern medicine, and " weak breath is inhaled " is cough, the asthma of today.Modern medicine finds that crocodile goods have obvious medicines and health protection effect.Studies have found that esturaine crocodile scale and shell extract has restraining effect to the tumour of mouse.Investigator has developed Alligator Zhikegao by a certain percentage with crocodile, Grosvenor Momordica and Radix Glycyrrhizae, find its to mouse have obvious antibechic, eliminate the phlegm, anti-inflammatory and immunoregulation effect.
In order to resist extraneous pressure, in long-term evolutionary process, crocodile a large amount of antibacterial peptide matters of having evolved out.Many crocodile antibacterial peptides of finding at present have certain bacteriostatic action to bacterial strains such as intestinal bacteria, pneumococcuss, but be there is not to toxic action in the normal histocyte of Mammals, it is as the substitute of antibiotics, at clinical, food and feed industry, has very high using value.From crocodile body, directly extract antibacterial peptide method complicated, output is very micro-; Adopt chemical synthesis to prepare antibacterial peptide cost too high, all possible without practical application.Therefore utilize gene recombination technology in microorganism, directly to express crocodile antibacterial peptide and just become the outlet addressing this problem.
Summary of the invention
For above-mentioned defect of the prior art, one object of the present invention is to provide a kind of America alligator Cathelicidin-AM antibacterial peptide, another object of the present invention is to provide a kind of purposes of nucleotide sequence and this antibacterial peptide of this antibacterial peptide of encoding.
On the one hand, the invention provides a kind of America alligator Cathelicidin-AM antibacterial peptide, it is characterized in that described polypeptide is selected from lower group:
(a) polypeptide of SEQ ID NO:2 aminoacid sequence;
(b) SEQ ID NO:2 aminoacid sequence is through modifying the polypeptide derivative of resulting functional equivalent;
(c) SEQ ID NO:2 aminoacid sequence is through replacement, the disappearance of one or more amino-acid residues or add the derivative polypeptide of formed functional equivalent.
Second aspect, the invention provides also a kind of polynucleotide, it is characterized in that the nucleotide sequence of described polynucleotide is selected from lower group:
(a) polynucleotide of polypeptide as claimed in claim 1 of encoding;
(b) polynucleotide complementary with polynucleotide (a).
Preferably, polypeptide or its complementary sequence of aminoacid sequence shown in described polynucleotide encoding SEQ ID NO:2.
More preferably, the sequence of described polynucleotide is selected from lower group a kind of:
(a) sequence of 17-139 position in SEQ ID NO:1;
(b) due to the degeneracy of genetic code derived from the sequence of polynucleotide sequence (a);
(c) polynucleotide sequence (a) and complementary sequence (b).
The third aspect, the present invention also provides also a kind of expression vector, it is characterized in that described expression vector contains at least above-mentioned polynucleotide described in any one.
Fourth aspect, the present invention also provides a kind of genetically engineered host cell, it is characterized in that described host cell contains above-mentioned expression vector, or in its genome, is integrated with at least above-mentioned polynucleotide described in any one.
Preferably, described genetically engineered host cell transforms subtilis by expression vector claimed in claim 5 and obtains.
The 5th aspect, the present invention also provides a kind of and has prepared as the method for above-mentioned America alligator Cathelicidin-AM antibacterial peptide, it is characterized in that described polypeptide synthesizes by mechanochemical method or generates by gene recombination method, described gene recombination method comprises the following steps:
1), in suitable substratum, expression vector claimed in claim 5 is transformed or transfection host cell;
2) screening positive clone;
3) abduction delivering antibacterial peptide as claimed in claim 1;
4) recovering condition substratum or cell extract,
5) from the described substratum that obtains or cell extract is separated and purifying described in polypeptide.
The 6th aspect, the present invention also provides the purposes of above-mentioned America alligator Cathelicidin-AM antibacterial peptide in the medicine composition of preparing fodder additives, sanitas, agricultural biological control agent or control infected by microbes.
The 7th aspect, it is a kind of for controlling the medicine composition of infected by microbes that the present invention also provides, and it is characterized in that described pharmaceutical composition comprises antibacterial peptide claimed in claim 1.
Eight aspect, the present invention also provides a kind of fodder additives, it is characterized in that described fodder additives comprises antibacterial peptide claimed in claim 1.
The 9th aspect, the present invention also provides a kind of sanitas, it is characterized in that described sanitas comprises antibacterial peptide claimed in claim 1.
The tenth aspect, the present invention also provides a kind of agricultural biological control agent, it is characterized in that described agricultural biological control agent comprises antibacterial peptide claimed in claim 1.
America of the present invention alligator Cathelicidin-AM antibacterial peptide has anti-microbial activity, can kill bacterium, fungi, virus and protozoon.These antibacterial peptides have effect to any organism with cytolemma or lipid bilayer component generally.This antibacterial peptide is to be applied in the mankind and/or livestock medicine, for animals or as the active compound of agricultural, food and industrial reagent.
In addition, antibacterial peptide of the present invention and polynucleotide sequence thereof also have following purposes:
1. immune animal after this Cathelicidin-AM antibacterial peptide and albumin coupling, can prepare the antibody with Cathelicidin-AM antibacterial peptide specific binding.This antibody can be for detection of the existence of the antibacterial peptide in solution.
2. by SQ ID NO:1 or the polynucleotide (comprise DNA or RNA) complementary with it, or its fragment, carry out after mark, can pass through the technology such as Southern, Northern, gene chip, detect the existence of the antibacterial peptide gene fragment in Animal genome, or detect the situation of transcribing of antibacterial peptide gene.
3. according to SQ ID NO:1 or polynucleotide complementary with it design primer, in can detecting antibacterial peptide gene each be organized in animal body by reverse transcription-polymerase chain reaction (RT-PCR), the situation of transcribing of different developmental phases.
Accompanying drawing explanation
Fig. 1 is America alligator mucous epithelium total tissue RNA electrophorogram.
Fig. 2 is the bacterium colony PCR electrophorogram of alligator mucous epithelium tissue cDNA library, America recombinant plasmid.Wherein: M.DNA mark; 1. blank; 2-5.PCR object band.
Fig. 3 is the PCR electrophoresis result of America alligator Cathelicidin-AM antibacterial peptide gene.Wherein: M.DNA mark; 1-4.Cathelicidin-AM antibacterial peptide gene PCR band.
Fig. 4 is P
43promotor and nprB signal peptide gene PCR electrophorogram.Wherein: M.DNA mark; 1-2.P
43the band of promotor; The band of 3-4.nprB signal peptide.
Fig. 5 is that overlapping extension amplification and SOE merge electrophorogram.Wherein: M.DNA mark; 1.nprB and P
43merge fragment; 2.P
43-SP-cathAM merges fragment.
Fig. 6 is the recombinant expression plasmid pHY43N-cathAM design of graphics of Cathelicidin-AM antibacterial peptide.
Fig. 7 is that the positive colony of pHY43N-cathAM detects electrophorogram.Wherein: M.DNA mark; 1. positive colony; 2. blank; 3. negative control.
Fig. 8 is the SDS-PAGE electrophorogram of America alligator Cathelicidin-AM antibacterial peptide recombination expression product.Wherein: 1. protein labeling; 2. the pHY43N-cathAM WB800 thalline that does not add induction; 3. the Cathelicidin-AM antibacterial peptide that adds pHY43N-cathAM WB800 thalline 4. purifying of induction.
Fig. 9 is America alligator Cathelicidin-AM antibacterial peptide ion exchange analysis purifying figure.
Figure 10 is the fungistatic effect of America alligator Cathelicidin-AM antibacterial peptide to colon bacillus.
Figure 11 is the fungistatic effect of America alligator Cathelicidin-AM antibacterial peptide to Salmonella typhimurtum.
Figure 12 is the fungistatic effect of America alligator Cathelicidin-AM antibacterial peptide to C type clostridium perfringens.
Figure 13 is the fungistatic effect of America alligator Cathelicidin-AM antibacterial peptide to hemophilus influenzae.
Figure 14 is the fungistatic effect of America alligator Cathelicidin-AM antibacterial peptide to Vibrio parahemolyticus.
Figure 15 is the fungistatic effect of America alligator Cathelicidin-AM antibacterial peptide to campylobacter jejuni.
Figure 16 is the fungistatic effect of America alligator Cathelicidin-AM antibacterial peptide to streptococcus aureus.
Figure 17 is the fungistatic effect of America alligator Cathelicidin-AM antibacterial peptide to Listeria Monocytogenes.
Figure 18 is the fungistatic effect of America alligator Cathelicidin-AM antibacterial peptide to streptococcus pneumoniae.
Embodiment
The present invention passes through to build America alligator skin histology cDNA library, and by checking order and screening, has obtained the cDNA of a kind of America alligator Cathelicidin-AM antibacterial peptide.Meanwhile, by artificial synthesis (mechanochemical method) and gene recombination method, obtained respectively the coded polypeptide fragment of this antibacterial peptide cDNA.In embodiments of the invention, this Cathelicidin-AM antibacterial peptide polypeptide contains 41 amino acid, most preferably is the aminoacid sequence that contains in SQ ID NO:2 1 to 41.This antibacterial peptide aminoacid sequence is compared through Protein Data Bank, find no any identical Cathelicidin mature peptide.
Polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, synthetic polypeptide, preferably recombinant polypeptide.In the present invention, term " derivative " refers to and substantially keeps biological function or the active polypeptide that Cathelicidin-AM antibacterial peptide of the present invention is identical.Polypeptide fragment of the present invention, derivative can be that (i) has one or more conservative or substituted polypeptide of non-conservation amino-acid residue (preferably conservative amino acid residue), or (ii) in one or more amino-acid residues, there is the polypeptide of substituted radical, or (iii) mature polypeptide and another compound merge formed polypeptide, or (iv) additional aminoacid sequence is fused to this peptide sequence and the polypeptide that forms (as leader sequence or secretion sequence or be used for the sequence of this polypeptide of purifying).These derivatives and analogue belong to the scope of well known to a person skilled in the art.
In the present invention, also comprise and having and variant form Cathelicidin-AM antibacterial peptide identical function, SEQ ID NO.2 sequence.These variant forms comprise (but being not limited to): one or more amino acid whose disappearances, insertion and/or replacement, and add one or several amino acid at C-terminal and/or N-terminal.For example, in the art, while replacing with the close or similar amino acid of performance, conventionally can not change the function of protein.Again such as, at C-terminal and/or N-terminal, add one or several amino acid and conventionally also can not change the function of protein.Variant form also comprises: homologous sequence, conservative property varient etc.The present invention also provides other polypeptide, as the fusion rotein that comprises this Cathelicidin-AM antibacterial peptide or its fragment.
Polynucleotide of the present invention can be strand or double-stranded.The coding region sequence of encoding mature polypeptide can be identical with the coding region sequence shown in SEQ ID NO:1 or the varient of degeneracy.Polynucleotide of the present invention also comprise the complementary sequence of the coding region sequence of encoding mature polypeptide, for example the complementary sequence SQ ID NO:3 of SEQ ID NO:1.
The polynucleotide of coding America alligator Cathelicidin-AM antibacterial peptide comprise: the encoding sequence of an encoding mature polypeptide; The encoding sequence of mature polypeptide and various additional code sequence; The encoding sequence of mature polypeptide (with optional additional code sequence) and non-coding sequence.
The invention still further relates to the varient of above-mentioned polynucleotide, its coding has the polypeptide of identical aminoacid sequence or the analogue of polypeptide and derivative with the present invention.These nucleotide diversity bodies comprise and replace varient, deletion mutation body and insert varient.As known in the art, polynucleotide varient is the replacement form of polynucleotide, can be from not changing in fact the function of the polypeptide of its coding.
The present invention also relates to the carrier that comprises polynucleotide of the present invention, and the host cell producing through genetically engineered with carrier of the present invention or this antibacterial peptide encoding sequence, and the method that produces polypeptide of the present invention through recombinant technology.
In the present invention, the polynucleotide sequence of America alligator Cathelicidin-AM antibacterial peptide can be inserted in recombinant expression vector.Term " expression vector " refers to bacterial plasmid well known in the art, phage, yeast plasmid, vegetable cell virus, mammalian cell is viral or other carriers.In a word, as long as can copy in host and stablize, any plasmid and carrier can be used.
In the present invention, host cell can be prokaryotic cell prokaryocyte, as bacterial cell; Or the eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as vegetable cell.Representative example has: intestinal bacteria, subtilis; Fungal cell is as yeast; Vegetable cell; Insect cell etc.Host cell of the present invention is preferably subtilis, because subtilis conventionally can be directly as fodder additives, when using subtilis as host cell expression antibacterial peptide of the present invention, and while intending this antibacterial peptide to be applied to fodder additives, do not need this antibacterial peptide to carry out separation and purification, can be directly using the bacillus subtilis bacteria culture fluid after abduction delivering as fodder additives, the Cathelicidin-AM antibacterial peptide of doing invention is contained in the inside.
Cathelicidin-AM antibacterial peptide of the present invention can be synthetic by conventional chemical solid phase method, and C is held to amidation.The present invention is preferably by conventional recombinant DNA technology and gives expression to recombinant C athelicidin-AM antibacterial peptide.In general there are following steps:
1), in suitable substratum, the expression vector building is transformed or transfection host cell;
2) screening positive clone;
3) abduction delivering Cathelicidin-AM antibacterial peptide of the present invention antibacterial peptide;
4) recovering condition substratum or cell extract,
5) from the described substratum that obtains or cell extract is separated and purifying described in polypeptide.
Antibacterial peptide in the present embodiment has very strong restraining effect (referring to embodiment five, table 1) to multiple gram positive bacterium and gram negative bacterium.The bacterial classifications such as the intestinal bacteria of choosing in the present embodiment, streptococcus aureus are considered to the model animals of Gram-positive and negative bacteria in the art, therefore to the biological activity of these model bacteriums, can be considered to the biological activity to whole Gram-positive and negative bacteria family.Therefore, Cathelicidin-AM antibacterial peptide of the present invention can be used as sterilization reagent or antibacterial reagent is used for stoping infection, kill microorganisms or suppresses microorganism growth and other function, and therefore can effectively treat or control the infection being caused by these microorganisms or the pollution causing.This antibacterial peptide can also be used to fodder additives, agricultural biological control agent etc., increases the resistibility of livestock product to microorganism, for livestock product provide new antibacterial approach, is with a wide range of applications.
Meanwhile, Cathelicidin-AM antibacterial peptide of the present invention also can be as medicine composition, is applicable to the mankind, livestock, agricultural and medicine company and uses, and it comprises acceptable carrier on one or more and the medicine in antibacterial peptide of the present invention.These pharmaceutical compositions can be according to as known in the art, prepare and administration for example oral, injection or topical administration apply to control and/or suppress to comprise the infection of the wide region microorganism of Gram-positive and negative bacteria.
Below in conjunction with embodiment, further illustrate content of the present invention, but embodiment should not be construed as limitation of the present invention.Without departing from the spirit and substance of the case in the present invention, the modification that method of the present invention, step or condition are done or replacement, 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.
Embodiment mono-: the solid state chemistry synthesis method of Cathelicidin-AM antibacterial peptide
According to coding America alligator Cathelicidin antibacterial peptide gene, infer Cathelicidin-AM mature peptide aminoacid sequence, according to this Cathelicidin-AM antibacterial peptide of standard solid phase peptide synthesis program synthetic.Synthetic peptide, through anti-phase-high pressure liquid chromatography (HPLC) (RP-HPLC, Column:X Bridge 4.6 * 250mm) desalination, purifying, adopts acetonitrile/water/trifluoroacetic acid system wash-out (Pump A:0.07% trifluoroacetic in 100% water; Pump B:0.05% trifluoroacetic in 100% acetonitrile), adopt electrospray ionization mass spectrum (ESI-MS) to measure its molecular weight.Sample purity >=96%, its theoretical molecular is 4546, actual measurement molecular weight is 4547.70, basically identical.Synthetic Cathelicidin-AM antibacterial peptide is dissolved in to sterilizing distilled water, for activity, detects.
Embodiment bis-: America alligator Cathelicidin-AM antibacterial peptide cDNA obtains
The inventor uses the Smart of TaKaRa company
tMcDNA library builds test kit, built America alligator skin histology cDNA library, by random picking cloning and sequencing, and compare by the sequence in BLAST and international gene pool (GenBank) recording sequence, obtained the cDNA of Cathelicidin-AM antibacterial peptide.
1. the extraction of America alligator mucous epithelium total tissue RNA
Get America alligator mucous epithelium and be organized in cryopreservation tube, America alligator is discharged thereupon, cryopreservation tube is placed in liquid nitrogen and transports laboratory back, is stored in liquid nitrogen container standby.
Get mucous epithelium and organize 0.1g, liquid nitrogen cryogenics is ground into powder.Add 2mL RNAiso Reagen room temperature standing to melting, then be ground to lysate and become transparence.4 ℃, the centrifugal 5min of 12000r/min.In supernatant liquor, add 0.2mL chloroform, fully emulsified solution is after milky white shape, in 4 ℃, the centrifugal 15min of 12000r/min, and isopropanol precipitating, 4 ℃, the centrifugal 10min collecting precipitation of 12000r/min.DEPC processes 75% ethanolic soln washing of water preparation.Drying at room temperature, RNase-free water dissolution precipitated rna, detects and can see 28S and l8S two bands (electrophoresis result is shown in Fig. 1) clearly through 1% agarose gel electrophoresis; With OD
260/ OD
280ratio is identified the purity of total RNA, and its ratio is 1.95;-80 ℃ of preservations.
2. the first chain cDNA's is synthetic
(1) thermally denature of RNA:
Total RNA(0.25 μ g/ μ L) 2 μ L
SMART IV nucleic acid oligomer 2 μ L
CDS III/3 ' PCR primer 1 μ L
Above composition is mixed, on whizzer, get rid of, hatch sex change 5min in 65 ℃, be put on ice immediately.
(2) reaction solution configuration:
(3) reverse transcription reaction:
Reaction solution is mixed gently, proceed as follows after adding 1 mineral oil: room temperature 10min; 42 ℃ of constant temperature 1h; Ice bath 5min; Synthetic cDNA the first chain.Instantaneously carry out next step operation or be placed in-20 ℃ saving backup after centrifugal.
3.LD-PCR synthetic double chain cDNA
CDNA the first chain synthesizing of take is template, carries out pcr amplification.PCR reaction system is as follows:
Reaction solution is mixed gently, on whizzer, get rid of.Add 2 mineral oil, reaction tubes is placed on the PCR instrument that is preheated to 95 ℃, carry out LD-PCR.
Reaction conditions: 95 ℃ of denaturation 5min; 95 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ of extension lmin, circulate 30 times altogether; After 72 ℃ of extension 5min, finish reaction.
Amplified production carries out electrophoretic analysis with 1.5% sepharose, voltage 4v/cm, and electrophoresis 1h, observes under gel imaging instrument.
The protease K digesting of 4.LD-PCR product
The double-stranded cDNA(2 μ of 50 μ L g), add 2 μ L Proteinase Ks (20 μ g/ μ L), 45 ℃ of water-bath 20min, with enzyme remaining in sample digestion.Add 50 μ L deionized waters, 100 μ L phenol are imitated alcohol (phenol: imitative: alcohol=50:49:1) the centrifugal 5min of 14000r/min, get upper strata water, add wherein after the centrifugal 5min of mixture 14000r/min of 100 μ L chloroforms and primary isoamyl alcohol, get upper strata water and add wherein 10 μ L sodium acetates (3mol/L), 1.3 μ L glycogens (20 μ g/ μ L), 95% ethanol of 2.5 times of volumes, under 14000r/min, rapid centrifugal 20min, drying precipitated in super clean bench.
5.cDNA carries out Sfi I enzyme and cuts
Carry out Sfi I (A, B) enzyme and cut, in the precipitation upwards obtaining in step, add following reagent:
Reaction solution is mixed gently, on whizzer, get rid of, 50 ℃ of insulation 2h.Add 2 μ L xylidene(s)s (1%), mix gently standby.
6.cDNA fractional separation
Press the operation of test kit operation instruction, the double-stranded cDNA that 100 μ L Sfi I (A, B) enzymes were cut and the mixture of xylidene(s), by CHROMA SPIN-400 post fractional separation, the collection tube that cDNA fragment after separation is greater than to 300bp merges, and standby after being further purified.
The connection of 7.cDNA fragment and carrier pDNR-LIB
Double-stranded cDNA fragment and Sfi I (A through identical, B) the ligation volume ratio carrier pDNR-LIB(1 μ L that enzyme is cut) adopts respectively 0.5:1,1:1 and 1.5:1, with this, test joint efficiency, cDNA remaining after test is connected with optimal condition, and under the effect of T4 DNA ligase, 16 ℃ of connections are spent the night.Again purifying connects product, to remove ion wherein, for electricity transforms, prepares.
8. the preparation of bacterium DH5 α competent cell
The mono-colony inoculation of DH5 α on picking flat board in 3mL LB nutrient solution, 37 ℃ of incubated overnight.Get this nutrient solution and be inoculated in fresh LB nutrient solution by 1/100 volume, 37 ℃ of 200r/min thermal agitations are cultured to OD
600it is 0.4 left and right.Erlenmeyer flask is taken out and puts immediately ice bath 15min.Bacterium liquid is transferred to sterilizing and in the 50mL of precooling centrifuge tube, 4 ℃, the centrifugal 10min of 3000r/min.Abandoning supernatant, adds the resuspended thalline of aseptic double-distilled water 10mL of precooling.Bacterium liquid in two centrifuge tubes is merged, 4 ℃, the centrifugal 10min of 3000r/min.Abandoning supernatant, adds the 0.1M CaCl of precooling
2the resuspended thalline of 10mL, ice bath 15min.4 ℃, the centrifugal 10min of 3000r/min.Abandoning supernatant as far as possible, then adds the 0.1M CaCl of 2mL precooling
2, re-suspended cell gently.Above-mentioned bacterium liquid is sub-packed in 1.5mL centrifuge tube to every pipe 200 μ L.4 ℃ are used after depositing 24h.Deposit the requirement that can meet conversion for a week for 4 ℃, glycerol adding to 15% is positioned over-80 ℃ and can preserves the long period.
9. the conversion of recombinant plasmid and screening
By connecting product, add in competent cell prepared by 100 μ L, on ice standing 30min.42 ℃ of thermal shock 45s, put back to 1min in ice immediately.Add 890 μ L LB substratum, after 37 ℃ of standing 10min, then on 37 ℃ of shaking tables shaking culture 60min, thalline is recovered and expresses resistant gene.On the selectivity flat board that contains paraxin (100 μ g/mL), be coated with 10 μ L IPTG(20mg/mL) and 40 μ L X-gal(200mg/mL), after absorbing completely, it is coated with 200 μ L bacterium liquid, just put 60min for 37 ℃, after liquid all absorbs, be inverted the dull and stereotyped 18h that continues to cultivate.Adopt α-complementary method to carry out blue hickie preliminary screening to recombinant plasmid.
10. the evaluation of recombinant plasmid
White colony with on aseptic toothpick picking LB flat board, is inoculated in (100pg/mL paraxin) in 5mL LB liquid nutrient medium, and 37 ℃ of overnight incubation are carried out PCR evaluation, enzyme to bacterium liquid and cut and identify and order-checking is identified.The PCR of recombinant plasmid identifies: the bacterium liquid of the white colony cultivated of take carries out PCR evaluation as template.The primer of PCR is to contain M13 ± sequence on M13+ and M13-(pDNR-LIB carrier), pcr amplification condition: 94 ℃ of sex change 5min; 94 ℃ of 2min, 53 ℃ of 1min, 72 ℃ of 2min, carry out 30 circulations; 72 ℃ of insulation 20min.1% agarose gel electrophoresis detects recombination fraction and inserts cDNA clip size (electrophoresis result is shown in Fig. 2).
The order-checking of recombinant plasmid is identified: PCR is identified to correct recombinant plasmid, send order-checking company to check order.
11. amplification library titer determinations
Get the 1 original library of μ L (cumulative volume 1mL), carry out respectively 1:1000,1:10000, after 1:100000 dilution, respectively gets 1 μ L and adds with coating on the LB flat board containing paraxin after appropriate LB substratum dilution, cultivates 18h counting clone number.
Library titre (cfu/mL)=clone number * extension rate * 10
3
Original library is coated with on several LB flat boards with amplification library, amplification library titre is in kind measured.With LB substratum, wash bacterium colony on lower flat board, add the glycerine after sterilizing, be sub-packed in centrifuge tube ,-80 ℃ of preservations.
The sequential analysis of 12. antibacterial peptide gene cDNA
Utilize BLAST instrument, carry out homology comparison with the database of GenBank.The cDNA that finds a sequence and other Cathelicidin antibacterial peptide has certain homology, and confirming as is by analysis America alligator Cathelicidin-AM antibacterial peptide cDNA sequence, and this sequence is as shown in SQ ID NO:1.
Embodiment tri-: recombinant expressed in subtilis of America alligator Cathelicidin antibacterial peptide
1. according to two terminal sequences of 17-139 position polynucleotide in SQ ID NO:1, meter primer is as follows:
P1：5’-GTGGGGTTCAAGAAAGGCCTG-3’；
P2:5 '-CCCAAGCTT CTAGAACTTGCCAAGGAACTTGGC-3 ' (introducing Hind III restriction enzyme site).
Take pET30a-cathAM plasmid as template amplification Cathelicidin-AM gene, and PCR system is as follows:
Response procedures: 94 ℃ of denaturation 3min; 94 ℃ of 30s of sex change, the 57 ℃ of 30s that anneal, extend 72 ℃ of 1min, 35 circulations; 72 ℃ are extended 10min.1% agarose gel electrophoresis detects PCR product (electrophoresis result is shown in Fig. 3).
2. take Bacillus subtillis genomic dna as template, according to sequence known in Genbank, establish primer amplification P
43promoter sequence, introduces EcoR I restriction enzyme site in primer P3, the primer of design is as follows:
P3：5’-CGGAATTCCTTGTAGAGCTCAGCATTATTG-3’；
P4：5’-GTGTACATTCCTCTCTTTATAATGGTACCGCTATCAC-3’。
Take subtilis genome as the template P that increases
43gene, PCR reaction system is as follows:
Response procedures: 94 ℃ of denaturation 3min; 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 30S, 35 circulations; 72 ℃ are extended 10min.(electrophoresis result is shown in Fig. 4).
3. take Bacillus subtillis genomic dna as template, the signal coding sequence according to primers amplification neutral protease gene nprB known in Genbank changes initiator codon (TTG) into ATG in P5, and the primer of design is as follows:
P5：5’-AAGAGAGGAATGTACACATGCGCAACTTGACCAAG-3’；
P6：5’-TTGAACCCCACTGAGGCATGTGTTAC-3’。
Take subtilis genome as the template nprB signal peptide gene that increases, and PCR reaction system is as follows:
Response procedures: 94 ℃ of denaturation 3min; 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 30S, 35 circulations; 72 ℃ are extended 10min(electrophoresis result and see Fig. 4).
4. with the P after purifying
43promotor and nprB signal peptide PCR product mixtures are template, take P3, P6 as primer, utilize overlapping extension (SOE) amplification, by P
43promotor and nprB signal coding sequence merge, and obtain P
43+ nprB; Product after purifying reclaims reclaims product with cathAM to be mixed as template, take P3, P2 as primer, utilizes overlapping extension (SOE) amplification, by P
43promotor and nprB signal coding sequence and peaT gene order merge, and obtain P
43+ nprB+cathAM.(electrophoresis result is shown in Fig. 5).
P
43it is as follows that+nprB merges PCR reaction system:
Response procedures: 94 ℃ of denaturation 3min; 94 ℃ of 30S, 55 ℃ of 30s, 72 ℃ of 30S, 40 circulations; 72 ℃ of 10min.
P
43it is as follows that+nprB+cathAM merges PCR reaction system:
Response procedures: 94 ℃ of denaturation 3min; 94 ℃ of 30S, 55 ℃ of 30s, 72 ℃ of 1min, 40 circulations; 72 ℃ are extended 10min.
5.P
43+ nprB+cathAM is connected with pMDl8-T simple carrier, transforms bacillus coli DH 5 alpha competent cell, prepares positive colony, and selects primer P3/P2 to carry out bacterium liquid PCR and carry out screening positive clone.
P
43the ligation system that+nprB+cathAM merges PCR product and pMDl8-T simple carrier is as follows, 16 ℃ of connections of spending the night.
PCR product 4.0 μ L
pMD18-T simple 1.0μL
Solution 4.0μL
Cumulative volume 10.0 μ L
6. from positive colony, extract plasmid, by the P that contains extracting
43t vector plasmid and the pHY300-PLK plasmid of+nprB+cathAM carry out double digestion.The double digestion object fragment being recovered to is connected, and the shuttle expression carrier pHY43N-cathAM(building process that obtains restructuring refers to Fig. 6).To connect product and transform bacillus coli DH 5 alpha competent cell, and carry out positive colony detection, and extract plasmid pHY43N-cathAM and carry out double digestion evaluation (electrophoresis result is shown in Fig. 7).
Positive colony detection reaction system is as follows:
Response procedures: 94 ℃ of denaturation 3min; 94 ℃ of 30s, 57 ℃ of 30s, 72 ℃ of 1min, 35 circulations; 72 ℃ are extended 10min.
Contain P
43t vector plasmid and the pHY300-PLK plasmid of+nprB+cathAM carry out double digestion by following reaction system:
37 ℃, reaction 3h.
The enzyme being recovered to is cut to object fragment, according to following reaction system, connect, 16 ℃ of connections of spending the night, obtain the shuttle expression carrier pHY43N-cathAM recombinating.
By the pHY43N-cathAM plasmid extracting from identify correct positive colony, according to following reaction system, 37 ℃ of water-baths, react 3h, carry out enzyme and cut evaluation:
7. take subtilis WB800 as recipient bacterium, the method transforming by electric shock, the recombinant expression vector pHY43N-cathAM building is proceeded in subtilis, adopt PCR, RT-PCR and the enzyme method such as cut to detect conversion results, obtained recombinant bacterial strain WB800-cathAM.Utilize SDS-PAGE to detect the secreting, expressing situation (see figure 8) of Cathelicidin-AM antibacterial peptide in subtilis WB800.
Embodiment tetra-: the separation and purification of restructuring America alligator Cathelicidin-AM antibacterial peptide
The fermented supernatant fluid that contains restructuring America alligator Cathelicidin-AM antibacterial peptide by obtaining, changes after level pad (25mM PBS, pH6.0) upper CM sepharose FF cationic exchange coloum into through Sephadex G-25 post.Collect percolation peak, with aforementioned level pad, wash post to plateau.Then, with solution A (25mM PBS, pH8.0) and solution B (the 25mM PBS that contains 1M NaCl, pH8.0) mixed solution linear gradient elution, collect each elution peak, with Tricine/Tris SDS-PAGE electrophoresis (silver dyes), analyze (elution protocol after optimization is for using respectively the PBS eluant solution of 100,200,500mM NaCl).Result shows as Fig. 9, and desired polypeptides, at the 25mM PBS that contains 200mM NaCl, is eluted in pH8.0.Finally can obtain purity at more than 95% restructuring America alligator Cathelicidin-AM antibacterial peptide, yield is 400 μ L/L substratum.
Embodiment five: the bacteriostatic activity of restructuring America alligator Cathelicidin-AM antibacterial peptide detects
Employing standard agar hole diffusion process detects the In Vitro Bacteriostatic of restructuring America alligator Cathelicidin-AM antibacterial peptide, and compares with traditional microbiotic.
The indicator strain that bacteriostatic test is used: colon bacillus (Escherichia coli CMCC44102); Colon bacillus (Escherichia coli ATCC25922); Salmonella typhimurtum (Salmonella typhimurium CMCC50115); C type clostridium perfringens (Clostridium perfringens type C C59-2); Hemophilus influenzae (Haemophilus influenzae ATCC49247); Vibrio parahemolyticus (Vibrio Parahemolyticus ATCC17802); Campylobacter jejuni (Campylobacter jejuni ATCC29428); Streptococcus aureus (Staphyloccocus aureus CMCC26003); Listeria Monocytogenes (Listeria monocytogenes ATCC19114); Streptococcus pneumoniae (Streptococcus pneumoniae ATCC49619).10 kinds of bacterial strains are our unit and preserve.
Bacteriostatic test concrete steps: single bacterium colony that use transfering loop is distinguished 10 kinds of bacteriums of picking from bacteria culture medium is in the liquid nutrient medium of 3-5rnL, 37 ℃ or 28 ℃, 250r/min shaking culture 3-5 hour, the OD value of bacterium liquid is measured in timing, and bacterial concentration is controlled to the identical order of magnitude.Draw each 30 μ L of cultured bacterium liquid and be placed in 100mL Erlenmeyer flask, add 30mL through melting and being cooled to the solid medium of 45-50 ℃, vibration shakes up rapidly, make thalline dispersed, pour into immediately in the culture dish that diameter is 90mm * 90mm, solidify and with the punch tool that internal diameter is 4mm * 4mm, make a call to 6 holes afterwards.Wherein, be numbered in l, 3, every hole of 5 and add 20 μ L testing samples, be numbered in every hole of 2,4,6 and add 20 μ L empty carrier transformant expressing protein (abduction delivering 3h cracking supernatant liquor) negative controls.At room temperature after sample spreads completely, be placed in 37 ℃ or 28 ℃ of incubators and cultivate 24-72h, observe and measure antibacterial circle diameter (experimental result is in Table 1), and carry out statistical study (seeing Figure 10-18).
The bacteriostatic activity of table 1 restructuring America alligator Cathelicidin-AM antibacterial peptide to indicator
Note: A, B, C represent 3 culture dish of replicate(determination).1,2,3 represent 3 parallel inhibition zones (mm) size on each culture dish.
Embodiment six: the mensuration of Cathelicidin-AM antibacterial peptide minimal inhibitory concentration (MIC)
With liquid coubling dilution, determine the minimal inhibitory concentration (MIC) of restructuring America alligator Cathelicidin-AM antibacterial peptide.Indicator strain used: colon bacillus (Escherichia coli CMCC44102); Colon bacillus (Escherichia coli ATCC25922); Salmonella typhimurtum (Salmonella typhimurium CMCC50115); C type clostridium perfringens (Clostridium perfringens type C C59-2); Hemophilus influenzae (Haemophilus influenzae ATCC49247); Vibrio parahemolyticus (Vibrio Parahemolyticus ATCC17802); Campylobacter jejuni (Campylobacter jejuni ATCC29428); Streptococcus aureus (Staphyloccocus aureus CMCC26003); Listeria Monocytogenes (Listeria monocytogenes ATCC19114); Streptococcus pneumoniae (Streptococcus pneumoniae ATCC49619).10 kinds of bacterial strains are our unit and preserve.Operate as follows:
(1) activation culture of bacterial classification: by each inoculation in liquid LB or beef-protein medium, 24h(37 ℃ of shaking table enlarged culturing, 150r/min), utilizing counting method of blood cell that each bacterial strain is mixed with to concentration with sterilized water is 10
6the bacteria suspension of cfu/ml.
(2) adjustment bacterium is dense, makes containing viable count, to reach l * 10 in every milliliters of liquid substratum
6cfu/mL, is layered on 96 well culture plates, 100 μ L/ holes.The recombinant C athelicidin-AM antibacterial peptide solution 10 μ L/ holes that add water doubling dilution, 37 ℃, 120r/min shake slowly cultivates 16h, surveys OD
600.The minimum Cathelicidin-AM antibacterial peptide concentration of bacteria growing inhibiting is that MIC(experimental result is in Table 2).
Table 2 restructuring America alligator Cathelicidin-AM antibacterial peptide is to the minimal inhibitory concentration of indicator (MIC)
Note: "+" represents positive, has colony growth; " one " represents negative, without colony growth.
Embodiment seven: Cathelicidin-AM antibacterial peptide C end amination derivative fungistatic effect
The Cathelicidin-AM antibacterial peptide C end of synthetic is carried out to amination modification, adopt standard agar hole diffusion process to detect its bacteriostatic activity in vitro, and compare with traditional microbiotic.
The indicator strain that bacteriostatic test is used: colon bacillus (Escherichia coli CMCC44102); Colon bacillus (Escherichia coli ATCC25922); Salmonella typhimurtum (Salmonella typhimurium CMCC50115); C type clostridium perfringens (Clostridium perfringens type C C59-2); Hemophilus influenzae (Haemophilus influenzae ATCC49247); Vibrio parahemolyticus (Vibrio Parahemolyticus ATCC17802); Campylobacter jejuni (Campylobacter jejuni ATCC29428); Streptococcus aureus (Staphyloccocus aureus CMCC26003); Listeria Monocytogenes (Listeria monocytogenes ATCC19114); Streptococcus pneumoniae (Streptococcus pneumoniae ATCC49619).10 kinds of bacterial strains are our unit and preserve.
Bacteriostatic test concrete steps: single bacterium colony that use transfering loop is distinguished 10 kinds of bacteriums of picking from bacteria culture medium is in the liquid nutrient medium of 3-5rnL, 37 ℃ or 28 ℃, 250r/min shaking culture 3-5 hour, the OD value of bacterium liquid is measured in timing, and bacterial concentration is controlled to the identical order of magnitude.Draw each 30 μ L of cultured bacterium liquid and be placed in 100mL Erlenmeyer flask, add 30mL through melting and being cooled to the solid medium of 45-50 ℃, vibration shakes up rapidly, make thalline dispersed, pour into immediately in the culture dish that diameter is 90mm * 90mm, solidify and with the punch tool that internal diameter is 4mm * 4mm, make a call to 6 holes afterwards.Wherein, be numbered in l, 3, every hole of 5 and add 20 μ L testing samples, be numbered in every hole of 2,4,6 and add 20 μ L empty carrier transformant expressing protein (abduction delivering 3h cracking supernatant liquor) negative controls.At room temperature after sample spreads completely, be placed in 37 ℃ or 28 ℃ of incubators and cultivate 24-72h, observe and measure antibacterial circle diameter (experimental result is in Table 3), and carry out statistical study, analytical results shows, Cathelicidin-AM antibacterial peptide C end is carried out to, after amination modification, still have bacteriostatic activity.
The bacteriostatic activity of table 3 Cathelicidin-AM antibacterial peptide C end amination modified derivative to indicator
Note: A, B, C represent 3 culture dish of replicate(determination).1,2,3 represent 3 parallel inhibition zones (mm) size on each culture dish.
Embodiment eight: Cathelicidin-AM antibacterial peptide aminoacid replacement derivative fungistatic effect
When the Cathelicidin-AM of synthetic antibacterial peptide, the Lys of the 8th in SEQ ID NO:2 aminoacid sequence polypeptide, the Lys of the 31st are replaced with Pro, Leu respectively.Employing standard agar hole diffusion process detects the In Vitro Bacteriostatic of Cathelicidin-AM antibacterial peptide aminoacid replacement derivative, and compares with traditional microbiotic.
The indicator strain that bacteriostatic test is used: colon bacillus (Escherichia coli CMCC44102); Colon bacillus (Escherichia coli ATCC25922); Salmonella typhimurtum (Salmonella typhimurium CMCC50115); C type clostridium perfringens (Clostridium perfringens type C C59-2); Hemophilus influenzae (Haemophilus influenzae ATCC49247); Vibrio parahemolyticus (Vibrio Parahemolyticus ATCC17802); Campylobacter jejuni (Campylobacter jejuni ATCC29428); Streptococcus aureus (Staphyloccocus aureus CMCC26003); Listeria Monocytogenes (Listeria monocytogenes ATCC19114); Streptococcus pneumoniae (Streptococcus pneumoniae ATCC49619).10 kinds of bacterial strains are our unit and preserve.
Bacteriostatic test concrete steps: single bacterium colony that use transfering loop is distinguished 10 kinds of bacteriums of picking from bacteria culture medium is in the liquid nutrient medium of 3-5rnL, 37 ℃ or 28 ℃, 250r/min shaking culture 3-5 hour, the OD value of bacterium liquid is measured in timing, and bacterial concentration is controlled to the identical order of magnitude.Draw each 30 μ L of cultured bacterium liquid and be placed in 100mL Erlenmeyer flask, add 30mL through melting and being cooled to the solid medium of 45-50 ℃, vibration shakes up rapidly, make thalline dispersed, pour into immediately in the culture dish that diameter is 90mm * 90mm, solidify and with the punch tool that internal diameter is 4mm * 4mm, make a call to 6 holes afterwards.Wherein, be numbered in l, 3, every hole of 5 and add 20 μ L testing samples, be numbered in every hole of 2,4,6 and add 20 μ L empty carrier transformant expressing protein (abduction delivering 3h cracking supernatant liquor) negative controls.At room temperature after sample spreads completely, be placed in 37 ℃ or 28 ℃ of incubators and cultivate 24-72h, observe and measure antibacterial circle diameter (experimental result is in Table 4), and carry out statistical study.Analytical results shows, Cathelicidin-AM antibacterial peptide is carried out to, after aminoacid replacement, still have bacteriostatic activity.
The bacteriostatic activity of table 4 Cathelicidin-AM antibacterial peptide aminoacid replacement derivative to indicator
Note: A, B, C represent 3 culture dish of replicate(determination).1,2,3 represent 3 parallel inhibition zones (mm) size on each culture dish.
Embodiment nine: the impact of Cathelicidin-AM antibacterial peptide on piglet blood immune indexes
120 of the DLY piglets that after selection wean, (28 age in days) body weight is 7kg, are divided into control group and test group at random, and wherein control group does not add ginkgo engineering alexin, and test group is added 0.02%Cathelicidin-AM antibacterial peptide; Every group of 5 repetitions, 12 piglets of every repetition, trial period 21d.After off-test, often repeat to get 3 and approach equal body weight piglet and butcher, blood sampling is centrifugal in 2500r/min, gets serum, measures seroimmunity index (test-results is in Table 5).
The impact of table 5 Cathelicidin-AM antibacterial peptide on piglet blood immune indexes
As shown in Table 5: compare with control group, add Cathelicidin-AM antibacterial peptide group albumin level and total protein level and improve respectively 19.97% and 14.97%, difference is all remarkable; Other haematogenic immunity index changes as follows respectively: IgG level improves 13.03%, IgM level and improves 12.27%, IgA level raising 7.89%; Immunological Markers for Cell-Mediated Immunity aspect: blood T lymphocyte transformation rate improves 8.25%(P > 0.05), blood IL-2 content improves 15.75%; Experiment results proved, antibacterial peptide of the present invention significantly improves the immune indexes of piglet.
Embodiment ten: Cathelicidin-AM antibacterial peptide is on microbe population impact in grice diarrhoea and ight soil
Select 90 8kg weanling pigs, be divided at random 3 groups, every group of 3 repetitions, 10 pigs of every repetition, schedule to last the feeding experiment of 52d, add Cathelicidin-AM antibacterial peptide to microbe population impact in grice diarrhoea and ight soil in Research foundation daily ration.A group is control group, the basal diet of feeding; B group is for adding Cathelicidin-AM antibacterial peptide of the present invention, and duration of test adds in basal diet with 0.05%; C group is microbiotic group, according to medication instruction, uses (test-results is in Table 6).
Table 6 Cathelicidin-AM antibacterial peptide is on affecting microbe population in grice diarrhoea and ight soil
As seen from Table 6: due to illness control group A rejects 4, case fatality rate is 13.33%, and antibacterial peptide group B and microbiotic group C surviving rate are 100%.Compare with control group, add Cathelicidin-AM antibacterial peptide grice diarrhoea rate and reduce, effect can compare favourably with microbiotic.Compare with microbiotic group with control group, add Cathelicidin-AM antibacterial peptide, in ight soil, intestinal bacteria obviously reduce, and milk-acid bacteria and bifidus bacillus obviously increase.Illustrate that Cathelicidin-AM antibacterial peptide has certain effect to preventing post-weaning diarrhea.
Embodiment 11: the application of Cathelicidin-AM antibacterial peptide in chicken intestinal tract disease
150 7 age in days broiler chicken are divided into tri-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 A group is control group, in whole trial period, do not do any treatment and process, B group is for adding Cathelicidin-AM antibacterial peptide of the present invention, and duration of test adds in feed with 25mg/kg.BW, early, late each 1 time, mix continuously and take 3d, the processing of normally feeding afterwards for 3 days, C group is antibiotic treatment group, with aminobenzylpenicillin, 50mg/kg.BW gavages, early, evening each 1 time, continuous 3d, the processing of normally feeding afterwards for 3 days.The whole test period is 10d, the ill and death condition of observed and recorded chicken, and trial period, finishes laggard row data statistic analysis, in Table 7
The impact of table 7 Cathelicidin-AM antibacterial peptide on chicken intestinal tract disease
Test-results shows, Cathelicidin-AM antibacterial peptide 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, is more conducive to food safety, is antibiotic ideal substitute.
Claims (11)
1. an America alligator Cathelicidin-AM antibacterial peptide, is characterized in that, described polypeptide is selected from lower group:
(a) polypeptide of SEQ ID NO:2 aminoacid sequence;
(b) SEQ ID NO:2 aminoacid sequence is through modifying the polypeptide derivative of resulting functional equivalent;
(c) SEQ ID NO:2 aminoacid sequence is through replacement, the disappearance of one or more amino-acid residues or add the derivative polypeptide of formed functional equivalent.
2. polynucleotide, is characterized in that, the nucleotide sequence of described polynucleotide is selected from lower group:
(a) polynucleotide of polypeptide as claimed in claim 1 of encoding;
(b) polynucleotide complementary with polynucleotide (a).
3. polynucleotide as claimed in claim 2, is characterized in that: polypeptide or its complementary sequence of aminoacid sequence shown in described polynucleotide encoding SEQ ID NO:2.
4. polynucleotide as claimed in claim 3, is characterized in that, the sequence of described polynucleotide is selected from lower group a kind of:
(a) sequence of 17-139 position in SEQ ID NO:1;
(b) due to the degeneracy of genetic code derived from the sequence of polynucleotide sequence (a);
(c) polynucleotide sequence (a) and complementary sequence (b).
5. an expression vector, is characterized in that: described expression vector contains at least one polynucleotide as described in any one in claim 2,3,4.
6. a genetically engineered host cell, is characterized in that: described host cell contains expression vector claimed in claim 5, or in its genome, is integrated with at least one polynucleotide as described in any one in claim 2,3,4.
7. genetically engineered host cell as claimed in claim 6, is characterized in that: described host cell transforms subtilis by expression vector claimed in claim 5 and obtains.
8. a method of preparing Cathelicidin-AM antibacterial peptide as claimed in claim 1, is characterized in that: described polypeptide synthesizes by mechanochemical method or generates by gene recombination method, and described gene recombination method comprises the following steps:
1), in suitable substratum, expression vector claimed in claim 5 is transformed or transfection host cell;
2) screening positive clone;
3) abduction delivering antibacterial peptide as claimed in claim 1;
4) recovering condition substratum or cell extract,
5) from the described substratum that obtains or cell extract is separated and purifying described in polypeptide.
9. America claimed in claim 1 alligator Cathelicidin-AM antibacterial peptide is being prepared fodder additives, sanitas, agricultural biological control agent or is being controlled the purposes in the medicine composition of infected by microbes.
10. for controlling a medicine composition for infected by microbes, it is characterized in that: described pharmaceutical composition comprises antibacterial peptide claimed in claim 1.
11. 1 kinds of fodder additivess, is characterized in that: described fodder additives comprises antibacterial peptide claimed in claim 1.
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| JUAN HAO ET AL.: "Molecular cloning, recombinant expression and antibacterial activity analysis of hepcidin from Simensis crocodile(Crocodylus siamensis)", 《COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY,PARTB》 * |
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