CN101775068A - Novel natural antibacterial peptides, and coding sequence and uses thereof - Google Patents
Novel natural antibacterial peptides, and coding sequence and uses thereof Download PDFInfo
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Abstract
The invention discloses novel natural antibacterial peptides, and a coding sequence and uses thereof. The antibacterial peptides can effectively and outstandingly prohibit the infection of a plurality of harmful pathogens. The antibacterial peptides, as natural antibacterial peptides, are mainly human-sourced and have lower risk of immunological rejection in the process of clinical application.
Description
Technical field
The present invention relates to biotechnology and field of medicaments, specifically, the present invention relates to new natural antibacterial peptide, and the polynucleotide of the described antibacterial peptide of encoding.The invention still further relates to preparation method, purposes and the composition of described antibacterial peptide.
Background technology
Antibiotic generation has been made huge contribution to the protection human health, but along with antibiotic extensive and life-time service, the continuous generation of Resistant strain and increasing anaphylaxis etc. force people to begin to seek the antiseptic-germicide of a new generation.Tellurian biological process evolution of long period of time has formed the mechanism of resisting external microbiological attack gradually, and living organism can be by the invasion and attack of secretory antibody and various defense molecule combating microorganisms.Defense molecule comprises (Chen Xiaozhang, prince, Yang Guanqun such as various cytokines, chemokine, enzyme and antibacterial peptide; External secretion physiology; Beijing: Science Press, 2003527-540).Wherein miscellaneous antibacterial peptide has been subjected to people's extensive attention, they are a kind of polypeptides matters with strong anti-microbial effect that produce in the organism, and has an incomparable superiority of Penicillin antibiotics, promptly can not induce the generation of drug resistance strain, what have also has a parasiticide, virus, the function of cancer cells.Up to now, comprised the multiple antibacterial active peptide of discovery in insect, animal, plant and the prokaryotic organism at multiple biology.It is very considerable as the application prospect that medicine had.
The alkaline small polypeptide that natural antibacterial peptide normally is made up of many amino-acid residues of 30-60, good water solubility, molecular weight is approximately 4000-10000 dalton.Most of antibacterial peptide has thermostability, and iso-electric point shows stronger positively charged ion feature greater than 7.The antibacterial peptide that has simultaneously also possesses the ability of opposing trypsinase or pepsin hydrolysis.Many antibacterial peptides have hydrophobic region and electrically charged zone, and these constructional features are for the significance that is formed with of the anti-microbial activity mechanism of antibacterial peptide.Studies show that, be that the anti-microbial effect and the Na ion concentration in the solution of part antibacterial peptide exists negative correlation (M.J.Goldman etc. at least; Humanbeta-defensin-1 is a salt-sensitive antibiotic in lung that is inactivated in cysticfibrosis, Cell 88 (1997) 553-560.), some antibacterial peptide loses anti-microbial effect (I.Nagaoka etc. when 150mM NaCl; Synergistic actions of antibacterial neutrophil defensins andcathelicidins, Inflamm Res 49 (2000) 73-79).
Form according to the amino acid of antibacterial peptide, it can be fallen into 5 types: (1) strand does not have the alpha-helix of cysteine residues, or two sections structures that alpha-helix is formed that connected by random coil, as Cecropins (J.Fink, the A.Boman etc. that are separated to the earliest; Design, synthesis and antibacterial activity of cecropin-likemodel peptides, Int J Pept Protein Res 33 (1989) 412-421.).(2) be rich in some amino-acid residue but do not contain the antibacterial peptide such as the Apidaecins (M.J.Clague etc. of halfcystine; A comparative studyof band 3 aggregation in erythrocyte membranes by melittin and other cationicagents, Biochim Biophys Acta 980 (1989) 93-99.).(3) contain the antimicrobial polypeptide of 1 disulfide linkage, as derive from the Bactenecin (K.Ando of ox neutrophil leucocyte, S.Natori, Inhibitory effect ofsarcotoxin IIA, an antibacterial protein of Sarcophaga peregrina, on growth ofEscherichia coli, J Biochem (Tokyo) 103 (1988) 735-739).(4) has the antibacterial peptide alexin (Defensin) of beta sheet structure.(5) contain special organic group antibacterial peptide such as Lantibiotics (J.N.Hansen, Antibiotics synthesized by posttranslational modification, Annu RevMicrobiol 47 (1993) 535-564) (lantibiotics).
The antibacterial peptide function it is generally acknowledged that from present result of study the antibacterial peptide sterilization mechanism mainly is the cytolemma that acts on bacterium, destroys its integrity and produces the perforation phenomenon, and it is outer and dead to cause entocyte to overflow born of the same parents.At first invested the bacterial film surface by electrostatic attraction, hydrophobic C end inserts the interior hydrophobic region of film and changes the conformation of film, and a plurality of antibacterial peptides form ionic channel and cause some ionic to be overflowed and death on film.Also there is the scholar to think that antibacterial peptide acts on membranin and causes cohesion, inactivation and ionic channel, causes the membrane permeability change and cause death, also have the scholar to propose the problems such as synergy whether antibacterial peptide exists specific membrane receptor and have or not other factor.The mechanism of action of different classes of antibacterial peptide may be different.
The antibacterial peptide majority has characteristics such as strong basicity, thermostability and broad-spectrum antimicrobial.Some antibacterial peptide all has strong lethal effect to part fungi, protozoon, virus and cancer cells etc.
1, antibacterial peptide is to the lethal effect of bacterium
Antibacterial peptide has lethal effect to bacterium, and some antibacterial peptide all has lethal effect such as Ceropin (C.Lowenberger etc. to Gram-negative and positive bacteria; Antimicrobial activity spectrum, cDNAcloning, and mRNA expression of a newly isolated member of the cecropin familyfrom the mosquito vector Aedes aegypti, J Biol Chem 274 (1999) 20092-20097), and the Andropin that exists in the fruit bat male imago reproductive tract, only act on Gram-negative bacteria, invalid to gram-positive microorganism.The anti-microbial effect mechanism of antibacterial peptide has several different hypothesis.Electromotive force relies on the passage hypothesis and thinks Cecropin, Magainins, antibacterial peptides such as Defensins can form parents' spirane structure and combine by electrostatic attraction by self positively charged and surperficial electronegative bacterial cell membrane of institute, and then act on the cytolemma of bacterium, depend on transmembrane potential and on film, form ionic channel (S.Cociancich etc.; Insect defensin, an inducible antibacterial peptide, forms voltage-dependent channels inMicrococcus luteus, J Biol Chem 268 (1993) 19239-19245), and K+ is separated out fast, then cause necrocytosis (M.Okada, S.Natori, Ionophore activity of sarcotoxin I, abactericidal protein of Sarcophaga peregrina, Biochem J 229 (1985) 453-458).Lockey etc. observe directly the duct that antibacterial peptide causes by Electronic Speculum on cytolemma, the formation that relies on passage for electromotive force provides direct evidence; Protein denaturation hypothesis (M.Okada, S.Natori, Ionophore activityof sarcotoxin I, a bactericidal protein of Sarcophaga peregrina, Biochem J 229 (1985) 453-458) think, antibacterial peptide acts on membranin, causes the protein condenses inactivation, and the cytolemma sex change forms ionic channel.Antibacterial peptide that has in addition such as Thanatin (P.Fehlbaum etc.; Structure-activityanalysis of thanatin, a 21-residue inducible insect defense peptide with sequencehomology to frog skin antimicrobial peptides, Proc Natl Acad Sci USA 93 (1996) 1221-1225) may be by suppressing the respiration killing bacteria of bacterium, but concrete machine-processed not clear.Also have the research report, antibacterial peptide can be suppressed the growth of bacterium by disturbing transcribing of bacterial gene, as Attacin (A.Carlsson etc.; Attacin, an antibacterial protein from Hyalophoracecropia, inhibits synthesis of outer membrane proteins in Escherichia coli byinterfering with omp gene transcription, Infect Immun 59 (1991) 3040-3045) can disturb Bacillus coli cells outer membrane protein Omp C, Omp F, Omp A and Lam B gene transcription, these protein contents are reduced, and the growth of bacterium is suppressed.Sarcotoxins II (K.Ando, S.Natori, Inhibitory effect of sarcotoxin IIA, an antibacterial protein of Sarcophagaperegrina, on growth of Escherichia coli, J Biochem (Tokyo) 103 (1988) 735-739) can suppress the formation of bacteria cell wall, make bacterium can not keep normal cellular form and growth retardation.
2, antibacterial peptide is to the lethal effect of fungi
Antibacterial peptide may have antibacterium and antimycotic activity simultaneously, as PGQ, the Dermaseptin of batrachia, and rabbit alexin NP-1, plant alexin.Thionine (Thionins) has lethal effect to the various plants pathomycete.Cecropin A and analogue thereof such as cecropin-melittin T1249 has certain lethal effect to the fungi of infected insect.Relevant (the L.Cavallarin etc. of 11 aminoacid sequences that research report Cecropins N end alpha helical region territory is arranged with anti-mycotic activity; Cecropin A-derived peptides are potent inhibitors offungal plant pathogens, Mol Plant Microbe Interact 11 (1998) 218-227).Not clear for the antimycotic mechanism of action of antibacterial peptide at present, there is the research report to infer antibacterial peptide Ib-AMP1 (D.G.Lee etc.; Antifungal mechanism of a cysteine-rich antimicrobial peptide, Ib-AMP 1, fromImpatiens balsamina against Candida albicans:Biotechnology Letters, 21 (1999), 1047-1050), LL-37 (M.D.Howell, J.F.Jones, K.O.Kisich, J.E.Streib, R.L.Gallo, D.Y.Leung, Selective killing of vaccinia virus by LL-37:implications for eczemavaccinatum, J Immunol 172 (2004) 1763-1767) may work by the variation that causes fungal cell's process.
3, antibacterial peptide is to the protozoacide lethal effect
Some antibacterial peptide can effectively kill and parasitize the mankind or the intravital parasite of animal.The antibacterial peptide Magainirls of Shive-I, frog skin can kill plasmodium.And the Cecropin/melittin T1249 can kill Lai Shiman flagellate (Sun Chao, Wang Hui, Sun Bo, Peng Xuexian; The peptide antibiotics progress; The Chinese Pharmacological circular, 2000; 16 (6): 605-609).Tussah antibacterial peptide D also has lethal effect to Trichomonas vaginalis, but mechanism of action it be unclear that.
4, antibacterial peptide is to the lethal effect of virus
Multiple antibacterial peptide has antiviral effect (L.Zhang etc.; Contribution of humanalpha- defensin 1,2, and 3 to the anti-HIV-1 activity of CD8 antiviral factor, Science 298 (2002) 995-1000).The antibacterial peptide that has can be by playing a role with direct combination of outer virionic membrane, and as the effect to simplexvirus of α-antibiotic, Modelin-1, Polyphemusins is to the effect of HIV virus.The antibacterial peptide that has can suppress the breeding of virus, and as Cecropin A, Melitiin expresses the propagation that suppresses HIV-1 virus by repressor gene under inferior toxicity concentration.Antibacterial peptide that also has such as Meliltin can be by the assembling of viral interference to virus generation effects.
5, antibacterial peptide is to the lethal effect of cancer cells
Some antibacterial peptide such as Magainin, Cecropin A, Melittin, α- alexin 1,2 all have the selective killing effect of pair cancer cells.Antibacterial peptide has the principle of the selective killing effect of cancer cells: 1. the hypermetabolism of cancer cells causes the change of cell membrane potential, easily by antibacterial peptide identification combination, and then suppresses its growth.2. the cancer cell membrane outside surface contains higher acidic phospholipid and can combine with the antibacterial peptide of alkalescence.3. antibacterial peptide activates the antioncogenes such as P53 in the tumour cell, the apoptosis of inducing tumor cell.4. regulatory factor required when antibacterial peptide and tumour take place interacts, and seals its avtive spot, thereby suppresses growth of tumor.
Because the antibacterial peptide kind is many, have the characteristic of the strong and wide spectrum of anti-microbial effect, and the less advantage that Resistant strain occurs, make it be expected to become anti-infectious important drugs.Simultaneously, at other biological function that some antibacterial peptides had, that antibacterial peptide also may be applied to will be antitumor, genital system infection, treatment of diseases such as sterile.Synthetic, the expression and the purifying process of antibacterial peptide are constantly developed.Chemical process has been used to study the antibacterial peptide analogue, with increased antimicrobial activity or minimizing cytotoxicity, but macromolecular antibacterial peptide, chemical synthesis process expense costliness, the strategy of gene engineering expression is expected to address this problem.
To sum up, this area also is necessary to find new antibacterial peptide, with exploitation for effectively medicine such as antibiotic, antiviral or anticancer.
Summary of the invention
The object of the present invention is to provide new natural antibacterial peptide, its encoding sequence and purposes.
In a first aspect of the present invention, a kind of isolated polypeptide is provided, described polypeptide is selected from down group:
(a) polypeptide of aminoacid sequence shown in SEQ ID NO:n; Or
(b) disappearance, insertion or the replacement of the aminoacid sequence shown in the SEQ ID NO:n through 1-5 amino-acid residue formed, and have the polypeptide polypeptides derived that suppresses the pathogenic agent function by aminoacid sequence shown in the SEQ IDNO:n;
Wherein, n is the positive integer that is selected from 1-7.
In another preference, described polypeptide is the polypeptide of aminoacid sequence shown in SEQ ID NO:n.
In a second aspect of the present invention, provide a kind of isolating polynucleotide, the described polypeptide of described polynucleotide encoding.
In another preference, described polynucleotide are selected from down group:
(1) as SEQ ID NO:(n+7) shown in the polynucleotide of nucleotide sequence; Or
(2) with the polynucleotide of polynucleotide (1) complementary (preferred complementary fully).
In a third aspect of the present invention, a kind of carrier is provided, described carrier contains described polynucleotide.
In a fourth aspect of the present invention, a kind of genetically engineered cell is provided, described cell contains in described carrier or its genome and is integrated with described polynucleotide.
In a fifth aspect of the present invention, a kind of method for preparing described polypeptide is provided, described method comprises:
(i) under conditions suitable for the expression, cultivate described cell; With
(ii) isolate described polypeptide.
In a sixth aspect of the present invention, the purposes of described polypeptide is provided, be used for the composition that preparation suppresses pathogenic agent (as bacterium, fungi, protozoon etc.).
In a seventh aspect of the present invention, a kind of composition is provided, described composition contains:
(A) described one or more polypeptide of significant quantity; With
(B) pharmaceutically acceptable carrier.
In a eighth aspect of the present invention, provide a kind of non-therapeutic ground to suppress the method for pathogenic agent, described method comprises: described one or more polypeptide that need to suppress the object significant quantity of pathogenic agent.
In another preference, described need to suppress pathogenic agent to as if having the mammalian subject of described pathogenic agent, or have the place or the article of described pathogenic agent.
Others of the present invention are because the disclosure of this paper is conspicuous to those skilled in the art.
Description of drawings
Fig. 1. contain the synoptic diagram of the plasmid of intein (Intein) and antibacterial peptide encoding gene.
Fig. 2. antibacterial peptide PCR identifies figure, and swimming lane 1:DNA Marker is followed successively by from top to bottom: 500bp, 400bp, 300bp, 200bp, 100bp, swimming lane 2-8 is followed successively by: hBD-134, hBD-116, hBD-114, hBD-113, hBD-110, hBD-spage, mBD-12.
Fig. 3. antibacterial peptide fusion protein is expressed electrophoresis and is identified figure, and swimming lane 1:IPTG induces preceding bacterium whole protein; Swimming lane 2; IPTG induces back bacterium whole protein; Swimming lane 3:IPTG induces white protein on the bacterium broken wall of back; Swimming lane 4: albumen Marker, molecular weight is followed successively by from top to bottom: 97.4KD, 66.2KD, 43KD, 31KD, 20KD.
Fig. 4. protein electrophoresis is identified figure behind the antibacterial peptide purifying, swimming lane 1: albumen Marker, molecular weight is followed successively by from top to bottom: 60KD, 45KD, 34KD, 16KD, 7KD, 4KD; Swimming lane 2: antibacterial peptide fusion protein is through the combination of chitin post, albumen behind buffer B 2 wash-outs.
Fig. 5. intestinal bacteria survival rate and different antibacterial peptide graph of a relation action time.
Fig. 6. Candida albicans survival rate and different antibacterial peptide figure action time.
The comparison of Fig. 7 antibacterial peptide of the present invention and antibacterial peptide Bin1b bacteriostatic activity.
Embodiment
The inventor is through deep research, found one group to have the natural antibacterial peptide that suppresses the pathogenic agent function, verified described antibacterial peptide suppresses the effect excellence of pathogenic agent, can suppress the infection of multiple pathogenic agent, comprises the infection that causes at by the antibiotics resistance pathogenic agent.Finished the present invention on this basis.
The inventor is at first by some protein active function prediction website or softwares, as: UniProtKB/Swiss-Prot and SignalP 3.0Server-prediction prediction have the active natural polypeptides sequence of antibacterial peptide, means by gene engineering expression obtain target protein again, and carry out repetition test screening and wherein have the albumen of bacteriostatic activity, have the active new natural antibacterial peptide of antibacterial peptide thereby obtain one group.
As used herein, described " antibacterial peptide " is meant polypeptide (as table 1) or its derived peptide with aminoacid sequence shown in the SEQ ID NO:n, and it has the function that suppresses pathogenic agent; Wherein, n is the positive integer that is selected from 1-7 (promptly 1,2,3,4,5,6 or 7).Among the present invention, term " polypeptide ", " albumen " are used interchangeably.
Table 1
| ??SEQ?ID??NO： | Antibacterial peptide | Aminoacid sequence (N end → C end) |
| ??1 | ??hBD-134 | ??GINSLSSEMHKKCYKNGICRLECYESEMLVAYCMFQLECCV??KGNPAP |
| ??2 | ??hBD-116 | ??GLFRSHNGKSREPWNPCELYQGMCRNACREYEIQYLTCPND??QKCCLKLSVKITSSKNVKEDYDSNSNLSVTNSSSYSHI |
| ??3 | ??hBD-114 | ??DRCTKRYGRCKRDCLESEKQIDICSLPRKICCTEKLYEEDDM??F |
| ??4 | ??hBD-113 | ??GPSVPQKKTREVAERKRECQLVRGACKPECNSWEYVYYYC??NVNPCCAVWEYQKPIINKITSKLHQK |
| ??5 | ??hBD-110 | ??NFEPKYRFERCEKVRGICKTFCDDVEYDYGYCIKWRSQCCV |
| ??SEQ?ID??NO： | Antibacterial peptide | Aminoacid sequence (N end → C end) |
| ??6 | ??hBD-spage??(hBD-SP) | ??DVPPGIRNTICHMQQGICRLFFCHSGEKKRDICSDPWNRCCV??SNTDEEGKEKPEMDGRSGI |
| ??7 | ??mBD-12 | ??GLEYSQSFPGGEIAVCETCRLGRGKCRRTCIESEKIAGWCKL??NFFCCRERI |
As used herein, " isolating " is meant that material separates (if natural substance, primal environment promptly is a natural surroundings) from its primal environment.For example, polynucleotide and polypeptide under the native state in the active somatic cell do not have separation and purification, if but other materials that same polynucleotide and polypeptide exist with native state separately, then for separation and purification.
Pathogenic agent of the present invention comprises various for the deleterious biology of Mammals (comprising the people), for example is easy to Mammals is caused (comprising the people) biology of infectious diseases.Described pathogenic agent includes but not limited to: virus, and bacterium, fungi, protozoon, or carry virus, bacterium, fungi or protozoacide harmful organism etc.More special, described pathogenic agent is bacterium or fungi, or carries the harmful organism of described bacterium or fungi.
Polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, synthetic polypeptide, preferred recombinant polypeptide.Polypeptide of the present invention can be the product of natural purifying, or the product of chemosynthesis, or uses recombinant technology to produce from protokaryon or eucaryon host (for example, bacterium, yeast, higher plant, insect and mammalian cell).The host used according to the recombinant production scheme, polypeptide of the present invention can be glycosylated, maybe can be nonglycosylated.
The present invention also comprises fragment, derivative and the analogue of described antibacterial peptide.As used herein, term " segment ", " derivative " are meant with " analogue " and keep identical biological function of natural polypeptides of the present invention or active polypeptide basically.Polypeptide fragments of the present invention, derivative and analogue can be: (1) has one or more conservative or substituted polypeptide of non-conservation amino-acid residue (preferred conservative amino acid residue), or (2) have the polypeptide of substituted radical in one or more amino-acid residues, or (3) mature polypeptide and another compound are (such as the compound that prolongs the polypeptide transformation period, polyoxyethylene glycol for example) merges formed polypeptide, or (4) additional aminoacid sequence is blended in this peptide sequence and the polypeptide that forms (as leader sequence or secretion sequence or be used for the peptide sequence or the proteinogen sequence of purifying, or with the fusion rotein of the pulsating formation of antigen I gG).These segments, derivative and analogue belong to the known scope of those skilled in the art.
The special antibacterial peptide analogue of one class be in other Mammalss (as ox, sheep, rabbit, dog, monkey, mouse etc.) with the albumen of the albumen homology shown in the SEQ ID NO.n (n is selected from the positive integer of 1-7).The encoding sequence of the homologous protein of these other species can obtain by the method for hybridizing or increase, and then obtain these homologous proteins by conventional recombination method according to sequence disclosed by the invention.
Among the present invention, term " antibacterial peptide " also comprises having the pathogenic agent of inhibition variant form function, SEQ ID NO.n (n is selected from the positive integer of 1-7) sequence.These variant forms comprise (but being not limited to): several (are generally 1-10, preferably 1-5,1-3 more preferably) amino acid whose disappearance, insertion and/or replacement, and add one or several at C-terminal and/or N-terminal and (be generally in 10, preferably being in 5, more preferably is in 3) amino acid.For example, in the art, when replacing, can not change proteinic function usually with the close or similar amino acid of performance.Again such as, add one or several amino acid at C-terminal and/or N-terminal and also can not change proteinic function usually.
Among the present invention, this histone conservative property variation polypeptide is meant with aminoacid sequence shown in the SEQ ID NO.n (n is selected from the positive integer of 1-7) to be compared, have 10 at the most, preferably at the most 5, more preferably 3 amino acid is replaced by similar performance or close amino acid and is formed polypeptide at the most.These conservative property variation polypeptide preferably carry out the amino acid replacement according to table 2 and produce.
Table 2
| Initial residue | Representational replacement | The preferred replacement |
| ??Ala(A) | ??Val；Leu；Ile | ??Val |
| ??Arg(R) | ??Lys；Gln；Asn | ??Lys |
| ??Asn(N) | ??Gln；His；Lys；Arg | ??Gln |
| ??Asp(D) | ??Glu | ??Glu |
| ??Cys(C) | ??Ser | ??Ser |
| ??Gln(Q) | ??Asn | ??Asn |
| ??Glu(E) | ??Asp | ??Asp |
| ??Gly(G) | ??Pro；Ala | ??Ala |
| ??His(H) | ??Asn；Gln；Lys；Arg | ??Arg |
| ??Ile(I) | ??Leu；Val；Met；Ala；Phe | ??Leu |
| ??Leu(L) | ??Ile；Val；Met；Ala；Phe | ??Ile |
| ??Lys(K) | ??Arg；Gln；Asn | ??Arg |
| ??Met(M) | ??Leu；Phe；Ile | ??Leu |
| ??Phe(F) | ??Leu；Val；Ile；Ala；Tyr | ??Leu |
| ??Pro(P) | ??Ala | ??Ala |
| ??Ser(S) | ??Thr | ??Thr |
| Initial residue | Representational replacement | The preferred replacement |
| ??Thr(T) | ??Ser | ??Ser |
| ??Trp(W) | ??Tyr；Phe | ??Tyr |
| ??Tyr(Y) | ??Trp；Phe；Thr；Ser | ??Phe |
| ??Val(V) | ??Ile；Leu；Met；Phe；Ala | ??Leu |
The present invention also comprises modified antibacterial peptide, and (the not changing primary structure usually) form of modification comprises: the chemically derived form such as the acetylize or carboxylated of the polypeptide that body is interior or external.Modification also comprises glycosylation, carries out glycosylation modified and polypeptide that produce in the procedure of processing as those in the synthetic and processing of polypeptide or further.This modification can be carried out glycosylated enzyme (as mammiferous glycosylase or deglycosylating enzyme) and finishes by polypeptide is exposed to.Modified forms also comprises have the phosphorylated amino acid residue sequence of (as Tyrosine O-phosphate, phosphoserine, phosphothreonine).Thereby also comprise the polypeptide that has been improved its anti-proteolysis performance or optimized solubility property by modifying.
Polynucleotide of the present invention can be dna form or rna form.Dna form comprises the DNA of cDNA, genomic dna or synthetic.DNA can be strand or double-stranded.DNA can be coding strand or noncoding strand.The coding region sequence of encoding mature polypeptide can with SEQ ID NO:(n+7) shown in the varient of the identical or degeneracy of coding region sequence (as table 3).As used herein, " varient of degeneracy " is meant that in the present invention coding has the protein of SEQ ID NO:n, but with SEQ ID NO:(n+7) in the differentiated nucleotide sequence of corresponding encoded region sequence.
Table 3
| ??SEQ?ID??NO： | Antibacterial peptide | Polynucleotide sequence (5-3) |
| ??8 | ??hBD-134 | ??GGTATAAATTCATTATCATCAGAAATGCACAAGAAATGCTATAAAAA??TGGCATCTGCAGACTTGAATGCTATGAGAGTGAAATGTTAGTTGCCT??ACTGTATGTTTCAGCTGGAGTGCTGTGTCAAAGGAAATCCTGCACCC??TGA |
| ??9 | ??hBD-116 | ??GGCCTGTTCAGATCCCACAATGGCAAGAGCCGAGAGCCTTGGAATCC??ATGTGAGCTTTACCAAGGCATGTGCAGAAACGCCTGCAGAGAATATG??AAATCCAATACTTAACCTGCCCAAATGATCAAAAGTGCTGCCTGAAA??CTTTCTGTGAAAATAACCAGTTCTAAAAATGTGAAGGAGGATTACGA??CTCTAACTCCAACTTGTCAGTTACAAACAGTTCAAGCTACTCTCACAT??TTGA |
| ??SEQ?ID??NO： | Antibacterial peptide | Polynucleotide sequence (5-3) |
| ??10 | ??hBD-114 | ??GATCGTTGCACCAAACGTTACGGTCGTTGTAAAAGAGACTGTCTTGA??GAGTGAAAAGCAAATAGACATATGTTCCTTACCAAGAAAAATTTGCT??GCACTGAGAAATTGTATGAAGAAGATGATATGTTTTGA |
| ??11 | ??hBD-113 | ??GGTCCATCAGTTCCACAGAAAAAAACAAGAGAAGTTGCAGAGAGAA??AAAGAGAATGTCAGCTTGTTCGTGGTGCTTGCAAGCCGGAATGCAAC??AGCTGGGAATATGTATATTATTACTGCAATGTTAACCCCTGCTGTGCG??GTATGGGAATACCAAAAGCCAATCATTAACAAAATCACTAGTAAACT??CCATCAAAAATAA |
| ??12 | ??hBD-110 | ??AATTTTGAACCAAAGTATAGATTTGAAAGATGCGAAAAAGTGAGAG??GAATATGTAAAACGTTTTGTGATGATGTTGAGTATGATTATGGATACT??GCATTAAATGGAAAGAAGTCAGTGCTGCGTAT |
| ??13 | ??hBD-spage | ??GATGTTCCACCGGGAATTAGAAATACCATCTGCCATATGCAGCAAGG??GATCTGCAGACTTTTTTTCTGCCATTCTGGTGAGAAAAAGCGTGACAT??TTGCTCTGATCCCTGGAATAGGTGTTGCGTATCAAATACAGATGAAG??AAGGAAAAGAGAAACCAGAGATGGATGGCAGATCTGGGATCTAA |
| ??14 | ??mBD-12 | ??GGACTTGAGTATTCCCAGTCATTTCCAGGAGGGGAGATTGCTGTGTG??CGAGACATGCAGGCTCGGCCGGGGAAAATGCAGGAGAACATGCATA??GAGAGTGAGAAGATTGCGGGCTGGTGCAAGCTGAACTTCTTCTGCTG??TCGAGAGAGGATCTGA |
Term " polynucleotide of coded polypeptide " can be the polynucleotide that comprise the antibacterial peptide of the present invention of encoding, and also can be the polynucleotide that also comprise additional code and/or 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 segment, analogue and the derivative of polypeptide with the present invention.The varient of these polynucleotide can be the allelic variant of natural generation or the varient that non-natural takes place.These nucleotide diversity bodies comprise and replace varient, deletion mutation body and insert varient.As known in the art, allelic variant is a kind of replacement form of polynucleotide, and it may be replacement, disappearance or the insertion of one or more Nucleotide, but this variation can be from not changing its encoded polypeptides function in fact.
The invention still further relates to and above-mentioned sequence hybridization and two sequences between have at least 50%, preferably at least 70%, the polynucleotide of at least 80% homogeny more preferably.The present invention be more particularly directed under stringent condition and the interfertile polynucleotide of polynucleotide of the present invention.In the present invention, " stringent condition " is meant: (1) than hybridization under low ionic strength and the comparatively high temps and wash-out, as 0.2 * SSC, and 0.1%SDS, 60 ℃; Or (2) hybridization the time is added with denaturing agent, as 50% (v/v) methane amide, 0.1% calf serum/0.1%Ficoll, 42 ℃ etc.; Or (3) only at the homogeny between the two sequences at least more than 90%, be more preferably 95% and just hybridize when above.And the polypeptide of interfertile polynucleotide encoding has identical biological function and activity with the antibacterial peptide shown in the SEQ ID NO:n.
Polynucleotide of the present invention can obtain with the method for pcr amplification method, recombination method or synthetic usually.For the pcr amplification method, can be disclosed according to the present invention about nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and must relevant sequence.Described recombination method normally is cloned into carrier with described polynucleotide, changes cell again over to, separates obtaining relevant sequence then from the host cell after the propagation by ordinary method.Can synthesize relevant sequence by the method for synthetic in addition.
At present, can be fully obtain the dna sequence dna of code book invention albumen (or its fragment, or derivatives thereof) by chemosynthesis.This dna sequence dna can be introduced in various existing dna moleculars as known in the art (or as carrier) and the cell then.In addition, also can will suddenly change and introduce in the protein sequence of the present invention by chemosynthesis.
The present invention also relates to comprise the carrier of polynucleotide of the present invention, and the host cell that produces through genetically engineered of carrier of the present invention or antibacterial peptide encoding sequence, and the method for producing polypeptide of the present invention through recombinant technology.
Recombinant DNA technology by routine (Science, 1984,224:1431), can utilize polynucleotide sequence of the present invention to express or produce the antibacterial peptide of reorganization.In general following steps are arranged:
(1), or transforms or the transduction proper host cell with the recombinant expression vector that contains these polynucleotide with the polynucleotide (or varient) of encoding antimicrobial peptide of the present invention;
(2) in suitable medium, cultivate host cell; With
(3) separation, protein purification from substratum or cell.
Among the present invention, the polynucleotide of the described antibacterial peptide of encoding can be inserted in the recombinant expression vector.Term " recombinant expression vector " refers to that bacterial plasmid well known in the art, yeast plasmid, vegetable cell virus, mammalian cell virus are as adenovirus, retrovirus or other carriers.In a word, as long as can duplicate in host and stablize, any plasmid and carrier can be used.A key character of expression vector is to contain replication orgin, promotor, marker gene and translation controlling elements usually.
Method well-known to those having ordinary skill in the art can be used to make up and contains described polynucleotide sequence and suitable transcribing/the translate expression vector of control signal.These methods comprise extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technology of body etc.Described dna sequence dna can effectively be connected on the suitable promotor in the expression vector, and is synthetic to instruct mRNA.In addition, expression vector preferably comprises one or more selected markers, to be provided for selecting the phenotypic character of transformed host cells, cultivate Tetrahydrofolate dehydrogenase, neomycin resistance and the green fluorescent protein (GFP) of usefulness as eukaryotic cell, or be used for colibacillary tsiklomitsin or amicillin resistance.
Comprise the polynucleotide of the above-mentioned described antibacterial peptide of coding and the carrier of suitable promotor or control sequence, can be used to transform appropriate host cell, so that it can marking protein.
Host cell can be a prokaryotic cell prokaryocyte, as bacterial cell; Or eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as mammalian cell.Representative example has: intestinal bacteria, yeast etc.At different host cells, also can carry out codon optimizedly to described polynucleotide sequence, to obtain improved expression effect, codon optimized technology is well known in the art.
Persons skilled in the art all know how to select appropriate carriers, promotor, enhanser and host cell.
Can carry out with routine techniques well known to those skilled in the art with the recombinant DNA transformed host cell.When the host was prokaryotic organism such as intestinal bacteria, the competent cell that can absorb DNA can be used CaCl in exponential growth after date results
2Method is handled, and used step is well-known in this area.Another kind method is to use MgCl
2If desired, transforming also the method for available electroporation carries out.When the host is an eukaryote, can select following DNA transfection method for use: coprecipitation of calcium phosphate method, conventional mechanical method such as microinjection, electroporation, liposome packing etc.
The transformant that obtains can be cultivated with ordinary method, to express described antibacterial peptide.According to used host cell, used substratum can be selected from various conventional substratum in the cultivation.Under the condition that is suitable for the host cell growth, cultivate.
Described antibacterial peptide can or be secreted into the extracellular at cell inner expression.Can utilize its physics, the separating by various separation methods with other characteristic and the albumen of purification of Recombinant of chemistry.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation handles, with protein precipitant handle (salt analysis method), centrifugal, the broken bacterium of infiltration, superly handle, the combination of super centrifugal, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods.
The present invention also provides a kind of checking antibacterial peptide active method.This method comprises the method for antibacterial peptide dilution, the method for bacterium dilution, and antibacterial peptide and bacteriological action time and definite active method of antibacterial peptide.
Antibacterial peptide of the present invention is of use in many ways.These purposes include, but is not limited to: directly screen antibody, polypeptide or other part that promotes or resist described antibacterial peptide function as chemoprophylaxis or treatment pathogenic infection relative disease and being used to.The peptide molecule that can stimulate described antibacterial peptide function that can be used for seeking therapeutic value with the recombinant antibacterial peptide screening peptide library of expressing.
When using antibacterial peptide of the present invention, also can use other medicaments simultaneously, as antibiotic such as penicillin.
The present invention also provides a kind of composition (preferred pharmaceutical compositions), and it contains antibacterial peptide of the present invention (or its agonist) and the pharmaceutically acceptable carrier or the vehicle of safe and effective amount.This class carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.Pharmaceutical preparation should be complementary with administering mode.Pharmaceutical composition of the present invention can be made into the injection form, for example is prepared by ordinary method with the physiological saline or the aqueous solution that contains glucose and other assistant agents.Pharmaceutical composition such as tablet and capsule can be prepared by ordinary method.Pharmaceutical composition such as injection, solution, tablet and capsule should be made under aseptic condition.The dosage of activeconstituents is the treatment significant quantity, for example every day about 1 microgram/kg body weight-Yue 5 mg/kg body weight.In addition, polypeptide of the present invention also can use with the other treatment agent.
When making pharmaceutical composition, be that antibacterial peptide of the present invention (or its agonist) with safe and effective amount is applied to Mammals (comprising the people), wherein this safe and effective amount is usually at least about 1 microgram/kg body weight, and in most of the cases be no more than about 8 mg/kg body weight, preferably this dosage is about 10 micrograms/kg body weight-Yue 1 mg/kg body weight.Certainly, concrete dosage also should be considered factors such as route of administration, patient health situation, and these all are within the skilled practitioners skill.In addition, the nucleotide sequence of described antibacterial peptide also can be used for multiple therapeutic purpose.
Antibacterial peptide has a multiple bioactive natural molecule as a kind of, and its biological function also will have new expansion.Along with deepening continuously and the continuous development of technology of research, antibacterial peptide will be increasing to the mankind's contribution.
Advantage of the present invention mainly is:
(1) antibacterial peptide of the present invention is a natural antibacterial peptide, and majority is the people source, has the risk of lower immunological rejection in process of clinical application.
(2) antibacterial peptide of the present invention has good sterilization effect for fungi, has broad application prospects at present more thorny clinically deep fungal infection (infection that causes as Candida albicans) disease; And the antifungal drug of using all has the liver renal toxicity at present, and people of the present invention source natural antibacterial peptide can not produce similar liver renal toxicity.
(3) similar antibacterial peptide is compared in antibacterial peptide of the present invention and the prior art, and its antibacterial vigor has bigger increase.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: lab guide (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise per-cent and umber calculate by weight.
The structure of embodiment 1. peptide expression plasmids
The inventor has selected a kind of prokaryotic expression carrier of albumen intron mediation purifying, i.e. pTWIN1 expression vector (available from NEB company) for use.Its advantage is: it is a kind of posttranslational modification incident that protein is sheared, and it will insert the intermediary protein intron (Intein) of precursor protein and shear, and with normal peptide bond both sides protein and peptide chain be coupled together.In this process, do not need the effect of coenzyme or cofactor, only need four step intramolecular reactions.Intein and flanking sequence thereof can be used for protein purification by oneself's cutting of sudden change generation high degree of specificity, protein connects and protein cyclisation reaction.PTWIN 1 carrier merges at the N-terminal of Intein to be had the chitin calmodulin binding domain CaM (chitin binding domain, CBD), it can make the Intein fusion rotein be adsorbed on the chitin pearl.When the C-terminal that target protein is fused to Intein is expressed, be 7 in pH value, temperature is under 25 ℃ the condition, and Intein is induced between itself and target protein to carry out from cutting, and like this, target protein just is released.Belong to disposable purification system based on Intein from the protein purification system of cutting, this system need not to use proteolytic enzyme, and the non-specific enzymolysis of having avoided the proteolytic enzyme cutting to be brought also need not to be further purified removal proteolytic enzyme, is the good method of a timing-saving and economic.
Obtain the dna sequence dna of antibacterial peptide coding region with the PCR method amplification.PCR is template with the human genome, increases with La Taq enzyme (TaKaRa), and primer is as shown in table 4.
Table 4
| Primer (5 → 3) | ??SEQ?ID??NO： | |
| ??hBD-116 | ??AAAGCTCTTCTAACggcctgttcagatcccacaat??AAAGGATCCtcaaatgtgagagtagcttga | ??15??16 |
| ??hBD-134 | ??AAAGCTCTTCTAACggtataaattcattatcatca??AAAGGATCCtcagggtgcaggatttccttt | ??17??18 |
| ??hBD-110 | ??aaaGCTCTTCTAACaattttgaaccaaagtatag??aaaCTGCAGtcatacgcagcactgacttc | ??19??20 |
| ??hBD-113 | ??aaaGCTCTTCTAACggtccatcagttccacagaa??aaaCTGCAGtcatttttgatggagtttacta | ??21??22 |
| Primer (5 → 3) | ??SEQ?ID??NO： | |
| ??hBD-114 | ??aaaGCTCTTCTAACgatcgttgcaccaaacgttac??aaaCTGCAGtcaaaacatatcatcttcttc | ??23??24 |
| ??hBD-SP | ??aaaGCTCTTCTAACgatgttccaccgggaattagaaata??aaaCTGCAGttagatcccagatctgccatccatc | ??25??26 |
| ??mBD-12 | ??aaaGCTCTTCTAACggacttgagtattcccagtc??aaaCTGCAGtcagatcctctctcgacagc | ??27??28 |
The PCR program is: 95 ℃ of 2min, 94 ℃ of 30s, 60 ℃ of 30s, 72 ℃ of 30s, 30 circulations, 72 ℃ of 5min, 16 ℃ of forever.Pcr amplification to dna fragmentation two ends the restriction enzyme site HBD-116 and the HBD-134 that are added with cloning vector and need be BstQ I and BamH I, HBD-110, HBD-113, HBD-114, HBD-SP and mBD-12 are BstQ I and Pst I, and the PCR product cuts back to close on the BstQ I and BamH I or BstQ I and Pst I site of being cloned into the pTWIN1 carrier through enzyme.Enzyme is cut and is checked order and identify correct plasmid called after: pTWIN1-116, pTWIN1-134, pTWIN1-110, pTWIN1-113, pTWIN1-114, pTWIN1-SP, pTWIN1-12 respectively, and the plasmid synoptic diagram is seen Fig. 1.
The conversion and the evaluation of embodiment 2. peptide expression plasmids
From-70 ℃ of refrigerators, get 200 μ l competent cell E.coli ER2566 bacterial strain (New EnglandBiolabs) suspensions, under the room temperature it is thawed, put on ice immediately after thawing.Add plasmid DNA solution (content is no more than 50ng, and volume is no more than 10 μ l), shake up gently, place 30 minutes on ice after.Thermal shock 90 seconds or 37 ℃ of water-baths are 5 minutes in 42 ℃ of water-baths, place rapidly behind the thermal shock cooled on ice 3-5 minute.Xiang Guanzhong adds 1ml LB liquid nutrient medium (not containing Amp), and 37 ℃ of shaking culture are 1 hour behind the mixing, makes bacterium the restore normal growth state and the antibiotics resistance gene (Amp) of expression plasmid coding.Get 100 μ l after above-mentioned bacterium liquid shaken up and coat on the screening flat board that contains Amp, face up and place half an hour, treat that bacterium liquid is absorbed the back by substratum fully and is inverted culture dish, cultivated 16-24 hour for 37 ℃.
The picking mono-clonal was cultivated 8-16 hour for 37 ℃ in 3ml LB substratum, and bacterium liquid is used for the PCR evaluation and order-checking is identified.What the pcr amplification rear electrophoresis was identified the results are shown in Figure 2, and sequencing result is correct.
The preparation of embodiment 3. antimicrobial peptide proteins
Picking mono-clonal from the flat board, be inoculated in the 3ml LB test tube that contains 0.1 μ g/ml, 37 ℃ of 200rpm overnight incubation, get the 1ml bacterium, be transferred to and contain the shaking in the bottle of 100ml LB, 37 ℃ of 200rpm cultivate 3hr, survey OD600 to about the 0.5-0.7, and the IPTG that adds 0.5mM spends the night in 25 ℃ of 200rpm inducing culture.
4 ℃ of centrifugal 10min of 100ml bacterium liquid 12000rpm of overnight incubation are abandoned supernatant, in precipitation, add 10ml dissolving buffer B 1 (20mM Tris-HCl, 50mM Nacl, 1mM EDTA, pH 8.5) and 0.15%Tween20 and 20 μ M PMSF, ultrasonication, ultrasonic time 8s, 15s intermittently, energy 300W, total time 10min, 4 ℃ of centrifugal 15min of 12000rpm get supernatant after ultrasonic 2 times.
The adding of 10ml supernatant is contained in the pillar of 4ml chitin filler, pillar is earlier with 20ml buffer B 1 washing balance, ultrasonic broken wall supernatant is in conjunction with the chitin filler after 2 hours, be circulated throughout post 5-10 time, behind 20ml B1 washing pillar, add 4ml elution buffer B2 (20mM Tris-HCl, 50Mm Nacl, 1Mm EDTA, pH 7.0) in conjunction with cutting, the liquid that the 4ml after 25 ℃ of combination cuttings are spent the night contains antibacterial peptide is used to do bacteriostatic experiment.Because the antimicrobial peptide protein size is similar, the preparation method is identical, and the inventor is an example with hBD-spage protein expression purification result, sees Fig. 3 and Fig. 4.
Earlier bacterium is diluted to 10
3Cfu/5 μ l, the B2 that 5 μ l bacterium and 50 μ l is contained antibacterial peptide mixes, and handles the different times for 37 ℃, coated plate, 37 ℃ of overnight incubation, the dull and stereotyped clone's number of going up of number, calculate the survival rate of different treatment time:
Antibacterial peptide the results are shown in Figure 5 for the bacteriostatic experiment of intestinal bacteria E.coli, and antibacterial peptide concentration is 0.3mg/ml, and bacteria concentration is 10
3Cfu/5 μ l, be 6 hours action time, per hour the survival rate of bacterium is surveyed in sampling, antibacterial through 6 hours, colibacillary survival rate is all below 10%.
Antibacterial peptide the results are shown in Figure 6 for the bacteriostatic experiment of Candida albicans Candida albicans, and antibacterial peptide concentration is 0.3mg/ml, and bacteria concentration is 10
3Cfu/5 μ l, be 6 hours action time, per hour the survival rate of bacterium is surveyed in sampling, antibacterial through 6 hours, colibacillary survival rate is all below 10%.
The bacteriostatic activity of embodiment 5. antibacterial peptides relatively
Several antibacterial peptides of the present invention and antibacterial peptide of the prior art (antibacterial peptide Bin1b (referring to application number 200410018102.2)) are carried out antibacterial vigor relatively, adopt the bacteriostatic experiment of intestinal bacteria E.coli.Antibacterial peptide concentration is 0.4mg/ml, and bacteria concentration is 10
3Cfu/5 μ l, be 4 hours action time, surveys the survival rate of bacterium every sampling in 1 hour.
The results are shown in Figure 7, as can be seen from the figure, antibacterial peptide of the present invention all significantly is better than Bin1b for the bacteriostatic action of intestinal bacteria E.coli.
In all documents that the present invention mentions are all asked in basis, quote as a reference, quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art of the present technique can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table
＜110〉Shanghai Nanfangmoshi Biological Sci-Tech Dev Co., Ltd.; Model animals research centre, south, Shanghai
＜120〉new natural antibacterial peptide, its encoding sequence and purposes
<130>083733
<160>28
<170>PatentIn?version?3.3
<210>1
<211>47
<212>PRT
＜213〉homo sapiens (Homo Sapiens)
<400>1
Gly?Ile?Asn?Ser?Leu?Ser?Ser?Glu?Met?His?Lys?Lys?Cys?Tyr?Lys?Asn
1???????????????5???????????????????10??????????????????15
Gly?Ile?Cys?Arg?Leu?Glu?Cys?Tyr?Glu?Ser?Glu?Met?Leu?Val?Ala?Tyr
20??????????????????25??????????????????30
Cys?Met?Phe?Gln?Leu?Glu?Cys?Cys?Val?Lys?Gly?Asn?Pro?Ala?Pro
35??????????????????40??????????????????45
<210>2
<211>79
<212>PRT
＜213〉homo sapiens (Homo Sapiens)
<400>2
Gly?Leu?Phe?Arg?Ser?His?Asn?Gly?Lys?Ser?Arg?Glu?Pro?Trp?Asn?Pro
1???????????????5???????????????????10??????????????????15
Cys?Glu?Leu?Tyr?Gln?Gly?Met?Cys?Arg?Asn?Ala?Cys?Arg?Glu?Tyr?Glu
20??????????????????25??????????????????30
Ile?Gln?Tyr?Leu?Thr?Cys?Pro?Asn?Asp?Gln?Lys?Cys?Cys?Leu?Lys?Leu
35??????????????????40??????????????????45
Ser?Val?Lys?Ile?Thr?Ser?Ser?Lys?Asn?Val?Lys?Glu?Asp?Tyr?Asp?Ser
50??????????????????55??????????????????60
Asn?Ser?Asn?Leu?Ser?Val?Thr?Asn?Ser?Ser?Ser?Tyr?Ser?His?Ile
65??????????????????70??????????????????75
<210>3
<211>43
<212>PRT
＜213〉homo sapiens (Homo Sapiens)
<400>3
Asp?Arg?Cys?Thr?Lys?Arg?Tyr?Gly?Arg?Cys?Lys?Arg?Asp?Cys?Leu?Glu
1???????????????5???????????????????10??????????????????15
Ser?Glu?Lys?Gln?Ile?Asp?Ile?Cys?Ser?Leu?Pro?Arg?Lys?Ile?Cys?Cys
20??????????????????25??????????????????30
Thr?Glu?Lys?Leu?Tyr?Glu?Glu?Asp?Asp?Met?Phe
35??????????????????40
<210>4
<211>66
<212>PRT
＜213〉homo sapiens (Homo Sapiens)
<400>4
Gly?Pro?Ser?Val?Pro?Gln?Lys?Lys?Thr?Arg?Glu?Val?Ala?Glu?Arg?Lys
1???????????????5???????????????????10??????????????????15
Arg?Glu?Cys?Gln?Leu?Val?Arg?Gly?Ala?Cys?Lys?Pro?Glu?Cys?Asn?Ser
20??????????????????25??????????????????30
Trp?Glu?Tyr?Val?Tyr?Tyr?Tyr?Cys?Asn?Val?Asn?Pro?Cys?Cys?Ala?Val
35??????????????????40??????????????????45
Trp?Glu?Tyr?Gln?Lys?Pro?Ile?Ile?Asn?Lys?Ile?Thr?Ser?Lys?Leu?His
50??????????????????55??????????????????60
Gln?Lys
65
<210>5
<211>41
<212>PRT
＜213〉homo sapiens (Homo Sapiens)
<400>5
Asn?Phe?Glu?Pro?Lys?Tyr?Arg?Phe?Glu?Arg?Cys?Glu?Lys?Val?Arg?Gly
1???????????????5???????????????????10??????????????????15
Ile?Cys?Lys?Thr?Phe?Cys?Asp?Asp?Val?Glu?Tyr?Asp?Tyr?Gly?Tyr?Cys
20??????????????????25??????????????????30
Ile?Lys?Trp?Arg?Ser?Gln?Cys?Cys?Val
35??????????????????40
<210>6
<211>61
<212>PRT
＜213〉homo sapiens (Homo Sapiens)
<400>6
Asp?Val?Pro?Pro?Gly?Ile?Arg?Asn?Thr?Ile?Cys?His?Met?Gln?Gln?Gly
1???????????????5???????????????????10??????????????????15
Ile?Cys?Arg?Leu?Phe?Phe?Cys?His?Ser?Gly?Glu?Lys?Lys?Arg?Asp?Ile
20??????????????????25??????????????????30
Cys?Ser?Asp?Pro?Trp?Asn?Arg?Cys?Cys?Val?Ser?Asn?Thr?Asp?Glu?Glu
35??????????????????40??????????????????45
Gly?Lys?Glu?Lys?Pro?Glu?Met?Asp?Gly?Arg?Ser?Gly?Ile
50??????????????????55??????????????????60
<210>7
<211>51
<212>PRT
＜213〉mouse (Muc musculus)
<400>7
Gly?Leu?Glu?Tyr?Ser?Gln?Ser?Phe?Pro?Gly?Gly?Glu?Ile?Ala?Val?Cys
1???????????????5???????????????????10??????????????????15
Glu?Thr?Cys?Arg?Leu?Gly?Arg?Gly?Lys?Cys?Arg?Arg?Thr?Cys?Ile?Glu
20??????????????????25??????????????????30
Ser?Glu?Lys?Ile?Ala?Gly?Trp?Cys?Lys?Leu?Asn?Phe?Phe?Cys?Cys?Arg
35??????????????????40??????????????????45
Glu?Arg?Ile
50
<210>8
<211>144
<212>DNA
＜213〉homo sapiens (Homo Sapiens)
<400>8
ggtataaatt?cattatcatc?agaaatgcac?aagaaatgct?ataaaaatgg?catctgcaga?????60
cttgaatgct?atgagagtga?aatgttagtt?gcctactgta?tgtttcagct?ggagtgctgt????120
gtcaaaggaa?atcctgcacc?ctga???????????????????????????????????????????144
<210>9
<211>240
<212>DNA
＜213〉homo sapiens (Homo Sapiens)
<400>9
ggcctgttca?gatcccacaa?tggcaagagc?cgagagcctt?ggaatccatg?tgagctttac?????60
caaggcatgt?gcagaaacgc?ctgcagagaa?tatgaaatcc?aatacttaac?ctgcccaaat????120
gatcaaaagt?gctgcctgaa?actttctgtg?aaaataacca?gttctaaaaa?tgtgaaggag????180
gattacgact?ctaactccaa?cttgtcagtt?acaaacagtt?caagctactc?tcacatttga????240
<210>10
<211>132
<212>DNA
＜213〉homo sapiens (Homo Sapiens)
<400>10
gatcgttgca?ccaaacgtta?cggtcgttgt?aaaagagact?gtcttgagag?tgaaaagcaa????60
atagacatat?gttccttacc?aagaaaaatt?tgctgcactg?agaaattgta?tgaagaagat???120
gatatgtttt?ga???????????????????????????????????????????????????????132
<210>11
<211>201
<212>DNA
＜213〉homo sapiens (Homo Sapiens)
<400>11
ggtccatcag?ttccacagaa?aaaaacaaga?gaagttgcag?agagaaaaag?agaatgtcag?????60
cttgttcgtg?gtgcttgcaa?gccggaatgc?aacagctggg?aatatgtata?ttattactgc????120
aatgttaacc?cctgctgtgc?ggtatgggaa?taccaaaagc?caatcattaa?caaaatcact????180
agtaaactcc?atcaaaaata?a??????????????????????????????????????????????201
<210>12
<211>126
<212>DNA
＜213〉homo sapiens (Homo Sapiens)
<400>12
aattttgaac?caaagtatag?atttgaaaga?tgcgaaaaag?tgagaggaat?atgtaaaacg?????60
ttttgtgatg?atgttgagta?tgattatgga?tactgcatta?aatggaaaga?agtcagtgct????120
gcgtat???????????????????????????????????????????????????????????????126
<210>13
<211>186
<212>DNA
＜213〉homo sapiens (Homo Sapiens)
<400>13
gatgttccac?cgggaattag?aaataccatc?tgccatatgc?agcaagggat?ctgcagactt?????60
tttttctgcc?attctggtga?gaaaaagcgt?gacatttgct?ctgatccctg?gaataggtgt????120
tgcgtatcaa?atacagatga?agaaggaaaa?gagaaaccag?agatggatgg?cagatctggg????180
atctaa???????????????????????????????????????????????????????????????186
<210>14
<211>156
<212>DNA
＜213〉mouse (Muc musculus)
<400>14
ggacttgagt?attcccagtc?atttccagga?ggggagattg?ctgtgtgcga?gacatgcagg?????60
ctcggccggg?gaaaatgcag?gagaacatgc?atagagagtg?agaagattgc?gggctggtgc????120
aagctgaact?tcttctgctg?tcgagagagg?atctga??????????????????????????????156
<210>15
<211>35
<212>DNA
＜213〉artificial sequence
<221>misc_feature
＜223〉primer
<400>15
aaagctcttc?taacggcctg?ttcagatccc?acaat???????????????????????????????35
<210>16
<211>30
<212>DNA
＜213〉artificial sequence
<221>misc_feature
＜223〉primer
<400>16
aaaggatcct?caaatgtgag?agtagcttga?????????????????????????????????????30
<210>17
<211>35
<212>DNA
＜213〉artificial sequence
<221>misc_feature
＜223〉primer
<400>17
aaagctcttc?taacggtata?aattcattat?catca???????????????????????????????35
<210>18
<211>30
<212>DNA
＜213〉artificial sequence
<221>misc_feature
＜223〉primer
<400>18
aaaggatcct?cagggtgcag?gatttccttt?????????????????????????????????????30
<210>19
<211>34
<212>DNA
＜213〉artificial sequence
<221>misc_feature
＜223〉primer
<400>19
aaagctcttc?taacaatttt?gaaccaaagt?atag????????????????????????????????34
<210>20
<211>29
<212>DNA
＜213〉artificial sequence
<221>misc_feature
＜223〉primer
<400>20
aaactgcagt?catacgcagc?actgacttc??????????????????????????????????????29
<210>21
<211>34
<212>DNA
＜213〉artificial sequence
<221>misc_feature
＜223〉primer
<400>21
aaagctcttc?taacggtcca?tcagttccac?agaa????????????????????????????????34
<210>22
<211>31
<212>DNA
＜213〉artificial sequence
<221>misc_feature
＜223〉primer
<400>22
aaactgcagt?catttttgat?ggagtttact?a???????????????????????????????????31
<210>23
<211>35
<212>DNA
＜213〉artificial sequence
<221>misc_feature
＜223〉primer
<400>23
aaagctcttc?taacgatcgt?tgcaccaaac?gttac???????????????????????????????35
<210>24
<211>30
<212>DNA
＜213〉artificial sequence
<221>misc_feature
＜223〉primer
<400>24
aaactgcagt?caaaacatat?catcttcttc?????????????????????????????????????30
<210>25
<211>39
<212>DNA
＜213〉artificial sequence
<221>misc_feature
＜223〉primer
<400>25
aaagctcttc?taacgatgtt?ccaccgggaa?ttagaaata???????????????????????????39
<210>26
<211>34
<212>DNA
＜213〉artificial sequence
<221>misc_feature
＜223〉primer
<400>26
aaactgcagt?tagatcccag?atctgccatc?catc????????????????????????????????34
<210>27
<211>34
<212>DNA
＜213〉artificial sequence
<221>misc_feature
＜223〉primer
<400>27
aaagctcttc?taacggactt?gagtattccc?agtc????????????????????????????????34
<210>28
<211>29
<212>DNA
＜213〉artificial sequence
<221>misc_feature
＜223〉primer
<400>28
aaactgcagt?cagatcctct?ctcgacagc??????????????????????????????????????29
Claims (10)
1. an isolated polypeptide is characterized in that, described polypeptide is selected from down group:
(a) polypeptide of aminoacid sequence shown in SEQ ID NO:n; Or
(b) disappearance, insertion or the replacement of the aminoacid sequence shown in the SEQ ID NO:n through 1-5 amino-acid residue formed, and have the polypeptide polypeptides derived that suppresses the pathogenic agent function by aminoacid sequence shown in the SEQ ID NO:n;
Wherein, n is the positive integer that is selected from 1-7.
2. polypeptide as claimed in claim 1 is characterized in that, described polypeptide is the polypeptide of aminoacid sequence shown in SEQ ID NO:n.
3. isolating polynucleotide is characterized in that, the described polypeptide of described polynucleotide encoding claim 1.
4. polynucleotide as claimed in claim 3 is characterized in that, described polynucleotide are selected from down group:
(1) as SEQ ID NO:(n+7) shown in the polynucleotide of nucleotide sequence; Or
(2) with polynucleotide (1) complementary polynucleotide.
5. a carrier is characterized in that, described carrier contains the arbitrary described polynucleotide of claim 3-4.
6. a genetically engineered cell is characterized in that, described cell contains and is integrated with the arbitrary described polynucleotide of claim 3-4 in the described carrier of claim 5 or its genome.
7. a method for preparing the described polypeptide of claim 1 is characterized in that, described method comprises:
(i) under conditions suitable for the expression, cultivate the described cell of claim 6; With
(ii) isolate the described polypeptide of claim 1.
8. the purposes of the described polypeptide of claim 1 is used to prepare the composition that suppresses pathogenic agent.
9. a composition is characterized in that, described composition contains:
(A) described one or more polypeptide of the claim 1 of significant quantity; With
(B) pharmaceutically acceptable carrier.
10. a non-therapeutic ground suppresses the method for pathogenic agent, it is characterized in that described method comprises: described one or more polypeptide of claim 1 that need to suppress the object significant quantity of pathogenic agent.
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| CN103360467A (en) * | 2013-07-16 | 2013-10-23 | 青岛科技大学 | Preparation method of broad-spectrum antibacterial polypeptide |
| CN103360464A (en) * | 2013-07-17 | 2013-10-23 | 武汉摩尔生物科技有限公司 | Polypeptide, DNA molecule encoding polypeptide, vector, preparation method and application thereof |
| CN103382219A (en) * | 2013-07-16 | 2013-11-06 | 青岛科技大学 | Preparation method of antitumor polypeptide |
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| CN113045628A (en) * | 2021-03-23 | 2021-06-29 | 山东大学 | Application of antibacterial peptide or variant thereof in preparation of antibacterial product |
| CN114196631A (en) * | 2020-09-02 | 2022-03-18 | 江苏申琅生物科技有限公司 | Antibacterial functional polypeptide and preparation method and application thereof |
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| CN103360467A (en) * | 2013-07-16 | 2013-10-23 | 青岛科技大学 | Preparation method of broad-spectrum antibacterial polypeptide |
| CN103382219A (en) * | 2013-07-16 | 2013-11-06 | 青岛科技大学 | Preparation method of antitumor polypeptide |
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| CN103360464A (en) * | 2013-07-17 | 2013-10-23 | 武汉摩尔生物科技有限公司 | Polypeptide, DNA molecule encoding polypeptide, vector, preparation method and application thereof |
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| CN109415412A (en) * | 2016-06-23 | 2019-03-01 | 建国大学校产学协力团 | There is with antibiotic to Gram-negative Multidrug resistant bacteria the antibacterial peptide and application thereof of building performance antibacterial effect |
| CN109517046A (en) * | 2018-11-28 | 2019-03-26 | 福建师范大学 | A kind of antibacterial polypeptide and its application based on outer membrane protein generting machanism |
| CN109517046B (en) * | 2018-11-28 | 2021-09-14 | 福建师范大学 | Antibacterial polypeptide based on outer membrane protein generation mechanism and application thereof |
| CN114196631A (en) * | 2020-09-02 | 2022-03-18 | 江苏申琅生物科技有限公司 | Antibacterial functional polypeptide and preparation method and application thereof |
| CN113045628A (en) * | 2021-03-23 | 2021-06-29 | 山东大学 | Application of antibacterial peptide or variant thereof in preparation of antibacterial product |
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