CN104109198A - Derived polypeptides formed by modifying structures of frog skin antibacterial peptides AR-23 and application of derived polypeptides - Google Patents
Derived polypeptides formed by modifying structures of frog skin antibacterial peptides AR-23 and application of derived polypeptides Download PDFInfo
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Abstract
The invention discloses a group of derived polypeptides formed by modifying the structures of frog skin antibacterial peptides AR-23 and an application of the derived polypeptides. The group of derived polypeptides are named as A1-A7, and have amino acid sequences, i.e., respectively SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6 and SEQ ID No.7. The experiment proves that compared with natural antibacterial peptides, according to the derived polypeptides formed by modifying the structures of frog skin antibacterial peptides AR-23, the selective toxicity of the derived polypeptides to bacteria is high while the toxicity of the derived polypeptides to human cells is obviously decreased, so that the influences of the derived polypeptides to somatic cells, red blood cells and the like are very low. The polypeptides A1-A7 have broad-spectrum killing effects on gram-positive bacteria or gram-negative bacteria, so that the polypeptides A1-A7 can be used for treating diseases caused by the infections of the gram-positive bacteria or the gram-negative bacteria resistant to antibiotics.
Description
Technical field
The present invention relates to derivative polypeptide and application that antibacterial frog skin peptide AR-23 in biological technical field changes structure.
Background technology
The resistance of bacterial antibiotic has become a significant problem that threatens human health.Antibacterial peptide (Antibacterial Peptide) is the defensive peptide class active substance of the exogenous pathogenic agent invasion and attack of the antagonism of organism generation pathogenic effects, it is the important component part of immunity of organism defence system, there is little, the thermally-stabilised and good water solubility of molecular weight, the features such as broad-spectrum antimicrobial (Andrea G, Giovanna P, Silvia F.N.Antimicrobial peptides:an overview of a promising class of therapeutics.Central European Journal of Biology, 2007,2 (1): 1-33).
Natural antibacterial peptide is generally comprised of 10-50 amino acid, and C end is rich in hydrophilic amino acid, and N end, containing more hydrophobic residue, has amphiphilic structure.Research shows, antibacterial peptide can be formed hole at bacterium surface, be caused cytolemma disintegration by " stave ", " looping pit " and " felt rug " isotype, finally causes bacterium dead.Because of its Antibacterial Mechanism completely different from microbiotic, be expected to overcome pathogenic bacteria to antibiotic multidrug resistant, do you there is vast potential for future development, yet as microbiotic, also there is its problem larger to eukaryotic toxicity (Brogden KA.Antimicrobial peptides:pore formers or metabolic inhibitors in bacteria in antibacterial peptide? Nat Rev Microbiol.2005; 3 (3): 238-50).
Melittin is the main component of European honeybee Apis mellifera bee venom, has the typical structure of antibacterial peptide.Melittin is comprised of 26 amino acid, and 1-20 position residue is mainly by hydrophobic amino acid.Crystal study shows, it is a kind of structure (Terwilliger T C & Eisenberg D.The structure of Melittin.J Biol Chem.1982 of the spiral-bending-spiral being comprised of 2 α spiral fragments; 257,6016 – 6022), wherein 1 – 10 amino acids residues and 13 – 26 amino acids residues form respectively 2 sections of spirals, and 11 – 12 residues form bending.Although, melittin exists with random curling monomeric form, but when peptide concentration increases, or add after salt, it can form the tetramer (the Tatham AS with spirane structure, Hider RC, Drake AF.The effect of counterions on melittin aggregation.Biochem is J.1983Jun1; 211 (3): 683-6).Show with the repercussion study of cytolemma, melittin is with barrel-stave mechanism (Papo N, Shai Y.Exploring peptide membrane interaction using surface plasmon resonance:differentiation between pore formation versus membrane disruption by lytic peptides.Biochemistry.2003Jan21; 42 (2): 458-66) mode (Ladokhin AS, the White SH.'Detergent-like'permeabilization of anionic lipid vesicles by melittin.Biochim Biophys Acta.2001Oct1 through carpet with similar sanitising agent; 1514 (2): 253-60) pass through cytolemma.Because melittin all has very strong affinity to various microbial films, thereby it all has cytotoxicity (Hancock RE widely to bacterium, fungi, virus and mammalian cell, Sahl HG.Antimicrobial and host-defense peptides as new anti-infective therapeutic strategies.Nat Biotechnol, 2006; 24 (12): 1551-1557).The unselected cell toxicity of Melittin can be brought out comparatively severe side effect, has had a strong impact on it as antibacterials application prospect.Research shows, in the 1-20 amino acid of melittin, Leu-6, Lys-7, Val-8, Leu-9, Leu-13, Leu-16, Ile-17, the amino acid such as Trp-19 and Ile-20 is to antibacterial and hemolytic action is all extremely important, deleting any one amino acid all can cause hemolytic activity sharply to decline, and cause (the Blondelle SE that weakens of anti-microbial activity, Houghten RA.Hemolytic and antimicrobial activities of the twenty-four individual omission analogues of melittin.Biochemistry.1991May14, 30 (19): 4671-8).On the 6 – 20 amino acids residues of melittin, there is a leucine zipper motif (leucine zipper motif) forming with 6 leucines or Isoleucine in the discoveries such as Asthana, (Asthana N, Yadav SP, Ghosh JK.J Biol Chem.2004Dec31; 279 (53): 55042-50).Pandey substitutes Leu-9 and/or Leu-16 with Ala, also obtains similar result (Pandey BK, Ahmad A, Asthana N.Biochemistry.2010Sep14; 49 (36): 7920-9).
Present inventor finds all there is stronger anti-microbial effect from the melittin related peptides AR-23 of the Ta Shi frog (Rana tagoi) with from the RV-23 of the red leg frog in California (Rana draytonii) in the research in early stage, from hemolysis rate curve, compare with melittin, AR-23 and RV-23 cytotoxicity be lower (Urban E obviously, Nagy E, Pal T, Sonnevend A, Conlon JM.Activities of Four Frog Skin-Derived Antimicrobial Peptides (Temporin-1dra, Temporin-1va and the Melittin-Related Peptides AR-23and RV-23) against Anaerobic Bacteria.Int J Antimicrob Agents, 2007, 29:317-321).AR-23 and RV-23 form by 23 amino-acid residues.Wherein, AR-23 and melittin have 78% homology, have the structure with the on all four leucine zipper of melittin, but its C end only has 3 amino acid.The leucine of RV-23 and Isoleucine number and melittin are just the same, and different is the structure that it does not form leucine zipper.By contrast, its anti-microbial activity of RV-23 and melittin and AR-23 are more or less the same, but toxicity is far below melittin and AR-23.Be badly in need of at present a class novel antimicrobial peptide, this antibacterial Toplink greatly reduces its toxicity to human cell when retaining its fungistatic effect, i.e. the antibacterial peptide of high-efficiency low-toxicity.
Summary of the invention
The object of this invention is to provide the derivative polypeptide that one group of antibacterial frog skin peptide AR-23 changes structure.These antibacterial frog skin peptides AR-23 changes the derivative polypeptide of structure and compares with natural antibacterial peptide, bacterium is had to higher selective toxicity, be that they have the bacteriostatic action identical or stronger with AR-23, and the mankind's cytotoxicity is obviously reduced, the impact that somatocyte, red corpuscle etc. is caused is extremely low.
Antibacterial frog skin peptide AR-23 provided by the present invention changes the derivative polypeptide of structure, and title is respectively A1-A7, and wherein, A7 is that aminoacid sequence is the polypeptide of SEQ ID No.1.
Wherein, SEQ ID No.1 is comprised of 23 amino-acid residues.
Aforementioned polypeptides can synthetic, also can first synthesize its encoding gene, then carries out biological expression and obtain.The encoding gene of aforementioned polypeptides can be by lacking the codon of one or several amino-acid residue in the DNA sequence dna shown in SEQ ID No.1, and/or the missense mutation of carrying out one or several base pair obtains.
A2-A6 is the derivative polypeptide of A7, and A2-A6 also belongs to protection scope of the present invention.
The derivative polypeptide of A7 provided by the present invention is the polypeptide that the 1st of SEQ ID No.1, the 8th and the 17th amino acids residue are obtained through the replacement of one or two amino-acid residue and/or disappearance and/or interpolation.
In above-mentioned derivative polypeptide, the derivative that described derivative polypeptide obtains for A7 being carried out in these three kinds of following M1-M3 two kinds or a kind of sudden change:
M1, the 1st amino acids residue of SEQ ID No.1 is sported to Ala, keep the constant polypeptide obtaining of other amino-acid residue of SEQ ID No.1;
M2, the 8th amino acids residue of SEQ ID No.1 is sported to Ala, keep the constant polypeptide obtaining of other amino-acid residue of SEQ ID No.1;
M3, the 17th amino acids residue of SEQ ID No.1 is sported to Ile, keep the constant polypeptide obtaining of other amino-acid residue of SEQ ID No.1.
In above-mentioned derivative polypeptide, described derivative polypeptide is following C1)-C6) in any:
C1) aminoacid sequence is the derivative polypeptide of SEQ ID No.2;
C2) aminoacid sequence is the derivative polypeptide of SEQ ID No.3;
C3) aminoacid sequence is the derivative polypeptide of SEQ ID No.4;
C4) aminoacid sequence is the derivative polypeptide of SEQ ID No.5;
C5) aminoacid sequence is the derivative polypeptide of SEQ ID No.6;
C6) aminoacid sequence is the derivative polypeptide of SEQ ID No.7.
Wherein, SEQ ID No.2 is comprised of 23 amino-acid residues, is the 1st of SEQ ID No.1 and the 8th 's Arg is sported respectively to the derivative polypeptide that Ala obtains, and this derivative polypeptide name is called polypeptide A 3; SEQ ID No.3 is comprised of 23 amino-acid residues, is the Arg of the 8th of SEQ ID No.1 is sported to the derivative polypeptide that Ala obtains, and this derivative polypeptide name is called polypeptide A 5; SEQ ID No.4 is comprised of 23 amino-acid residues, is the Arg of the 1st of SEQ ID No.1 is sported to the derivative polypeptide that Ala obtains, and this derivative polypeptide name is called polypeptide A 6; SEQ ID No.5 is comprised of 23 amino-acid residues, is the 1st Arg of SEQ ID No.1 sported to Ala and the Lys of the 17th is sported to the derivative polypeptide that Ile obtains, and this derivative polypeptide name is called polypeptide A 2; SEQ ID No.6 is comprised of 23 amino-acid residues, is the Lys of the 17th of SEQ ID No.1 is sported to the derivative polypeptide that Ile obtains, and this derivative polypeptide name is called polypeptide A 4; SEQ ID No.7 is comprised of 23 amino-acid residues, is the Arg of the 8th of SEQ ID No.1 is sported to Ala and the Lys of the 17th is sported to the derivative polypeptide that Ile obtains, and this derivative polypeptide name is called polypeptide A 1.
In above-mentioned derivative polypeptide, described derivative polypeptide can synthetic, also can first synthesize its encoding gene, then carries out biological expression and obtain.
The biomaterial relevant to A1-A7 also belongs to protection scope of the present invention.
The biomaterial relevant to A1-A7 provided by the present invention is following B1) to B16) in any:
B1) nucleic acid molecule of coding A1-A7;
B2) contain B1) expression cassette of described nucleic acid molecule;
B3) contain B1) recombinant vectors of described nucleic acid molecule;
B4) contain B2) recombinant vectors of described expression cassette;
B5) contain B1) recombinant microorganism of described nucleic acid molecule;
B6) contain B2) recombinant microorganism of described expression cassette;
B7) contain B3) recombinant microorganism of described recombinant vectors;
B8) contain B4) recombinant microorganism of described recombinant vectors;
B9) contain B1) transgenetic animal cell of described nucleic acid molecule system;
B10) contain B2) transgenetic animal cell of described expression cassette system;
B11) contain B3) transgenetic animal cell of described recombinant vectors system;
B12) contain B4) transgenetic animal cell of described recombinant vectors system;
B13) contain B1) transgenic plant cells of described nucleic acid molecule system;
B14) contain B2) transgenic plant cells of described expression cassette system;
B15) contain B3) transgenic plant cells of described recombinant vectors system;
B16) contain B4) transgenic plant cells of described recombinant vectors system.
In above-mentioned biomaterial, described nucleic acid molecule is following 1) or 2) or 3) or 4) shown in nucleic acid molecule:
1) DNA molecular of coding A1-A7;
2) the cDNA molecule of coding A1-A7
3) with 1) or 2) nucleotide sequence that limits has 75% or 75% above identity, and cDNA molecule or the genomic dna molecule of the A1-A7 that encodes;
4) under stringent condition with 1) or 2) nucleotide sequence hybridization that limits, and cDNA molecule or the genomic dna molecule of coding A1-A7.
Those of ordinary skills can adopt known method at an easy rate, and for example the method for orthogenesis and point mutation, suddenlys change to the nucleotide sequence of coding A1-A7 of the present invention.Those,, through manually modified, have and the nucleotide sequence 75% of A1-A7 of the present invention or the Nucleotide of higher identity, and need only coding A1-A7 and there is bacteriostatic action, be to be all derived from nucleotide sequence of the present invention and to be equal to sequence of the present invention.
Term used herein " identity " refers to the sequence similarity with natural acid sequence." identity " comprises with the nucleotide sequence of coding of the present invention A1-A7 having 75% or higher, or 85% or higher, or 90% or higher, or 95% or the nucleotide sequence of higher identity.Identity can be with the naked eye or computer software evaluate.Use computer software, the identity between two or more sequences can use per-cent (%) to represent, it can be used for evaluating the identity between correlated series.
In above-mentioned biomaterial, described stringent condition is at 2 * SSC, in the solution of 0.1%SDS, hybridizes and wash film 2 times at 68 ℃, and each 5min again in 0.5 * SSC, in the solution of 0.1%SDS, is hybridized and washes film 2 times at 68 ℃, each 15min.
Above-mentioned 75% or 75% above identity, can be more than 80%, 85%, 90% or 95% identity.
In above-mentioned biomaterial, B2) expression cassette of the described nucleic acid molecule that contains coding A7 or described derivative polypeptide, refer to the DNA that can express A1-A7 in host cell, this DNA not only can comprise the promotor that starts A1-A7 genetic transcription, also can comprise the terminator that stops A1-A7 genetic transcription.Further, described expression cassette also can comprise enhancer sequence.
The recombinant vectors that available existing expression vector establishment contains A1-A7 expression casette.
In above-mentioned biomaterial, described carrier can be plasmid, glutinous grain, phage or virus vector.
In above-mentioned biomaterial, B5)-B8) described microorganism can be yeast, bacterium, algae or fungi, as intestinal bacteria.
In above-mentioned biomaterial, B9)-B16) described transgenic plant cells system and transgenetic animal cell are not comprise reproductive material.
A1-A7 or above-mentioned B1) to B16) in the application of any biomaterial in preparing antibacterial peptide, also belong to protection scope of the present invention.
In above-mentioned application, the pathogenic bacteria that described antibacterial peptide suppresses is bacterium.
In above-mentioned application, described bacterium is gram-positive microorganism or Gram-negative bacteria, as intestinal bacteria, Pseudomonas aeruginosa, dysentery bacterium, staphylococcus aureus, staphylococcus epidermidis or streptococcus pneumoniae.
A1-A7 or above-mentioned B1) to B16) in any biomaterial in preparation, prevent and/or treat the medicine of bacterial disease or the application in vaccine.
In above-mentioned application, described bacterium is gram-positive microorganism or Gram-negative bacteria, as intestinal bacteria, Pseudomonas aeruginosa, dysentery bacterium, staphylococcus aureus, staphylococcus epidermidis or streptococcus pneumoniae.
Experiment showed, and compare natural antibacterial peptide AR-23, polypeptide A 1-A7 of the present invention all has reduction in various degree to the molten broken ability of red corpuscle, and wherein, polypeptide A 7 and A3, A5 and A6 reduce particularly remarkable to the molten broken ability of red corpuscle.When antibacterial peptide and peptide concentration are 12.5 μ M, the hemolysis rate of AR-23 almost reaches 100%, and now the hemolysis rate of polypeptide A 7 and A3, A5 and A6 is 0, the hemolysis rate of A1, A2 and A4 is respectively 88.83%, 42.05% and 36.03%, when antibacterial peptide concentration reaches 100 μ M, the hemolysis rate of AR-23 is 99.19%, almost reach 100%, the hemolysis rate of polypeptide A 7 and A3, A5 and A6 is respectively 7.74%, 28.71%, 18.04% and 22.83%, illustrates that polypeptide A 7 can reduce it to erythrocytic hemolytic action.The therapeutic index of polypeptide A 7 is 2.83, far away higher than the therapeutic index of AR-23 (therapeutic index is 0.07), RV-23 (therapeutic index is 0.42) and melittin (therapeutic index is 0.15).Hela cell survival rate is 50% (IC
50) time, the concentration of polypeptide A 7, A3, A5 and A6 not only promotes more compared with natural antibacterial peptide AR-23, and in natural antibacterial peptide RV-23 and melittin, also has greatly improved, and illustrates that polypeptide A 7, A3, A5 and A6 reduce the virulence of Hela cell.Polypeptide A 7 and the colibacillary combination rate of fluorescein TAMRA mark are 97.32%, AR-23 and the colibacillary combination rate of fluorescein TAMRA mark are 33.73%, show that polypeptide A 7 and colibacillary binding ability increase compared with cecropin A R-23 and colibacillary binding ability.When the cecropin A R-23 of TAMRA mark and RV-23 born of the same parents' extracellular concentration are 6 μ M, the two all can enter Hela cell, and the polypeptide A 7 of TAMRA mark does not enter Hela cell, and the toxicity of 7 pairs of Hela cells of polypeptide A is less.Polypeptide A 1-A7 compares with natural antibacterial peptide, and bacterium is had to higher selective toxicity, and they have the bacteriostatic action identical or stronger with AR-23, and the mankind's cytotoxicity is obviously reduced, and the impact that somatocyte, red corpuscle etc. is caused is extremely low.The synthetic polypeptide A 1-A7 of the present invention has the lethal effect of wide spectrum to gram-positive microorganism or Gram-negative bacteria, can be used for the treatment of the disease that the gram-positive microorganism of antibiotics resistance or gram positive bacterial infection are caused.
Accompanying drawing explanation
Fig. 1 is the erythrocytic hemolysis rate of polypeptide A 7, A1, A2, A3, A4, A5, A6, AR-23, RV-23 and the melittin of different concns.
Fig. 2 is the survival rate of the Hela cell of the polypeptide A 7 of different concns, derivative polypeptide A 1, A2, A3, A4, A5, A6, AR-23, RV-23 and melittin.
Fig. 3 is polypeptide A 7, AR-23 and the RV-23 of fluorescein TAMRA mark and the binding ability of intestinal bacteria and streptococcus aureus.
Fig. 4 is the penetrativity that polypeptide A 7, AR-23 and the RV-23 of TAMRA mark penetrates Hela cell.
Embodiment
Below in conjunction with embodiment, the present invention is further described in detail, the embodiment providing is only in order to illustrate the present invention, rather than in order to limit the scope of the invention.
Experimental technique in following embodiment, if no special instructions, is ordinary method.
In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
Pathogenic bacteria in following enforcement: intestinal bacteria, Pseudomonas aeruginosa, streptococcus aureus and staphylococcus epidermidis Jun Laiyu China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), wherein, intestinal bacteria are numbered 1.3373 CGMCC's, Pseudomonas aeruginosa is numbered 1.2620 CGMCC's, streptococcus aureus is numbered 1.2910 CGMCC's, and staphylococcus epidermidis is numbered 1.4260 CGMCC's.
Embodiment 1, polypeptide A 1-A7's is synthetic
The application changes structure to AR-23 sequence, and that has synthesized antibacterial frog skin peptide AR-23 that title is respectively A1-A7 changes the derivative polypeptide of structure, and as shown in table 1, the aminoacid sequence of A7 is SEQ ID No.1 in sequence table.From aminoacid sequence, can find out, SEQ ID No.2 is comprised of 23 amino-acid residues, is the 1st of SEQ ID No.1 and the 8th 's Arg is sported respectively to the derivative polypeptide that Ala obtains, by this derivative polypeptide called after A3; SEQ ID No.3 is comprised of 23 amino-acid residues, is the Arg of the 8th of SEQ ID No.1 is sported to the derivative polypeptide that Ala obtains, by this derivative polypeptide called after A5; SEQ ID No.4 is comprised of 23 amino-acid residues, is the Arg of the 1st of SEQ ID No.1 is sported to the derivative polypeptide that Ala obtains, by this derivative polypeptide called after A6; SEQ ID No.5 is comprised of 23 amino-acid residues, is the 1st Arg of SEQ ID No.1 sported to Ala and the Lys of the 17th is sported to the derivative polypeptide that Ile obtains, by this derivative polypeptide called after A2; SEQ ID No.6 is comprised of 23 amino-acid residues, is the Lys of the 17th of SEQ ID No.1 is sported to the derivative polypeptide that Ile obtains, by this derivative polypeptide called after A4; SEQ ID No.7 is comprised of 23 amino-acid residues, is the Arg of the 8th of SEQ ID No.1 is sported to Ala and the Lys of the 17th is sported to the derivative polypeptide that Ile obtains, by this derivative polypeptide called after A1.
Chemically synthesized polypeptide A1-A7, their purity all reaches 90% left and right.
Chemosynthesis polypeptide A R-23, melittin and RV-23 in contrast, the sequence of AR-23, melittin and RV-23 is in Table 1, and their purity all reaches 90% left and right.
Table 1, newly with the antibacterial frog skin peptide derivative aminoacid sequence becoming
Embodiment 2, the evaluation of polypeptide A 1-A7 to the molten broken ability to function of red corpuscle
In triplicate, each experimental technique repeating is as follows in experiment:
Freshman red corpuscle washs three times with PBS damping fluid, and with PBS damping fluid, is made into the red blood cell suspension of 1.25% (v/v).With the PBS damping fluid of different volumes, dissolve polypeptide A 7, A1, A2, A3, A4, A5 and the A6 that embodiment 1 obtains respectively, be made into polypeptide A 7 solution, A1 solution, A2 solution, A3 solution, A4 solution, A5 solution and the A6 solution of different concns., with AR-23, RV-23 and melittin, test as a comparison meanwhile, be made into AR-23 solution, RV-23 solution and the melittin solution of different concns.Polypeptide A 7 solution, A1 solution, A2 solution, A3 solution, A4 solution, A5 solution, A6 solution, AR-23 solution, RV-23 solution and the melittin solution of different concns are respectively got 40 μ L, join in the above-mentioned red blood cell suspension of 160 μ L, mix, obtain the red blood cell suspension of polypeptide A 7, A1, A2, A3, A4, A5, A6, AR-23, RV-23 and the melittin of following different concns: A7 final concentration is the red blood cell suspension of 100 μ M, 50 μ M, 25 μ M, 12.5 μ M, 6.25 μ M, 3.13 μ M, 1.56 μ M and 0.78 μ M; A1 final concentration is the red blood cell suspension of 100 μ M, 50 μ M, 25 μ M, 12.5 μ M, 6.25 μ M, 3.13 μ M, 1.56 μ M and 0.78 μ M; A2 final concentration is the red blood cell suspension of 100 μ M, 50 μ M, 25 μ M, 12.5 μ M, 6.25 μ M, 3.13 μ M, 1.56 μ M and 0.78 μ M; A3 final concentration is the red blood cell suspension of 100 μ M, 50 μ M, 25 μ M, 12.5 μ M, 6.25 μ M, 3.13 μ M, 1.56 μ M and 0.78 μ M; A4 final concentration is the red blood cell suspension of 100 μ M, 50 μ M, 25 μ M, 12.5 μ M, 6.25 μ M, 3.13 μ M, 1.56 μ M and 0.78 μ M; A5 final concentration is the red blood cell suspension of 100 μ M, 50 μ M, 25 μ M, 12.5 μ M, 6.25 μ M, 3.13 μ M, 1.56 μ M and 0.78 μ M; A6 final concentration is the red blood cell suspension of 100 μ M, 50 μ M, 25 μ M, 12.5 μ M, 6.25 μ M, 3.13 μ M, 1.56 μ M and 0.78 μ M; AR-23 final concentration is the red blood cell suspension of 100 μ M, 50 μ M, 25 μ M, 12.5 μ M, 6.25 μ M, 3.13 μ M, 1.56 μ M and 0.78 μ M; RV-23 final concentration is the red blood cell suspension of 100 μ M, 50 μ M, 25 μ M, 12.5 μ M, 6.25 μ M, 3.13 μ M, 1.56 μ M and 0.78 μ M; Melittin final concentration is the red blood cell suspension of 100 μ M, 50 μ M, 25 μ M, 12.5 μ M, 6.25 μ M, 3.13 μ M, 1.56 μ M and 0.78 μ M.In the red blood cell suspension of polypeptide A 7, A1, A2, A3, A4, A5, A6, AR-23, RV-23 and the melittin of above-mentioned different concns, erythrocytic final concentration is 1% (v/v).Simultaneously, the positive contrast of red blood cell suspension that the TritonX-100 content of take is the TritonX-100 of 1% (v/v) as 0.1% (volume percent), red corpuscle content, with the negative contrast of red blood cell suspension of 1% (v/v) that directly obtain with the red blood cell suspension of PBS damping fluid dilution 1.25% (v/v).
The red blood cell suspension of the polypeptide A of above-mentioned different concns 7, A1, A2, A3, A4, A5, A6, AR-23, RV-23 and melittin and positive control and negative control are hatched 1 hour respectively at 37 ℃, the centrifugal 5min of 800 * g, abandon precipitation, obtain polypeptide A 7, A1, A2, A3, A4, A5, A6, AR-23, RV-23 and the melittin of different concns and the reaction solution of erythrocytic reaction solution and positive control and negative control.Get in above-mentioned reaction solution to 96 orifice plate of 100 μ L, detect the OD of above-mentioned reaction solution
450value, and calculate erythrocytic hemolysis rate.Erythrocytic hemolysis rate calculation formula is: (OD
450 experiments-OD
450 negative controls)/(OD
450 positive controls-OD
450 negative controls) * 100%, gained hemolysis rate is greater than 1% and has been considered as haemolysis, and the erythrocytic hemolysis rate of polypeptide A 7, A1, A2, A3, A4, A5, A6, AR-23, RV-23 and the melittin of different concns is in Table 2 and Fig. 1.
When antibacterial peptide concentration is 12.5 μ M, the hemolysis rate of AR-23 almost reaches 100%, and now the hemolysis rate of polypeptide A 7, A3, A5 and A6 is 0, the hemolysis rate of A1, A2 and A4 is respectively 88.83%, 42.05% and 36.03%, when antibacterial peptide concentration reaches 100 μ M, the hemolysis rate of AR-23 is 99.19%, and the hemolysis rate of polypeptide A 7, A3, A5 and A6 is respectively 7.74%, 28.71%, 18.04% and 22.83%.Presentation of results polypeptide A 7 can reduce it to a certain extent to erythrocytic hemolytic action.
The minimum hemolytic concentration of polypeptide A 7, A1, A2, A3, A4, A5, A6, AR-23, RV-23 and melittin the results are shown in Table 2.
Result demonstration, polypeptide A 7 of the present invention and A1-A6 thereof all have molten broken ability in various degree to red corpuscle, and wherein polypeptide A 7, A3, A5 and A6 are lower to the molten broken ability of red corpuscle, and A1, A2 and A4 are slightly high to the molten broken ability of red corpuscle.Compare AR-23, polypeptide A 7 and A1-A6 thereof all have reduction in various degree to the molten broken ability of red corpuscle, and wherein, polypeptide A 7 and A3 thereof, A5 and A6 reduce particularly remarkable to the molten broken ability of red corpuscle.
Embodiment 3, the minimal inhibitory concentration of polypeptide A 1-A7 to different bacterium
Press micro-dilution method and identify the minimal inhibitory concentration of polypeptide A 1-A7 to different bacterium with 96 orifice plates.In triplicate, the method that at every turn repeats experiment is as follows in experiment:
Intestinal bacteria (Chinese common micro-organisms DSMZ, 1.3373), Pseudomonas aeruginosa (Chinese common micro-organisms DSMZ numbering:, 1.2620), streptococcus aureus (Chinese common micro-organisms DSMZ numbering:, 1.2910) and staphylococcus epidermidis (Chinese common micro-organisms DSMZ numbering:, numbering: 1.4260) streak inoculation is dull and stereotyped to LB respectively, and 37 spend night.Choose respectively single bacterium colony of intestinal bacteria, Pseudomonas aeruginosa, streptococcus aureus and staphylococcus epidermidis in common LB substratum, under 37 degree, 200rpm, cultivate, grow to logarithmic phase to intestinal bacteria, Pseudomonas aeruginosa, streptococcus aureus and staphylococcus epidermidis, with sterilized water, dilute the bacterium liquid to 10 of the logarithmic phase of intestinal bacteria, Pseudomonas aeruginosa, streptococcus aureus and staphylococcus epidermidis
6polypeptide A 7 solution, A1 solution, A2 solution, A3 solution, A4 solution, A5 solution, A6 solution, AR-23 solution, RV-23 solution and the melittin solution of CFU/mL different concns is respectively got 50 μ L in 96 orifice plates, get altogether 4 times, in polypeptide A 7 solution, A1 solution, A2 solution, A3 solution, A4 solution, A5 solution, A6 solution, AR-23 solution, RV-23 solution and the melittin solution of getting for the 1st time, add respectively 50 μ L10
6the Escherichia coli bacteria liquid of CFU/mL adds respectively 50 μ L10 in polypeptide A 7 solution, A1 solution, A2 solution, A3 solution, A4 solution, A5 solution, A6 solution, AR-23 solution, RV-23 solution and the melittin solution of getting for the 2nd time
6the Pseudomonas aeruginosa bacterium liquid of CFU/mL adds respectively 50 μ L10 in polypeptide A 7 solution, A1 solution, A2 solution, A3 solution, A4 solution, A5 solution, A6 solution, AR-23 solution, RV-23 solution and the melittin solution of getting for the 3rd time
6the streptococcus aureus bacterium liquid of CFU/mL, respectively adds 50 μ L10s in polypeptide A 7 solution, A1 solution, A2 solution, A3 solution, A4 solution, A5 solution, A6 solution, AR-23 solution, RV-23 solution and the melittin solution of getting in the 4th
6the staphylococcus epidermidis bacterium liquid of CFU/mL.Obtain following different concns A7, A1, A2, A3, A4, A5, A6, AR-23, the Escherichia coli bacteria liquid of any polypeptide in RV-23 and melittin: 100 μ M, 50 μ M, 25 μ M, 12.5 μ M, 6.25 μ M, 3.13 μ M, 1.56 μ M and 0.78 μ M, following different concns A7, A1, A2, A3, A4, A5, A6, AR-23, the Pseudomonas aeruginosa bacterium liquid of any polypeptide in RV-23 and melittin: 100 μ M, 50 μ M, 25 μ M, 12.5 μ M, 6.25 μ M, 3.13 μ M, 1.56 μ M and 0.78 μ M, following different concns A7, A1, A2, A3, A4, A5, A6, AR-23, the streptococcus aureus bacterium liquid of any polypeptide in RV-23 and melittin: 100 μ M, 50 μ M, 25 μ M, 12.5 μ M, 6.25 μ M, 3.13 μ M, 1.56 μ M and 0.78 μ M, and following different concns A7, A1, A2, A3, A4, A5, A6, AR-23, the staphylococcus epidermidis bacterium liquid of any polypeptide in RV-23 and melittin: 100 μ M, 50 μ M, 25 μ M, 12.5 μ M, 6.25 μ M, 3.13 μ M, 1.56 μ M and 0.78 μ M.
Above-mentioned bacterium liquid is hatched 20 hours in 37 degree, by multi-functional microplate reader, detect its OD600, minimal inhibitory concentration is defined as to the concentration of the polypeptide A 1-A7 of complete bacteria growing inhibiting, when the concentration of polypeptide A 1-A7 is less than this concentration, can not bacteria growing inhibiting.
According to the minimum hemolytic concentration (in Table 3) of polypeptide A 7, A1, A2, A3, A4, A5, A6, AR-23, RV-23 and melittin, with it, minimal inhibitory concentration of intestinal bacteria, Pseudomonas aeruginosa, streptococcus aureus and staphylococcus epidermidis (μ M) is calculated the therapeutic index of polypeptide A 7, A1, A2, A3, A4, A5, A6, AR-23, RV-23 and melittin, therapeutic index=minimum hemolytic concentration/geometric mean, the results are shown in Table 3.
Polypeptide A 7, A1, A2 and A4 have reduction in various degree to Gram-negative bacteria minimal inhibitory concentration compared with natural antibacterial peptide AR-23, A4 has reduction to gram-positive microorganism minimal inhibitory concentration compared with natural antibacterial peptide melittin, A2 has reduction to Gram-negative bacteria minimal inhibitory concentration compared with natural antibacterial peptide AR-23, A1 and A2 have reduction to gram-positive microorganism minimal inhibitory concentration compared with natural antibacterial peptide RV-23, A1, the comprehensive antibacterial situation of A2 and A4 has reduction in various degree compared with natural antibacterial peptide AR-23, the comprehensive antibacterial situation of A2 has reduction in various degree compared with natural antibacterial peptide RV-23.
The therapeutic index of polypeptide A 7 is far away higher than the therapeutic index of AR-23, RV-23 and melittin, and the therapeutic index of A3, A5, A6 and A2 is also all higher than the therapeutic index of AR-23, RV-23 and melittin.The therapeutic index of A4 is higher than the therapeutic index of AR-23 and melittin, and the therapeutic index of A1 is higher than the therapeutic index of AR-23.
The therapeutic index of table 3, polypeptide A 1-A7
Embodiment 4, the polypeptide A 1-A7 detection to the toxic action of cervical cancer cell (Hela)
In triplicate, the method that at every turn repeats experiment is as follows in experiment:
HeLa cell (US mode culture collection warehousing (ATCC)) is cultivated in recovery, by HeLa cell seeding in 96 orifice plates, 5 * 10
3individual/hole, overnight incubation in 37 ℃ of thermostat containers.When cell converges rate and reaches 70-80%, in the enchylema in 96 one of them hole of orifice plate, add 20 μ L PBS damping fluids as positive control, in all the other enchylema of 96 orifice plates, add polypeptide A 7 solution of PBS damping fluid doubling dilution for 20 μ L, A1 solution, A2 solution, A3 solution, A4 solution, A5 solution, A6 solution, AR-23 solution, RV-23 solution or melittin solution, make polypeptide A 7, A1, A2, A3, A4, A5, A6, AR-23, the final concentration of RV-23 and melittin is following concentration respectively: 50 μ M, 25 μ M, 12.5 μ M, 6.25 μ M, 3.13 μ M, 1.56 μ M and 0.78 μ M.Obtain altogether the HeLa cell suspending liquid of polypeptide A 7, A1, A2, A3, A4, A5, A6, AR-23, RV-23 and the melittin of following different concns: polypeptide A 7 final concentrations are the HeLa cell suspending liquid of 50 μ M, 25 μ M, 12.5 μ M, 6.25 μ M, 3.13 μ M, 1.56 μ M and 0.78 μ M; A1 final concentration is the HeLa cell suspending liquid of 50 μ M, 25 μ M, 12.5 μ M, 6.25 μ M, 3.13 μ M, 1.56 μ M and 0.78 μ M; A2 final concentration is the HeLa cell suspending liquid of 50 μ M, 25 μ M, 12.5 μ M, 6.25 μ M, 3.13 μ M, 1.56 μ M and 0.78 μ M; A3 final concentration is the HeLa cell suspending liquid of 50 μ M, 25 μ M, 12.5 μ M, 6.25 μ M, 3.13 μ M, 1.56 μ M and 0.78 μ M; A4 final concentration is the HeLa cell suspending liquid of 50 μ M, 25 μ M, 12.5 μ M, 6.25 μ M, 3.13 μ M, 1.56 μ M and 0.78 μ M; A5 final concentration is the HeLa cell suspending liquid of 50 μ M, 25 μ M, 12.5 μ M, 6.25 μ M, 3.13 μ M, 1.56 μ M and 0.78 μ M; A6 final concentration is the HeLa cell suspending liquid of 50 μ M, 25 μ M, 12.5 μ M, 6.25 μ M, 3.13 μ M, 1.56 μ M and 0.78 μ M; AR-23 final concentration is the HeLa cell suspending liquid of 50 μ M, 25 μ M, 12.5 μ M, 6.25 μ M, 3.13 μ M, 1.56 μ M and 0.78 μ M; RV-23 final concentration is the HeLa cell suspending liquid of 50 μ M, 25 μ M, 12.5 μ M, 6.25 μ M, 3.13 μ M, 1.56 μ M and 0.78 μ M; Melittin final concentration is the HeLa cell suspending liquid of 50 μ M, 25 μ M, 12.5 μ M, 6.25 μ M, 3.13 μ M, 1.56 μ M and 0.78 μ M.
Above-mentioned HeLa cell suspending liquid is placed in to 37 ℃ and hatches 24h, then to every hole, add 20 μ L3-(4, 5-dimethylthiazole-2)-2, (3-(4 for 5-phenylbenzene tetrazole bromine salt, 5 – dimethyl – 2 – thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide, MTT) (sigma company product) (5.0mg/mL), in 37 ℃ of thermostat containers, continue to hatch 4h, hatch after end, carefully discard nutrient solution, in cell, add 150 μ L dimethyl sulfoxide (DMSO) (Dimethyl sulfoxide, DMSO), in 37 ℃, place 10min, then with vibrator, fully mix, obtain reacted HeLa cell suspending liquid.Use the HeLa cell suspending liquid OD after multi-functional microplate reader assaying reaction
490value.
According to the method described above, by 3-(4,5-dimethylthiazole-2)-2, (3-(4 for 5-phenylbenzene tetrazole bromine salt, 5 – dimethyl – 2 – thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide, MTT) replace with PBS, all the other steps are identical, and the result obtaining is as negative control.
HeLa cell survival rate calculation formula is: (OD
490 experiments-OD
490 negative controls)/(OD
490 positive controls-OD
490 negative controls) * 100%, take HeLa cell survival rate as 50% (IC
50) standard as judgement polypeptide A 1-A7 to the toxicity size of Hela cell, experimental result is in Table 4 and Fig. 2.
Experimental result demonstration, Hela cell survival rate is 50% (IC
50) time, the concentration of polypeptide A 7, A2, A3, A4, A5 and A6 all increases compared with natural antibacterial peptide AR-23, especially the concentration of polypeptide A 7, A3, A5 and A6 not only promotes more compared with natural antibacterial peptide AR-23, and in natural antibacterial peptide RV-23 and melittin, also have greatly improved, illustrate that polypeptide A 7, A3, A5 and A6 reduce the virulence of Hela cell.
Table 4, Hela cell survival rate are 50% (IC
50) time the concentration (μ M) of polypeptide A 1-A7
| Polypeptide | AR-23 | A1 | A2 | A3 | A4 | A5 | A6 | A7 | RV-23 | melittin |
| IC 50 | 4.73 | 4.27 | 5.31 | 36.87 | 6.74 | 38.84 | 49.39 | 46.51 | 8.09 | 2.03 |
The binding ability of embodiment 5, polypeptide A 7, AR-23 and RV-23 and intestinal bacteria and streptococcus aureus detects
In triplicate, the method that at every turn repeats experiment is as follows in experiment:
N end by fluorescein TAMRA mark to polypeptide A 7, AR-23 and RV-23.By intestinal bacteria (Chinese common micro-organisms DSMZ, 1.3373) and streptococcus aureus (Chinese common micro-organisms DSMZ numbering:, numbering: 1.2910) cultivate under 37 degree, 200rpm in LB liquid nutrient medium, while arriving intestinal bacteria and staphylococcus aureus growth to logarithmic phase, it is 0.5 that bacterium liquid is diluted to OD600.Get intestinal bacteria after 200 μ L dilutions and streptococcus aureus bacterium liquid in EP pipe, the polypeptide A 7, AR-23 and the RV-23 that add fluorescein TAMRA mark, make its final concentration be 20 μ M, 37 degree are hatched 30min, obtain reacted intestinal bacteria and streptococcus aureus.With twice of the intestinal bacteria after PBS damping fluid washing reaction and streptococcus aureus, according to the excitation wavelength of fluorescein TAMRA and wavelength of transmitted light, in flow cytometer, detect the binding ability (excitation wavelength of fluorescein TAMRA is 560nm, and wavelength of transmitted light is 582nm) of polypeptide A 7, AR-23 and RV-23 and intestinal bacteria and streptococcus aureus.According to the method described above, polypeptide A 7, AR-23 and the RV-23 of fluorescein TAMRA mark are replaced with to PBS damping fluid, other steps are constant, and in contrast, result as shown in Figure 3 for the result obtaining.
Result shows, polypeptide A 7 and the colibacillary combination rate of fluorescein TAMRA mark are 97.32%, the AR-23 of fluorescein TAMRA mark and colibacillary combination rate are 33.73%, and the RV-23 of fluorescein TAMRA mark and colibacillary combination rate are 80.33%; The polypeptide A 7 of fluorescein TAMRA mark is 96.1% with the combination rate of streptococcus aureus, the AR-23 of fluorescein TAMRA mark and the combination rate of streptococcus aureus are 98.47%, and the RV-23 of fluorescein TAMRA mark and the combination rate of streptococcus aureus are 98.85%.Result shows, polypeptide A 7 with colibacillary binding ability compared with cecropin A R-23 and the increase of colibacillary binding ability.
The penetrativity that embodiment 6, polypeptide A 7, AR-23 and RV-23 penetrate HeLa cell detects
In triplicate, the method that at every turn repeats experiment is as follows in experiment:
TAMRA is marked in polypeptide A 7, AR-23 and RV-23.Hela cell (US mode culture collection warehousing (ATCC)) is planted in 8 vestibule chamber culture dish to every hole 10
4individual cell, cultivated after 24 hours, and polypeptide A 7, AR-23 and RV-23 to adding respectively TAMRA mark in bacterium liquid, make its final concentration be 6 μ M, continued to cultivate 30min, obtained the Hela cell after polypeptide A 7, AR-23 and the RV-23 with TAMRA mark.Twice of PBS damping fluid washing for above-mentioned Hela cell, to the DAPI dye liquor 100 μ L that add 300nM in the Hela cell with after PBS damping fluid washing, after reaction 1-2min, sucking-off dye liquor, and wash Hela cell one time with PBS damping fluid, by the fixing 30min of 2% paraformaldehyde for the Hela cell after washing, then under laser confocal microscope, observe.According to the method described above, the polypeptide A of TAMRA mark 7, AR-23 and RV-23 are replaced with to PBS damping fluid, other steps are constant, and in contrast, result as shown in Figure 4 for the result obtaining.
Result shows, when the cecropin A R-23 of TAMRA mark and RV-23 born of the same parents' extracellular concentration are 6 μ M, the two all can enter Hela cell (red fluorescence), and have no red fluorescence in the Hela cell of polypeptide A 7 and contrast, explanation does not enter Hela cell when polypeptide A 7 concentration are 6 μ M, and the toxicity of 7 pairs of Hela cells of polypeptide A is less.
Claims (9)
1. polypeptide, for aminoacid sequence is the polypeptide A 7 of SEQ ID No.1.
2. the derivative polypeptide of polypeptide described in claim 1, the derivative polypeptide of its aminoacid sequence for the 1st of SEQ ID No.1, the 8th and the 17th amino acids residue are obtained through the replacement of one or two amino-acid residue and/or disappearance and/or interpolation.
3. derivative polypeptide according to claim 2, is characterized in that: the derivative that described derivative polypeptide obtains for polypeptide described in claim 1 being carried out in these three kinds of following M1-M3 two kinds or a kind of sudden change:
M1, the 1st amino acids residue of SEQ ID No.1 is sported to Ala, keep the constant polypeptide obtaining of other amino-acid residue of SEQ ID No.1;
M2, the 8th amino acids residue of SEQ ID No.1 is sported to Ala, keep the constant polypeptide obtaining of other amino-acid residue of SEQ ID No.1;
M3, the 17th amino acids residue of SEQ ID No.1 is sported to Ile, keep the constant polypeptide obtaining of other amino-acid residue of SEQ ID No.1.
4. according to the derivative polypeptide described in claim 2 or 3, it is characterized in that: described derivative polypeptide is following C1)-C6) in any:
C1) aminoacid sequence is the derivative polypeptide of SEQ ID No.2;
C2) aminoacid sequence is the derivative polypeptide of SEQ ID No.3;
C3) aminoacid sequence is the derivative polypeptide of SEQ ID No.4;
C4) aminoacid sequence is the derivative polypeptide of SEQ ID No.5;
C5) aminoacid sequence is the derivative polypeptide of SEQ ID No.6;
C6) aminoacid sequence is the derivative polypeptide of SEQ ID No.7.
To polypeptide described in claim 1 or with claim 2-4 in the relevant biomaterial of arbitrary described derivative polypeptide, be following B1) to B16) in any:
B1) nucleic acid molecule of arbitrary described derivative polypeptide in polypeptide or claim 2-4 described in coding claim 1;
B2) contain B1) expression cassette of described nucleic acid molecule;
B3) contain B1) recombinant vectors of described nucleic acid molecule;
B4) contain B2) recombinant vectors of described expression cassette;
B5) contain B1) recombinant microorganism of described nucleic acid molecule;
B6) contain B2) recombinant microorganism of described expression cassette;
B7) contain B3) recombinant microorganism of described recombinant vectors;
B8) contain B4) recombinant microorganism of described recombinant vectors;
B9) contain B1) transgenetic animal cell of described nucleic acid molecule system;
B10) contain B2) transgenetic animal cell of described expression cassette system;
B11) contain B3) transgenetic animal cell of described recombinant vectors system;
B12) contain B4) transgenetic animal cell of described recombinant vectors system;
B13) contain B1) transgenic plant cells of described nucleic acid molecule system;
B14) contain B2) transgenic plant cells of described expression cassette system;
B15) contain B3) transgenic plant cells of described recombinant vectors system;
B16) contain B4) transgenic plant cells of described recombinant vectors system.
6. the application of biomaterial in preparing antibacterial peptide described in arbitrary described derivative polypeptide or claim 5 in polypeptide or claim 2-4 described in claim 1.
7. application according to claim 6, is characterized in that: the pathogenic bacteria that described antibacterial peptide suppresses is bacterium.
Described in claim 1 in polypeptide or claim 2-4 described in arbitrary described derivative polypeptide or claim 5 biomaterial in preparation, prevent and/or treat the medicine of bacterial disease or the application in vaccine.
9. according to arbitrary described application in claim 7-8, it is characterized in that: described bacterium is gram-positive microorganism or Gram-negative bacteria.
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| CN102206264A (en) * | 2011-04-28 | 2011-10-05 | 中国科学院成都生物研究所 | O.jingdongensis sp. antibacterial peptide and preparation method and use thereof |
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| CN102206264A (en) * | 2011-04-28 | 2011-10-05 | 中国科学院成都生物研究所 | O.jingdongensis sp. antibacterial peptide and preparation method and use thereof |
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| EDIT URBÁN ET AL，: "Activities of four frog skin-derived antimicrobial peptides (temporin-1DRa, temporin-1Va and the melittin-related peptides AR-23 and RV-23) against anaerobic bacteria", 《INTERNATIONAL JOURNAL OF ANTIMICROBIAL AGENTS》 * |
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| CN106632647B (en) * | 2017-02-10 | 2020-05-05 | 中国人民解放军军事医学科学院野战输血研究所 | Polypeptide modified by antibacterial peptide and application thereof in cell transfection |
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