CN100485038C - Miniaturized Staphylococcus aureus polypeptide of against drug-resistance and its uses and preparation method - Google Patents
Miniaturized Staphylococcus aureus polypeptide of against drug-resistance and its uses and preparation method Download PDFInfo
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- CN100485038C CN100485038C CNB2005100216613A CN200510021661A CN100485038C CN 100485038 C CN100485038 C CN 100485038C CN B2005100216613 A CNB2005100216613 A CN B2005100216613A CN 200510021661 A CN200510021661 A CN 200510021661A CN 100485038 C CN100485038 C CN 100485038C
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Abstract
The invention discloses a midget anti drug-tolerance staphylococcus aureus polypeptide comprised of passage structure domain to form ion channel colicine and staphylococcus aureu pheromone, as well as opposite coded nucleotide sequence and recombination plasmid contained said sequence. Wherein, the plupeptide can not induce bacteria to generate trsditional drug-tolerance by the mechnism that it forms directly ion channel on cell film of target cell to kill bacteria. This invention has more single antimicrobial spectrum without effect to Gram-negative bacteria of escherichia coli, shows at least 500 times effect to pheneazonecillin, and can be used as synergist to beta-lactam antibiotics.
Description
Technical field
The present invention relates to a kind of gene, recombinant plasmid, polypeptide and application thereof and preparation method of Miniaturized Staphylococcus aureus polypeptide of against drug resistance of reorganization.
Background technology
Infectation of bacteria is human life and healthy main threat, since sulfanilamide (SN) and penicillin appearance, the antibiotic that the mankind invent successively is mainly with synthetic by suppressing bacterial cell wall, and nucleic acid of inhibition or interference bacterium and proteinic metabolism and route of synthesis reach antibiotic purpose.Yet these antibiotic approach lure that easily bacterium undergos mutation and produce antibiotic resistance into.Therefore people are being devoted to the antibiotic of development of new always.
Directly forming ionic channel and cause bacterium death on bacterial membrane is one of more promising antibiotic exploitation direction.Constructed the antibacterial engineering polypeptide of artificial combination according to this design concept contriver, this invention patent applied for, and obtain Patent Office of State Intellectual Property Office and authorize (patent No. is ZL01128836.1).Yet the anti-Staphylococcus aureus polypeptide in the above-mentioned patent working example still has the some shortcomings part on 26S Proteasome Structure and Function: the N-terminal signal structure of colicin Ia territory still is retained in this antimicrobial polypeptide, cause (1) antimicrobial polypeptide to have broad-spectrum antibacterial action like this, both anti-streptococcus aureus, Chinese People's Anti-Japanese Military and Political College enterobacteria class Gram-negative bacteria again; (2) greatly then immunogenicity just might be bigger for polypeptide molecular weight, and is unfavorable to drug safety.
Summary of the invention
One object of the present invention is to provide a kind of gene, recombinant plasmid, polypeptide of novel reorganization Miniaturized Staphylococcus aureus polypeptide of against drug resistance; This peptide species can be special kill the resistance streptococcus aureus and can not injure the human normal cell.Compare with the disclosed artificial combination antibacterial engineering polypeptide of ZL01128836.1, antimicrobial spectrum is more single-minded, and it only kills streptococcus aureus, and does not kill the coliform bacteria Gram-negative bacteria, and fungicidal effectiveness is stronger.This polypeptide itself is again the false mediator of streptococcus aureus density adjusting and controlling growth system (Quorum-sensing regulating system), Signal Regulation-gene expression system of streptococcus aureus capable of blocking, thus the secretion of resistance staphylotoxin and beta-lactamase can effectively be reduced.A further object of the present invention provides the preparation method of above-mentioned reorganization Miniaturized Staphylococcus aureus polypeptide of against drug resistance.
Technical scheme of the present invention is as follows:
The gene of coding streptococcus aureus pheromone Agr D1 is operably connected with colicin channel architecture domain gene, thus the nucleotide sequence of acquisition express recombinant Miniaturized Staphylococcus aureus polypeptide of against drug resistance.The gene of coding streptococcus aureus pheromone Agr D1 is the described nucleotide sequence of SEQ ID NO.2 in the sequence table.The optional self energy in colicin channel architecture territory forms the colicin El of ionic channel, Ia, Ib, A, B and N, in a preferred embodiment of the invention, the gene with above-mentioned streptococcus aureus pheromone Agr D1 forms the described nucleotide sequence as SEQ ID NO.4 in the sequence table (colicin Ia channel architecture territory-streptococcus aureus pheromone Agr D1) with the carboxyl end groups of colicin Ia channel architecture territory (SEQ ID NO.1) because of being connected.In another preferred embodiment of the present invention, the gene of above-mentioned streptococcus aureus pheromone Agr D1 is connected with the aminoterminal gene in colicin Ia channel architecture territory (SEQID NO.1) and forms described nucleotide sequence as SEQ ID NO.6 in the sequence table (streptococcus aureus pheromone Agr D1-colicin Ia channel architecture territory).
Recombinant plasmid of the present invention is that carboxyl terminal or aminoterminal that aforesaid nucleotide sequence of encoding streptococcus aureus pheromone Agr D1 inserts colicin channel architecture territory through double-stranded oligonucleotide point mutation technology are formed recombinant plasmid of the present invention.If select colicin Ia for use, after the 626th amino acids of the nucleotide sequence of the streptococcus aureus pheromone of then will encode Agr D1 insertion Ia channel architecture domain gene before (carboxyl terminal) or the 451st amino acids (aminoterminal) and form all recombinant plasmids of the present invention.The original commercial plasmid pET-15b of construction recombination plasmid comes from Novagen company, colicin Ia channel architecture territory and corresponding Immunity protein gene have been loaded therein by the contriver, at streptococcus aureus pheromone Agr D1 gene design several to primer, its sequence is seen embodiment 1 and embodiment 2.Utilize double-stranded oligonucleotide point mutation technology, obtain recombinant plasmid of the present invention according to the medicine-chest operation of Strategene company.
The preparation method of Miniaturized Staphylococcus aureus polypeptide of against drug resistance of the present invention is that the above-mentioned recombinant plasmid that will obtain is transfected in the colibacillus engineering and obtains to produce the engineering bacteria cell of reorganization antimicrobial polypeptide, promptly obtains Staphylococcus aureus polypeptide of against drug resistance of the present invention with the CM column separating purification.
The present invention has proved that by experiment described Staphylococcus aureus polypeptide of against drug resistance can be to the secretion of overriding resistance infection of staphylococcus aureus and inhibition streptococcus aureus beta-lactamase, both can make separately to be used for killing existing antibiotic is produced chemical sproof resistant organism, can be used as the toughener drug combination of β-lactam antibiotics such as penicillins and cynnematin again.So just can or prevent the resistance infection of staphylococcus aureus that a kind of new drug is provided for treatment, for β-lactam antibiotic provides a kind of brand-new synergistic agent to the beta-lactamase that antimicrobial agent produces, provide a kind of brand-new inhibitor for reducing bacteriotoxic toxic action clinically.Can make the pharmaceutical composition that is suitable for clinical use by polypeptide of the present invention is added pharmaceutically receivable carrier or vehicle or optional additional components.
Staphylococcus aureus polypeptide of against drug resistance of the present invention is compared to the advantage of traditional antibiotic, it can not induce bacterium to produce traditional resistance, its mechanism is that recombinant polypeptide of the present invention is killing bacteria by directly forming ionic channel on the after birth of target bacteria, the ionic channel that recombinant polypeptide of the present invention forms cause leak and cause bacterium death this than short duration in, it is damaged that bacterium is difficult to utilize mutator gene one protein expression mode to repair the geometry that the antimicrobial polypeptide ionic channel causes on its cytolemma.Compare with the disclosed artificial combination antibacterial engineering polypeptide of ZL01128836.1, the antimicrobial spectrum of Staphylococcus aureus polypeptide of against drug resistance of the present invention is more single-minded, only kill streptococcus aureus, and do not kill the coliform bacteria Gram-negative bacteria, and fungicidal effectiveness is stronger, and Staphylococcus aureus polypeptide of against drug resistance is 500 times of Oxazacillin result of treatment at least to the result of treatment of overriding resistance infection of staphylococcus aureus in an embodiment of the present invention.Staphylococcus aureus polypeptide of against drug resistance of the present invention has dual-use function: not only sterilization but also suppress the bacteriotoxin secretion, therapeutic value clinically will beat by miles existing antibiotic.While has similar active artificial combination antibacterial engineering polypeptide (MW70,000) and compares and will weaken greatly because molecular weight diminishes, and its immunogenicity and ZL01128836.1 patent are disclosed, thereby has increased the security of drug use.
Description of drawings
Fig. 1 is the structural representation that contains the recombinant plasmid pCHCSACOM1 of streptococcus aureus pheromone Agr D1 gene and colicin Ia channel architecture domain gene, and wherein streptococcus aureus pheromone Agr D1 is connected on the 626th amino acids of colicin Ia.
Fig. 2 is the structural representation that contains the recombinant plasmid pCHCSACOM2 of streptococcus aureus pheromone Agr D1 gene and colicin Ia channel architecture domain gene, and wherein streptococcus aureus pheromone Agr D1 is connected on the 451st amino acids of colicin Ia.
Fig. 3 illustrates the structure of the antimicrobial polypeptide that comprises 184 amino-acid residues and the Miniaturized Staphylococcus aureus polypeptide of against drug resistance 15%SDS-PAGE electrophoresis result of preparation, A. the antimicrobial polypeptide structural representation that contains 184 amino-acid residues, a. the colicin Ia channel architecture territory of containing 175 amino-acid residues (D451-I626), b. contains the streptococcus aureus pheromone Agr D1 of 8 amino-acid residues; B.15%SDS-PAGE electrophoresis result figure, a molecular weight marker (standard protein); B. wild-type colicin Ia, molecular weight about 70,000; C. Zhi Bei Miniaturized Staphylococcus aureus polypeptide of against drug resistance COM1, molecular weight about 26,000; D. Zhi Bei Miniaturized Staphylococcus aureus polypeptide of against drug resistance COM2, molecular weight about 26,000.
Fig. 4 is the inhibition experimental result picture to the streptococcus aureus of penicillin sensitivity, this figure shows that the growth of the responsive streptococcus aureus of penicillin can only be suppressed by Oxazacillin, the described artificial combination antibacterial engineering polypeptide of ZL01128836.1 (MW70,000) and Miniaturized Staphylococcus aureus polypeptide of against drug resistance of the present invention.Control among the figure, contrast; Oxa, Oxazacillin; Ia WT, wild-type colicin Ia; Ph-SA, the described artificial combination antibacterial engineering polypeptide of ZL01128836.1 (MW70,000); COM1, colicin Ia channel architecture territory carboxyl terminal has connected the anti-Staphylococcus aureus polypeptide of streptococcus aureus pheromone Agr D1; COM2, colicin Ia channel architecture territory aminoterminal has connected the anti-Staphylococcus aureus polypeptide of streptococcus aureus pheromone Agr D1.The drug dose of each experimental group: Oxazacillin, 9 μ g/ml are roughly equal to 22nM/ml; Wild-type colicin Ia, 7 μ g/ml are roughly equal to 0.1nM/ml; Ph-SA, 7 μ g/ml are roughly equal to 0.1nM/ml; COM1 and COM2 are 2 μ g/ml, are roughly equal to 0.1nM/ml.Ordinate zou is the A595nm optical density value, and X-coordinate is a bacterial growth hour.
Fig. 5 is the inhibition experimental result to methicillin-resistant staphylococcus aureus, and this figure shows that the growth of methicillin-resistant staphylococcus aureus can only effectively suppress by Miniaturized Staphylococcus aureus polypeptide of against drug resistance described by the present invention.Control among the figure, contrast; Oxa, Oxazacillin; Ia WT, wild-type colicin Ia; Ph-SA, the described artificial combination antibacterial engineering polypeptide of ZL01128836.1 (MW70,000); COM1, colicin Ia channel architecture territory carboxyl terminal has connected the anti-Staphylococcus aureus polypeptide of streptococcus aureus pheromone Agr D1; COM2, colicin Ia channel architecture territory aminoterminal has connected the anti-Staphylococcus aureus polypeptide of streptococcus aureus pheromone Agr D1.The drug dose of each experimental group: Oxazacillin, 9 μ g/ml are roughly equal to 22nM/ml; Wild-type colicin Ia, 7 μ g/ml are roughly equal to 0.1nM/ml; Ph-SA, 7 μ g/ml are roughly equal to 0.1nM/ml; COM1 and COM2 are 2 μ g/ml, are roughly equal to 0.1nM/ml.Ordinate zou is the A595nm optical density value, and X-coordinate is a bacterial growth hour.
Fig. 6 is to colibacillary inhibition experimental result, shows that Miniaturized Staphylococcus aureus polypeptide of against drug resistance of the present invention can not suppress colibacillary growth.Control among the figure, contrast; Ia WT, wild-type colicin Ia; Ph-SA, the described artificial combination antibacterial engineering polypeptide of ZL01128836.1 (MW70,000); COM1, colicin Ia channel architecture territory carboxyl terminal has connected the anti-Staphylococcus aureus polypeptide of streptococcus aureus pheromone Agr D1.The drug dose of each experimental group: wild-type colicin Ia, 7 μ g/ml are roughly equal to 0.1nM/ml; Ph-SA, 7 μ g/ml are roughly equal to 0.1nM/ml; COM1 2 μ g/ml are roughly equal to 0.1nM/ml.Ordinate zou is the A595nm optical density value, and X-coordinate is a bacterial growth hour.
Fig. 7 suppresses experimental result picture to resistance streptococcus aureus beta-lactamase excretory, Control among the figure, contrast; PEN, penicillin; COM1, colicin Ia channel architecture territory carboxyl terminal has connected the anti-Staphylococcus aureus polypeptide of streptococcus aureus pheromone Agr D1; PEN+COM1, penicillin+Miniaturized Staphylococcus aureus polypeptide of against drug resistance; Each group adds dosage and sees that embodiment illustrates.The result shows that Miniaturized Staphylococcus aureus polypeptide of against drug resistance of the present invention can suppress the secretion of resistance streptococcus aureus beta-lactamase.Fig. 7 A ordinate zou is the A595nm optical density value, and Fig. 7 B ordinate zou is the Nitrocefin reading value, and X-coordinate is bacterial growth hour.
Fig. 8 suppresses experimental result picture to resistance streptococcus aureus δ-hemolysin excretory, Control among the figure, contrast; PEN, Benzylpenicillin sodium; COM1, colicin Ia channel architecture territory carboxyl terminal has connected the anti-Staphylococcus aureus polypeptide of streptococcus aureus pheromone Agr D1.Each group adds dosage and sees that embodiment illustrates.The result shows that Miniaturized Staphylococcus aureus polypeptide of against drug resistance of the present invention can suppress resistance streptococcus aureus δ-hemolysin secretion.Ordinate zou is the HPLC reading value, and X-coordinate is a bacterial growth hour.
Fig. 9 is to the synergism experiment of β-Lactam antibiotics endogenous protective effect figure as a result; a is a control group among the figure; b is the Benzylpenicillin sodium group, and c is half amount Benzylpenicillin sodium and partly measures anti-Staphylococcus aureus polypeptide group of the present invention that d is an anti-Staphylococcus aureus polypeptide group of the present invention.Each group adds dosage and asks for an interview the embodiment explanation.The result shows that half amount Miniaturized Staphylococcus aureus polypeptide of against drug resistance can effectively strengthen the endogenous protective effect of penicillin to antimicrobial agent.Ordinate zou is a surviving animals numerical value, X-coordinate be the survival time (my god).
Figure 10 is the mouse liver after Miniaturized Staphylococcus aureus polypeptide of against drug resistance of the present invention was handled on the 30th, kidney, the tissue slice of spleen; The a hepatic tissue section, the section of b nephridial tissue, c spleen tissue slice, HE dyeing, 100x.
Embodiment
The structure of the plasmid of embodiment 1 expression Miniaturized Staphylococcus aureus polypeptide of against drug resistance and the preparation of reorganization Miniaturized Staphylococcus aureus polypeptide of against drug resistance
Original plasmid is the commercial plasmid of pET-15b (the plasmid size 6.3kb that has loaded colicin Ia channel architecture territory and Immunity protein gene, available from Novagen company, said gene is loaded by Huaxi Hospital Attached to Sichuan Univ transplantation immunity laboratory), through double-stranded oligonucleotide point mutation technology (QuickChange
TMKit, Strategene company) will encode after the gene (SEQ ID NO.2 in the sequence table) of streptococcus aureus pheromone Agr D1 is inserted into the I626 site of colicin Ia channel architecture domain gene, prepare a kind of recombinant plasmid pCHCSACOM1 (as shown in Figure 1) of antimicrobial polypeptide.Recombinant plasmid is transfected in the E.coli TG1 engineering bacteria and prepares polypeptide, obtains the described Miniaturized Staphylococcus aureus polypeptide of against drug resistance of SEQ ID NO.5 in the sequence table.Continue after embodiment in be called " antimicrobial polypeptide 1 (COM1) ".
The sudden change program is undertaken by Strategene QuickChange Site-Directed Mutagenesis Kit (catalog#200518) medicine-chest handbook:
1, prepares the point mutation reactant
5ul 10 x buffer
2ul (10ng) wild-type colicin Ia plasmid
1.25ul (125ng) She Ji 5 '-3 ' oligonucleotide primer
1.25ul (125ng) She Ji 3 '-5 ' oligonucleotide primer
1ul dNTP
Distilled water 50ul
1ul pfu
(except that plasmid, primer and distilled water, being reagent that medicine-chest is equipped with)
2, carry out pcr amplification, amplification condition: 95 ℃ of sex change, 35 seconds, anneal 53 ℃, 70 seconds, extend 68 ℃, totally 20 circulations in 17 fens;
3, add (37 ℃, 1 hour) behind the Dpn1 restriction endonuclease 1ul digestion mother body D NA chain, get 1ul reactant and XL1-Blue competent cell 50ul ice and incubated 30 minutes, 42 ℃ of capable thermal shockings 45 seconds, were inserted in the ice 2 minutes again;
4, add NZY and train 220rpm behind the basic 0.5ml, 37 ℃ were shaken bacterium after 1 hour, got 50-100ul reactant bed board (LB training base adds 1% agar and adds the 50ug/ml penbritin, and 37 ℃ are spent the night);
5, choose bacterium after 18 hours, follow Qiagene, companies such as Gibco various commercial extract the plasmid medicine-chests extract plasmids all can, order-checking is determined to suddenly change successfully;
6, the TG1 engineering bacteria competent cell 50ul ice of plasmid 50ng and preparation was incubated 30 fens, 42 ℃, thermal shocking in 90 seconds, get 50-100ul reactant adding LB and train basic 0.5ml, 220rpm, 37 ℃ are shaken bacterium bed board after 1 hour (LB training base adds 1% agar and adds the 50ug/ml penbritin) and hatch picking colony after 18 hours for 37 ℃;
7, increase bacterium in a large number, 8-16 rises FB training base, 250rpm, 37 ℃, 6-8 hour; The centrifugation thalline, 4 ℃, 6000g, 20 minutes, get 50mM Borate, 2mM EDTA, 2mM DTT, pH9.0,4 ℃ of 50-80ml suspension thalline, row ultrasonication (4 ℃, 400W, 2 minutes) behind the adding 0.2M PMSF 250ul, high speed centrifugation precipitates broken thalline (4 ℃, 75000g, 1.5 hours), get supernatant and add the Vetstrep 5,000,000 deposit D NA of unit, and behind the high speed centrifugation (4 ℃, 30000g, 10 minutes) get supernatant and pack the dialysis tubing of molecular weight 15,000 in 50mM Borate, 2mM EDTA, 2mM DTT, pH9.0 is after 4 ℃ of solution dialysed overnight, (4 ℃, 30000g, 10 minutes) are splined on CM post (Amersham Biosciences) with supernatant behind the high speed centrifugation, through 0.2M NaCl, 50mM Borate, 2mM EDTA, 2mM DTT, pH9.0 can obtain the antimicrobial polypeptide of recombinating behind 4 ℃ of solution linear gradient elutions, through 10mM sodium phosphate, 0.2MNaCl, 2mM EDTA, pH7.4, standby after 4 ℃ of dialysis.
Be listed as the designed concrete double-stranded oligonucleotide sequence of streptococcus aureus pheromone Agr D1 gene of preparation pCHCSACOM1 plasmid below:
pCHCSACOM1
1、5’-3’
gcg aat aag ttc tgg ggt att TAT TCC ACC TGT GAT TTT ATA ATG taa ata aaa tat aag aca ggc
3’-5’
gcc tgt ctt ata ttt tat tta CAT TAT AAAATC ACA GGT GGAATA aat acc cca gaa ctt att cgc
The structure of the plasmid of embodiment 2 expression Miniaturized Staphylococcus aureus polypeptide of against drug resistance and the preparation of reorganization Miniaturized Staphylococcus aureus polypeptide of against drug resistance
Original plasmid is the commercial plasmid of pET-15b (the plasmid size 6.3kb that has loaded colicin Ia channel architecture territory and Immunity protein gene, available from Novagen company, said gene is loaded by Huaxi Hospital Attached to Sichuan Univ transplantation immunity laboratory), through double-stranded oligonucleotide point mutation technology (QuickChange
TMKit, Strategene company) will encode before the gene (the described nucleotide sequence of SEQ ID NO.2 in the sequence table) of streptococcus aureus pheromone Agr D1 is inserted into the D451 site of colicin Ia channel architecture domain gene, prepare a kind of recombinant plasmid pCHCSACOM2 (as shown in Figure 2) of antimicrobial polypeptide.Recombinant plasmid is transfected in the E.coli TG1 engineering bacteria and prepares polypeptide, obtains the described Miniaturized Staphylococcus aureus polypeptide of against drug resistance of SEQ ID NO7 in the sequence table, continue after embodiment in be called " antimicrobial polypeptide 2 (COM2) ".
The sudden change program is undertaken by Strategene QuickChange Site-Directed Mutagenesis Kit (catalog#200518) medicine-chest handbook:
1, prepares the point mutation reactant
5ul 10 x buffer
2ul (10ng) wild-type colicin Ia plasmid
1.26ul (125ng) She Ji 5 '-3 ' oligonucleotide primer
1.25ul (125ng) She Ji 3 '-5 ' oligonucleotide primer
1ul dNTP
Distilled water 50ul
1ul pfu
(except that plasmid, primer and distilled water, being reagent that medicine-chest is equipped with)
2, carry out pcr amplification, amplification condition: 95 ℃ of sex change, 35 seconds, anneal 53 ℃, 70 seconds, extend 68 ℃, totally 20 circulations in 17 fens;
3, add (37 ℃, 1 hour) behind the Dpnl restriction endonuclease 1ul digestion mother body D NA chain, get 1ul reactant and XL1-Blue competent cell 50ul ice and incubated 30 minutes, 42 ℃ of capable thermal shockings 45 seconds, were inserted in the ice 2 minutes again;
4, add NZY and train 220rpm behind the basic 0.5ml, 37 ℃ were shaken bacterium after 1 hour, got 50-100ul reactant bed board (LB training base adds 1% agar and adds the 50ug/ml penbritin, and 37 ℃ are spent the night);
5, choose bacterium after 18 hours, follow Qiagene, companies such as Gibco various commercial extract the plasmid medicine-chests extract plasmids all can, order-checking is determined to suddenly change successfully;
6, the TG1 engineering bacteria competent cell 50ul ice of plasmid 50ng and preparation was incubated 30 fens, 42 ℃, thermal shocking in 90 seconds, get 50-100ul reactant adding LB and train basic 0.5ml, 220rpm, 37 ℃ are shaken bacterium bed board after 1 hour (LB training base adds 1% agar and adds the 50ug/ml penbritin) and hatch picking colony after 18 hours for 37 ℃;
7, increase bacterium in a large number, 8-16 rises FB training base, 250rpm, 37 ℃, 6-8 hour; The centrifugation thalline, 4 ℃, 6000g, 20 minutes, get 50mM Borate, 2mM EDTA, 2mM DTT, pH9.0,4 ℃ of 50-80ml suspension thalline, row ultrasonication (4 ℃, 400W, 2 minutes) behind the adding 0.2M PMSF 250ul, high speed centrifugation precipitates broken thalline (4 ℃, 75000g, 1.5 hours), get supernatant and add the Vetstrep 5,000,000 deposit D NA of unit, and behind the high speed centrifugation (4 ℃, 30000g, 10 minutes) get supernatant and pack the dialysis tubing of molecular weight 15,000 in 50mM Borate, 2mM EDTA, 2mM DTT, pH9.0 is after 4 ℃ of solution dialysed overnight, (4 ℃, 30000g, 10 minutes) are splined on CM post (Amersham Biosciences) with supernatant behind the high speed centrifugation, through 0.2M NaCl, 50mM Borate, 2mM EDTA, 2mM DTT, pH9.0 can obtain the antimicrobial polypeptide of recombinating behind 4 ℃ of solution linear gradient elutions, through 10mM sodium phosphate, 0.2MNaCl, 2mM EDTA, pH7.4, standby after 4 ℃ of dialysis.
The double-stranded oligonucleotide sequence of the streptococcus aureus pheromone Agr D1 gene of preparation pCHCSACOM2 plasmid is pressed the method design of embodiment 1.
The body outer suppressioning test of the responsive streptococcus aureus of 3 pairs of penicillin of embodiment
Bacterium is the USS bacterial strain, the responsive streptococcus aureus of ATCC 25923 penicillin, bacterium liquid 5 microlitres (10
8CFU/ml level bacterium amount) adds 1% Tryptones, 1% NaCl, 0.5% yeast, 0.5% glucose, 1%HK
2PO
4Nutrient solution 10ml in, prepare 6 groups altogether.First group adds 0.2M NaCl+10mM PBS damping fluid (antimicrobial polypeptide and the control drug amount of liquid that add in amount and the experimental group are identical) in contrast; The 2nd group adds Oxazacillin, and 9 μ g/ml are roughly equal to 22nM/ml; The 3rd group adds wild-type colicin Ia7 μ g/ml, is roughly equal to 0.1nM/ml (it is 0.2MNaCl+10mM PBS damping fluid that colicin is preserved liquid); The 4th group adds the disclosed antibacterial engineering polypeptide Ph-SA of ZL01128836.1, and 7 μ g/ml are roughly equal to 0.1nM/ml (preserving liquid is 0.2M NaCl+10mM PBS damping fluid); The 5th group adds the Miniaturized Staphylococcus aureus polypeptide of against drug resistance COM1 (embodiment 1 preparation) that colicin Ia channel architecture territory carboxyl terminal has connected streptococcus aureus pheromone Agr D1,2 μ g/ml, be roughly equal to 0.1nM/ml (preserving liquid is 0.2M NaCl+10mM PBS damping fluid), the 6th group adds the Miniaturized Staphylococcus aureus polypeptide of against drug resistance COM2 (embodiment 2 preparations) that colicin channel architecture territory aminoterminal has connected streptococcus aureus pheromone Agr D1,2 μ g/ml are roughly equal to 0.1nM/ml (preserving liquid is 0.2M NaCI+10mM PBS damping fluid).
The above-mentioned liquid of respectively organizing places the 100ml Erlenmeyer flask, 200rpm, 37 ℃ of growths, the 100ul that per hour samples adds in the 96 hole enzyme plates through spectrophotometer (A595nm) colorimetric bacteria tested growth turbidity, the growth curve of bacteria that draws comes the inhibitory effect of comparison antimicrobial polypeptide, the result shows that the growth of the responsive streptococcus aureus of penicillin can only be suppressed by Oxazacillin, ZL 01128836.1 disclosed antibacterial engineering polypeptide and Miniaturized Staphylococcus aureus polypeptide of against drug resistance of the present invention as shown in Figure 4.
The body outer suppressioning test of 4 pairs of methicillin-resistant staphylococcus aureus of embodiment
Bacterium is the USS bacterial strain, ATCC BAA-42 methicillin-resistant staphylococcus aureus, bacterium liquid 5 microlitres (10
8CFU/ml level bacterium amount) adds 1% Tryptones, 1% NaCl, 0.5% yeast, 0.5% glucose, 1% HK
2PO
4Nutrient solution 10ml in, prepare 6 groups altogether.First group adds 0.2M NaCl+10mM PBS damping fluid (antimicrobial polypeptide and the control drug amount of liquid that add in amount and the experimental group are identical) in contrast; Second group adds Oxazacillin, and 9 μ g/ml are roughly equal to 22nM/ml; The 3rd group adds wild-type colicin Ia 7 μ g/ml, is roughly equal to 0.1nM/ml (it is 0.2MNaCl+10mM PBS damping fluid that colicin is preserved liquid); The 4th group adds ZL 01128836.1 disclosed antibacterial engineering polypeptide Ph-SA, and 7 μ g/ml are roughly equal to 0.1nM/ml (preserving liquid is 0.2M NaCI+10mM PBS damping fluid); The 5th group adds the Miniaturized Staphylococcus aureus polypeptide of against drug resistance COM1 (embodiment 1 preparation) that colicin Ia channel architecture territory carboxyl terminal has connected streptococcus aureus pheromone Agr D1,2 μ g/ml are roughly equal to 0.1nM/ml (preserving liquid is 0.2M NaCl+10mM PBS damping fluid); The 6th group adds the Miniaturized Staphylococcus aureus polypeptide of against drug resistance COM2 (embodiment 2 preparations) that colicin Ia channel architecture territory aminoterminal has connected streptococcus aureus pheromone Agr D1,2 μ g/ml are roughly equal to 0.1nM/ml (preserving liquid is 0.2M NaCl+10mM PBS damping fluid).
The above-mentioned liquid of respectively organizing places the 100ml Erlenmeyer flask, 200rpm, 37 ℃ of growths, the 100ul that per hour samples adds in the 96 hole enzyme plates through spectrophotometer (A595nm) colorimetric bacteria tested growth turbidity, the growth curve of bacteria that draws comes the inhibitory effect of comparison antimicrobial polypeptide, the result as shown in Figure 5, under same concentrations, the disclosed antibacterial engineering polypeptide of wild-type colicin Ia and ZL01128836.1 all can not effectively suppress the growth of methicillin-resistant staphylococcus aureus; Concentration can not effectively suppress the growth of methicillin-resistant staphylococcus aureus greater than the Oxazacillin of 220 times of Miniaturized Staphylococcus aureus polypeptide of against drug resistance; The growth of methicillin-resistant staphylococcus aureus can only be suppressed by COM1 (embodiment 1 preparation) Miniaturized Staphylococcus aureus polypeptide of against drug resistance.
5 pairs of colibacillary antibacterial activity in vitro tests of embodiment
Bacterium is the R361 intestinal bacteria, bacterium liquid 5 microlitres (10
8CFU/ml level bacterium amount) adds among 1% Tryptones, 1% NaCl, the 0.5% zymic LB nutrient solution 10ml, prepare 6 groups altogether.First group add 0.2M NaCl+10mM PBS damping fluid (
The amount withThe pharmaceutical liquid amount that adds in the experimental group is identical) in contrast; Second group adds wild-type colicin Ia7 μ g/ml, is roughly equal to 0.1nM/ml (it is 0.2M NaCl+10mM PBS damping fluid that colicin is preserved liquid); The 3rd group adds ZL 01128836.1 disclosed antibacterial engineering polypeptide Ph-SA, and 7 μ g/ml are roughly equal to 0.1nM/ml (preserving liquid is 0.2M NaCl+10mM PBS damping fluid); The 4th group adds the Miniaturized Staphylococcus aureus polypeptide of against drug resistance COM1 (embodiment 1 preparation) that colicin Ia channel architecture territory carboxyl terminal has connected streptococcus aureus pheromone Agr D1,2 μ g/ml are roughly equal to 0.1nM/ml (preserving liquid is 0.2M NaCl+10mM PBS damping fluid).
The above-mentioned liquid of respectively organizing places the 100ml Erlenmeyer flask, 200rpm, 37 ℃ of growths, the 100ul that per hour samples adds in the 96 hole enzyme plates through spectrophotometer (A595nm) colorimetric bacteria tested growth turbidity, the growth curve of bacteria that draws comes the relatively inhibitory effect of each group, the result shows that colibacillary growth can only be suppressed by wild-type colicin Ia and ZL 01128836.1 disclosed antibacterial engineering polypeptide as shown in Figure 6.And Miniaturized Staphylococcus aureus polypeptide of against drug resistance of the present invention can not suppress colibacillary growth.
The vitro inhibition activity test of 6 pairs of resistance streptococcus aureuses of embodiment beta-lactamase excretory
Bacterium is the USS bacterial strain, and ATCC29213 produces beta-lactamase streptococcus aureus, bacterium liquid 5 microlitres (10
8CFU/ml level bacterium amount) adds 1% Tryptones, 1%NaCl, 0.5% yeast, 0.5% glucose, 1%HK
2PO
4Nutrient solution 10ml in the 100ml Erlenmeyer flask, 200rpm, 37 ℃ grow to optical density value 0.3 (A595nm), prepare 4 groups altogether.First group adds 0.2M NaCl+10mM PBS damping fluid (the pharmaceutical liquid amount that adds in amount and each experimental group is identical) in contrast; Second group adds Benzylpenicillin sodium 10 μ g/ml; The 3rd group adds the Miniaturized Staphylococcus aureus polypeptide of against drug resistance COM1 (embodiment 1 preparation) that colicin Ia channel architecture territory carboxyl terminal has connected streptococcus aureus pheromone Agr D1,5 μ g/ml (preserving liquid is 0.2M NaCl+10mM PBS damping fluid); The 4th group adds Miniaturized Staphylococcus aureus polypeptide of against drug resistance COM1 (embodiment 1 preparation) 2.5 μ g/ml and the Benzylpenicillin sodium 5 μ g/ml (preserving liquid is 0.2M NaCl+10mM PBS damping fluid) that colicin Ia channel architecture territory carboxyl terminal has connected streptococcus aureus pheromone Agr D1.Measured in per 1 hour and organize the optical density value of bacterium at the 595nm place, its beta-lactam enzymic activity of detection by quantitative also compared with the control simultaneously.Enzymic activity detects to getting the centrifugal back PBS (pH7.2 of bacterium liquid, concentration 0.05M) resuspended thalline, hatch in 37 ℃ with staphylococcus lysozyme (lysostaphin) then and obtained enzyme extract in 1 hour, suitably dilution back and 0.01mol/ml Nitrocefin are in 37 ℃ of reactions, read the OD value under the 386nm wavelength, and calculate enzymic activity according to the drop-out value of OD in 5 minutes.The result shows that Miniaturized Staphylococcus aureus polypeptide of against drug resistance of the present invention has suppressed the secretion of streptococcus aureus beta-lactamase effectively as shown in Figure 7; And Benzylpenicillin sodium stimulates streptococcus aureus secretion beta-lactamase.
The vitro inhibition activity test of 7 pairs of resistance streptococcus aureus δ-hemolysin excretory of embodiment
Bacterium is the USS bacterial strain, ATCC BAA-42 methicillin-resistant staphylococcus aureus, bacterium liquid 5 microlitres (10
8CFU/ml level bacterium amount) adds 1% Tryptones, 1% NaCl, 0.5% yeast, 0.5% glucose, 1% HK
2PO
4Nutrient solution 10ml in the 100ml Erlenmeyer flask, 200rpm, 37 ℃ grow to optical density value 0.2 (A595nm), prepare 3 groups altogether.First group adds 0.2M NaCl+10mM PBS damping fluid (antimicrobial polypeptide and the control drug amount of liquid that add in amount and the experimental group are identical) in contrast; Second group adds Benzylpenicillin sodium 5 μ g/ml, is roughly equal to 0.1nM/ml; The 3rd group adds the Miniaturized Staphylococcus aureus polypeptide of against drug resistance COM1 (embodiment 1 preparation) that colicin Ia channel architecture territory carboxyl terminal has connected streptococcus aureus pheromone Agr D1,2 μ g/ml are roughly equal to 0.1nM/ml (preserving liquid is 0.2M NaCl+10mM PBS damping fluid).The back continues to cultivate, respectively at after the dosing 1,2,3,4,5, respectively got 1ml bacterium liquid in 6,7 hours, 19, centrifugal 5 minutes of 000g, supernatant is got 600 μ l filter carrying out HPLC and is analyzed through 0.22 μ m membrane filtration, and mobile phase A is 0.1% trifluoroacetic acid (TFA)/10% second fine (acetonitrile), Mobile phase B is 0.1% TFA/90% acetonitrile, volumetric flow rate 1ml/min is behind the last sample, earlier with 11%B wash-out foreign protein, B with 11%-100% carries out linear gradient elution then, the variance analysis of The data replicate measurement.The result shows that Miniaturized Staphylococcus aureus polypeptide of against drug resistance of the present invention has suppressed the secretion of streptococcus aureus δ-hemolysin effectively as shown in Figure 8.
The antibacterial activity in vitro test of embodiment 8 Miniaturized Staphylococcus aureus polypeptide of against drug resistance
1, medicine
Miniaturized Staphylococcus aureus polypeptide of against drug resistance COM1 (embodiment 1 preparation), cefazolin for injection, benzylpenicillin sodium for injection, oxacillin for inj, vancocin vial, Ph-SA (ZL 01128836.1 disclosed antibacterial engineering polypeptide).
Above sample sterilized water dissolved dilution, medicine final concentration are 256mg/L, 128mg/L, 64mg/L, 32mg/L, 16mg/L, 8mg/L, 4mg/ml, 2mg/L, 1mg/ml, 0.5mg/L, 0.25mg/L, 0.125mg/L, 0.06mg/L, 0.03mg/L, 0.015mg/L.
2, bacterium
The clinical isolates strain: the test bacterial strain uses therefor is 2002 from Sichuan, the Beijing area clinical separation pathogenic bacterium of collecting, all bacterial classifications are being collected isolating unit (clinical laboratory of Huaxi Medical Univ and Medical University Of Chongqing's contagious department Bacteriology Room) all after identifying, use after identify again with the API system in Huaxi Hospital Attached to Sichuan Univ transplantation immunity laboratory again.
Golden yellow staphylococcus 128 strains (74 strains of MRSA bacterial strain, 54 strains of MSSA bacterial strain), staphylococcus epidermidis 39 strains (14 strains of MRSE bacterial strain, 25 strains of MSSE bacterial strain), faecalis 22 strains, intestinal bacteria 20 strains, pseudomonas aeruginosa 15 strains, totally 224 strains.
3, result
| Bacterial strain | Medicine MIC50 MIC90 MIC scope |
| MRSA (74) | COM1 8 16 0.125-32 Cephazolins〉128 256 2-〉256 penicillin〉128〉128 4-256 Oxazacillins〉128〉128 4-256 vancomycins, 0.5 8 0.03-32 Ph-SA, 16 64 0.005-128 |
| MSSA (54) | COM1 0.5 4 0.00125-32 Cephazolins 0.25 0.5 0.025-〉128 penicillin 16〉32 0.0015-128 Oxazacillins, 0.125 0.5 0.0015-8 vancomycin, 0.5 1 0.03-4 Ph-SA, 0.5 8 0.05-32 |
| MRSE (14) | COM1 16 32 0.0125-64 Cephazolins 128〉256 0.25-〉256 penicillin〉128〉128 8-256 Oxazacillins 128〉128 4-〉256 vancomycins, 0.5 1 0.03-16 Ph-SA〉64〉64 0.25-128 |
| MSSE (25) | COM1 48 0.00125-32 Cephazolins 0.25 128 0.0125-〉128 penicillin, 16 128 4-256 Oxazacillins, 0.125 1 0.004-2 vancomycin, 0.5 1 0.00125-8 Ph-SA〉1 32 0.005-64 |
| Faecalis (22) | COM1 32 64 0.25-64 Cephazolins 32 128 2-〉256 penicillin 64〉64 4-256 Oxazacillins 32〉128 4-256 |
| Intestinal bacteria (20) | COM1 64 128 16-128 Cephazolins〉128 256 2-〉256 penicillin〉128〉128 4-256 Oxazacillins〉128〉128 4-256 vancomycins 128〉128 2-128 Ph-SA, 16 64 0.005-128 |
| Pseudomonas aeruginosa (15) | COM1 128〉128 64-256 Cephazolins〉128 256 128-〉256 penicillin〉128〉128〉128 Oxazacillins〉128〉128〉128 vancomycins 128〉128 64-〉128 Ph-SA 8〉64 2-128 |
The MRSA antibacterial activity in vitro compares: Oxazacillin MIC90 measured value is bigger 8 times than Miniaturized Staphylococcus aureus polypeptide of against drug resistance measured value of the present invention, and is bigger 16 times than vancomycin measured value.Miniaturized Staphylococcus aureus polypeptide of against drug resistance molecular weight of the present invention (26,000) is 19 times of vancomycin molecular weight (1,400), 65 times of Oxazacillin molecular weight (400).By marking of same medicine molecule number in the unit volume, Miniaturized Staphylococcus aureus polypeptide of against drug resistance then of the present invention is equivalent to vancomycin 8 times to the MRSA antibacterial activity in vitro to the MRSA antibacterial activity in vitro, be equivalent to Oxazacillin 520 times to the MRSA antibacterial activity in vitro, be several times as much as the disclosed antibacterial engineering polypeptide of ZL01128836.1 (molecular weight 70,000) to the MRSA antibacterial activity in vitro.
Protection test in the animal body of embodiment 9 Miniaturized Staphylococcus aureus polypeptide of against drug resistance
1, experiment material
(1) medicine
Miniaturized Staphylococcus aureus polypeptide of against drug resistance COM1 (embodiment 1 preparation), cefazolin for injection, benzylpenicillin sodium for injection, oxacillin for inj, vancocin vial, medicine injection final concentration is 10mg/kg, 5mg/kg, 2.5mg/kg.
(2) bacterium
ATCC BAA-42 methicillin-resistant staphylococcus aureus (MRSA), ATCC 29213 penicillin resistant streptococcus aureuses (producing the penicillinase strain), ATCC 700802 vancomycin-resistant enterococcus (VRE).
2, experimental technique
120 of kunming mices, male and female half and half, body weight 18-22 grams, random packet is that ATCCBAA-42 methicillin-resistant staphylococcus aureus (MRSA) is injected in the abdominal cavity respectively, ATCC 29213 penicillin resistant streptococcus aureuses and ATCC 700802 vancomycin-resistant enterococcus (VRE) three big groups, wherein penicillin, Cephazolin, Oxazacillin, vancomycin and Miniaturized Staphylococcus aureus polypeptide of against drug resistance COM1 test group of the present invention are respectively 10 of every group of mouse, 10 of control groups.(0.5ml, 5.2 x 10 behind the bacterium of abdominal injection lethal dose
6CFU/ml), each drug test group mouse from the medicine of intravenous injection 10mg/kg, 5mg/kg, 2.5mg/kg dosage once, per 24 hours observationss, 7-14 days continuously, with the positive result of dead mouse.
3, result
10mg/kg, 5mg/kg, 2.5mg/kg drug dose group result show, Miniaturized Staphylococcus aureus polypeptide of against drug resistance COM1 group mouse survival rate has remained the constant gap with vancomycin group mouse survival rate in 3 dosage groups, therefore test middle-size and small-sizeization Staphylococcus aureus polypeptide of against drug resistance COM1 in vivo the result of treatment of methicillin-resistant staphylococcus aureus resistance is much better than vancomycin, this is because Miniaturized Staphylococcus aureus polypeptide of against drug resistance COM1 has sterilization and suppresses bacteriotoxin excretory dual-use function.Therefore this medicine will have the incomparable result of treatment of existing antibiotic when clinical application.Other each antibiotic group mouse death rate 〉=80% has not possessed comparability; Control group is all dead in 48 hours.Experimental result sees the following form.
By result in the table as can be seen: in the test of SA mouse endogenous protective; the mortality ratio minimum (30%) of Miniaturized Staphylococcus aureus polypeptide of against drug resistance COM1 under minimum dose (2.5mg/kg) condition; other is respectively organized mortality ratio and is respectively; penicillin group 80%; Cephazolin group 100%; Oxazacillin group 100%, vancomycin group 60%, control group 100%.This result shows, the protection ratio of Miniaturized Staphylococcus aureus polypeptide of against drug resistance of the present invention the highest (70%); The protection ratio of vancomycin take second place (40%).
The synergism test of 10 couples of β of embodiment-Lactam antibiotics endogenous protective effect
1, experiment material
(1) medicine
Miniaturized Staphylococcus aureus polypeptide of against drug resistance COM1 of the present invention (embodiment 1 preparation), Benzylpenicillin sodium.
(2) bacterium
ATCC 29213 penicillin resistant streptococcus aureuses (producing the penicillinase strain).
2, experimental technique
20 of kunming mices, male and female half and half, body weight 18-22 grams, the random packet abdominal cavity is injected ATCC 29213 penicillin resistant streptococcus aureuses (0.5ml, 4.8 x 10 respectively
5CFU/ml).The bacterium of abdominal injection lethal dose is after 1 hour, each drug test group mouse from the medicine of intravenous injection corresponding dosage once, per 24 hours observationss, 7-14 days continuously, with the positive result of dead mouse.A is control group (0.9%NaCl among Fig. 9, n=5), b is Benzylpenicillin sodium group (10.75mg/kg, n=5), c is half amount Benzylpenicillin sodium and partly measures Miniaturized Staphylococcus aureus polypeptide of against drug resistance group of the present invention (5.17mg/kg Miniaturized Staphylococcus aureus polypeptide of against drug resistance+5.37mg/kg Benzylpenicillin sodium, n=5), d be Miniaturized Staphylococcus aureus polypeptide of against drug resistance group of the present invention (10.35mg/kg, n=5).
In the test of mouse endogenous protective; the mortality ratio of Miniaturized Staphylococcus aureus polypeptide of against drug resistance of the present invention minimum (0%); other is respectively organized mortality ratio and is respectively, and penicillin group 60% is partly measured Benzylpenicillin sodium and partly measured Miniaturized Staphylococcus aureus polypeptide of against drug resistance group 20% of the present invention.This result shows that half amount Miniaturized Staphylococcus aureus polypeptide of against drug resistance makes the protection ratio of half amount Benzylpenicillin sodium improve 2 times than the protection ratio of full dose Benzylpenicillin sodium.
The test of embodiment 11 Miniaturized Staphylococcus aureus polypeptide of against drug resistance toxicity in vivo
10 of kunming mices, male and female half and half, body weight 18-22 grams, every day was injected 10mg/kg Miniaturized Staphylococcus aureus polypeptide of against drug resistance COM1 of the present invention totally 20 days in the abdominal cavity, and all mouse all survive, weight increase, get sections observation such as liver, kidney and spleen after the execution, do not see morphological abnormalities, detail as per accompanying drawing 10.The toxic side effect of existing antibacterials does not appear in this presentation of results Miniaturized Staphylococcus aureus polypeptide of against drug resistance of the present invention in the present embodiment test.
SEQUENCE LISTING
＜110〉Huaxi Hospital Attached to Sichuan Univ
＜120〉Miniaturized Staphylococcus aureus polypeptide of against drug resistance and application thereof and preparation method
<160>7
<170>PatentIn Version 3.2
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<212>DNA
<213>Escherichia coli
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＜223〉colicin Ia channel architecture domain gene
<400>1
<210>2
<211>24
<212>DNA
<213>
<220>
<222>(1).....(24)
＜223〉streptococcus aureus pheromone D1 gene
<400>2
<210>3
<211>8
<212>PRT
＜213〉artificial sequence
＜223〉streptococcus aureus pheromone D1 polypeptid acid sequence
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＜213〉artificial sequence
<220>
<221>CDS
<222>(1).....(552)
＜223〉gene 1 of coding Staphylococcus aureus polypeptide of against drug resistance
<400>4
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＜213〉artificial sequence
＜223〉the Miniaturized Staphylococcus aureus polypeptide of against drug resistance aminoacid sequence 1
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＜213〉artificial sequence
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＜223〉gene 2 of coding Staphylococcus aureus polypeptide of against drug resistance
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＜213〉artificial sequence
＜223〉the Miniaturized Staphylococcus aureus polypeptide of against drug resistance aminoacid sequence 2
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Claims (6)
1, a kind of gene of the Miniaturized Staphylococcus aureus polypeptide of against drug resistance of encoding, it is characterized in that this gene by the gene in coding colicin channel architecture territory and the genomic constitution of coding streptococcus aureus pheromone, it is the described nucleotide sequence of SEQ ID NO.4 in the sequence table.
2, a kind of recombinant plasmid of the Miniaturized Staphylococcus aureus polypeptide of against drug resistance gene of encoding is characterized in that it contains the described gene of claim 1.
3, a kind of Miniaturized Staphylococcus aureus polypeptide of against drug resistance is characterized in that it is the described aminoacid sequence of SEQ ID NO.5 in the sequence table.
4, the application of the described polypeptide of claim 3 in the medicine of preparation treatment or prevention resistance infection of staphylococcus aureus.
5, the application of the described polypeptide of claim 3 in preparation β-lactam antibiotic synergistic agent and bacteriotoxin inhibitor.
6, the preparation method of the described polypeptide of a kind of claim 3 is characterized in that may further comprise the steps:
The gene of coding streptococcus aureus pheromone operationally is connected the recombinant plasmid that obtains coding reorganization Miniaturized Staphylococcus aureus polypeptide of against drug resistance gene with colicin channel architecture domain gene, the recombinant plasmid that obtains is transfected in the colibacillus engineering and obtains to produce the engineering bacteria cell of reorganization antimicrobial polypeptide, increase bacterium, centrifugation thalline, ultrasonication, high speed centrifugation then and precipitate broken thalline, get supernatant, promptly obtain the described Staphylococcus aureus polypeptide of against drug resistance of claim 3 with the CM column separating purification.
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| WO2007083175A1 (en) * | 2006-01-17 | 2007-07-26 | West China Hospital, Sichuan University | Antiviral bifunctional molecules, methods of construction and methods of treating virus-induced cancer therewith |
| CN101643501B (en) * | 2008-11-07 | 2012-06-20 | 畿晋庆三联（北京）生物技术有限公司 | Novel antibiotic and nucleotide sequence, preparation method and application thereof |
| CN101633699B (en) * | 2009-09-02 | 2012-02-15 | 畿晋庆堂国际生物技术有限公司 | New type antibiotic containing an antibody analog, preparation method and application method thereof |
| US8431687B2 (en) * | 2010-05-05 | 2013-04-30 | New York University | Staphylococcus aureus leukocidins, therapeutic compositions, and uses thereof |
| CN106729610B (en) * | 2016-12-16 | 2021-01-29 | 湖北工业大学 | Application of N15 polypeptide in inhibiting staphylococcus aureus inhibitor |
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| CN1341604A (en) * | 2001-09-11 | 2002-03-27 | 四川大学华西医院 | Artificial combined antibacterial engineering polypeptide and its preparation method |
| CN1403484A (en) * | 2002-10-11 | 2003-03-19 | 浙江大学 | Prepn and application of transmembrane staphylococcus aureus toxin A fusion protein |
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| CN1341604A (en) * | 2001-09-11 | 2002-03-27 | 四川大学华西医院 | Artificial combined antibacterial engineering polypeptide and its preparation method |
| CN1403484A (en) * | 2002-10-11 | 2003-03-19 | 浙江大学 | Prepn and application of transmembrane staphylococcus aureus toxin A fusion protein |
Non-Patent Citations (1)
| Title |
|---|
| 抗耐药金黄色葡萄球菌工程肽--一种人工构建的抗菌蛋白质分子机器. 廖颖等.四川大学学报医学版，第34卷第4期. 2003 * |
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