CN1148379C - Antibiotic/antiendotoxic mimic peptide and its preparation and application - Google Patents
Antibiotic/antiendotoxic mimic peptide and its preparation and application Download PDFInfo
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- CN1148379C CN1148379C CNB011045914A CN01104591A CN1148379C CN 1148379 C CN1148379 C CN 1148379C CN B011045914 A CNB011045914 A CN B011045914A CN 01104591 A CN01104591 A CN 01104591A CN 1148379 C CN1148379 C CN 1148379C
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Abstract
The present invention discloses 66 kinds of antibiotic and antiendotoxic stimulated peptide which is stimulated, designed and artificially synthesized by a computer. The molecular design of polypeptide is simulated by the assistance of the computer according to the space conformational analysis and the physicochemical property analysis of three LOOP regions of a BPIN terminal to design a polypeptide molecule having neutralization LPS activity. The antibiotic and antiendotoxic stimulated peptide is synthesized by a solid phase polypeptide chemical synthesis method. The antibiotic and antiendotoxic stimulated peptide of the present invention has the whole or part of biological functions of BPI and can be used for treating G<minus>bacterial infection and human endotoxemia. The present invention has the characteristics of small molecular weight and stable and repeated production.
Description
Technical field
The present invention relates to antibiotic/antiendotoxic mimic peptide, its preparation method and in pharmaceutically application, the antibiotic/antiendotoxic mimic peptide by computer simulation design, synthetic particularly, and be used for the treatment of G
-Infectation of bacteria, people's endotoxemia.
Background technology
Gram-negative (G
-) bacillus Sepsis and shock be to cause one of clinical patient main causes of death, particularly with serious intracellular toxin (Lipopolysaccharide, endotoxin, LPS) G of mass formed by blood stasis
-The infection of bacillus.G
-The bacteremic case fatality rate of bacterium is 20-30%, and by microbemia development and the Sepsis of coming suffer a shock its case fatality rate can be up to 50-80%.And, still do not have effective measure clinically at the treatment of endotoxemia, and therefore cause the treatment of infectious diseases more thorny, therefore studying its pathogeny and seeking therapy measure is the focus that clinical medicine is paid close attention to always.
In recent years, along with to G
-Going deep into of coli infections research is present in G
-Endotoxic effect comes into one's own day by day on the bacillus adventitia.LPS is considered to the Sepsis shock patient cause of death--property inflammatory response syndrome out of control (Systemic inflammatory response syndrome.SIRS) and compensatory anti-inflammatory syndrome (Compensatory anti-inflammatory response syndrome, promotor CARS).Enter its main biological action of intracellular toxin in the blood comprise the following aspects 1. with can induce, discharge multiple inflammatory mediator such as TNF, IL-1 etc. after scavenger cell contacts, directly or indirectly bring into play part and systemic effect by these media.2. while intracellular toxin molecule also can combine with multiple tissue, parenchyma and directly cause cell injury.3. activate blood coagulation, fibrinolytic, complement system, bring out D1C.4. act on the a-acceptor, impel suprarenal gland catecholamine secretion, cause microcirculation disturbance, plasma extravasation, until shock and other taking place as heating, oligoleukocythemia, Shwartzman reaction etc.In the inflammatory reaction network of complexity, regulation measure at various inflammatory factors such as TNF, IL-1 also constantly proposes respectively, but the result is disappointing, and it is inseparable that its major cause is that various inflammatory factor interacts, and only can not deal with problems at certain or several cytokines.Therefore catch the principal aspect of a contradiction--LPS, it is significant to seek the breakthrough point of dealing with problems.
Both at home and abroad about endotoxic discovering: intracellular toxin is present in G
-On the cytolemma of bacillus, its main chemical compositions is a lipopolysaccharides.Its basic comprising is the O-specific side chains, core polysaccharide, lipoid A three parts.Wherein lipoid A is the active centre of toxin.Main biological action induces, discharges multiple inflammatory mediator such as TNF, IL-1 etc. for stimulating monokaryon-phagocytic cell system, by these medium activated inflammatory reaction networks and direct or indirect performance part and systemic effect.
Over surplus in the of nearly ten year, in the world at G
-Endotoxemia that coli infections is followed and the study on prevention of SIRS, mainly contain how anti-, the monoclonal antibody of antiendotoxin core glycolipid (Lipid A), various anti-cytokine antibodies and receptor antagonist, as anti-TNF antibodies, anti-IL-8 I (IL-1) antibody, anti-IL-6 antibodies and NO antagonist etc.In animal experiment study, above-mentioned preparation has the certain protection effect to the visceral organ injury due to the Sepsis, does not obtain drugs approved by FDA but all cut because of uncertain therapeutic efficacy in clinical trial.
Bactericidal properties/power/permeability increasing protein (Bactericihcidal/permeability increasing protein, BPI) be positively charged positive protein matter in a kind of people of being present in and the Mammals PMN azurophilic granule, the about 55kDa of molecular weight is made up of 456 amino acid, has the G of killing
-Bacterium and in and the ability of LPS, be to promise to be most at present to be applied to clinical preparation, the concern of gone abroad numerous scholars and pharmacy corporation.Confirmed that at present BPI is to many G
-Bacterium comprises performance germicidal actions such as Colibacter, Pseudomonas aeruginosa, salmonella, Shigella, Rhodopseudomonas, Klebsiella, proteus, serratia, eisseria; BPI also can with G
-LPS specific combination on the bacterial membrane, the activity of the free LPS of inhibition.Terminal 1-199 the amino-acid residue fragment of BPI N-(BPI23) has all biological activity of complete BPI, and its allosteric body BPI21 is except that the biological activity with BPI23, to part G
+Coccus such as drug-resistant staphylococcus aureus, suis have fungicidal activity, and structure is more stable.Recombinant BPI 23/21 by U.S. development has entered clinical study, and preliminary clinical study shows that BPI can obviously reduce bacterial count in the blood, improves cardiac index, art pO2, blood pressure, reduction plasma endotoxin level, the animal survival rate that improves.External recombinant BPI 23 (rBPI23 sees 1992, Infect.Immun.60:4754-4761) and the development research of mutant (rBPI21) entered the III clinical trial phase, PRELIMINARY RESULTS proves that rBPI23 and rBPI21 are to serious G
-Infectation of bacteria has significant curative effect.But rBPI23 and rBPI21 are to G
-The treatment of infectation of bacteria and LPS mass formed by blood stasis exists that dosage is big, cost is high, the problem that is difficult to promote.
Chinese patent application 94191894 and 94194835 relates to the biologically active peptides and the application thereof of the functional zone of bactericidal properties permeability-increasing protein, the aminoacid sequence behaviour sterilization/aminoacid sequence of enhancing permeability protein (BPI) functional zone that has or the peptide of its subsequence are wherein disclosed, and the varient of this sequence or its subsequence, they have the biologic activity of at least a BPI, as heparin in conjunction with the neutralization of, heparin, LBS in conjunction with, LPS neutralization or fungicidal activity, and provide the peptide that is used for various therepic use and the medicinal compositions of this peptide.
Although more, still there is not a kind of medicine that can be used for the treatment of endotoxemia safely and effectively clinically to endotoxic research.Therefore, thus reaching the purpose that reduce to infect mortality ratio in focusing on and on the intracellular toxin has very important significance.
Summary of the invention
The object of the present invention is to provide a series of antibiotic/antiendotoxic mimic peptides, have sterilization and in and the LPS activity.
Another object of the present invention is to adopt the solid-phase polypeptide chemical synthesis process, synthetic above-mentioned antibiotic, antiendotoxic mimic peptide, the peptide molecule that obtains having biologic activity.
A further object of the present invention is to study antibiotic/antiendotoxic mimic peptide and is used for the treatment of G
-Infectation of bacteria, people's endotoxemia are so that explore the endotoxic novel drugs that can sterilization can neutralize again.
To achieve these goals, the technical solution used in the present invention is: by at present known in the nature biotechnology body, exist have a material in conjunction with activity of endotoxin, as BPI, LBP, LAF etc., it is carried out homology analysis, on the basis of BPI three-dimensional structure, infer the structure-function relationship that it is possible by The study of computer simulation, and under area of computer aided, carry out the molecular designing of mimic peptide.Endotoxin binding protein BPI of finder and rabbit and the homology of LBP are the highest; Primary sequence hydrophilic and hydrophobic analysis to the BPI molecule finds that the primary sequence of BPI molecule mainly is made up of hydrophobic amino acid; Three-dimensional structural analysis to the BPI molecule is found, BPI molecule (456aa) is boomerang shape (boomerang-shaped) albumen of a 13.5nm * 3.5nm * 3.5nm size, its structural domain by two structural similitudies is formed, between by the chain connection of 21 peptides of a proline(Pro) enrichment, and measured the tomograph of BPI molecule N end LOOP; In addition, the present invention has also carried out computational analysis to the surface electrostatic gesture of BPI molecule, finds that 3 LOOP districts of N end have concentrated many positive electricity amino acid, and its surface electrostatic gesture positive electricity distributes more remarkable; Its primary structure and even tertiary structure are analyzed comparison, find out with intracellular toxin bonded structural domain be LOOP district, N end structure territory, under area of computer aided, carry out the molecular designing of mimic peptide, board design go out to have stronger sterilization and in and the active peptide molecule of LPS.
The solid-phase polypeptide chemical synthesis process that the present invention adopts Merrifield to create; the method of amino-acids by Fmoc protection or the method for amino-acids synthetic of Tboc protection or carry out the synthetic of antibiotic/antiendotoxic mimic peptide of the present invention by automatic Peptide synthesizer obtain the antibiotic/antiendotoxic mimic polypeptide of a series of the present invention's designs.Polypeptide palpus process high-pressure liquid phase (HPLC) purifying that obtains, and through mass spectrum evaluation and biological activity evaluation.
The present invention also the activity that produces of person monocytic cell's tumour necrosis factor (TNF-a) and the interleukin 6 (IL-6) by the inside and outside fungicidal activity of measuring antibiotic/antiendotoxic mimic polypeptide of the present invention, external anti-endotoxin activity, vitro inhibition endotaxin induction, internally the toxin attacks mouse dead provide protection, to the therapeutic action of rat endotoxemia, simulating peptide to the dead provide protection of intestinal bacteria attack mouse, simulating peptide to the pyemic therapeutic action of rat be used for the treatment of G so that study it
-Infectation of bacteria and people's endotoxemia.
The present invention is antibiotic/antiendotoxin peptide (being antibiotic/antiendotoxic mimic peptide), and its type is an amino acid, and topological framework is linear, and molecule type is a peptide, and the sequence of molecule is made up of an amino acid whose word brevity code, and is optional from following sequence peptide:
KSKVGFLQLLFHKKLRGSLTKLKG，
KSKVGWLILLFHKK，
KTKVGFLIQLFHKK，
KTKVGFLIQLFHKK，
KTKVGFLIQLFHKK，
KGKVGFLIQLFHKK，
KTKGGFLQLLFHKK，
KGKGGFLQLLFHKK，
KSKGGFLQLLFHKK，
KGKGGWLIQLFHKK，
KSKGGWLIQLFHKK，
KTKGGWLIQLFHKK，
KGKVGFLQLLFHKK，
KTKVGFLQLLFHKK，
KSKVGWLQLLFHKK，
KGKVGWLQLLFHKK，
KTKVGWLQLLFHKK，
KSKSGFLQLLFHKK，
KGKGGFLIQLFHKK, or
KSKHLGKAQKRFLK。
Wherein, preferably following sequence peptide:
KSKVGFLQLLFHKKLRGSLTKLKG，
KTKVGFLIQLFHKK，
KTKGGFLQLLFHKK，
KGKGGFLQLLFHKK，
KSKGGFLQLLFHKK，
KGKGGWLIQLFHKK，
KSKGGWLIQLFHKK，
KTKGGWLIQLFHKK，
KGKVGFLQLLFHKK，
KTKVGFLQLLFHKK，
KTKVGWLQLLFHKK，
KSKVGWLQLLFHKK，
KSKSGFLQLLFHKK，
KGKGGFLIQLFHKK, or
KSKHLGKAQKRFLK。
Of the present invention antibiotic/preparation method of antiendotoxin peptide is: the molecular designing of carrying out mimic peptide under area of computer aided, board design go out to have stronger sterilization and in and the active peptide molecule of LPS, synthetic with Merrifield solid-phase polypeptide chemical synthesis process, the solid-phase polypeptide chemical synthesis process is:
A. peptide resin is synthetic
Take by weighing an amount of Fmoc-L-Lys-PEG-PS resin or other polypeptide synthetic resins and pour in the synthetic post, add solvent-swollen, take by weighing an amount of Fmoc-amino acid successively in suitable container according to the aminoacid sequence of antibiotic/antiendotoxic mimic peptide of the present invention.Solid state chemistry synthetic method according to the Merrifield invention holds beginning to carry out successively according to the aminoacid sequence of antibiotic/antiendotoxic mimic peptide of the present invention from C, in synthetic post, add the DMF Deblock liquid that contains 20% hexahydropyridine and take off peptide resin Fmoc blocking group, add in the synthetic post after adding an amount of DMF dissolving Fmoc-amino acid, the DMF solution that adds an amount of diisopropylethylamine and azepine benzo triazole methyl urea hexafluorophosphate more respectively carries out coupling reaction in post, peptide resin in the synthetic post in the coupled back Fmoc-amino acid of DMF flush away remnants, preparation process such as rare gas element dries up, so circulation is finished the peptide chain formation of whole antibiotic/antiendotoxic mimic peptide and is obtained its peptide resin; From synthetic post, take out synthetic antibiotic/the antiendotoxin peptide resin, put freeze-drying in the freeze drier, it is standby to obtain peptide resin;
B. the cracking of peptide resin
Get above-mentioned peptide resin in container, add lysate: 4.5ml trifluoroacetic acid+0.25ml thio phenyl methyl ether+0.1ml methyl-phenoxide+0.15ml 1,2-7 two mercaptan, in room temperature lucifuge reaction 2 hours, filter, filtrate adds in advance in the refrigerated anhydrous diethyl ether after room temperature concentrates, freezing spending the night, centrifugal, abandon supernatant liquor, precipitation is dissolved with an amount of Glacial acetic acid, dry in the freezing rearmounted freeze drier, get thick peptide;
The purifying of c. thick peptide
Get the thick peptide of BNEP and add an amount of no heat source water dissolving, obtain sample solution, filter and collect sample filtrate; With acetonitrile and there is not heat source water 0.45u membrane filtration, add 0.1% trifluoroacetic acid respectively, mixing is used the C18 reversed-phase column, mobile phase A: the 100%H2O+0.1% trifluoroacetic acid; Mobile phase B: 100% second is fine+0.1% trifluoroacetic acid, from 0 to 15 minute gradient elution purifying, the smart peptide of BNEP.
Of the present invention antibiotic/the antiendotoxin peptide is in the application that is being used for preparing kill bacteria medicine and treatment endotoxemia medicine.
Be detailed description of the present invention below.
The used workstation of computer simulation is SGI Indigo among the present invention
2Series Solid Impact R1000 graphics workstation, simulator is a Biosym/MSI molecular simulation system.All working all carries out on INSIGHT II workplatform, and it is DelPhi that electrostatic potential is calculated used software.It is the FASIA program that the protein primary sequence is searched for used software, the protein sequence storehouse be EMBL (European Molecular Biology Laboratory) SWISS-PROT protein sequence storehouse (Release34, October1996).
The present invention infers the structure-function relationship that it is possible by The study of computer simulation on the basis of BPI three-dimensional structure, and carries out the molecular designing of mimic peptide under area of computer aided.Show by the primary sequence hydrophilic and hydrophobic analysis of homology analysis, BPI molecule, the three-dimensional structural analysis of BPI molecule, the analog results such as surface electrostatic gesture computational analysis of BPI molecule, the biological function district of BPI mainly is positioned at three LOOP districts of N end, and the main mode of action of itself and acceptor is an electrostatic interaction.Therefore according to the simulating peptide design that the space conformation analysis and the physicochemical property analysis in three LOOP districts of BPI N end are formulated be: 1. two ends are the spirane structure that strong positive potential distributes; 2. the corner spirane structure that distributes of positive potential; 3. the strong spiral tendency of folds structure that distributes of positive potential; 4.BPIN the improved-type structure of end LOOP district native peptides section.Obtain being fit to antibiotic/antiendotoxic mimic peptide of the present invention by computer simulation.The shortest person of antibiotic/antiendotoxic mimic peptide sequence of the present invention is 7 amino acid polypeptides, and the elder of sequence is 28 amino acid polypeptides; Type is an amino acid; Topological framework is linear; Molecule type is a peptide; The sequence of molecule is made up of an amino acid whose word brevity code.As shown in table 1.
The sequence of table 1 66 antibiotic/antiendotoxic mimic peptide molecules of the present invention
Numbered sequence theoretical value measured value
BNEP9600139： KSKAQKRFLK MW：1233.5 1233
BNEP9600257： KIKGGFLFHKK MW：1302.6 1302.4
BNEP9600258： KRPSVTSPRK MW：1155.3 1356
BNEP9600301： KSKVLGK MW：758.9 761
BNEP9600307： KSKVLGKAQKRFLK MW：1931.0 1630.5
BNEP9600308： KSKVLGKRFLK MW：1303.6 1302.8
BNEP9700031： KGKVLGKAQKRFLK MW：1600.9 1602
BNEP9700032： KGKVLGKFLK MW：1117.4 1118.1
BNEP9700055： KGKVLGK MW：728.9 728.5
BNEP9700145： KTKVLGK MW：773.0 773.0
BNEP9700146： KLKVLGK MW：785.0 784.2
BNEP9701047： RRAKVFIKM MW：1148.4 1150.1
BNEP9800431： RLKLILKSK MW：1098.4 1100.1
BNFP9800433： KSVLILWKSK MW：1201.5 1202.5
BNEP9800637： KSKVGILLQLVRKR MW：1638.0 1637.5
BNEP9800638： KSKFVILLQLGHKFK MW：1786.2 1786.4
BNEP9800723： KSKVGWLIQLFHKKKTKGGWLIQLFHKK MW：3378.0 3380
BNEP9800892： KIKHLGKKSKVGFLQLLFHKK MW：2478.0 2482.3
BNEP9800895： KIKHLGKKTKGGWLIQLFHKK MW：2489.0 2490.1
BNEP9800993： KKVGWLIQLFHKKIE MW：1876.2 1875.5
BNEP9801013： KSKVGFLQLLFHKKLRGSLTKLKG MW：2727.3 2728.0
BNEP9801042： SKVGWLILLFHKKIE MW：1811.2 1810.1
BNEP9801043： KSKVGWLILLFHKK MW：1697.0 1696.1
BNEP9801047： KIKHLGLFHKK MW：1348.1 1385.0
BNEP9801087： KSKVGFLIQLFHKK MW：1673.0 1675.1
BNEP9801095： KTKVGFLIQLFHKK MW：1687.0 1685.1
BNEP9801161： KGKVGFLIQLFHKK MW：1643.0 1645.2
BNEP9801174： KSKGGWVLQLLFHKK MW：1670.0 1668.4
BNEP9801175： KGKGGWLQLLFHKK MW：1639.0 1642.0
BNEP9801286： KTKGGFLQLLFHKK MW：1643.0 1641.2
BNEP9801287： KGKGGFLQLLFHKK MW：1600.9 1600.5
BNEP9801289： KSKGGFLQLLFHKK MW：1630.9 1629
BNEP9901311： KGKGGWLIQLFHKK MW：1639.9 1641.5
BNEP9901312： KSKGGWLIQLFHKK MW：1670.0 1669.5
BNEP9901315： KTKGGWLIQLFHKK MW：1684.0 1682.8
BNEP9901317： KGKVGFLQLLFHKK MW：1643.0 1643.0
BNEP9901318： KTKVGFLQLLFHKK MW：1687.0 1688.5
BNEP9901319： KSKVGWLQLLFHKK MW：1712.0 1715.8
BNEP9901320： KGKVGWLQLLFHKK MW：1682.0 1688.2
BNEP9901321： KTKVGWLQLLFHKK MW：1726.1 1729.5
BNEP9901496： KIKHLGKAQKRFLK MW：1695.1 1692.5
BNEP9901501： KSKSGFLQLLFHKK MW：1661.0 1661.5
BNEP9901534： KSKAAWLLQLFHKK MW：1698.0 1689.7
BNEP9901536： KSKSAFLLQLFGKK MW：1594.9 1596.4
BNEP9901537： KSKAAFLQLAFKSK MW：1566.9 1568.3
BNEP9901538： RSKAAFLQLAFKSK MW：1594.9 1596.1
BNEP9901539： RSKSAFLLQLFGKK MW：1622.9 1621.4
BNEP9901541： RSKSAFLLQLFGKR MW：1650.9 1653.4
BNEP9901595： RSKAAFLQLAFKSR MW：1622.9 1622.8
BNEP9901602： FCKIKHLGKCY MW：1339.6 1338.5
BNEP9901603： FCKIKHLGKCR MW：1332.6 1335.4
BNEP2001606： KCKIKHLGKCR MW：1313.6 1315.2
BNEP2001627： FCKIKHLGKCKK MW：1432.8 1435.7
BNEP2001628： KCKIKHLGKCKK MW：1413.8 1417.8
BNEP2001629： KCKIKHLGKCY MW：1320.6 1324.7
BNEP2001630： FCKIKHLGKCR MW：1332.6 1332.6
BNEP2001671： KCAQKRFLKMCK MW：1483.9 1485.2
BNEP2001677： YCAQKRFLKMCY MW：1553.9 1552.8
BNEP2001678： RCAQKRFLKMCY MW：1546.9 1548.1
BNEP2001681： KCKNLQRPLKLNQKCR MW：1970.3 1972.5
BNEP2001695： KGKGGFLIQLFHKK MW：1600.9 1602.1
BNEP2001708： RRPLIQLVKK MW：1250.5 1253.4
BNEP2001756： KKKILQLLKK MW：1239.6 1242.1
BNEP2001757： RRPILPRR MW：1063.3 1063.8
BNEP2001758： KSKHLGKAQKRFLK MW：1669.0 1671.8
BNEP2001784： KIKVLFHKSK MW：1227.5 1228.6
Antibiotic/antiendotoxic mimic peptide of the present invention can also be in above-mentioned 66 polypeptide, at least two polypeptide that identical or different peptide is connected to form.
Antibiotic/antiendotoxic mimic peptide of the present invention can also be end group or side chain through above-mentioned 66 polypeptide of chemically modified, as the salt of the acceptable above-mentioned antibiotic/antiendotoxic mimic peptide of biology, ester, acid amides, heterocycle, alkyl etc.
The solid-phase polypeptide chemical synthesis process (Merrifield RB.1963 J Am ChemSoc.85:2149) that Merrifield creates has developed into a kind of sophisticated well-known solid-phase polypeptide chemical synthesising technology.Use this technology, can synthesize antibiotic/antiendotoxic mimic peptide of the present invention, be specially:
1. peptide resin is synthetic
Taking by weighing an amount of Fmoc-L-Lys-PEG-PS resin (or other polypeptide synthetic resins) pours in the synthetic post, add solvent-swollen, take by weighing an amount of Fmoc-amino acid successively in suitable container according to the aminoacid sequence of antibiotic/antiendotoxic mimic peptide of the present invention.Solid state chemistry synthetic method according to the Merrifield invention holds beginning to carry out successively according to the aminoacid sequence of antibiotic/antiendotoxic mimic peptide of the present invention from C, in synthetic post, add the DMF Deblock liquid that contains 20% hexahydropyridine and take off peptide resin Fmoc blocking group, add in the synthetic post after adding an amount of DMF dissolving Fmoc-amino acid, the DMF solution that adds an amount of diisopropylethylamine (DIPEA) and azepine benzo triazole methyl urea hexafluorophosphate (HATU) more respectively carries out coupling reaction in post, peptide resin in the synthetic post in the coupled back Fmoc-amino acid of DMF flush away remnants, preparation process such as rare gas element dries up, so circulation is finished the peptide chain formation of whole antibiotic/antiendotoxic mimic peptide and is obtained its peptide resin; Take out synthetic antibiotic/antiendotoxic mimic peptide resin from synthetic post, put freeze-drying in the freeze drier, it is standby to obtain peptide resin.
2. the cracking of peptide resin
Get above-mentioned peptide resin in container, add lysate: 4.5ml trifluoroacetic acid+0.25ml thio phenyl methyl ether+0.1ml methyl-phenoxide+0.15ml 1, in room temperature lucifuge reaction 2 hours, filter, filtrate adds in advance in the refrigerated anhydrous diethyl ether after room temperature concentrates, freezing spending the night, centrifugal, abandon supernatant liquor, precipitation is dissolved with an amount of Glacial acetic acid, dry in the freezing rearmounted freeze drier, get thick peptide.
3. the purifying of thick peptide
Get the thick peptide of BNEP and add an amount of no heat source water dissolving, obtain sample solution, filter and collect sample filtrate; With acetonitrile and there is not heat source water 0.45u membrane filtration, add 0.1% trifluoroacetic acid respectively, mixing is used the C18 reversed-phase column, mobile phase A: the 100%H2O+0.1% trifluoroacetic acid; Mobile phase B: 100% second is fine+0.1% trifluoroacetic acid, 0 → 15min gradient elution purifying, the smart peptide of BNEP.
Antibiotic/antiendotoxic mimic peptide of the present invention also can adopt suitable other chemistry of the present invention, instrument or biochemical method synthetic.
Internal and external test shows, antibiotic/antiendotoxic mimic peptide of the present invention external to E.coli β
+Bacteriums such as-HI, E.coli J5, E.coli V517, MSSA25923, PSPNC49619, PA103 and intestinal bacteria all have fungicidal activity in various degree; Experimentation on animals shows that antibiotic/antiendotoxic mimic peptide of the present invention can obviously reduce TNF-α peak concentration in the serum, has the effect that prolongs the Sepsis survival time of animals, improves the animal survival rate; External, antibiotic/antiendotoxic mimic peptide of the present invention to LPS external all have neutralizing effect is arranged in various degree, different BNEP there are differences the neutralizing effect of LPS, and the secretion of person monocytic cell's tumour TNF-α of endotaxin induction and IL-6 is all had in various degree restraining effect; Animal experiment shows; the internal toxin attacks mouse of antibiotic/antiendotoxic mimic peptide of the present invention all has provide protection in various degree; the generation of the rat TNF-α of endotaxin mediate and IL-6 is all had in various degree restraining effect, the rat endotoxemia is had therapeutic action.Can be used for the treatment of G
-Infectation of bacteria and people's endotoxemia.
Antibiotic/antiendotoxic mimic peptide of the present invention can be aided with medicinal diluent, adjuvant and carrier and make medicinal compositions, as tablet, pulvis, pill, solution or suspension agent, by sucking, inject or other mode administrations, is used for the treatment of G
-Infectation of bacteria and endotoxemia; Antibiotic/antiendotoxic mimic peptide of the present invention can also be compound with other drug, is used for the treatment of G
-Infectation of bacteria and endotoxemia.
Antibiotic/antiendotoxic mimic peptide of the present invention is used for the treatment of G as thinner, adjuvant and carrier
-Infectation of bacteria and people's endotoxemia.
At least two identical or different antibiotic/antiendotoxic mimic peptides of the present invention are connected to a peptide, are used for the treatment of G
-Infectation of bacteria and endotoxemia.
In addition, at least two identical or different antibiotic/antiendotoxic mimic peptides of the present invention are connected to a peptide, can be used as thinner, adjuvant and carrier and are used for the treatment of G
-Infectation of bacteria.
Description of drawings
The multiple sequence comparing result of the primary sequence of Fig. 1 .BPI and homologous protein thereof
The primary sequence hydrophilic and hydrophobic of Fig. 2 .BPI molecule is analyzed
The three-dimensional structural analysis of Fig. 3 .BPI molecule
The three-dimensional structural analysis of Fig. 4 .BPI molecule
The three-dimensional structural analysis of Fig. 5 .BPI molecule N and C end LOOP
The tomograph of Fig. 6 .BPI molecule N end LOOP
The surface electrostatic gesture of Fig. 7 .BPI molecule distributes
Fig. 8. the body outer disinfecting activity of antibiotic/antiendotoxic mimic peptide BNEP9800892
Fig. 9. antibiotic/antiendotoxic mimic peptide is to the colibacillary body outer disinfecting activity of J5
Figure 10. killing colon bacillus activity in the antibiotic/antiendotoxic mimic peptide body
Figure 11. antibiotic/antiendotoxic mimic peptide reduces the activity of serum TNF in the body-a concentration
Figure 12. the active qualitative experiment of the external anti-endotoxin of antibiotic/antiendotoxic mimic peptide
Figure 13. the external anti-endotoxin active level experiment of antibiotic/antiendotoxic mimic peptide
Figure 14. antibiotic/antiendotoxic mimic peptide influences endogenous toxic material to LPS inductive THP-1 cell TNF-a excretory
Plain irritation cell is various dose simulating peptide inhibition TNF-a secretion situation after 2 hours
Figure 15. antibiotic/antiendotoxic mimic peptide influences intracellular toxin to LPS inductive TFP-1 cell IL-6 excretory
Irritation cell is various dose simulating peptide inhibition IL-6 secretion situation after 2 hours
Figure 16. different antibiotic/antiendotoxic mimic peptides discharge the inhibition situation to LPS inductive rat TNF-a
Figure 17. different antibiotic/antiendotoxic mimic peptides discharge the inhibition situation to LPS inductive rat IL-6
The mass spectroscopy figure of Figure 18 .BNEP9801042 peptide sample
The mass spectroscopy figure of Figure 19 .BNEP9901537 peptide sample
The mass spectroscopy figure of Figure 20 .BNEP9901536 peptide sample
Embodiment
Be specific embodiments of the invention below, described embodiment is used to describe the present invention, rather than restriction the present invention.
1. homology analysis
For more deep understanding have in conjunction with the biological function of activity of endotoxin BPI molecule and and protein three-dimensional structure between dependency, the present invention is that probe carries out the homology search and the homologous protein sequence that finds is carried out the multiple sequence contrast in PDB storehouse and SWISS-PROT sequence library with the primary sequence of BPI molecule.6 homologous protein sequences have been found that, they are respectively the BPI molecules of ox, the phospholipid transfer protein of people and mouse (PhospholipidTransfer Protein PLTP) molecule, cholesterol fat transfer protein (the CholesterylEster Transfer Protein CETP) molecule of the endotoxin binding protein of people and rabbit (Lipopolysacchride Binding Protein LBP) molecule and rabbit, as shown in Figure 1, wherein the homology of BPI and LBP is the highest.List the result of sequence alignment that BPI probe and 6 kinds have the protein sequence of obvious homology among Fig. 1.
2.BPI the primary sequence hydrophilic and hydrophobic of molecule is analyzed
The primary sequence hydrophilic and hydrophobic analysis of BPI molecule as shown in Figure 2, X-coordinate wherein is the sequence label of amino-acid residue, ordinate zou is the hydrophobicity score value.Horizontal line is above to be hydrophilic region, and horizontal line is following to be hydrophobic region.As seen the primary sequence of BPI molecule mainly is made up of hydrophobic amino acid.
3.BPI the three-dimensional structural analysis of molecule
See Fig. 3-5, according to the BPI three-dimensional structure of measuring, total length BPI molecule (456aa) is boomerang shape (boomerang-shaped) albumen of a 13.5nm * 3.5nm * 3.5nm size.Its structural domain by two structural similitudies is formed, i.e. N end structure territory (1-229) and C end structure territory (251-456), and 21 peptide linker by a proline(Pro) enrichment between two structural domains connect.Two structural domains form three structural units, central βZhe Die sheet and N end C end two barrel-like structures (10-193 and 260-421), each barrel-like structure comprises three secondary building units, a short α spiral, an antiparallel βZhe Die sheet and a long α spiral that comprises 5 folding strand.The structure superposition of two structural domains shows that the two relative orientation angle is 173 °, 169 C alpha atoms be 0.3mn apart from mean-squared departure (RMSD, root-mean-square deviation), but the conformation in the LOOP district of two structural domains differs bigger.The conformational difference in LOOP district may reflect the difference on two structural domain functions, and the C end does not possess this activity because N end structure territory has the LPS binding ability.LOOP district, N end structure territory is the petal-shaped composition by the partial amino-acid series that lays respectively in the 17th～45,65～99 and 142～169 zones, sees Fig. 6.Although BPI molecule N end structure territory is very similar with C end structure territory, the homology residue per-cent less than 20% of its aminoacid sequence.Therefore this uncommon structural similarity of two structural domains may also have more further meaning, i.e. their total probably certain present also undiscovered biological functions.
4.BPI the surface electrostatic gesture computational analysis of molecule
Electrostatic potential is calculated and is found, it mainly is negative potential that the electrostatic potential of the C end (250～465) of BPI molecule distributes, and N end (1～230) then is mainly positive potential, sees Fig. 7.Interaction between the albumen mainly realizes by surface residue, therefore we the surface electrostatic gesture distribution situation of weight analysis BPI molecule, the solvent of BPI molecule and surface generate with the Connolly method, be that the probe molecule (water molecules size) of 0.14nm rolls on the surface of BPI molecule promptly with a radius, the formed surface, center of rolling middle probe molecule is the solvent-accessible surface of BPI molecule, and the surface electrostatic gesture of BPI molecule distributes and remains that N rectifies, the C end is negative.The positive electrostatic potential of light color representative district among the figure, Fig. 7 is seen in the negative electrostatic potential district of dark representative.Particularly 3 LOOP districts of N end have concentrated many positive electricity amino acid, and its surface electrostatic gesture positive electricity distributes more remarkable.
Present embodiment relates to the molecular designing and the synthetic method of antibiotic/antiendotoxic mimic peptide
The present invention infers the structure-function relationship that it is possible by The study of computer simulation on the basis of BP1 three-dimensional structure, and carries out the molecular designing of mimic peptide under area of computer aided.Analog result shows that the biological function district of BPI mainly is positioned at three LOOP districts of N end, and the main mode of action of itself and acceptor is an electrostatic interaction.Therefore according to the simulating peptide design that the space conformation analysis and the physicochemical property analysis in three LOOP districts of BPI N end are formulated be: 1. two ends are the spirane structure that strong positive potential distributes; 2. the corner spirane structure that distributes of positive potential; 3. the strong spiral tendency of folds structure that distributes of positive potential; 4.BPIN the improved-type structure of end LOOP district native peptides section.
By computer simulation, the present invention obtains 66 antibiotic/antiendotoxic mimic peptide molecules altogether, and sequence sees Table 1.
Embodiment 3
With BNEP 9901315 is example, and the synthetic method of antibiotic/antiendotoxic mimic polypeptide of the present invention is described.
Taking by weighing Fmoc-L-Lys-PEG-PS resin (or other polypeptide synthetic resins) 0.05mmol pours in the synthetic post, add refining dimethyl formamide (DMF) the swelling 30min of about 15ml, other gets 1.0mmol diisopropylethylamine (DIPEA) 50ml (8.7mlDIPEA+41.3ml DMF), 0.5mmol azepine benzo triazole methyl urea hexafluorophosphate (HATU) 50ml (9.5gHATU+40.5ml DMF) and the DMF (Deblock liquid) that contains 20% hexahydropyridine add respectively in the reagent bottle in right amount, take by weighing the Fmoc-amino acid of 2mmol successively in suitable container by BNEP 9901315 aminoacid sequences again.Successively carry out synthetic post add Deblock liquid according to BNEP 9901315 aminoacid sequences from the beginning of C end according to the solid state chemistry synthetic method of merrifield invention and take off peptide resin Fmoc blocking group, add and add in the synthetic post behind an amount of DMF dissolving Fmoc-amino acid, add an amount of DIPEA, HATU more respectively and in post, carry out peptide resin in coupling reaction, the synthetic post in coupled back, so circulate and finish the peptide chain formation of whole BNEP 9901315 and obtain its peptide resin with preparation process such as Fmoc-amino acid, the rare gas element of DMF flush away remnants dry up; Take out BNEP 9901315 peptide resins from synthetic post, put freeze-drying in the freeze drier, it is standby to obtain peptide resin 0.391g.
2.BNEP9901315 the cracking of peptide resin
Get above-mentioned peptide resin 0.391g in suitable container, add 5ml lysate (4.5ml trifluoroacetic acid+0.25ml thio phenyl methyl ether+0.1mI methyl-phenoxide+0.15 ml 1,2 dithioglycol), in room temperature lucifuge reaction 2 hours, to filter, filtrate is after room temperature is concentrated into 1/2 volume, being added dropwise to 10 times measures in advance in the refrigerated anhydrous diethyl ether, freezing spending the night, centrifugal, abandon supernatant, precipitation is dissolved with an amount of Glacial acetic acid, dry in the freezing rearmounted freeze drier, get thick peptide 73.9mg, yield is 87.97%.
3.BNEP9901315 the purifying of thick peptide
Get the thick peptide of BNEP and add an amount of no heat source water dissolving, obtain the sample solution of 0.5mg/ml,, collect filtrate in a small beaker with needle-based strainer filtered sample liquid; With acetonitrile and there is not heat source water 0.45u membrane filtration, add 0.1% trifluoroacetic acid respectively, mixing, carry out purifying by following chromatographic condition:
Post: C18 reversed-phase column
Mobile phase A: 100%H2O+0.1% trifluoroacetic acid
Mobile phase B: 100% second is fine+0.1% trifluoroacetic acid (0 → 15min gradient elution)
Get the smart peptide 58.85mg of BNEP9901315, yield is 79.63%.
Embodiment 4-70
With reference to other 65 kinds of antibiotic/antiendotoxic mimic polypeptide of listing in the synthetic table 1 of the method for embodiment 3, mass spectrum the results are shown in Table 1.Figure 18-20 is the mass spectroscopy figure of BNEP9801042, BNEP9901537 and BNEP9901536 peptide sample.
Embodiment 71
The inside and outside fungicidal activity of antibiotic/antiendotoxic mimic polypeptide of the present invention
1. the body outer disinfecting activity of antibiotic/antiendotoxic mimic polypeptide of the present invention
1.1 experimental technique
With E.coli β
+-HI, E.coli J5, E.coli V517, MSSA25923, bacteriums such as PSPNC49619, PA103 are inoculated in respectively in the M-H substratum, cultivate 18 hours for 37 ℃, transfer in the fresh culture and cultivate 4 hours, adjust bacterial concentration to 10 with the M-H substratum
5Cfu/ml.
Respectively manage concentration behind the table 2 antibiotic/antiendotoxic mimic peptide sample doubling dilution
| The | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | Control tube |
| Bacterium liquid (10 5cfu/ml)(ml) | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 |
| Simulating peptide sample (μ g/ml) | 128 | 64 | 32 | 16 | 8 | 4 | 2 | 1 | 0.5 | 0.25 | 0.125 |
Getting 11 in test tube is 1 group and numbering, and except that first pipe adds the bacterial suspension 2ml, all the other every pipes add bacterium liquid 1ml; Add sample 128 μ g to be determined in first pipe, doubly be diluted to the 11st with bacterium liquid etc. behind the mixing and manage, every pipe sample concentration sees Table 2.Hatch for 37 ℃ and respectively get 10 μ l after 30 minutes and cry and evenly coat on the M-H solid medium 37 ℃, cultivate and carry out enumeration after 18～24 hours.
1.2 experimental result
The result shows, antibiotic/antiendotoxic mimic peptide of the present invention all has in various degree fungicidal activity to used bacterium, and different bacterium exists than big-difference the susceptibility of BNEP; Different BNEP also there are differences (seeing Fig. 8-9) to the fungicidal activity of intestinal bacteria J5.
2. fungicidal activity in the body of antibiotic/antiendotoxic mimic polypeptide of the present invention
2.1 experimental technique
2.1.1 big white mouse weigh the back intramuscular injection 1% vetanarcol (0.5ml/kg body weight), treat the animal holonarcosis after, the sterilization rat tail vein, intravenous injection intestinal bacteria J5, administered dose are 2 * 10
8The cfu/kg body weight, this Escherichia coli bacteria liquid cumulative volume is 1ml, injection finishes in 5 minutes.At random 250 big white mouse are divided into 5 groups: (1) simple Sepsis group (abbreviation control group): only give intestinal bacteria and physiological saline; (2) Sepsis simulating peptide treatment group gives antibiotic/antiendotoxic mimic peptide 20mg/kg body weight simultaneously except that giving intestinal bacteria.Observed the animal survival rate in 0,1,2,3,4,5 hour respectively after giving bacterium or bacterium+simulating peptide, the survival rat lives and takes off Vena cava blood extremely.
2.1.2 the processing of blood preparation
(1) 10 μ l fresh blood is evenly coated the M-H solid medium after diluting certain multiple with physiological saline, cultivates after 18～24 hours for 37 ℃ and makes bacterial colony count.
(2) anticoagulation not: centrifugal immediately, 2000rpm, 15 minutes, get serum, place in-70 ℃ of refrigerators to be measured.
(3) anticoagulation: after treating hemopexis, 2000rpm, 15 minutes, get blood plasma, put in-70 ℃ of refrigerators to be measured.
2.1.3 experimental index
(1) observation of animal dis motility rate: put each treated animal survival condition when observing each mutually.
(2) whole blood bacterial count: get fresh blood 10 μ l, behind the physiological saline gradient dilution, draw 10 μ l, evenly coat on the M-H flat board, hatched 18～24 hours, and did enumeration for 37 ℃.
(3) mensuration of serum TNF-α concentration: by rat TNF-α elisa kit description operation (TNF-α examination U.S. Biosource company product).
2.2 experimental result
(1) antibiotic/antiendotoxic mimic polypeptide of the present invention is to the influence of rat Sepsis animal survival rate
The intestinal bacteria J5 bacterium amount that gives rat in this experiment is 2 * 10
8The cfu/kg body weight, the bacterium amount is very big, the i.e. all death in 4～5 hours of general rat, giving the simulating peptide amount is the 20mg/kg body weight.As seen, antibiotic/antiendotoxic mimic peptide of the present invention has the effect that prolongs survival time of animals, improves the animal survival rate from table 3.
Table 3 antibiotic/antiendotoxic mimic peptide of the present invention is to the influence of rat Sepsis animal survival rate
Time survival rate (%)
(hour) control group 9,901,315 9,901,289 9,901,501 9901286
1.0 100 100 100 100 100
2.0 100 100 100 100 100
3.0 67 100 100 100 100
4.0 20 90 100 100 100
5.0 0 80 90 90 100
(2) whole blood intestinal bacteria counting
After heavy dose of intestinal bacteria J5 injects rat vein, the bacterial count of rat whole blood reaches peak value very soon, descend immediately, bacterial count is 1/4 of a control group only in the antibiotic/antiendotoxic mimic peptide treatment treated animal whole blood, illustrates that antibiotic/antiendotoxic mimic peptide has very strong effect to see Figure 10 to reducing the whole blood bacterial number.
3. the mensuration of serum TNF-α concentration
Serum TNF-α concentration peaks after 2 hours at injection intestinal bacteria J5, and antibiotic/antiendotoxic mimic peptide of the present invention can obviously reduce TNF-α peak concentration, and makes TNF-α concentration always remain at low levels (Figure 11).
Embodiment 72
In the inside and outside of antibiotic/antiendotoxic mimic polypeptide of the present invention and the LPS activity
In 1 antibiotic/antiendotoxic mimic polypeptide of the present invention external and the LPS activity
1.1 experimental technique
LPS 0111:B4 1000pg is added in 10 the former glass test tubees that reduce phlegm and internal heat, add antibiotic/antiendotoxic mimic peptide sample to be identified 100 μ g more respectively, add pyrogen-free physiological saline to final volume 1ml, other establishes reagent contrast and blank pipe, hatch after 30 minutes for 37 ℃ and operate by quantitative matrix development process tachypleus amebocyte lysate box (Japanese Seikagaku Kogyo Co. Ltd.) specification sheets, reaction finishes back DU-7Beckman ultraviolet spectrophotometer 545nm colorimetric estimation OD value.
Carry out the making of typical curve the same period: get LPS 1000pg in first pipe, to wait times gradient dilution 10 pipes, LPS measures the same.
1.2 experimental result
The result shows, antibiotic/antiendotoxic mimic peptide of the present invention to LPS external all have neutralizing effect is arranged in various degree, different BNEP there are differences (legend 12-13) to the neutralizing effect of LPS.
2. the ability that produces of person monocytic cell's tumour necrosis factor of simulating peptide vitro inhibition endotaxin induction (TNF-α) and interleukin 6 (IL-6)
2.1 experimental technique
Adopt RPMI1640 cultivator monocyte strain (THP-I), testing and adjusting cell concn the day before yesterday is 2 * 10
5/ ml is inoculated in 48 well culture plates, establishes (1) control group respectively: adopt LPS 0111:B4 2ng/ml irritation cell; (2) treatment group: get 11 in test tube and numbering, add respectively and wait the doubly testing sample of dilution, concentration is from 128 μ g/ml to 0.125 μ g/ml; Every then Guan Zhongzai adds LPS 0111:B4 respectively, and making its final concentration is 1ng/ml; Hatch after 30 minutes for 37 ℃ and be added on respectively in 48 well culture plates, 37 ℃, 5%CO
2The following cultivation; Got supernatant in 0,1,2,4,6,8,12 hour and measure TNF-α and IL-6 concentration (TNF-α and IL-6 test kit are respectively French Diaclone company, U.S. Biosource company product) respectively at cultivating the back.
2.2 result
Antibiotic/antiendotoxic mimic peptide of the present invention all has in various degree restraining effect (legend 14-15) to the secretion of person monocytic cell's tumour TNF-α of endotaxin induction and IL-6.
3. the provide protection of the internal toxin attacks dead mouse of antibiotic/antiendotoxic mimic peptide of the present invention
3.1 experimental technique
100 of cleaning level Kunming mouses, male and female half and half, body weight 18-22g is divided into 10 groups at random: be respectively control group, dexamethasone treatment group and 7 simulating peptide treatment group.Control group: simple tail vein injection LPSO111:B420mg/kg; Dexamethasone treatment group: tail vein injection LPSO111:B4 20mg/kg; Back injection dexamethasone boron 10mg/kg; Simulating peptide treatment group: injection simulating peptide 5mg/kg behind the tail vein injection LPS 20mg/kg.All animals administer volumes are 0.2ml/20g, are administered once altogether.
Observation index is 7 days mortality ratio of animal.
3.2 result
The internal toxin attacks mouse of antibiotic/antiendotoxic mimic peptide of the present invention all has provide protection in various degree, sees Table 4.
The provide protection of the internal toxin attacks mouse of table 4 antibiotic/antiendotoxic mimic peptide of the present invention
| Grouping | Dosage (mg/kg) | Laboratory animal (only) | Dead animal (only) | The P value |
| LPS | - | 10 | 10 | |
| | 10 | 10 | 2 | ＜0.01 |
| | 5 | 10 | 0 | ＜0.01 |
| | 5 | 10 | 1 | ＜0.01 |
| BNEP9901317 | 5 | 10 | 0 | ＞0.01 |
| BNEP9901315 | 5 | 10 | 5 | ＞0.05 |
| BNEP9901161 | 5 | 10 | 4 | ＞0.05 |
| BNEP9901321 | 5 | 10 | 1 | ＞0.01 |
| BNEP9801286 | 5 | 10 | 3 | ＞0.05 |
| BNEP9901174 | 5 | 10 | 6 | ＞0.05 |
| BNEP9801013 | 5 | 10 | 1 | ＜0.01 |
4. antibiotic/antiendotoxic mimic peptide of the present invention is to the therapeutic action of rat endotoxemia
4.1 experimental technique
Adopting rat to keep somewhere cardiac catheterization carries out administration and gets blood
A: rat is kept somewhere cardiac catheterization: after the animal via 1% vetanarcol 4mg/100g body weight intraperitoneal anesthesia, every animal is measured the distance of cervical incision to heart earlier, to determine intubation length, skin of neck is after conventional preserved skin, sterilization, be about the transverse incision of 0.5cm to the mid point work one of heart in neck outside lower jaw, expose row vein intubate behind the external jugular vein, conduit is fixed after subcutaneous tunnel is drawn to the neck and be fixing, injection 12500u/100ml heparin 0.2ml anti-freezing in the pipe.
B: administration and get blood
Put 28 of 24 hours Wistar rats of heart catheter, the cleaning level, male and female half and half, body weight 220-260g is divided into 7 groups at random, 4 of every treated animals.Control group: careful tube injection LPS O111:B4 10mg/kg; The simulating peptide treatment group; Earlier careful tube injection LPS O111:B4 10mg/kg, back injection simulating peptide 10mg/kg.All animals all are administered once altogether.TNF-α, IL-6 (TNF-α and IL-6 test kit are respectively French Diaclone company, U.S. Biosource company product) are surveyed in blood sampling in 0,1,2,4,6,12,24,48 hour behind injectable drug.
4.2 result
Antibiotic/antiendotoxic mimic peptide of the present invention all has in various degree restraining effect (legend 16-17) to the generation of the rat TNF-α of endotaxin mediate and IL-6.
Antibiotic/antiendotoxic mimic peptide of the present invention, it is the BPI simulating peptide that molecular weight is little, can stablize duplication of production, antibiotic/antiendotoxic mimic peptide of the present invention, it is the active polypeptide molecule, biological function with all or part of BPI, as toxicity isoreactivity in sterilization and the neutralization, be a kind of extremely promising treatment G
-The medicine of infectation of bacteria, people's endotoxemia has been filled up the blank in this field.
Claims (5)
1. antibiotic/the antiendotoxin peptide, its type is an amino acid, and topological framework is linear, and molecule type is a peptide, and the sequence of molecule is made up of an amino acid whose word brevity code, and is optional from following sequence peptide:
KSKVGFLQLLFHKKLRGSLTKLKG，
KSKVGWLILLFHKK，
KTKVGFLIQLFHKK，
KTKVGFLIQLFHKK，
KTKVGFLIQLFHKK，
KGKVGFLIQLFHKK，
KTKGGFLQLLFHKK，
KGKGGFLQLLFHKK，
KSKGGFLQLLFHKK，
KGKGGWLIQLFHKK，
KSKGGWLIQLFHKK，
KTKGGWLIQLFHKK，
KGKVGFLQLLFHKK，
KTKVGFLQLLFHKK，
KSKVGWLQLLFHKK，
KGKVGWLQLLFHKK，
KTKVGWLQLLFHKK，
KSKSGFLQLLFHKK，
KGKGGFLIQLFHKK, or
KSKHLGKAQKRFLK。
2. according to claim 1 antibiotic/the antiendotoxin peptide, it is characterized in that, be selected from following sequence peptide:
KSKVGFLQLLFHKKLRGSLTKLKG，
KTKVGFLIQLFHKK，
KTKGGFLQLLFHKK，
KGKGGFLQLLFHKK，
KSKGGFLQLLFHKK，
KGKGGWLIQLFHKK，
KSKGGWLIQLFHKK，
KTKGGWLIQLFHKK，
KGKVGFLQLLFHKK，
KTKVGFLQLLFHKK，
KTKVGWLQLLFHKK，
KSKVGWLQLLFHKK，
KSKSGFLQLLFHKK，
KGKGGFLIQLFHKK, or
KSKHLGKAQKRFLK。
3. prepare claim 1 described antibiotic/method of antiendotoxin peptide, it is characterized in that, under area of computer aided, carry out the molecular designing of mimic peptide, board design go out to have stronger sterilization and in and the active peptide molecule of LPS, synthetic with Merrifield solid-phase polypeptide chemical synthesis process, the solid-phase polypeptide chemical synthesis process is:
A. peptide resin is synthetic
Take by weighing an amount of Fmoc-L-Lys-PEG-PS resin or other polypeptide synthetic resins and pour in the synthetic post, add solvent-swollen, take by weighing an amount of Fmoc-amino acid successively in suitable container according to the aminoacid sequence of antibiotic/antiendotoxic mimic peptide of the present invention.Solid state chemistry synthetic method according to the Merrifield invention holds beginning to carry out successively according to the aminoacid sequence of antibiotic/antiendotoxic mimic peptide of the present invention from C, in synthetic post, add the DMF Deblock liquid that contains 20% hexahydropyridine and take off peptide resin Fmoc blocking group, add in the synthetic post after adding an amount of DMF dissolving Fmoc-amino acid, the DMF solution that adds an amount of diisopropylethylamine and azepine benzo triazole methyl urea hexafluorophosphate more respectively carries out coupling reaction in post, peptide resin in the synthetic post in the coupled back Fmoc-amino acid of DMF flush away remnants, preparation process such as rare gas element dries up, so circulation is finished the peptide chain formation of whole antibiotic/antiendotoxic mimic peptide and is obtained its peptide resin; From synthetic post, take out synthetic antibiotic/the antiendotoxin peptide resin, put freeze-drying in the freeze drier, it is standby to obtain peptide resin;
B. the cracking of peptide resin
Get above-mentioned peptide resin in container, add lysate: 4.5ml trifluoroacetic acid+0.25ml thio phenyl methyl ether+0.1ml methyl-phenoxide+0.15ml 1, in room temperature lucifuge reaction 2 hours, filter, filtrate adds in advance in the refrigerated anhydrous diethyl ether after room temperature concentrates, freezing spending the night, centrifugal, abandon supernatant liquor, precipitation is dissolved with an amount of Glacial acetic acid, dry in the freezing rearmounted freeze drier, get thick peptide;
The purifying of c. thick peptide
Get the thick peptide of BNEP and add an amount of no heat source water dissolving, obtain sample solution, filter and collect sample filtrate; With acetonitrile and there is not heat source water 0.45u membrane filtration, add 0.1% trifluoroacetic acid respectively, mixing is used the C18 reversed-phase column, mobile phase A: the 100%H2O+0.1% trifluoroacetic acid; Mobile phase B: 100% second is fine+0.1% trifluoroacetic acid, from 0 to 15 minute gradient elution purifying, the smart peptide of BNEP.
Claim 1 described antibiotic/the antiendotoxin peptide is in the application that is used for preparing the kill bacteria medicine.
Claim 1 described antibiotic/the antiendotoxin peptide is in the application that is being used for preparing treatment endotoxemia medicine.
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