CN106188233B - A kind of polypeptide that treating S. aureus L-forms mastitis for milk cows and preparation process - Google Patents
A kind of polypeptide that treating S. aureus L-forms mastitis for milk cows and preparation process Download PDFInfo
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- CN106188233B CN106188233B CN201610567471.XA CN201610567471A CN106188233B CN 106188233 B CN106188233 B CN 106188233B CN 201610567471 A CN201610567471 A CN 201610567471A CN 106188233 B CN106188233 B CN 106188233B
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- C—CHEMISTRY; METALLURGY
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- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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Abstract
The present invention provides a kind of polypeptide for treating S. aureus L-forms mastitis for milk cows and preparation process, provide a kind of new peptide material: pentapeptide, Leu-Pro-Arg-Asp-Ala can significantly inhibit S. aureus L-forms sorting enzymatic activity, reduce bacterial surface protein anchoring, it hinders bacterial adhesion to colonize, there is preferable therapeutic effect to S. aureus L-forms mammitis.Since internal polypeptide degradation is amino acid, the latter is consistent with the basic composition unit of dairy products, complete noresidue；In addition, polypeptide mainly using S. aureus L-forms sorting enzyme as target, is selection pressure to bacterium, is not easy in-ductive drug -tolerance.
Description
Technical field
The present invention provides a kind of polypeptide for treating S. aureus L-forms mastitis for milk cows, for a kind of new polypeptide, can be used for treating gold
Portugal's bacterium mastitis for milk cows disease；The invention also discloses the preparation processes of the polypeptide, belong to biopharmaceutical technology.
Background technique
Sorting enzyme (Sortase) is most of Gram-positive pathogens bacterium (such as streptococcus and staphylococcus aureus) table
The key protein of face pathogenicity proteins anchoring can recognize surface protein of the catalysis containing " LPXTG " sequence and be anchored to it carefully
Cell wall, it is indispensable to the pathogenicity of pathogen, and its structure is highly conserved in Gram-positive pathogens bacterium.Mastitis for milk cows
It is the important diseases for endangering dairy production, causes heavy economic losses and seriously endanger public health.Every, China disease at present
The loss of ox is about 2000-3600 member, annual because economic loss caused by mammitis is up to 30,000,000,000 yuan.Mastitis for milk cows
Pathogenic bacteria are mainly Gram-positive pathogenic bacterium, including S. aureus L-forms and streptococcus etc., and wherein S. aureus L-forms are the weights of mastitis for milk cows
Pathogenic bacteria are wanted, are distributed widely in nature, it is stronger to the tolerance of external environment.It is directed to the treatment master of mastitis for milk cows at present
Will be based on antibiotic treatment, but antibiotic treatment not only promotes the occurrence and development of bacterial drug resistance, antibiotic residue
Seriously threaten public food safety.Pathogenic bacteria resistance to drugs is got worse and the appearance of national dairy products safety standard etc. is compeled to be essential
Want drug resistance low and the newtype drug of drug residue free or low-residual.
Summary of the invention
The present invention provides a kind of polypeptide for treating S. aureus L-forms mastitis for milk cows, can be significant for a kind of new polypeptide compound
S. aureus L-forms are inhibited to sort enzymatic activity.
Invention further provides a kind of preparation processes of polypeptide for treating S. aureus L-forms mastitis for milk cows, are suitable for industry
Metaplasia produces.
A kind of polypeptide for treating S. aureus L-forms mastitis for milk cows described in the present invention, referred to as: pentapeptide；Leu-Pro-Arg-Asp-
Ala；Its molecular structure is as follows:
Molecular formula is C24H42N8O8；Molecular weight is 570.31
A kind of preparation process of polypeptide that treating S. aureus L-forms mastitis for milk cows described in the present invention, comprising the following steps:
1) peptide bond coupling reaction is generated
Weigh 2.56g(1mmol) Rink Amide MBHA resin, it is fitted into the reaction flask of automatic synthesizer, Fmoc protection
Amino acid derivativges (Fmoc-Ala-OH, Fmoc-Asp (OtBu)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Pro-OH,
Fmoc-Leu-OH is equivalent), coupling reagent, DMF be respectively charged into each solvent bottle (Fmoc- amino acid derivativges and coupling reagent
It is excessive relative to 4 times of resin (mole)) start to synthesize, specific synthetic parameters are as follows: reaction temperature is 25 DEG C, the coupling reaction time
It is set to 50 minutes, the deprotection time is 10 minutes (5min × 2 time), and coupling reaction and deprotection reaction solvent add charge and be
50ml, resin washing times are 6 times, each 1min.After the last one amino acid couplings, mPEG-SC washes resin 5 with methanol
It is secondary, it is 30 minutes dry to be passed through nitrogen.
2) it is deprotected and cuts peptide-resin
Deprotection cutting reagent is TFA/EDT/ thioanisole/anisole (92.5: 2.5: 2.5: 2.5) (volume ratio), is taken off
It is 60ml that protection cutting reagent, which adds charge, in N2It reacts 1 hour, filters under protection, at 25 DEG C, collect filtrate.Resin is cut again
30 minutes, merge collection liquid.
3) it precipitates
Cold anhydrous ether 600ml is added into reaction solution, is precipitated at -20 DEG C 20 minutes, then with 8000rpm centrifugation 10
Minute, precipitating washes secondary, vacuum drying with 60ml anhydrous ether again, obtains pentapeptide crude product 560mg, and thick peptide yield is 98.2%.
4) purifying of thick peptide
HPLC purification process: thick peptide is configured to the aqueous solution of 100mg/ml, sample introduction 10ml, Mobile phase B is from 5% to 15%
Gradient elution 30min collects product peak.The sample solution vacuum distillation of collection is removed into acetonitrile, vacuum freezedrying.
Mobile phase: A phase: water, 0.1%TFA, B phase: 90% acetonitrile solution, 0.1%TFA
Gradient: the concentration of B is from 5% to 15%
Elution time: 10 min
Detection wavelength: 210 nm
Flow velocity: 10ml/min
Purification process: pentapeptide crude product is configured to the aqueous solution that concentration is 100 mg/ml, and 10 ml of sample introduction, Mobile phase B is from 5%
To 15% gradient elution, 30 min, product peak is collected.The sample solution vacuum distillation of collection is removed into acetonitrile, vacuum freezedrying.
5) polyethyleneglycol modified pentapeptide
Pentapeptide is dissolved in the aqueous solution for being configured to 1-10 mg/ml in distilled water, is with phosphate buffer (PBS) pH
5.5-8.5 is diluted to 5 times of volumes, and the acetonitrile solution of above-mentioned 20 mg/ml reactive polyethylene glycol, reactive polyethylene glycol is then added
With pentapeptide molar ratio be 5-20:1.After room temperature reaction 1-4 hours, is terminated and reacted with glycine, use reversed-phase high performance liquid chromatography
Purifying, main peak eluent rotate reduced pressure at 40 DEG C, and concentrate freeze-drying obtains polyethylene glycol modified product.
6) Mass Spectrometer Method determines the sequence of synthesis pentapeptide.
A kind of polypeptide for treating S. aureus L-forms mastitis for milk cows described in the present invention, by sorting enzyme active suppression test, glimmering
Signal test, cell invasion test and mouse S. aureus L-forms mammitis therapeutic test confirm that polypeptide sorts enzymatic activity to S. aureus L-forms
Inhibiting effect and its therapeutic effect to S. aureus L-forms mammitis.
The positive effect of the present invention is: provide a kind of new peptide material: pentapeptide, Leu-Pro-Arg-Asp-Ala,
S. aureus L-forms sorting enzymatic activity can be significantly inhibited, bacterial surface protein anchoring is reduced, hinders bacterial adhesion to colonize, to S. aureus L-forms
Mammitis has preferable therapeutic effect.Since internal polypeptide degradation is amino acid, the basic composition unit of the latter and dairy products
Unanimously, complete noresidue；In addition, polypeptide mainly using S. aureus L-forms sorting enzyme as target, is selection pressure to bacterium, is not easy to induce resistance to
Pharmacological property.
Detailed description of the invention
Fig. 1 ~ Fig. 3 is the inhibiting effect that 1 ~ embodiment of embodiment, 3 polypeptide sorts enzymatic activity to S. aureus L-forms；
Fig. 4 ~ Fig. 6 is the influence after 1 ~ embodiment of embodiment, 3 polypeptide is handled to the anchoring of S. aureus L-forms surface protein；
Fig. 7 ~ Fig. 9 is influence of 1 ~ embodiment of embodiment, 3 polypeptide to bacteria attack host cell J774；
Figure 10 ~ Figure 12 is that 1 ~ embodiment of embodiment, 3 polypeptide inhibits to colonize in S. aureus L-forms breast tissue；
Figure 13 ~ Figure 15 is that 1 ~ embodiment of embodiment, 3 polypeptide reduces inflammatory factor in S. aureus L-forms breast tissue.
Specific embodiment
By following embodiment further illustrate description the present invention, do not limit the invention in any way, without departing substantially from
Under the premise of technical solution of the invention, easy to accomplish any of those of ordinary skill in the art made for the present invention changes
Dynamic or change is fallen within scope of the presently claimed invention.
Embodiment 1
Peptide systhesis
Weigh 2.56g(1mmol) Rink Amide MBHA resin, it is fitted into the reaction flask of automatic synthesizer, Fmoc protection
Amino acid derivativges (Fmoc-Ala-OH, Fmoc-Asp (OtBu)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Pro-OH,
Fmoc-Leu-OH is equivalent), coupling reagent (Py-BOP Reagent and HOBt Hydrate), DMF be respectively charged into each solvent bottle
In (Fmoc- amino acid derivativges and coupling reagent are excessive relative to 4 times of resin (mole)) start to synthesize, specific synthetic parameters are such as
Under: reaction temperature is 25 DEG C, and the coupling reaction time is set to 50 minutes, and the deprotection time is 10 minutes (5min × 2 time), and coupling is anti-
It answers and deprotection reaction solvent adds charge as 50ml, resin washing times are 6 times, each 1min.The last one amino acid couplings
After, mPEG-SC is washed resin 5 times with methanol, is passed through dry 30 min of nitrogen.Deprotection cutting reagent is TFA/EDT/ benzene first
Thioether/anisole (92.5: 2.5: 2.5: 2.5) (volume ratio), it is 60ml that deprotection cutting reagent, which adds charge, in N2Under protection,
1 h is reacted at 25 DEG C, is filtered, and filtrate is collected.Resin is cut 30 minutes again, merges collection liquid.Using the anhydrous ether precipitation method and
HPLC method obtains purification desired polypeptides；Molecular formula is C24H42N8O8；Molecular weight is 570.31.
Polypeptide of the present invention is shown in Fig. 1 to the inhibiting effect of S. aureus L-forms sorting enzymatic activity；S. aureus L-forms surface protein is anchored
Influence diagram 4；Fig. 7 is shown in influence to bacteria attack host cell J774；Inhibit to colonize in S. aureus L-forms breast tissue and sees Figure 10；Drop
Inflammatory factor is shown in Figure 13 in low S. aureus L-forms breast tissue.
Embodiment 2
Peptide systhesis
Weigh 2.56g(1mmol) Rink Amide MBHA resin, it is fitted into the reaction flask of automatic synthesizer, Fmoc protection
Amino acid derivativges (Fmoc-Ala-OH, Fmoc-Asp (OtBu)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Pro-OH,
Fmoc-Leu-OH is equivalent), coupling reagent (Py-BOP Reagent and HOBt Hydrate), DMF be respectively charged into each solvent bottle
In (Fmoc- amino acid derivativges and coupling reagent are excessive relative to 4 times of resin (mole)) start to synthesize, specific synthetic parameters are such as
Under: reaction temperature is 28 DEG C, and the coupling reaction time is set to 40 minutes, and the deprotection time is 15 minutes (5min × 3 time), and coupling is anti-
It answers and deprotection reaction solvent adds charge as 50ml, resin washing times are 6 times, each 1min.The last one amino acid couplings
After, mPEG-SC is washed resin 5 times with methanol, is passed through dry 30 min of nitrogen.Deprotection cutting reagent is TFA/EDT/ benzene first
Thioether/anisole (92.5: 2.5: 2.5: 2.5) (volume ratio), it is 60ml that deprotection cutting reagent, which adds charge, in N2Under protection,
1 h is reacted at 25 DEG C, is filtered, and filtrate is collected.Resin is cut 30 minutes again, merges collection liquid.Using the anhydrous ether precipitation method and
HPLC method obtains purification desired polypeptides.Molecular formula is C24H42N8O8；Molecular weight is 570.31.
Polypeptide of the present invention is shown in Fig. 2 to the inhibiting effect of S. aureus L-forms sorting enzymatic activity；S. aureus L-forms surface protein is anchored
Influence diagram 5；Fig. 8 is shown in influence to bacteria attack host cell J774；Inhibit to colonize in S. aureus L-forms breast tissue and sees Figure 10；Drop
Inflammatory factor is shown in Figure 14 in low S. aureus L-forms breast tissue.
Embodiment 3
Peptide systhesis
Weigh 2.56g(1mmol) Rink Amide MBHA resin, it is fitted into the reaction flask of automatic synthesizer, Fmoc protection
Amino acid derivativges (Fmoc-Ala-OH, Fmoc-Asp (OtBu)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Pro-OH,
Fmoc-Leu-OH is equivalent), coupling reagent (Py-BOP Reagent and HOBt Hydrate), DMF be respectively charged into each solvent bottle
In (Fmoc- amino acid derivativges and coupling reagent are excessive relative to 4 times of resin (mole)) start to synthesize, specific synthetic parameters are such as
Under: reaction temperature is 22 DEG C, and the coupling reaction time is set to 80 minutes, and the deprotection time is 10 minutes (5min × 2 time), and coupling is anti-
It answers and deprotection reaction solvent adds charge as 50ml, resin washing times are 6 times, each 1min.The last one amino acid couplings
After, mPEG-SC is washed resin 5 times with methanol, is passed through dry 30 min of nitrogen.Deprotection cutting reagent is TFA/EDT/ benzene first
Thioether/anisole (92.5: 2.5: 2.5: 2.5) (volume ratio), it is 60ml that deprotection cutting reagent, which adds charge, in N2Under protection,
1 h is reacted at 25 DEG C, is filtered, and filtrate is collected.Resin is cut 30 minutes again, merges collection liquid.Using the anhydrous ether precipitation method and
HPLC method obtains purification desired polypeptides.Molecular formula is C24H42N8O8；Molecular weight is 570.31.
Polypeptide of the present invention is shown in Fig. 3 to the inhibiting effect of S. aureus L-forms sorting enzymatic activity；S. aureus L-forms surface protein is anchored
Influence diagram 6；Fig. 9 is shown in influence to bacteria attack host cell J774；Inhibit to colonize in S. aureus L-forms breast tissue and sees Figure 10；Drop
Inflammatory factor is shown in Figure 15 in low S. aureus L-forms breast tissue.
Pass through the medical treatment activity of polypeptide described in following present invention:
1, sorting inhibition of enzyme activity experiment
Sorting enzyme (4 mM) and three embodiment polypeptides of various concentration (3.125 μ Μ are differed to 25 μ Μ) is purified to be incubated for altogether
30 min(37 DEG C), the fluorescent peptide substrate (Dabcyl-QALPTTGEE-Edans) (10 μM) modified is added and is incubated for 1 h again
(37 DEG C).Using fluorescence intensity in microplate reader detection reaction system, (excitation wavelength and launch wavelength are respectively 350 nm and 520
Nm).The experimental results showed that sorting enzymatic activity is remarkably decreased, and dosage is presented in this effect after three embodiment polypeptides are handled
Dependence, it is 10.61 μ Μ (Fig. 1 ~ Fig. 3) that polypeptide, which inhibits the active IC50 of sorting enzyme,.
, fluorescent marker test
S. aureus L-forms USA300 and its sorting enzyme knock out sub- USA300 △ SrtA and three embodiment polypeptides co-culture 2.5 h
Thallus is collected in (37 DEG C), centrifugation (3000 rpm, 10 min), three times using PBS cleaning thallus, the resistance to surface albumen of FITC label
A antibody marks bacterial surface protein, and bacterium surface after the three embodiment polypeptide processing of application confocal laser scanning microscope
The influence of albumen anchoring.The experimental results showed that sorting enzyme knocks out the anchoring that sub- USA300 △ SrtA shows no surface protein A, through three
The anchoring (Fig. 4 ~ Fig. 6) for showing albumin A can be significantly reduced after a embodiment polypeptide processing.
, cell invasion test
Host cell J774 and the S. aureus L-forms of three embodiment polypeptide processing are co-cultured in the DMEM containing 10% fetal calf serum
(37 DEG C, 1 h) in culture medium.The termination invasion of 300 μ g/mL gentamicins are added to test, lytic cell, coated plate after doubling dilution
Bacterium colony counts, and analyzes the influence under three embodiment polypeptide processing to bacteria attack host cell.The result shows that three embodiments
Polypeptide can significantly inhibit bacteria attack host cell J774(Fig. 7 ~ Fig. 9).
, mouse S. aureus L-forms mastitis treatment test
Lactation female rat (BALB/c) establishes mouse S. aureus L-forms mazoitis model through milk duct injection S. aureus L-forms, while through latex dust
It is administered (50 mg/kg) (three embodiment polypeptides)；It is administered that (three embodiments are more during successive treatment again every 12 h
Peptide).48 h after infection, cervical dislocation put to death infecting mouse and win breast tissue, carry out tissue bacterium colony and colonize number analysis and group
Tissue inflammation factorial analysis.(figure is colonized in S. aureus L-forms tissue the experimental results showed that significantly inhibiting after three embodiment polypeptide therapeutics
10 ~ 12) and reduce tissue in Inflammatory Factors Contents (Figure 13 ~ 15).
<110>Jilin University
<120>
<140>
<160> 1
<210> 1
<211>
<212>polypeptide
<213>pentapeptide
<400> 1
Leu-Pro-Arg -Asp-Ala
Claims (2)
1. a kind of polypeptide for treating S. aureus L-forms mastitis for milk cows, referred to as: pentapeptide, Leu-Pro-Arg-Asp-Ala, molecular structure
It is as follows:
Molecular formula is C24H42N8O8；Molecular weight is 570.31.
2. a kind of preparation process for the polypeptide for treating S. aureus L-forms mastitis for milk cows as described in claim 1, comprising the following steps:
1) peptide bond coupling reaction is generated
2.56g Rink Amide MBHA resin is weighed, is fitted into the reaction flask of automatic synthesizer, fmoc-protected amino acid spreads out
Biology, coupling reagent, DMF are respectively charged into each solvent bottle, wherein Fmoc- amino acid derivativges and coupling reagent are relative to tree
4 times of rouge, that is, be 4 mol, start to synthesize, specific synthetic parameters are as follows: reaction temperature is 25 DEG C, and the coupling reaction time is set to 50
Minute, the deprotection time is 5min × 2 time, and it is 50ml, resin washing times that coupling reaction and deprotection reaction solvent, which add charge,
It is 6 times, each 1min；It after the last one amino acid couplings, is washed resin 5 times with methanol, it is 30 minutes dry to be passed through nitrogen；
The amino acid derivativges are as follows: Fmoc-Ala-OH, Fmoc-Asp (OtBu)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-
Pro-OH, Fmoc-Leu-OH equivalent weigh；
The coupling reagent are as follows: Py-BOP Reagent and HOBt Hydrate；
2) it is deprotected and cuts peptide-resin
It is 92.5: 2.5: 2.5: 2.5 that deprotection cutting reagent is TFA/EDT/ thioanisole/anisole by volume, deprotection
It is 60ml that cutting reagent, which adds charge, in N2It reacts 1 hour, filters under protection, at 25 DEG C, collect filtrate；Resin cuts 30 points again
Clock merges collection liquid；
3) it precipitates
Cold anhydrous ether 600ml is added into reaction solution, is precipitated at -20 DEG C 20 minutes, is then centrifuged 10 points with 8000rpm
Clock, precipitating wash secondary, vacuum drying with 60ml anhydrous ether again；
4) purifying of thick peptide
HPLC purification process: thick peptide is configured to the aqueous solution of 100mg/ml, sample introduction 10ml, Mobile phase B gradient from 5% to 15%
30min is eluted, product peak is collected；The sample solution vacuum distillation of collection is removed into acetonitrile, vacuum freezedrying；
Mobile phase: A phase: water, 0.1%TFA, B phase: 90% acetonitrile solution, 0.1%TFA；
Gradient: the concentration of B is from 5% to 15%；
Elution time: 10 min；
Detection wavelength: 210 nm；
Flow velocity: 10ml/min；
5) polyethyleneglycol modified pentapeptide
Pentapeptide is dissolved in the aqueous solution for being configured to 1-10 mg/ml in distilled water, is 5.5-8.5 dilution with phosphate buffer pH
To 5 times of volumes, the acetonitrile solution of 20 mg/ml reactive polyethylene glycols is then added, reactive polyethylene glycol and pentapeptide molar ratio are 5-
20:1；After room temperature reaction 1-4 hours, is terminated and reacted with glycine, purified with reversed-phase high performance liquid chromatography, main peak eluent is 40
It rotates and is concentrated under reduced pressure at DEG C, concentrate freeze-drying obtains polyethylene glycol modified product；
6) Mass Spectrometer Method determines the sequence of synthesis pentapeptide.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1520881A (en) * | 2003-02-13 | 2004-08-18 | 上海高科联合生物技术研发有限公司 | Sterilizing preparation for preventing and curing bovine mastitis and preparing process thereof |
| CN1970750A (en) * | 2006-12-11 | 2007-05-30 | 中国人民解放军军事医学科学院军事兽医研究所 | Garget-resistant milk cow breeding method |
| CN102524558A (en) * | 2010-12-27 | 2012-07-04 | 内蒙古伊利实业集团股份有限公司 | Feed additive for treating dairy cattle recessive mastitis |
| CN103386115A (en) * | 2012-05-11 | 2013-11-13 | 浙江华尔成生物药业股份有限公司 | Application of thymopentin in preparing medicine used for treating mastitis |
-
2016
- 2016-07-19 CN CN201610567471.XA patent/CN106188233B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1520881A (en) * | 2003-02-13 | 2004-08-18 | 上海高科联合生物技术研发有限公司 | Sterilizing preparation for preventing and curing bovine mastitis and preparing process thereof |
| CN1970750A (en) * | 2006-12-11 | 2007-05-30 | 中国人民解放军军事医学科学院军事兽医研究所 | Garget-resistant milk cow breeding method |
| CN102524558A (en) * | 2010-12-27 | 2012-07-04 | 内蒙古伊利实业集团股份有限公司 | Feed additive for treating dairy cattle recessive mastitis |
| CN103386115A (en) * | 2012-05-11 | 2013-11-13 | 浙江华尔成生物药业股份有限公司 | Application of thymopentin in preparing medicine used for treating mastitis |
Non-Patent Citations (1)
| Title |
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| 一种抗革兰氏阳性致病菌新型靶酶;罗立新 等;《生物化学与生物物理进展》;20060930;第33卷(第9期);第828-833页 |
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