CN1763081A - Antibiotic dipeptide and its chemical modifier, its preparation method and uses - Google Patents
Antibiotic dipeptide and its chemical modifier, its preparation method and uses Download PDFInfo
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- CN1763081A CN1763081A CN 200510037303 CN200510037303A CN1763081A CN 1763081 A CN1763081 A CN 1763081A CN 200510037303 CN200510037303 CN 200510037303 CN 200510037303 A CN200510037303 A CN 200510037303A CN 1763081 A CN1763081 A CN 1763081A
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Abstract
The present invention discloses one kind of antibacterial dipeptide and its chemical modifier, preparation process and application. The antibacterial dipeptide has DNA sequence of TCTAAT and amino acid sequence of Ser Asn. During yeast expression, it has one serine glycosylated site, and the transformant fermenting product and the synthesized dipeptide have the effects of resisting hay bacillus, Bacilluspyocyaneus, Staphylococcus aureus, etc. The dipeptide through alpha-amino radical acetylation has molecule expression of Ac-Ser-Asn, and the modifier has inhibition on hay bacillus, Bacilluspyocyaneus and Staphylococcus aureus. The dipeptide of the present invention may be used as precursor for developing new antibiotics effective on Bacilluspyocyaneus, Staphylococcus aureus, etc. In addition, owing to the powerful inhibition on hydrophilic aeromonad, the present invention may find application in the field of aquatic cultivation.
Description
Technical field
The present invention relates to the genetically engineered field, be specifically related to a kind of antibiotic dipeptide and chemical modification object thereof, preparation method and application.
Background technology
Penicillin has successfully solved the difficult problem of infection of staphylococcus aureus clinically as antibacterials the earliest, and the Macrolide of Wen Shiing, aminoglycoside antibiotics make pneumonia, phthisical mortality ratio reduce by 80% again subsequently.Have been found that many kinds of microbiotic up to now, they are played a great role in the treatment infectious diseases, have prolonged human predicted life greatly.
Most microbiotic are synthetic by microorganism, wherein 2/3rds produced by streptomycete.According to antibiotic antibiotic mechanism and character, it can be divided into five big classes: β-Nei Xiananleikangshengsu, Macrolide, aminoglycosides, quinolones and chloromycetin, clindamycin tetracyclines.Various antibiotic antibacterial effects, majority is bacteriostatic action, germicidal action of minority tool or bacteriolysis.The antibiotic mechanism of action can reduce: suppress the synthetic of nucleic acid; Synthesizing of arrestin matter; Change the permeability of cytolemma; The formation of interference cell wall; Act on energy metabolism system or as metabolic antagonist.
The performance of early stage bacterial resistance is mainly certain bacterium to certain class medicine resistance.From the later stage eighties to the nineties, having occurred very refractory multi-drug resistant bacteria in the gram-positive cocci infects, the drug-fast multidrug resistant gram-positive cocci of this height except that indivedual microbiotic almost to all antibacterials resistance all, form very big threat to clinical, caused the shock in the whole world and the attention of height.The genetic mechanism of bacterial drug resistance and branch give the basis can reduce following six aspects: one, the antibacterials deactivating enzyme produced of bacterium and cause resistance; Two, the enzyme (albumen) of bacterium production is protected the antibacterials target site and resistance; Three, bacterium obtains function replacement albumen (enzyme) and resistance; Four, the bacterial cell membrane permeability changes causes resistance; Five, initiatively the pumping function of bacterium and cause resistance; Six, the antibacterials target site of bacterium changes and causes resistance.
Some small peptides of coding inhibition or kill bacteria are called antibacterial peptide in the genome of many biologies.Antibacterial peptide has characteristics such as anti-microbial activity is strong, distribution is wide.But the general length of antibacterial peptide surpasses 10 amino acid, has certain immunogenicity and relatively poor biological accessibility.The good drug molecule amount of general biological accessibility is all below 500 dalton.If can develop the antibiotic oligopeptides of small molecules, then can fully excavate the antibiotic resource that makes new advances.So, develop antibiotic oligopeptides human beings'health, herding production, aquaculture etc. had great importance.
Summary of the invention
The objective of the invention is to overcome existing microbiotic and have chemical sproof problem, provide a sharp anti-microbial activity high antibiotic dipeptide.
Another object of the present invention provides the application of above-mentioned antibiotic dipeptide on the preparation antibacterials.
Further purpose of the present invention provides the chemical modification object of above-mentioned antibiotic dipeptide.
Further again purpose of the present invention provides the application of chemical modification object on the preparation antibacterials of above-mentioned antibiotic dipeptide.
The objective of the invention is to realize: make up the fusion dna fragment of yeast α mating factor leading peptide and dipeptides, be used for the secreting, expressing of dipeptides by following technical measures; Insert the yeast saccharomyces cerevisiae carrier that contains inducible promoter with EcoR I and Xba I double digestion; Recombinate with pastoris genomic dna after conversion for the linear DNA of avoiding not connecting, it is carried out digestion process with exonuclease III and Semen Phaseoli radiati Germinatus nuclease; Electricity swashs transformed saccharomyces cerevisiae behind the purifying; Transformant is forwarded to semi-lactosi abduction delivering flat board, covers the observation inhibition zone after the overnight growth with the upper strata substratum that contains bacterium; Or with surveying bacteriostasis rate behind the liquid nutrient medium abduction delivering; Positive colony is carried out pcr amplification, order-checking; Chemosynthesis part antibacterial peptide is used the agar diffusion method detection of active.
Antibiotic dipeptide of the present invention, dna sequence dna are TCTAAT, and aminoacid sequence is Ser Asn.Have a Serine glycosylation site when yeast expression, the tunning of transformant has the effect of anti-Bacillus subtilus, Pseudomonas aeruginosa, streptococcus aureus, Aeromonas hydrophila etc.The present invention utilizes the polypeptide automatic DNA synthesizer DNA to synthesize above-mentioned dipeptides not modified and through modifying.The not modified and acetylizad dipeptides molecular formula of process alpha-amino group end is respectively Ser-Asn, Ac-Ser-Asn.Antibiotic dipeptide alpha-amino group end acetylation modification thing Ac-Ser-Asn is inhibited to Bacillus subtilus, Pseudomonas aeruginosa, streptococcus aureus.
Beneficial effect of the present invention: the isolating antibiotic dipeptide of the present invention has the effect of anti-Bacillus subtilus, Pseudomonas aeruginosa, streptococcus aureus, Aeromonas hydrophila etc.The only remaining vancomycin of multidrug resistant clinically at present streptococcus aureus can be used, and has also occurred the bacterial strain to this antibiotics resistance at present.The dipeptides that the present invention finds can be as lead compound, to develop effective new antibiotic such as streptococcus aureus, Pseudomonas aeruginosa.In addition, because its strongly inhibited effect to Aeromonas hydrophila can be applied in aquaculture field.
Description of drawings
Cover the antibacterial effect figure of streptococcus aureus behind the positive transformant abduction delivering of Fig. 1 a;
Fig. 1 b is the design sketch that covers streptococcus aureus behind the empty carrier transformant abduction delivering, is negative contrast;
Cover the antibacterial effect figure of Bacillus subtilus behind the positive transformant abduction delivering of Fig. 2 a;
Fig. 2 b is the design sketch that covers Bacillus subtilus behind the empty carrier transformant abduction delivering, is negative contrast;
Cover the antibacterial effect figure of Aeromonas hydrophila behind the positive transformant abduction delivering of Fig. 3 a;
Fig. 3 b is the design sketch that covers Aeromonas hydrophila behind the empty carrier transformant abduction delivering, is negative contrast;
Fig. 4 is that 95.4% antibiotic dipeptide Ac-Ser-Asn point sample 100 μ g are at the formed antibacterial loop graph of the substratum that contains Bacillus subtilus for purity; Sterilized water is as negative contrast;
Fig. 5 is that full-automatic synthetic, purity are that 95.4% antibiotic dipeptide Ac-Ser-Asn point sample 100 μ g are at the formed antibacterial loop graph of the substratum that contains streptococcus aureus; 0.1 the conduct of μ l 100mg/ml penbritin is over against photograph, sterilized water is as negative contrast.
Embodiment
Embodiment 1
(1) makes up the fusion dna fragment of yeast α mating factor leading peptide and dipeptides, be used for the secreting, expressing of dipeptides.5 ' primer is＜1〉GCGAATTCAAAAATGAGATTTCCTTCAA.3 ' the primer of coding Ser-Asn is＜2〉CGTCTAGATCAATTAGATCTTTTCTCGAGAGAT, and 3 ' primer of other dipeptides of encoding is by the similar principles design.With having the active Pfu archaeal dna polymerase amplification of high-fidelity.The amplification template usage quantity is every 1ml PCR reaction solution 50ng plasmid pPICZ α A (the Invitrogen product contains yeast α mating factor leading peptide dna sequence dna).Amplification condition is: 94 ℃ of pre-sex change in 5 minutes; 94 ℃ 30 seconds, 46 ℃ 30 seconds, 72 ℃ 20 seconds, 30 circulations; 72 ℃ of 5 minutes benefits are flat.Amplified production precipitates 7 minutes with 1/10 pH5.22.5M NaAc and 2.5 times of absolute ethanol in 13000rpm.Use 70% washing with alcohol, 13000rpm precipitation 3 minutes.Hair dryer dries up.Be suspended in the sterile distilled water.Spend the night with EcoR I and Xba I double digestion, cumulative volume is 30 μ l.70 ℃ of heat inactivations 20 minutes.
(2) 5 μ g pYES2/CT (Invitrogen product) spend the night with EcoR I and Xba I double digestion, and cumulative volume is 30 μ l.70 ℃ of heat inactivations 20 minutes.Get the pYES2/CT of 6.5 μ l double digestions, add the PCR product of 6 μ l double digestions, 1.5 μ l 10X ligase enzyme damping fluids, 1 μ l T4 dna ligase, 16 ℃ of connections are spent the night.
(3) recombinate with pastoris genomic dna after conversion for the linear DNA of avoiding not connecting, need it to be carried out digestion process with nuclease.Connect the PCR purification column purifying (see product description) of product with Qiagen company.With 30 μ l H
2The O wash-out.Add 3.5 μ l 10X exonuclease III damping fluids and 1 μ l (20 unit) exonuclease III, handled 1 hour for 37 ℃.70 ℃ of inactivations 15 minutes.Add 3.5 μ l 10X Semen Phaseoli radiati Germinatus nuclease damping fluids and 30 μ l H
2O adds 1 μ l (20 unit) Semen Phaseoli radiati Germinatus nuclease again, handles 1 hour 20 minutes for 37 ℃.With the PCR purification column purifying of Qiagen company, with 13 μ l H
2The O wash-out.Getting 10 μ l does electric sharp.
(4) at first prepare the yeast competent cell.Yeast saccharomyces cerevisiae bacterial classification INVSc1 inoculation 200ml YPD with incubated overnight.30 ℃ of shake-flask culture are divided in the 50ml centrifuge tube to OD value 1.3 with culture, 5000rpm, and 4 ℃ are centrifugal 5 minutes.Remove supernatant.Be resuspended in cumulative volume 100ml Lithium Acetate/dithiothreitol (DTT) pretreatment fluid, 25 ℃ are incubated 1 hour.Be resuspended in the sterilized water of cumulative volume 200ml ice precooling.5000rpm, 4 ℃ centrifugal 5 minutes.Remove supernatant.Be resuspended in the sterilized water of cumulative volume 100ml ice precooling.5000rpm, 4 ℃ centrifugal 5 minutes.Remove supernatant.Be resuspended in the 1mol/L sorbyl alcohol of 20ml ice precooling.5000rpm, 4 ℃ centrifugal 5 minutes.Remove supernatant.Be resuspended in the 1mol/L sorbyl alcohol of 0.2ml ice precooling.Ice bath.The DNA (cumulative volume is 10 μ l) and the 20 μ g ssDNA (salmon sperm dna) that add 80 μ l yeast suspensions and 100ng in each sterile tube, mixing gently is transferred to the aseptic electricity of 0.2-cm and swashs cup, 10 ℃ of water-baths 5 minutes.1.8 doing electricity, kilovolt, 25 microfarads, 200 ohm swash.Adding 650 μ l 1mol/L sorbyl alcohols at once swashs in the cup to electricity.Mixing is transferred to culture tube gently, adds 650 μ l 2X YPD/1mol/L sorbyl alcohols, and 30 ℃ of shaking tables were cultivated 1 hour after the mixing.
(5) electricity is swashed mixture coating glucose/sorbyl alcohol and select flat board, in 30 ℃ of growths 3 days.Being forwarded to the glucose that does not contain sorbyl alcohol selects dull and stereotyped.
(6) be forwarded to glycerine/semi-lactosi abduction delivering flat board, in 30 ℃ of growths 1 day.Propose inoculation the day before yesterday and be used to detect various bacteria such as the active streptococcus aureus of antibacterial peptide, in 37 ℃ of overnight growth.
(7) respectively the bacteriums such as streptococcus aureus of 5 μ l overnight growth are added to 0.7% agarose/LB that 50 ℃ of autoclavings of 10ml are crossed.Being poured on length gently has on the glycerine/semi-lactosi abduction delivering flat board of yeast transformant.Leave standstill and solidified in 10 minutes.Make a call to an aperture in the place that does not have transformant with rifle head point, add 0.5 μ l50mg/ml penbritin, put super clean bench 5 minutes as over against photograph.Be inverted overnight growth at 37 ℃ of incubators.Observe inhibition zone, record is taken pictures.The dna sequence dna of positive colony amalgamation and expression is TCTAAT, and aminoacid sequence is Ser-Asn, and fungistatic effect is seen Fig. 1,2,3.This explanation antibiotic dipeptide Ser-Asn is inhibited to Aeromonas hydrophila, Bacillus subtilus, streptococcus aureus.
(8) insert fragment from the yeast transformant amplification.Get and grow in the yeast 1.5ml that glucose is selected logarithmic growth late period of substratum, centrifugal 15 seconds of 13000rpm removes supernatant.With the distilled water washing once, centrifugal 15 seconds of 13000rpm.Precipitation is suspended in 30 μ lTE damping fluids.On boiling water, boil 3min.The centrifugal 1min of 13000rpm.Supernatant is stored in-20 ℃.Get 3 μ l and be used for being PCR.According to carrier pYES2/CT sequences Design upstream and downstream primer.<3>CCTCTATACTTTAACGTCAAGGAGAAAAAACCCCGGATCG。<4>GGCGTGAATGTAAGCGTGACATAACTAATTACATGATGCG。Amplification condition is: 95 ℃ of pre-sex change in 5 minutes; 94 ℃ 30 seconds, 68 ℃ 2 minutes, 72 ℃ 30 seconds, 4 circulations; 94 ℃ 30 seconds, 68 ℃ 40 seconds, 72 ℃ 30 seconds, 41 circulations; 72 ℃ of 5 minutes benefits are flat.Select the Taq archaeal dna polymerase and the Pfu archaeal dna polymerase of Shanghai Bo Ya company for use.100 μ l reaction adds 5 Taq of unit archaeal dna polymerases and 2 Pfu of unit archaeal dna polymerases.Amplified fragments is long to be 570bp.Order-checking.
Used Lithium Acetate/dithiothreitol (DTT) pretreatment fluid prescription is (every 100ml): 0.1M Lithium Acetate (final concentration), 10mM Tris (final concentration), pH7.5,1mM EDTA (final concentration) adds the 10m1 0.1M dithiothreitol (DTT) of filtration sterilization to the above-mentioned solution of 90ml (final concentration is 10mM) behind the autoclaving.
It is (every liter) that used glucose/sorbyl alcohol is selected the plate culture medium prescription: and 1.7g YNB (no amino acid yeast nitrogen, Amresco, Inc.), 1M sorbyl alcohol, 10g glucose, 30mg leucine, 20mg Histidine, 30mg tryptophane, 20g agar.Autoclaving.Fall dull and stereotyped.
The used glucose that does not contain sorbyl alcohol selects dull and stereotyped prescription to be (every liter): and 1.7g YNB (no amino acid yeast nitrogen, Amresco, Inc.), 10g glucose, 30mg leucine, 20mg Histidine, 30mg tryptophane, 20g agar.Autoclaving.Fall dull and stereotyped.
The dull and stereotyped prescription of used glycerine/semi-lactosi abduction delivering is (every liter): (Amresco, Inc.), adjust pH is 4.1 to 1.7g YNB, 30ml glycerine, 30mg leucine, 20mg Histidine, 30mg tryptophane, 20g agar.Autoclaving.The final concentration that adds filtration sterilization is 0.5% semi-lactosi.Fall dull and stereotyped.
Used LB prescription is (500ml): 5g peptone, 2.5g yeast extract, 5g sodium-chlor, distilled water, pH7.0.
Embodiment 2
Synthesize dipeptides Ser-Asn in Shanghai Bo Ya company with Peptide synthesizer.Utilize Fmoc/tBu solid-phase polypeptide synthesis method synthetic.Take off Fmoc with 20% hexahydropyridine, dimethyl formamide.Dimethyl formamide, methyl alcohol, washed with dichloromethane.Crude product is through preparation HPLC purifying, and is freezing, gets pure product.Take by weighing the dipeptides of suitable weight, add water, boiled 8 minutes.
Embodiment 3
Utilize Fmoc/tBu solid-phase polypeptide synthesis method synthetic through the acetylizad dipeptides Ac-Ser-Asn of aminoterminal by the biochemical (Shanghai) Co., Ltd. of gill.Use Fmoc-Asn (trt)-Wang resin.Take off Fmoc with 20% hexahydropyridine, dimethyl formamide.Dimethyl formamide, methyl alcohol, washed with dichloromethane.Crude product is through preparation HPLC purifying, and is freezing, gets pure product.Take by weighing the dipeptides of suitable weight, add water, boiled 8 minutes.
Embodiment 4
The bacterial classification (Bacillus subtilus, streptococcus aureus) of 5 μ l overnight growth is added to 0.7% agarose/LB that 50 ℃ of left and right sides autoclavings of 10ml are crossed respectively, dull and stereotyped, leave standstill and solidified in 10 minutes.Beat a plurality of apertures with rifle head point, an aperture adds 100 μ g dipeptides Ac-Ser-Asn (ACSN), and an aperture adds the negative contrast of entry conduct, and a dull and stereotyped aperture of part adds the conduct of 0.1 μ l 50mg/ml penbritin over against photograph.Put super clean bench 5 minutes.Be inverted overnight growth at 37 ℃ of incubators.Observe inhibition zone, record is taken pictures.The results are shown in Figure 4,5.
Embodiment 5
The bacterial classification (Pseudomonas aeruginosa, Bacillus subtilus, streptococcus aureus) of 5 μ l overnight growth is added to 0.7% agarose/LB that 50 ℃ of left and right sides autoclavings of 10ml are crossed respectively, dull and stereotyped, leave standstill and solidified in 10 minutes.Beat a plurality of apertures with rifle head point, two apertures add 100 μ g dipeptides Ser-Asn and Ac-Ser-Asn (ACSN) respectively, and an aperture adds the negative contrast of entry conduct, and a dull and stereotyped aperture of part adds the conduct of 0.1 μ l 50mg/ml penbritin over against photograph.Put super clean bench 5 minutes.Be inverted overnight growth at 37 ℃ of incubators.Observe inhibition zone, survey antibacterial circle diameter.As shown in table 1.This has illustrated that Ac-Ser-Asn can effectively suppress the growth of streptococcus aureus, Bacillus subtilus, Pseudomonas aeruginosa or the like bacterial classification, has better antibacterial activity.Ser-Asn also has anti-microbial activity to Bacillus subtilus, Pseudomonas aeruginosa.
Table 1
Embodiment 6
The bacterial classification (Pseudomonas aeruginosa, Bacillus subtilus, streptococcus aureus) of 5 μ l overnight growth is added to 0.7% agarose/LB that 50 ℃ of left and right sides autoclavings of 10ml are crossed respectively, dull and stereotyped, leave standstill and solidified in 10 minutes.Beat a plurality of apertures with rifle head point, two apertures add 150 μ g dipeptides Ser-Asn and Ac-Ser-Asn (ACSN) respectively, and an aperture adds the negative contrast of entry conduct, and a dull and stereotyped aperture of part adds the conduct of 0.1 μ l 50mg/ml penbritin over against photograph.Put super clean bench 5 minutes.Be inverted overnight growth at 37 ℃ of incubators.Observe inhibition zone, survey antibacterial circle diameter.
Table 2
As shown in table 2.This has illustrated that Ac-Ser-Asn can effectively suppress the growth of streptococcus aureus, Bacillus subtilus, Pseudomonas aeruginosa or the like bacterial classification, has the excellent antibiotic activity.The antibacterial effect that the Ac-Ser-Asn consumption is big is better than the little antibacterial effect of consumption.Ser-Asn also has anti-microbial activity to Bacillus subtilus, Pseudomonas aeruginosa.
Claims (7)
1, a kind of antibiotic dipeptide, dna sequence dna are TCTAAT, and aminoacid sequence is Ser Asn.
2, the chemical modification object of the described antibiotic dipeptide of claim 1.
3, the chemical modification object of antibiotic dipeptide as claimed in claim 2 is characterized in that described chemical modification object is the acetylizad antibiotic dipeptide of aminoterminal, and aminoacid sequence is: Ac-Ser Asn.
4, the application of the described antibiotic dipeptide of claim 1 in the preparation antibacterials.
5, the application of the chemical modification object of the described antibiotic dipeptide of claim 2 in the preparation antibacterials.
6, application as claimed in claim 4 is characterized in that the application of described antibiotic dipeptide in the medicine of the anti-Aeromonas hydrophila of preparation, Bacillus subtilus, Pseudomonas aeruginosa, streptococcus aureus.
7, application as claimed in claim 5 is characterized in that the application of chemical modification object in the medicine of the anti-Bacillus subtilus of preparation, Pseudomonas aeruginosa, streptococcus aureus of described antibiotic dipeptide.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102618551A (en) * | 2012-03-15 | 2012-08-01 | 安徽希普生物科技有限公司 | Method for using brewer's yeast to express antibacterial peptide G13 |
| CN110506884A (en) * | 2019-08-30 | 2019-11-29 | 江苏大学 | A kind of double lysine antibacterial agents and preparation method and application |
| CN110583956A (en) * | 2019-08-30 | 2019-12-20 | 江苏大学 | Double-histidine antibacterial agent, preparation method and application |
| CN110637966A (en) * | 2019-08-30 | 2020-01-03 | 江苏大学 | Cold plasma treated bis-arginine antibacterial agent, preparation method and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1294258C (en) * | 2004-01-16 | 2007-01-10 | 刘秋云 | Method for separating antibiotic peptide and separated antibiotic peptide |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102618551A (en) * | 2012-03-15 | 2012-08-01 | 安徽希普生物科技有限公司 | Method for using brewer's yeast to express antibacterial peptide G13 |
| CN110506884A (en) * | 2019-08-30 | 2019-11-29 | 江苏大学 | A kind of double lysine antibacterial agents and preparation method and application |
| CN110583956A (en) * | 2019-08-30 | 2019-12-20 | 江苏大学 | Double-histidine antibacterial agent, preparation method and application |
| CN110637966A (en) * | 2019-08-30 | 2020-01-03 | 江苏大学 | Cold plasma treated bis-arginine antibacterial agent, preparation method and application |
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