CN109517046A - A kind of antibacterial polypeptide and its application based on outer membrane protein generting machanism - Google Patents
A kind of antibacterial polypeptide and its application based on outer membrane protein generting machanism Download PDFInfo
- Publication number
- CN109517046A CN109517046A CN201811433991.7A CN201811433991A CN109517046A CN 109517046 A CN109517046 A CN 109517046A CN 201811433991 A CN201811433991 A CN 201811433991A CN 109517046 A CN109517046 A CN 109517046A
- Authority
- CN
- China
- Prior art keywords
- ompf
- polypeptide
- outer membrane
- hand cleanser
- escherichia coli
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
- C07K14/245—Escherichia (G)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
- A61Q17/005—Antimicrobial preparations
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/10—Washing or bathing preparations
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Organic Chemistry (AREA)
- Dermatology (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Birds (AREA)
- Epidemiology (AREA)
- Medicinal Chemistry (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a kind of antibacterial polypeptides based on outer membrane protein generting machanism, are named as OmpF-16s, amino acid sequence is as shown in SEQ ID NO.1.By the gene cloning of OmpF-16s on pBAD carrier, the inducible protein expression on the plate containing arabinose (ara), SDS and EDTA, the results showed that can not survive after the bacterial expression polypeptide in the case where outer membrane interferes screening conditions.Company's synthesis polypeptide is further transferred to, Antimicrobial test is carried out, the results showed that polypeptide of the present invention has good bactericidal effect under the auxiliary of EDTA, to Escherichia coli.Further increase by one section in the N-terminal of OmpF-16s and lead peptide sequence, obtain polypeptide L-OmpF-16s, for amino acid sequence as shown in SEQ ID NO.2, Antimicrobial test shows that it has good bactericidal effect in simple aqueous medium.Hand cleanser sterilization experiment further is carried out with L-OmpF-16s, L-OmpF-16s is added after hand cleanser is diluted 800 times, the hand cleanser after dilution still has good bactericidal effect.
Description
Technical field
The invention belongs to the antibacterial technical fields of polypeptide in biotechnology, and in particular to one kind generates machine based on outer membrane protein
The antibacterial polypeptide of system and its application.
Technical background
It is existing at present many studies demonstrate that, when some protein function, it is only necessary to partial function region it is complete,
The complete of full sequence structure is not needed.By way of the partial function segment of only synthetic proteins matter, this can also be played
The corresponding effect of protein, purifying hardly possible cumbersome and at high cost so as to avoid technique existing for gene engineering expression protein
The disadvantages of.From since the 21th century, China's polypeptide industry continues to remain the situation of fast development.Peptide systhesis company is numerous and confused
It establishes, synthesis and extractive technique are continuously improved, and more and more polypeptide products realize industrialization, this is the function of polypeptide and answers
It is laid a good foundation with research.At present polypeptide application be concentrated mainly on polypeptide drugs, polypeptide drugs carrier, tissue engineering material and
Polypeptide nutrition food etc..With the improvement of living standards, requirement of the people for health, safety is also continuously improved, selecting
Another day people increasingly tend to that selection component is natural, the product small to human injury when articles.Active peptides have many excellent
Characteristic, therefore polypeptide is not applied only to food, medicine aspect in recent years, before tempting application is also shown in the fields such as chemical industry
Scape.
The cell envelope of Gram-E. coli is by between the film between intercellular membrane, epicyte and intracellular outer membrane
Matter region is constituted.Outer membrane protein is also known as fenestra road albumen, is the main component of outer membrane.The important outer membrane protein of Escherichia coli has
OmpC, OmpF and PhoE etc., their transmembrane lipid bilayers, wherein outer membrane protein OmpF is the most important outer membrane of Escherichia coli
One of albumen, the ion channel that it is formed are Escherichia coli and the extraneous important channel for carrying out mass exchange, can make molecular weight
Water-soluble substances no more than 600Da pass through outer membrane by the ion channel.
The generation of outer membrane protein need to undergo the process of a series of complex.The new polypeptide chain of outer membrane protein is in cytoplasm
Ribosome biogenesis after, by Sec movement system across bacterium inner membrance reach film interstitial areas, film interstitial areas is hydrophilic
Property environment, newborn outer membrane protein is easy to that false folding occurs in the environment, and film interstitial quality control factor can assist
It helps newborn outer membrane protein to pass through the film interstitial space for lacking ATP, be finally positioned at bacterial outer membrane and completed just in bacterial outer membrane
True folding, assembling and slotting membrane process.SurA is most important film interstitial molecular chaperones, SurA in outer membrane protein generating process
Knockout will lead to bacterial outer membrane and generate serious defect, make it to the quick of the substances such as SDS-EDTA, ovobiocin and bile salt
Perception enhancing.There are some researches prove when the interaction of the new polypeptide chain of SurA and outer membrane protein, only with the N-terminal of newborn outer membrane protein
It interacts with C-terminal, when SurA combines an improper outer membrane protein, this albumen not can be carried out subsequent normal slotting
Film, SurA would not be come out by " liberation ", be in occupied state, and other outer membrane proteins cannot be assisted to complete to insert film,
So that bacterium is in the state for being similar to SurA knockout strain, therefore some small peptides can be designed according to the generating process of outer membrane protein,
The generation for interfering Bacterial outer membrane proteins, to achieve the purpose that sterilization.
Summary of the invention
The purpose of the present invention is to provide a kind of antibacterial polypeptide based on outer membrane protein generting machanism and its applications.
To achieve the above object, the invention adopts the following technical scheme:
The present invention devises a kind of polypeptide with bacteriostasis according to Gram-negative bacterial outer membrane protein OmpF, is named as
OmpF-16s is made of 42 amino acid, and amino acid sequence is as shown in SEQ ID NO.1.
The gene order of OmpF-16s is cloned on pBAD carrier (pBAD/Myc-His C) by the present invention first, uses large intestine
Bacillus expression system inducing expression polypeptide, there is similar to SurA knockout strain bacterium after finding the endogenous expression OmpF-16s of Induction of bacterial
Phenotype, i.e. the phenotype of bacterium is that cell membrane is more fragile, to extraneous environment sensitive, can not be deposited in the case where outer membrane interferes screening conditions
It is living.
The inducer that Induction of bacterial expresses albumen is arabinose.The concentration of arabinose concretely 0.0002% (m/
w).The outer membrane interference screening conditions are that SDS and EDTA is added simultaneously in LB plate.The concentration of the SDS is 0.5% (m/
W), the concentration of EDTA is 1mM.
Further, the amino acid sequence synthesis polypeptide of OmpF-16s is pressed by Peptide systhesis company, (purity of polypeptide is greater than
80%) Antimicrobial test is carried out, the experimental results showed that, polypeptide OmpF-16s of the present invention, under conditions of EDTA auxiliary, centainly
The polypeptide of concentration has preferable bactericidal effect to Gram-negative bacteria.
The concentration of the EDTA concretely 1mM.
The Gram-negative bacteria concretely Escherichia coli (such as Escherichia coli BW25113).
The final concentration of the polypeptide concretely 25uM, 50uM, 75uM, 100uM.
It is demonstrated experimentally that be directed to Escherichia coli, the present invention in polypeptide OmpF-16s with compare (be not added polypeptide handle sample
Product) it compares, there is the bactericidal effect of the 2-5 order of magnitude, i.e. germicidal efficiency after addition polypeptide OmpF-16s is more than 99%, and highest can
Up to 99.999%.
Further, the present invention has added one section can assist what polypeptide entered bacterial cell to lead peptide sequence in the N-terminal of OmpF-16s,
It is named as L-OmpF-16s, amino acid sequence is as shown in SEQ ID NO.2.
The amino acid sequence of peptide is led as shown in SEQ ID NO.3.
L-OmpF-16s does not need the auxiliary of EDTA, can be directly entered bacterial cell, i.e., only needs just to have in an aqueous medium
Good extracorporeal disinfecting effect.Antimicrobial test shows polypeptide L-OmpF-16s, (sample of polypeptide processing is not added with compareing
With the sample for leading peptide processing containing same concentrations) it compares, only need to be in water environment, the polypeptide of very low concentrations is to Gram-negative bacteria
Just there is fabulous bactericidal effect, and sterilization conditions ingredient is more simple, safety.
The Gram-negative bacteria concretely Escherichia coli (such as Escherichia coli BW25113).
The polypeptide and lead the final concentration of peptide concretely 20uM, 30uM.
Antibacterial polypeptide of the invention has good antibacterial or bactericidal effect, and can be used for preparing has antibacterial or biocidal efficacies
Articles for washing.
Since some chemical substances in the hand cleanser with bacteriostasis may stimulate hand skin, for children and
Skin be easy allergy crowd injury it is larger, to solve this problem, the present invention in the hand cleanser that certain multiple is diluted with water,
Certain density polypeptide is added, makes the hand cleanser containing low concentration chemistry sterilization component that there is good bactericidal effect.
The hand cleanser concretely blue moon aloetic bacteriostatic hand cleanser.
It is described that hand cleanser is diluted to certain multiple, concretely dilute 800 times (w/w).
The concentration of the L-OmpF-16s concretely 50uM.
The results show, hand cleanser dilute certain multiple after, the hand cleanser containing polypeptide (L-OmpF-16s) with compare
(containing lead the hand cleanser of peptide with without containing the hand cleanser of polypeptide) compare, have the bactericidal effect of the 3-4 order of magnitude, i.e. addition L-
Bactericidal effect after OmpF-16s is more than 99.99%.The various chemical components that script in hand cleanser can be reduced in the application, make
Hand cleanser is more safe and effective.
Detailed description of the invention
Fig. 1 is the phenotype induced after the endogenous expression OmpF-16s of Escherichia coli, and three rows are respectively three kinds different flat in figure
Plate, wherein it is 10 that each grid, which is dilution,3Bacterium colony figure.
Fig. 2 is that the OmpF-16s of various concentration handles the bactericidal effect of Escherichia coli.In figure, the 1st row sample be without appoint
Where reason Escherichia coli, the 2nd row be through being free of polypeptide the processed sample of DMSO solvent, the 3rd, 4,5,6 rows be with dense
Degree is respectively the sample of the polypeptide processing of 100uM, 75uM, 50uM, 25uM.Wherein, 6 bacterium colony figures under each processing from the right side to
A left side is respectively that dilution is respectively 105、104、103、102, 10, the bacterium colony figure under 1.
The bactericidal effect for the processing Escherichia coli that Fig. 3 is the L-OmpF-16s of various concentration.In figure, the 1st row sample is not
Escherichia coli through any processing, the 2nd row are the processed samples of DMSO solvent by being free of polypeptide, and the 3rd, 4 be to use respectively
The sample for leading peptide and L-OmpF-16s processing that concentration is 20uM.5,6 be with concentration be respectively that 30uM leads peptide and L-OmpF-
The sample of 16s processing.Wherein, it is respectively 10 that 6 bacterium colony figures under each processing are respectively dilution from right to left5、104、103、
102, 10, the bacterium colony figure under 1.
Fig. 4 is the bactericidal effect figure that polypeptide enhances low chemical component hand cleanser.In figure, the 1st row sample is without any place
The sample of the Escherichia coli of reason, the 2nd, 3,4 rows be the processed sample of hand cleanser after 800 times of dilution, wherein the 2nd row
Hand cleanser contains the DMSO solvent of 1ul, and the hand cleanser of the 3rd row contains the peptide of leading of 1ul, and leads the final concentration of 50uM of peptide, the 4th row
Hand cleanser contains the L-OmpF-16s of 1ul, the final concentration of 50uM of polypeptide.Wherein, 6 bacterium colony figures under each processing from the right side to
A left side is respectively that dilution is respectively 105、104、103、102, 10, the bacterium colony figure under 1.
Specific embodiment
The present invention is further described in detail With reference to embodiment, and the embodiment provided is only for explaining
The bright present invention, the range being not intended to be limiting of the invention.
Experimental method in following embodiments is unless otherwise specified conventional method.Material as used in the following examples
Material, reagent, instrument etc., are commercially available unless otherwise specified.Quantitative test in following embodiments, is respectively provided with
Three repeated experiments, results are averaged.
The E. coli K-12BW25113 public in following embodiments can obtain the biology material from applicant
Material, the biomaterial are only attached most importance to used in the related experiment of duplicate invention, not can be used as other purposes and are used (E.coli K-
12BW25113 is the widely used Escherichia coli reference culture of biology laboratory, and bibliography can use Baba T.et al.
(2006)Construction of Escherichia coli K-12in-frame,single-gene knockout
mutants:the Keio collection.Mol Systems Biol 2(1),1-11,doi:10.1038/
msb4100050.)
The present invention devises a kind of polypeptide with bacteriostasis according to Gram-negative bacterial outer membrane protein OmpF, is named as
OmpF-16s is made of 42 amino acid, and amino acid sequence is as shown in SEQ ID NO.1.
Further, the present invention has added one section can assist what polypeptide entered bacterial cell to lead peptide sequence in the N-terminal of OmpF-16s,
It is named as L-OmpF-16s, amino acid sequence is as shown in SEQ ID NO.2.
The amino acid sequence of peptide is led as shown in SEQ ID NO.3.
The endogenous expression OmpF-16s Phenotypic Observation of 1 Escherichia coli of embodiment
1, by the gene cloning of OmpF-16s on pBAD carrier.
2, the plasmid built is transferred in Δ OmpF competent cell.Competent cell is put from -80 DEG C of refrigerator taking-ups
In ice chest, be in competent cell be added when not exclusively thawing 1uL to Pignus pignoris grain, ice bath 30min, after ice bath plus
The LB liquid medium of 1ml, which is put in 37 DEG C of shaking tables, restores 60min.Then the bacterium of conversion is coated in flat containing ammonia benzyl resistance
On plate, 12 to 16 hours of culture in 37 DEG C of constant incubator are placed on, picking positive colony is cultivated to plateau, uses 1.5mL
Sterilized EP pipe packing be used as seed liquor.
3, reactivated bacteria.The Escherichia coli that -80 DEG C of refrigerators are stored in 20% glycerol are drawn respectively, and OmpF knocks out strain (Δ
OmpF), Sura knocks out strain (Δ SurA, chlorampenicol resistant), OmpF covering OmpF knocks out strain (Δ OmpF+POmpF, ammonia benzyl resistance)
10 μ l of bacterium solution, addition 20ml LB liquid medium (configuration ingredient: 1L culture medium 10g containing tryptone, yeast extract 5g,
NaCl 10g, remaining is water；High pressure steam sterilization), 37 DEG C of shaking tables (220rpm) were incubated overnight to plateau, with going out for 1.5mL
The EP pipe packing of bacterium is crossed as seed liquor.
4, the seed liquor obtained using step 2 and step 3 is taken seed liquor to dilute 100 times and is inoculated in 2ml LB liquid as mother liquor
In culture medium, while similarly to meet the inoculation of bacterium ratio WT, Δ SurA, Δ OmpF, Δ
OmpF+POmpF, Δ OmpF+-POmpF-16s, (OD value is 0.5 left for 37 DEG C of shaking table (220rpm) cultures to logarithmic phase
It is right)；
5, take each bacterium solution in step 4, by bacterium solution dilute 1000 times of difference drops on normal LB plate, SDS-EDTA puts down
Plate, SDS-EDTA-ara plate, wherein the content of SDS is 0.5% (m/w), and the content of EDTA is 1mM, ara (arabinose)
Content be 0.0002%, after the plate put is placed in 37 DEG C of incubator cultures 8-12 hours, observe the survival condition of bacterium.Knot
Fruit is as shown in Figure 1.
The results show that the induction endogenous expression OmpF-16s of Escherichia coli, bacterium nothing on the plate of outer membrane interference screening
It is similar to knock out the phenotype of strain with SurA for method survival.And wild-type e. coli, OmpF knock out strain and endogenous expression OmpF overall length
Escherichia coli can be with normal growth.
The extracorporeal disinfecting of 2 polypeptide OmpF-16s of embodiment is tested
1, Escherichia coli reference culture (E.coli BW25113) is activated.It draws and is stored in -80 DEG C of refrigerators with 20% glycerol
10 μ l of bacterium solution, be added 20ml LB liquid medium (configuration ingredient: 1L culture medium 10g containing tryptone, yeast extract 5g,
NaCl 10g, remaining is water；High pressure steam sterilization), 37 DEG C of shaking tables (220rpm) were incubated overnight to plateau, with going out for 1.5mL
The EP pipe packing of bacterium is crossed as seed liquor.It takes seed liquor to dilute 100 times and is inoculated in 2ml LB liquid medium, 37 DEG C of shaking tables
To logarithmic phase, (OD value is 0.5-0.65) for (220rpm) culture；
2,6 sterilizing EP pipes are taken, every pipe dispenses the logarithmic phase bacterium solution that 100 μ l steps 1 obtain, wherein a pipe is placed on 4 DEG C
It chromatographs and is used as experiment contrast in cabinet.Other 5 pipe is placed in 13000rpm in rapid centrifugation machine and is centrifuged 4min minutes, abandons supernatant；
3, it is resuspended with 0.9% Nacl of 100ul, 13000rpm is centrifuged 4min minutes, abandons supernatant；
4, it is resuspended with the treatment fluid of 99ul (EDTA aqueous solution, EDTA concentration are 1mM), in 5 processing samples, one of them
The DMSO of 1ul is added in sample as processing control, in addition 4 samples add the certain density OmpF-16s polypeptide of 1ul molten respectively
Liquid, making the final concentration of OmpF-16s polypeptide in system is respectively 25uM, 50uM, 75uM, 100uM.5 minutes are stood after mixing.
5, it stands after five minutes, the treatment fluid in bacterium solution step 4 is successively diluted according to 10 times every time of gradient, each
Dilution takes 5 μ l bacterium solution drops on LB solid medium hexagonal mesh plate, after being placed in 37 DEG C of incubator cultures 8-12 hours, observation
The survival condition of bacterium.As a result as shown in Figure 2 and Table 1.
The OmpF-16s polypeptide of 1 various concentration of table handles the survival rate and germicidal efficiency relatively of lower Escherichia coli
Note: the survival containing polypeptide sample when opposite germicidal efficiency=handled but be free of survival rate/processing of polypeptide sample
Rate
The results show that by the OmpF-16s polypeptide solution (25uM, 50uM, 75uM, 100uM) of Escherichia coli various concentration
Processing, is compared, 2,3,5,5 orders of magnitude are respectively increased in the death rate of Escherichia coli with the processing sample without polypeptide.As it can be seen that more
Peptide OmpF-16s has good bactericidal effect in vitro.
The extracorporeal disinfecting of the certain density polypeptide L-OmpF-16s of embodiment 3 is tested
1, Escherichia coli reference culture (E.coli BW25113) is activated.It draws and is stored in -80 DEG C of refrigerators with 20% glycerol
10 μ l of bacterium solution, be added 20ml LB liquid medium (configuration ingredient: 1L culture medium 10g containing tryptone, yeast extract 5g,
NaCl 10g, remaining is water；High pressure steam sterilization), 37 DEG C of shaking tables (220rpm) were incubated overnight to plateau, with going out for 1.5mL
The EP pipe packing of bacterium is crossed as seed liquor.It takes seed liquor to dilute 100 times and is inoculated in 2ml LB liquid medium, 37 DEG C of shaking tables
To logarithmic phase, (OD value is 0.45-0.65) for (220rpm) culture；
2,6 sterilizing EP pipes are taken, every pipe dispenses the logarithmic phase bacterium solution that 100 μ l steps 1 obtain, wherein a pipe is placed on 4 DEG C
It chromatographs and is used as experiment contrast in cabinet.Other 5 pipe is placed in 13000rpm in rapid centrifugation machine and is centrifuged 4min minutes, abandons supernatant；
3, it is resuspended with 0.9% Nacl of 100ul, 13000rpm is centrifuged 4min minutes, abandons supernatant；
4, it is resuspended with the aqua sterilisa of 99ul, in 5 processing samples, the DMSO of 1ul is added in one of sample as at
Reason control, 2 samples add 1ul is certain density to lead peptide solution respectively, make the final concentration that peptide is led in system be respectively 20uM,
30uM.Other 2 samples add the certain density polypeptide L-OmpF-16s solution of 1ul respectively, make polypeptide L-OmpF-16s in system
Final concentration be respectively 20uM, 30uM, stand 5 minutes after mixing.
5, it stands after five minutes, the treatment fluid in bacterium solution step 4 is successively diluted according to 10 times every time of gradient, each
Dilution takes 5 μ l bacterium solution drops on LB solid medium hexagonal mesh plate, after being placed in 37 DEG C of incubator cultures 8-12 hours, observation
The survival condition of bacterium.As a result as shown in figure 3 and table 2.
The L-OmpF-16s polypeptide of 2 various concentration of table handles the survival rate and germicidal efficiency relatively of lower Escherichia coli
Note: the sample containing polypeptide when survival rate/processing of opposite germicidal efficiency=handled but the sample without polypeptide
Survival rate
The results show that the polypeptide L-OmpF-16s aqueous solution (20uM, 30uM) of Escherichia coli various concentration is handled, and
Processing sample without polypeptide is compared, and 4 to 5 orders of magnitude can be improved in the death rate of Escherichia coli.As it can be seen that polypeptide L-OmpF-16s
There is good bactericidal effect in vitro.
Application example of the 4 polypeptide L-OmpF-16s of embodiment in hand cleanser
1, Escherichia coli reference culture (E.coli BW25113) is activated.It draws and is stored in -80 DEG C of refrigerators with 20% glycerol
10 μ l of bacterium solution, be added 20ml LB liquid medium (configuration ingredient: 1L culture medium 10g containing tryptone, yeast extract 5g,
NaCl 10g, remaining is water；High pressure steam sterilization), 37 DEG C of shaking tables (250rpm) were incubated overnight to plateau, with going out for 1.5mL
The EP pipe packing of bacterium is crossed as seed liquor.It takes seed liquor to dilute 100 times and is inoculated in 2ml LB liquid medium, 37 DEG C of shaking tables
Logarithmic phase is arrived in (220rpm) culture.
2,4 sterilizing EP pipes, number 1,2,3,4 are taken.Every pipe dispenses 100 μ l bacterium solutions, wherein No. 1 pipe is placed on 4 DEG C of floor
It analyses and is used as experiment contrast in cabinet, in addition 3 pipes are placed in 13000rpm in rapid centrifugation machine and are centrifuged 4 minutes, abandon supernatant；
3,2,3, No. 4 pipes use 0.9% Nacl of 100ul to be resuspended respectively, and 13000rpm is centrifuged 4 minutes, abandon supernatant；
4, after abandoning supernatant, 2,3, No. 4 pipes use the hand cleanser of 100ul to be resuspended respectively.Wherein, contain 1ul's in No. 2 hand cleanser
DMSO leads peptide containing 1ul in No. 3 hand cleanser, leads the final concentration of 50uM of peptide, contain the more of 1ul in No. 4 hand cleanser
Peptide, the final concentration of 50uM of polypeptide stand 5 minutes at room temperature；
5, it stands after five minutes, bacterium solution is successively diluted according to 10 times every time of gradient with the mixed solution containing step 4, often
A dilution takes 5 μ l bacterium solution drops on LB solid medium hexagonal mesh plate, after being placed in 37 DEG C of incubator cultures 8-12 hours, sees
The survival condition of bacterium is examined, as a result as shown in Fig. 4 and table 3.
Table 3
Note: opposite germicidal efficiency=handled but survival rate/hand cleanser processing of the hand cleanser processing sample without polypeptide
When the sample containing polypeptide survival rate
The results show that after hand cleanser is diluted 800 times, that is, greatly dilute the various sterilizations of script in hand cleanser
Ingredient, the processing sample containing polypeptide L-OmpF-16s in hand cleanser, compared with the processing sample without polypeptide, Escherichia coli
4 orders of magnitude can be improved in the death rate.As it can be seen that polypeptide L-OmpF-16s of the present invention in the application can replace traditional chemical sterilization at
Point, keep product composition simple and bactericidal effect is significant.
Sequence table
<110>Fujian Normal University
<120>a kind of antibacterial polypeptide and its application based on outer membrane protein generting machanism
<130> 2018
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 42
<212> PRT
<213>artificial sequence (Artifical sequence)
<400> 1
Met Met Lys Arg Asn Ile Leu Ala Val Ile Val Pro Ala Leu Leu Val
1 5 10 15
Ala Gly Thr Ala Asn Ala Ala Glu Ile Tyr Asn Lys Asp Gly Asn Lys
20 25 30
Val Asp Leu Tyr Gly Lys Ala Val Gly Leu
35 40
<210> 2
<211> 55
<212> PRT
<213>artificial sequence (Artifical sequ)
<400> 2
Lys Phe Phe Lys Phe Phe Lys Phe Phe Lys Gly Ser Gly Met Met Lys
1 5 10 15
Arg Asn Ile Leu Ala Val Ile Val Pro Ala Leu Leu Val Ala Gly Thr
20 25 30
Ala Asn Ala Ala Glu Ile Tyr Asn Lys Asp Gly Asn Lys Val Asp Leu
35 40 45
Tyr Gly Lys Ala Val Gly Leu
50 55
<210> 3
<211> 13
<212> PRT
<213>artificial sequence (Artifical sequence)
<400> 3
Lys Phe Phe Lys Phe Phe Lys Phe Phe Lys Gly Ser Gly
1 5 10
Claims (5)
1. a kind of antibacterial polypeptide based on outer membrane protein generting machanism, it is characterised in that: its amino acid sequence such as SEQ ID NO.1
It is shown.
2. a kind of contain the antibacterial polypeptide for leading peptide, it is characterised in that: its amino acid sequence is as shown in SEQ ID NO.2.
3. antibacterial polypeptide as claimed in claim 1 or 2 is inhibiting or is killing the application in Gram-negative bacteria.
4. application according to claim 3, it is characterised in that: the Gram-negative bacteria is Escherichia coli.
5. application according to claim 3, it is characterised in that: the antibacterial polypeptide is used to prepare with antibacterial or sterilization function
The articles for washing of effect.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811433991.7A CN109517046B (en) | 2018-11-28 | 2018-11-28 | Antibacterial polypeptide based on outer membrane protein generation mechanism and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811433991.7A CN109517046B (en) | 2018-11-28 | 2018-11-28 | Antibacterial polypeptide based on outer membrane protein generation mechanism and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109517046A true CN109517046A (en) | 2019-03-26 |
| CN109517046B CN109517046B (en) | 2021-09-14 |
Family
ID=65794516
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811433991.7A Active CN109517046B (en) | 2018-11-28 | 2018-11-28 | Antibacterial polypeptide based on outer membrane protein generation mechanism and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109517046B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999029342A1 (en) * | 1997-12-11 | 1999-06-17 | Medeva Europe Limited | Vaccines containing attenuated bacteria |
| CN101775068A (en) * | 2009-01-14 | 2010-07-14 | 上海南方模式生物科技发展有限公司 | Novel natural antibacterial peptides, and coding sequence and uses thereof |
| CN102481369A (en) * | 2009-06-16 | 2012-05-30 | 东海大学 | Anti-gram-negative bacteria agent |
| CN108129555A (en) * | 2017-09-11 | 2018-06-08 | 中国医学科学院病原生物学研究所 | Specifically bind the design of polypeptide of six type excretory system immune protein of pseudomonas aeruginosa and its verification of antibacterial activity |
-
2018
- 2018-11-28 CN CN201811433991.7A patent/CN109517046B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999029342A1 (en) * | 1997-12-11 | 1999-06-17 | Medeva Europe Limited | Vaccines containing attenuated bacteria |
| CN101775068A (en) * | 2009-01-14 | 2010-07-14 | 上海南方模式生物科技发展有限公司 | Novel natural antibacterial peptides, and coding sequence and uses thereof |
| CN102481369A (en) * | 2009-06-16 | 2012-05-30 | 东海大学 | Anti-gram-negative bacteria agent |
| CN108129555A (en) * | 2017-09-11 | 2018-06-08 | 中国医学科学院病原生物学研究所 | Specifically bind the design of polypeptide of six type excretory system immune protein of pseudomonas aeruginosa and its verification of antibacterial activity |
Non-Patent Citations (2)
| Title |
|---|
| BEHRENS, S等: "The SurA periplasmic PPlase lacking its parvulin domains functions in vivo and has chaperone activity", 《EMBO JOURNAL》 * |
| 谭咪乐等: "将大肠杆菌OmpA信号肽改造成为抗菌肽的研究", 《内科》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109517046B (en) | 2021-09-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Kondorosi et al. | A paradigm for endosymbiotic life: cell differentiation of Rhizobium bacteria provoked by host plant factors | |
| JP4484941B2 (en) | Short peptides having biological activity and methods of using the peptides | |
| Makovitzki et al. | Inhibition of fungal and bacterial plant pathogens in vitro and in planta with ultrashort cationic lipopeptides | |
| Sorensen et al. | In vitro antifungal and fungicidal activities and erythrocyte toxicities of cyclic lipodepsinonapeptides produced by Pseudomonas syringae pv. syringae | |
| Booth et al. | Recent progress on the application of 2H solid-state NMR to probe the interaction of antimicrobial peptides with intact bacteria | |
| Jiang et al. | Effects of hydrophobicity on the antifungal activity of α‐helical antimicrobial peptides | |
| Mohammad et al. | Probiotic properties of bacteria isolated from bee bread of stingless bee Heterotrigona itama | |
| JPH03503522A (en) | Inhibition of eukaryotic pathogens and neoplasms and stimulation of fibroblasts and lymphocytes by lytic peptides | |
| Srihongthong et al. | Complete amino acid sequence of globin chains and biological activity of fragmented crocodile hemoglobin (Crocodylus siamensis) | |
| CN111315393A (en) | Compositions for use in agriculture and related methods | |
| Sila et al. | Antibacterial activity of new peptides from barbel protein hydrolysates and mode of action via a membrane damage mechanism against Listeria monocytogenes | |
| Burrows et al. | Activity of novel non-amphipathic cationic antimicrobial peptides against Candida species | |
| CN103333225A (en) | Antibacterial peptide, preparation method and applications thereof | |
| CN112760253A (en) | Lactobacillus plantarum, antibacterial peptide and application thereof | |
| CN107252475B (en) | The application of natural host defense peptide Alligatorin4 | |
| Rodrigues et al. | Adevonin, a novel synthetic antimicrobial peptide designed from the Adenanthera pavonina trypsin inhibitor (ApTI) sequence | |
| CN107142226A (en) | A kind of Bacillus subtillis ATR2 and preparation method and application | |
| CN108026155A (en) | New antimicrobial peptide, their variation and purposes | |
| Riciluca et al. | Rondonin: antimicrobial properties and mechanism of action | |
| CN104177485A (en) | Yangtze alligator antimicrobial peptide Alligatorin 6 and application thereof | |
| Yin et al. | A specific antimicrobial protein CAP-1 from Pseudomonas sp. isolated from the jellyfish Cyanea capillata | |
| Wang et al. | Effect of extracted housefly pupae peptide mixture on chilled pork preservation | |
| CN109517046A (en) | A kind of antibacterial polypeptide and its application based on outer membrane protein generting machanism | |
| Vasilchenko et al. | Purification of a novel bacteriocin-like inhibitory substance produced by Enterococcus faecium ICIS 8 and characterization of its mode of action | |
| Van Rensburg | The tyrocidines in the creation of antimicrobial cellulose and sterilizing materials |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |