CN109517046B

CN109517046B - Antibacterial polypeptide based on outer membrane protein generation mechanism and application thereof

Info

Publication number: CN109517046B
Application number: CN201811433991.7A
Authority: CN
Inventors: 付新苗; 王妍; 张爽
Original assignee: Fujian Normal University
Current assignee: Fujian Normal University
Priority date: 2018-11-28
Filing date: 2018-11-28
Publication date: 2021-09-14
Anticipated expiration: 2038-11-28
Also published as: CN109517046A

Abstract

The invention discloses an antibacterial polypeptide based on an outer membrane protein production mechanism, which is named as OmpF-16s, and the amino acid sequence of the antibacterial polypeptide is shown as SEQ ID NO. 1. The OmpF-16s gene was cloned in a pBAD vector and protein expression was induced on plates containing arabinose (ara), SDS and EDTA, indicating that the bacteria were not viable under conditions of outer membrane interference screening after expressing the polypeptide. The polypeptide is further synthesized by companies to carry out in-vitro antibacterial experiments, and the result shows that the polypeptide has good bactericidal effect on escherichia coli with the assistance of EDTA. And further adding a peptide leader sequence at the N end of OmpF-16s to obtain the polypeptide L-OmpF-16s, wherein the amino acid sequence of the polypeptide L-OmpF-16s is shown in SEQ ID NO.2, and in-vitro antibacterial experiments show that the polypeptide L-OmpF-16s has good bactericidal effect in a simple aqueous medium. And further performing a hand sanitizer sterilization experiment by using L-OmpF-16s, diluting the hand sanitizer by 800 times, and adding the diluted hand sanitizer into the L-OmpF-16s, wherein the diluted hand sanitizer still has a good sterilization effect.

Description

Antibacterial polypeptide based on outer membrane protein generation mechanism and application thereof

Technical Field

The invention belongs to the technical field of polypeptide bacteriostasis in biotechnology, and particularly relates to an antibacterial polypeptide based on an outer membrane protein generation mechanism and application thereof.

Technical Field

Many studies have now demonstrated that some proteins function by only partially completing their functional domains, and not by completely completing their entire sequence structure. The corresponding efficacy of the protein can be exerted by only synthesizing partial functional fragments of the protein, thereby avoiding the defects of complicated process, high cost, difficult purification and the like existing in the expression of the protein by genetic engineering. Since the 21 st century, the polypeptide industry in China continues to keep a rapid development situation. Polypeptide synthesis companies are built in succession, synthesis and extraction technologies are continuously improved, and more polypeptide products are industrialized, so that a foundation is laid for function and application research of polypeptides. At present, the application of the polypeptide mainly focuses on the aspects of polypeptide drugs, polypeptide drug carriers, tissue engineering materials, polypeptide nutritious foods and the like. With the improvement of living standard, the requirements of people on health and safety are continuously improved, and people tend to select products with natural components and little harm to human bodies when selecting daily necessities. The active polypeptide has a plurality of excellent characteristics, so that the polypeptide not only can be applied to the aspects of food and medicine, but also has attractive application prospect in the fields of chemical industry and the like in recent years.

The cell envelope of gram-negative E.coli is composed of the inner cell membrane, the outer cell membrane and the intermembranous region between the inner and outer cell membranes. The outer membrane protein, also known as the porin, is the major component of the outer membrane. Important outer membrane proteins of Escherichia coli include OmpC, OmpF, PhoE and the like, which span a membrane lipid bilayer, wherein the outer membrane protein OmpF is one of the most important outer membrane proteins of Escherichia coli, and an ion channel is formed, which is an important channel for exchanging substances between Escherichia coli and the outside and enables water-soluble substances with molecular weight not more than 600Da to pass through the outer membrane.

The production of outer membrane proteins is subject to a complex series of processes. After ribosome synthesis in cytoplasm, the nascent peptide chain of the outer membrane protein crosses the inner membrane of the bacterium through a Sec transport system to reach a membrane-mesenchymal region, the membrane-mesenchymal region is a hydrophilic environment, the nascent outer membrane protein is easy to be misfolded in the environment, and a membrane-mesenchymal quality control factor can assist the nascent outer membrane protein to cross an ATP-deficient membrane-mesenchymal space, finally to be positioned on the outer membrane of the bacterium and complete correct folding, assembly and membrane insertion processes on the outer membrane of the bacterium. The SurA is the most important membrane-mediated chaperone in the process of outer membrane protein production, and the knockout of the SurA can cause serious defects on the outer membrane of bacteria, so that the sensitivity of the SurA to substances such as SDS-EDTA, neomycin, bile salts and the like is enhanced. Researches prove that when the SurA interacts with a nascent peptide chain of an outer membrane protein, the SurA interacts with the N end and the C end of the nascent outer membrane protein only, when the SurA is combined with an abnormal outer membrane protein, the protein cannot be subjected to subsequent normal membrane insertion, the SurA cannot be liberated, and in an occupied state, the SurA cannot assist other outer membrane proteins to complete membrane insertion, so that bacteria are in a state similar to a SurA knockout strain, therefore, some short peptides can be designed according to the generation process of the outer membrane protein to interfere the generation of the outer membrane protein of the bacteria, and the aim of sterilization is fulfilled.

Disclosure of Invention

The invention aims to provide an antibacterial polypeptide based on an outer membrane protein production mechanism and application thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention designs a polypeptide with bacteriostatic action according to gram-negative bacteria outer membrane protein OmpF, which is named as OmpF-16s and consists of 42 amino acids, and the amino acid sequence of the polypeptide is shown in SEQ ID NO. 1.

According to the invention, firstly, the gene sequence of OmpF-16s is cloned on a pBAD vector (pBAD/Myc-His C), an escherichia coli expression system is used for inducing and expressing polypeptide, and the fact that after the OmpF-16s is endogenously expressed by induced bacteria, the bacteria have a phenotype similar to a SurA knockout strain is found, namely, the phenotype of the bacteria is that cell membranes are fragile, sensitive to the external environment and cannot survive under the condition of external membrane interference screening.

The inducer for inducing the bacteria to express the protein is arabinose. The concentration of arabinose may specifically be 0.0002% (m/w). The outer membrane interference screening condition is that SDS and EDTA are added into an LB plate at the same time. The concentration of SDS was 0.5% (m/w) and the concentration of EDTA was 1 mM.

Furthermore, the polypeptide is synthesized by a polypeptide synthesis company according to the amino acid sequence of OmpF-16s, (the purity of the polypeptide is more than 80 percent) and is subjected to in vitro antibacterial experiments, and the experimental result shows that the polypeptide OmpF-16s has better bactericidal effect on gram-negative bacteria by the polypeptide with a certain concentration under the condition of EDTA assistance.

The concentration of EDTA may be specifically 1 mM.

The gram-negative bacteria can be specifically Escherichia coli (such as Escherichia coli BW 25113).

The final concentration of the polypeptide may specifically be 25uM, 50uM, 75uM, 100 uM.

Experiments prove that the polypeptide OmpF-16s has 2-5 orders of magnitude of sterilization effect compared with a control (a sample without polypeptide treatment) aiming at escherichia coli, namely the sterilization efficiency after the polypeptide OmpF-16s is added exceeds 99 percent, and the highest sterilization efficiency can reach 99.999 percent.

Furthermore, the invention adds a peptide guide sequence which can assist the polypeptide to enter bacterial cells at the N end of OmpF-16s, is named as L-OmpF-16s, and the amino acid sequence of the peptide guide sequence is shown in SEQ ID NO. 2.

The amino acid sequence of the leader peptide is shown in SEQ ID NO. 3.

L-OmpF-16s can directly enter bacterial cells without the assistance of EDTA, namely, the L-OmpF-16s only has good in-vitro bactericidal effect in an aqueous medium. In vitro antibacterial experiments show that compared with the control (a sample without polypeptide treatment and a sample containing the same concentration of peptide guide treatment), the polypeptide L-OmpF-16s has excellent bactericidal effect on gram-negative bacteria only by the polypeptide with very low concentration in a water environment, and the components of the bactericidal condition are simpler and safer.

The final concentration of the polypeptide and the leader peptide may specifically be 20uM, 30 uM.

The antibacterial polypeptide has good antibacterial or bactericidal effect, and can be used for preparing washing products with antibacterial or bactericidal efficacy.

In order to solve the problem that some chemical substances in the hand sanitizer with the bacteriostatic action can stimulate the skin of hands and cause great harm to children and people with skin allergy, the hand sanitizer disclosed by the invention is added with the polypeptide with a certain concentration into the hand sanitizer diluted by water by a certain multiple, so that the hand sanitizer containing the low-concentration chemical sterilization components has a good sterilization effect.

The hand sanitizer can be specifically a bacteriostatic hand sanitizer containing aloe vera.

The hand sanitizer is diluted by a certain time, specifically, the dilution is 800 times (w/w).

The concentration of L-OmpF-16s may be specifically 50 uM.

Experimental results prove that after the hand sanitizer is diluted by a certain multiple, the hand sanitizer containing the polypeptide (L-OmpF-16s) has 3-4 orders of magnitude of sterilization effect compared with a control (the hand sanitizer containing the peptide and the hand sanitizer containing no polypeptide), namely the sterilization effect after the L-OmpF-16s is added is over 99.99%. In application, various original chemical components in the hand sanitizer can be reduced, so that the hand sanitizer is safer and more effective.

Drawings

FIG. 1 shows the phenotype of E.coli after induction of endogenous OmpF-16s expression, three rows of three different plates, each at a dilution of 10³A colony map of (a).

FIG. 2 shows the bactericidal effect of OmpF-16s treatment on E.coli at different concentrations. In the figure, the sample on line 1 is E.coli without any treatment, the sample on line 2 is a sample treated with a DMSO solvent without polypeptide, and the samples on lines 3, 4, 5, and 6 are samples treated with polypeptides at concentrations of 100uM, 75uM, 50uM, and 25uM, respectively. Wherein, the dilution degree of each 6 colony images under each treatment is respectively 10 from right to left⁵、10⁴、10³、10²10, 1.

FIG. 3 shows the bactericidal effect of L-OmpF-16s treatment on E.coli at different concentrations. In the figure, line 1 is E.coli without any treatment, line 2 is a sample treated with a DMSO solvent without a polypeptide, and lines 3 and 4 are samples treated with a 20uM leader peptide and L-OmpF-16s, respectively. 5. 6 are samples treated with leader peptide at a concentration of 30uM and L-OmpF-16s, respectively. Wherein, the dilution degree of each 6 colony images under each treatment is respectively 10 from right to left⁵、10⁴、10³、10²10, 1.

FIG. 4 is a graph showing the bactericidal effect of the polypeptide enhanced low chemical composition hand sanitizer. In the figure, line 1 is a sample of E.coli without any treatment, and lines 2, 3 and 4 are samples treated with 800-fold diluted soap, wherein the soap in line 2 contains 1ul of DMSO solvent, and the soap in line 3 contains 1ul of the peptide, the peptideThe final concentration was 50uM, the hand sanitizer in line 4 contained 1ul of L-OmpF-16s, and the final concentration of the polypeptide was 50 uM. Wherein, the dilution degree of each 6 colony images under each treatment is respectively 10 from right to left⁵、10⁴、10³、10²10, 1.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention.

The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified. In the quantitative tests in the following examples, three replicates were set up and the results averaged.

Coli K-12BW25113 in the following examples this biological material is available to the public from the applicant only for use in repeating experiments relating to the present invention and not for other uses (E. coli K-12BW25113 is a standard strain of E.coli widely used in biological laboratories and reference is made to Baba T.et al (2006) Construction of Escherichia coli K-12in-frame, single-gene knock-out variants: the Keio collection. mol Systems Biol 2(1),1-11, doi:10.1038/msb4100050.)

The amino acid sequence of the leader peptide is shown in SEQ ID NO. 3.

EXAMPLE 1 Observation of E.coli endogenously expressed OmpF-16s phenotype

1. The OmpF-16s gene was cloned into the pBAD vector.

2. The constructed plasmid was transferred into Δ OmpF competent cells. Taking out the competent cells from a refrigerator at minus 80 ℃ and placing the competent cells in an ice box, adding 1uL of plasmid to be transferred when the competent cells are not completely melted, carrying out ice bath for 30min, adding 1ml of LB liquid culture medium after the ice bath, and putting the mixture in a shaking table at 37 ℃ for recovering for 60 min. The transformed bacteria were then spread on a plate containing ampicillin resistance, cultured in a 37 ℃ incubator for 12 to 16 hours, positive clones were picked up, cultured to the plateau stage, and dispensed as seed liquid using 1.5mL sterilized EP tubes.

3. The bacteria are activated. Escherichia coli preserved in a refrigerator at-80 ℃ with 20% glycerol, 10. mu.l of OmpF-knockout (Δ OmpF), Sura-knockout (Δ SurA, chloramphenicol resistance), and OmpF-complementation OmpF-knockout (Δ OmpF + POmpF, ampicillin resistance) bacterial solution were aspirated separately, 20mL of LB liquid medium (formulation: 1L medium containing 10g of tryptone, 5g of yeast extract, 10g of NaCl, and the balance water) was added thereto, autoclaved, cultured overnight at 37 ℃ in a shaker (220rpm) until the plateau, and dispensed as a seed solution using 1.5mL of sterilized EP tube.

4. Taking the seed liquid obtained in the step 2 and the step 3 as mother liquid, diluting the seed liquid by 100 times, inoculating the diluted seed liquid into 2ml of LB liquid culture medium, and simultaneously inoculating WT, delta SurA, delta OmpF and delta in the same inoculation proportion

OmpF + POmpF,. DELTA.OmpF + -POmpF-16s, and cultured in a shaker (220rpm) at 37 ℃ until the logarithmic phase (OD value of about 0.5);

5. and (4) taking each bacterial liquid in the step (4), diluting the bacterial liquid by 1000 times, respectively dripping the diluted bacterial liquid on a normal LB plate, an SDS-EDTA plate and an SDS-EDTA-ara plate, wherein the content of SDS is 0.5% (m/w), the content of EDTA is 1mM, and the content of ara (arabinose) is 0.0002%, and observing the survival condition of bacteria after the dripped plates are placed in a 37 ℃ incubator for culturing for 8-12 hours. The results are shown in FIG. 1.

The results showed that, by inducing endogenous expression of OmpF-16s in E.coli, the bacterium failed to survive on the outer membrane interference-screened plate, similar to the phenotype of the SurA knockout strain. While wild type escherichia coli, an OmpF knockout strain and escherichia coli endogenously expressing OmpF full length can grow normally.

EXAMPLE 2in vitro Sterilization test of the polypeptide OmpF-16s

1. Standard strain of activated escherichia coli (e.coli BW 25113). 10. mu.l of the bacterial solution stored in a refrigerator at-80 ℃ with 20% glycerol was aspirated, 20mL of LB liquid medium (formulation: 1L of medium containing 10g of tryptone, 5g of yeast extract, 10g of NaCl, and the balance water; steam sterilization under high pressure) was added, the mixture was cultured overnight in a shaker (220rpm) at 37 ℃ until the plateau, and 1.5mL of sterilized EP tube was dispensed as a seed solution. Diluting the seed liquid by 100 times, inoculating to 2ml LB liquid culture medium, culturing at 37 deg.C with shaking table (220rpm) to logarithmic phase (OD value of 0.5-0.65);

2. taking 6 sterilized EP tubes, subpackaging 100 mu l of the logarithmic phase bacterial liquid obtained in the step 1 in each tube, and placing one tube in a chromatography cabinet at 4 ℃ as an experimental control. The other 5 tubes are placed in a rapid centrifuge for 4min at 13000rpm, and the supernatant is discarded;

3. resuspend with 100ul 0.9% NaCl, centrifuge at 13000rpm for 4min, discard the supernatant;

4. 99ul of treatment solution (EDTA aqueous solution, EDTA concentration 1mM) was used for resuspension, and 1ul of DMSO was added to one of 5 treated samples as a treatment control, and 1ul of OmpF-16s polypeptide solution was added to the other 4 samples, respectively, so that the final concentrations of OmpF-16s polypeptide in the system were 25uM, 50uM, 75uM, and 100uM, respectively. After mixing, the mixture was left to stand for 5 minutes.

5. And (3) standing for 5 minutes, sequentially diluting the bacterial liquid with the treatment liquid in the step (4) according to a gradient of 10 times each time, dripping 5 mu l of the bacterial liquid at each dilution on an LB solid culture medium six-square-grid flat plate, culturing for 8-12 hours in an incubator at 37 ℃, and observing the survival condition of the bacteria. The results are shown in FIG. 2 and Table 1.

TABLE 1 survival and relative bactericidal efficiency of E.coli treated with OmpF-16s polypeptide at different concentrations

Note: relative bactericidal efficiency-survival of treated but polypeptide-free samples/survival of polypeptide-containing samples at treatment

The results showed that the mortality of E.coli was increased by 2, 3, 5 orders of magnitude, respectively, when E.coli was treated with different concentrations of OmpF-16s polypeptide solutions (25uM, 50uM, 75uM, 100uM) compared to the treated samples without the polypeptide. As can be seen, the polypeptide OmpF-16s has good bactericidal effect in vitro.

EXAMPLE 3 in vitro Sterilization test for polypeptide L-OmpF-16s at a concentration

1. Standard strain of activated escherichia coli (e.coli BW 25113). 10. mu.l of the bacterial solution stored in a refrigerator at-80 ℃ with 20% glycerol was aspirated, 20mL of LB liquid medium (formulation: 1L of medium containing 10g of tryptone, 5g of yeast extract, 10g of NaCl, and the balance water; steam sterilization under high pressure) was added, the mixture was cultured overnight in a shaker (220rpm) at 37 ℃ until the plateau, and 1.5mL of sterilized EP tube was dispensed as a seed solution. Diluting the seed liquid by 100 times, inoculating to 2ml LB liquid culture medium, culturing at 37 deg.C with shaking table (220rpm) to logarithmic phase (OD value of 0.45-0.65);

4. 99ul of sterile water was used for resuspension, and 5 samples were treated, wherein 1ul of DMSO was added to one sample as a control, and 1ul of a peptide-targeting solution was added to each of 2 samples, so that the final concentrations of the peptides in the system were 20uM and 30uM, respectively. And adding 1ul of polypeptide L-OmpF-16s solution with a certain concentration into the other 2 samples respectively to ensure that the final concentrations of the polypeptide L-OmpF-16s in the system are respectively 20uM and 30uM, uniformly mixing, and standing for 5 minutes.

5. And (3) standing for 5 minutes, sequentially diluting the bacterial liquid with the treatment liquid in the step (4) according to a gradient of 10 times each time, dripping 5 mu l of the bacterial liquid at each dilution on an LB solid culture medium six-square-grid flat plate, culturing for 8-12 hours in an incubator at 37 ℃, and observing the survival condition of the bacteria. The results are shown in FIG. 3 and Table 2.

TABLE 2 survival and relative bactericidal efficiency of E.coli treated with different concentrations of L-OmpF-16s polypeptide

Note: relative bactericidal efficiency-survival of treated, but polypeptide-free samples/survival of samples containing polypeptides upon treatment

The results show that the mortality of E.coli can be increased by 4 to 5 orders of magnitude when E.coli is treated with different concentrations of the polypeptide L-OmpF-16s in water (20uM, 30uM) compared to the treated samples without the polypeptide. As can be seen, the polypeptide L-OmpF-16s has good bactericidal effect in vitro.

EXAMPLE 4 application example of the polypeptide L-OmpF-16s in a hand sanitizer

1. Standard strain of activated escherichia coli (e.coli BW 25113). 10. mu.l of the bacterial solution stored in a refrigerator at-80 ℃ with 20% glycerol was aspirated, 20mL of LB liquid medium (formulation: 1L of medium containing 10g of tryptone, 5g of yeast extract, 10g of NaCl, and the balance water; steam sterilization under high pressure) was added, the mixture was cultured overnight in a shaker (250rpm) at 37 ℃ until the plateau, and 1.5mL of sterilized EP tube was dispensed as a seed solution. The seed solution was diluted 100 times and inoculated into 2ml of LB liquid medium, and cultured on a shaker (220rpm) at 37 ℃ until logarithmic phase.

2. 4 sterilized EP tubes were numbered 1, 2, 3, 4. Each tube is separately filled with 100 mul of bacterial liquid, wherein the tube No.1 is placed in a chromatography cabinet at 4 ℃ as an experimental control, the other tube No.3 is placed in a rapid centrifuge for centrifugation at 13000rpm for 4 minutes, and the supernatant is discarded;

3. tubes No.2, 3 and 4 were resuspended in 100ul of 0.9% NaCl, centrifuged at 13000rpm for 4 minutes, and the supernatant was discarded;

4. after discarding the supernatant, tubes 2, 3, and 4 were resuspended in 100ul of each hand cleanser. Wherein, the No.2 hand sanitizer contains 1ul of DMSO, the No.3 hand sanitizer contains 1ul of peptide, the final concentration of the peptide is 50uM, the No. 4 hand sanitizer contains 1ul of polypeptide, the final concentration of the polypeptide is 50uM, and the hand sanitizer is kept for 5 minutes at room temperature;

5. after standing for 5 minutes, the bacterial suspension was diluted with the mixed solution containing step 4 in a gradient of 10 times each time, 5. mu.l of the bacterial suspension was dropped on a LB solid medium six-grid plate for each dilution, and after culturing in an incubator at 37 ℃ for 8 to 12 hours, the survival of the bacteria was observed, and the results are shown in FIG. 4 and Table 3.

TABLE 3

Note: relative bactericidal efficiency-survival of treated but polypeptide-free liquid soap treated samples/survival of samples containing polypeptides upon liquid soap treatment

The results show that after the hand sanitizer is diluted by 800 times, namely various original bactericidal components in the hand sanitizer are greatly diluted, the death rate of escherichia coli can be improved by 4 orders of magnitude compared with that of a treatment sample without the polypeptide in the hand sanitizer containing the polypeptide L-OmpF-16 s. Therefore, the polypeptide L-OmpF-16s can replace the traditional chemical sterilization component in application, so that the product has simple components and remarkable sterilization effect.

Sequence listing

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Claims

1. An antimicrobial polypeptide based on the outer membrane protein production machinery, characterized in that: the amino acid sequence is shown in SEQ ID NO. 1.

2. An antimicrobial polypeptide comprising a depsipeptide, wherein: the amino acid sequence is shown in SEQ ID NO. 2.

3. Use of a polypeptide according to claim 1 or 2 for inhibiting or killing gram-negative bacteria for non-therapeutic purposes.

4. Use according to claim 3, characterized in that: the gram-negative bacteria are escherichia coli.

5. Use of a polypeptide according to claim 1 or 2 for the preparation of a washing product having bacteriostatic or bactericidal activity.