阅读说明：本技术 基于乳铁蛋白的仿生抗菌功能多肽及制备方法和应用 (Lactoferrin-based bionic antibacterial functional polypeptide and preparation method and application thereof ) 是由 张凌琳 罗俊元 姜文韬 王琨 王雨霏 冯泽宁 于 2020-12-24 设计创作，主要内容包括：本发明公开了基于乳铁蛋白的仿生抗菌功能多肽及其制备方法和应用,属于生物技术领域,解决了现有技术中天然抗菌肽序列和结构较复杂、提纯生产成本高、体内代谢可能被干扰的问题。本发明的多肽包含如SEQ ID No.1所示的氨基酸序列,进一步地包含如SEQ ID No.2所示的氨基酸序列。本发明公开了多肽在制备具有包括但不仅限于杀菌功能药物中的应用。本发明的多肽分子量小,结构简单稳定,仿生效果好,具有良好的抗菌效果及安全性；其制备方法简单,生产成本低,易于推广。(The invention discloses a lactoferrin based bionic antibacterial functional polypeptide and a preparation method and application thereof, belongs to the technical field of biology, and solves the problems that in the prior art, a natural antibacterial peptide sequence and structure are complex, the purification production cost is high, and the in vivo metabolism is possibly interfered. The polypeptide of the invention comprises an amino acid sequence shown as SEQ ID No.1, and further comprises an amino acid sequence shown as SEQ ID No. 2. The invention discloses application of polypeptide in preparing medicines with bactericidal function. The polypeptide has the advantages of small molecular weight, simple and stable structure, good bionic effect, good antibacterial effect and safety; the preparation method is simple, low in production cost and easy to popularize.)

1. The bionic antibacterial functional polypeptide based on lactoferrin is characterized by comprising an amino acid sequence shown as SEQ ID No. 1.

2. A lactoferrin based biomimetic antibacterial function polypeptide according to claim 1, comprising the amino acid sequence as shown in SEQ ID No. 2.

3. A biomimetic lactoferrin based on functional antibacterial polypeptide according to claim 1 or 2, wherein the C-terminal of the polypeptide is amidated and modified, or is pharmaceutically or acceptable salt or ester thereof.

4. A biomimetic lactoferrin based on functional antibacterial polypeptide according to claim 3, wherein the pharmaceutically acceptable salt is hydrochloride, sulfate, acetate, mesylate, succinate, fumarate, citrate, malate or organic amine salt.

5. A method for preparing the polypeptide with antibacterial function according to any one of claims 1 to 4, which comprises the following steps: according to the prepared polypeptide sequence, amidating the first amino group, protecting the amino group by Fmoc, connecting the amino group to Wang resin serving as a solid-phase carrier, and removing the amino protecting group; then reacting the second amino acid with the amino group protected by Fmoc with the amino group of the first amino acid connected with the solid phase carrier under the activation action of a condensing agent to form a peptide bond; repeating the peptide bond forming reaction to enable the peptide chain to grow from the C end to the N end until the last amino acid is accessed, and obtaining the target polypeptide after cutting.

6. A pharmaceutical composition comprising the polypeptide of any one of claims 1-5, and a pharmaceutically acceptable carrier and/or excipient.

7. The pharmaceutical composition of claim 6, wherein the formulation comprises a liquid formulation, a solid formulation, or a semi-solid formulation.

8. The pharmaceutical composition of claim 7, wherein the liquid formulation comprises a solution or an injection, the solid formulation comprises a tablet or a capsule, and the semi-solid formulation comprises an ointment or a gel.

9. Use of the polypeptide of any one of claims 1-4 in the manufacture of a medicament having bactericidal properties, including but not limited to.

10. The use of claim 9, wherein the bacteria include, but are not limited to, cariogenic bacteria.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a lactoferrin-based bionic antibacterial functional polypeptide, and a preparation method and application thereof.

Background

Caries is a chronic infectious disease that occurs on hard dental tissue and is the most common oral disease in humans. Caries can lead to demineralization of the hard tissues of the tooth body, and unapproved caries can progress to pulposis, periapical disease and even tooth loss. Because the caries has high incidence rate and wide epidemic areas and seriously affects the oral cavity and the whole body health, the world health organization classifies the caries as one of three major non-infectious diseases for human prevention and treatment, which are second only to cardiovascular diseases and tumors.

The pathogenic factors of caries include four factors of bacterial plaque biomembrane, diet, host susceptibility and time, wherein the bacterial plaque biomembrane is the most important initiating factor of caries. Common cariogenic bacteria mainly comprise streptococcus mutans, lactobacillus acidophilus and actinomyces viscosus, which can adhere to the tooth surface to form dental plaque organisms, and can metabolize sugar to produce acid when a human body ingests a high-sugar diet, so that demineralization of hard tissues of the tooth body is caused, and further caries is caused. Therefore, infection control of cariogenic bacteria is an important principle for the prevention and treatment of caries.

Classical anticariogenic agents can inhibit the action of cariogenic bacteria to some extent, thereby inhibiting the onset and progression of caries, however, these agents have shown some limitations in long-term clinical practice. Fluoride is recognized as the most effective anticaries agent at present, which inhibits the progression of caries by promoting remineralization and inhibiting the action of cariogenic bacteria metabolism. However, with the widespread use of various fluoride preparations, the emergence of fluorine-resistant strains, fluorodentia and fluoroossicles has made their limitations increasingly prominent in caries prevention. In addition, antibacterial drugs such as chlorhexidine and tetracycline can obviously inhibit the activity of cariogenic microorganisms, but long-term use of the drugs can also show the defects of dysbacteriosis, appearance of drug-resistant bacteria, staining of tooth bodies and the like. In view of the above problems, other caries-preventing drugs and methods having definite antibacterial effects and low side effects should be actively sought.

The antibacterial peptide is a polypeptide substance with antibacterial activity widely existing in animals and plants in nature, generally consists of 12-50 amino acid residues, most of the antibacterial peptide is cationic antibacterial peptide, and more than two thousand of natural antibacterial peptides are found at present. Compared with the traditional antibacterial preparation, the antibacterial composition has the advantages of low molecular weight, wide antibacterial spectrum, unique antibacterial mechanism, low toxic and side effects, low drug resistance and the like, is expected to overcome the defects of the traditional antibacterial preparation, and is expected to become a new generation of antibacterial drugs. However, natural antibacterial peptides also have the defects of relatively complex sequence and structure, high purification and production cost, possible interference of in vivo metabolism and the like, so that the natural antibacterial peptides are limited in clinical application.

Therefore, it is an urgent need to solve the problems of the technical personnel in the field to provide an antibacterial functional polypeptide, which has simple preparation method, small molecular weight and simple and stable structure.

Disclosure of Invention

Lactoferrin is a multifunctional endogenous immune protein in vivo, and retains free iron ions in body fluid through iron chelation, thereby playing roles in multiple biological functions such as antibiosis, anti-inflammation, anti-tumor and the like. Lactoferrin is an important component of saliva and acquired membranes and plays an important role in inhibiting the adhesion of cariogenic bacteria, plaque formation and the progression of caries. It can form iron chelate to capture the iron ion necessary for bacterial growth to inhibit bacteria, and can also kill cariogenic bacteria directly. In addition, the active cariogenic bacteria can stimulate the secretion of lactoferrin in the body to be increased, the increase of the content of the lactoferrin can be cooperated with other proteins to inhibit the cariogenic action of bacteria, and the physiological stress mechanism of the body is met.

The invention aims to provide a bionic antibacterial functional polypeptide based on lactoferrin, which has small molecular weight, stable structure and better antibacterial activity than a parent peptide.

The second purpose of the invention is to provide a preparation method of the bionic antibacterial functional polypeptide.

The invention also aims to provide a pharmaceutical composition containing the bionic antibacterial functional polypeptide.

The fourth purpose of the invention is to provide the application of the bionic antibacterial functional polypeptide.

The technical scheme adopted by the invention is as follows:

the bionic antibacterial functional polypeptide based on lactoferrin and pharmaceutically acceptable salts thereof are characterized in that the polypeptide comprises an amino acid sequence shown as SEQ ID No. 1.

Further, the polypeptide comprises an amino acid sequence shown as SEQ ID No. 2.

Preferably, the polypeptide is C-terminal amidated and modified, or is a pharmaceutically or acceptable salt or ester thereof.

In the technical scheme of the invention, the pharmaceutically acceptable salt is hydrochloride, sulfate, acetate, methanesulfonate, succinate, fumarate, citrate, malate or organic amine salt.

The preparation method of the antibacterial functional polypeptide comprises the following steps: according to the prepared polypeptide sequence, amidating the first amino group, protecting the amino group by Fmoc, connecting the amino group to Wang resin serving as a solid-phase carrier, and removing the amino protecting group; then reacting the second amino acid with the amino group protected by Fmoc with the amino group of the first amino acid connected with the solid phase carrier under the activation action of a condensing agent to form a peptide bond; repeating the peptide bond forming reaction to enable the peptide chain to grow from the C end to the N end until the last amino acid is accessed, and obtaining the target polypeptide after cutting.

The pharmaceutical composition contains the polypeptide and a pharmaceutically acceptable carrier and/or auxiliary material. The "pharmaceutically acceptable carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic agent is administered and which is, within the scope of sound medical judgment, suitable for contact with the tissues of humans and/or other animals without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit/risk ratio.

In the technical scheme of the invention, the preparation comprises a liquid preparation, a solid preparation or a semisolid preparation.

Preferably, the liquid preparation comprises a solution or an injection, the solid preparation comprises a tablet or a capsule, and the semisolid preparation comprises an ointment or a gel.

The polypeptide of the invention is applied to the preparation of medicines with bactericidal function, including but not limited to.

Preferably, the bacteria include, but are not limited to, cariogenic bacteria.

Compared with the prior art, the invention has the following beneficial effects:

the bionic functional polypeptide based on lactoferrin has the advantages of small molecular weight, simple and stable structure, good bionic effect, good antibacterial effect and safety, low hemolysis and controllable cytotoxicity, can kill common cariogenic bacteria in the oral cavity at lower concentration in short time.

The bionic functional polypeptide based on lactoferrin has the advantages of simple preparation method, low production cost, easy popularization and industrialization realization, can effectively reduce the occurrence of bacterial drug resistance, and has great potential in the direction of replacing antibiotics to become mainstream antibacterial drugs.

Drawings

FIG. 1-1 is a graph showing the bactericidal kinetics of LF-1 against S.mutans;

FIG. 1-2 is a graph showing the bactericidal kinetics of LF-1 against Actinomycetes viscosus;

FIGS. 1-3 are graphs showing the bactericidal kinetics of LF-2 against S.mutans;

FIGS. 1-4 are graphs showing the bactericidal kinetics of LF-2 against Actinomycetes viscosus;

FIG. 2-1 is a graph showing the biofilm inhibitory effect of LF-1 on Streptococcus mutans;

FIG. 2-2 is a graph showing the biofilm inhibitory effect of LF-2 on Streptococcus mutans;

FIG. 3-1 is a transmission electron micrograph of the bactericidal effect of LF-1 and LF-2 on Streptococcus mutans;

FIG. 3-2 is a transmission electron micrograph of the bactericidal effect of LF-1 and LF-2 on Lactobacillus acidophilus;

FIG. 3-3 is a transmission electron micrograph of the bactericidal effect of LF-1 and LF-2 on Actinomycetes viscosus;

FIG. 4-1 is a circular dichroism map of LF-1;

FIG. 4-2 is a circular dichroism map of LF-2;

FIGS. 4-3 are graphs of LF-1 and LF-2 for circular dichroism analysis of LF-1;

FIG. 5-1 is a graph showing the effect of LF-1 on proliferation of human gingival fibroblasts;

FIG. 5-2 is a graph showing the effect of LF-2 on proliferation of human gingival fibroblasts.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to specific examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Example 1

The embodiment provides a lactoferrin-based biomimetic antibacterial functional polypeptide LF-1, and the amino acid sequence is shown as follows:

SEQ ID No.1：WKLLRKAWKLLRKA。

the preparation method comprises the following steps:

1. Fmoc-His (Trt) -Wang Resin is selected as Resin (carrier);

2. the resin was fully swollen with DCM (dichloromethane);

3. removing the Fmoc-protecting group with appropriate concentration of DBLK (piperidine + DMF);

4. washing with DMF for several times to remove DBLK;

5. weighing a proper condensing agent and an activating agent (HBTU, NMM) and a second Fmoc-protected amino acid (Fomc-Leu-OH) at the C terminal for coupling;

6. ninhydrin detection to ensure complete ligation;

7. washing with DMF for several times to remove residual residues and activator condensing agent;

8. coupling according to the amino acid sequence of SEQ ID No.1, wherein the method refers to the steps 3-7;

9. removing the final Fmoc-protecting group by adopting the method of 3-4 after all amino acids are connected;

10. using TFA cutting fluid to perform cracking, and removing resin and amino acid protecting groups to obtain a crude product;

11. the product is confirmed to be correct by mass spectrum (the molecular weight of SEQ ID No.1 is 1810.23 and is in accordance with a theoretical value);

12. the crude product is sent to purification and separation to improve the purity.

Example 2

The embodiment provides a lactoferrin-based biomimetic antibacterial functional polypeptide LF-2, and the amino acid sequence is shown as follows:

SEQ ID No.2：GKLIWKLLRKAWKLLRKA。

compared with the preparation steps of the embodiment 1, the preparation steps are different in that the step 8 is as follows: the coupling is carried out according to the amino acid sequence of SEQ ID No.2, and the rest conditions are the same.

Wherein, the product is confirmed to be correct by mass spectrometry (the molecular weight of SEQ ID No.2 is 2221.88 which accords with the theoretical value).

Example 3

The embodiment discloses the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) determination of lactoferrin bionic antibacterial function-based polypeptides LF-1 and LF-2.

The minimum inhibitory concentration mic (minimum inhibitory concentration) refers to the lowest concentration of an antibacterial agent that inhibits the growth of bacteria in a culture medium in an in vitro assay. Minimum bactericidal concentration mbc (minimum bacterial concentration) refers to the minimum concentration of an antimicrobial drug that reduces viable bacterial growth by more than 99% in an in vitro test. MIC and MBC are indexes of the antibacterial activity of the medicine, and show the capability of the medicine for inhibiting and killing pathogenic microorganisms. The bacteria used in the experiments were Streptococcus mutans (UA 159), Streptococcus sanguis (ATCC 10556), Streptococcus Gracillus (DL 1), Lactobacillus acidophilus (ATCC 14931), Lactobacillus casei (ATCC 393), Lactobacillus fermentum (933ATCC 8), Actinomyces viscosus (ATCC 15987), Actinomyces naeslundii (ATCC 12104).

The MIC and MBC determination experiment steps are as follows:

1. single colony was picked up in 10mL BHI liquid medium at 37 ℃ constant temperature anaerobic culture tank (80% N)₂，10％H₂，10％CO₂) The culture was carried out overnight.

2. 100 mul of bacterial liquid is sucked into 10mL of BHI liquid culture medium and placed in a constant-temperature anaerobic culture tank (80% N)₂，10％H₂，10％CO₂) After subculturing for 2-3 generations, taking the bacteria liquid in the middle logarithmic growth phase and diluting the bacteria liquid to 2 multiplied by 10⁶CFU/mL is ready for use.

3. The polypeptides were added to U-shaped 96-well plates at 20. mu.L per well using a 2-fold dilution method.

4. 80 mu L of BHI culture medium and 100 mu L of bacterial liquid are added into each hole, so that the final concentration of the polypeptide is 256-0.5 mu mol/L.

5. An antibacterial peptide template (WA-7, sequence: WKLLRKA as shown in SEQ ID No. 3) and an antibacterial peptide parent peptide (WR-17, sequence: WKLLSKAQEKFGKNKSR as shown in SEQ ID No. 4) are used as drug controls, and sterile deionized water is used as a negative control.

6. The 96-well plate is placed in a constant temperature anaerobic culture tank (80% N) at 37 DEG C₂，10％H₂，10％CO₂) And culturing for 24 h.

7. MIC is the lowest clarified polypeptide concentration in the well plate.

8. Sucking 100 μ L bacterial liquid from the clear hole, spreading on BHI plate, and culturing in 37 deg.C constant-temperature anaerobic culture tank (80% N)₂，10％H₂，10％CO₂) The culture was carried out overnight for 48 h.

9. MBC is the lowest polypeptide concentration for growth of a sterile colony on the plate.

10. The above experiment was repeated at least 3 times.

The resulting data are shown in table 1: both polypeptides have certain inhibition effect on common oral bacteria. LF-1 showed specific antibacterial effects against S.mutans compared to other oral bacteria, with MIC and MBC of 8 and 16 (. mu.mol/L), respectively. LF-2 showed broad-spectrum antibacterial activity against common oral bacteria with MIC and MBC of 16 and 32 (. mu. mol/L) against S.mutans, respectively.

TABLE 1 MIC and MBC (. mu. mol/L) of antibacterial functional Polypeptides against common bacteria in the oral cavity

Example 4

The embodiment discloses a test of the sterilization kinetics of common oral cariogenic bacteria based on lactoferrin bionic antibacterial functional polypeptides LF-1 and LF-2.

The bactericidal kinetics is an index reflecting the relationship between the antibacterial capacity and the action time of the medicament. The experimental bacteria are mainly oral cariogenic bacteria, Streptococcus mutans (UA 159) and Actinomyces viscosus (ATCC 15987). The determination steps are as follows:

1. single colony was picked up in 10mL BHI liquid medium at 37 ℃ constant temperature anaerobic culture tank (80% N)₂，10％H₂，10％CO₂) The culture was carried out overnight.

2. 100 mul of bacterial liquid is sucked into 10mL of BHI liquid culture medium and placed in a constant-temperature anaerobic culture tank (80% N) at 37 DEG C₂，10％H₂，10％CO₂) After subculturing for 2-3 generations, taking the bacteria liquid in the middle logarithmic growth phase and diluting the bacteria liquid to 2 multiplied by 10⁶CFU/mL is ready for use.

3. The polypeptides were diluted with sterile deionized water and added to 24-well plates at 100. mu.L per well.

4. 400 mul BHI culture medium and 500 mul bacterial liquid are added into each hole, the final concentration of the polypeptide is 4MBC, 2MBC and MBC, and sterile deionized water is used as negative control.

5. The 24-hole plate is placed in a constant-temperature anaerobic culture tank (80% N) at 37 DEG C₂，10％H₂，10％CO₂) And (5) culturing.

6. At each time point (0min, 1min, 5min, 10min, 20min, 30min, 1h, 2h, 4h), 100 mu L of bacterial liquid is sucked, diluted and coated on a BHI plate, and the colony count is calculated after overnight culture at 37 ℃ for 48 h;

8. the experiment was repeated 3 times.

The experimental results are shown in FIGS. 1-1 to 1-4: time is plotted on the abscissa, and the ordinate represents the number of colonies per ml, and the change in the number of colonies with increasing time indicates the growth of the bacteria. As can be seen from the figure, when the concentration of the polypeptide is located at the concentration of MBC, the polypeptide can kill bacteria within 30-60 minutes, and the higher the concentration of the polypeptide is, the shorter the sterilization time is. Particularly, the sterilizing effect of LF-2 is stronger than that of LF-1 under the same conditions. The high-concentration antibacterial peptide can kill bacteria effectively in a short time, has strong sterilizing capability and short time, and has considerable application prospect.

Example 5

The embodiment discloses the influence of lactoferrin-based biomimetic antibacterial functional polypeptides LF-1 and LF-2 on a streptococcus mutans biological membrane.

The minimum biofilm inhibition concentration MBIC (minimum biofilm inhibition concentration) is an index reflecting the biofilm formation resistance of a drug. Wherein the MBIC₉₀Refers to the lowest concentration of the antimicrobial agent that inhibits greater than 90% of biofilm formation in the culture medium in an in vitro assay. The experimental bacterium is Streptococcus mutans (UA 159). The determination steps are as follows:

1. single colony was picked up in 10mL BHI liquid medium at 37 ℃ constant temperature anaerobic culture tank (80% N)₂，10％H₂，10％CO₂) The culture was carried out overnight.

2. 100 mul of bacterial liquid is sucked into 10mL of BHI liquid culture medium and placed in a constant-temperature anaerobic culture tank (80% N)₂，10％H₂，10％CO₂) After subculturing for 2-3 generations, taking the bacteria liquid in the middle logarithmic growth phase and diluting the bacteria liquid to 2 multiplied by 10⁶CFU/mL is ready for use.

3. The polypeptide was dissolved in sterile distilled water and added to a 24-well plate using a 2-fold dilution method, 100. mu.L per well.

4. 400 mu L of BHIS (BHI containing 2% of sucrose) culture medium and 500 mu L of bacterial liquid are added into each hole, so that the final concentration of the polypeptide is 256-0.5 mu mol/L, and sterile deionized water is used as a negative control.

5. The 24-hole plate is placed in a constant-temperature anaerobic culture tank (80% N) at 37 DEG C₂，10％H₂，10％CO₂) Culturing for 24 h;

6. the culture solution was aspirated off, and the amount of biofilm formed was detected: washing with PBS twice, fixing with methanol for 15min, dyeing with 0.1% crystal violet for 5min, decolorizing with 33% glacial acetic acid for 30min, and measuring absorbance at 595 nm;

7、MBIC₉₀the lowest polypeptide concentration is the one with absorbance reduced by more than 90% compared with the negative control group.

8. The experiment was repeated at least 3 times.

The results are shown in FIGS. 2-1 to 2-2: the abscissa represents the drug concentration and the ordinate represents the absorbance, which reflects the amount of biofilm, the higher the absorbance, the more the amount of biofilm. The amount of biomembrane formed by the drug group is obviously reduced compared with that of the negative control group, and the difference has statistical significance (P)<0.05), sterile water negative control group had no effect on biofilm formation. The reduction of the formation of the biofilm by the LF-1 group with the drug concentration of 8 mu mol/L and the LF-2 group with the drug concentration of 16 mu mol/L is more than 90 percent of that of the negative control group, and the MBIC of the polypeptide can be seen₉₀Respectively 8. mu. mol/L (LF-1) and 16. mu. mol/L (LF-2).

Example 6

The embodiment discloses transmission electron microscope observation of the sterilization effect of lactoferrin bionic antibacterial function-based polypeptides LF-1 and LF-2 on common oral cariogenic bacteria.

The experimental bacteria are mainly oral cariogenic bacteria, namely Streptococcus mutans (UA 159), Lactobacillus acidophilus (ATCC 14931) and Actinomyces viscosus (ATCC 15987). The experimental steps are as follows:

1. single colony was picked up in 10mL BHI liquid medium at 37 ℃ constant temperature anaerobic culture tank (80% N)₂，10％H₂，10％CO₂) The culture was carried out overnight.

2. 100 mul of bacterial liquid is sucked into 10mL of BHI liquid culture medium and placed in a constant-temperature anaerobic culture tank (80% N)₂，10％H₂，10％CO₂) After subculturing for 2-3 generations, taking the bacteria liquid in the middle logarithmic growth phase and diluting the bacteria liquid to 2 multiplied by 10⁹CFU/mL is ready for use.

3. The polypeptide was diluted with sterile deionized water and added to a15 mL centrifuge tube, 1mL per tube.

4. 4mL of BHI culture medium and 5mL of bacterial liquid are added into each tube, so that the final concentration of the polypeptide is 64 mu mol/L, and sterile deionized water is used as a negative control.

5. Placing the centrifuge tube in a 37 deg.C constant temperature anaerobic culture tank (80% N)₂，10％H₂，10％CO₂) And culturing for 24 h.

6. The bacterial pellets were collected by centrifugation at 4,500 Xg for 5min, washed 2 times with PBS buffer solution, and fixed overnight at 4 ℃ using 2.5% glutaraldehyde solution.

7. Dehydration was performed using an ethanol gradient (35%, 50%, 75% treatment for 30min, 90% and 100% treatment for 30min2 times).

8. Embedding the sample, curing in an oven, and slicing.

9. 3% uranium acetate-lead citrate double staining, and observing by a transmission electron microscope.

The results are shown in FIGS. 3-1 to 3-3: the death form of perforation and fragmentation of common cariogenic bacteria can be observed under a transmission electron microscope, microscopically, the cell walls of the bacteria are mainly damaged after the bacteria are treated by the polypeptide, the alpha-helical structure sterilization principle of the polypeptide is met, and the bacteria are cracked and finally die due to the fact that the alpha-helical structure perforates the cell walls of the bacteria after the polypeptide contacts the bacteria.

Example 7

The embodiment discloses circular dichroism spectrum analysis of lactoferrin bionic antibacterial functional polypeptide LF-1 and LF-2.

Circular dichroism (CD for short) spectroscopy is a rapid, simple and accurate method for studying protein conformation in dilute solution. This experiment was used to measure the structure of polypeptides to verify the validity of their design principle. As can be seen from a review of the literature, circular dichroism spectroscopy of the α -helical structure shows a positive band near 192nm at the UV wavelength and two negative characteristic shoulder bands at 222 and 208 nm. The experimental instrument used was a Jasco J-1500CD Spectrometer (Japan) which was performed as follows:

under the condition of 25 ℃, the light transmission length of the container is measured to be 1mm, and the wavelength range of the ultraviolet light is 190 nm-240 nm. Each sample was scanned 10 times to flattenAnd (4) average value. The sample polypeptide was 100. mu. mol/L in PBS (20mmol/L), SDS (25mmol/L) in PBS and TFE (50% v/v) in PBS, respectively. The obtained data is used for calculating the molar ellipticity through a formulaThe plotted graph is shown in FIG. 4.

As shown in FIGS. 4-1-4-2, the polypeptide spectrogram has a typical alpha-helical structure in the presence of a surfactant (SDS) and a common chemical solvent (TFE); as can be seen from FIGS. 4-3, the ratio of alpha helices of both polypeptides was more than 50% in the pseudo-membrane environment SDS solution. The results show that the secondary structure of the polypeptide in the solution is alpha helix, the design principle of the polypeptide is verified, and the bactericidal capability of the polypeptide is structurally explained and proved.

Example 8

The embodiment discloses a hemolytic experiment based on lactoferrin bionic antibacterial function polypeptides LF-1 and LF-2, which comprises the following steps:

1. defiberized aseptic sheep blood was selected, centrifuged at 3000rpm for 15 minutes, washed with 1 time PBS solution (20mmol/L), and sheep red blood cells were collected, and the above procedure was repeated 3 times.

2. Preparing a red blood cell suspension: blood cells were suspended in a 1-fold PBS solution at a ratio of 20% (v/v).

3. A20% suspension of red blood cells was diluted 1:20 in PBS to form a final 2mmol/L hemoglobin solution before use.

4. Using a 96-well plate, 100. mu.L of red blood cell suspension and 100. mu.L of polypeptide PBS solution are added into a single well, and the final concentration of the polypeptide is 256-1. mu. mol/L by gradient dilution. 1% Tween-20 WAs used as a positive control, 1-fold PBS solution WAs used as a negative control, and an antimicrobial peptide template (WA-7) and an antimicrobial peptide parent peptide (WR-17) were used as drug controls.

5. Incubate at 37 ℃ for 1h and experiment was repeated at least 3 times.

6. And absorbing the supernatant of the solution, and measuring the absorbance value at 450nm to judge the hemolysis.

The results are shown in table 2: the higher the absorbance of the obtained solution, the higher the reaction solutionThe stronger the blood effect, and calculate the corresponding hemolysis rate and HC₅₀(concentration of polypeptide that results in hemolysis of 50% erythrocytes). The resulting data are plotted in the following table. LF-1 showed very low hemolysis rate at high concentration (256. mu. mol/L). HC of LF-2₅₀Between 256-128. mu. mol/L, it is indicated that the hemolysis effect is acceptable at a concentration of polypeptide below 128. mu. mol/L. The template peptide (WA-7) and the parent peptide (WR-17) did not exhibit significant hemolysis. Therefore, LF-1 and LF-2 have better clinical value.

TABLE 2 hemolytic comparison of biomimetic antibacterial functional polypeptides

Example 9

The embodiment discloses the influence of lactoferrin bionic antibacterial function-based polypeptides LF-1 and LF-2 on the proliferation of human gingival fibroblasts.

After approval by the institute of medical and ethics of Sichuan university, gingival fibroblasts (hereinafter abbreviated as HGFs) were purchased at Beijing Beinana institute of Biotechnology and creation, and are referred to as "HGFs". Cell culture and passage were performed according to the instructions and passed to generations 4-6 for experiments.

1. HGFs were seeded in 96-well plates at 5000 cells per well in DMEM medium containing double antibody, 10% fetal bovine serum in CO₂Incubator (5% CO)₂And culturing in 95% air at 100% humidity at 37 ℃ for 24-48 h until the coverage area of the cells is about 50%.

2. And (3) carrying out gradient dilution on the polypeptide, wherein the concentration range is 256-1 mu mol/L, the concentration of each group is 3 multiple holes, and treating the cells for 1 h. After the treatment, the supernatant was discarded, washed with pre-warmed PBS, and then added with 100. mu.L of DMEM medium to continue the culture.

3. Add 10. mu.L of CCK-8 solution, polypeptide treated and untreated (negative) to each wellControl) in CO₂Incubator (5% CO)₂Incubation at constant temperature of 95% air, 100% humidity, 37 ℃ for 1-4 h until the solution exhibits appropriate color.

4. Mix gently on a shaker and read the values at 450nm using a microplate reader.

The absorbance of the liquid in each well is higher, which indicates that the cell number is higher and the cell proliferation is better. The obtained data are shown in FIGS. 5-1-5-2: for LF-1, high concentration (more than 128. mu. mol/L) polypeptide treatment has a great influence on cell proliferation, but after small concentration (less than or equal to 64. mu. mol/L) polypeptide treatment, the cell survival rate can be more than 80%, and the polypeptide with the concentration of 32. mu. mol/L and below is basically non-cytotoxic (P is more than 0.05), and the concentration can quickly kill common cariogenic bacteria such as S.mutans, and the like, so the method has strong clinical application value. LF-2 has a concentration-dependent effect on the proliferative activity of cells, and lower concentrations have less effect on the proliferative activity of cells. The concentration of the MBC in the oral cavity cariogenic bacteria is 16-32 mu mol/L, and the influence on cell proliferation can be accepted at the concentration.

In conclusion, the functional polypeptide with good antibacterial capacity is designed based on the antibacterial sequences of natural lactoferrin and the polypeptide by means of a bionic idea, and a new ideal way is provided for preventing and treating caries. The bionic antibacterial functional polypeptide shows good antibacterial effect, and can kill common cariogenic bacteria in the oral cavity at lower concentration in a short time. Meanwhile, the molecular weight is small, the structure is clear and stable, the hemolytic property is low, and the cytotoxicity to human gingiva fibroblast in vitro is in a controllable range. Therefore, the polypeptide has important research value in the fields of caries prevention and treatment and antibiosis.

The foregoing is merely a preferred embodiment of this invention, which is intended to be illustrative, not limiting; those skilled in the art will appreciate that many variations, modifications, and even equivalent variations are possible within the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

<110> Sichuan university

<120> bionic antibacterial functional polypeptide based on lactoferrin, preparation method and application

<130> 20201221

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<170> PatentIn version 3.3

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