A strain of Bacillus Velezii CB6 and its antimicrobial protein and its application

Abstract

The present invention discloses a strain of Bacillus Velez CB6 and the antimicrobial protein produced and its application, belonging to the field of microbial application technology. The deposit number of the Bacillus Velez CB6 is CGMCC NO.30515. The strain has a good antagonistic effect on Gram-negative bacteria such as multidrug-resistant Escherichia coli. The antimicrobial protein CB6-E prepared by fermentation of the strain shows good antimicrobial activity against a variety of Gram-negative bacteria, and has good safety for a variety of animals. CB6-E has a good therapeutic effect on mice and wax moths infected with polymyxin-resistant and carbapenem-resistant Escherichia coli. The antimicrobial protein CB6-E prepared by fermentation of Bacillus Velez CB6 of the present invention is expected to enrich the current existing antimicrobial drug library and provide new hope for the treatment of multidrug-resistant Gram-negative bacterial infections.

Description

Technical Field

[0001] The invention relates to the technical field of microbial application, in particular to a strain of Bacillus Velezii CB6 and the antibacterial protein produced thereby and application thereof. Background Art

[0002] With the continuous emergence and widespread spread of multidrug-resistant bacteria, especially the discovery and spread of transferable resistance genes of pan-resistant pathogens. Multidrug-resistant Escherichia coli B2 (Escherichia coli, E. coli B2) is the main pathogen that causes intestinal infections or urinary tract infections in humans and animals. It is resistant to multiple antibiotics, such as polymyxins and carbapenems, and is one of the most common pathogens among Gram-negative bacteria. In the phylogenetic group of Escherichia coli, the E. coli B2 group contains both extraintestinal pathogenic Escherichia coli (EXPC) and enteropathogenic Escherichia coli (EPEC) strains. At present, E. coli B2 has become an important pathogen that endangers the health of humans and animals. With the increasing resistance of the bacteria to commonly used antibiotics in clinical practice, it has caused serious economic losses to humans and animal husbandry, and also brought huge challenges to clinical anti-infection treatment.

[0003] Bacillus is a common type of bacteria that can produce a variety of substances with antibacterial activity during its growth process, such as antibiotics, bacteriocins, and antimicrobial proteins, which can kill or inhibit other bacteria, help Bacillus gain growth advantages in competition, and protect its living environment from other bacteria. Antimicrobial proteins are a type of natural defense molecules that exist in a variety of organisms. They have good antimicrobial activity, a broad antimicrobial spectrum, and safety and stability against microorganisms (such as bacteria, fungi, and viruses). They have therapeutic potential for human diseases and are considered to be an important resource for the development of new antimicrobial drugs. At present, although the antimicrobial proteins produced by microbial metabolism can produce broad-spectrum resistance to a variety of pathogens including Gram-negative and Gram-positive bacteria, there are no new antimicrobial protein drugs for Gram-negative bacteria, especially super-resistant bacteria resistant to polymyxins and carbapenems, due to the serious harm and high drug resistance of Gram-negative pathogens in current clinics.

[0004] Based on this, exploring antimicrobial proteins that target resistant Gram-negative bacteria such as polymyxin-resistant and carbapenem-resistant Escherichia coli is of great significance for clinical anti-infection treatment and enriching the current antimicrobial drug library. Summary of the invention

[0005] The purpose of the present invention is to provide a strain of Bacillus Velez CB6 and the antibacterial protein produced thereby and applications thereof, so as to solve the problems existing in the above-mentioned prior art. The present invention independently isolates a strain of Bacillus Velez CB6 having good antagonistic effect on Gram-negative bacteria such as multidrug-resistant Escherichia coli. The antibacterial protein CB6-E prepared by fermentation of the bacterium shows good antibacterial activity against a variety of Gram-negative bacteria and has good safety for a variety of animals. It is expected to enrich the current antibacterial drug library and provide new hope for the treatment of multidrug-resistant Gram-negative bacteria infection.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a bacteriocin-producing Bacillus velezensis CB6, which was deposited in the General Microbiology Center of China National Committee for the Preservation of Microorganisms on May 7, 2024, with the deposit number of CGMCCNO.30515.

[0008] The present invention also provides an antibacterial protein CB6-E for inhibiting Gram-negative bacteria. The antibacterial protein CB6-E is obtained by fermenting the Bacillus Velezii CB6, and the amino acid sequence is shown in SEQ ID NO.1.

[0009] The present invention also provides a method for preparing the antibacterial protein CB6-E, comprising the steps of inoculating and fermenting the Bacillus Velez subtilis CB6, extracting and purifying the fermentation product by ammonium sulfate precipitation, dialysis interception and chromatography purification, respectively, to obtain the bacteriocin CB6-E.

[0010] Furthermore, the fermentation temperature is 28° C., the fermentation time is 48 hours, and the initial pH of the fermentation is 7.5.

[0011] Furthermore, the fermentation is a tremor fermentation of the Bacillus Velez subtilis CB6 at a rotation speed of 180 rpm.

[0012] Furthermore, the saturation degree of the ammonium sulfate is 90%.

[0013] The present invention also provides the use of the Bacillus Velezii CB6 or the antibacterial protein CB6-E in the preparation of a drug for inhibiting Gram-negative bacteria.

[0014] Furthermore, the Gram-negative bacteria are multidrug-resistant Gram-negative bacteria.

[0015] Furthermore, the multi-drug resistant Gram-negative bacteria are polymyxin-resistant and carbapenem-resistant Escherichia coli.

[0016] The present invention also provides a drug for inhibiting Gram-negative bacteria, wherein the active ingredient is the antibacterial protein CB6-E.

[0017] The present invention discloses the following technical effects:

[0018] The present invention independently separated a strain of Bacillus Velezii CB6 which has good antagonistic effect on Gram-negative bacteria such as multidrug-resistant Escherichia coli, and obtained the antibacterial protein CB6-E through "ammonium sulfate sedimentation - 500Da dialysis bag interception - dextran agarose cross-linked gel chromatography column purification - DEAE-sephdex ion column further purification". The antibacterial protein CB6-E has broad-spectrum antibacterial activity against a variety of Gram-negative bacteria, especially the strongest bactericidal activity against resistant E. coli B2, Salmonella and Pseudomonas aeruginosa, with an MIC of 32 μg / mL.

[0019] The antimicrobial protein CB6-E obtained by the present invention shows good antimicrobial activity against a variety of Gram-negative bacteria, has good safety for a variety of animals, and has good therapeutic effects on mice and wax moths infected with multidrug-resistant Escherichia coli. 20 mg / kg of the antimicrobial protein CB6-E can increase the survival rates of wax moth larvae and mice infected with multidrug-resistant Escherichia coli by 60% and 80%, respectively, and has the potential to become a new anti-Gram-negative bacteria drug.

[0020] The present invention uses a strain of Escherichia coli B2 resistant to polymyxin and carbapenems as an indicator bacteria to screen a novel broad-spectrum antibacterial drug against Gram-negative bacteria. Escherichia coli B2 is a standard strain of multi-drug resistance (resistance to polymyxin and carbapenems), and has stronger drug resistance than other drug-resistant bacteria. The Bacillus Velezii CB6 screened by using the strain as an indicator bacteria has a stronger antibacterial effect on drug-resistant Gram-negative bacteria and a better effect.

[0021] The antibacterial protein CB6-E prepared by fermenting Bacillus velez CB6 in the present invention is expected to enrich the current antibacterial drug library and provide new hope for treating infections caused by multidrug-resistant Gram-negative bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 The effect of different liquid volumes on the antibacterial activity of the fermentation supernatant of Bacillus Velez CB6;

[0024] Figure 2 The effect of different inoculum amounts on the antibacterial activity of the fermentation supernatant of Bacillus velez CB6;

[0025] Figure 3 The effect of different culture temperatures on the antibacterial activity of the fermentation supernatant of Bacillus velez CB6;

[0026] Figure 4 The effect of different rotation speeds on the antibacterial activity of the fermentation supernatant of Bacillus Velez CB6;

[0027] Figure 5 The effect of different initial pH values ​​of the culture medium on the antibacterial activity of the fermentation supernatant of Bacillus Velez CB6;

[0028] Figure 6 The effect of different culture time on the antibacterial activity of the fermentation supernatant of Bacillus velez CB6;

[0029] Figure 7 The effect of different saturation of ammonium sulfate on the antibacterial activity of the fermentation supernatant of Bacillus Velez CB6;

[0030] Figure 8 This is the chromatogram after purification using a dextran agarose cross-linked chromatography column;

[0031] Fig. 9 Chromatogram after purification using DEAE-sephdex ion column;

[0032] Fig.10 The results of 10% polyacrylamide gel electrophoresis;

[0033] Fig.11 This is the killing curve of the antimicrobial protein CB6-E against multi-drug resistant Escherichia coli;

[0034] Fig.12 is the survival rate of G. mellonella larvae;

[0035] Fig.13 is the survival rate of mice;

[0036] Fig.14 This is a picture of mouse lung tissue observation;

[0037] Fig.15 is the bacterial load of each organ of the mouse;

[0038] Fig.16 This is the histopathological analysis of various organs of mice. Scale bar: 1:100. DETAILED DESCRIPTION

[0039] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0040] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0041] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0042] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0043] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0044] The indicator bacteria used for screening strains and determining antibacterial activity in the embodiments of the present invention are all polymyxin-resistant and carbapenem-resistant Escherichia coli (E. coli) B2, which is a model strain of multidrug resistance and has stronger drug resistance than other drug-resistant bacteria. It was donated by China Agricultural University and stored in the Veterinary Pharmacology Laboratory of Jilin Agricultural University. Other clinical isolates were isolated and obtained by the Veterinary Pharmacology Laboratory of Jilin Agricultural University. It has been experimentally verified that clinical isolates show strong resistance to antibiotics such as carbapenems and polymyxins.

[0045] Example 1 Isolation, screening and identification of strain CB6

[0046] 1. Sample collection and isolation and initial screening of strain CB6

[0047] The soil samples rich in secondary metabolites from Changbai Mountain were diluted 10 times in a row with PBS buffer (pH 7.4). 3 and 10 4 Take 100 μL of soil dilution from each soil sample and spread it on LB agar medium containing multi-drug resistant E. coli B2, and culture at 37°C for 12 hours. Pick a single colony with an inhibition zone on the LB plate, transfer it to LB medium for culture, then use an inoculation loop to dip the cultured bacterial solution and streak it on LB solid medium for purification culture, pick the purified single colony, and transfer it to a sterile LB test tube broth medium. Culture at 37°C, 180r / min overnight, take the culture solution and store it in 80% glycerol, and freeze it at -80°C for later use.

[0048] 2. 16S rRNA Identification of Antagonistic Strains

[0049] The genome of the antagonist was extracted using a genomic DNA extraction kit, and the 16S rRNA gene of strain CB6 was amplified by PCR. After identification by agarose gel electrophoresis, it was sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. The 16S rRNA sequencing sequence was submitted to the GenBank database for BLAST comparison analysis. The phylogenetic tree was constructed using MEGA software, and the results showed that the antagonists obtained in the initial screening belonged to the same branch as Bacillus velezensis and had the closest affinity. Therefore, the antagonists obtained in the initial screening were identified as Bacillus velezensis.

[0050] 3. Rescreening of Antagonistic Strains

[0051] The antagonistic bacterial suspension identified by 16S rRNA sequencing was inoculated into LB broth at a ratio of 1% (V / V), cultured at 37°C, 180r / mL for 24h, centrifuged at 8000×g for 60min, filtered and sterilized with a 0.22μm microporous filter membrane, and the Oxford cup was vertically inserted into the solid culture medium coated with multi-drug resistant Escherichia coli B2 by agar diffusion method, 200μL of sterile antagonistic bacterial fermentation supernatant was added, and it was allowed to stand at 4°C for a period of time. After the supernatant was fully infiltrated into the agar culture medium, it was cultured in a constant temperature incubator at 37°C for 12h, and the diameter of the inhibition zone of the antagonistic bacterial fermentation supernatant was determined. The colony with the largest diameter of the inhibition zone was selected and recorded as strain CB6. The above-mentioned 16S rRNA sequencing identification shows that the strain belongs to Bacillus velezensis. The strain was deposited in the China General Microbiological Culture Collection Center on May 7, 2024, with its address at Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO: 30515.

[0052] Example 2 Optimization of fermentation conditions for antimicrobial protein production by Bacillus Velez CB6 using orthogonal experiments

[0053] The indicator bacteria used in the following determinations of antibacterial activity were all multidrug-resistant E. coli B2.

[0054] The following agar diffusion method was used to determine the antibacterial activity. The specific steps were as follows: Use a sterile inoculation loop to dip the multidrug-resistant E. coli B2 solution into the LB agar plate and perform dot-line inoculation. The next day, pick a single colony and inoculate it into a 2.0 mL EP tube containing LB medium. Place it in a 37°C shaker and culture it until the OD 600 The value reached 0.5. Spread 10 6 For the indicator bacteria of CFU, add 3 μL of antagonistic bacteria solution on the plate containing the indicator bacteria, let it stand at room temperature for 30 minutes, and place it in a 37°C constant temperature incubator for 12 hours after the bacterial solution is completely absorbed. Observe and record the diameter of the inhibition zone every other day.

[0055] Microsoft Office Excel 2018 software was used for data statistics, SPSS Statistics 26 was used for method analysis (ANOVA), Duncan's method was used for significance test, and the difference was expressed by letters, with a significance level of p < 0.05.

[0056] 1. Effect of changes in the volume of culture bottle fluid on antibacterial activity

[0057] Prepare 6 500mL conical flasks, add 50mL, 100mL, 150mL, 200mL, 250mL and 300mL of LB liquid culture medium respectively and sterilize by high pressure, inoculate Bacillus Velez CB6 with 1% inoculum, place the conical flask in a 37℃ constant temperature shaker, and culture at 180rpm for 48h. After 48h, collect the fermentation liquid, concentrate it 15 times and verify its antibacterial activity, measure the diameter of the antibacterial zone, and determine the optimal liquid volume of the culture bottle.

[0058] The results are as follows Figure 1 As shown in the figure, the volume of the culture bottle has little effect on the production of antibacterial active substances in the fermentation supernatant of Bacillus Velez. When the volume of the culture bottle increases, the change in the diameter of the inhibition zone is not significant, and there is no significant change in the diameter of the inhibition zone. In order to collect more fermentation liquid in subsequent experiments, the volume of the culture bottle is set to 300mL.

[0059] 2. Effect of changes in the inoculum size of antagonistic strains on antibacterial active substances

[0060] Prepare 5 conical flasks, add 300mL of liquid culture medium, and then inoculate at 1%, 2%, 3%, 4% and 5% by volume, respectively. Place the conical flasks in a 37℃ shaker for 48h at 180rpm. After 48h, collect the fermentation liquid for concentration and verify its antibacterial zone diameter to determine the optimal inoculation amount. The results are shown in Figure 2 As shown in the figure, with the increase of inoculation amount, the diameter of the inhibition zone did not change significantly, which showed that different inoculation amounts had little effect on the production of antibacterial active substances in the fermentation supernatant of Bacillus Velez. Therefore, the inoculation amount was set at 1%.

[0061] 3. Effect of temperature change on antibacterial active substances

[0062] Prepare 5 conical flasks, add 300mL of liquid culture medium, and then inoculate at 1% inoculation volume. Place the conical flasks in a shaker at 16℃, 20℃, 24℃, 28℃ and 37℃ for 48h at a speed of 180rpm. After 48h, collect the fermentation liquid for concentration and verify its antibacterial zone diameter to determine the optimal culture temperature. The results are as follows Figure 3 As shown in the figure, with the increase of culture temperature, the diameter of the inhibition zone increases and the antibacterial activity increases. The variance analysis results show that different culture temperatures have a significant effect on the antibacterial activity. When the culture temperature is 16℃ or 20℃, the diameter of the inhibition zone is small. With the increase of culture temperature, the change of the inhibition diameter is more significant. Therefore, 37℃ is selected as the optimal culture temperature for subsequent orthogonal experiments.

[0063] 4. Effect of speed change on antibacterial active substances

[0064] Prepare 5 conical flasks, add 300mL liquid culture medium, and then inoculate them at 1% inoculation volume. Place the conical flasks in a 37℃ shaker for 48h, with the speeds of 50, 100, 150, 180, and 200rpm / min. After 48h, collect the fermentation liquid for concentration and verify its antibacterial zone diameter to determine the optimal speed. The results are as follows Figure 4 As shown in the figure, with the increase of culture speed, the diameter of the inhibition zone increases and the antibacterial activity increases. The results of variance analysis show that different speeds have a significant effect on the antibacterial activity. When the speed is 50rpm or 100rpm, the diameter of the inhibition zone is small. When the speed is 180rpm, the diameter of the inhibition zone is the largest. When the speed is increased to 200rpm, the diameter of the inhibition zone decreases. Therefore, the speed of 180rpm was selected for subsequent orthogonal experiments.

[0065] 5. Effect of changes in culture medium pH on antibacterial active substances

[0066] Prepare 5 conical flasks, add 300mL of liquid culture medium, adjust the initial pH of the culture medium to 5, 5.5, 6, 6.5 and 7.5 respectively, then inoculate at 1% inoculum, place the conical flasks in a 37℃ shaker for 48h, and culture at 180rpm. After 48h, collect the fermentation liquid for concentration and verify its antibacterial zone diameter to determine the optimal pH value of the culture medium. Figure 5 As shown in the figure, when the pH value of the culture medium is changed, the diameter of the inhibition zone changes accordingly. The results show that the pH value of the culture medium has a significant effect on the antibacterial activity. When the pH value of the culture medium is 5 or 5.5, the diameter of the inhibition zone is small and the antibacterial activity is the weakest. When the pH value of the culture medium is 6.5, the diameter of the inhibition zone is the largest and the antibacterial activity is the strongest. Therefore, the pH value of 6.5 was selected for the subsequent orthogonal experiment.

[0067] 6. Effect of changes in culture time on the active substances of antibacterial substances

[0068] Prepare 5 conical flasks, add 300mL liquid culture medium, and then inoculate at 1% inoculation volume, place the conical flasks in a 37℃ shaker at 180rpm, and culture for 12h, 24h, 36h, 48h and 60h, respectively. Collect the fermentation liquid, concentrate it and verify its antibacterial zone diameter to determine the optimal culture time. Figure 6 As shown in the figure, with the increase of culture time, the diameter of the inhibition zone increases and the antibacterial activity increases. The results show that the different culture times have a significant effect on the antibacterial activity. When the culture time is 12h, 24h or 36h, the diameter of the inhibition zone is small, and when the culture time is 48h, the diameter of the inhibition zone is the largest, so the culture time of 48h was selected for the subsequent orthogonal experiment.

[0069] An orthogonal experiment as shown in Table 1 was designed to screen the fermentation culture conditions with the best antibacterial activity.

[0070] Table 1 Orthogonal experiment

[0071]

[0072]

[0073] As shown in Table 1, the optimal culture conditions for the production of antimicrobial protein by Bacillus Velez CB6 were finally determined to be: liquid volume 300 mL, initial pH = 7.5, inoculation amount 1%, culture temperature 28°C, culture time 48 h, and culture speed 180 rpm.

[0074] Example 3 Preparation of antimicrobial protein CB6-E

[0075] 1. Preparation of Fermentation Supernatant

[0076] Bacillus Velez CB6 was placed in a conical flask containing 300 mL of LB medium, inoculated with an inoculum size of 1%, and cultured in a shaker at 28°C and 180 rpm for 48 hours, and then centrifuged at 8500 rpm for 30 minutes at 4°C. The bacterial fermentation supernatant was collected and sterilized by filtering through a 0.22 μm filter membrane, concentrated 15 times using a rotary evaporator, and then sterilized by filtering through a 0.22 μm filter membrane again. The sterile fermentation concentrated supernatant was obtained, and its antibacterial activity was detected by agar diffusion method.

[0077] 2. Extraction of Crude Antimicrobial Protein CB6-E

[0078] At 4°C, 50%, 60%, 70%, 80%, 90% and 100% ammonium sulfate were added to the sterile fermentation broth in several times, stirred slowly for 12 hours with a magnetic stirrer, and then allowed to stand overnight. The precipitated antimicrobial protein was collected by centrifugation at 4°C and 8500 rpm for 30 minutes. The protein was reconstituted with sterile PBS buffer, and the reconstituted protein was filtered through a 500Da dialysis bag to remove ammonium sulfate, and sterilized by filtering through a 0.22μm microporous filter membrane to obtain the antimicrobial protein CB6-E crude extract. The antibacterial diameter of the antimicrobial protein CB6-E crude extract was detected by agar diffusion method.

[0079] The results of precipitation of antibacterial crude protein by ammonium sulfate with different saturations are as follows Figure 7 As shown in the figure, when the saturation of ammonium sulfate increases, the diameter of the inhibition zone increases and the amount of antibacterial protein increases. When the saturation of ammonium sulfate reaches 90%, the diameter of the inhibition zone is the largest. Therefore, ammonium sulfate with a saturation of 90% will be used to extract crude protein.

[0080] 3. Purification of antimicrobial protein CB6-E using dextran agarose cross-linked gel chromatography column

[0081] The obtained antimicrobial protein CB6-E crude extract was purified using a dextran agarose cross-linked gel chromatography column (column inner diameter: 16 mm × 1 m, filler: 75 PG). Proteins of different molecular weights were separated at a flow rate of 0.5 mL / min, and the effluent was collected according to the absorbance value at 280 nm of the UV detector. Each single peak was collected, and the activity of the antimicrobial protein was verified by the agar diffusion method. The results are shown in FIG. Figure 8 shown.

[0082] 4. Purification of antimicrobial protein CB6-E using DEAE-sephdex ion column

[0083] The antibacterial active product collected in the first step of purification was further purified by DEAE-sephdex (10mm×300mm, filler model is Shanghai Boglong DEAE Bestarose HP) purification column. The sample was loaded with a disposable sterile syringe after the sterile Tris-Hcl balanced ion column, and the binding protein was eluted step by step with different concentrations of NaCl (0.1M, 0.2M, 0.3M, 0.4M, 0.5M, 0.8M and 1M). The effluent was collected according to the absorbance value at 280nm of the ultraviolet detector. Each single peak was collected in one tube. A part of the sample was taken to verify the antibacterial activity of the antibacterial protein CB6-E by the agar diffusion method. The results are shown as follows: Fig. 9 Another part of the sample was taken and the molecular weight and band uniformity of the antimicrobial protein CB6-E were detected by 10% polyacrylamide gel electrophoresis. Fig.10 shown.

[0084] After secondary mass spectrometry analysis, the amino acid sequence of the antibacterial protein CB6-E is shown as SQE ID NO.1.

[0085] SQE ID NO.1:

[0086] MSSNKLTTSWGAPVGDNQNSMTAGDRGPALIQDVHLLEKLAHFNRERVPERVVHAKGAGAHGYFEVTNDVTKYTKAAFLSEVGKRTPLFIRFSTVAGELGSSDTVRDPRGFAVKFYTEEG NYDIVGNNTPVFFIRDAIKFPDFIHTQKRDPRTHLKNPTAVWDFWSLSPESLHQVTILMSDRGIPATLRHMHGFGSHTFKWTNDKGEGVWIKYHFKTEQGVKNLDVNTAAKIAGENPDYHT EDLFNAIENGDFPAWKLYVQIMPLEDANTYRFDPFDVTKVWSQKDYPLIEVGRMVLNRNPENYFAEVEQATFSPGTLVPGVDVSPDKMLQGRLFAYHDAHRYRVGANHQALPINRSRNEV KNYQRDGQMRFDDNGGRSVYYEPNSFGGPKESPEDKQAAYPVSGFADSVSYNHHDHYTQAGDLYRLMSEEERARLVANIVSAMKPVEKEEIKLRQIGHFYKADPEYGRRVAEGLGLPSPK.

[0087] Example 4 Determination of MIC of antimicrobial protein CB6-E against different drug-resistant bacteria

[0088] This example is to determine the minimum inhibitory concentration of the antimicrobial protein CB6-E against different drug-resistant bacteria. The MIC of the antimicrobial protein CB6-E against E.coli B2, Pseudomonas aeruginosa, Shigella, Salmonella, Acinetobacter baumannii, Klebsiella pneumoniae, Staphylococcus aureus (Staphylococcus aureus ATCC25923) and methicillin-resistant Staphylococcus aureus (MRSA) was determined.

[0089] The specific method is as follows:

[0090] The purified antibacterial protein CB6-E was freeze-dried and weighed to 2.048 g. 1 mL of sterile PBS (pH 6.5) was added, gently mixed, and sterilized by filtration using a 0.22 μM microporous filter membrane.

[0091] The indicator bacteria in Table 2 were inoculated into LB liquid culture medium and cultured in a shaking incubator at 37°C until OD 600 was 0.5, and then the indicator bacteria in Table 2 were diluted 1000 times.

[0092] Add 100 μL of LB liquid culture medium to wells 1 to 12 of a sterile 96-well plate, add 100 μL of the antimicrobial protein CB6E to well 1, pipette and mix well from well 1 and pipette 100 μL to well 2, dilute to well 10 in sequence as described above, and then pipette 100 μL of the diluted indicator bacteria in Table 2 and add them to wells 1 to 10 in sequence. Add only 100 μL of LB culture medium to well 11 as a negative control, and add 100 μL of LB and 100 μL of E.coli B2 bacterial solution to well 12 as a positive control.

[0093] Place the 96-well plate in a 37°C incubator and culture for about 12 hours (the time varies according to the logarithmic growth phase of different indicator bacteria), and use an enzyme reader to measure the OD 600 The absorbance values ​​are shown in Table 2.

[0094] Table 2 MIC determination of antimicrobial protein CB6-E against different drug-resistant bacteria

[0095]

[0096] As shown in Table 2, the antimicrobial protein CB6-E has no antibacterial activity against four drug-resistant bacteria, namely Acinetobacter baumannii, Klebsiella pneumoniae, Staphylococcus aureus and methicillin-resistant Staphylococcus aureus. The MIC value for Shigella is 64 μg / mL, and the MIC value for three drug-resistant bacteria, namely Escherichia coli B2, Pseudomonas aeruginosa and Salmonella, is 32 μg / mL.

[0097] Example 5 Time-killing curve of antimicrobial protein CB6-E against Escherichia coli B2

[0098] This example is to determine the time-killing curve of the antimicrobial protein CB6-E against Escherichia coli B2.

[0099] 1. Preparation of Escherichia coli B2 Bacterial Suspension

[0100] Take out E. coli B2 from the -80℃ freezer, streak it on the LB agar plate, and then place it in a 37℃ incubator for 12h. Pick a single colony and inoculate it in LB medium and culture it on a 37℃ shaker until its optical density (OD 600nm ) reached 0.5, a 1000-fold dilution was performed to make the colony forming unit 10 5 CFU / mL of bacterial solution.

[0101] 2. Determination of sterilization curve

[0102] 10 5 The bacterial suspension of CFU / mL was mixed with the antimicrobial protein CB6-E at the minimum inhibitory concentration and then placed in a shaker at 37°C. Every 4 hours, 100 μL of the mixture was diluted several times, and then 100 μL of the dilutions of different concentrations were spread on the agar plate. The plate was placed in an incubator at 37°C for overnight culture, and then the colonies were counted.

[0103] The results are as follows Fig.11 As shown in the figure, compared with the PBS group, the number of Escherichia coli B2 treated with antimicrobial proteins was significantly reduced over time. Under 1×MIC treatment, the number of bacteria decreased by about 2 orders of magnitude within 24 hours, under 2×MIC treatment, the number of bacteria decreased by 3 orders of magnitude within 24 hours, and under 4×MIC treatment, the number of bacteria decreased by 4 orders of magnitude within 24 hours. However, under different doses of antimicrobial protein CB6-E treatment, the bacteria were not completely killed within 24 hours.

[0104] Example 6 Greater wax moth protection experiment

[0105] Twenty-five larvae of G. mellonella were kept overnight at room temperature and randomly divided into five groups, with five larvae in each group. The five groups of larvae were injected with 10 μL of Escherichia coli B2 solution (2×10 8 CFU / mL), and 1 hour later, 3 groups of wax moth larvae were injected with 5mg / kg, 10mg / kg and 20mg / kg of antimicrobial protein CB6-E at the right ventral foot for treatment. The 4th group of wax moth larvae were treated with 10μL polymyxin B as a positive control group. The 5th group of wax moth larvae were treated with 10μL sterile PBS as a negative control group. The survival of wax moth larvae was observed every day for 48 hours and the survival of wax moth larvae was recorded. Fig.12As shown, compared with the control group, after treatment with 5 mg / kg of antimicrobial protein, the survival rate of the wax moth larvae within 48 hours was 40%, after treatment with 10 mg / kg of antimicrobial protein, the survival rate of the wax moth larvae within 48 hours was 40%, after treatment with 20 mg / kg of antimicrobial protein, the survival rate of the wax moth larvae within 48 hours was 60%.

[0106] Example 7 Mouse protection experiment

[0107] Fifty 4-week-old Kunming mice (half male and half female) were randomly divided into 5 groups, with 10 mice in each group.

[0108] Group 1: Bacteria group (5×10 8 CFU / mL) + antimicrobial protein CB6-E (5 mg / kg, 100 μL)

[0109] Group 2: Bacteria group (5×10 8 CFU / mL) + antimicrobial protein CB6-E (10 mg / kg, 100 μL)

[0110] Group 3: Bacteria group (5×10 8 CFU / mL) + antimicrobial protein CB6-E (20 mg / kg, 100 μL)

[0111] Group 4: Bacteria group (5×10 8 CFU / mL)+polymyxin B (100μL)

[0112] Group 5: Bacteria group (5×10 8 CFU / mL)+sterile PBS (100μL)

[0113] The mice in the five groups were intraperitoneally injected with 100 μL of Escherichia coli B2 suspension (5×10 8 CFU / mL), and 1 hour later, 5mg / kg, 10mg / kg and 20mg / kg of antimicrobial protein CB6-E were given for treatment. The mice were observed for 5 days and the survival of the mice was recorded. After 5 days, the mice were dissected, the lung changes were examined and photographed. Then the heart, liver, spleen, lung and kidney were taken, and some organs were washed with sterile saline, ground and diluted in multiples for colony counting. The other part was rinsed with sterile saline, fixed with paraformaldehyde, and the samples were sent to Sevier Biotechnology Co., Ltd. for HE staining.

[0114] After 5 days, the survival of mice in each group was as follows Fig.13As shown, compared with the fifth group, after treatment with 5 mg / kg of antimicrobial protein CB6-E, the survival rate of mice was 60% within 2 days, 40% within 3 days, and 20% within 4 days; after treatment with 10 mg / kg of antimicrobial protein CB6-E, the survival rate of mice was 80% within 2 days, and 60% within 4 days; after treatment with 20 mg / kg of antimicrobial protein CB6-E, the survival rate of mice was 80% within 4 days.

[0115] Lung tissues of mice in each group Fig.14 As shown, the lungs of the mice in the fifth group and the treatment group were red and swollen, and bleeding appeared on the surface. After treatment with different concentrations of the antimicrobial protein CB6-E (5 mg / kg, 10 mg / kg and 20 mg / kg), the redness and swelling on the surface decreased, the bleeding spots decreased, and it was observed that the pathological phenomenon was significantly alleviated.

[0116] The bacterial load of each organ of mice is as follows Fig.15 As shown, compared with the fourth group of mice, the bacterial load in the organs of mice treated with CB6-E was significantly reduced, and the bacterial content in each organ could be reduced by about 4-5 orders of magnitude. The experimental results show that different doses of the antimicrobial protein CB6-E can significantly reduce the bacterial load in the organs of the infected mice and have a good therapeutic effect on the infected mice.

[0117] Organ histopathological analysis Fig.16 As shown in the figure, the liver of mice infected with E. coli B2 was severely vacuolated and had obvious inflammatory infiltration. The heart, spleen, lungs and kidneys showed hemorrhages, punctate necrosis, dark red cytoplasm, interstitial hemorrhage, granular chromatin and other histopathological changes. After treatment with the antimicrobial protein CB6-E, the pathological changes in various tissues and organs of the infected mice were alleviated.

[0118] The above results indicate that the antimicrobial protein CB6-E can alleviate the pathological lesions of mouse organs caused by infection with Escherichia coli B2, and has a good therapeutic effect on damage to mouse tissues and organs.

[0119] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of an antibacterial protein CB6-E obtained by fermenting a bacteriocin-producing Bacillus velezensis CB6 in the preparation of a drug for inhibiting Gram-negative bacteria, wherein the Gram-negative bacteria are multidrug-resistant Escherichia coli B2, Shigella, Pseudomonas aeruginosa and Salmonella; The accession number of the Bacillus Velez CB6 is CGMCC NO.30515; The amino acid sequence of the antibacterial protein CB6-E is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: The Gram-negative bacteria are multidrug-resistant Escherichia coli B2, Pseudomonas aeruginosa and Salmonella.

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