KR102178334B1 - Method for preparing bacterial ghosts by using sulfuric acid, bacterial ghosts prepared by the same and uses thereof - Google Patents

Abstract

The present invention relates to a method for producing a bacterial ghost using anaerobic gram-positive bacteria and sulfuric acid, a bacterial ghost prepared by the method, and a use thereof.
Therefore, the method of producing a bacterial ghost using sulfuric acid according to the present invention is effective as a method for producing a bacterial ghost that can save time and cost while being simple, since it can only suppress the growth of the bacteria without substantially altering the outer film of the bacteria. In particular, the present invention is more effective as a method for producing bacterial ghosts since it can provide optimum conditions for treating the sulfuric acid. In addition, the bacterial ghost produced by the production method of the present invention can be used as a vaccine or foreign antigen carrier.

Description

Method for preparing bacterial ghosts using sulfuric acid, bacterial ghosts prepared by the above method, and uses thereof {Method for preparing bacterial ghosts by using sulfuric acid, bacterial ghosts prepared by the same and uses thereof}

The present invention relates to a method for producing a bacterial ghost using anaerobic gram-positive bacteria and sulfuric acid, a bacterial ghost prepared by the method, and a use thereof.

Bacterial ghosts can be simply defined as structures that completely maintain the shape of the envelope, although the inside is empty by removing the components (cytoplasmic content) within the bacterial cell. In addition, bacterial ghosts are not considered genetically modified organisms (GMOs) because they are dead cells that lack genetic material.

Since the bacterial ghost maintains the outer membrane of the live cell, it retains the antigenic determinant of the outer membrane of the live cell as it is, and thus exhibits functionally similar effects to the live vaccine. In particular, in the treatment of bacterial infections, vaccines using bacterial ghosts (ghost vaccines) do not inhibit the induction of immunity due to a large amount of non-specific cytoplasm, so that the intrinsic immunity and the adaptive immunity system can be maintained without the addition of adjuvants. Can be easily stimulated. In addition, inactivated bacterial ghosts can not only adhere to special tissues or cells of animals, humans, or plants, but can also be introduced into a delivery system that can effectively deliver recombinant antigens or nucleic acids to target cells. (delivery system).

The most common method for producing such a bacterial ghost is an E protein-mediated lysis method, which is a method of expressing a plasmid cloned from the bacteriophage ΦX174 lysis gene E into a Gram-negative bacteria. Transformed bacteria inhibit the E gene and then express E protein according to temperature change. The synthesized E protein forms a transmembrane tunnel in the bacterial cell membrane and cell wall without causing physicochemical damage to the bacterial surface structure. Release the composition (non-patent paper 0001). However, such methods require a multi-step molecular biology technique, high cost, and long manufacturing time. Therefore, research on a simple manufacturing technique for mass production of bacterial ghosts, a low-cost production cost, and a new manufacturing method that can save time are being conducted.

A method for producing a bacterial ghost by treating chemical substances such as SDS, sodium hydroxide (NaOH), and hydrogen peroxide (H ₂ O ₂ ) using the recent Gram-negative bacteria E. coli ( E. coli BL21 (DE3) pLysS) as a model (non-patent literature 0004), or E. coli ( E. coli DH5α) as a model, the minimum of ammonium sulfate {(NH ₄ ) ₂ SO ₄ }, calcium chloride (CaCl ₂ ), and ethylenediaminetetraacetic acid (EDTA), which are chemicals that affect the cell wall A method for producing bacterial ghosts (patent document 0004) based on the minimum inhibitory concentration (MIC) has been developed. In addition, recently, Gram-negative bacteria, Salmonella enterica Enteriditis, and Gram-positive bacteria, Staphylococcus aureus , were prepared as a bacterial ghost by treatment with sodium hydroxide, and then immunized to experimental animals and induce immunity. Studies proving the potential of vaccines have been reported (non-patent paper 0005 and non-patent paper 0006).

However, there is a possibility that some epitope proteins present in the outer membrane of the bacterial ghost may be released by basic substances such as sodium hydroxide, and in that case, the efficacy as a vaccine may be degraded. As a result of the acid-positive and gram acids Listeria monocytogenes (Listeria monocytogenes ) was developed to produce a bacterial ghost (Patent Document 0005). However, no research or patent has been disclosed for bacterial ghosts using sulfuric acid.

Republic of Korea Patent Publication No. 10-0273847, 2000. 12. 15. Announcement Republic of Korea Patent Publication No. 10-0765357, 2007. 10. 10. Announcement US 7968323 B2, published on June 28, 2011 Republic of Korea Patent Publication No. 10-1449628, 2014. 10. 02. Announcement Korean Patent Publication No. 10-1825439, 2018. 01. 30. Announcement

Haidinger, W. et al., Appl. Environ. Microbiol. 2003, Vol. 69(1), pPropionibacterium 468-474 Ebensen, T. et al., J. Immunol. 2004, Vol. 172, pPropionibacterium 6858-6865 Konopa, G. & Taylor, K., Biochim. Biophys. Acta. 1975, Vol. 399(2), pPropionibacterium 460-467 Amara, A. A. et al., The Scientific World Journal, 2013, Vol. 2013, Article ID 545741, 7 pages, doi:10.1155/2013/545741 Vinod, N. et al., Vaccine, 2014, Vol. 32, pPropionibacterium 3249-3255, doi:10.1016/j.vaccine. 2014.03.090 Vinod, N. et al., Infection and Immunity, 2015, Vol. 83, pPropionibacterium 2957-2965, doi:10.1155/2013/545741

Accordingly, the present inventors have made diligent efforts to provide a method for producing bacterial ghosts that are simple and can save time and cost while being able to produce bacterial ghosts in large quantities. When sulfuric acid is used, the outer membrane of bacteria is maintained at the same level as live bacteria. While inhibiting the growth of bacteria, it was confirmed that the bacterial ghost can be effectively produced, and the present invention was completed.

Accordingly, an object of the present invention is to provide a method for producing a bacterial ghost using sulfuric acid and a bacterial ghost prepared by the method.

Another object of the present invention is to provide a vaccine composition for the prevention or treatment of anaerobic Gram-positive bacteria infection or a composition for a foreign antigen carrier comprising the bacterial ghost.

In order to achieve the above object, the present invention comprises the steps of: i) inoculating and culturing anaerobic Gram-positive bacteria in a medium;

ii) preparing a suspension by separating and obtaining the bacteria cultured in step i) and inoculating them in a new medium; And

iii) culturing after treating the suspension of step ii) with sulfuric acid;

It provides a method for producing a bacterial ghost comprising a.

According to a preferred embodiment of the present invention, anaerobic gram-positive bacteria is Propionibacterium sludge tumefaciens (Propionibacterium) spp., Streptococcus (Streptococcus) in bacteria, enterococci (Enterococcus) genus bacteria, Lactobacillus Cocos (Lactococcus) of step i) the group consisting of spp., flow Pocono stock (Leuconostoc) spp., Phedi OKO kusu (Pediococcus) spp., Clostridium (Clostridium) in bacteria, lactic acid bacillus (Lactobacillus) in bacteria and Bifidobacterium (Bifidobacterium) spp. It may be any one or more selected from.

According to a preferred embodiment of the present invention, the concentration of bacteria in the suspension of step ii) may be 10 ³ to 10 ⁹ CFU/ml.

According to a preferred embodiment of the present invention, the pH of the suspension in step iii) may be 0.1 to 1.5.

According to a preferred embodiment of the present invention, sulfuric acid in step iii) may be treated to a minimum inhibitory concentration.

According to a preferred embodiment of the present invention, the minimum inhibitory concentration of sulfuric acid may be 5 to 100 mg/ml.

According to a preferred embodiment of the present invention, the culture in step iii) may be performed for at least 5 minutes.

According to a preferred embodiment of the present invention, the cultivation in step iii) may be performed at 25°C to 50°C.

The present invention also provides a bacterial ghost prepared by the above production method and a vaccine composition for preventing or treating anaerobic gram-positive bacteria infection or a composition for a foreign antigen carrier comprising the bacterial ghost.

According to a preferred embodiment of the present invention, the anaerobic Gram-positive bacteria is Propionibacterium sludge tumefaciens (Propionibacterium) spp., Streptococcus (Streptococcus) in bacteria, enterococci (Enterococcus) genus bacteria, Lactobacillus Cocos (Lactococcus) in bacteria, acids Any selected from the group consisting of the genus Leuconostoc , the genus Pediococcus , the genus Clostridium , the genus Lactobacillus , and the bacteria of the genus Bifidobacterium . There may be more than one.

Therefore, the method of producing a bacterial ghost using sulfuric acid according to the present invention is effective as a method for producing a bacterial ghost that can save time and cost while being simple, since it can only suppress the growth of the bacteria without substantially altering the outer film of the bacteria. In particular, the present invention is more effective as a method for producing bacterial ghosts since it can provide optimum conditions for treating the sulfuric acid. In addition, the bacterial ghost produced by the production method of the present invention can be used as a vaccine or foreign antigen carrier.

1 shows the minimum inhibitory concentration (MIC) of a chemical agent that inhibits the proliferation of Propionibacterium acnes by a two-fold broth dilution method (A: sodium hydroxide treatment group, B: hydrochloric acid treatment group And C: sulfuric acid treatment group; concentration of chemicals treated in each test tube of each treatment group: (C) no treatment (control), (1) 50 mg/ml, (2) 25 mg/ml, (3) 12.5 mg/ Ml, (4) 6.25 mg/ml, (5) 3.125 mg/ml, (6) 1.5625 mg/ml, (7) 0.78125 mg/ml, (8) 0.390625 mg/ml, (9) 0.1953125 mg/ml) . It was confirmed that the MIC of sodium hydroxide, hydrochloric acid, and sulfuric acid were 25 mg/ml, 12.5 mg/ml and 50 mg/ml, respectively.
Fig. 2 shows the presence or absence of colony formation in each treatment group of [Fig. 1] (A: sodium hydroxide treatment group, B: hydrochloric acid treatment group and C: sulfuric acid treatment group; each treatment medium in each treatment group) One sample: (C): a sample obtained by diluting the untreated control of [Fig. 1] to 1/1000, (1): test tube No. 1 sample of [Fig. 1], (2): test tube No. 2 of [Fig. 1] Sample, (3): sample 3 of [Fig. 1] and (4): sample 4 of [Fig. 1]). It was confirmed that colonies were formed at concentrations below the minimum inhibitory concentration in all treatment groups.
FIG. 3 shows the results of agarose electrophoresis of genomic DNA extracted from Propionibacterium acnes ghost prepared 60 minutes after treatment with a chemical agent at a minimum inhibitory concentration (M: protein molecular weight marker, lane 1: Untreated control, lane 2: sodium hydroxide, lane 3: hydrochloric acid, lane 4: sulfuric acid). DNA was detected in the untreated control group, but it was confirmed that DNA was not detected in the chemical treatment group.
4 is a propionibacterium acnes ghost (PAG) prepared by treating each of sodium hydroxide (NaOH), hydrochloric acid (HCl) or sulfuric acid (HCl) for 60 minutes and untreated (Control ( P.acnes ), Control ( E. coli )) of a genomic DNA sample extracted from Propionibacterium acnes with a primer specific to Propionibacterium acnes and a real-time PCR. It was confirmed that DNA was amplified only in ghosts prepared by treatment with no treatment group (Control ( P. acnes )) and sodium hydroxide, and DNA was not amplified in the rest of the experimental groups.
5 shows the results of scanning electron microscopy analysis of Propionibacterium acnes ghost prepared by treating a chemical agent with a minimum inhibitory concentration for 60 minutes. The point indicated by the arrow is a transmembrane tunnel formed by dissolving the membrane of Propionibacterium acnes bacteria (A: sodium hydroxide, B: hydrochloric acid, C: sulfuric acid, D: untreated control). When treated with sodium hydroxide or hydrochloric acid, the outer membrane of the bacteria was severely deformed, whereas when treated with sulfuric acid, it was confirmed that the outer membrane had almost the same level as that of the untreated control.
6 shows the presence or absence of colony formation according to the passage of 15, 30, 45, 60 and 90 minutes after treatment with sulfuric acid (50 mg/ml) of a minimum inhibitory concentration. It was confirmed that no colonies were formed after 15 minutes elapsed after the sulfuric acid treatment.

Hereinafter, the present invention will be described in more detail.

As described above, conventional methods for producing bacterial ghosts have a problem that they are difficult and require a lot of cost and time, and there is a limitation in that the outer membrane of the produced bacterial ghost is severely deformed when using a simple chemical substance. In order to overcome the above limitation, there is a need for a study on a method for producing a bacterial ghost that is simple and can save time and cost.

The method for producing a bacterial ghost using sulfuric acid according to the present invention can inhibit the growth of bacteria without damaging the outer membrane of the bacteria, and when the sulfuric acid is treated under optimal conditions, it is possible to more effectively produce a bacterial ghost. In addition, the bacterial ghost prepared by the above production method may be provided as a vaccine or foreign antigen carrier.

The present inventors tried to prepare a bacterial ghost by treating anaerobic Gram-positive bacteria with chemicals such as sodium hydroxide (NaOH), hydrochloric acid (HCl), or sulfuric acid (H ₂ SO ₄ ), respectively. The minimum inhibition concentration (MIC) required to prepare bacterial ghosts with the above chemicals was 25 mg/ml, 12.5 mg/ml, and 50 mg/ml, respectively ([Fig. 1] and [Fig. 2]). , Bacterial ghosts prepared by treating the chemicals with MIC for 60 minutes, respectively, did not detect DNA remaining therein ([Fig. 3] and [Fig. 4]).

As a result of analyzing the surface of bacterial ghosts prepared by treating chemicals such as sodium hydroxide (NaOH), hydrochloric acid (HCl) or sulfuric acid (H ₂ SO ₄ ) for 60 minutes, sulfuric acid compared to the case of treatment with sodium hydroxide or hydrochloric acid. In the case of the bacterial ghost prepared by processing, it was confirmed that the appearance was maintained at almost the same level as that of the live bacteria (FIG. 5). Accordingly, the present inventors tried to determine the time required to prepare a bacterial ghost using sulfuric acid, and it was confirmed that this was 15 minutes (FIG. 6).

Accordingly, the present invention comprises the steps of i) inoculating and culturing anaerobic Gram-positive bacteria in a medium;

ii) preparing a suspension by separating and obtaining the bacteria cultured in step i) and inoculating them in a new medium; And

iii) culturing after treating the suspension of step ii) with sulfuric acid;

It is possible to provide a method for producing a bacterial ghost, including a bacterial ghost, and a bacterial ghost prepared by the method.

The anaerobic Gram-positive bacteria of step i) of the present invention is preferably carried out after culturing for 68 to 76 hours in an incubator, more preferably 70 to 74 hours, and most preferably It is preferably carried out after 72 hours incubation. The cell walls of bacteria in the exponential growth phase during the bacterial growth phase are sensitive to chemicals, but the bacteria cultured for longer than the above time have elastic cell walls, so it is difficult to form a dissolved tunnel structure using chemicals. It can be more effective.

The anaerobic Gram-positive bacteria of the step i) of the present invention include bacteria of the genus Propionibacterium , bacteria of the genus Streptococcus , bacteria of the genus Enterococcus , bacteria of the genus Lactococcus , and leukonostok ( not less than Leuconostoc) spp., Phedi OKO kusu (Pediococcus) spp., Clostridium (Clostridium) in bacteria, lactic acid bacillus (Lactobacillus) in bacteria and Bifidobacterium (any one selected from the group consisting of Bifidobacterium) spp. It is preferable, and more preferably, it is preferable that it is a fungus of the genus Propionibacterium .

The propionibacterium ( Propionibacterium ) genus can be used in the present invention without limitation, but preferably Propionibacterium acidifaciens , Propionibacterium acidipropionici , Propionibacterium acnes, Propionibacterium australiense , Propionibacterium avidum , Propionibacterium cyclohexanicum , Propionibacterium damnosum , Propionibacterium freudenreichii , Propionibacterium granulosum , Propionibacterium jensenii , Propionibacterium lymphophilum , Propionibacterium microaerophilic , Propionibacterium namnetense , Propionibacterium olivae And Propionibacterium It is preferably any one or more selected from the group consisting of propionicus , Propionibacterium thoenii , and more preferably Propionibacterium acnes ( Propionibacterium acnes ) is preferred.

The concentration of the bacteria in the suspension of step ii) of the present invention is preferably 10 ³ to 10 ⁹ CFU/ml, more preferably 10 ⁴ to 10 ⁸ CFU/ml, and most preferably 10 ⁵ It is preferably in the range of 10 ⁷ CFU/ml.

The pH of the suspension in step iii) of the present invention is preferably 0.1 to 1.5, more preferably 0.3 to 1.2, and most preferably 0.5 to 1.

In step iii) of the present invention, sulfuric acid may be treated with a minimum inhibitory concentration, and the minimum inhibitory concentration of sulfuric acid is preferably 5 to 100 mg/ml, but more preferably 20 to 80 mg/ml, and Most preferably, it is preferably 40 to 60 mg/ml. If the sulfuric acid is treated below the minimum inhibitory concentration, the dissolution rate is not perfect and there may be surviving bacteria. If the sulfuric acid is treated above the minimum inhibitory concentration, the bacteria's outer membrane structure is damaged and the perfect tunnel structure cannot be formed. It is impossible to produce ghosts.

In the step iii) of the present invention, the minimum time of incubation is preferably 10 to 20 minutes, more preferably 12 to 18 minutes, and most preferably 14 to 16 minutes or more. In order to produce a safer bacterial ghost, it is preferable to incubate for at least 60 minutes, and in the case of culturing for longer than that, the culture time may be adjusted by a person skilled in the art according to the purpose of producing the bacterial ghost.

In the step iii) of the present invention, the culture is preferably performed at 25°C to 50°C, more preferably 30°C to 45°C, and most preferably 35°C to 40°C. .

That is, in the step iii) of the present invention, when the pH of the suspension, the concentration of sulfuric acid to be treated, and the time and temperature for incubation after treatment with sulfuric acid are combined under optimal conditions, they act comprehensively to create a synergistic effect. An effective method for producing bacterial ghosts can be provided.

In addition, the present invention can provide a vaccine composition for preventing or treating anaerobic Gram-positive bacteria infection or a composition for a foreign antigen carrier comprising the bacterial ghost.

The anaerobic Gram-positive bacteria of the present invention are the bacteria of the genus Propionibacterium , the bacteria of the genus Streptococcus , the bacteria of the genus Enterococcus , the bacteria of the genus Lactococcus , the bacteria of the genus Leuconostoc , Pediococcus ( Pediococcus ) genus bacteria, Clostridium ( Clostridium ) genus bacteria, lactic acid bacillus ( Lactobacillus ) genus bacteria and Bifidobacterium ( Bifidobacterium ) is preferably any one or more selected from the group consisting of bacteria.

The Propionibacterium (Propionibacterium) Genus may be applicable without limitation, but preferablyPropionibacterium acidifaciens , Propionibacterium acidipropionici , Propionibacterium acnes , Propionibacterium australiense , Propionibacterium avidum , Propionibacterium cyclohexanicum , Propionibacterium damnosum , Propionibacterium freudenreichii , Propionibacterium granulosum , Propionibacterium jensenii , Propionibacterium lymphophilum , Propionibacterium microaerophilic , Propionibacterium namnetense , Propionibacterium olivae And Propionibacterium propionicus , Propionibacterium thoenii It is preferably any one or more selected from the group consisting of, more preferably Propionibacterium acnes (Propionibacterium acnes) Is preferred.

Hereinafter, the present invention will be described in more detail by examples. However, the present invention is not limited by the following examples, and it is apparent to those of ordinary skill in the art that various modifications or modifications can be made within the idea and scope of the present invention. If there are no other definitions in the technical and scientific terms used at this time, they have meanings that are generally understood by those of ordinary skill in the technical field to which this invention belongs. In addition, repeated descriptions of the same technical configuration and operation as in the prior art may be omitted.

Anaerobic gram-positive bacteria

Propionibacterium acnes , an anaerobic Gram-positive bacteria acnes ) was inoculated into a BHI (brain heart infusion) medium, and cultured with shaking for one day at 37°C, 200 rpm, and anaerobic conditions. The growth and lysis of Acnes bacteria were measured at OD 600nm using a Biochrom Libra S22 spectrophotometer.

Determination of the minimum inhibitory concentration of chemicals

In the case of producing a bacterial ghost by treating a strain with a chemical agent, the minimum inhibition concentration (MIC) of the chemical agents to be treated was determined by confirming the proliferation and viability of the strain.

Specifically, the MIC value was measured using a two-fold broth dilution method (Penna et al., Infec. Dis. 2001, Vol. 1, pPropionibacterium 1-8). First, sodium hydroxide hydrochloric acid and sulfuric acid 50 mg/ml were serially diluted (serial dilution; 50 mg/ml, 25 mg/ml, 12.5 mg/ml, 6.25 mg/ml, 3.125 mg/ml, 1.5625 mg/ml, 0.78125 mg) /Ml, 0.390625 mg/ml and 0.1953125 mg/ml) and propionibacterium acnes 10 ⁶ CFU/ml were added to the inoculated BHI liquid medium, and cultured at 37°C for 18 hours to confirm the proliferation capacity of the strain. In addition, in order to confirm the viability of the strain, each concentration-treated group was plated on 5 BHI agar medium, and cultured at 37°C for 72 hours, and then viable colonies were observed.

Chemical Minimum inhibitory concentration ( MIC , mg/ml) Medium pH NaOH 25 13.5 HCl 12.5 1.3 H ₂ SO ₄ 50 0.7

As a result, as shown in [Fig. 1] and [Table 1], the MIC of sodium hydroxide (A) is 25 mg/ml (2), hydrochloric acid (B) as the concentration at which the strain does not grow and a cloudy and clear medium appears. The MIC of was 12.5 mg/ml (3), and the MIC of sulfuric acid (C) was 50 mg/ml (1).

In addition, MIC of sodium hydroxide (A), hydrochloric acid (B), or sulfuric acid (C) compared to the untreated group (C) in which the strain culture solution not treated with each chemical agent was diluted to 1/1000 as shown in [Fig. 2] It was confirmed that no colonies were formed in the medium treated with the above concentration.

bacteria ghost Verification

<3-1> Bacterial ghost production

The Propionibacterium acnes bacterium (hereinafter, Acnes bacterium) of [Example 1] was cultured in BHI liquid medium for 72 hours, centrifuged at 10,000 g for 10 minutes, and the precipitate was harvested to obtain phosphate-buffered saline (PBS, pH 7.0) Washed with a buffer solution, and the concentration of Acnes bacteria was adjusted to 10 ⁶ CFU/ml. 50 mg/ml of sulfuric acid was added to 2 ml of the bacterial suspension and incubated at 37° C. for 60 minutes. After the dissolution of Acnes bacteria was completed, the bacteria were washed twice with a buffer solution of PBS and centrifuged at 10,000 g for 15 minutes to harvest bacterial ghosts.

<3-2> DNA detection- Agarose Gel( agarose gel) DNA analysis

In order to analyze the presence of genomic DNA in the bacterial ghost, a 1% agarose gel containing ethidium bromide (EtBr 0.5g/ml, w/v) was loaded with the bacterial ghost from the MIC for 60 minutes and subjected to electrophoresis.

As a result, as shown in Fig. 3, agarose gel electrophoresis analysis results, (2) sodium hydroxide MIC 60 minutes treatment group, (3) hydrochloric acid MIC 60 minutes treatment group, and (4) sulfuric acid MIC 60 minutes treatment group remaining in bacterial ghost Although no DNA was observed at all, (1) genomic DNA bands were observed in the untreated area of Acnes. That is, it was confirmed that all bacterial ghosts prepared by simply treating MIC of sodium hydroxide, hydrochloric acid and sulfuric acid did not contain genomic DNA.

<3-3> DNA detection-Real-time PCR amplification analysis

Further, in Example <3-2>, it was attempted to more reliably prove whether genomic DNA exists in the bacterial ghost.

Specifically, manufacturers using a commercial extraction kit (iNtRON Biotechnology, Korea) from bacterial ghosts without treatment (positive control), TE buffer (negative control), sodium hydroxide MIC 60 minutes, hydrochloric acid MIC 60 minutes, sulfuric acid MIC 60 minutes Genomic DNA was extracted according to the method of. Using the DNA sample as a template, a DNA fragment of 16s rRNA was amplified by a real-time PCR (TaqMan Probe Detection, Stratagene Mx3000P real time PCR) method with an Acnes-specific primer shown in Table 2 below (total reaction solution (20 μl): 1 μl template DNA (5 ng), 1 μl forward and reverse primers (10 pmol/μl), 10 μl 2x HS Prime qPCR Premix with UDG (GeNet Bio, Korea), 7 μl sterile distilled water; reaction temperature: 3 minutes at 50°C (1 cycle), 10 minutes at 95°C (1 cycle) and 10 seconds at 95°C-30 seconds at 60°C (40 cycles)).

Name Standing heat Sequence number Acnes bacteria
Specific primer Forward direction 5’-GGA ATT CCA CGT GTA GCG GTG AAA T-3’ One Reverse 5’-GAC TAC CAG GGT ATC TAA TCC TGT TTG-3’ 2 taqman probe 5’-AAC ACC AGT GGC GAA GGC GA-3’ 3

As a result, it was confirmed that a large amount of DNA was amplified in the MIC treatment group of sodium hydroxide as shown in [FIG. 4], unlike in Example <3-2> or [FIG. 3]. On the other hand, no DNA was amplified in the hydrochloric acid and sulfuric acid MIC treatment. As a result, it was found that hydrochloric acid and sulfuric acid were more effective than sodium hydroxide for the production of bacterial ghosts.

Bacterial Ghost Surface Analysis

Bacterial ghost prepared by treating sodium hydroxide, hydrochloric acid, and sulfuric acid with MIC for 60 minutes, respectively, was fixed in a PBS solution to which 2.5% glutaraldehyde was added at 4°C for 2 hours, washed with the same solution, and then added to a 1% osmium tetroxide (OsO4) solution. It was fixed at 4°C for 1.5 hours. After that, the bacterial ghost was dehydrated with ethanol diluted stepwise, dried with liquefied carbon dioxide, and coated with gold using a polaron high-resoultion sputter. Bacterial ghosts of each treatment prepared by the above method were observed with a Hitachi S-4800 FESEM (Field Emission Scaning Electron Microscope) Scanning electron mircroscopy (SEM).

As a result, it was confirmed that the outer membrane shape was not maintained and dissolved or decomposed in the sodium hydroxide (A) or hydrochloric acid MIC treatment group (B) compared to the untreated group (D) as shown in [Fig. 5] to have a bacterial ghost form. . On the other hand, it was confirmed that the sulfuric acid MIC treatment zone (C) maintained a distinct dissolved tunnel structure and a perfect outer film shape. As a result, it was found that sulfuric acid was more effective than sodium hydroxide and hydrochloric acid for the production of bacterial ghosts, so sulfuric acid was selected.

bacteria ghost Determine the minimum production time

It was attempted to determine the minimum time required to produce a bacterial ghost by the above method.

Specifically, after harvesting the bacterial ghosts at 15 minute intervals (15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes) after MIC treatment with sulfuric acid, the dissolution rate was measured. To measure the number of colony forming units (CFU) of Acnes bacteria targeting the prepared bacterial ghost, a smear method, a standard analysis method, was performed. Each treatment was plated on 5 BHI agar medium, cultured at 37°C, and then the number of colonies to survive and grow was measured.

As a result, as shown in Fig. 6, no surviving colonies were observed after 15 minutes of sulfuric acid MIC treatment. Therefore, it can be seen that the minimum time required to completely produce the bacterial ghost is 15 minutes.

<110> PAICHAI UNIVERSITY INDUSTRY-ACADEMIC COOPERATION FOUNDATION <120> Method for preparing bacterial ghosts by using sulfuric acid, bacterial ghosts prepared by the same and uses thereof <130> 1065278 <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> acnes_F <400> 1 ggaattccac gtgtagcggt gaaat 25 <210> 2 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> acnes_R <400> 2 gactaccagg gtatctaatc ctgtttg 27 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> taqman probe <400> 3 aacaccagtg gcgaaggcga 20

Claims (12)

i) inoculating and culturing the anaerobic Gram-positive bacteria of the genus Propionibacterium in a medium;
ii) preparing a suspension having a concentration of 10 ⁵ to 10 ⁷ CFU/ml by separating and obtaining the bacteria cultured in step i) and inoculating it in a new medium; And
iii) treating the suspension of step ii) with sulfuric acid at a concentration of 40 to 60 mg/ml and then incubating at 35 to 40°C for 10 to 20 minutes;
Bacterial ghost production method comprising a.
delete delete delete delete delete delete delete Bacterial ghost prepared by the method of claim 1.
A vaccine composition for preventing or treating bacterial infections of the genus Propionibacterium , which is an anaerobic Gram-positive bacterium comprising the bacterial ghost of claim 9.
delete A composition for a foreign antigen carrier comprising the bacterial ghost of claim 9.

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