JP4902845B2 - Killed lactic acid bacteria preparation with enhanced immune function and method for producing the same - Google Patents

Description

  The present invention relates to a killed lactic acid bacterium preparation with enhanced immune function and a method for producing the same, and more specifically, the lactic acid bacterium is cultured in a culture medium containing a surfactant and a carbonate and then killed. The present invention relates to a killed lactic acid bacteria preparation with enhanced immune function and a method for producing the same.

  A technique for attaching and expressing a desired protein on the cell surface of a microorganism is called a cell surface display technique. This cell surface expression technology is a technology for expressing a target protein on the surface using surface proteins of microorganisms such as bacteria and yeast as a surface anchoring motif, producing a recombinant live vaccine, and producing a peptide / antibody library. And a technique having a wide range of applications such as screening, whole cell absorbent, and whole cell biocatalyst. Since this technology has a very wide range of applications depending on the type of protein expressed on the cell surface, it can be said that the potential for industrial application is extremely high.

  For successful cell surface expression techniques, the surface expression matrix is most important. The core of this technology is to select and develop a matrix that can effectively express the target protein on the cell surface. A surface-expressing matrix having the following properties is preferably used. First, there is a secretory signal that allows passage through the inner membrane to send the target protein to the cell surface, and second, a label that allows the target protein to be stably attached to the surface of the outer membrane. Third, it is expressed in a large amount on the cell surface but has little effect on cell growth. Fourth, it can change the three-dimensional structure of the target protein regardless of the size of the protein. It can be expressed stably without waking up. However, at present, a surface-expressing matrix that satisfies all of the above conditions has not yet been developed.

  There are four types of surface-expressing matrixes known and used so far, such as surface engineered proteins such as extracellular membrane proteins, lipoproteins, secretory proteins, and flagellar proteins. In the case of surface expression in Gram negative bacteria, LamB, PhoE (Charbit et al., J. Immunol., 139: 1658, 1987; Agterberg et al., Vaccine, 8:85, 1990), OmpA, etc. The proteins present in the extracellular membrane of the protein are mainly used to become lipoproteins, TraT (Felici et al., J. Mol. Biol., 222: 301, 1991), PAL (peptidoglycan associated lipoprotein) (Fuchs et al.,). Bio / Technology, 9: 1369, 1991) and Lpp (Francisco et al., Proc. Natl. Acad. Sci. US A, 489: 2713, 1992) has also been used. Furthermore, pilis such as FimA and fimbriae FimH adhesion factor (adhesin) and other pili proteins (Hedegaard et al., Gene, 85: 115, 1989), PapA pill subunits and the like. ) Proteins and the like are used as cell surface expressed matrixes.

  Other than these, ice nucleation protein (Jung et al., Nat. Biotechnol., 16: 576, 1998; Jung et al., Enzyme Microb. Technol., 22: 348, 1998; Lee et. al., Nat. Biotechnol., 18: 645, 2000), Klebsiella oxytoca pullulanase (Kornacker et al., Mol. Microl., 4: 1101, 1990), Neiseria (Neisa). IgA protease (Klauser et al., EMBO J., 9: 1991, 1990), AID which is an adhesion factor of Escherichia coli -1, there is a report that VirG protein Shigella, such as a fusion protein of Lpp and OmpA are used as a surface anchoring motif.

  When Gram positive bacteria are used, protein A and FnBPB proteins derived from Staphylococcus aureus, surface coat proteins of lactic acid bacteria, and Ml protein derived from Streptococcus pyogenes D et al., Proc. Natl. Acad. Sci. USA., 92: 6868, 1995), S layer protein EA1 of Bacillus anthracis, CotB of Bacillus subtilis, etc. are used as surface expression matrixes. It has been.

  The present inventors have used a novel complex expression gene (pgsBCA) derived from poly (γ-glutamic acid) derived from a Bacillus genus strain as a new surface expression matrix, a novel vector that efficiently expresses a target protein on the surface of a microorganism, and the vector Has developed a method for expressing a large amount of target protein on the surface of microorganisms transformed in Korea (Korea Patent Registration No. 0469800). Studies have been attempted to stably express pathogen antigens or antigenic determinants in a mass-produceable bacterium using genetic engineering methods using the surface-expressed matrix described in the patent. In particular, when the target immunogen is expressed on the surface of non-pathogenic bacteria and administered orally, it causes a more sustained and stronger immune response than vaccines using traditional attenuated pathogenic bacteria and viruses It has been reported that it can.

  Lactic acid bacteria used as surface-expressing host cells in the present invention are safe (GRAS: generally recognized) that have played an important role in the production of fermented foods, etc., while having a deep relationship with our diet since ancient times. as safe) and widely distributed in nature from agricultural products to animal bodies. This type of lactic acid bacterium has an inhibitory action against intestinal harmful bacteria and a cleansing action, a function of reducing blood cholesterol, an increase in nutritional value, an action of suppressing infection of pathogens, an action of improving cirrhosis of liver, It has effects such as cancer action, anti-aging action, and immunopotentiating action (immunity enhancing action through macrophage activation), and its application fields are becoming widespread. It has been tried to be used as a vaccine vehicle in terms of the safety of lactic acid bacteria and used in fermented foods. Unmethylated CpG DNA, lipoteichoic acid, peptidoglycan, etc., which are abundant constituents contained in cells Plays a role as an immune enhancer, and its usefulness is highly appreciated. Furthermore, lactic acid bacteria are resistant to bile acids and gastric acids and can transmit antigens up to the intestine, and thus have many advantages in that they can induce mucosal immunity in the intestine (Trends Biotechnol.,). 20: 508, 2002).

  As described above, lactic acid bacteria on which a specific target protein is surface-expressed have been actively developed for use in various fields of application and academic research, but culture processes and preparations for mass production necessary for industrialization. The current situation is that there is an extremely shortage of technological development for the conversion process. In particular, in the case of lactic acid bacteria in which the protein is expressed on the surface, a phenomenon of a decrease in the growth rate that appears to be caused by the expression and insertion of a large amount of the target protein into the cell membrane appears, and the yield of the cells at the maximum culture also It has the disadvantage that it is much lower than general lactic acid bacteria. Furthermore, the surface-expressed protein may be degraded by a proteolytic system that is well known to lactic acid bacteria in connection with the degradation of casein. In fact, since the amount of surface-expressed protein tends to continuously decrease after the middle stage of culture, it has been listed as a problem that must be solved in the mass production process for commercialization (Antonie Van Leeuwenhoek, 76: 139, 1999). In addition to these problems, transformed lactic acid bacteria whose surface is expressed by a specific target protein contains a plasmid vector having a recombinant gene encoding the target protein, thus solving problems that may occur during commercialization. At present, the introduction of a process for this purpose is also desired.

  For this reason, as a result of diligent efforts to solve the problems of the prior art as described above and enhance the immune function of lactic acid bacteria preparations, the inventors added surfactants and carbonates to the basic medium of lactic acid bacteria. And culturing while maintaining the pH of the culture solution at 6.0 to 7.0 during the culture, and discovering that the lactic acid bacterium preparation killed has enhanced immune function compared to viable bacteria. It came to be completed.

  Therefore, an object of the present invention is to provide a killed lactic acid bacteria preparation with enhanced immune function and a method for producing the same.

  The present invention provides a method for producing a killed lactic acid bacteria preparation with enhanced immune function, comprising: (a) culturing lactic acid bacteria; and (b) heat treating the lactic acid bacteria culture solution.

  In the present invention, the culture medium used for the culture further contains 0.1 to 1% by weight of a surfactant and 0.01 to 0.1% by weight of a carbonate, Polysorbate 80. In addition, the step (a) is performed while maintaining the pH at 6.0 to 7.0, and the heat treatment is performed at 80 to 120 ° C. for 5 to 30 minutes.

  In the present invention, the lactic acid bacterium is transformed with a microorganism surface expression vector containing any one or more genes selected from the group consisting of PgsA, PgsB and PgsC and a gene encoding the target protein. The target protein is preferably an antigen, peptide or enzyme, and more preferably an antigen.

  The present invention also provides an immunological preparation containing the killed lactic acid bacterium preparation produced by the above method as an active ingredient.

  Other features and implementations of the invention will become more apparent from the following detailed description and claims.

  In the present invention, first, an α-amylase gene and a vector (pJT1-PGsA-amylase and pJT1) each containing a gene encoding a part of an α-amylase gene and PEDS (Porcine Epidemic Diarhea Virus Spike protein) are used. -PGsA-PEDSs).

  The vector thus prepared was transformed with Lactobacillus caustic, and cultured while maintaining the pH at 6.0 to 7.0 in a medium containing surfactant (particularly polysorbate 80) and carbonate. .

  After culturing lactic acid bacteria, the number of viable bacteria in the culture, measurement of amylase activity, and Western blotting of the target protein resulted in the addition of surfactant and / or carbonate to the surface expression of the viable bacteria and target protein. The amount increased and the surface expression level and stability of the target protein increased due to pH-corrected culture.

  On the other hand, as a result of examining the heat treatment temperature of the lactic acid bacteria cultured according to the present invention, the number of survivors over time, and the presence of the recombinant gene-containing plasmid in the surface-expressed lactic acid bacteria, the lactic acid bacteria cultured according to the present invention were heat treated at 100 ° C. for 20 minutes. It can be confirmed that the viable bacteria present in the culture medium are removed, the recombinant gene-containing plasmid present in the cells of the transformed lactic acid bacteria is removed, and the immune function is enhanced by killing. It was.

  That is, in the present invention, wild-type lactic acid bacteria or target proteins are expressed on the surface while maintaining the pH at 6.0 to 7.0 using a culture medium to which a surfactant (particularly polysorbate 80) and carbonate are added. After cultivating the transformed lactic acid bacteria, heat treatment is performed at 100 ° C. for 20 minutes to kill them, resulting in removal of viable bacteria or recombinant gene-containing plasmids inside the bacteria and enhanced immune function It was possible to produce a lactic acid bacteria preparation. Furthermore, the culture method of the present invention was able to increase not only the mass production of lactic acid bacteria but also the surface expression level and stability of the target protein.

EXAMPLES Hereinafter, the present invention will be described in detail with reference to examples. Those skilled in the art will appreciate that these examples are merely illustrative of the invention and that the scope of the invention is not limited by these examples.

Example 1 Production of α-Amylase Surface Expression Vector and Lactic Acid Bacterium Transformant 107 bp ldh promoter (Sungmin F. Kim) corresponding to SEQ ID NO: 1 which is a promoter of LDH (lactate dehydrogenase) gene derived from Lactobacillus casei et al. Appl. Environ. Microbiol., 57: 2413, 1991), the promoter was inserted into a vector having a replication origin, RepA, capable of replicating in both E. coli and causative bacteria. Later, pgsA, a surface expression matrix derived from Bacillus, was introduced downstream of the promoter, and BamHI and XbaI restriction enzyme sites into which the target gene could be inserted were added to the C-terminus of pgsA. The pJT1-PgsA vector was produced. The selection marker for holding the vector contains an erythromycin resistance gene (FIG. 1).

Using the genome as a template as a gene derived from Streptococcus bovis (ATCC 700410), an α-amylase gene-containing DNA fragment was obtained through PCR using the primers of SEQ ID NOs: 2 and 3.
Sequence number 2: 5'-tct gga tcc gat gaa ca gtg tca atg-3 '
SEQ ID NO: 3: 5′-cag tatta tct aga tta ttt tag ccc atc-3 ′

  The obtained DNA fragment contains a sequence encoding the remaining 703 amino acids excluding the 39 N-terminal amino acid sites that are secretion signals of extracellular α-amylase (extracellular α-amylase), This is a 2,130 bp PCR product containing BamHI and XbaI restriction enzyme sites at both ends.

  By inserting the α-amylase gene-containing DNA fragment into the C-terminus of PgsA of the pJT1-PgsA vector using the BamHI and XbaI restriction enzyme sites, a vector pJT1- that can surface-express a PgsA-α-amylase fusion protein in a lactic acid bacterial host. PgsA-amylase was produced (FIG. 1).

  After transforming caustic bacteria (Lactobacillus casei: KCTC 3109) as a lactic acid bacterium into the α-amylase surface expression vector pJT1-PgsA-amylase, the obtained caustic fungus transformant must have a transmitted plasmid. It was confirmed. The amount of surface-expressed α-amylase was confirmed by two methods: activity measurement and Western blot.

Example 2 Production of PEDSc Surface Expression Vector and Lactic Acid Bacterium Transformant PEDSc is a part of spike protein (Spike protein; S) which is one of the antigenic proteins of porcine epidemic diarrhea virus (PEDV) It is. In order to synthesize the gene for PEDSc, PCR using primers of SEQ ID NO: 4 and SEQ ID NO: 5 was performed. Through the PCR, two primers were annealed without template and amplified, and a PEDSc gene-containing DNA fragment in which BamHI and XbaI restriction enzyme sites were inserted at both ends was synthesized.
Sequence number 4: 5'-tct gga tcc tgt ttt tca ggt tgt tgt agg ggt cct aga ctt caa-3 '
SEQ ID NO: 5: 5′-tta tct aga tta gac ctt ttt aa aagc ttc gta agg ttt aag tct agg-3 ′

  The PgsA-PEDSc fusion protein can be surface-expressed in a lactic acid bacteria host by inserting a DNA fragment containing PEDSc gene into the C-terminus of PgsA of the pJT1-PgsA vector prepared according to Example 1 using the BamHI and XbaI restriction enzyme sites. The vector pJT1-PgsA-PEDSc was produced (FIG. 2).

  After transforming caustic fungus into PEDSc surface expression vector pJT1-PgsA-PEDSc, it was confirmed that the obtained caustic fungal transformant had a transferred plasmid. The surface expressed PgsA-PEDSc fusion protein was confirmed by Western blotting.

Example 3 Effect of Surfactant on Target Protein Expression and Growth of Lactic Acid Bacteria After culturing the lactic acid bacteria transformant (PEDSc surface-expressing lactic acid bacteria) of Example 2 in a medium containing surfactant polysorbate 80, the viable cell count and surface Experiments for measuring the expression level of the expressed protein confirmed the effect of the surfactant on the growth of the lactic acid bacteria expressing the surface of the antigen protein, the final viable cell count, and the expression level of the antigen protein.

  Basic medium used for cultivation of caustic bacteria (1% casein hydrolyzate, 1.5% yeast extract, 2% dextrose, 0.2% ammonium citrate, 0.5% sodium acetate, 0.01% After adding surfactant polysorbate 80 0.1, 0.2, 0.5, 1.0% to magnesium sulfate, 0.05% manganese sulfate and 0.2% dipotassium phosphate), 121 ° C., Sterilization was performed for 10 minutes.

  Each sterilized medium to which surfactant polysorbate 80 is added according to concentration is placed in a 3 L fermentor in 2.0 L portions, and 5% (v / v) of PEDSc surface-expressing lactic acid bacteria is passed through two stages of seed culture of 5 ml and 100 ml medium ) After inoculation, the cells were cultured at 30 ° C for 24 hours.

  The number of viable bacteria and the expression level of the surface expressed protein were compared at the time of culturing for 24 hours. What was cultured in the culture medium which did not add surfactant polysorbate 80 was used as a control group.

  As shown in Table 1, the higher the concentration of the surfactant added, the higher the number of viable bacteria in the culture solution was observed. When 1.0% of the surfactant was added, the maximum value was compared with the control group. About 1.8 times higher viable count. Since a certain amount of PgsA-PEDSc fusion protein per cell is surface-expressed, an increase in the number of viable bacteria means an increase in the fusion protein, and from this, surface expression is carried out in the culture medium by adding a surfactant. It was confirmed that the amount of PgsA-PEDSc fusion protein increased.

  By collecting the bacterial cells at the time of culturing for 24 hours when the number of viable cells was measured and performing Western blotting, the surface expression level of PEDSc expressed by being fused with PgsA was confirmed.

All the cells of PgsA-PEDSc surface-expressing bacteria recovered in the experimental group were adjusted to the same cell concentration, and a certain amount was extracted from these to denature the protein to prepare a sample, which was used as an SDS-polyacrylamide gel. After analysis by electrophoresis, the fractionated protein was transferred to a PVDF (polyvinylidene-difluoride membranes; Bio-Rad) membrane. The protein-transferred PVDF membrane was blocked by shaking for 1 hour in a blocking buffer solution (50 mM Tris-HCl, 5% skim milk, pH 8.0), and then the rabbit-derived polyclonal primary against PgsA was blocked in the blocking buffer solution. The antibody was diluted 1000 times and allowed to react for 12 hours. After completion of the reaction, the membrane was washed with a buffer solution, and a secondary antibody against a rabbit conjugated with biotin in a blocking buffer solution was diluted 1000 times and reacted for 4 hours. The membrane after the reaction was washed with a buffer solution, reacted with avidin-biotin for 1 hour, and then washed again. Substrate (H ₂ 0 ₂ ) and a coloring reagent (DAB) were added to the washed membrane to develop color, thereby confirming specific binding between the specific antibody against PgsA and the fusion protein (FIG. 3).

  As a result, as shown in FIG. 3, a PgsA-PEDSc fusion protein of about 45.9 kDa was detected, and the amount detected was proportionally increased according to the concentration of the surfactant added to the medium.

  From the above results, it was confirmed that when a constant concentration of a surfactant was added to the medium, both the maximum growth value of the lactic acid bacteria on which the target protein was surface expressed and the surface expression level of the protein per cell increased.

Example 4 Effect of Carbonate on Target Protein Expression and Growth of Lactic Acid Bacteria After culturing the lactic acid bacteria transformant (PEDSc surface-expressing lactic acid bacteria) of Example 2 in a carbonate-containing medium, the number of viable bacteria and expression of surface expressed proteins By carrying out a quantity measurement experiment, the effect of carbonate on the growth of the surface-expressed lactic acid bacteria of the antigen protein and the final viable cell count and the expression level of the antigen protein was confirmed.

  In a medium in which 0.5% of a surfactant is added to the basic medium used for cultivation of caustic fungi, and 0.01%, 0.05% and 0.1% of carbonate are added to the basic medium. After culturing the transformants for 24 hours, the number of viable bacteria at the maximum growth point and the expression level of the surface expressed protein were compared by the method as described above. Those cultured in a medium without added carbonate were used as a control group.

  As a result of collecting the culture solution at the time of culturing for 24 hours and confirming the number of viable bacteria in the culture solution, as shown in Table 2, the higher the concentration of carbonate added, the more viable bacteria in the culture solution, When 0.1% of carbonate was added, the maximum viable cell count was about 8.1 times higher than that of the control group. Furthermore, it was also confirmed that the amount of PgsA-PEDSc fusion protein surface-expressed in the culture medium increased by the addition of carbonate (Table 2).

  As shown in FIG. 4, it was confirmed that the amount of the PgsA-PEDSc fusion protein specifically detected by the antibody against PgsA increases proportionally according to the concentration of the carbonate added to the medium.

  From the above results, it is confirmed that when a constant concentration of surfactant and carbonate is added to the medium, both the maximum growth value of the lactic acid bacteria on which the target protein is surface expressed and the surface expression of the protein per cell increase. I was able to.

Example 5: Effect of pH-corrected culture on stable surface expression of target protein Surface expression occurs when culturing lactic acid bacteria while maintaining the pH of the culture solution at 6.0 to 7.0 and when not cultivating. In order to confirm the effect on the expression level of the protein, the amylase activity was measured and Western blotting was performed during the culture using the transformant of the α-amylase surface-expressing lactic acid bacterium according to Example 1.

  Full-scale culture was performed in a 3 L fermentor according to the culture method. After the start of full-scale culture, the culture solution is collected every 4 hours from the lapse of 5 hours, and the viable cell count, pH change, amylase activity measurement and Western blotting are performed. The culture medium is a basic medium (1% casein hydrolyzed). Degradation product, 1.5% yeast extract, 2% glucose, 0.2% ammonium citrate, 0.5% sodium acetate, 0.01% magnesium sulfate, 0.05% manganese sulfate and 0.2% phosphoric acid 2 Potassium) was added with 0.5% surfactant and 0.1% carbonate, and the antibiotic erythromycin was cultured to a final concentration of 16 μg / ml. Ammonia water, KOH or NaOH was used for pH correction.

  An activity measurement kit (Kikoman Co., Tokyo, Japan) was used to measure the activity of the enzyme of amylase expressed on the surface of lactic acid bacteria. As a substrate, N3-G5-β-CNP (2-chloro-4-nitrophenyl-65-azido-65-deoxy-β-maltopentaside) was used.

  The culture solution was centrifuged to recover only the cells, washed twice with PBS buffer solution, suspended in 100 μl of the same buffer solution, and reacted with 400 μl of the substrate-containing solution at 37 ° C. for 10 minutes. Subsequently, 800 μl of a reaction stop solution was added to complete the reaction, and the absorbance at 400 nm was measured. One unit of activity is necessary for producing 1 μmole of CNP (2-chloro-4-nitrophenol) having an absorbance at 400 nm from N3-G5-β-CNP at 37 ° C. for 1 minute. Defined as the amount of enzyme.

  In FIG. 5, a shows the activity of amylase at the time of pH correction and non-correction culture in units per 1 ml of culture solution, and compares the change in the activity of total amylase in the culture solution per 1 ml at each measurement time point. b shows the activity of amylase by dividing the unit per 1 ml of the culture solution by the OD value of the culture solution at the time of measurement, and comparing the change in the amylase activity of a certain amount of cells at each time of measurement. It is.

  As shown in FIG. 5, in the non-pH-corrected experimental group, the amylase activity gradually decreases from 10 to 15 hours after the start of the culture, whereas in the pH-corrected experimental group, the activity of the whole culture solution is the culture. It was found that it increased continuously up to 20 hours later, and the activity of amylase per certain amount of microbial cells tended to increase in a small amount without decreasing, and showed a stable expression level. At the time of 25 hours, which is the end of the culture, the amylase activity as a whole per ml of culture solution is about 19.2 times higher than that of the non-pH-corrected culture experimental group, and a certain amount of cells. The activity of amylase per hit was 17.7 times higher. Since the activity is proportional to the amount of surface-expressed amylase, it was found from the above results that the surface expression amount of the protein is increased by the pH correction culture.

  Furthermore, the western blot method was performed using the culture solution sample collect | recovered for every time point using the amylase specific antibody according to said method (FIG. 6). As a result, as shown in FIG. 6, the change in the amount of the surface-expressed PgsA-amylase fusion protein detected by the amylase-specific antibody showed the same tendency as the activity measurement result shown in FIG. In the case of non-pH-corrected culture, the expression level gradually decreased with the passage of culture time, and in the case of pH-corrected culture, it was confirmed that the expression level was continuously high even when the culture time passed.

  From the above results, it was found that not only a quantitative increase in the protein expressed on the cell surface but also a stable retention was achieved by pH-corrected culture.

Example 6: Death of surface-expressed lactic acid bacteria through heat treatment The heat treatment temperature for the lactic acid bacteria culture solution, the number of remaining viable bacteria over time, and the presence or absence of the recombinant gene-containing plasmid in the surface-expressed lactic acid bacteria were confirmed to establish killing conditions did.

The number of viable bacteria is confirmed 2-3 days after smearing the killed culture solution on the MRS solid medium, and the presence or absence of the plasmid is determined after recovering the lactic acid bacteria of the killed culture solution and washing with water. PCR was performed using the primers of SEQ ID NO: 6 at the N-terminal site and SEQ ID NO: 7 at the C-terminal site of the internal site of the erythromycin resistance gene contained in the plasmid using This was confirmed by whether a PCR product was detected.
SEQ ID NO: 6: 5′-gtg tgt tga tag tgc agt atc-3 ′
SEQ ID NO: 7: 5′-ccg tag gcg cta ggg acc tct tatta gc-3 ′

  The lactic acid bacteria culture solution is heat-treated at 100 ° C. for 20 minutes through the examination of the treatment temperature and treatment time within a range in which denaturation occurs on the surface-expressed proteins and bacterial cells by the heat treatment and the intended efficacy can be prevented from disappearing. As a result, it was confirmed that viable bacteria present in the culture solution were removed. In addition, the recombinant gene containing plasmid contained in the transformed lactic acid bacteria was not detected (FIG. 7).

Example 7: Immunity-enhancing effect of killed lactic acid bacteria The immune-enhancing effect of lactic acid bacteria that had undergone the process of killing bacteria and viable lactic acid bacteria that had not undergone the process of killing sterilization was expressed through dendritic cell stimulation (maturation). ELISA kits for cytokines IL-10 and IL-12 p70 secreted from cells (measured using human IL-10 Duose ELISA development system, human IL-12 p70 Duoset ELISA system, R & D systems).

Dendritic cells were obtained from human blood samples for laboratory stimulation of dendritic cells. Mononuclear cells were separated from human blood samples by Ficoll-Hypaque gradient separation (d = 1.077 g / ml) and 1% w / v tissue-culture grade bovine serum albumin ( RPMI 1640 medium supplemented with Bovine Serum Albumin) was added and cultured in a CO ₂ incubator at 37 ° C. for one day to remove the attached cells. After selecting the non-adherent cells by the above process, the cell culture solution to which 100 ng / ml of GM-CSF and 10 ng / ml of IL-4 were added was added, and then cultured in a CO ₂ incubator in the same manner for 6 days. Immature dendritic cells were prepared.

After immature dendritic cells prepared in this manner were placed in a 24-well plate at 5 × 10 ⁴ cells / well, a group in which only dendritic cells were cultured as a negative control group, a known lipo group as a positive control group A group to which polysaccharide (LPS) was added at a concentration of 100 ng / ml, a group to which 1 × 10 ³ viable bacteria were added, a group to which 1 × 10 ³ caustic bacteria after the killing process were added, 1 A group to which × 10 ³ living bacteria of PEDSc surface-expressing caustic bacteria were added, a group to which 1 × 10 ³ PEDSc surface-expressing caustic bacteria after the killing step were added, were cultured under the same conditions for 3 days. After the culture, the culture supernatant treated with each sample was collected, and the secretion induction ability of human IL-10 and human IL-12 p70 secreted in the culture supernatant was measured using an ELISA kit.

(1) Human IL-10 ELISA
In 96 wells prepared (preliminarily coated with anti-human IL-10 monoclonal antibody), 50 μl of IL-10 standard solution (standard solution) and 50 μl of culture supernatant were added and reacted at room temperature for 2 hours. Then, after washing 5 times with a washing buffer solution (300 μl / well), 100 μl of anti-human IL-10 polyclonal antibody to which biotin as the primary antibody was bound was added and reacted at room temperature for 1 hour, and the washing buffer solution ( 300 μl / well). Next, 100 μl of the secondary antibody, avidin-horseradish peroxidase complex, was added and reacted at room temperature for 30 minutes, washed 7 times, and then reacted with TMB coloring solution for 30 minutes. The color development was then stopped with 50 μl of reaction termination solution. Then, the amount of IL-10 secreted was measured at 450 nm using an ELISA reader.

(2) Human IL-12 p70 ELISA
In 96 wells prepared (pre-coated with anti-human IL-12 p70 monoclonal antibody), 50 μl of IL-10 p70 standard solution (standard solution) and 50 μl of the culture supernatant were added and allowed to react at room temperature for 2 hours. Subsequently, after washing 5 times with a washing buffer (300 μl / well), 100 μl of anti-human IL-12 p70 polyclonal antibody to which biotin as the primary antibody was bound was added and reacted at room temperature for 1 hour. Wash 5 times with (300 μl / well). Next, 100 μl of the secondary antibody, avidin-horseradish peroxidase complex, was added, reacted at room temperature for 30 minutes, washed 7 times, and reacted with TMB coloring solution for 30 minutes. The color development was then stopped with 50 μl of reaction termination solution. Then, the amount of IL-12 p70 secretion was analyzed by measurement with an ELISA reader at 450 nm.

  As a result, as shown in FIG. 8, the killed bacteria-added group and the killed PEDSc surface-expressed bacteria-added group had higher IL-10 values from dendritic cells than the other groups. And induction of IL-12 p70 secretion. From this result, it was found that killed lactic acid bacteria had a better effect than alive lactic acid bacteria in terms of immune enhancement, especially in the induction of dendritic cell maturation.

  Although specific portions of the contents of the present invention have been described in detail above, these specific descriptions are merely preferred embodiments for those having ordinary skill in the art, and thus the scope of the present invention. It is self-evident that is not limited. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

  The present invention has the effect of providing a killed lactic acid bacteria preparation with enhanced immune function and a method for producing the same, including a step of heat-treating a lactic acid bacteria culture solution. According to the present invention, it is possible to produce a lactic acid bacteria preparation having not only an effect of preventing functional damage of a surface-expressed target protein but also an enhanced immune enhancing effect. A killed lactic acid bacterium produced according to the method of the present invention exhibits an improved immune enhancing effect compared to a living bacterium and is mass-produced, and is therefore effective for feed additives, veterinary drugs or vaccines. .

FIG. 1 is a gene map of pJT1-PgsA-amylase, which is a transfer vector that surface-expresses α-amylase protein. FIG. 2 is a gene map of pJT1-PgsA-PEDSc, which is a transfer vector that surface-expresses PEDSc protein. FIG. 3 shows the results of confirming the amount of PgsA-PEDSc fusion protein expressed in PEDSc surface-expressing lactic acid bacteria cultured in a surfactant-containing medium by a Western blot method using a specific antibody against PgsA. It is. FIG. 4 is a diagram showing the results of confirming the amount of PgsA-PEDSc fusion protein expressed in PEDSc surface-expressing lactic acid bacteria cultured in a carbonate-containing medium by Western blotting using a specific antibody against PgsA. FIG. 5 is a diagram comparing the growth of amylase surface-expressing lactic acid bacteria and the change in amylase enzyme activity (unit / ml) during pH-corrected and non-corrected cultures. FIG. 6 is a diagram showing the results of confirming the amount of PgsA-amylase fusion protein expressed in amylase surface-expressing lactic acid bacteria during pH correction and non-correction culture by Western blotting using a specific antibody against PgsA. FIG. 7 is an agarose gel photograph in which the presence or absence of a recombinant gene-containing plasmid in lactic acid bacteria transformed with a surface expression vector in the killed and untreated group according to the present invention was confirmed. FIG. 8 shows the effect of stimulating human dendritic cells (maturation of dendritic cells) by the stimulation of dendritic cells in the wild type lactic acid bacteria group and the lactic acid bacteria group transformed into the surface expression vector. It is a figure which shows the result confirmed from the quantity of the cytokine made.

Claims (2)

(A) including at least one gene selected from the group consisting of PgsA, PgsB and PgsC, and a gene encoding any one target protein selected from the group consisting of an antigen, a peptide and an enzyme A lactic acid bacterium transformed with a vector for expression on a microorganism surface was adjusted to a pH of 6.0 to 7.0 in a medium containing 0.1 to 1% by weight of polysorbate 80 and 0.01 to 0.1% by weight of carbonate. And (b) producing a lactic acid bacteria culture solution on which the antigen, peptide or enzyme is surface-expressed at 80 to 120 ° C. at 5 to 30. A method of producing a killed lactic acid bacteria preparation with enhanced immune function, comprising a step of heat-treating for minutes . The method according to claim 1, wherein the lactic acid bacterium is transformed with a vector for expression on the surface of a microorganism comprising a porcine epidemic diarrhea virus spike protein (PEDSc) and PgsA .

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