WO2007055437A1 - Killed lactic acid bacteria preparation having enhanced immunity and method for preparing the same - Google Patents
Killed lactic acid bacteria preparation having enhanced immunity and method for preparing the same Download PDFInfo
- Publication number
- WO2007055437A1 WO2007055437A1 PCT/KR2005/004159 KR2005004159W WO2007055437A1 WO 2007055437 A1 WO2007055437 A1 WO 2007055437A1 KR 2005004159 W KR2005004159 W KR 2005004159W WO 2007055437 A1 WO2007055437 A1 WO 2007055437A1
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- Prior art keywords
- lactic acid
- acid bacteria
- culturing
- bacteria
- expressed
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Classifications
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/20011—Coronaviridae
- C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
Abstract
The present invention relates to killed lactic acid bacteria preparation having enhanced immunity and a method for preparing the same, and more particularly, relates to killed lactic acid bacteria preparation having enhanced immunity and a method for preparing the same, the method comprising the steps of culturing lactic acid bacteria in a culture medium containing surfactant and carbonate and killing. According to the present invention, it is possible to produce lactic acid bacteria preparation that has enhanced immunity effect, as well as, the effect of preventing functional damage of surface-expressed target protein, with high concentration rate. Since the killed lactic acid bacteria prepared according to the present invention shows improved immunity enhancing effect and enables mass production compared to live bacteria, it is useful as feed additives, animal drugs or vaccines etc.
Description
Killed Lactic Acid Bacteria Preparation Having Enhanced Immunity and Method for Preparing the Same
TECHNICAL FIELD
The present invention relates to a killed lactic acid bacteria preparation having enhanced immunity and a method for preparing the same, and more specifically, relates to a killed lactic acid bacteria preparation having enhanced immunity and a method for preparing the same, the method comprising culturing the lactic acid bacteria in a culture medium containing surfactant and carbonate and killing.
BACKGROUND ART
The technology of expressing a desired protein by attaching it onto the cell surface of a microorganism is called cell surface display technology. The cell surface display technology uses surface proteins of microorganisms, such as bacteria or yeast as a surface anchoring motif to express a target protein on the surface and has an application scope including production of recombinant live vaccines, construction of peptide/antibody library for screening, whole cell absorbent, whole cell biotransformation catalyst, and the like. The application scope of this technology is very diversified according to the kind of proteins to be expressed on the cell surface. Therefore, the cell surface display technology has tremendous potential in industrial applicability.
For successful cell surface display technology, the surface anchoring motif is the most important. It is the core of this technology to select and develop a motif capable of expressing a foreign protein on the cell surface effectively. A surface anchoring motif having the following properties is preferable. First, it should
have a secretion signal to help a target protein to pass through the cellular inner membrane so that it can be transferred to the cell surface. Second, it should have a target signal to help a target protein to be stably fixed on the surface of the cellular outer membrane. Third, it can be expressed in a large quantity on a cell surface but does not affect growth of the cell. Fourth, it has nothing to do with protein size and can express a target protein without change in the three- dimensional structure of the target protein. However, a surface anchoring motif satisfying the foregoing requirements has not yet been developed.
The surface anchoring motives which have been known and used so far are largely classified into four types of cell outer membrane proteins, lipoproteins, secretory proteins and surface organ proteins such as flagella protein. In the case of surface anchoring on gram negative bacteria, proteins existing on the cellular outer membrane, such as LamB, PhoE (Charbit et al., J. Immunol., 139:1658, 1987; Agterberg et al., Vaccine, 8:85, 1990), OmpA, and the like have been mainly used, lipoproteins, such as TraT (Felici et al., J. MoI. 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. USA, 489:2713, 1992) have been used. Also, Fimbriae proteins, such as FimA or FimH adhesion of type 1 fimbriae (Hedegaard et al., Gene, 85:115, 1989) and pili proteins, such as PapA pilu subunit have been used as a cell surface anchoring motif.
In addition, it has been reported that 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), pullulanase of Klebsiela oxytoca (Kornacker et al., MoI. Microl., 4:1101, 1990), IgA protease of Neiseria (Klauser et al., EMBO J., 9:1991, 1990), AIDA-I which is adhesion of E. coli, VirG protein of shigella, a fusion protein of Lpp and OmpA were used as a surface anchoring motif.
When gram positive bacteria is used, protein A and FnBPB protein derived from
Staphylococcus aureus, a surface coat protein of lactic acid bacteria, M6 protein derived from Streptococcus pyogenes (Medaglini, D et al., Proc. Natl. Acad. Sci. USA., 92:6868, 1995), S-layer protein EAl of Bacillus anthracis, CotB of Bacillus subtilis, and the like are used as a surface anchoring motif.
The present inventors have developed a novel vector for effectively expressing a target protein on the cell surface of a microorganism using poly-gamma-glutamate synthetase complex (pgsBCA) derived from the genus Bacillus strain as a novel surface anchoring motif and a method for mass-expressing a target protein on the surface of a microorganism transformed with the vector (Korean Patent No. 0469800). Researches have been conducted to stably express a pathogenic antigen or an antigen determining group in bacteria suitable for mass-production by genetic engineering method using the surface anchoring motives in the aforementioned Patent. Particularly, it has been reported that when a target immunogen is expressed on the surface of non-pathogenic bacteria to be orally administered, more sustained and stronger immune response can be induced, as compared to conventional vaccines using attenuated pathogenic bacteria or viruses.
The lactic acid bacteria used as a host cell of surface anchoring in the present invention are GRAS(generally recognized as safe) bacteria which have been playing an important role in the production of fermented food etc., maintaining close relationship with our dietary life for a long time and is widely distributed in nature through out the agricultural products and the body of animals. These lactic acid bacteria have a repression effect and a cleansing effect on pathogenic bacteria in the intestines, a decrease effect on blood cholesterol, an increase in nutritional value, a repression effect on virus infection, an improving effect on liver cirrhosis, anticancer effect, anti-aging effect, immunity enhancing effect(immunity enhancing effect by macrophage activation) etc., so application fields are getting larger. Because of the fact that the lactic acid bacteria have safety and are used in fermented food, application to vaccine vehicles is attempted, and a great number of
components, unmethylated CpG DNA, lipoteichoic acid, peptidoglycan etc., contained in the cell play a role as an adjuvant, thus the utility is appreciated more highly. Also, lactic acid bacteria have an advantage in that it shows resistance to bile acid and gastric acid, so it is possible to deliver an antigen to the intestines, and thus can induce mucosal immunity in the intestines(7re«ds Biotechnol, 20:508, 2002).
As described in the foregoing, application development in various application fields and scientific research of lactic acid bacteria having a certain target protein expressed on the surface, are being actively conducted, however, technology of culturing process and preparation process etc. for mass production needed for industrialization lacks development. Especially, in the case of lactic acid bacteria having proteins expressed on the surface, it has disadvantages in that it shows growth rate decline assumed to be caused by target proteins largely expressed and thus inserted into cell membrane, and strain recovery rate in maximum culturing becomes significantly lower compared to normal lactic acid bacteria. Also, surface anchored protein can be decomposed by proteolytic system of lactic acid bacteria, well known related with decomposition of casein. Actually, as mid-stage of culturing passes, the amount of surface anchored protein tends to decrease continuously, thus it is an issue problem to be solved for mass production process for manufacture by all means(Antonie Van Leeuwenhoek, 76:139, 1999). Besides these problems, in the case of transformed lactic acid bacteria having a certain target protein expressed on the surface, it comprises a plasmid vector containing a recombinant gene encoding the target protein, thus it is necessary to introduce the process to solve the problems that might occur when manufactured.
DISCLOSURE OF THE INVENTION
Thus, the present inventors have made extensive efforts to solve the above
problems of prior art and to enhance the immunity of lactic acid bacteria preparation, and as a result, found that the immunity of lactic acid bacteria preparation obtained by adding surfactant and carbonate to a basic medium of lactic acid bacteria and culturing while maintaining the pH of culture broth within 6.0~7.0 to kill is enhanced compared to live bacteria, thereby completing the present invention.
Therefore, the object of the present invention is to provide killed lactic acid bacteria preparation having enhanced immunity and a method for preparing the same.
Other features and embodiments of the present invention will be more fully apparent from the following detailed description and appended claims.
BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 is a genetic map of p JT 1-Pgs A- Amylase, a transfer vector surface- expressing α- Amylase protein.
FIG. 2 is a genetic map of pJTl-PgsA-PEDSc, a transfer vector surface-expressing PEDSc protein.
FIG. 3 is a result showing the amount of PgsA-PEDSc fusion protein expressed on PEDSc surface-expressing lactic acid bacteria cultured in a surfactant-added medium by western blot using a specific antibody to PgsA.
FIG. 4 is a result showing the amount of PgsA-PEDSc fusion protein expressed on PEDSc surface expressing lactic acid bacteria cultured in a carbonate-added medium by western blot using a specific antibody to PgsA.
FIG. 5 is showing a comparison of the growth of lactic acid bacteria surface- expressing amylase and the change of amylase enzyme activity(unit/ml) between the culturing with pH correction and the culturing with non- correction.
FIG. 6 is a result showing the amount of PgsA- Amylase fusion protein expressed on amylase surface expressing lactic acid bacteria between the culturing with pH correction and the culturing with non-correction by western blot using a specific antibody to PgsA.
FIG. 7 is a photograph of agarose gel showing whether a plasmid containing a recombinant gene is present in lactic acid bacteria transformed with surface expressing vector in killed group and non-killed group.
FIG. 8 is a result showing the effect of human dendritic cell simulation(dendritic cell maturation) according to whether killing treatment of the wild type lactic acid bacteria group and the lactic acid bacteria group transformed with surface expressing vector, is performed, which is confirmed by the amount of cytokine excreted by dendritic cell simulation.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a method for preparing killed lactic acid bacteria preparation having enhanced immunity, the method comprising the steps: (a) culturing a lactic acid bacteria; and
(b) heat treating the lactic acid bacteria culture broth.
In the present invention, the medium used in said culturing preferably contains 0.1~l wt% additional surfactant and 0.01~0.1 wt% additional carbonate, and said surfactant is preferably polysorbate 80. Also, said step(a) is preferably performed,
while maintaining pH level of 6.0~7.0, and said heat treatment is preferably performed at 80~120°C for 5~30 minutes.
In the present invention, said lactic acid bacteria is preferably transformed with a microorganism surface anchoring vector, comprising any one or more of genes selected from the group consisting of PgsA, PgsB and PgsC and a gene encoding a target protein, and said target protein is preferably an antigen, a peptide and an enzyme, and more preferably an antigen.
The present invention also provides an immune preparation containing the killed lactic acid bacteria preparation produced by said method as an active ingredient.
In the present invention, vectors(pJTl -PGs A- Amylase and pJTl -PGsA-PEDSs) containing α- Amylase gene and a gene encoding part of PEDS(Porcine Epidemic Diarrhea Virus Spike protein), respectively, were first constructed using PCR and cloning technology.
Lactobacillus casei was transformed with said constructed vector and cultured in a medium containing surfactant(particularly, polysorbate 80) and carbonate while maintaining the pH level between 6.0—7.0.
After culturing the lactic acid bacteria, the number of live bacteria in the medium, and amylase activity were measured and western blot on a target protein was performed, as a result, it was shown that the number of live bacteria and the amount of surface expression of the target protein increased by adding surfactant and/or carbonate, and stability and the amount of surface expression of the target protein increased by the culturing with pH correction.
Meanwhile, as a result of examining the number of survived bacteria and the absence or presence of the plasmid containing a recombinant gene in surface-
expressing lactic acid bacteria according to the temperature and time of heat treatment of the inventive lactic acid bacteria, it was confirmed that when the lactic acid bacteria cultured according to the present invention were treated with heat at 100°C for 20 minutes, live cells existing in the medium were eliminated, and the plasmid containing the recombinant gene existing in the cells of transformed lactic acid bacteria was eliminated, and immunity was enhanced by killing.
In other words, in the present invention, a wild type lactic acid bacteria or a transformed lactic acid bacteria having a target protein expressed on the surface were cultured while maintaining pH level of 6.0~7.0 using a culture medium containing surfactant(particularly, polysorbate 80) and carbonate and heat treated at 100°C for 20 minutes to kill the bacteria, and as a result, live bacteria or the plasmid containing a recombinant gene inside bacteria is eliminated, thus making it possible to prepare the killed lactic acid bacteria preparation having enhanced immunity. Also, culturing method of the present invention could achieve an increase in stability and the amount of surface expression of target protein, as well as, mass production of lactic acid bacteria.
Examples
Hereinafter, the present invention will be described in more detail by specific examples. However, the present invention is not limited to these examples, and it is obvious to those of ordinary skill in the field of the present invention that numerous variations or modifications could be made within the spirit and scope of the present invention.
Example 1. Construction of α- Amylase surface expression vector and lactic acid bacteria transformant
To be expressed by 107 bp of ldh promoter (Sungmin F. Kim et al. Appl. Environ.
Microbiol, 57:2413, 1991) corresponding to SEQ ID NO:1 which is a promoter in LDH(lactate dehydrogenase) gene derived from lactobacillus casei, said promoter was inserted into a vector having RepA replicable both in coliform bacteria and lactobacillus casei as a replication origin, and then pgsA, surface anchoring motif derived from bacillus was introduced at the downstream of the promoter, followed by adding BamHI, Xbal restriction enzyme site where a target gene could be inserted into C-terminal end of PgsA, thus constructing pJTl-PgsA vector. The vector contains erythromycin-resistant gene as a selective marker (FIG. 1).
Using a gene derived from Streptococcus bovis (ATCC 700410) as a template, and primers SEQ ID NOs: 2 and 3, PCR was performed to obtain a DNA fragment containing α-amylase gene.
SEQ ID NO: 2: 5 ' -tct gga tec gat gaa caa gtg tea atg-3 ' SEQ ID NO: 3: 5 ' -cag tta tct aga tta ttt tag ccc atc-3 '
The obtained DNA fragment is 2,130 bp of PCR product which contains sequences encoding the rest of 703 amino acids except 39 N-end amino acid sites which is secretion signal of extracellular α-amylase and contains BamHI and Xbal restriction enzyme sites at both ends, respectively.
Vector pJTl -Pgs A- Amylase capable of surface expressing PgsA-α-amylase- conjugated protein in lactic acid bacteria was constructed by inserting DNA fragment containing α-amylase gene into PgsA C-terminal end of p JTl -PgsA vector using said BamHI and Xbal restriction enzyme sites(FIG. 1).
Lactic acid bacteria, lactobacillus casei (KCTC 3109) was transformed with vector pJTl -PgsA- Amylase for surface-expressing α-amylase to confirm that the obtained lactobacillus casei transformant possesses delivered plasmid. The amount of α-amylase surface expressed was measured by activity measurement and western blot.
Example 2. Construction of PEDSc surface expression vector and lactic acid bacteria transformant
PEDSc is one part of spike protein(S) which is one of antigen proteins of porcine epidemic diarrhea virus (PEDV). To synthesize said PEDSc gene, PCR was performed using primers of SEQ ID NO: 4 and SEQ ID NO: 5. By said PCR, two primers were annealed, and then amplified without a template to synthesize DNA fragment containing PEDSc gene having BamHI and Xbal restriction enzyme sites inserted at both ends. SEQ ID NO: 4: 5 '-tot gga tec tgt ttt tea ggt tgt tgt agg ggt cct aga ctt caa-3 ' SEQ ID NO: 5: 5'-tta tct aga tta gac ctt ttc aaa age ttc gta agg ttg aag tct agg-3'
Vector pJTl -Pgs A-PEDSc capable of surface-expressing PgsA-PEDSc-conjugated protein in lactic acid bacteria was constructed by inserting DNA fragment containing PEDSc gene into C-terminal end of Pgs A of pJTl-PgsA vector constructed in example 1 using said BamHI and Xbal restriction enzyme sites(FIG. 2).
Lactic acid bacteria, lactobacillus casei was transformed with vector pJTl-PgsA- PEDSc for surface-expressing α-amylase to confirm that the obtained lactobacillus casei transformant possesses delivered plasmid. The amount of surface-expressed PgsA-PEDSc-conjugated protein was confirmed by western blot.
Example 3. The effect of surfactant on expression of target protein and growth of lactic acid bacteria
The effect of surfactant on growth of lactic acid bacteria having antigen protein surface-expressed, the number of live bacteria counted finally and the amount of antigen protein expression was examined by culturing lactic acid bacteria transformant(lactic acid bacteria having PEDSc surface-expressed) of example 2 in
a medium supplemented with surfactant, polysorbate 80, and performing measurement experiment for the number of live bacteria and the amount of surface- expressed proteins.
Each 0.1, 0.2, 0.5, 1.0% of surfactant, polysorbate 80 was added to a basic medium(l% casein hydrolysate, 1.5% yeast extract, 2% destrose, 0.2% ammonium citrate, 0.5% sodium acetate, 0.01% magnesium sulfate, 0.05% manganese sulfate and 0.2% dipotassium phosphate) used in the culturing of lactobacillus casei to sterilize for 10 minutes at 121 °C .
2.0L of each sterilized medium supplemented with surfactant, polysorbate 80 at each concentration was put in 3 L fermenter to perform two step- culture in 5 ml and 100 ml media, and then 5%(v/v) of lactic acid bacteria having PEDSc expressed on the surface was inoculated, followed by culturing for 24 hours at 30 °C .
At 24 hour after the culturing, the number of live bacteria and the amount of surface-expressed proteins were compared. Those cultured in a medium without surfactant polysorbate 80 was used as a control group.
As shown in Table 1, as the concentration of added surfactant increases, a higher number of live cells in the medium was observed and when 1.0% of surfactant is added, the number of live bacteria is about 1.8 times higher, which is the highest compared to the control group. As a certain amount of PgsA-PEDSc fusion protein per cell are surface-expressed, an increase in the number of live bacteria shows the increase of fusion protein, thereby showing that the amount of surface- expressed PgsA-PEDSc fusion protein in a medium increases by adding the surfactant.
Table 1
At 24 hours after the culturing, the amount of PEDSc fused with PgsA and expressed on the surface was measured by recovering bacteria and performing western blot.
After adjusting the concentrations of whole cells of lactobacillus casei having PgsA-PEDSc expressed on the surface recovered from the test group the same, a given amount of these was extracted and protein was denatured to prepare experimental sample, and then the sample was analyzed with SDS-polyacrylamide gel electrophoresis, followed by transferring the fractionated proteins onto a PVDF (polyvinylidene-difluoride membranes, Bio-Rad) membrane. The resulting PVDF membrane was stirred in a blocking buffer (50 ml Tris HCl, 5% skim milk, pH 8.0) for one hour to block, and then allowed to react for 12 hours with polyclonal primary antibody derived from a rabbit against PgsA, diluted 1,000 times with the blocking buffer. After completing the reaction, the resulting membrane was washed with buffer solution and allowed to react for 4 hours with biotin-conjugated secondary antibody, diluted 1,000 times with the blocking buffer. The membrane after the reaction was then washed again with the buffer and an avidin-biotin system was used for one hour and washed. The washed membrane was colored by adding substrate(H202) and dye(DAB), which identified a specific binding between specific antibodies against the PgsA and the above fusion proteins(FIG. 3).
As a result, as shown in FIG. 3, about 45.9 kDa of PgsA-PEDSc fusion protein was detected, and the detected amount increased in proportion to the concentration of added surfactant.
From the above results, when adding a given concentration of surfactant to a medium, it was confirmed that the maximum growth value of lactic acid bacteria having target proteins expressed on the surface and the amount of surface- expressed proteins per bacteria were increased.
Example 4. The effect of carbonate on expression of target protein and growth of lactic acid bacteria
The effect of carbonate on growth of lactic acid bacteria having antigen proteins expressed on the surface, the number of live bacteria counted finally and the amount of antigen protein expression was confirmed by culturing lactic acid bacteria transformant(lactic acid bacteria having PEDSc expressed on the surface) of example 2 in a medium added with carbonate and performing measurement experiment for the number of live bacteria and the amount of surface-expressed proteins.
Using said basic medium used in the culturing of lactobacillus casei, which is added with 0.5% of surfactant, as a basic medium, each 0.01, 0.05, 0.1% of carbonate was added in the basic media, respectively, and then transformant was cultured in each medium for 24 hours to compare the number of live bacteria and the amount of surface-expressed proteins at the maximum growth rate by the same method discribed above. Those cultured in a medium without carbonate was used as a control group.
At 24 hours after the culturing, the culture broth was recovered and examined for the number of live bacteria, and as a result, as shown in Table 2, as the
concentration of added carbonate increased, the number of live bacteria in the medium increased, and when 0.1% of carbonate is added, about 8.1 times higher number of live cells could be obtained, which is the highest compared to the control group. Also, it could be seen that the amount of surface-expressed PgsA-PEDSc fusion protein in the medium was increased by adding the carbonate(Table 2).
Table 2
As shown in FIG. 4, the amount of PgsA-PEDSc fusion protein specifically detected by the antibody against PgsA increased in proportion to the concentration of added carbonate.
From the above results, when adding a given concentration of surfactant and carbonate to a medium, it was confirmed that the maximum growth value of lactic acid bacteria having target proteins expressed on the surface and the amount of surface-expressed proteins per bacteria were increased.
Example 5. Effect of pH correction culture on stable surface expression of target protein
In order to examine the effect on the amount of surface-expressed proteins in the case of culturing while maintaining the pH of culture broth between 6.0~7.0 and in the case without maintaining the pH between 6.0~7.0 during the culturing period of lactic acid bacteria, measurement of amylase activity and western blotting was performed during the culturing period using lactic acid bacteria transformant
having α-amylase expressed on the surface according to example 1. Culturing was performed in a 3 L fermentor according to said culturing method. After a lapse of 5 hours of culturing, culture broth was recovered at 4-hour interval, measured for the number of live bacteria, a change in pH and subjected to amylase activity measurement and western blotting, and 0.5% of surfactant and 0.1% of carbonate were added to a basic medium(l% casein hydrolysate, 1.5% yeast extract, 2% destrose, 0.2% ammonium citrate, 0.5% sodium acetate, 0.01% magnesium sulfate, 0.05% manganese sulfate and 0.2% dipotassium phosphate) to use as a culture medium, and the culturing was carried out using antibiotic, erythromycin at a final concentration of 16 μg/ml. For pH correction, KOH or NaOH was used.
To measure enzyme activity of amylase expressed on lactic acid bacteria surface, a kit for measuring activity (Kikkoman Co., Tokyo, Japan) was used. N3-G5-β- CNP (2-chloro-4-nitrophenyl 65-azido-65-deoxy-β-maltopentaoside) was used as a substrate.
Only bacteria were recovered by centrifuging the culture broth to wash 2 times with PBS buffer solution, suspended with lOOμl of the same buffer solution and allowed to react with 400μl of a solution containing a substrate at 37 °C for 10 minutes, and then the reaction was completed by adding 800μl of a reaction-termination solution to measure absorbance at 400nm. 1 unit of activity was defined as the amount of enzyme needed for producing lμmole of CNP (2-chloro-4-nitrophenol) showing absorbance at 400nm from N3-G5-β-CNP for 1 minute at 37 °C .
In FIG. 5, 'a' shows a comparison between changes in whole amylase activity in culture broth per ml at each measuring point by indicating amylase enzyme activity in culturing with pH correction and non-correction with unit per culture broth ml and 'b' indicates amylase enzyme activity by dividing unit per ImI of culture broth into OD value of culture broth at measuring point, which is a comparison between changes in amylase activity that a certain amount of bacteria have at each
measuring point.
As shown in FIG. 5, whereas amylase activity gradually started to decrease 10~15 hours after the initiation of culturing in the pH non-correction test group, the whole activity of culture broth continuously increased till 20 hours after culturing in the pH correction test group, and amylase activity per a given amount of bacteria also didn't decrease and tended to increase in small amount, showing stable expression amount. At 25 hours after completing the culturing, the activity of whole amylase per culture broth ml showed about 19.2 times higher, and the activity of amylase per a given amount of bacteria showed about 17.7 times higher in culturing with pH correction compared to culturing with pH non-correction. As the activity is in proportion to the amount of surface-expressed amylase, from the above results, it could be seen that the amount of surface-expressed proteins increased by culturing with pH correction.
Also, using the culture broth sample recovered at each culturing point, western blotting using amylase specific antibody according to said method was performed (FIG. 6). As a result, as shown in FIG. 6, a change in the amount of the surface- expressed PgsA-Amylase fusion protein detected by amylase specific antibody showed the same tendency as the activity measurement result shown in FIG. 5. In the case of culturing with pH non-correction, the amount of expression gradually decreased with the lapse of time, and in the case of culturing with pH correction, even though the culturing time lapsed, it was confirmed to continuously show high expression rate.
From the above results, it could be seen that stable preservation as well as an increase of quantity of proteins expressed on the surface of bacteria could be accomplished by culturing with pH correction.
Example 6. Killing of surface expressing lactic acid bacteria by heat treatment
Killing condition was established by confirming the number of remaining live bacteria and whether a plasmid containing a recombinant gene in surface expressing lactic acid bacteria remains, according to the temperature and time of heat treatment for lactic acid bacteria culture broth.
The number of live bacteria was examined 2~3 days after killed culture broth had been spread on MRS solid medium, and whether the plasmid remains, was examined by recovering lactic acid bacteria of killed culture broth to wash it with water, and subjecting inner part of erythromycin-resistant gene contained in the plasmid to PCR using the washed lactic acid bacteria as template and primers of N- end region of SEQ ID NO: 6 and C- end region of SEQ ID NO: 7 to determine whether l,156bp of PCR product is detected. SEQ ID NO: 6: 5 '-gtg tgt tga tag tgc agt atc-3 '
SEQ ID NO: 7: 5'-ccg tag gcg eta ggg ace tct tta gc-3'
By determining the temperature and time of treatment in the range where the disappearance of desired effect due to denaturation of surface expressed protein and bacteria by heat treatment can be prevented, lactic acid bacteria culture broth was treated with heat at 100°C for 20 minutes, and as a result, live bacteria existing in the medium was found removed, and the plasmid containing a recombinant gene in the transformed lactic acid bacteria was not detected (FIG. 7).
Example 7. Immunity enhancing effect of killed lactic acid bacteria
Immunity enhancing effect of lactic acid bacteria treated with killing process and live lactic acid bacteria without treating with killing process was measured using ELISA kit(Human IL-10 Duoset ELISA Development system, Human IL- 12 ρ70 Duoset ELISA Development system, R&D systems) for respective cytokine IL-10
and IL- 12 p70, which is secreted from dendritic cells by dendritic cell stimulation(maturity) .
The dendritic cells were obtained from human blood sample to experiment dendritic cell stimulation laboratorially. Precise monocyte was isolated from human blood sample with Ficoll-Hypaque gradients(d= 1.077 g/ml), added with
RPMI 1640 medium supplemented with 1% w/v tissue-cultured bovine serum albumin and cultured in 37°C CO2 CuItUrUIg device for one day to remove adhered cells. After selecting cells which are not adhered by the above process, cell culture broth supplemented with GM-CSF 100ng/ml and IL-4 10ng/ml was placed in the incubator and cultured in CO2 culturing device for 6 days to prepare premature dendritic cells.
After putting each 5x 104 cell/well of the above prepared premature dendritic cells in 24 well plate, a group in which only dendritic cells are cultured as a negative control group, a group added with well known lipopolysaccharide(LPS) at a concentration of 100ng/ml as a positive control group, a group added with IxIO3 of
Lactobacillus casei live bacteria, a group added with IxIO3 of Lactobacillus casei after killing process, a group added with IxIO3 of Lactobacillus casei live bacteria having PEDSc expressed on the surface, and a group added with IxIO3 of
Lactobacillus casei bacteria having PEDSc expressed on the surface after killing process etc. were cultured under the same conditions for 3 days. After the culturing, the ability of inducing secretion of human IL-IO and human IL- 12 p70 existing in culture supernatant were measured by recovering culture supernatant treated with each sample using ELISA kit.
(1) Human IL-IO ELISA
50μl of IL-IO standard solution and 50μl of culture supernatant were put into prepared 96 well(precoated with anti-human IL-10, monoclonal antibody) and allowed to react for 2 hours at room temperature. The resulting product was
washed 5 times with washing buffer solution(300 μl/well), added with 100 μl of anti-human IL-IO multiclonal antibody bonded with biotin which is a primary antibody, and allowed to react for 1 hour at room temperature to wash 5 times with washing buffer solution(300 μl/well). After that, the resulting product was added with lOOμl of avidin-horseradish peroxidase conjugate which is a secondary antibody and allowed to react for 30 minutes at room temperature to wash 7 times, and then allowed to react with TMB dye solution for 30 minutes, followed by fixing the dye with 50μl of stop solution. The excretion amount of IL-10 was measured using ELISA reader at 450nm.
(2) Human IL-12 p70 ELISA
50μl of IL-10 p70 standard solution and 50μl of culture supernatant were put into prepared 96 well (precoated with anti-human IL-12 p70, monoclonal antibody) and allowed to react for 2 hours at room temperature. The resulting product was washed 5 times with washing buffer solution(300 μl/well), added with lOOμl of anti-human IL-12 p70 multiclonal antibody bonded with biotin which is a primary antibody and allowed to react for 1 hour at room temperature, and then washed 5 times with washing buffer solution(300 μl/well). After that, the resulting product was added with lOOμl of avidin-horseradish peroxidase conjugate which is a secondary antibody and allowed to react for 30 minutes at room temperature to wash 7 times, and then allowed to react with TMB dye solution for 30 minutes, followed by fixing the dye with 50μl of stop solution. The excretion amount of IL-12 p70 was measured using ELISA reader at 450nm.
As a result, as shown in FIG. 8, it was confirmed that the group added with killed Lactobacillus casei and the group added with killed Lactobacillus casei having PEDSc expressed on the surface has high inducibility of IL-10 and IL-12 p70 excretions from the dendritic cells compared to other groups. From these results, it could be seen that killed lactic acid bacteria have excellent effect of enhancing immunity, especially inducing maturity of dendritic cells compared to live lactic
acid bacteria.
Although the present invention has been described in detail with reference to the specific features, it will be apparent to those skilled in the art that this description is solely for a preferred embodiment and does not limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.
INDUSTRIALAPPLICABILITY
The present invention has an effect of providing killed lactic acid bacteria preparation having enhanced immunity and a method for preparing the same, comprising heat treating the lactic acid bacteria culture broth. According to the present invention, it is possible to prepare lactic acid bacteria preparation that has enhanced immunity effect, as well as, the effect of preventing functional damage of surface-expressed target protein. Since the killed lactic acid bacteria prepared according to the present invention shows improved immunity enhancing effect compared to live bacteria and enables mass production, it is useful as feed additives, animal drugs or vaccines etc.
Claims
1. A method for preparing killed lactic acid bacteria preparation having enhanced immunity, the method comprising the steps:
(a) culturing lactic acid bacteria; and
(b) heat treating the lactic acid bacteria culture broth.
2. The method according to claim 1, wherein the medium used in said culturing additionally contains 0.1~l wt% surfactant and 0.01~0.1 wt% carbonate.
3. The method according to claim 2, wherein said surfactant is polysorbate 80.
4. The method according to claim 1, wherein step (a) is performed while maintaining the pH level between 6.0~7.0.
5. The method according to claim 1, wherein said heat treatment is performed at 80-120 °C for 5-30 minutes.
6. The method according to claim 1, wherein said lactic acid bacteria is transformed with a microorganism surface anchoring vector containing any one or more of genes selected from the group consisting of PgsA, PgsB and PgsC, and a gene encoding a target protein.
7. The method according to claim 6, wherein said target protein is any one selected from the group consisting of an antigen, a peptide and an enzyme.
8. The method according to claim 7, wherein said target protein is an antigen.
9. An immune preparation containing the killed lactic acid bacteria preparation prepared by the method of any one claim among claims 1 to 8 as an active ingredient.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020050107860A KR100578395B1 (en) | 2005-11-11 | 2005-11-11 | A killed lactic acid bacteria preparation with enhanced immunity and method for preparing the same |
| KR10-2005-0107860 | 2005-11-11 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2007055437A1 true WO2007055437A1 (en) | 2007-05-18 |
Family
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/KR2005/004159 WO2007055437A1 (en) | 2005-11-11 | 2005-12-06 | Killed lactic acid bacteria preparation having enhanced immunity and method for preparing the same |
Country Status (3)
| Country | Link |
|---|---|
| JP (1) | JP4902845B2 (en) |
| KR (1) | KR100578395B1 (en) |
| WO (1) | WO2007055437A1 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2251022A1 (en) * | 2009-05-11 | 2010-11-17 | Nestec S.A. | Non-replicating micro-organisms and their immune boosting effect |
| EP3541944A4 (en) * | 2018-01-09 | 2020-10-21 | Cell Biotech Co., Ltd. | Gene expression cassette and expression vector including the same |
| CN112760336A (en) * | 2020-12-30 | 2021-05-07 | 广州辉园苑医药科技有限公司 | Expression system and surface display system of epitope peptide and construction method thereof |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2009078995A (en) * | 2007-09-26 | 2009-04-16 | Genolac Bl:Kk | Immuno-vaccine for periodontal disease |
| EP2251020A1 (en) * | 2009-05-11 | 2010-11-17 | Nestec S.A. | Short-time high temperature treatment generates microbial preparations with anti-inflammatory profiles |
| KR101369219B1 (en) | 2010-11-04 | 2014-03-03 | 정명준 | Antibacterial tyndalized lactic acid bacteria and preparing method thereof |
| CN102989010A (en) * | 2012-12-04 | 2013-03-27 | 山东信得科技股份有限公司 | Porcine epidemic diarrhea virus genetic engineering subunit oral vaccine |
| JP2014210747A (en) | 2013-04-19 | 2014-11-13 | アンジェスＭｇ株式会社 | Oral vaccine having improved cell-mediated immunity induction potency |
| WO2019022463A2 (en) | 2017-07-24 | 2019-01-31 | (주)제이비바이오텍 | Immunogenic system and animal vaccine comprising same |
| CN111575223B (en) * | 2020-05-20 | 2022-04-15 | 江南大学 | Method for reducing secretion of surface substances of lactobacillus rhamnosus |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR19990003300A (en) * | 1997-06-25 | 1999-01-15 | 김경환 | Medium composition for high concentration culture of bifidus bacteria |
| WO2003014360A1 (en) * | 2001-08-10 | 2003-02-20 | Bioleaders Corporation | SURFACE EXPRESSION VECTORS HAVING pgsBCA, THE GENE CODING POLY-GAMMA-GLUTAMATE SYNTHETASE, AND A METHOD FOR EXPRESSION OF TARGET PROTEIN AT THE SURFACE OF MICROORGANISM USING THE VECTOR |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP3993322B2 (en) * | 1998-09-30 | 2007-10-17 | 雪印乳業株式会社 | Lactic acid bacteria growth promoter and use thereof |
| JP3472804B2 (en) * | 2000-05-24 | 2003-12-02 | 独立行政法人食品総合研究所 | Novel bacteriocin and method for producing the same |
-
2005
- 2005-11-11 KR KR1020050107860A patent/KR100578395B1/en active IP Right Grant
- 2005-12-06 WO PCT/KR2005/004159 patent/WO2007055437A1/en active Application Filing
-
2006
- 2006-01-20 JP JP2006012977A patent/JP4902845B2/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR19990003300A (en) * | 1997-06-25 | 1999-01-15 | 김경환 | Medium composition for high concentration culture of bifidus bacteria |
| WO2003014360A1 (en) * | 2001-08-10 | 2003-02-20 | Bioleaders Corporation | SURFACE EXPRESSION VECTORS HAVING pgsBCA, THE GENE CODING POLY-GAMMA-GLUTAMATE SYNTHETASE, AND A METHOD FOR EXPRESSION OF TARGET PROTEIN AT THE SURFACE OF MICROORGANISM USING THE VECTOR |
Non-Patent Citations (1)
| Title |
|---|
| MATSUGUCHI T. ET AL.: "Lipoteichoic acids from Lactobacillus strains elicit strong tumor necrosis factor alpha-inducing activities in macrophages through Toll-Like Receptor 2", CLINICAL AND DIAGNOSTICS LABORATORY IMMUNOLOGY, vol. 10, no. 2, 2003, pages 259 - 266 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2251022A1 (en) * | 2009-05-11 | 2010-11-17 | Nestec S.A. | Non-replicating micro-organisms and their immune boosting effect |
| WO2010130660A1 (en) * | 2009-05-11 | 2010-11-18 | Nestec S.A. | Non-replicating micro-organisms and their immune boosting effect |
| CN102724987A (en) * | 2009-05-11 | 2012-10-10 | 雀巢产品技术援助有限公司 | Non-replicating micro-organisms and their immune boosting effect |
| EP3541944A4 (en) * | 2018-01-09 | 2020-10-21 | Cell Biotech Co., Ltd. | Gene expression cassette and expression vector including the same |
| CN112760336A (en) * | 2020-12-30 | 2021-05-07 | 广州辉园苑医药科技有限公司 | Expression system and surface display system of epitope peptide and construction method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| KR100578395B1 (en) | 2006-05-10 |
| JP4902845B2 (en) | 2012-03-21 |
| JP2007131610A (en) | 2007-05-31 |
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