JPH0632630B2 - Method for producing protein antigen produced by pathogenic bacteria in livestock and poultry - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing a protein antigen produced by a pathogenic bacterium of livestock (poultry) used for infectious disease diagnosis.

(Prior Art) The recent development of the livestock industry has been remarkable, and especially for the purpose of improving productivity, the scale expansion by multi-breeding is surprising. However, along with this, chronic diseases pose a major threat to livestock management as a factor that impedes productivity improvement, and there is an urgent need to clean up these diseases. Therefore, diagnosis of the disease (mainly infectious disease) is important. In particular, a diagnostic method using a protein antigen produced by a pathogenic bacterium of domestic animals (poultry) is preferably adopted as a simple and accurate diagnostic method for the infectious disease, and a large amount of this protein antigen is produced. However, as a conventional manufacturing method, there is the following manufacturing method through a liquid culture method. That is, first, pathogenic bacteria are cultivated in a liquid medium, and separated by centrifugation or sterilization into bacterial cells and a supernatant or liquid containing the protein antigen produced in the medium, and the supernatant or The solution is concentrated and purified to a certain concentration using the vacuum concentration method and the ultrafiltration method (using several ultrafiltration membranes with different molecular weight cutoffs), or by salting out using salts such as ammonium sulfate. Then, methods such as collecting and using the protein antigen-containing liquid are taken.

Another method is as follows. That is, a semipermeable membrane bag is suspended in a liquid medium, and a bacterial solution (bacteria suspended in purified water, physiological saline, or a buffer solution) is placed in the semipermeable membrane bag, and a liquid medium containing an external liquid is added. After stirring and culturing with a magnetic stirrer, etc., the latter half of the culture permeable bag is taken out and separated by centrifugation into the bacterial cells and the supernatant containing the protein antigen produced in the bacterial solution, and the supernatant is separated. It is a method of collecting the antigenic substance by packing it in a semipermeable membrane tube and concentrating it with a concentrating agent.

(Problems to be Solved by the Invention) The above conventional manufacturing methods have the following drawbacks. That is,
When performing the above-mentioned ultrafiltration, viscous substances such as mucopolysaccharides produced by cultured bacteria or animal / fish meat extract, peptone in the medium, or various high contents such as blood and serum components of animal / livestock etc. It takes a lot of time because the ultrafiltration does not proceed smoothly due to the molecular weight component, and since this operation is repeated twice or more, the concentration / purification process is complicated and the required protein antigen substance is required. Is also adsorbed on the ultrapermeate, so the recovery of the antigenic substance is also poor.

Further, when the protein antigen is recovered from the supernatant obtained by the above centrifugation or the liquid obtained by the eradication by a salting out method using salts such as ammonium sulfate, it takes a long time to add a large amount of salts little by little. After salting out, centrifugation must be carried out, the precipitate must be dissolved with a small amount of purified water, packed in a semipermeable membrane tube, and the salts must be removed by dialysis. For that purpose, it is necessary to use a large amount of purified water, physiological saline, or a buffer solution, and the separation, purification, and recovery steps are complicated and take a lot of time, and inactivation due to protein denaturation of the antigenic material after recovery is also observed. To be

Further, in the above liquid medium, a semipermeable membrane bag is suspended, and in the case of culturing by inserting a bacterial solution in the semipermeable membrane, the production amount of the protein antigenic substance per amount of the liquid medium used is small, and The recovered liquid is contaminated with low-molecular weight dyes contained in the medium,
Since these substances are removed, it is difficult to remove them even by dialysis with purified water, physiological saline solution, buffer solution or the like, which causes inconvenience that a highly accurate antigen product cannot be obtained.

(Means for Solving Problems) The present invention eliminates the above-mentioned drawbacks of the conventional method, and achieves a high yield and high quality in a short time with a significantly simple manufacturing process as compared with the conventional method. Livestock (fowl) adapted to produce protein antigens
The method for producing a protein antigen produced by the pathogenic bacterium of claim 1, wherein a semipermeable membrane that allows nutrient components in the medium to pass but does not pass the protein antigen is attached to the semisolid medium, Apply the culture fluid of pathogenic bacteria of livestock (poultry) to
After the main culture was performed to produce the protein antigen on the semipermeable membrane, the semipermeable membrane was peeled from the semisolid medium together with the produced protein antigen, and then the semipermeable membrane was washed to obtain The present invention is characterized in that the antigen is separated from the microbial cells and collected from the washing solution (Example).

According to the invention, a semi-solid medium is used, a representative of which is:
It is generally known as an agar medium. The agar medium for pathogenic bacteria includes peptones, beef (fish) meat extract, yeast extract, salts, blood such as cotton sheep, horses, chickens, cows, pigs and rabbits, or saccharides such as serum, butter sugar and starch,
Alternatively, L-cysteine hydrochloride, hemin, etc. are appropriately selected and blended, and 1 to 2% of agar is added thereto, and the mixture is heated and dissolved, and is autoclaved mainly at 110 ° C to 121 ° C for 10 to 15 minutes), 45 ~
It is cooled to 50 ° C and then poured into a sterile Petri dish in an amount of 15 to 20 ml to be solidified. When blood or serum is blended, the composition other than this is sterilized by high pressure and cooled, and then blood or serum is added and well stirred and mixed to obtain an agar medium. Next, a semipermeable membrane is attached on the semisolid medium having a predetermined composition thus prepared. As the semipermeable membrane, one that does not allow passage of protein antigens produced by pathogenic bacteria of livestock and poultry is used. The molecular weight of the protein antigen is 10,000-200,00
0, and most of them have a molecular weight of 20,000 as a subunit.
Since it is ~ 50,000, the range of molecular weight cutoff is 3,500 ~ 5
It is common to use the one of 0,000. Then, a culture solution of pathogenic bacteria of livestock (poultry) is applied onto the semipermeable membrane. Examples of the pathogenic bacterium include, for example, the genus Actinomyces, Bordetella.
Genus, Pasteurella genus, Fusobacterium genus, Actinobacillus
The bacterium belonging to the genus lus) and the genus Haemophillus is used.

Specific examples of such pathogenic bacteria include, for example, Streptococcus equisimiles.
lis), Streptococcus s
uis), Staphylococcu
s hyicus subsp.hyicus), Staphylococcus aureus, Actinomyces pyogenes, Bordetella
Bordetella bronchiseptica,
Haemophilus parasuis,
Actinobaci
llus pleuropneumoniae), Pasteurella martosida (P
asteurella multocida), Fusobacterium necrophorum, etc., and protein antigens produced by these bacteria and their target animals are shown in Table 1 below.

That is, on a semipermeable membrane with a specific cut-off molecular weight pasted on the surface of a semi-solid medium that has been sterilized in advance, a certain amount of a fresh culture solution obtained by pre-culturing a pathogenic bacterium that produces a specific protein antigen of interest. Place and apply the bacterial solution evenly. This is cultivated for 18 hours to 14 days at a culturing temperature of 25 to 40 ° C. under the desired culturing conditions such as aerobic, anaerobic, and carbon dioxide substitution depending on the bacterial species (main culturing). In this culture, a polymeric substance such as a protein contained in the semi-solid medium passes through the semi-permeable membrane and does not come into contact with the transplanted bacterium, but salts in the medium,
The nutritional components of vitamins, peptides, amino acids, and other low molecular weight substances pass through the semipermeable membrane and are supplied to the bacterium, and the nutrients cause the bacterium to grow. As the bacterium grows, the bacterium produces a protein antigen substance of a type suitable for the bacterium.
However, since the produced antigenic substance passes through the semipermeable membrane because it is a polymer and does not diffuse into the medium, the bacterial cells and the protein antigenic substance produced by the bacterium and low molecular weight substances are not formed on the semipermeable membrane. Medium components remain.

Many of the pigments in the medium do not pass through the semipermeable membrane because they are bound to high molecular substances such as proteins. Since a small amount of free dye is fixed by agar, it is prevented from passing through the semipermeable membrane and being mixed with the antigen substance side.

After this main culture, the semipermeable membrane on the medium surface is stripped off, the residue such as the antigenic substance adhering to it is washed off with a buffer solution, and the washing solution is centrifuged to contain the bacterial cells and protein antigenic substance. Separate into supernatant. Centrifugation is 3,000-15,
If the operation is performed at 000 rpm for 10 to 60 minutes, the bacterial cells will remain as a bacterial mass on the bottom of the centrifuge tube, so only the supernatant liquid is collected.
The recovered supernatant contains protein antigenic substances, low-molecular weight medium components, buffer salts, and the like. This supernatant liquid was packed in a semipermeable membrane tube, and commercially available from A Daiichi Kogyo Kagaku Co., Ltd.
G gum, polyethylene glycol # 60 manufactured by Wako Pure Chemical Industries, Ltd.
Concentration with a concentrating agent such as 00 removes the low molecular weight medium components of the medium components, and thus a solution with a high concentration of protein antigen can be obtained.

Further, the concentrated protein antigen solution may be put into a semipermeable membrane tube and repeatedly dialyzed with purified water to remove low molecular weights such as salts to obtain a protein antigen substance having higher purity. The recovered concentrated solution, that is, the antigen, is frozen by a method in which the activity unit is determined by the box titration method, and a diluent (buffer solution) is added so that a certain unit antigen is obtained, and the protein antigen is not denatured or inactivated. After drying, a diagnostic protein antigen is obtained. The diagnostic antigen of the present invention thus obtained can be stably stored for a long period of time in a cool and dark place.

Incidentally, the culture method of separating the pathogenic bacterium strain and producing the inoculum or original bacterium used in the main culture of the present method by pure culture may be any conventionally used culture method, such as liquid culture and semi-solid culture. Etc., the medium composition thereof may be the same as that of the semi-solid medium described above.

Strains of the specified pathogenic bacteria can be easily obtained from the Veterinary Biologics Association, the Ministry of Agriculture, Forestry and Fisheries, Animal Health Test Center, etc., and those isolated and fixed from livestock affected by each infectious disease should be used. You can use it.

Next, specific examples of the present invention will be described.

Example Actinomyces pyogenes strain (use one that has been distributed from the Ministry of Agriculture, Forestry and Fisheries Animal Health Laboratory, or isolate this bacterium from Actinomyces pyogenes infected pigs,
Then, a strain with high protease-producing ability may be selected and used) to inoculate a blood agar medium which is a semi-solid medium and cultured under anaerobic conditions at 37 ° C for 1 to 2 days. The resulting colonies are transplanted again to a fresh blood agar medium and anaerobically cultured at 37 ° C. for 1 to 2 days to obtain inoculum. Up to this point, the method is the same as the conventional method.

This inoculum is cultured in a modified VL agar medium having the composition shown in the table below or a modified VL liquid medium having a composition excluding the agar in the table to prepare the original bacterium.

That is, this modified inoculum was inoculated into the modified liquid medium, cultured under anaerobic conditions at 37 ° C for 1 to 2 days, and then aseptically centrifuged (3,000 to
After centrifugation at 8,000 rpm for 10 to 30 minutes, the centrifugation supernatant is discarded, and the precipitated bacterial cells are suspended in sterile phosphate buffered saline (pH 7.0 to 7.2) (sterile physiological saline or sterile purified water may be used). Get the original fungus. Alternatively, the above-mentioned inoculum is further applied to the modified VL agar medium which is further made into a semisolid medium, and anaerobically cultured at 37 ° C. for 1 to 2 days, and the moss is sterilized with phosphate buffered saline (pH 7.0). ~
7.2) (Sterile physiological saline or sterile purified water may be used)
The cells are then suspended again and again 1 to 2 times to wash the cells (centrifugation is 3,000 to 8,000 rpm for 10 to 30 minutes), and then sterile phosphate buffered saline (pH 7.0 to 7.2).
Suspend in sterile physiological saline or sterile purified water, and use this as the original bacterium.

Then, separately, pre-sterilized semipermeable membrane (cut off molecular weight
8,000 to 10,000 dialysis membrane tubes) are aseptically attached to the semipermeable membrane of the modified VL agar medium (medium having the same composition as above), which is a semisolid medium. 0.1 ml of suspended culture fluid (wet bacterium amount about 10 mg / ml)
Is evenly applied, and then anaerobically cultured at 37 ° C. for 1 to 2 days (main culture). After the main culture, peel off the semipermeable membrane on the medium surface,
The cells are washed with 0.1 M Tris / HCl buffer (pH 8.0), and the washed solution is centrifuged (8,000 rpm, 30 minutes) to separate the cells and the supernatant containing the protease antigen. Since the cells remain as a cell mass on the bottom of the centrifuge tube, only the supernatant is collected.
The recovered supernatant contains protease antigens, low molecular weight medium components, buffer components, and the like. This supernatant is packed in a semipermeable membrane tube (molecular weight cut-off of 8,000 to 20,000), treated with a condensing agent (AG gum or polyethylene glycol # 6,000, etc.) in a cool dark room (about 4 ° C) and concentrated to obtain a high purity. Obtain the protease antigen. The activity unit of the recovered protease antigen solution was determined by Box Titration. (The antigenic activity (sedimentation test in agar gel) was 1:32 to 1:62. No dye was found in the solution.) 0.1M Tris-hydrochloric acid so as to become a constant unit antigen Buffer solution pH8.0 was added and freeze-dried by a vacuum freeze-drying method in which the protease antigen was not denatured or inactivated to obtain a diagnostic protease antigen.

Comparative Example 1 A semipermeable membrane bag was suspended in a modified VL liquid medium in which the amount of each medium component dissolved in purified water 3 was dissolved in 2.7 purified water, and the semipermeable membrane bag was obtained in Example 1 above. Put 300 ml of suspension of Actinomyces pyogenes, which is the same original bacteria, and after culture with carbon dioxide, take out the semipermeable membrane bag and centrifuge to separate the cells and Actinomyces pyogenes. Was separated into a supernatant containing a protease (proteolytic enzyme) antigen produced by, and the supernatant was collected and concentrated to obtain a protease antigen solution showing an antigen activity (precipitation test in agar gel) of 1:32 or more. Only a small amount of about 5 to 15 ml was obtained. Moreover, low-molecular weight pigments contained in the medium were mixed. Even if they were dialyzed against purified water, physiological saline solution, buffer solution or the like to remove these substances, they were not removed.

Comparative Example 2 The original strain of the modified VL liquid culture of the Actinomyces pyogenes obtained according to Example 1 was inoculated in about 17 times the amount of the modified VL liquid medium, carbon dioxide-cultured, and centrifuged. Therefore, the cells and the supernatant containing the protease (proteolytic enzyme) antigen produced in the medium are separated, and the supernatant is packed in a semipermeable membrane tube and concentrated to about 1/10 with a concentrating agent. After salting out with a buffer solution, the supernatant was subjected to an ultrafiltration method using two ultrafiltration membranes with different molecular weight cutoffs (molecular weight cutoffs: 300,000 and 10,000) to bring the protease antigen to a constant concentration. Concentrated and purified. The number of manufacturing days required 11 to 15 days.

2 as compared with 6 to 7 days required in the production method of the present invention in Example 1
It took more than double the number of days.

Comparative Test Example 1 For a protein antigen preparation (diagnostic protease antigen) of the product of the present invention obtained in Example 1 and the conventional product obtained in Comparative Example 2, one vial of 32 times amount was prepared. It was diluted with distilled water and the absorption wavelengths were compared and examined. The results are shown in the attached drawings. This figure shows an ultraviolet-visible absorption diagram of a protease antigen for diagnosing Actinomyces pyogenes infectious disease. From this figure, the product of the present invention (dashed line) shows no absorption in the visible wavelength region and is colorless. Although it is a transparent solution, the conventional product (solid line) shows a considerable absorption, indicating that it is a colored solution.

Comparative Test Example 2 The yield of the protease antigen solution was compared between the production method of the present invention shown in Example 1 and the conventional production method shown in Comparative Example 2.

The results are shown in Table 2 below.

As is clear from the above table, the method of the present invention was applied to about 18 to 2 of the final recovered antigen solution per medium in 6 trials.
The amount of the conventional method was about 9 to 11 ml, while the yield of the method of the present invention was about 1.9 times higher than that of 0 ml. Further, in comparison of proteins, the method of the present invention is 16.668 mg to 18.68 mg, and the conventional method is 7.1.
It was 82 mg to 8.745 mg, and the number of the method of the present invention was about 2.2 times as large as the average.

(Effects of the Invention) As described above, according to the present invention, a semipermeable membrane that allows nutrient components in the medium to pass therethrough but does not allow protein antigens to pass through is attached to the semisolid medium, and the livestock is attached to the semipermeable membrane. , (Poultry) pathogenic bacterium culture liquid was applied and main culture was performed, so that the protein antigen produced on the semipermeable membrane was mixed with impurities such as polymer substances and pigments in the medium. Can be obtained without mixing, and the productivity can be improved. Further, after the main culture, the semipermeable membrane is peeled from the semisolid medium, the semipermeable membrane is washed, and the protein antigen is recovered from the obtained washing solution, so that the concentration of the produced protein antigen solution is higher than that in the conventional method. Further, there are effects such that purification can be easily carried out, separation, recovery and purification operations are facilitated, and a protein antigen of high quality can be produced in a short time and in a high yield.

[Brief description of drawings]

The drawing is a graph showing the UV-visible absorption characteristic curves of protein antigen products obtained by this method as well as the conventional method.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical indication (C12P 21/00 C12R 1:44) (C12P 21/00 C12R 1:46) (C12P 21/00 C12R 1:01)

Claims (3)

[Claims] 1. A semi-permeable medium is attached with a semi-permeable membrane that allows nutrient components in the medium to pass through but does not pass protein antigens.
A culture solution of a pathogenic bacterium of domestic animals (poultry) is applied onto the semipermeable membrane, main culture is performed, and a protein antigen is produced on the semipermeable membrane, and then the semipermeable membrane is produced by the produced protein. A method for producing a protein antigen, which comprises peeling from an semi-solid medium together with an antigen, then washing the semi-permeable membrane, and separating and recovering the antigen from bacterial cells from the resulting washing solution. 2. The method according to claim 1, wherein the semipermeable membrane has a drawing molecular weight of 3,500 to 50,000. 3. The pathogenic bacterium belongs to the genus Actinomyces, the genus Bordetella, the genus Pasteurella, the genus Staphylococcus, the genus Streptococcus, the genus Fusobacterium, the genus Actinobacillus and the genus Haemophilus. The manufacturing method according to 1.