JP2969147B2 - Sialic acid-specific hemagglutinin from B. bronchiseptica and its purification - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention is remarkably used as an antigen for a biological preparation such as a vaccine or a diagnostic agent for a bronchial septic bacterium (also called Bordetella bronchiseptica or Bb bacterium) infection such as swine atrophic rhinitis. The present invention relates to an effective sialic acid-specific hemagglutinin, a vaccine and a diagnostic agent containing the same, and a method for purifying the sialic acid-specific hemagglutinin.

[0002]

BACKGROUND OF THE INVENTION Bronchial sepsis is a pathogen in livestock, poultry and wildlife, causing a variety of respiratory diseases. In pigs, bronchiseptic bacillus causes atrophic rhinitis and the like, and may kill young pigs due to infection in their newborns. Atrophic rhinitis is a chronic disease characterized by atrophic lesions of the turbinate bones, which, when advanced, cause facial deformation such as shortening of the maxilla and bending of the nose. This disease is highly contagious and spreads to all heads in multi-headed breeding, and many studies have been conducted because it causes enormous economic loss due to delayed growth, reduced feed efficiency, induction of secondary infection, etc. Was.

[0003] Atrophic rhinitis is caused by toxin-producing Pasteurella multocida (hereinafter also referred to as Pm bacterium) in addition to the above-mentioned bronchiseptic bacterium. Both produce cutaneous necrotic toxins that can form turbinate lesions, but the toxins differ in antigenicity and the lesions induced are heterogeneous. Note that P
M bacteria hardly colonize the normal porcine nasal mucosa, and usually the nasal mucosa can be colonized by infection with bronchiseptic bacteria. As vaccines for preventing this disease, killed bacteria of bronchiseptic bacteria, live attenuated bacteria of bronchiseptic bacteria, killed bacteria of Pm bacteria, inactivated skin necrotic toxins of Pm bacteria, and the like have been used. However, although these vaccines are effective in alleviating the symptoms of the disease and reducing the lesion, they cannot inhibit the infection of the causative bacteria.

On the other hand, one of the protective antigens of B. bronchiseptica is
Sialic acid-specific hemagglutinin, which is one of the sialic acid-specific hemagglutinins, is capable of agglutinating erythrocytes by specifically binding to sialic acid-containing sugar chains on the surface of bovine erythrocyte membrane (Ishikawa a
nd Isayama, Infection and Immunity, 55, 1607-1609,
1987).

[0005] Bronchial sepsis is presumed to possess a plurality of hemagglutinins (Ishikawa and Isayama, Journ).
al of Medical Microbiology, 26, 205-209, 1988),
Agglutinin having affinity for bovine erythrocytes has been reported to date by Ohgitani et al. (Vaccine, 9, 653-658, 1991) and Sakurai et al. (Journal of Medical Microbiology,
39, 388-392, 1993). However, the hemagglutinin reported by Ohgitani et al. Is a fine fibrous structure (2 × 150 nm), and the presence or absence of sialic acid binding specificity has not been clarified. Furthermore, the purification method is complicated (after concentration of the culture supernatant, density gradient centrifugation, ion exchange chromatography, gel filtration chromatography, affinity chromatography for removing endotoxin, and high performance liquid chromatography twice). Perform) and the recovery is low (0.68%).

On the other hand, hemagglutinin having sialic acid binding ability reported by Sakurai et al. Was purified by affinity chromatography (using bovine submaxillary mucin as a ligand), but the affinity capacity of the affinity gel was small. (The peak absorbance at 280 nm of the eluate when a column having a bed volume of 2 ml is used is about 0.03.
5) Not only the purification efficiency is poor, but also the purity is low (6,15
3 hemagglutination units / mg protein). The shape and properties of the purified sample are unknown.

[0007]

That is, there has been a strong demand for an antigen which is remarkably effective as an antigen for a biological product such as a vaccine or a diagnostic agent for a bronchiseptic infection.

Accordingly, an object of the present invention is to provide a sialic acid-specific hemagglutinin which is excellent in protection against infection against bronchiseptic bacillus and is extremely effective as a vaccine or diagnostic agent for bronchiseptic bacillus infection. It is.

Another object of the present invention is to provide a vaccine against B. bronchiseptica infection, which has a strong protective effect against B. bronchiseptica. Another object of the present invention is to
An object of the present invention is to provide a diagnostic agent for bronchisepsis infection capable of accurately and easily diagnosing bronchisepsis infection.

[0010] Another object of the present invention is to provide a sialic acid-specific hemagglutinin derived from bronchisepsis for use in the production of biological products such as vaccines or diagnostic agents for bronchisepsis infection. It is an object of the present invention to establish a simple, efficient and high-purity purification method.

[0011]

The object of the present invention has been achieved by the following constitutions. (1) A sialic acid-specific hemagglutinin derived from B. bronchiseptica, which has a vesicle-like morphology having a diameter of 7 to 280 nm in an aqueous solution and is a non-pilus membrane-like structure. (2) A vaccine against bronchial sepsis infection, which comprises the sialic acid-specific hemagglutinin derived from bronchisepsis described in (1). (3) The vaccine against B. bronchiseptica infection according to (2), further comprising an adjuvant. (4) A diagnostic agent for bronchisepsis infection, comprising the sialic acid-specific hemagglutinin derived from bronchisepsis described in (1) as an antigen. (5) The sialic acid-specific hemagglutinin contained in the bronchiseptic bacterium culture supernatant is adsorbed on an insoluble support having the erythrocyte membrane component of cow or sheep immobilized thereon, and the adsorbed sialic acid-specific hemagglutinin is chaotropic. A method for purifying sialic acid-specific hemagglutinin derived from bronchiseptica, which is eluted with a buffer containing ions and having an ionic strength of 0.5 or more. (6) The above-mentioned (5), wherein the insoluble support is cyanogen bromide-activated agarose or cyanogen bromide-activated cellulose on which a red blood cell membrane component of cow or sheep is immobilized.
2. A method for purifying sialic acid-specific hemagglutinin derived from B. bronchiseptica according to item 1.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION The sialic acid-specific hemagglutinin of the present invention is derived from a bronchiseptic bacterium, has a vesicular form having a diameter of 7 to 280 nm in an aqueous solution, and has a non-pilus membrane. Like structure. In the present invention, the vesicular form refers to a vesicle among a plurality of hemagglutinins possessed by B. bronchiseptica. The morphology observed with a transmission electron microscope after negative staining is shown in FIG. 1 (for example, relatively large and relatively small ones are indicated by arrows). The sialic acid-specific hemagglutinin of the present invention is derived from a non-pilus membrane-like structure localized on the surface layer of bronchiseptic bacteria and is contained in the culture supernatant (see FIG. 1). 2: In order to show the localization site of the sialic acid-specific hemagglutinin of the present invention in the bronchiseptic bacterium, an ultrathin section of the bacterium was treated with an anti-sialic acid-specific hemagglutinin antibody and a gold-labeled secondary antibody. (The black spots of the arrows are the localized sites). The sialic acid-specific hemagglutinin of the present invention agglutinates red blood cells of cattle and sheep, but does not agglutinate red blood cells of goats, horses, pigs, dogs, rabbits, guinea pigs, chickens and geese.

In the present invention, the aqueous solution may contain salts and the like. However, it does not include chaotropic ions and surfactants as described below. Analysis of the sialic acid-specific hemagglutinin of the present invention by electrophoresis (SDS-PAGE) reveals that the sialic acid-specific hemagglutinin is composed of various types of protein molecules (lane 1 in FIG. 3). As the bronchial sepsis bacterium that can be used in the present invention, A19 strain, B-1 strain,
Bb-111 strain, H-16 strain and the like. Among them, the A19 strain is preferable in that a high hemagglutination value (HA value) can be obtained. Sources of these strains include the Association for Animal Biologicals and the like.

In the present invention, the vaccine containing the sialic acid-specific hemagglutinin has an excellent protective effect against B. bronchiseptica and is 10 times higher than that of the conventional inactivated vaccine for porcine Bordetella infection. It has the above immunization properties. The content of the sialic acid-specific hemagglutinin in the vaccine of the present invention in the vaccine was as follows: HA value: 3
2 to 512, protein content: 80 to 1280 µg, more preferably HA value: 128 to 256, and protein content: 160 to 640 µg. In the present invention, the method for producing a vaccine containing the sialic acid-specific hemagglutinin includes purifying the sialic acid-specific hemagglutinin,
If necessary, an additive is added, and an adjuvant or the like is added to the suspension or freeze-dried product.

The above additives include carbohydrates (sorbitol, lactose, mannitol, starch, sucrose, glucose, dextran, etc.), casein digest,
It is a buffer such as a phosphate buffer, and examples of the adjuvant include aluminum hydroxide, aluminum phosphate, aluminum oxide, oil emulsions, saponins, vitamins, etc., alone or in combination of two or more thereof.
It is preferable that the vaccine of the present invention contains the adjuvant as described above, since a stronger protective effect can be obtained. Examples of the amount of the adjuvant to be added include an oil emulsion of 20 to 50%, and an aluminum gel with an Al amount of 0.2 to 1%.

[0016] The vaccine of the present invention may be mixed with a vaccine against other infectious diseases to make a mixed multivalent vaccine.

The dose of the vaccine of the present invention to pigs is appropriately determined depending on the age, weight, administration method and the like of pigs.
A specific guideline is 0.5 to 2 ml / pig.

When the vaccine of the present invention is administered to pigs for immunization, the vaccines are administered through the nose and mouth of the pig or intramuscularly, subcutaneously,
This can be done by intraperitoneal injection. When the vaccine of the present invention is involved in respiratory tract infections such as rhinotracheitis, it is preferably administered via the nose. Thereby, immunity is locally imparted at an early stage in the mucosal epithelium of the nose, which is a natural infection route, and the onset by bronchial sepsis can be prevented.

The animals to which the vaccine of the present invention can be used include all animals that can be infected with B. bronchiseptica. Preferably used for so-called domestic animals.

[0020] The diagnostic agent of the present invention is a diagnostic agent for bronchisepsis infection comprising the sialic acid-specific hemagglutinin derived from the bronchiseptic bacterium of the present invention as an antigen. Examples of the form of the diagnostic agent include those in which sialic acid-specific hemagglutinin is adsorbed on an insoluble carrier, those in which sialic acid-specific hemagglutinin is immobilized in a well of a 96-well microplate, and the like.

As a specific method of using the diagnostic agent of the present invention, a method in which the sialic acid-specific hemagglutinin is adsorbed on an insoluble carrier such as polystyrene particles, bentonite particles, kaolin particles, and collodion particles is used as the diagnostic agent. A so-called rapid agglutination method which can be used for diagnosis by detecting the presence or absence of agglutination as a result of an antigen-antibody reaction by mixing the diagnostic agent with the serum of a subject animal is preferred. Here, the amount of sialic acid-specific hemagglutinin adsorbed on the carrier is 80 to
A 150 μg protein / ml carrier suspension is preferred.

In another embodiment, sialic acid-specific hemagglutinin is immobilized on the wells of a 96-well polystyrene microplate, and the antibody is detected by enzyme immunoassay using the serum of the subject animal as a subject. As a specific method using the diagnostic agent of the present invention, among these, the rapid aggregation method is preferable.

As a method for purifying the sialic acid-specific hemagglutinin derived from the bronchiseptic bacterium of the present invention, any method can be used as long as it can obtain the one having the above-mentioned form. The purification method of the present invention is preferable in that a simple and high-purity product can be obtained.

In the purification method of the present invention, the method for preparing the culture supernatant of bronchiseptic bacterium is not particularly limited, but shaking culture (500 rpm) in a predetermined liquid medium at 37 ° C. for 3 days. It is obtained by doing. As a medium for bronchial sepsis, a liquid medium such as brain heart infusion broth and heart infusion broth is suitable.

Examples of the insoluble support for immobilizing the erythrocyte membrane component of cattle or sheep used for adsorbing sialic acid-specific hemagglutinin include the following 1) and 2). 1) Use of an agarose-based support, a cellulose-based support, a cross-linked dextran-based support, a cross-linked polyacrylamide-based support, porous silica beads, or the like, activated by various methods or into which various spacers are introduced. it can. In the present invention, agarose and cellulose activated with cyanogen bromide in a conventional manner are preferred. Cyanogen bromide-activated microgranular cellulose is suitable for mass production because it has good operability and is inexpensive. In this case, the particle size of the finely divided cellulose is preferably 25 to 75 μm.
Here, the insoluble support on which the red blood cell membrane component of cattle or sheep is immobilized is obtained by lysing red blood cells of cattle or sheep, extracting the membrane component by ultrasonic treatment, and immersing the support in the liquid containing the membrane component. By attaching the membrane component to the support. The amount of the bovine or sheep erythrocyte membrane component bound to the support was 5 to 10 mg protein / ml agarose gel,
1 mg protein / ml cellulose is preferred.

2) Cattle or sheep erythrocytes fixed with glutaraldehyde, formalin or the like can be used. Examples of the insoluble support for adsorbing sialic acid-specific hemagglutinin include the insoluble support in the form of 1) above.
This is preferable because the operation is simple.

The buffer as an eluate for eluting the sialic acid-specific hemagglutinin adsorbed in the above 1) or 2) is as follows:
It contains at least chaotropic ions, has an ionic strength of 0.5 or more, and has a buffering action. Sources of the chaotropic ions include urea, guanidine hydrochloride, NaClO ₄ , and NaSCN. Of these, urea is preferred. The concentration of the chaotropic ion in the eluate is preferably 0.5 to 4M. The ionic strength is 0.5 or more, preferably 0.5 to 4.0. To adjust the ionic strength, sodium chloride, potassium chloride or the like is added. As the addition amount,
0.5-4M is preferred.

As the eluate, a solution having a buffer action is used.
The buffer is preferably an acidic buffer, and specifically, an acetate buffer, a phosphate buffer, a glycine / hydrochloric acid buffer, or the like can be used. The pH of the eluate is 3.0-10.
0 is preferable, 3.5 to 7.0 is more preferable, and 4.0 is preferable.
-5.0 is more preferable. The method for adsorbing sialic acid-specific hemagglutinin to the insoluble support having the red blood cell membrane component immobilized thereon, and the method for eluting the adsorbed sialic acid-specific hemagglutinin may be any method. A method is preferred in which the insoluble support is filled in a container such as a column, and the culture supernatant of B. bronchiseptica is refluxed on the column.

Preferred embodiments of the purification method of the present invention are described below. (A) Cattle or sheep red blood cells are lysed to prepare a red blood cell membrane suspension, and the membrane components are extracted by ultrasonication. (B) An affinity gel is prepared by immobilizing the erythrocyte membrane extract on cyanogen bromide-activated agarose gel or cellulose, and packed in a column. (C) The culture supernatant of B. bronchiseptica is added to the column, and the sialic acid-specific hemagglutinin contained in the culture supernatant is adsorbed on the affinity gel. (D) An acidic buffer containing urea and sodium chloride is added to the column to elute the sialic acid-specific hemagglutinin adsorbed on the affinity gel.

(1) Cattle or sheep erythrocytes are fixed with glutaraldehyde. (2) adding the culture supernatant of bronchiseptic bacilli to fixed erythrocytes,
Sialic acid-specific hemagglutinin contained in the culture supernatant is adsorbed to fixed bovine or sheep erythrocytes. (3) An acidic buffer containing urea and sodium chloride is added to the fixed erythrocytes to which the sialic acid-specific hemagglutinin has been adsorbed, and the sialic acid-specific hemagglutinin is eluted from the fixed cattle or sheep erythrocytes.

Hereinafter, the purification method of the present invention will be described in more detail. (A) Preparation method of bovine erythrocyte membrane extract 10 mM phosphate buffered saline (PBS, pH 7.1)
Erythrocytes (packed erythrocytes) washed 10m
1 with 400 mM of 5 mM phosphate buffer (PB, pH 8.0)
After lysing the obtained erythrocyte membrane with PB, the erythrocyte membrane is suspended in PB so that the total volume becomes 20 ml (red blood cell membrane suspension). The erythrocyte membrane suspension was subjected to ultrasonic treatment (20 kHz, 1 minute) to extract membrane components, and the centrifuged supernatant was subjected to 0.1 mL.
Dialysis against 1 M sodium bicarbonate-0.5 M sodium chloride solution (CB, pH 8.3) (red blood cell membrane extract).

(B) Preparation method of affinity gel Cyanogen bromide-activated agarose gel (manufactured by Pharmacia Biotech, CNBr-activated Sepharose 4B) washed with 1 mM hydrochloric acid was added to 20 ml of C
Wash with B and transfer to 20 ml of erythrocyte membrane extract. After shaking for 2 hours to immobilize the erythrocyte membrane extract (BSE) on the agarose gel, the gel is washed with 50 ml of CB. Gel 5
0 ml of a 1 M monoethanolamine-hydrochloric acid solution (pH 8.
0) and allowed to stand for 2 hours to block remaining active groups. Wash alternately 5 times with 50 ml each of 0.1 M acetic acid-0.5 M sodium chloride solution (pH 4.0) and CB (BSE-agarose).

(C) Method for Preparing Bronchiseptic Bacterial Culture Supernatant A bronchiseptic bacterium (strain A19, etc.) that produces sialic acid-specific hemagglutinin is added to a liquid medium such as Brainheart Infusion Broth at 37 ° C. for 3 days. After culturing with shaking, the culture supernatant is recovered by centrifugation.

(D) Affinity Chromatography The above BSE-agarose was packed in a column, and PBS (p
H8.0). Bronchial sepsis culture supernatant 5
After 0 ml is refluxed for 2 hours on the BSE-agarose column to adsorb sialic acid-specific hemagglutinin to BSE-agarose, the column is washed with PBS (pH 8.0) to remove non-adsorbed fractions. . 2M urea as eluate
3M sodium chloride-0.1M acetate buffer (pH 4.
The sialic acid-specific hemagglutinin adsorbed on BSE-agarose is eluted using 5). Fractions having a hemagglutination value (HA value) of 16 or more were collected, and PBS (pH 8.
Dialysis to 0) to remove urea. Here, the measurement of the HA value is based on a hemagglutination reaction using an eluate fraction dilution (25 μl) and an equal amount of glutaraldehyde-fixed bovine erythrocytes (0.5% suspension).

[0035]

EXAMPLES Hereinafter, the contents of the present invention will be described in detail using examples, but the contents of the present invention are not limited thereto. 1) Purification of the sialic acid-specific hemagglutinin of the present invention (1) Affinity chromatography According to the above-mentioned method, 50 ml of culture supernatant of bronchiseptic bacillus A19 strain having an HA value of 128 was applied to a bed volume of 10 ml.
Was added to the above BSE-agarose column, and the above 2M urea-3M sodium chloride-0.1M acetate buffer (pH
It was eluted with the eluate of 4.5). At that time, the sialic acid-specific hemagglutinin had an absorbance of 0.692 (A ₂₈₀ , FIG. 4).
(Line A in FIG. 4) was eluted as a peak showing an HA value of 1,024 (HA titer, line B in FIG. 4) (FIG. 4).
A sample obtained by collecting fractions having an HA value of 16 or more has a specific activity of 246,000 hemagglutinating units (HAunits) per mg of protein, a 620-fold purification degree, and a high recovery (34.2). %) (Table 1 below)
Purified sample 1).

[0036]

[Table 1]

(2) Sialic acid binding specificity of the purified sample In order to clarify the sialic acid binding specificity of the purified sample 1,
Bovine erythrocytes for neuraminidase ( Arthrobacter ureafacie
( derived from ns ) to remove sialic acid contained in erythrocyte surface sugar chains. Since untreated erythrocytes were strongly agglutinated by purified sample 1 but neuraminidase-treated erythrocytes were not agglutinated (Table 2 below), purified sample 1 could specifically bind to sialic acid-containing sugar chains. It became clear.

[0038]

[Table 2]

(3) Morphology of purified sialic acid-specific hemagglutinin It is clear from the lane 1 in FIG. 3 that the sialic acid-specific hemagglutinin of the purified sample 1 is composed of various types of protein molecules. is there. The lane 1 in FIG.
This is the result of DS-PAGE. Further, the purified sample 1 was negatively stained with uranium acetate as it was, and observed with a transmission electron microscope. The sialic acid-specific hemagglutinin of the purified sample 1 had a vesicular form with a diameter of 7 to 280 nm (arrow in FIG. 1). Further, in order to examine the localization of the sialic acid-specific hemagglutinin of the present invention in cells, an ultrathin section of bronchiseptic bacterium was used using an anti-sialic acid-specific hemagglutinin antibody and a gold-labeled secondary antibody. The result of immunogold staining and observation with a transmission electron microscope is shown in FIG. It was confirmed by the distribution of gold colloid particles that the sialic acid-specific hemagglutinin of the present invention was derived from a non-pilus membrane-like structure localized on the surface layer of bronchiseptic bacteria. . In addition, the research report about the hemagglutinin derived from the bronchiseptic bacterium which has the form of the said FIG. 1 has not been performed until now. Furthermore, it is an unreported substance as a bacterial cell constituent purified from bronchial sepsis and confirmed in morphology. The various sizes of the purified sialic acid-specific hemagglutinin are presumed to be due to the repeated association of unit particles formed free from the cells.

(4) Purification Method Using Cyanogen Bromide Activated Microgranular Cellulose In place of the above BSE-agarose column, microgranular cellulose (particle size: 25 to 75 μm) (Whatman Co., CC31) was used as a carrier. The gel was activated by the cyanogen bromide method to prepare an affinity gel, and a column having a bed volume of 4 ml was prepared. 40 ml of the culture supernatant of bronchiseptic bacillus A19 strain having an HA value of 64 was added to the column, and sialic acid-specific hemagglutinin was purified. At this time, 16 or more H
A sample obtained by collecting fractions having an A value was 1 m of protein.
It has a specific activity of 204,800 HA units per gram and a 1,300-fold purification degree, and has a high recovery rate (60%).
%)showed that. Also, N-hydroxysuccinimide-activated agarose (manufactured by Bio-Rad, Affi-Gel
10 gel). FIG. 5 shows a purification separation pattern by affinity gel prepared using the above-described cyanogen bromide-activated microgranulated cellulose and N-hydroxysuccinimide-activated agarose. It can be seen that the one using microgranular cellulose has a higher HA value for each fraction than the one using agarose, and is also excellent in specific activity, purification degree and recovery.

The A19 strain, B-1 strain, Bb-111 strain purified in the same manner as described above using the microgranulated cellulose described above.
SDS- of sialic acid-specific hemagglutinin derived from strain culture supernatant
FIG. 6 shows the PAGE pattern. Although there was a difference in the concentration of the protein band depending on the strain of origin, the pattern was consistent. The HA titer of the culture supernatant was highest for the A19 strain.

(5) Purification Method Using Fixed Bovine Erythrocytes Sialic acid-specific hemagglutinin contained in bronchisepsis culture supernatant was adsorbed to glutaraldehyde-fixed bovine erythrocytes. Specifically, culture supernatant 3 was added to 50 μl of fixed erythrocytes.
After adding 0 ml and stirring, the non-adsorbed fraction was removed by washing with PBS, and the sialic acid-specific hemagglutinin adsorbed on the fixed erythrocytes was eluted with the eluate used in the above (1) and purified. According to this method, the recovery rate is rather low (2
8.8%) and a highly purified (2,040-fold) sialic acid-specific hemagglutinin is obtained (purified sample 2 in Table 1 above). The constituent proteins of the purified sample show the same SDS-PAGE profile as the sample purified by BSE-agarose (lane 2 in FIG. 3).

2) Efficacy test using pigs inoculated with the above purified sialic acid-specific hemagglutinin As a test animal, a 5-week-old anti-bronchiseptica antibody-negative pig was used. The sialic acid-specific hemagglutinin (hereinafter also referred to as SSHA) used in the efficacy test was purified by the method using the affinity chromatography of the above (1), and had an HA value of 32 with an O / W adjuvant.
It diluted and adjusted so that it might be set to ~ 256, and the antigen solution for inoculation was created. The inoculation method is 2 ml of each antigen solution having the above HA value.
Was inoculated twice into the ear root muscle of a pig at two week intervals. The test schedule is shown in FIG. As a comparative example, S
Pigs inoculated only with O / W adjuvant without SHA were used. The following tests 1 to 3 were performed under the above conditions.

(Test 1) In order to determine the amount of bronchiseptic bacterium used for the attack, 10 ³ , 10 viable A19 strains were used.
Intranasal challenge was performed at ⁵ , 10 ⁷ pigs / pig. As a result, it was found that the minimum bacterial amount at which bronchial sepsis was continuously isolated from the nasal cavity from the first week after the challenge was 10 ⁵ cells / pig.

(Test 2) After inoculation with the SSHA of the present invention, intranasal challenge was performed with 10 ⁵ live bacteria of A19 strain / one pig as described above. The amount of inoculated SSHA to the test pig is the HA value, which is No33: 256 No34: 128 No35: 64 No36: 32 No37: 0 (inoculated only with O / W adjuvant). Anti-SSHA serum hemagglutination-inhibition (HI) antibody titers of pig Nos. 33 to 37 up to 4 weeks after the start of the test, ELI
SA antibody and agglutination (AGG) antibody titers were measured. The results are shown in Table 3 below. The anti-SSHA serum HI antibody titer is measured by adding an equal amount of 2-fold serially diluted swine serum and 8-unit adjusted SSHA, and reacting at 37 ° C. for 30 minutes. After completion of the reaction, an equal amount of glutaraldehyde-fixed bovine erythrocytes (0.5% suspension) was added to a mixture of the diluted serum and SSHA.
After reacting at 7 ° C for 2 hours, the mixture is allowed to stand at 4 ° C overnight, and the HI antibody titer is determined based on the presence or absence of aggregation suppression. The assay method for the ELISA antibody was as follows: 100 ng was added to each well of a 96-well microplate.
After adding protein SHAA, the mixture is allowed to stand at 4 ° C. overnight, and then blocked with 1% bovine serum albumin to prepare a reaction plate. A 100-fold diluted swine serum is added to the reaction plate, reacted at 37 ° C. for 1 hour, and washed with a washing solution containing Tween 80. An anti-porcine IgG peroxidase-labeled antibody is added, and the mixture is reacted at 37 ° C. for 1 hour and washed. After adding a chromogenic substrate solution and reacting at 25 ° C. for 15 minutes in a dark place, a stop solution (2N sulfuric acid) is added, the absorbance at 450 nm is measured, and an ELISA antibody is determined using the reference serum as a control. AGG antibody titer is measured using AR antigen "Kitaken" (manufactured by Kitasato Institute).

[0046]

[Table 3]

As shown in Table 3, pigs Nos. 33 to 36 inoculated with the SHA of the present invention were compared with the control pigs of No. 37.
, The anti-SSHA serum HI antibody titer, ELISA antibody and AGG antibody titers all increased, and reached a plateau three weeks after the start of the test.

Further, in pigs Nos. 33 to 37, the A19 strain was challenged 4 weeks after the start of the test, and the anti-SSHA serum HI antibody titer and the ELISA antibody up to the 9th week were measured in the same manner as described above. FIG. 8 shows the result. In pigs Nos. 33 and 35 inoculated with the SSHA of the present invention, the HI antibody titer and the ELISA antibody titer already reached a plateau by 4 weeks after the challenge, and no remarkable increase was observed even after the challenge.
Pig Nos. 34 and 36 also changed in the same manner. In pig No. 37, the HI antibody titer did not increase after the challenge.

Next, the results of isolation of bacteria (bronchial sepsis) from nasal swabs in swine Nos.
The autopsy results at week (9 weeks after the start of the test) are shown in Table 4 below. + Indicates that the bacterium was isolated, and-indicates that the bacterium was not isolated.

[0050]

[Table 4]

From the results in Table 4, in the pig No. 37 as a control, bronchial sepsis was continuously isolated from the first week after the challenge. In contrast, pig N inoculated with the SHA of the present invention
In o35 and 36, bronchiseptic bacilli were isolated from the first to fourth weeks after the challenge, but were not isolated at the fifth week.
In pig Nos. 33 and 34, bronchial sepsis was not isolated at all. No nasal turbinate lesions were observed in any of the test pigs. Therefore, it can be understood that the vaccine containing the SSHA of the present invention can impart sufficient immunity to pigs and has an excellent protective effect against infection against bronchial sepsis.

(Test 3) As described above, the SSH of the present invention
After inoculation the vaccine containing the A, A19 Kabuseikin 10 ¹⁰ / pig 1 head and Pasteurella multocida D-type (ATCC 129
(48 strains) The nasal cavity was challenged for 2 consecutive days with 10 ¹⁰ viable bacteria / one pig. The amount of inoculated SSHA into the test pigs is H
No. A: No. 143: 256 No. 144: 128 No. 147: 0 (only inoculated with O / W adjuvant) No. 148: 0 (only inoculated with O / W adjuvant).

After inoculating the test swine with the vaccine containing SSHA, the pigs were challenged as described above on the fourth week, and the anti-S
SHA serum HI antibody titer and ELISA antibody were measured as described above. FIG. 9 shows the result. SSHA of the present invention
In the pigs Nos. 143 and 144 inoculated with E. coli, the HI antibody titer and the ELISA antibody titer already reached the plateau four weeks after the start of the test in which the challenge was carried out, as in Test 2 above.
Only a slight increase in the LISA antibody value was observed. In contrast, the control pigs 147 and 148 had anti-SSH
No increase in the serum HI antibody titer was observed, and only the ELISA antibody rapidly increased and reached a high value after the challenge. In addition, Table 5 and FIGS. 10 and 11 show the results of isolation of bacteria (Bb bacteria and Pm bacteria) from nasal swabs after the challenge and the results of autopsy at 5 weeks after the challenge (9 weeks after the start of the test). + Indicates that the bacterium was isolated, and-indicates that the bacterium was not isolated.

[0054]

[Table 5]

As shown in Table 5, the pigs No. 143 and 144 and the control pigs Nos. 147 and 148 vaccinated with the vaccine containing the SHA of the present invention all showed B at 5 weeks after the challenge.
B bacteria were isolated and Pm bacteria were not isolated. Further, as shown in FIG. 10, in the pig Nos. 143 and 144 inoculated with the vaccine containing the SHA of the present invention, mild atrophy was observed in the turbinates of the abdomen. As shown in FIG. 11, the control pig N
In o147, loss of the left nasal turbinate, right nasal turbinate, and extremely severe lesions of the nasal septum were observed. In pig No. 148, both abdominal turbinate atrophy and severe lesions of the nasal septum were observed. Therefore, a vaccine comprising the SHA of the present invention
It can be seen that sufficient immunity can be imparted to pigs and that they have an excellent effect of suppressing the formation of turbinate atrophy lesions.

Diagnostic agent containing SSHA of the present invention (rapid aggregation method) Latex particles (material: polystyrene, particle size: 0.9 μm)
m, SDL59 manufactured by Takeda Pharmaceutical Co., Ltd.) obtained by a purification method using microgranular cellulose (HA value;
512-1024) was adsorbed using 50 mM glycine / sodium hydroxide buffer (pH 8.2), centrifuged, the supernatant was discarded, and 0.5 to 0% in a phosphate buffer containing 0.5% bovine serum albumin. The particles were suspended at 0.6% to coat the particles. The SSHA-conjugated latex particles (Lx) were reacted with the serum of pigs infected with Bb bacteria for 5 minutes on a glass plate, and the Lx agglutination titer of the serum was determined based on the presence or absence of Lx aggregation by the diluted serum. At the same time, using the same pig serum, the anti-SSHA serum HI antibody titer was measured in the same manner as described above. Lx agglutination value and HI
FIG. 12 shows the relationship with the antibody titer. There is a high correlation between the Lx agglutination titer and the HI antibody titer (r = 0.9421, n = 9
4) was obtained. Therefore, it can be seen that the diagnostic agent of the present invention is a highly accurate diagnostic agent capable of measuring an antibody in a short time. Incidentally, the ELISA method and the HI antibody measurement method each require half a day and two days.

The test pig was vaccinated with the A vaccine: SSHA (HA value 1).
28) + O / W adjuvant, B vaccine: formalin-inactivated Bb cells (pre-inactivated viable cell count of 10 ¹⁰ cells / ml or more) +
Two types of aluminum hydroxide gel adjuvant were inoculated twice at two week intervals, and control pigs were similarly inoculated only with O / W adjuvant. Thereafter, an attack was performed in the same manner as in the above (Test 2). Serum from pigs inoculated only with each of the vaccine and adjuvant was collected up to 9 weeks after inoculation, and the Lx agglutination titer was measured as described above. The result is shown in FIG. As shown in FIG. 13, the Lx agglutination value of the pigs vaccinated with the vaccine A of the present invention reached a plateau 3 to 4 weeks after the inoculation, as in the above-mentioned results. Therefore, it is understood that the diagnostic agent of the present invention has excellent accuracy. In addition, the Lx agglutination titer increased in the control pigs after the challenge, and the infectious antibodies were well detected. As described above, the accuracy and sensitivity of the rapid agglutination method using Lx is higher than the HI antibody measurement method shown in Tests 2 and 3, and
It can be determined that this is equivalent to the LISA method. Also, ELIS
It is understood that the operation is simpler than the method A and can be applied as a diagnostic agent. Furthermore, the vaccine A of the present invention induces a remarkably high Lx agglutination titer as compared with the inactivated cell vaccine, indicating that it has excellent immunogenicity.

[0058]

The object of the present invention is to provide a sialic acid-specific hemagglutinin having an excellent protective activity against infection against B. bronchiseptica. In addition, a vaccine against bronchiseptic infection that can impart sufficient immunity to bronchisepsis infection and has excellent protective activity against infection, as well as accurate bronchisepsis infection,
Another object of the present invention is to provide a diagnostic agent for bronchisepsis infection which can be easily diagnosed. According to the purification method of the present invention, it is possible to produce a high-purity vaccine antigen free of various harmful substances derived from bronchiseptic bacterium, and thus highly safe for the living body. Further, since a large amount of antigen can be recovered by a single-stage purification operation, it is possible to greatly reduce the cost required for producing vaccines or diagnostic agents.

[Brief description of the drawings]

FIG. 1 is a view showing an electron micrograph of a sialic acid-specific hemagglutinin of the present invention.

FIG. 2 is an electron micrograph showing the localization site of the sialic acid-specific hemagglutinin of the present invention in bacterial cells.

FIG. 3 shows the electrophoresis of the sialic acid-specific hemagglutinin of the present invention.
It is a figure which shows a dynamic (SDS-PAGE) profile.

FIG. 4 shows the elution profile of sialic acid-specific hemagglutinin adsorbed on a BSE-agarose column.

FIG. 5 is a diagram showing an elution profile of sialic acid-specific hemagglutinin adsorbed on an affinity gel column prepared using agarose and cellulose as an insoluble support.

FIG. 6 shows the electrophoresis of the sialic acid-specific hemagglutinin of the present invention.
It is a figure which shows a dynamic (SDS-PAGE) profile.

FIG. 7 is a diagram showing a test schedule for an efficacy test using pigs inoculated with the sialic acid-specific hemagglutinin of the present invention.

FIG. 8 is a graph showing changes in anti-SSHA serum HI antibody titer and ELISA antibody in swine-inoculated pigs.

FIG. 9 is a graph showing changes in anti-SSHA serum HI antibody titers and ELISA antibodies in swine-inoculated pigs.

FIG. 10 is a photograph showing an autopsy record photograph of a swine-vaccinated pig (immune group).

FIG. 11 is a photograph showing an autopsy record of a swine non-inoculated pig (control group).

FIG. 12 is a diagram showing the relationship between Lx aggregation value and HI value.

FIG. 13 shows the Lx agglutination value of vaccinated pigs.

────────────────────────────────────────────────── ─── Continuing from the front page (56) References Proteins, nucleic acids, and enzymes Enzyme editorial edition, “Recent Ayumi of Bacterial Toxin Research,” October 25, 1978) Kyoritsu Shuppan Co., Ltd. 164-183 Abstracts of the 104th Annual Meeting of the Japanese Society of Veterinary Medicine August 1987 p. 158 ▲ V ▼ 88 Vaccine, Vol. 9, (Sep. 1991) p. 653-658 J.C. Vet. Med. Sci. , Vol. 54, No. 1, (1992) p. 37-42 Abstracts of the 110th Annual Meeting of the Japanese Society of Veterinary Medicine October 1990 (Heisei 2) Excerpt “Kyoto Micro-Institute” AR Component Vaccine Product Brochure Microbial Chemistry Laboratory Inc. (58) Fields surveyed (Int. Cl. ⁶⁾ , DB name) A61K 39/10 BIOTECHABS (STN) CA (STN) MEDLINE (STN)

Claims (6)

(57) [Claims] 1. A sialic acid-specific hemagglutinin derived from B. bronchiseptica, which has a vesicle-like morphology having a diameter of 7 to 280 nm in an aqueous solution and is a non-pilus membrane-like structure. 2. A vaccine against bronchial sepsis infection, which comprises the sialic acid-specific hemagglutinin derived from B. bronchiseptica according to claim 1. 3. The vaccine against B. bronchiseptica infection according to claim 2, further comprising an adjuvant. 4. A diagnostic agent for bronchisepsis infection, comprising the sialic acid-specific hemagglutinin derived from bronchisepsis according to claim 1 as an antigen. 5. A sialic acid-specific hemagglutinin which is contained in a bronchiseptic bacterium culture supernatant and is adsorbed to an insoluble support having a cow or sheep erythrocyte membrane component immobilized thereon. A sialic acid-specific hemagglutinin derived from bronchiseptic bacterium, wherein the sialic acid is eluted with a buffer containing chaotropic ions and having an ionic strength of 0.5 or more. 6. The bronchial sepsis according to claim 5, wherein the insoluble support is cyanogen bromide-activated agarose or cyanogen bromide-activated cellulose on which a red blood cell membrane component of bovine or sheep is immobilized. A method for purifying a sialic acid-specific hemagglutinin derived from a bacterium.