KR20080082883A - An attenuated bordetella bronchiseptica (bbs) strain and method for producing thereof, an immunogenic composition comprising the strain and a method of treating or preventing bbs infection from mammals using the same - Google Patents

Abstract

An attenuated Bordetella bronchiseptica(BBS) strain is provided to exhibit immunogenicity and show attenuated toxicity, thereby being be used as a live vaccine. An immunogenic composition comprising the same is provided to induce production of an antibody against BBS toxin in case of being administered into mammal. An attenuated BBS strain is characterized in that an aroA gene is deleted therefrom through homologous recombination and is deposited as a deposition no. KCCM-10840P. An immunogenic composition for treating or preventing BBS infection of mammal comprises the attenuated BBS strain and a pharmaceutically acceptable carrier or an adjuvant. Further, the aroA gene has SEQ ID : NO. 1.

Description

An attenuated Bordetella bronchiseptica (BBS) strain and method for attenuating Bordetella bronchiseptica (KS) strain and a method of manufacturing the same, an immunogenic composition comprising the same and a method for treating Mammalian infectious disease of mammals using the same producing particular, an immunogenic composition comprising the strain and a method of treating or preventing BBS infection from mammals using the same}

1 is a schematic diagram illustrating a process of preparing a DNA insert for homologous recombination by amplifying kanamycin resistance gene (kan ^R ) from pKD13 helper plasmid by PCR using primers each containing 40mers at both ends of the BBS aroA gene.

Figure 2 is a schematic diagram of the verification PCR to determine whether the aroA locus is substituted with kan ^R.

Figure 3 is a restriction enzyme Eco to ensure that the Kan ^R containing a picture (A) and aroA ends amplified by the PCR Kan ^R containing the aroA both ends was amplified by using the PCR insert in a cloning vector of pGEM-T easy Figure (B) treated with RI.

Figure 4 shows the results of performing PCR verification 1 and 2, the PCR product of the expected size was produced to the size of 0.85kb and 1.2kb, respectively.

5 is a graph showing the results of measuring the bacterial counts of BBS isolated after inoculation of the BBS mutant strains and outdoor strains of the present invention through the nasal cavity.

Figure 6 is a graph comparing the anti-BBS antibody production inducing ability of the mutant strain and the outdoor strain of the present invention.

7 is a result of measuring the BBS separated in BAL and lung tissue by inoculation with the outdoor strain after immunization with the mutant strain and PBS of the present invention.

Figure 8 is a graph showing that the immunization using the mutant strain of the present invention, the number of mouse somatic cells (immune cells) in the BAL was measured almost the same compared to the negative control (PBS).

The invention Bordetella chevron kisep urticae (Bordetella bronchiseptica : Hereinafter, the present invention relates to an attenuated BBS strain from which the aroA gene of BBS) has been removed, an immunogenic composition comprising the same, a method for treating or preventing a BBS infection in a mammal using the same, and a method for producing an attenuated BBS strain.

Atropic rhimitis (AR) is a chronic respiratory disease of pigs in most pig farms ( Bordetella). bronchiseptica ), which causes inflammation of the nasal mucosa, and as the disease progresses gradually, the nasal mucosa and maxillary atrophy. Infection is a respiratory disease that causes large economic losses for the hog industry, resulting in lower feed efficiency and weight gain.

The disease is especially caused by Pasteurella multocidae type D ( Pasteurella). multocida type D) Symptoms worsen when this secondary infection occurs. Atrophic rhinitis-infected pigs can release pathogens in their runny nose, causing infection by direct contact through the tip of their nose and pathogens attached to dust. Once propagated, it adheres strongly to the nasal mucosa and secretes toxins, damaging the nasal mucosa, causing necrosis of the tissues, and atrophy of the nasal turbinate, resulting in a crooked nose. Hygiene, poor ventilation, and retention of toxic gases (ammonia gas) in pigs can speed up the spread of atrophic rhinitis and worsen symptoms.

Initially, it begins with a light sneeze, gradually excreting mucus pus from the nose, swelling and redness of the eyes, tears, dust attached to the eyes, resulting in black spots. In severe cases, bleeding from the nose, wrinkles in the nose and the skin, and from 4 to 5 weeks of age, facial distortions and crooked nose. From this time, respiratory diseases such as pandemic and pleural pneumonia increase. Chronic aging pigs usually lose 20% feed efficiency and weight gain.

It can be visually observed through coughing, nasal secretions, and clinical manifestations of crooked nose.

The serological diagnostic method is not effective because the antibodies are formed in almost all pigs, but the causal agent can be easily separated by culturing the tip of the nose or around the nasal cavity with a sterile swab using a selective medium that selectively cultures only BBS.

The most prophylactic vaccine against this disease is toxins of P. multocida toxin and the BBS bactericidal mixture.Because the bacterium vaccine is a toxin produced by BBS, demonecrotoxin Because there will be no production of Bs, prevention of BBS by vaccinated vaccines is never expected.

Therefore, it may be a new alternative to remove a gene (for example, aroA gene), which plays an important role in the metabolism of the bacterium, to produce attenuated lively toxic strains for use in vaccines.

Korean Patent Laid-Open Publication No. 2005-55837 describes chitosan microparticles for vaccine delivery for enhancing immune activity against swine atrophic rhinitis, but is different from the attenuated strain from which the aroA gene of the present invention has been removed.

In addition, Korean Laid-Open Patent Publication No. 2005-103215 discloses a dog vaccine against BBS, which is different from the present invention because it is a vaccine composition using p68 antigen.

The inventors have surprisingly found that the attenuated strain of BBS from which the aroA gene has been removed retains immunogenicity without being pathogenic, thus completing the present invention.

It is an object of the present invention to provide an attenuated BBS strain from which the aroA gene of BBS has been removed and a method for producing the same.

Another object of the present invention is to provide an immunogenic composition for treating or preventing a BBS infection comprising the strain.

Another object of the present invention is to provide a method for treating or preventing BBS infection of a mammal by administering the immunogenic composition to a mammal.

In order to achieve the object of the present invention, the present invention provides an attenuated BBS strain from which the aroA gene of BBS has been removed. The attenuated BBS strain is preferably Accession No. KCCM-10840P.

aroA is a gene involved in important metabolic processes of BBS, a causative agent of atrophic rhinitis, a swine respiratory disease, and preferably has the sequence of SEQ ID NO: 1.

In the present invention, “attenuated” is one in which the toxic strain is modified in such a way that it becomes a less toxic strain or weaker pathogen than before the modification.

The attenuated BBS strain of the present invention was prepared as follows. PCR amplification and cloning of kanamycin resistance gene (Kan ^R ) sequence including N-terminal 40mer and C-terminal 40mer among aroA gene sites involved in important metabolic process of BBS, a pathogen of swine respiratory disease The aroA locus was removed using the homologous recombination insert.

More specifically,

a) BBS aroA gene deletion For production of non-pathogenic mutants, a kanamycin resistance gene (Kan ^R ) sequence comprising N-terminal 40mer and C-terminal 40mer among aroA gene sites is amplified by PCR (see FIG. 1). That is, primers are synthesized by adding the N-terminal 40mer of the aroA gene to the 5'-primer at both ends of the primer used to amplify Kan ^R from the helper vector pKD13 into which the Kan ^R gene is inserted, and vice versa. synthesized by adding a 3'-primer 40mer and then, when the Kan ^R amplified by PCR using the two primers having the Kan ^R gene aroA region of each respective 40mer on both ends are amplified (Fig. 1).

b) The PCR product is cloned into the cloning vector pGEM-T easy and digested with EcoRI to prepare a linear DNA insert for homologous recombination and the desired insert is extracted by gel elution.

c) Electrocompetent cells are prepared by transforming pKD46 plasmid into BBS field strain by electroporation. The pKD46 helper vector expresses λ Red recombinase, an enzyme that causes homologous recombination, and causes locus replacement by homologous recombination in cells.

d) In order to introduce the pKD46 plasmid to induce λ Red recombinase in transformed BBS, L-arabinose was incubated in a medium prepared to have a final concentration of 10 mM and prepared for transformation of linear DNA for homologous recombination. do.

e) The linear DNA is transformed into electrocompetent cells by electroporation and selected from medium to which kanamycin has been added.

f) Confirm knockout of the gene of interest in the genomic DNA of the transformant by PCR.

In order to achieve another object of the present invention, the present invention is for the treatment or prevention of mammalian BBS infection, comprising attenuated BBS strain from which the aroA gene of BBS has been removed and a pharmaceutically acceptable carrier or adjuvant It provides an immunogenic composition.

The attenuated BBS strain is as described above. Preferably, the BBS strain is Accession No. KCCM-10840P.

By "immunogenic" is meant the ability of the composition to elicit an immune response in a mammal against a particular pathogen. The immune response is a cellular immune response that is primarily mediated by cytotoxic T-cells and cytokine-producing T-cells, or humoral immunity that activates B-cells after being mediated primarily by helper (helper) T-cells to produce antibodies May be a reaction.

The immunogenic compositions of the invention can be administered in an effective amount. An “effective amount” means an amount required to alleviate or eliminate the condition to be treated or prevented and may be appropriately selected by those skilled in the art. For example, the therapeutically or prophylactically effective amount is such that the attenuated BBS strain is 1 × ^{10 6} to 1 × ^{10 10} cfu / mL per day, preferably 1 × ^{10 7} to 1 × ^{10 9} cfu / mL per day, more preferably Can be administered in the range of 1 × 10 ⁷ to 1 × ^{10 8} cfu / mL per day.

The immunogenic composition of the present invention may further comprise a pharmaceutically acceptable carrier or adjuvant in addition to the attenuated BBS strain.

In the immunogenic composition of the present invention, the carrier is used as a meaning including a diluent, a buffer, a preservative, and the like. The carrier may include one or more components selected from the group consisting of excipients, disintegrants, binders and lubricants known in the art, but is not limited to these examples.

In addition, the adjuvant is a substance used to enhance the immunogenicity of antigens, and suitable adjuvant for use in the present invention include several adjuvant classes, such as mineral salts such as Alum, aluminum hydroxide, aluminum phosphate. And calcium phosphate; Surfactants and particulates such as nonionic block polymer surfactants (such as cholesterol), virosomes, saponins (such as Quil A, QS-21 and GPI-0100), proteosomes, immune stimulation Complexes, cocleates, quaternary amines (dimethyl dioctadecyl ammonium bromide (DDA)), abridine, vitamin A, vitamin E; Bacterial products such as RIBI Adjuvant System (Ribi Inc.), Mycobacterum phlei cell wall backbone (Detox®), Muramil Dipeptide (MDP) and Tripeptide (MTP), Monophos Poryl lipid A, Bacillus Calmete-Guerin, heat-labile E. coli enterotoxin, cholera toxin, trehalose dimicholate, CpG oligodeoxynucleotides; Cytokines and hormones such as interleukin (IL-1, IL-2, IL-6, IL-12, IL-15, IL-18), granulocyte colony stimulating factor, dehydroepiandrosterone, 1,25-dihydrate Roxy Vitamin D3; Polyanions such as dextran; Polyacrylics (eg, polymethylmethacrylate, Carbopol 934P); Carriers such as tetanus toxide, dipteria toxide, cholera toxin B subunit, mutant heat-labile enterotoxin (rmLT) of enterooxygenic Escherichia coli, heat shock protein; Oil-in-water emulsions such as AMPHIGEN® (Hydronics, USA); And water-in-oil emulsions, such as Freund's complete and incomplete adjuvants.

The immunogenic compositions of the invention may be in any form known in the art, for example, in the form of solutions and injections, but are not limited to these examples. In the case of solutions or injections, it may contain 10-40% propylene glycol if necessary, and an amount of sodium chloride sufficient to prevent hemolysis (eg about 1%). The solution or injection may include any diluent or buffer known in the art. In addition, the immunogenic composition of the present invention can be prepared immediately before use by lyophilizing the attenuated BBS strain in a container such as a vial and adding a carrier or adjuvant, saline, etc. required for injection before use. It may be.

In the immunogenic composition of the present invention, the mammal may be human, cow, pig, goat, dog, sheep and the like, but is not limited to these examples. Preferably, the mammal is a pig.

In the immunogenic composition of the present invention, the BBS infection may include any of the symptoms caused by BBS infection, for example, swine atrophy rhinitis.

The immunogenic compositions of the invention can be administered by any means of administration known in the art. For example, the administration can be administered directly to the subject intravenously, intramuscularly, orally, transdermal, mucosal, intranasal, intratracheal or subcutaneous. The administration may be to be administered systemically or locally.

The present invention also provides a method for treating or preventing BBS infection, comprising administering an immunogenic composition according to the invention to a mammal.

In the method of the present invention, the mammal is a non-human mammal, and may be preferably selected from cows, pigs, dogs, goats, and sheep, but is not limited to these examples. More preferably, the mammal is a pig.

In the method of the present invention, the BBS infection may include any of the symptoms caused by BBS infection, including, for example, swine atrophic rhinitis.

In the method of the present invention, the administration may be by any means of administration known in the art. For example, the administration may be administered directly to the subject intravenously, intramuscularly, orally, transdermal, mucosal, intranasal, intratracheal or subcutaneous. The administration may be to be administered systemically or locally.

In the method of the present invention, the composition of the present invention is administered in a therapeutically or prophylactically effective amount. The "therapeutically or prophylactically effective amount" may be appropriately selected by those skilled in the art in consideration of the severity of symptoms, sex, age and weight of the individual. Preferably, the therapeutically or prophylactically effective amount is such that the attenuated BBS strain is 1 × ^{10 6} to 1 × ^{10 10} cfu / mL per day, preferably 1 × ^{10 7} to 1 × ^{10 9} cfu / mL per day, more preferably May be administered in the range of 1 × 10 ⁷ to 1 × ^{10 8} cfu / mL per day, but is not limited to these examples.

In order to achieve another object of the present invention, the present invention provides a method for producing an attenuated BBS strain comprising the step of removing the aroA gene of BBS by homologous recombination.

Specifically, the present invention relates to a method for producing an attenuated strain by removing the aroA gene on the genome of BBS, which is one of the causes of mammalian respiratory disease. That is, the knock-out the aroA gene of the BBS and a selectable marker to the area as a technology to put to replace the kanamycin resistance gene (Kan ^R), and amplifies the Kan ^R containing 40 bp of the aroA gene at both ends by PCR transformed it to a BBS After conversion, attenuated strains were prepared by replacing Kan ^R at the aroA gene position by homologous recombination using λ Red recombinase.

In the method for producing a strain according to an embodiment of the present invention, the strain is accession number KCCM-10840P.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example

Example  1: BBS aroA  Gene removed Attenuated  Preparation of BBS Strains

(One) Disclosure strain  And plasmids

E. coli JM109 was purchased from Invitrogen (Carlsbad, CA, USA) and used, and the pGEM-T easy vector (Promega, Madison, Wis.) Was used for cloning. The red helper plasmid pKD46 (Genbank # AY048746) is a vector that produces red and gam proteins using the pBAD promoter, and when homologous recombination occurs when foreign DNA enters the cells. That is, it is a vector containing Red recombinase. Kan ^R , used as a selection marker, was cloned from the pKD13 helper vector (Genbank # AY048744). BBS standard strain was used by the National Veterinary Research and Quarantine Service.

(2) aroA  Kanamycin resistance gene comprising N- or C-terminal 40mer (Kan ^R ) Cloning

The configuration of the PCR primers is as follows. AroA-FRT-F (P1): 5 'containing a priming site of aroA N-terminal 40mer and pKD13 as a forward primer using a pKD13 helper vector as a template using the existing aroA gene information (GenBank database: AF182427) -ATG AGC GGA TTG GCA TAT CTC GAC CTG CCC GCG GCG CGC C-GT GTA GGC TGG AGC TGC TTC-3 '(SEQ ID NO: 2) was used and the reverse priming sites of aroA C-terminal 40mer and pKD13 were used as reverse primers. Including aroA-FRT-R (P2): 5'-TCA GTC CCG CGC GGC CAG CAG GCC CGC GTA CAC GTC GAA AAT TCC GGG GAT CCG TCG ACC-3 '(SEQ ID NO: 3) was used.

PCR conditions were carried out for 30 minutes at 94 ℃ for 5 minutes premodification, 94 ℃ / 30 s, 57 ℃ / 30 s, and 72 ℃ / 1 min, and the final extension was carried out for 72 ℃ / 5 min. PCR products were subjected to electrophoresis on 0.8% agarose gel. The transformed colonies were selected and subjected to enrichment and plasmid purification again, and then treated with Eco RI, a restriction enzyme, to confirm the insertion of inserts, and the entire construct was subjected to sequencing only to the identified constructs to reconfirm whether the desired base sequence was amplified. The 1.3 kb fraction treated with Eco RI was used as an insert for homologous recombination.

Kan ^R was amplified by PCR using pKD13 as a template using primers aroA-FRT-F and aroA-FRT-R. As shown in FIG. 3A, a 1.3kb PCR product was obtained, which was a cloning vector pGEM. After cloning in -T easy, the PCR product of 1.3 kb was cloned by treatment with restriction enzyme Eco RI (Fig. 3B).

(3) through homologous recombination aroA Gene locus  remove

5 ml of freshly prepared BBS broth was inoculated into 500 ml BHI broth (BBL ™, Becton, Dickinson and Company, Sparks, MD) and incubated until the OD value became 0.5. The bacteria were recovered by centrifugation for 15 minutes. It was concentrated to 2.5 ml of a cooled 10% glycerol solution to prepare competent cells for electroporation. PKD46 plasmid was transfected using Gene Pulser Xcell ™ electroporation system (Bio-Rad, Hercules, CA) to express λ Red recombinase in the cells, and then ampicillin and L-arabinose (final concentration 10 mM). It was added in 250ml SOB medium (20 g of bactotrypton per liter of distilled water, 5 g of Bakto yeast extract, prepared by sterilization with 0.5 g of NaCl) and incubated at 30 ° C. until the OD value became 0.5. Thereafter, the resultant was concentrated again with 2.5 ml of a cooled 10% glycerol solution to prepare electrocompetent cells for transforming the linear DNA for homologous recombination prepared earlier.

40 μl of electrocompetent cells were transformed by electroporation into 1 μl (100 ng) of insert DNA, and selected from LB medium to which 50 μg / ml kanamycin was added to select a bacterium from which aroA was removed.

Example  2: PCR  Verification

Colonies surviving in kanamycin selection medium acted as reporters to indicate that kan ^R was inserted, but once again, PCR was performed to verify by molecular biological methods. That is, PCR was performed as follows to determine whether kan ^R was substituted for the aroA locus (see FIG. 2). That is, in performing PCR using two kinds of primer sets, PCR verification number 1 (V1) is aro-FRT-F (P1) and kan-F primer (P4, 5'-ATGATTGAACAAGATGGATT-3 '(SEQ ID NO: 4). )), Validation 2 (V2) was performed using kan-R (P3, 5'-TCAGAAGAACTCGTCAAGAA-3 '(SEQ ID NO: 5)) and aroA-FRT-R (P2), respectively, of 0.85 kb and 1.2 kb, respectively. Amplification of the PCR product was performed.

As a result, it was confirmed that 0.85 kb of PCR product and 1.2 kb of PCR product were generated in verification 1, indicating that the aroA gene was successfully removed (FIG. 4).

The present inventors deposited the attenuated BBS strain obtained from the example as described above to the Korea Biotechnology Research Institute Gene Bank, an international depository institution under the Budapest Treaty, on February 8, 2007 (Accession No. KCCM-10840P).

Example  3: BBS mutationism of this invention Attenuation  Identification

To determine if the attenuated BBS mutant of the present invention was attenuated, the mutant and wild-type (W / T) of the present invention were co-cultured in BHI medium, followed by nasal cavity of 6-week-old BALB / c mice. 10 μl (1 × 10 ⁸ BBS cells / ml) of the culture medium were added thereto. Thereafter, mice were sacrificed at 1 day intervals to isolate BBS from bronchio-alveolar lavage (BAL) and lung tissue, and the number of bacteria was measured. As a result, the BBS mutant strains were all attenuated and attenuated within 1 day in the mouse nasal cavity, whereas the outdoor strains were released for 6 days after inoculation (FIG. 5).

Example  4: confirmation of antibody production induced by BBS mutant of the present invention

In order to determine whether the BBS mutant strain of the present invention induced the expression of antibodies to BBS similarly to the outdoor strain, 10 μl (1 × 10) of bacterial cultures were divided into three groups of 6-week-old BALB / c mice. ⁸ BBS cells / ml) were added, respectively, and the negative control group was administered with PBS (phosphate buffered saline). The experimental group was subjected to the second boosting (boosting) on the 14th and 28th day, one week after the final inoculation and blood was collected to prepare the serum. To detect anti-BBS IgG present in serum, anti-BBS cells treated with formalin as antigen and goat anti-mouse HRP conjugated antibody (Pierce, USA) as secondary antibody were anti- BBS antibodies were detected and reacted with 50 μl of substrate solution for color development. Substrate solution is 0.04% (w / v) o -phenylenediamine (Sigma) in phosphate-citrate buffer (pH 5.0, 24 mM citrate, 64 mM disodium hydrogen phosphate, 0.01% (v / v) hydrogen peroxide) , USA) was used. The color reaction was stopped by addition of 50 μl of 10% SDS solution and read at a wavelength of 405 nm with a Multiskan Ascent microplate reader (Thermo Labsysterms, USA). As a result, the mutant strain of the present invention induced the anti-BBS antibody similarly to the outdoor strain, it can be seen that even if the mutant strain production induction ability of the antibody is not changed (Fig. 6).

Example  5: Vaccine and Defense Experiments

The vaccine using the mutant strain of the present invention was tested to examine the survival rate (separation rate) and the induction of immune cells in the BAL after field challenge. That is, 10 μl (1 × 10 ⁸ BBS cells / ml) of bacterial cultures were added to the mouse nasal cavity, and the negative control group was administered with PBS. Both groups were boosted on the 14th and 28th day of inoculation and 3 days after the final inoculation to inoculate the same amount of outfielders to separate outfielders from BAL and lung tissue. As a result, in the group immunized with the mutant strain, separation of the outdoor strain was statistically significant (P <0.01) lower than that of the control group (FIG. 7).

On the other hand, in order to measure the number of mouse somatic cells (immune cells) in the BAL, in the same manner as the method described above, after measuring the cell number 3 days after the last challenge after inoculation using a hemochromometer (hematometer) as a result, Almost the same number of cells were measured in the immunized group (FIG. 8).

According to the attenuated BBS strain of the present invention, immunogenicity, but toxicity is weakened can be used as a live vaccine.

The immunogenic composition of the present invention can induce antibody production against BBS toxins when administered in mammals.

<110> INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY <120> An attenuated Bordetella bronchiseptica (BBS) strain and method          for producing particular, an immunogenic composition comprising the          strain and a method of treating or preventing BBS infection from          mammals using the same <160> 5 <170> KopatentIn 1.71 <210> 1 <211> 1329 <212> DNA <213> Bordetella bronchiseptica <400> 1 atgagcggat tggcatatct cgacctgccc gcggcgcgcc tggcgcgcgg cgaggtggcc 60 ctgccgggtt ccaagagcat ctccaacagg gtattgctgc tggccgcgct ggccgaaggc 120 agcaccgaaa tcacgggcct gctcgattcc gatgacaccc gcgtcatgct ggccgcgttg 180 cgccagctgg gcgtatcggt gggcgaggtg gccgatggcc gcgtgaccat cgaaggcgtg 240 gcgcgctttc cgaccgaaca ggccgagctg ttcctaggca acgccggcac cgcgttccgg 300 ccgctgaccg cggcgctggc gttgatgggc ggcgattacc gcctgtccgg cgtgccgcgc 360 atgcacgagc ggcccatcgg cgacctggtc gacgccttgc gccagttcgg cgccgggatc 420 gaatatctgg ggcaggcggg gtatccgccg ctgcgcatcg gcggcggcag cattcgcgtc 480 gacgggccgg tgcgggtgga gggctcggtg tccagccagt tcctgaccgc cttgctgatg 540 gccgcccccg tgctggctcg gcgcagcggc caggacatca ccatcgaggt ggtgggcgag 600 ctgatttcca aaccctatat cgagatcacg ctcaatctga tggcgcgttt tggcgtgtcg 660 gtgcggcgcg acggctggcg cgccttcacg atcgcgcgcg atgcggccta ccgcggcccg 720 ggccgcatgg cgatcgaggg cgatgcctcg acggcgtcgt acttcctggc cctgggcgcc 780 atcggcggcg ggccggtgcg cgtcaccggc gtgggcgagg acagcatcca gggcgacgtg 840 gcgttcgccg cgacgctggc ggcgatgggc gccgacgtgc gctatggccc gggctggatc 900 gagacgcgcg gcgtgcgggt ggccgagggc ggacgcctga aggcgttcga cgctgacttc 960 aacctgattc ccgacgccgc catgacggcc gcgacgctgg cgctgtacgc cgacggccca 1020 tgccgcctgc gcaacatcgg cagctggcgc gtcaaggaga ccgaccgcat ccacgccatg 1080 cacaccgagc tggagaagct gggggcgggc gtgcaaagcg gggcggactg gctggaggtg 1140 gcgccgccgg cgcccggcgg ctggcgcgac gcccacatcg gcacctggga cgaccaccgc 1200 atggccatgt gcttctcgct ggccgcgttc ggtccggctg cggtgcgcat cctggatccg 1260 ggttgcgtca gcaagacttt ccccgattat ttcgacgtgt acgcgggcct gctggccgcg 1320 cgggactga 1329 <210> 2 <211> 60 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 2 atgagcggat tggcatatct cgacctgccc gcggcgcgcc gtgtaggctg gagctgcttc 60                                                                           60 <210> 3 <211> 60 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 3 tcagtcccgc gcggccagca ggcccgcgta cacgtcgaaa attccgggga tccgtcgacc 60                                                                           60 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 4 atgattgaac aagatggatt 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 5 tcagaagaac tcgtcaagaa 20  

Claims (12)

Bordetella Bordetella Attenuated BBS strain from which aroA gene of bronchiseptica (BBS) was removed. The bronchiseptica strain according to claim 1, wherein the strain is Accession No. KCCM-10840P. According to claim 1, wherein the aroA gene is a Bronchiceptika strain, characterized in that having a nucleotide sequence of SEQ ID NO: 1. An immunogenic composition for treating or preventing BBS infection in a mammal, comprising the BBS attenuated strain of any one of claims 1 to 3 and a pharmaceutically acceptable carrier or adjuvant. The immunogenic composition of claim 4, wherein the mammal is a pig. The immunogenic composition according to claim 5, wherein the BBS infection is porcine atrophic rhinitis. A method of treating or preventing a BBS infection comprising administering to a mammal an immunogenic composition according to claim 4. 8. The method of claim 7, wherein said mammal is a non-human mammal. The method of claim 8, wherein the non-human mammal is a pig. 10. The method of claim 9, wherein the infection is porcine atrophic rhinitis. A method for producing an attenuated BBS strain comprising removing the aroA gene of BBS by homologous recombination. The method of claim 11, wherein the kanamycin resistance gene (Kan ^R ) sequence comprising 40 bp of both ends of the aroA gene of BBS, amplified by PCR, and transformed into BBS, homologous recombination using λ Red recombinase Method of producing an attenuated strain by substituting Kan ^R in the aroA gene position through.

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