KR20150086672A - Multivalent vaccine composition for preventing or treating infection by actinobacillus pleuropneumoniae, pasteurella multocida and porcine circovirus - Google Patents

Abstract

The present invention relates to a complex vaccine composition. The complex vaccine composition includes Actinobacollus pleuropueumoniae antigens of serotype-1, serotype-2, and serotype-5, Pasteurella multocida type-A antigens, and circovirus type-2. The multivalent vaccine composition is capable of simultaneously preventing the infection caused by Actinobacollus pleuropueumoniae, Pasteurella multocida type-A, and circovirus type-2.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a multivalent vaccine composition for preventing or treating infections of pigs by Actinobacillus flour pneumoniae, Pasteurella mutosidia, and a circovirus. [0002] MULTIVALENT VACCINE COMPOSITION FOR PREVENTING OR TREATING INFECTION BY ACTINOBACILLUS PLEUROPNEUMONIAE, PASTEURELLA MULTOCIDA AND PORCINE CIRCOVIRUS [

And to a multivalent vaccine composition for simultaneously preventing or treating infection by Actinobacillus flour pneumoniae, Pasteurella multocida A and Pseudorabies virus type 2.

According to the 2010 Survey of Disease Surveillance by the Swine Association, the highest incidence of pleural pneumonia (65%) among respiratory diseases has been reported for the past three years. Pleural pneumonia usually occurs in finishing pigs, causing acute mortality and delayed growth by chronic type. Up to now, 15 serotypes have been identified, but there is no cross - protective ability between serotypes. Currently, only 2 and 5 types Vaccines are being produced.

Korean Patent Application No. 1991-0006414, filed on Apr. 19, 1991, by Akzo Nobel, Inc., V. describes actinobacillus plurolonumonica subunit vaccine, and Chonbuk National University Industry & Korean Patent Application No. 2008-0138479 filed on March 31 presents a method for producing Apx IIA protein displayed on the surface of yeast cells and its use as a feed additive. Actinobacillus < RTI ID = 0.0 > pleuropneumoniae , APP) is a gram-negative bacterium, which is a highly contagious respiratory disease in weaned pigs and finishing pigs, and represents fibroplastic pleurisy and infarction pneumonia lesions. There are 15 species of serotypes to date. Depending on the serotype, the disease type varies from acupuncture to chronic type, and it spreads through the juice, secretions, direct contact with the pufferfish, exhibits high mortality and chronic atrophy, and the recovered money is very economically important It is a disease. APP is detected by alveolar macrophages of the alveoli and produces toxins such as Apx I, Apx II and Apx III according to the serotype and alveolar macrophages, endothelial cells, alveolar epithelial cells, alveolar walls, endothelial cells of capillaries It is known to exert toxicity on cells.

Korean Patent Application No. 2006-0135461, filed on December 27, 2006 by Chonnam National University, provides non-pathogenic attenuated strains of Pasteurella multocida and vaccines containing the same, and Sungkyunkwan University Industry & Korean Patent Application No. 2006-0134798, filed on December 27, 2005, describes a cholera vaccine using the recombinant outer membrane protein H of Pasteurella mutosidase strain. Pasteurella A type multocida type A, PMA) is a small egg - shaped bacterium that grows well in the blood medium and has a convex shape of the staphylococcus auricle and has a diameter of 1-2 ㎛. PMA is present in the upper respiratory mucosa of the health money. When the normal defensive function is weakened due to environmental deterioration, stress, other viral diseases, etc., PMA invades the bronchi and alveoli and induces a severe inflammatory condition. The direct contact of the infected pig oral or nasal secretion Or airborne. The most common symptom is bronchitis pneumonia, which causes bubbles in the trachea causing dyspnea. As the disease progresses, the bronchial exudate increases and thus the coughing is observed. The body temperature rises to 40-41 ° C, causing depression, anorexia and inflammatory exudates in the nose. In case of acute type, persistent dry cough and growth retardation are observed in 5 ~ 10 days after death.

Korean Patent Application No. 2008-0065488 filed on July 7, 2008 by the applicant of the present invention, Intervet International Inc., Korean Patent Application No. 2008-7007927, filed on April 1, 2008 by V., provides PCV-2 vaccine. Porcine circovirus type 2 (PCV2) is a single stranded circular DNA virus. It is a morphologically icosahedral symmetry virus. It has no coat and is about 15-17 nm in size. PCV2 shares approximately 80% sequence identity with porcine circovirus type 1 (PCV1). In contrast to PCV1, which is generally non-virulent, pigs infected with PCV2 usually exhibit symptoms referred to as PMWS (Post-weaning Multisystemic Wasting Syndrome). The PMWS is clinically characterized by wasting, pale skin, drowsiness, dyspnea, diarrhea and jaundice. In some infected pigs, a combination of all symptoms occurs, while in the other pigs only one or two of these symptoms are present. Microscopic lesions and gross lesions are seen in many tissues and organs at autopsy. Lymphatic organs are the most common lesion sites. The mortality of pigs infected with PCV2 can reach 80%. In addition to PMWS, PCV2 may also be used in the treatment of pseudorabies, porcine reproductive tract respiratory syndrome (PRRS), Glasser's disease, streptococcal meningitis, salmonellosis, postweaning colibacillosis, It may be accompanied by a number of other infections, including dietary hepatosis and suppurative bronchopneumonia.

Therefore, poultry pneumonia is caused by a complex causative agent, and since it is necessary to reduce the number of times of vaccination for efficient farm use, it is still required to develop a combined vaccine capable of protecting respiratory bacteria and viruses during the finishing period.

Accordingly, the present inventors have developed a vaccine composition of the present invention as a result of efforts to develop a multivalent vaccine having a protective effect against major pneumococci in pigs and exhibiting an enhanced vaccine efficacy.

Thus, it is an object of the present invention to provide a polyvalent vaccine composition having a protective ability against a major pneumococcal agent of a pig.

Therefore, the present invention provides a method for preventing infection by any one of the components of a polyvalent vaccine composition, which does not significantly interfere with the immunological action of the polyvalent vaccine composition, and its administration is inhibited by infection with porcine actinobacillus flourus mononitus, Which is capable of preventing and / or treating at the same time, an effective amount of a multivalent vaccine composition of the present invention.

The present invention also relates to a method of vaccinating pigs in need of prevention and / or treatment of pneumonia, including the administration of a polyvalent vaccine composition to pigs.

The present invention also relates to a method for producing a polynucleotide of the present invention which does not significantly interfere with the immunological action of any one component of the polyvalent vaccine composition, It is an object of the present invention to provide a multivalent vaccine composition for preventing and / or minimizing the severity of a disease caused by any one of infectious agents, or ameliorating symptoms of at least one of the diseases.

The present invention also relates to a method for preventing or treating diseases caused by one or more causative agents among infections caused by porcine actinobacillus flouromonas, pustularela multocida type A, and type 2 cervical virus by administering the polyvalent vaccine composition of the present invention to pigs Preventing, and / or minimizing the disease, or improving the symptoms of one or more of these diseases.

In one embodiment of the invention, a porcine actinobacillus floruni strain, comprising a porcine actinobacillus flour pneumoniae antigen, a Pasteurella multocida type A antigen, a cough virus type 2 antigen and a veterinarily acceptable adjuvant, There is provided a multivalent vaccine composition for providing simultaneous protection against infection by mononuclear cells, Pasteurella multocida type A, and type 2 cirrhosis virus. In one embodiment of the invention, Serotype 1 deposited with KACC91772P, Serotype 2 deposited with KACC91766P and Serotype 5 deposited with KACC91768P were each used in an amount of 2 x 10 ⁹ CFU. Another embodiment of the present invention further comprises Apx I, Apx II and Apx III, the major exotoxins of Actinobacillus fluorescens. The APP produces the so-called RTX toxin (RTX represents repetition within the toxin). The presence of these RTX toxins is a major pathogenic feature of APP, and all known RTX toxins exhibit some cytotoxic or cytolytic activity, the most important pathogenic factor being extracellular proteins, the Apx exotoxin.

Another embodiment of the present invention provides a polyvalent vaccine composition comprising 20 ㎍ each of recombinant rApx I (SEQ ID NO: 1), rApx II (SEQ ID NO: 2) and rApx III (SEQ ID NO: 3), respectively.

In one embodiment of the invention, the antigen deposited with KCTC12541BP as Pasteurella mutoside A form was used in an amount of 4 x 10 ⁹ CFU.

The porcine circovirus type 2 antigen may be ORF2, wherein ORF2 may comprise the nucleotide sequence shown in SEQ ID NO: 4. The CAKY98 deposited in another one embodiment, in a pig KCTC11150BP sseoko virus type 2 was used in an amount of 10 ^6.25 TCID _50.

Unless defined otherwise herein, the present invention can be performed by molecular techniques, microbiology, protein purification, protein engineering, and DNA sequencing and conventional techniques commonly used in the art of recombinant DNA within the skill of the art. These techniques are known to those skilled in the art and are described in many standardized textbooks and references.

Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Various scientific dictionaries describing the terms contained herein are well known and available in the art.

The singular forms used in this specification include plural objects unless the context clearly dictates otherwise. Also, unless otherwise indicated, nucleic acids are understood to be from left to right, 5 'to 3', respectively, and the amino acid sequence is understood to be from left to right carboxyl orientation.

The numerical ranges that may be used herein are interpreted to include numerical values defined in the above ranges. All maximum numerical limitations given throughout this specification include all lower numerical limitations, as the lower numerical limitations are explicitly stated. All minimum numerical limitations given throughout this specification include all higher numerical limitations as higher numerical limitations are explicitly stated. All numerical limitations given throughout this specification will include all better numerical ranges within the broader numerical ranges, as narrower numerical limitations are explicitly stated.

The titles set forth for purposes of illustration, including the names of the inventions herein, are to be understood as the reference of the specification in various aspects or in all aspects of the invention, and are not intended to limit the following embodiments.

The term " about "or" about "as used herein means a statistically significant range of a given value. The permissible variations encompassed by the terms " about "or" about " depend on the specific system under study and can be readily appreciated by those skilled in the art.

As used herein, the term "colony forming unit" or "CFU" refers to the number of organisms that can replicate in a given sample. This is based on the theory that colonies are derived from the replication of bacteria in pairs / clusters or single cells.

The term "veterinarily acceptable carrier" as used herein means a diluent, adjuvant, excipient, stabilizer, preservative or vehicle to be administered with the compound or composition. Such carriers may include sterile liquids such as water and oils, for example, balsilen, animal, plant or synthetic origin oils such as peanut oil, loam oil, mineral oil, jojoba oil and the like. Water or aqueous salt solutions, such as phosphate-buffered saline, Ringer's solution and aqueous dextrose and glycerol solutions, may also be used.

As used herein, the term "immunologically effective amount" refers to the amount of an immunostimulating agent, such as pig Actinobacillus flourneumoniae, parasuellulla multocida type A, and porcine actinobacillus flourneumoniae , Pasteurella multocida A type, and the type of cervicovirus type 2. The "amount of immunological effect" may vary depending on the species, breed, age, size and health status of the recipient animal. As used herein, an "immunologically effective amount" of living non-toxic pig Actinobacillus flourus mononitus, Pasteurella multocida type A and type 2 cirrhosis virus is defined as the amount of a suitable veterinarily acceptable adjuvant When used together, it enhances the immunity of the live pig Actinobacillus flour pneumoniae, Pasteurella multocidae A, and the type 2 cough virus 2, and thus enhances the immunity of pig actinobacillus flour pneumoniae, Refers to the amount of swine actinobacillus flourus monkey, pau stuurela mutoside type A and sukovirus type 2 sufficient to provide protective immunity against attack by type A and type 2 virus.

As used herein, the term "immunogenicity" means that live non-toxic pig Actinobacillus flourus monkey, pau stuurela mutoside type A, and type 2 circo virus can induce body fluids and / or cellular immune responses it means. The immune strain is also antigenic. An immunogenic composition refers to a composition capable of inducing body fluids and / or cellular immune responses when administered to an animal.

The term "immunogenic composition " as used herein relates to any pharmaceutical composition containing, for example, a microorganism, which can be used to induce an immune response in a mammal. The immune response may comprise a T cell response, a B cell response, or a reaction of both T cells and B cells. The composition may serve to sensitize the mammal by providing an antigen that binds to MHC molecules at the cell surface. In addition, antigen-specific T-lymphocytes or antibodies may be generated to enable protection of the immunized host. An "immunogenic composition" is a live attenuated composition comprising a whole microorganism or an immunogenic portion derived therefrom that induces a cell-mediated (T-cell) immune response or an antibody-mediated Or may contain the killed / inactivated vaccine, protect the animal from one or more symptoms associated with infection by the microorganism, or protect the animal from death due to infection by the microorganism.

The term "vaccine" or "vaccine composition" as used herein refers to a pharmaceutical composition comprising one or more immunogenic compositions that induce an immune response in an animal. The vaccine or vaccine composition may comprise one or more additional components that are capable of protecting the animal from infection-induced disease or possible death, and that enhance the immunological activity of the active ingredient. The vaccine or vaccine composition may comprise additional components typical of pharmaceutical compositions, for example, an adjuvant. The immunologically active component of the vaccine may be in its original form or may be an attenuated organism in a modified live vaccine or an inactivated organism in a suitable manner within a killed or inactivated vaccine or a fully live organism, Subunit vaccines comprising the source components, or genetically engineered, mutated or cloned vaccines prepared by methods known to those skilled in the art.

In the present invention, the term "veterinarily acceptable adjuvant" refers to a water-in-oil emulsion of aluminum hydroxide, saponin, dimethyldioctadecylammonium bromide (DDA), polyphosphazene or mineral oil, squalane, But are not limited to, aqueous emulsions. In one embodiment, Immulant-alpha was used as an adjuvant in an amount of 25%. The polyvalent vaccine composition of the present invention may further comprise a veterinarily acceptable diluent, isotonic agent, or stabilizer. The "veterinarily acceptable diluent" includes, but is not limited to, water, saline, dextrose, ethanol, or glycerol. The "veterinarily acceptable isotonic agent" includes, but is not limited to, sodium chloride, dextrose, mannitol, sorbitol and lactose. The "veterinarily acceptable stabilizer" may include, but is not limited to, albumin or an alkali salt of ethylenediaminetetraacetic acid.

The polyvalent vaccine composition according to the present invention can be administered intradermally, intravascularly or intravaginally. The systemic administration can be by intravenous, intravascular, intramuscular, intranasal, intraarterial, intraperitoneal, oral or intrathecal routes. In the case of topical administration, it may be administered directly or indirectly to the inside or nearby of the subcutaneous, intradermal, intradermal, intralobal, intrathecal, intrapulmonary or tissue to be treated (connective-, bone-, muscle-, nerve-, ≪ / RTI > Depending on the intended duration and effectiveness of the treatment, the compositions according to the invention may be administered in single or multiple doses, and intermittently, for example for days, weeks or months, at different doses on a daily basis.

The composition according to the present invention induces an immune response against porcine actinobacillus flourus monkey, paustulella mutosista type A, and type 2 cirrhosis virus 2 and prevents and / or prevents the severity of the disease caused by these causative agents Or ameliorate one or more symptoms associated with the disease. Thus, the multivalent vaccine composition of the present invention can minimize the number of immunizations required to provide protection against a number of pathogens, lower the administration cost, and increase the tolerance and application rate.

Figure 1 shows the composition of a combined vaccine comprising Actinobacillus flourus monkey, Pasteurella mirtusida type A and a co-virus type 2 antigen and a veterinarily acceptable adjuvant.
Figure 2 shows pleural pneumonia symptoms in the lungs (10 < ^{8 >} CFU / dose) inoculated according to Example 2. [

Hereinafter, various embodiments are provided to facilitate understanding of the present invention. The following examples are provided to facilitate understanding of the invention and are not intended to limit the scope of the invention.

Example  1: Preparation of vaccine

As shown in the following Table 1 and FIG. 1, a combination vaccine comprising a porcine actinobacillus flourus monkey antigen, a Pasteurella mutoside A type antigen, a circovirus type 2 antigen and a veterinarily acceptable adjuvant Three lots (T32APMX01, T32APMX02, T32APMX03) were prepared and used in this experiment.

Table 1. Composition of Compound Vaccine according to Example 1

Example  2: Evaluation of efficacy of vaccine

2.1. The mortality and mortality from the inoculation of pleural fluid pneumonia Lethal time  evaluation

Pleuropneumonia serotypes 1, 2, and 5 to step diluting the strain culture solution to confirm the pathogenic for each of 10 ^9, 10 ^8, 10 ^7, 10 ^6, 10 ^5, 10 ⁴ and 10 weeks of age finishing pigs and 2 rabbits in CFU / dose And the mortality and mortality time were observed. Subjects who did not die were euthanized after one week 's observation and autopsied and lung lesions were compared and observed. The results are shown in Table 2 below. Based on the pathogenicity confirmation data, the vaccine was also inoculated to the pigs vaccinated twice, and the protective effect against the attack inoculation was evaluated according to the following criteria (Table 3) and lung lesion score criteria.

Table 2. Mortality and mortality time according to serotype of pleural pneumonia attack inoculation

Table 3. Scores based on clinical symptoms

As can be seen in Table 2 and FIG. 2, the mortality rate decreased as the number of inoculation bacteria decreased. In the case of inoculation with 10 ⁸ CFU / dose or more, the mortality or mortality time was very short in acute within 18 hours and typical pleural pneumonia symptoms such as nasal bleeding and intracorporeal bacterial exudate appeared in the lung. On the other hand, when inoculated with 10 ⁴ to 10 ⁵ CFU / dose, survived for more than 142 hours (about 6 days), but respiratory symptoms such as depression, inhaled bubbles, intermittent vomiting and coarse breathing were observed.

2.2. Evaluation of clinical symptoms according to serotype 1 attack

The target animal efficacy test for serotype 1 was evaluated and the results are shown in Table 4 below. PBS treated group not vaccinated was used as a control group.

Table 4. Clinical symptom scores according to inoculation of pleural pneumonia type 1

^a Mean SD, n = 5; SPSS statistics ver.19 / one-way NOVA / SNK (student-newman-keuls), p <0.05.

As shown in Table 4, the vaccine group according to the present invention showed a statistically significant effect as compared with the control group.

Example  3: Stability test of compound vaccine

In order to confirm the stability of the combined vaccine prepared in Example 1, a combination vaccine of three kinds of lots was stored in a cold dark place at 2 to 8 ° C, and after the preparation, 3 months, 6 months and 9 months, Concentration test, aseptic test, preservative quantitative test, inactivation confirmation test, safety test, and potency test.

3.1. Characteristic test

The three-lot composite vaccine prepared according to Example 1 was tested for color, haziness, foreign body or odor according to General Test Method 1-11-20-01 of the National Test Standard for Veterinary Drug Biologics, Were examined for solubility, turbidity and color tone by gentle shaking. The results are shown in Table 5 below.

Table 5. Characteristics of Compound Vaccine according to Example 1

* MPM: Months Post Manufacture

** -: voice

As can be seen from Table 5 above, all three combined vaccines were a uniform aqueous suspension of yellowish white until 9 months, with some precipitation but shaking to an even liquid. Also, there was no foreign matter, no odor, the property of the contents was uniform, and no change was observed.

3.2. Hydrogen ion concentration test

The three-lot composite vaccine prepared according to Example 1 was measured for hydrogen ion concentration according to General Test Methods 1-11-20-03 of the National Test Standard for Veterinary Drug Biologics and the results are shown in Table 6 below .

Table 6. Hydrogen ion concentration test results of the combined vaccine according to Example 1

As can be seen from the above Table 6, all three combined vaccines satisfied the assay standard (pH 6.0 to 8.0) until 9 months.

3.3. Aseptic test

Three lots of the combined vaccine prepared according to Example 1 were tested in a nutrient agar (NA), a liquid nutrient medium (Nutrient) according to the General Tests 1-11-20-06 of the National Test Standard for Veterinary Drug Biologics broth, NB) and liquid thioglycollate medium (Thio). The cells were incubated at 22 ° C and 37 ° C for 7 days, and the presence or absence of the bacteria was observed. Respectively.

Table 7. Example  1 as a result of aseptic testing of a combined vaccine according to

* MPM: Months Post Manufacture

** -: No bacterial growth

NA: nutrient agar, NB: nutrient broth, Thio: fluid thioglycollate medium

As shown in Table 7 above, no growth of any bacteria was observed up to 9 months in all three combined vaccines.

3.4. Preservative Quantitative Test

Three lots of the combined vaccine prepared according to Example 1 were colorimetrically assayed for formalin content according to General Test Methods 1-11-20-09 of the National Test Standard for Veterinary Drug Biologics. 0.2 ml of Schiff reagent was added to 1 ml of each of three lots of the combined vaccine and the formalin standard solution, and the mixture was allowed to stand at room temperature for 30 minutes, and the absorbance was measured using a 520 nm spectrophotometer. The standard curve of the polynomial was prepared using the absorbance of the standard solution and the formalin concentration was calculated by correlation with the vaccine. The results are shown in Table 8 below.

Table 8. Preservative test results of the combined vaccine according to Example 1

As can be seen from the above Table 8, all three combined vaccines showed a formalin content of 0.3% or less up to 9 months.

3.5. Inactivation confirmation test

Three lots of the conjugate vaccine prepared according to Example 1 were stored in cold dark place at 2-8 ° C, and 2 ml of the vaccine was frozen and thawed for each lot immediately after the preparation, 3 months, 6 months, and 9 months Respectively. Then, the supernatant was centrifuged and dialyzed overnight at 4 ° C in phosphate buffered saline (PBS: pH 7.2 to 7.4) at 100 times or more of the vaccine. As a tissue culture cell, PK-15 cells (Chungnam University Univ., 1999) were proliferated in a flask (25 cm 2), and 1 ml of the dialyzed test material was inoculated and sensitized for 1 hour at 37 ° C. After 1 hour, a new cell-holding medium was added and cultured for 7 days. After the supernatant was removed, a fluorescent antibody method (FA) was used to confirm the growth of porcine circovirus. The results are shown in Table 9 below.

Table 9. Inactivation Confirmation Test Result of Compound Vaccine According to Example 1

As shown in Table 9 above, fluorescence due to circovirus proliferation was not observed in all three rounds of the combined vaccine until 9 months.

3.6. Safety test

Three lots of the combined vaccine prepared according to Example 1 were stored in cold dark place at 2-8 캜, and safety was confirmed in mice, guinea pigs and pigs immediately after the production, 3 months, 6 months and 9 months. Eight mice (males) weighing 15 to 20 g were prepared, and 1/4 mice (0.5 ml) were inoculated into 8 mouse abdominal cavities and observed for 7 days. The guinea pigs were divided into two groups: two guinea pigs (male) each weighing 300-350 g were inoculated into the subcutaneous and intraperitoneal cavities, one in two (2 ml), and the two were inoculated intradermally in 0.1 ml each for 7 days. The pigs did not inoculate PCV2-related vaccine in the past, and two of the pigs were inoculated intensively with two pigs aged 5 to 7 weeks (Doowon Farm, Jeongeup, Male). The hypersensitivity reaction was confirmed within 1 to 2 hours after the inoculation, and adverse effects such as pyogenic, necrotic, fever and skin lesions at the injection site were observed for 21 days. The results are shown in Table 10 below.

Table 10. Safety test of compound vaccine according to Example 1

* Number of abnormal animals / number of vaccinated animals

IP: Intraperitoneal, SC: Subcutaneous, ID: Intradermal, IM: Intramuscular

As can be seen from the above Table 10, 3 lots of conjugated vaccines were inoculated into mice, guinea pigs and pigs every 3 months after the production of the vaccine for 9 months, and all survived without any side effects.

4. Potency test

4.1 Pigs Circovirus  Type 2 antigen ( PCV2 )

Three lots of test vaccines were preserved in cold dark place at 2-8 ° C, and guinea pig titration test was performed on PCV2 immediately after the production, 3 months, 6 months and 9 months. Eight guinea pigs (male) weighing 300 ~ 350 g were weighed in each lot and subcutaneously injected with 1/2 of them. After 3 weeks, blood samples were collected with two vaccine-untreated control groups and subjected to sandwich indirect ELISA and IFA (immunofluorescence assay ) Antibody titers were measured. The results are shown in Table 11 below.

Table 11. ELISA and IFA titration test results for PCV2

MPM: Months post manufacture, OD: Optical Density

T / C: Test serum optical density / average of control

GMT ± SD: Geometric mean titer ± standard deviation

As shown in Table 11, the T / C value of the inoculated group OD mean (T) / control OD average (C) in the ELISA of the 3-lot test vaccine up to 9 months was not less than 2.0 as a result of the potency test on PCV2 . In addition, IFA antibody results were confirmed to be 128 times or more on average.

4.2 Pasteurella Mertoshida  A type PMA )

Three test lots of the vaccine were stored in cold dark place at 2-8 ° C, and the potency test of PMA was performed immediately after the production, 3 months, 6 months and 9 months later. Seventy healthy males (male) weighing 15 ~ 20g were selected for each lot, and the vaccine stock solution, 5 - fold and 25 - fold diluted solution were injected into 20 rats at intervals of 2 weeks , And 10 mice were inoculated with the vaccine without treatment. 10 days after the second inoculation, PMA 100 LD ₅₀ /0.2 ml was inoculated into peritoneal cavity and viability was observed for 10 days. The results are shown in Table 12 below.

Table 12. Potency test results for PMA

* Number of surviving animals / Number of test animals

As shown in Table 12, as a result of the potency test for PMA, the combined vaccines of three lots up to 9 months showed a survival rate of 80% for the undiluted solution, 50% for the 5-fold diluted solution group and 20% for the 25-fold diluted solution group .

4.3 Actino Bacillus Flor Newmani  ( APP )

4.3.1 APP  For serotype 1, 2 and 5 Potency test

Six male (2 kg) healthy rabbits not infected with APP were assigned to each of four rabbits, and one dose of the vaccine was inoculated into the muscle twice at intervals of two weeks. Two mice were treated without vaccination and treated as a control. Two weeks after the second inoculation, blood samples were collected from each of the APP type 1, 2 and 5 sera according to the National Test for Veterinary Medicines (Ministry of Agriculture, Forestry and Livestock Quarantine Administration, No. 2013-39).

Antigens for APP serotype 1, 2, and 5 were diluted to 3 μg / ml in coating buffer and dispensed into ELISA plates in a volume of 100 μl. The cells were adsorbed at 37 ° C. for 30 minutes and then left at 4 ° C. overnight . Antigen-adsorbed ELISA plates were incubated at room temperature for 30 minutes, then washed three times with PBS, and 100 ㎕ of blocking buffer was added to all wells followed by incubation at 37 캜 for 1 hour. After washing three times with washing buffer, 100 μl of PBS was dispensed into each plate well. A well was added to blank well, 100 μl of negative serum and positive serum were added to the first well of B and C And the sample serum was diluted 2-fold to 1,024 times from the D-well in the same manner. The ELISA plate was agitated at 125 rpm / 1 to 3 minutes and then placed at 37 DEG C for 1 hour. Then, 100 l of the anti-rabbit IgG HRP conjugate, which was washed with washing buffer three times and appropriately diluted with blocking buffer, was dispensed at 37 ° C for 1 hour and then washed three times with washing buffer. 100 [mu] l OPD substrate was dispensed into each well and allowed to stand at room temperature for 15 minutes before stopping the reaction with 50 [mu] l of final solution and measuring the absorbance (OD) of each well at 492 nm. The results are shown in Tables 13 to 15 below.

Table 13. Rabbit ELISA potency test results for APP serotype 1

MPM: Months post manufacture, GMT ± SD: Geometric mean ± standard deviation

Table 14. Rabbit ELISA potency test results for APP serotype 2

Table 15. Rabbit ELISA potency test results for APP serotype 5

As shown in Tables 13 to 15 above, the ELISA antibody titers for APP serotypes 1, 2 and 5 were 320-fold or more in all three-dose combination vaccines up to 9 months, respectively. On the other hand, it was confirmed to be less than 10 times in the control group.

4.3.2 rApx  I, rApx II  And rApx III For Potency test

Six healthy 2 kg rabbits (males) not infected with APP were assigned to each of four rabbits, and one dose of the vaccine was inoculated twice to the muscle at intervals of two weeks, and two mice were used as a control. Two weeks after the second inoculation, blood samples were collected and the rApx I, II, and III were purified and coated. The antibody titers to the toxin from the vaccinated rabbits were determined according to the National Test for Animal Products No. 2013-39). Antigens for rApx I, II, and III were each diluted to 1 μg / ml in coating buffer and dispensed into ELISA plates in 100 μl aliquots, adsorbed at 37 ° C for 30 minutes, and then placed at 4 ° C overnight. The antigen-adsorbed ELISA plate was allowed to stand at room temperature for 30 minutes, and then washed three times with PBS. Then, 100 μl of blocking buffer was added to all wells, followed by incubation at 37 ° C for 1 hour. Then, 100 μl of PBS was dispensed into each well of the plate, and then 100 μl of negative serum and positive serum were added to the first well of the A-well (Blankwell), B and C, And the sample serum was diluted 2-fold to 1,024 times from the D-well in the same manner. The ELISA plate was agitated at 125 rpm / 1 to 3 minutes and then placed at 37 DEG C for 1 hour. Then, 100 μl of the anti-rabbit IgG HRP conjugate diluted with blocking buffer was washed three times with washing buffer, and then placed at 37 ° C for 1 hour. Washed three times with washing buffer, and 100 [mu] l OPD substrate was dispensed into each well and allowed to stand at room temperature for 15 minutes. The reaction was stopped with 50 [mu] l of final solution and the absorbance (OD) of each well was measured at 492 nm. The results are shown in Tables 16 to 18 below.

Table 16. Rabbit ELISA potency test results for rApx I

MPM: Months post manufacture, GMT ± SD: geometric mean potency volume ± standard deviation

Table 17. Rabbit ELISA potency test results for rApx II

Table 18. Rabbit ELISA potency test results for rApx III

As shown in Tables 16-18 above, the ELISA antibody titers for purified toxins were> 320-fold in all three-lot vaccines up to nine months. On the other hand, it was confirmed to be less than 10 times in the control group.

Agricultural Biotechnology Research Institute KACC91772 20121214 Agricultural Biotechnology Research Institute KACC91766 20121212 Agricultural Biotechnology Research Institute KACC91768 20121212 Korea Biotechnology Research Institute KCTC12541 20140109 Korea Biotechnology Research Institute KCTC11150 20070110

<110> KOREA ANIMAL AND PLANT QUARANTINE AGENCY <120> MULTIVALENT VACCINE COMPOSITION FOR PREVENTING OR TREATING          INFECTION BY ACTINOBACILLUS PLEUROPNEUMONIAE, PASTEURELLA          MULTOCIDA AND PORCINE CIRCOVIRUS <130> IPDB-51932 <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 1023 <212> PRT <213> Artificial Sequence <220> <223> recombinant rApx I <400> 1 Met Ala Asn Ser Gln Leu Asp Arg Val Lys Gly Leu Ile Asp Ser Leu   1 5 10 15 Asn Gln His Thr Lys Ser Ala Ala Lys Ser Gly Ala Gly Ala Leu Lys              20 25 30 Asn Gly Leu Gly Gln Val Lys Gln Ala Gly Gln Lys Leu Ile Leu Tyr          35 40 45 Ile Pro Lys Asp Tyr Gln Ala Ser Thr Gly Ser Ser Leu Asn Asp Leu      50 55 60 Val Lys Ala Ala Glu Ala Leu Gly Ile Glu Val His Arg Ser Glu Lys  65 70 75 80 Asn Gly Thr Ala Leu Ala Lys Glu Leu Phe Gly Thr Thr Glu Lys Leu                  85 90 95 Leu Gly Phe Ser Glu Arg Gly Ile Ala Leu Phe Ala Pro Gln Phe Asp             100 105 110 Lys Leu Leu Asn Lys Asn Gln Lys Leu Ser Lys Ser Leu Gly Gly Ser         115 120 125 Ser Glu Ala Leu Gly Gln Arg Leu Asn Lys Thr Gln Thr Ala Leu Ser     130 135 140 Ala Leu Gln Ser Phe Leu Gly Thr Ala Ile Ala Gly Met Asp Leu Asp 145 150 155 160 Ser Leu Leu Arg Arg Arg Asn Gly Glu Asp Val Ser Gly Ser Glu                 165 170 175 Leu Ala Lys Ala Gly Val Asp Leu Ala Ala Gln Leu Val Asp Asn Ile             180 185 190 Ala Ser Ala Thr Gly Thr Val Asp Ala Phe Ala Glu Gln Leu Gly Lys         195 200 205 Leu Ala Met Pro Tyr Leu Thr Leu Ala Leu Ser Gly Leu Ala Ser Lys     210 215 220 Leu Asn Asn Leu Pro Asp Leu Ser Leu Ala Gly Pro Gly Phe Asp Ala 225 230 235 240 Val Ser Gly Ile Leu Ser Val Ser Ser Ala Ser Phe Ile Leu Ser Asn                 245 250 255 Lys Asp Ala Asp Ala Gly Thr Lys Ala Ala Ala Gly Ile Glu Ile Ser             260 265 270 Thr Lys Ile Leu Gly Asn Ile Gly Lys Ala Val Ser Gln Tyr Ile Ile         275 280 285 Ala Gln Arg Val Ala Ala Gly Leu Ser Thr Ala Ala Thr Gly Gly     290 295 300 Leu Ile Gly Ser Val Val Ala Leu Ala Ile Ser Pro Leu Ser Phe Leu 305 310 315 320 Asn Val Ala Asp Lys Phe Glu Arg Ala Lys Gln Leu Glu Gln Tyr Ser                 325 330 335 Glu Arg Phe Lys Lys Phe Gly Tyr Glu Gly Asp Ser Leu Leu Ala Ser             340 345 350 Phe Tyr Arg Glu Thr Gly Ala Ile Glu Ala Ala Leu Thr Thr Ile Asn         355 360 365 Ser Val Aly Ala Ala Aly Aly Aly Aly Thr Gly     370 375 380 Ser Leu Val Gly Ala Le Val Ser Ala Ile Thr Gly 385 390 395 400 Ile Ile Ser Gly Ile Leu Asp Ala Ser Lys Gln Ala Ile Phe Glu Arg                 405 410 415 Val Ala Thr Lys Leu Ala Asn Lys Ile Asp Glu Trp Glu Lys Lys His             420 425 430 Gly Lys Asn Tyr Phe Glu Asn Gly Tyr Asp Ala Arg His Ser Ala Phe         435 440 445 Leu Glu Asp Thr Phe Glu Leu Leu Ser Gln Tyr Asn Lys Glu Tyr Ser     450 455 460 Val Glu Arg Val Val Ala Ile Thr Gln Gln Arg Trp Asp Val Asn Ile 465 470 475 480 Gly Glu Leu Ala Gly Ile Thr Arg Lys Gly Ala Asp Ala Lys Ser Gly                 485 490 495 Lys Ala Tyr Val Asp Phe Phe Glu Glu Gly Lys Leu Leu Glu Lys Asp             500 505 510 Pro Asp Arg Phe Asp Lys Lys Val Phe Asp Pro Leu Glu Gly Lys Ile         515 520 525 Asp Leu Ser Ser Ile Asn Lys Thr Thr Leu Leu Lys Phe Ile Thr Pro     530 535 540 Val Phe Thr Ala Gly Glu Glu Ile Arg Glu Arg Lys Gln Thr Gly Lys 545 550 555 560 Tyr Glu Tyr Met Thr Glu Leu Phe Val Lys Gly Lys Glu Lys Trp Val                 565 570 575 Val Thr Gly Val Glu Ser Asn Ale Ile Tyr Asp Tyr Thr Asn Leu             580 585 590 Ile Gln Leu Ala Ile Asp Lys Lys Gly Glu Lys Arg Gln Val Thr Ile         595 600 605 Glu Ser His Leu Gly Glu Lys Asn Asp Arg Ile Tyr Leu Ser Ser Gly     610 615 620 Ser Ser Ile Val Tyr Ala Gly Asn Gly His Asp Val Ala Tyr Tyr Asp 625 630 635 640 Lys Thr Asp Thr Gly Tyr Leu Thr Phe Asp Gly Gln Ser Ala Gln Lys                 645 650 655 Ala Gly Glu Tyr Ile Val Thr Lys Glu Leu Lys Ala Asp Val Lys Val             660 665 670 Leu Lys Glu Val Val Lys Thr Gln Asp Ile Ser Val Gly Lys Thr Cys         675 680 685 Ser Glu Lys Leu Glu Tyr Arg Asp Tyr Glu Leu Ser Pro Phe Glu Leu     690 695 700 Gly Asn Gly Ile Arg Ala Lys Asp Glu Leu His Ser Val Glu Glu Ile 705 710 715 720 Ile Gly Ser Asn Arg Lys Asp Lys Phe Phe Gly Ser Arg Phe Thr Asp                 725 730 735 Ile Phe His Gly Ala Lys Gly Asp Asp Glu Ile Tyr Gly Asn Asp Gly             740 745 750 His Asp Ile Leu Tyr Gly Asp Asp Gly Asn Asp Val Ile His Gly Gly         755 760 765 Asp Gly Asn Asp His Leu Val Gly Gly Asn Gly Asn Asp Arg Leu Ile     770 775 780 Gly Gly Lys Gly Asn Asn Phe Leu Asn Gly Gly Asp Gly Asp Asp Glu 785 790 795 800 Leu Gln Val Phe Glu Gly Gln Tyr Asn Val Leu Leu Gly Gly Ala Gly                 805 810 815 Asn Asp Ile Leu Tyr Gly Ser Asp Gly Thr Asn Leu Phe Asp Gly Gly             820 825 830 Val Gly Asn Asp Lys Ile Tyr Gly Gly Leu Gly Lys Asp Ile Tyr Arg         835 840 845 Tyr Ser Lys Glu Tyr Gly Arg His Ile Ile Glu Lys Gly Gly Asp     850 855 860 Asp Asp Thr Leu Leu Leu Ser Asp Leu Ser Phe Lys Asp Val Gly Phe 865 870 875 880 Ile Arg Ile Gly Asp Asp Leu Leu Val Asn Lys Arg Ile Gly Gly Thr                 885 890 895 Leu Tyr Tyr His Glu Asp Tyr Asn Gly Asn Ala Leu Thr Ile Lys Asp             900 905 910 Trp Phe Lys Glu Gly Lys Glu Gly Gln Asn Asn Lys Ile Glu Lys Ile         915 920 925 Val Asp Lys Asp Gly Ala Tyr Val Leu Ser Gln Tyr Leu Thr Glu Leu     930 935 940 Thr Ala Pro Gly Arg Gly Ile Asn Tyr Phe Asn Gly Leu Glu Glu Lys 945 950 955 960 Leu Tyr Tyr Gly Glu Gly Tyr Asn Ala Leu Pro Gln Leu Arg Lys Asp                 965 970 975 Ile Glu Gln Ile Ser Ser Thr Gly Ala Leu Thr Gly Glu His Gly             980 985 990 Gln Val Leu Val Gly Ala Gly Gly Pro Leu Ala Tyr Ser Asn Ser Pro         995 1000 1005 Asn Ser Ile Pro Asn Ala Phe Ser Asn Tyr Leu Thr Gln Ser Ala    1010 1015 1020 <210> 2 <211> 956 <212> PRT <213> Artificial Sequence <220> <223> recombinant rApx II <400> 2 Met Ser Lys Ile Thr Leu Ser Ser Leu Lys Ser Ser Leu Gln Gln Gly   1 5 10 15 Leu Lys Asn Gly Lys Asn Lys Leu Asn Gln Ala Gly Thr Thr Leu Lys              20 25 30 Asn Gly Leu Thr Gln Thr Gly His Ser Leu Gln Asn Gly Ala Lys Lys          35 40 45 Leu Ile Leu Tyr Ile Pro Gln Gly Tyr Asp Ser Gly Gln Gly Asn Gly      50 55 60 Val Gln Asp Leu Val Lys Ala Asn Asp Leu Gly Ile Glu Val Trp  65 70 75 80 Arg Glu Glu Arg Ser Asn Leu Asp Ile Ala Lys Thr Ser Phe Asp Thr                  85 90 95 Thr Gln Lys Ile Leu Gly Phe Thr Asp Arg Gly Ile Val Leu Phe Ala             100 105 110 Pro Gln Leu Asp Asn Leu Leu Lys Lys Asn Pro Lys Ile Gly Asn Thr         115 120 125 Leu Gly Ser Ala Ser Ser Ser Ser Gln Asn Ile Gly Lys Ala Asn Thr     130 135 140 Val Leu Gly Gly Ile Gln Ser Ile Leu Gly Ser Val Leu Ser Gly Val 145 150 155 160 Asn Leu Asn Glu Leu Leu Gln Asn Lys Asp Pro Asn Gln Leu Glu Leu                 165 170 175 Ala Lys Ala Gly Leu Glu Leu Thr Asn Glu Leu Val Gly Asn Ile Ala             180 185 190 Ser Ser Val Gln Thr Val Asp Ala Phe Ala Glu Gln Ile Ser Lys Leu         195 200 205 Gly Ser His Leu Gln Asn Val Lys Gly Leu Gly Gly Leu Ser Asn Lys     210 215 220 Leu Gln Asn Leu Pro Asp Leu Gly Lys Ala Ser Leu Gly Leu Asp Ile 225 230 235 240 Ile Ser Gly Leu Leu Ser Gly Ala Ser Ala Gly Leu Ile Leu Ala Asp                 245 250 255 Lys Glu Ala Ser Thr Glu Lys Lys Ala Ala Ala Gly Val Glu Phe Ala             260 265 270 Asn Gln Ile Ile Gly Asn Val Thr Lys Ala Val Ser Ser Tyr Ile Leu         275 280 285 Ala Gln Arg Val Ala Ser Gly Leu Ser Ser Thr Gly Pro Ala     290 295 300 Leu Ile Ala Ser Thr Val Ala Leu Ala Val Ser Pro Leu Ser Phe Leu 305 310 315 320 Asn Val Ala Asp Lys Phe Lys Gln Ala Asp Leu Ile Lys Ser Tyr Ser                 325 330 335 Glu Arg Phe Gln Lys Leu Gly Tyr Asp Gly Asp Arg Leu Leu Ala Asp             340 345 350 Phe His Arg Glu Thr Gly Thr Ile Asp Ala Ser Val Thr Thr Ile Asn         355 360 365 Thr Ala Leu Ala Ala Ile Ser Gly     370 375 380 Ser Leu Val Gly Ala Pro Val Ala Leu Leu Val Ala Gly Val Thr Gly 385 390 395 400 Leu Ile Thr Thr Ile Leu Glu Tyr Ser Lys Gln Ala Met Phe Glu His                 405 410 415 Val Ala Asn Lys Val His Asp Arg Ile Val Glu Trp Glu Lys Lys His             420 425 430 Asn Lys Asn Tyr Phe Glu Gln Gly Tyr Asp Ser Arg His Leu Ala Asp         435 440 445 Leu Gln Asp Asn Met Lys Phe Leu Ile Asn Leu Asn Lys Glu Leu Gln     450 455 460 Ala Glu Arg Val Val Ala Ile Thr Gln Gln Arg Trp Asp Asn Gln Ile 465 470 475 480 Gly Asp Leu Ala Ala Ile Ser Arg Arg Thr Asp Lys Ile Ser Ser Gly                 485 490 495 Lys Ala Tyr Val Asp Ala Phe Glu Glu Gly Gln His Gln Ser Tyr Asp             500 505 510 Ser Ser Val Gln Leu Asp Asn Lys Asn Gly Ile Ile Asn Ile Ser Asn         515 520 525 Thr Asn Arg Lys Thr Gln Ser Val Leu Phe Arg Thr Pro Leu Leu Thr     530 535 540 Pro Gly Glu Glu Asn Arg Glu Arg Ile Gln Glu Gly Lys Asn Ser Tyr 545 550 555 560 Ile Thr Lys Leu His Ile Gln Arg Val Asp Ser Trp Thr Val Thr Asp                 565 570 575 Gly Asp Ala Ser Ser Ser Val Asp Phe Thr Asn Val Val Gln Arg Ile             580 585 590 Ala Val Lys Phe Asp Asp Ala Gly Asn Ile Ile Glu Ser Lys Asp Thr         595 600 605 Lys Ile Ile Ala Asn Leu Gly Ala Gly Asn Asp Asn Val Phe Val Gly     610 615 620 Ser Ser Thr Thr Val Ile Asp Gly Gly Asp Gly His Asp Arg Val His 625 630 635 640 Tyr Ser Arg Gly Glu Tyr Gly Ala Leu Val Ile Asp Ala Thr Ala Glu                 645 650 655 Thr Glu Lys Gly Ser Tyr Ser Val Lys Arg Tyr Val Gly Asp Ser Lys             660 665 670 Ala Leu His Glu Thr Ile Ala Thr His Gln Thr Asn Val Gly Asn Arg         675 680 685 Glu Glu Lys Ile Glu Tyr Arg Arg Glu Asp Asp Arg Phe His Thr Gly     690 695 700 Tyr Thr Val Thr Asp Ser Leu Lys Ser Val Glu Glu Ile Ile Gly Ser 705 710 715 720 Gln Phe Asn Asp Ile Phe Lys Gly Ser Gln Phe Asp Asp Val Phe His                 725 730 735 Gly Gly Asn Gly Val Asp Thr Ile Asp Gly Asn Asp Gly Asp Asp His             740 745 750 Leu Phe Gly Gly Asp Gly Asp Asp Val Ile Asp Gly Gly Asn Gly Asn         755 760 765 Asn Phe Leu Val Gly Gly Thr Gly Asn Asp Ile Ser Ser Gly Gly Lys     770 775 780 Asp Asn Asp Ile Tyr Val His Lys Thr Gly Asp Gly Asn Asp Ser Ile 785 790 795 800 Thr Asp Ser Gly Gly Gln Asp Lys Leu Ala Phe Ser Asp Val Asn Leu                 805 810 815 Lys Asp Leu Thr Phe Lys Lys Val Asp Ser Ser Leu Glu Ile Ile Asn             820 825 830 Gln Lys Gly Glu Lys Val Arg Ile Gly Asn Trp Phe Leu Glu Asp Asp         835 840 845 Leu Ala Ser Thr Val Ala Asn Tyr Lys Ala Thr Asn Asp Arg Lys Ile     850 855 860 Glu Glu Ile Ile Gly Lys Gly Gly Glu Glu Ile Thr Ser Glu Gln Val 865 870 875 880 Asp Lys Leu Ile Lys Glu Gly Asn Asn Gln Ile Ser Ala Glu Ala Leu                 885 890 895 Ser Lys Val Val Asn Asp Tyr Asn Thr Ser Lys Asp Arg Gln Asn Val             900 905 910 Ser Asn Ser Leu Ala Lys Leu Ile Ser Ser Val Gly Ser Phe Thr Ser         915 920 925 Ser Ser Asp Phe Arg Asn Asn Leu Gly Thr Tyr Val Ser Ser Ile     930 935 940 Asp Val Ser Asn Asn Ile Gln Leu Ala Arg Ala Ala 945 950 955 <210> 3 <211> 1052 <212> PRT <213> Artificial Sequence <220> <223> recombinant rApx III <400> 3 Met Ser Thr Trp Ser Ser Met Leu Ala Asp Leu Lys Lys Arg Ala Glu   1 5 10 15 Glu Ala Lys Arg Gln Ala Lys Lys Gly Tyr Asp Val Thr Lys Asn Gly              20 25 30 Leu Gln Tyr Gly Val Ser Gln Ala Lys Leu Gln Ala Leu Ala Ala Gly          35 40 45 Lys Ala Val Gln Lys Tyr Gly Asn Lys Leu Val Leu Val Ile Pro Lys      50 55 60 Glu Tyr Asp Gly Ser Val Gly Asn Gly Phe Phe Asp Leu Val Lys Ala  65 70 75 80 Ala Glu Glu Leu Gly Ile Gln Val Lys Tyr Val Asn Arg Asn Glu Leu                  85 90 95 Glu Val Ala His Lys Ser Leu Gly Thr Ala Asp Gln Phe Leu Gly Leu             100 105 110 Thr Glu Arg Gly Leu Thr Leu Phe Ala Pro Gln Leu Asp Gln Phe Leu         115 120 125 Gln Lys His Ser Lys Ile Ser Asn Val Val Gly Ser Ser Thr Gly Asp     130 135 140 Ala Val Ser Lys Leu Ala Lys Ser Gln Thr Ile Ile Ser Gly Ile Gln 145 150 155 160 Ser Val Leu Gly Thr Val Leu Ala Gly Ile Asn Leu Asn Glu Ala Ile                 165 170 175 Ile Ser Gly Gly Ser Glu Leu Glu Leu Ala Glu Ala Gly Val Ser Leu             180 185 190 Ala Ser Glu Leu Val Ser Asn Ile Ala Lys Gly Thr Thr Thr Ile Asp         195 200 205 Ala Phe Thr Thr Gln Ile Gln Asn Phe Gly Lys Leu Val Glu Asn Ala     210 215 220 Lys Gly Leu Gly Gly Val Gly Arg Gln Leu Gln Asn Ile Ser Gly Ser 225 230 235 240 Ala Leu Ser Lys Thr Gly Leu Gly Leu Asp Ile Ser Ser Leu Leu                 245 250 255 Ser Gly Val Thr Ala Ser Phe Ala Leu Ala Asn Lys Asn Ala Ser Thr             260 265 270 Ser Thr Lys Val Ala Gly Phe Glu Leu Ser Asn Gln Val Ile Gly         275 280 285 Gly Ile Thr Lys Ala Val Ser Ser Tyr Ile Leu Ala Gln Arg Leu Ala     290 295 300 Ala Gly Leu Ser Thr Thr Gly Pro Ala Ala Ala Leu Ile Ala Ser Ser 305 310 315 320 Ile Ser Leu Ala Ile Ser Pro Leu Ala Phe Leu Arg Val Ala Asp Asn                 325 330 335 Phe Asn Arg Ser Lys Glu Ile Gly Glu Phe Ala Glu Arg Phe Lys Lys             340 345 350 Leu Gly Tyr Asp Gly Asp Lys Leu Leu Ser Glu Phe Tyr His Glu Ala         355 360 365 Gly Thr Ile Asp Ala Ser Ile Thr Thr Ile Ser Thr Ala Leu Ser Ala     370 375 380 Ile Ala Gla Aly Ala Gla Ala Gla Ala Gla Ala 385 390 395 400 Pro Ile Thr Leu Leu Val Thr Gly Ile Thr Gly Leu Ile Ser Gly Ile                 405 410 415 Leu Glu Phe Ser Lys Gln Pro Met Leu Asp His Val Ala Ser Lys Ile             420 425 430 Gly Asn Lys Ile Asp Glu Trp Glu Lys Lys Tyr Gly Lys Asn Tyr Phe         435 440 445 Glu Asn Gly Tyr Asp Ala Arg His Lys Ala Phe Leu Glu Asp Ser Phe     450 455 460 Ser Leu Leu Ser Ser Phe Asn Lys Gln Tyr Glu Thr Glu Arg Ala Val 465 470 475 480 Leu Ile Thr Gln Gln Arg Trp Asp Glu Tyr Ile Gly Glu Leu Ala Gly                 485 490 495 Ile Thr Gly Lys Gly Asp Lys Leu Ser Ser Gly Lys Ala Tyr Val Asp             500 505 510 Tyr Phe Gln Glu Gly Lys Leu Leu Glu Lys Lys Pro Asp Asp Phe Ser         515 520 525 Lys Val Val Phe Asp Pro Thr Lys Gly Glu Ile Asp Ile Ser Asn Ser     530 535 540 Gln Thr Ser Thr Leu Leu Lys Phe Val Thr Pro Leu Leu Thr Pro Gly 545 550 555 560 Thr Glu Ser Arg Glu Arg Thr Gln Thr Gly Lys Tyr Glu Tyr Ile Thr                 565 570 575 Lys Leu Val Val Lys Gly Lys Asp Lys Trp Val Val Asn Gly Val Lys             580 585 590 Asp Lys Gly Ala Val Tyr Asp Tyr Thr Asn Leu Ile Gln His Ala His         595 600 605 Ile Ser Ser Ser Ala Arg Gly Glu Glu Tyr Arg Glu Val Arg Leu     610 615 620 Val Ser His Leu Gly Asn Gly Asn Asp Lys Val Phe Leu Ala Ala Gly 625 630 635 640 Ser Ala Glu Ile His Ala Gly Glu Gly His Asp Val Val Tyr Tyr Asp                 645 650 655 Lys Thr Asp Thr Gly Leu Leu Val Ile Asp Gly Thr Lys Ala Thr Glu             660 665 670 Gln Gly Arg Tyr Ser Val Thr Arg Glu Leu Ser Gly Ala Thr Lys Ile         675 680 685 Leu Arg Glu Val Ile Lys Asn Gln Lys Ser Ala Val Gly Lys Arg Glu     690 695 700 Glu Thr Leu Glu Tyr Arg Asp Tyr Glu Leu Thr Gln Ser Gly Asn Ser 705 710 715 720 Asn Leu Lys Ala His Asp Glu Leu His Ser Val Glu Glu Ile Ile Gly                 725 730 735 Ser Asn Gln Arg Asp Glu Phe Lys Gly Ser Lys Phe Arg Asp Ile Phe             740 745 750 His Gly Ala Asp Gly Asp Asp Leu Leu Asn Gly Asn Asp Gly Asp Asp         755 760 765 Ile Leu Tyr Gly Asp Lys Gly Asn Asp Glu Leu Arg Gly Asp Asn Gly     770 775 780 Asn Asp Gln Leu Tyr Gly Gly Glu Gly Asn Asp Lys Leu Leu Gly Gly 785 790 795 800 Asn Gly Asn Asn Tyr Leu Ser Gly Gly Asp Gly Asn Asp Glu Leu Gln                 805 810 815 Val Leu Gly Asn Gly Phe Asn Val Leu Arg Gly Gly Lys Gly Asp Asp             820 825 830 Lys Leu Tyr Gly Ser Ser Gly Ser Asp Leu Leu Asp Gly Gly Glu Gly         835 840 845 Asn Asp Tyr Leu Glu Gly Asp Gly Ser Asp Phe Tyr Val Tyr Arg     850 855 860 Ser Thr Ser Gly Asn His Thr Ile Tyr Asp Gln Gly Lys Ser Ser Asp 865 870 875 880 Leu Asp Lys Leu Tyr Leu Ser Asp Phe Ser Phe Asp Arg Leu Leu Val                 885 890 895 Glu Lys Val Asp Asp Asn Leu Val Leu Arg Ser Ser Gn Ser Ser His             900 905 910 Asn Asn Gly Val Leu Thr Ile Lys Asp Trp Phe Lys Glu Gly Asn Lys         915 920 925 Tyr Asn His Lys Ile Glu Gln Ile Val Asp Lys Asn Gly Arg Lys Leu     930 935 940 Thr Ala Glu Asn Leu Gly Thr Tyr Phe Lys Asn Ala Pro Lys Ala Asp 945 950 955 960 Asn Leu Leu Asn Tyr Ala Thr Lys Glu Asp Gln Asn Glu Ser Asn Leu                 965 970 975 Ser Ser Leu Lys Thr Glu Leu Ser Lys Ile Ile Thr Asn Ala Gly Asn             980 985 990 Phe Gly Val Ala Lys Gln Gly Asn Thr Gly Ile Asn Thr Ala Ala Leu         995 1000 1005 Asn Asn Glu Val Asn Lys Ile Ile Ser Ser Ala Asn Thr Phe Ala Thr    1010 1015 1020 Ser Gln Leu Gly Gly Ser Gly Met Gly Thr Leu Pro Ser Thr Asn Val 1025 1030 1035 1040 Asn Ser Met Met Leu Gly Asn Leu Ala Arg Ala Ala                1045 1050 <210> 4 <211> 702 <212> PRT <213> Porcine circovirus type-2 ORF2 <400> 4 Ala Thr Gly Ala Cys Gly Thr Ala Thr Cys Cys Ala Ala Gly Gly Ala   1 5 10 15 Gly Gly Cys Gly Thr Thr Ala Cys Cys Gly Gly Ala Gly Ala Ala Gly              20 25 30 Ala Ala Gly Ala Cys Ala Cys Cys Gly Cys Cys Cys Cys Cys Cys Gly Cys          35 40 45 Ala Gly Cys Cys Ala Thr Cys Thr Thr Gly Gly Cys Cys Ala Gly Ala      50 55 60 Thr Cys Cys Thr Cys Cys Gly Cys Cys Gys Cys Cys Gly Cys Cys Cys  65 70 75 80 Cys Thr Gly Gly Cys Thr Cys Gly Thr Cys Cys Ala Cys Cys Cys Cys                  85 90 95 Cys Gly Cys Cys Cys Cys Cys Gly Thr Thr Ala Cys Cys Gly Cys Thr             100 105 110 Gly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Thr Gly Gly         115 120 125 Cys Ala Thr Cys Thr Thr Cys Ala Ala Cys Ala Cys Cys Cys Gly Cys     130 135 140 Cys Thr Ala Thr Cys Cys Cys Gly Cys Ala Cys Cys Thr Thr Cys Gly 145 150 155 160 Gly Ala Thr Ala Thr Ala Cys Thr Ala Thr Cys Ala Ala Gly Cys Gly                 165 170 175 Ala Ala Cys Cys Ala Cys Ala Cys Gly             180 185 190 Cys Cys Cys Thr Cys Cys Thr Gly Gly Gly Cys Gly Gly Thr Gly Gly         195 200 205 Ala Cys Ala Thr Gly Ala Thr Gly Ala Gly Ala Thr Thr Cys Ala Ala     210 215 220 Thr Ala Thr Thr Ala Ala Thr Gly Ala Cys Thr Thr Thr Cys Thr Thr 225 230 235 240 Cys Cys Cys Cys Cys Ala Gly Gly Ala Gly Gly Gly Gly Gly Cys Thr                 245 250 255 Cys Ala Ala Ala Cys Cys Cys Cys Cys Cys Gly Cys Thr Cys Thr Gly Thr             260 265 270 Gly Cys Cys Cys Thr Thr Thr Gly Ala Ala Thr Ala Cys Thr Ala Cys         275 280 285 Ala Gly Ala Ala Thr Ala Ala Gly Ala Ala Ala Gly Gly Thr Thr Ala     290 295 300 Ala Gly Gly Thr Cys Gly Ala Ala Thr Thr Cys Thr Gly Gly Cys Cys 305 310 315 320 Cys Thr Gly Cys Thr Cys Cys Cys Cys Cys Gly Ala Thr Cys Ala Cys Cys                 325 330 335 Cys Ala Gly Gly Gly Thr Gly Ala Cys Ala Gly Gly Gly Gly Ala Gly             340 345 350 Thr Gly Gly Gly Cys Thr Cys Cys Ala Gly Thr Gly Cys Thr Gly Thr         355 360 365 Thr Ala Thr Thr Cys Thr Ala Gly Ala Thr Gly Ala Thr Ala Ala Cys     370 375 380 Thr Thr Thr Gly Thr Ala Ala Cys Ala Ala Ala Gly Gly Cys Cys Ala 385 390 395 400 Cys Ala Gly Cys Cys Cys Thr Cys Ala Cys Cys Thr Ala Thr Gly Ala                 405 410 415 Cys Cys Cys Cys Thr Ala Thr Gly Thr Ala Ala Ala Cys Thr Ala Cys             420 425 430 Thr Cys Cys Thr Cys Cys Cys Cys Cys Cys Ala Thr Ala Cys Cys Ala         435 440 445 Thr Ala Ala Cys Cys Cys Ala Gly Cys Cys Cys Thr Thr Cys Thr Cys     450 455 460 Cys Thr Ala Cys Cys Ala Cys Thr Cys Cys Cys Gly Cys Thr Ala Cys 465 470 475 480 Thr Thr Ala Cys Cys Cys Cys Cys Cys Cys Cys Thr Gly                 485 490 495 Thr Cys Cys Thr Ala Gly Ala Thr Thr Cys Cys Ala Cys Thr Ala Thr             500 505 510 Thr Gly Ala Thr Thr Ala Cys Thr Thr Cys Cys Ala Ala Cys Cys Ala         515 520 525 Ala Ala Cys Ala Ala Cys Ays Ala Ala Ala Gly Ala Ala Cys Cys     530 535 540 Ala Gly Cys Thr Gly Thr Gly Gly Cys Thr Gly Ala Cys Thr 545 550 555 560 Ala Cys Ala Ala Ala Cyr Thr Gly Cys Thr Gly Aly Ala Ala Thr                 565 570 575 Gly Thr Ala Gly Aly Cys Cys Aly Cys Gly Thr Ala Gly Gly Cys Cys             580 585 590 Thr Cys Gly Gly Cys Ala Cys Thr Gly Cys Gly Thr Thr Cys Gly Ala         595 600 605 Ala Ala Ala Cys Ala Gly Thr Ala Thr Ala Thr Ala Cys Gly Ala Cys     610 615 620 Cys Ala Gly Aly Ala Thr Ala Cys Ala Ala Thr Ala Thr Cys Cys 625 630 635 640 Gly Thr Gly Thr Ala Ala Cyr Cys Ala Thr Gly Thr Ala Thr Gly Thr                 645 650 655 Ala Cys Ala Ala Thr Thr Cyr Ala Gly Ala Ala Ala Thr Thr Thr             660 665 670 Ala Ala Thr Cys Thr Thr Ala Ala Ala Gly Ala Cys Cys Cys Cys Cys         675 680 685 Cys Ala Cys Thr Thr Ala Ala Cys Cys Cys Thr Thr Ala Ala     690 695 700

Claims (5)

(Accession No .: KACC91772P) antigen of serotype 1, porcine Actinobacillus florus pneumoniae (accession number: KACC91766P) antigen of serotype 2, porcine Actinobacillus fluruni of serotype 5 (Accession number: KCTC11550PP) antigen, a vaginal epithelium A type (accession number: KCTC12541BP) antigen, a sukavirus type 2 (accession number: KCTC11150BP) antigen, and a veterinarily acceptable adjuvant.
A vaccine composition for providing simultaneous protection against infection by swine Actinobacillus flurus pneumoniae, Pasteurella mutosidia type A and type 2 cirrhosis virus. The method according to claim 1,
Wherein the vaccinia composition further comprises rApx I, rApx II, and rApx III as the actinobacillus fluorescinol antigen. 3. The method according to claim 1 or 2,
Wherein the veterinarily acceptable adjuvant is Immulant-alpha. &Lt; RTI ID = 0.0 &gt; 18. &lt; / RTI &gt; 3. The method according to claim 1 or 2,
1 × 10 ² to 1 × 10 ¹⁵ CFU antigen of Actinobacillus flourus monkey (accession number: KACC91772P) serotype 1, 1 × 10 ² to 1 × 10 ¹⁵ CFU of Actinobacillus fluruni (Accession No: KACC91766P) antigen and 1 x 10 ² to 1 x 10 ¹⁵ CFU of Actinobacillus flourus mononey (Accession No .: KACC91768P) antigen of serotype 5, 1 x 10 ² to 1 x 10 ¹⁵ CFU Pseudotuberculosis virus type 2 (accession number: KCTC12541BP) antigen, 1 x 10 ² to 1 x 10 ¹⁵ TCID ₅₀ (50% tissue culture infectious dose 50) : &Lt; / RTI &gt; KCTC11150BP) antigen, and 5% emulant-alpha. 5. The method of claim 4,
Characterized in that said actinobacillus fluorescinol antigens further comprise 10 to 50 [mu] g rApx I, rApx II and rApx III, respectively.

Images