Novel vector for producing recombinant antigen associated with porcine wasting disease, and vaccine composition using same

Abstract

The present invention relates to: a vector capable of simultaneously producing an antigen of porcine circovirus 2d, recently prevalent in Korea, an antigen of porcine circovirus 2a, which has caused diseases from the past to the present, a porcine circovirus 2 cellular immunity recombinant protein, and a p65 recombinant protein of Mycoplasma hyopneuminiae, which causes porcine mycoplasma pneumonia; a vaccine against PCV2 and Mycoplasma, the vaccine comprising four types of antigens, Mycoplasma hyopneuminiae dead cells, and Mycoplasma hyorhinis dead cells produced by the vector; a method for producing same; and a use thereof.

Description

A novel vector for producing a recombinant antigen related to swine wasting disease, and a vaccine composition using the same

[0001] The present invention relates to an antigen production vector capable of simultaneously expressing four proteins, including a recombinant VLP antigen for circovirus 2d and 2a genotype viruses causing swine wasting disease, a cellular immune protein for porcine circovirus type 2, and a surface protein of swine epidemic pneumoniae, and a method for producing the same. In addition, the present invention relates to a vaccine composition for preventing and treating swine wasting disease, comprising a mixture of the four antigens produced by the antigen production vector and killed cells of two Mycoplasma species, and a method for producing the same.

[0002]

[0003] Porcine circovirus type 2 (PCV2) is a small, non-enveloped, circular single-stranded DNA virus belonging to the Circoviridea (family) and Circovirus (genus) and has three major open reading frames (ORFs).

[0004] PCV2 disease is characterized by clinical signs such as progressive weight loss, dyspnea, anemia, diarrhea, and jaundice. In acute cases, the mortality rate is approximately 10%, but in severe cases, it can reach 50%. Necropsy findings include lymphadenopathy, granulomatous hepatitis, nephritis, and interstitial pneumonia. PCV2 is associated with postweaning multisystemic wasting syndrome (PMWS) in weaned piglets, porcine dermatitis and nephropathy syndrome, porcine respiratory disease complex, and reproductive disorders. These diseases and syndromes negatively impact the global swine industry and are referred to as porcine circovirus associated disease (PCVAD) in the United States and Asia and porcine circovirus disease (PCVD) in Europe.

[0005] PCV2 is attracting attention as the primary causative agent of PCVAD. When PCV2 is infected alone, mild PMWS symptoms are observed, but when PCV2 is co-infected with porcine parvovirus (PPV) or porcine reproductive and respiratory syndrome virus (PRRSV), symptoms almost identical to postweaning multisystemic wasting syndrome (PMWS) in severe weaned piglets are reported to be reproducible. In addition to PMWS, PCV2 has been linked to various diseases such as proliferating and necrotizing pneumonia, sow abortion and mortality syndrome, and porcine dermatitis and nephropathy syndrome. Therefore, active research is being conducted on the various pathogenesis mechanisms of the pathogenicity of PCV2.

[0006] PCV2 is currently divided into eight major genotypes (PCV2a to 2h), of which PCV2d, PCV2a, and PCV2b genotypes are the primary pathogenic genotypes. PCV2d has been the predominant pathogenic genotype recently observed in pigs in Korea and the United States, while PCV2a has also been consistently pathogenic since 2010.

[0007] Mycoplasmal pneumoniae of swine (MPS) is a chronic respiratory epidemic pneumonia in pigs caused by Mycoplasma hyopneumoniea (Mhp). It is the most common bacterial respiratory disease in pigs. In countries with developed pig farming, approximately 35-50% of pigs are found to have lesions caused by Mhp. Pigs infected with MPS exhibit symptoms such as persistent dry cough, growth retardation, reduced feed efficiency, and immunosuppression. They have high morbidity and transmission rates, but very low mortality rates, causing significant economic losses to farms. MPS occurs across all age groups and increases susceptibility to secondary infections, leading to mixed infections with other bacteria or viruses. Mhp is easily transmitted to piglets when infected sows are infected through airborne transmission by infected sows or contact infection by infected cohabiting sows. It has been reported that infected weaned piglets newly entering the farm and infecting the existing farm account for about 80% of all infections. Mhp is the smallest bacterium that can grow and multiply independently, measuring about 0.2 to 0.5 μm in size, and is known to be highly species-specific and very slow in growth, making isolation, identification, and handling of the bacterium very difficult.

[0008] Mycoplasma hyorhinis (Mhr), similar to Mhp, colonizes the upper respiratory tract and proliferates in the nasal passages and tonsils of infected pigs. Mhr is also known to cause polyserositis in pigs, but it is also frequently found in pneumonic lesions in pigs showing signs of respiratory disease. Mhr causes mild bronchopneumonia or interstitial pneumonia in pigs exposed to the respiratory tract. Like Mhp, Mhr infection leads to economic losses in pig farming and poses a threat to pig farms. The interaction between PCV2 and M. hyorhinis induces more severe lung lesions than pigs infected with Mhr alone.

[0009] The inventors developed this vaccine to prevent economic losses to farms that have consistently suffered significant damage due to porcine circovirus type 2, porcine mycoplasma epidemic pneumonia, and porcine peritonitis.

[0010]

[0011] The present invention aims to provide a vector capable of simultaneously producing PCV2d, PCV2a, surface proteins of Mycoplasma hyopneumoniae (Mhp) and PCV2 cellular immune peptides.

[0012] In addition, the present invention aims to provide a vaccine and a method for producing the same, which simultaneously contain four types of recombinant antigens produced by the above vector, Mycoplasma hyopneumoniae (Mhp) killed cells, and Mycoplasma hyorhinis (Mhr) killed cells.

[0013] The present invention also provides a method for preventing and treating porcine circovirus disease, mycoplasma epidemic pneumonia and mycoplasma peritonitis, comprising a step of administering the vaccine composition.

[0014]

[0015] The present invention provides a method for producing a vector, characterized by including a step of inserting a T7 terminator having a base sequence of SEQ ID NO: 22 and a multiple cloning site (MCS) having a base sequence of SEQ ID NO: 23 into a vector having a base sequence of SEQ ID NO: 1.

[0016] In one embodiment, the vector may have the base sequence of SEQ ID NO: 2.

[0017] The present invention provides a method for producing a vector, characterized in that it comprises a step of converting a His tag in a vector having a base sequence of SEQ ID NO: 2 into at least one selected from the group consisting of an HA tag, a Strep tag, and a FLAG tag.

[0018] In one embodiment, the His tag may have the base sequence of SEQ ID NO: 24.

[0019] In one embodiment, the HA tag, Strep tag, and FLAG tag may each have a base sequence of SEQ ID NO: 3 to 5.

[0020] In one embodiment, the vector may have one of the base sequences of SEQ ID NOs: 6 to 8.

[0021] The present invention provides a method for producing a vector, characterized in that it comprises a step of introducing at least one selected from the group consisting of base sequences of SEQ ID NOs: 9 to 13 into a vector selected from the group consisting of base sequences of SEQ ID NOs: 2 and 6 to 8.

[0022] In one embodiment, the vector may have the base sequence of SEQ ID NO: 14.

[0023] In one embodiment, the manufacturing method may include a step of introducing a base sequence of SEQ ID NO: 10 into a vector having a base sequence of SEQ ID NO: 6.

[0024] In one embodiment, the vector may have the base sequence of SEQ ID NO: 15.

[0025] In one embodiment, the manufacturing method may include a step of introducing a base sequence of SEQ ID NO: 11 into a vector having a base sequence of SEQ ID NO: 7.

[0026] In one embodiment, the vector may have the base sequence of SEQ ID NO: 16.

[0027] In one embodiment, the manufacturing method may include a step of introducing a base sequence of SEQ ID NO: 12 into a vector having a base sequence of SEQ ID NO: 16.

[0028] In one embodiment, the vector may have the base sequence of SEQ ID NO: 17.

[0029] In one embodiment, the manufacturing method may include a step of introducing a base sequence of SEQ ID NO: 13 into a vector having a base sequence of SEQ ID NO: 8.

[0030] In one embodiment, the vector may have the base sequence of SEQ ID NO: 18.

[0031] In one embodiment, the vector may be characterized as being for producing a recombinant antigen associated with porcine wasting disease.

[0032] The present invention provides a method for producing a vector, characterized by comprising the steps of: obtaining a PCV2a base sequence from a vector having a base sequence of SEQ ID NO: 15; and introducing the PCV2a base sequence into a vector having a base sequence of SEQ ID NO: 14.

[0033] In one embodiment, the vector has the base sequence of SEQ ID NO: 19 and can obtain PCV2a and PCV2d.

[0034] The present invention provides a method for producing a vector, characterized by comprising the steps of: obtaining a T_ BLS base sequence from a vector having a base sequence of SEQ ID NO: 17; and introducing the T_ BLS base sequence into a vector having a base sequence of SEQ ID NO: 19.

[0035] In one embodiment, the vector has the base sequence of SEQ ID NO: 20 and can obtain PCV2a, PCV2d and T_ BLS.

[0036] The present invention provides a method for producing a vector, characterized by comprising the steps of: obtaining a p65 base sequence from a vector having a base sequence of SEQ ID NO: 18; and introducing the p65 base sequence into a vector having a base sequence of SEQ ID NO: 20.

[0037] In one embodiment, the vector has the base sequence of SEQ ID NO: 21 and can obtain PCV2a, PCV2d, T_ BLS and p65.

[0038] The present invention provides a vector for producing a recombinant antigen related to swine wasting disease, comprising any one of the base sequences of SEQ ID NOs: 2, 6 to 8, and 14 to 21.

[0039] In one embodiment, the vector may comprise any one of the base sequences of SEQ ID NOs: 19 to 21.

[0040] In one embodiment, the vector is capable of producing PCV2a and PCV2d.

[0041] In one embodiment, the vector may comprise the base sequence of SEQ ID NO: 21.

[0042] In one embodiment, the vector can produce PCV2a, PCV2d, T_ BLS and p65.

[0043] The present invention provides a multivalent peptide manufactured using the above vector.

[0044] In one embodiment, the multivalent peptides may be two or more selected from the group consisting of PCV2a, PCV2d, T_ BLS and p65.

[0045] The present invention provides a vaccine composition for preventing or treating swine wasting disease comprising the multi-balanced peptide.

[0046] In one embodiment, the vaccine composition may further comprise one or more of Mycoplasma hyopneumoniae (Mhp) cells and inactivated Mycoplasma hyorhinis (Mhr) cells.

[0047] In one embodiment, the vaccine composition may further comprise an immunostimulant.

[0048] In one embodiment, the vaccine composition can prevent or treat porcine circovirus type 2 infection, mycoplasma pneumonia, or mycoplasma peritonitis.

[0049] The present invention provides E. coli transformed with the above vector.

[0050]

[0051] The present invention relates to producing a vector capable of simultaneously producing PCV2 antigen, Mhp antigen, and PCV2 cellular immune peptide for porcine circovirus infection, mycoplasma swine epidemic pneumonia, and mycoplasma peritonitis occurring in Korea, and to producing a vaccine by mixing a recombinant antigen produced by the vector with two types of inactivated mycoplasma cells.

[0052] The vaccine of the present invention manufactured by the above manufacturing method induced cellular and humoral immune responses upon inoculation, and was confirmed to have reduced blood viremia and maintained mycoplasma distribution in the nasal cavity at a lower level than the control group for field pathogenic bacteria, and also to not cause serious lesions in tissues.

[0053] Therefore, the vaccine of the present invention can be usefully used for the prevention and treatment of porcine circovirus type 2 infection, mycoplasma epidemic pneumonia, and mycoplasma peritonitis.

[0054]

[0055] Figure 1 is a schematic diagram showing the modified MCS cassette structure of pHis-CS plasmid.

[0056] Figure 2 is a schematic diagram of the production process of PCM 4B plasmid.

[0057] Figure 3 is a photograph showing the results of a western blot experiment of antigens produced in E. coli transformed with PCM 4B plasmid.

[0058] Figure 4 is a graph showing changes in PCV2 antibodies over time after vaccination with five types of vaccines.

[0059] Figure 5 is a graph showing changes in p65 antibodies over time after vaccination with five types of vaccines.

[0060] Figure 6 is a graph showing changes in the number of IFN-γ secreting cells over time after vaccination with two types of vaccines.

[0061]

[0062] Hereinafter, with reference to the attached drawings, embodiments and examples of the present invention will be described in detail so that those skilled in the art can easily implement the present invention. However, the present invention may be implemented in various forms and is not limited to the embodiments and examples described herein.

[0063] Throughout this specification, whenever a part is said to "include" a component, this means that it may include other components, but not to the exclusion of other components, unless otherwise stated.

[0064] The term "PCV2" used in the present invention refers to porcine circovirus type 2 (PCV2), which is a circular single-stranded DNA virus belonging to the Circoviridea (family) and Circovirus (genus), having three major ORFs (open reading frames), and causing PCV2 disease characterized by clinical symptoms such as progressive weight loss, dyspnea, anemia, diarrhea, and jaundice.

[0065] The term "PCV2a" used in the present invention refers to one of the major genotypes of PCV2, which is pathogenic. The PCV2a genotype has been the main causative genotype of swine circovirus infection in Korea since the early 2000s. Although the incidence of this genotype has been decreasing since the use of the PCV2a vaccine from 2007 to 2010, it is still a genotype that occurs at a low frequency in Korea to this day.

[0066] The term "PCV2d" used in the present invention refers to one of the major genotypes of PCV2, which is pathogenic. Since the use of the vaccine against the PCV2a genotype in 2007-2010, the disease caused by the PCV2a genotype has decreased, while the disease caused by the PCV2d genotype has increased. Recently, more than 80% of swine circovirus infections have been confirmed to be of the PCV2d genotype.

[0067] The term "Mycoplasma hyopneumoniea" used in the present invention refers to a bacterium that causes Mycoplasmal pneumoniae of swine (MPS), which causes chronic respiratory epidemic pneumonia in pigs and is also referred to as "Mhp."

[0068] The term "Mycoplasma hyorhinis" used in the present invention is a bacterium that forms colonies in the upper respiratory tract similar to Mhp and proliferates in the nasal cavity and tonsils of infected pigs, and is known as a causative agent of polyserositis and arthritis in pigs, but is also frequently found in lesions of pigs showing signs of respiratory disease.

[0069]

[0070] The term "T cell epitope" used in the present invention refers to a protein related to cellular immunity within the orf1 and orf3 proteins of the PCV2 virus, and is also referred to as a "T cell epitope."

[0071]

[0072] The term "BLS" used in the present invention is Brucella lumazine synthase protein, which is reported to be an immune enhancing substance that stimulates the cellular immune system by stimulating Toll-like receptor 4 in the immune system.

[0073] The term "T_BLS" used in the present invention is a protein produced to improve the cellular immune response to PCV2 by combining parts of the orf1 and orf3 proteins that cause cellular immune stimulation of the PCV2 virus and the BLS protein.

[0074] The term "vector" as used herein may refer to a construct capable of delivering, and preferably expressing, one or more gene(s) or sequence(s) of interest into a host cell. Examples thereof include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid, or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells.

[0075] The vaccine composition of the present invention may be prepared in any suitable, pharmaceutically acceptable form, such as a ready-to-use solution or suspension, a concentrated stock solution suitable for dilution prior to administration, or a reconstitutable form, such as a lyophilized, freeze-dried, or frozen preparation.

[0076] The vaccine composition of the present invention may additionally include an adjuvant. An adjuvant is one or more substances that enhance an immune response to an antigen, and may be, for example, one or more selected from the group consisting of complete Freund's, incomplete Freund's, saponin, gel-like aluminum adjuvants, surface-active substances (e.g., lysolecithin, pluronic glycol, polyanions, peptides, oil or hydrocarbon emulsions, etc.), vegetable oils (cottonseed oil, peanut oil, corn oil, etc.), and vitamin E acetate, but is not limited thereto.

[0077] The term “immune response” as used in the present invention means a humoral immune response, a cellular immune response, or both.

[0078] The term "prevention" as used in the present invention means any act of inhibiting or delaying the onset of a disease by administering a composition, and "treatment" means any act of improving or beneficially changing the symptoms of a subject suspected of or suffering from a disease by administering a composition.

[0079] The present invention will be described in more detail through the following examples; however, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0080]

[0081] [Manufacturing Example 1]

[0082] pHis-CS plasmid construction

[0083]

[0084] Based on the pHis-parallel 1 plasmid (GenBank; AF097413) having the base sequence of sequence number 1 in Table 1 below, a novel plasmid was synthesized for the expression of two or more different proteins, and was named "pHis-CS plasmid."

[0085] The sequence of the above pHis-CS plasmid was confirmed to have the sequence of sequence number 2 in Table 1 below through base sequence analysis.

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092] The above pHis-CS plasmid was constructed using the following method. Specifically, as shown in Fig. 1, in order to insert multiple antigen genes into the genes of the existing pHis-parallel 1 plasmid, a new T7 terminator sequence shown in Table 1 and a new multiple cloning site (MCS) were inserted in front of the existing T7 terminator.

[0093] In order to express two or more different proteins, the protein expression gene is inserted from the NdeI to NotI restriction enzyme sites shown in Fig. 1, and then, for multivalent protein expression, another protein expression gene (T7 promoter, gene to be expressed, and T7 terminator gene base sequence) is used to create a primer suitable for the specific restriction enzyme sites from KpnI to XhoI shown in Fig. 1 using the in-fusion cloning method. For example, after creating a primer suitable for in-fusion using the In-Fusion Primer Design Tool (https: / takara.teselagen.com / # / DesignPag), cloning is performed according to the suggested protocol using the In-Fusion HD cloning kit. In order to express two or more proteins, primers suitable for the additionally created restriction enzyme sites (between KpnI and XhoI) are sequentially created, and then genes for the expression of two or more proteins can be in-fusion cloned.

[0094]

[0095] [Manufacturing Example 2]

[0096] Production of plasmids with substituted peptide tags

[0097]

[0098] In order to replace the His tag base sequence of the pHis-CS plasmid produced in Manufacturing Example 1 with another tag sequence, genes for HA (SEQ ID NO: 3), Strep (SEQ ID NO: 4), and FLAG (SEQ ID NO: 5) tags were synthesized as shown in Table 2 below.

[0099]

[0100]

[0101] The plasmids in which the His tag sequence in the pHis-CS plasmid was replaced with HA, Strep, and FLAG tags, respectively, were named pHA-VT3, pStrep-VT4, and pFLAG-VT1 plasmids.

[0102] The pHA-VT3 plasmid was prepared using the following method. After linearizing the pHis-CS plasmid by treating it with restriction enzymes NdeI and BamHI, primers were designed using the In-Fusion Primer Design Tool, and then in-fusion cloning was performed using the HA tag PCR product obtained by PCR and the linearized plasmid, ultimately obtaining the pHA-VT3 plasmid.

[0103] The above pStrep-VT4 plasmid was prepared by the following method. After linearizing the pHis-CS plasmid by treating it with restriction enzymes NdeI and NcoI, primers were designed using the In-Fusion Primer Design Tool, and then in-fusion cloning was performed using the Strep tag PCR product obtained by PCR and the linearized plasmid, and finally the pStrep-VT4 plasmid was obtained.

[0104] The above pFLAG-VT1 plasmid was prepared by the following method. After linearizing the pHis-CS plasmid by treating it with restriction enzymes NdeI and BamHI, primers were designed using the In-Fusion Primer Design Tool, and then in-fusion cloning was performed using the FLAG tag PCR product obtained by PCR and the linearized plasmid, and finally the pFLAG-VT1 plasmid was obtained.

[0105]

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114] [Manufacturing Example 3]

[0115] Construction of pHis-VT2-PCV2d, pHA-VT3-PCV2a, pStrep-VT4-T_BLS and pFLAG-VT1-p65 plasmids

[0116]

[0117] To construct pHis-VT2-PCV2d, pHA-VT3-PCV2a, pStrep-VT4-T_BLS, and pFLAG-VT1-p65 plasmids, genes for PCV2d orf2, PCV2a orf2, T-cell epitope peptide, BLS, and p65 having the base sequences shown in Table 4 below were designed using a codon optimizer and then synthesized.

[0118]

[0119]

[0120]

[0121]

[0122] First, pHis-VT2-PCV2d plasmid was constructed using the following method. pHis-CS plasmid was linearized by treating it with restriction enzymes BamHI and NotI, and then the synthesized PCV2d gene was infused into the linearized pHis-CS plasmid according to the protocol using an infusion primer design tool and an infusion cloning kit (In-fusion® HD cloning kit, TaKaRa), thereby constructing pHis-VT2-PCV2d plasmid (SEQ ID NO: 14).

[0123] pHA-VT3 plasmid was also linearized by treatment with restriction enzymes BamHI and NotI, and the synthesized PCV2a gene was infused into the linearized pHA-VT3 plasmid using the infusion primer design tool and infusion cloning kit (In-fusion® HD cloning kit, TaKaRa) according to the manufacturer's protocol, thereby producing pHA-VT3-PCV2a plasmid (SEQ ID NO: 15).

[0124] After designing the T_cell epitope peptide base sequence to include a linker (GGGSGGGS) and GGATCC, which is a recognition sequence of BamHI restriction enzyme, at the end, a T_cell epitope peptide base sequence including the linker and recognition sequence was produced.

[0125] After linearizing the pStrep-VT4 plasmid with the restriction enzyme NcoI, the T_cell epitope base including the linker and recognition sequence was infused into the linearized pStrep-VT4 plasmid using the infusion primer design tool and the infusion cloning kit (In-fusion® HD cloning kit, TaKaRa) according to the manufacturer's protocol, thereby constructing the pStrep-VT4-T_cell epitope plasmid (SEQ ID NO: 16).

[0126] Afterwards, the pStrep-VT4-T_cell_epitope plasmid was linearized with restriction enzymes BamHI and NotI, and the synthesized BLS gene was infused into the linearized pStrep-VT4-T_cell_epitope plasmid using the infusion primer design tool and infusion cloning kit (In-fusion® HD cloning kit, TaKaRa) according to the manufacturer's protocol, thereby constructing pStrep-VT4-T_BLS plasmid (SEQ ID NO: 17).

[0127] After linearizing the pFLAG-VT1 plasmid with restriction enzymes BamHI and NotI, the synthesized p65 gene was infused into the linearized pFLAG-VT1 plasmid using the Infusion primer design tool and infusion cloning kit (In-fusion® HD cloning kit, TaKaRa) according to the manufacturer's protocol, thereby constructing pFLAG-VT1-p65 plasmid (SEQ ID NO: 18).

[0128] The plasmids produced above are shown in Table 5 below.

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

[0141] [Manufacturing Example 4]

[0142] Production of bivalent to tetravalent plasmids

[0143]

[0144] For cloning, the pHis-VT2-PCV2d plasmid (SEQ ID NO: 14) was linearized by treating it with Kpnl restriction enzyme. Then, in-fusion primers for the region including the T7 promoter, PCV2a gene, and T7 terminator gene in the pHA-VT3-PCV2a plasmid were designed, and PCR was performed to obtain the PCR product of the region. Finally, in-fusion cloning was performed using the linearized pHis-VT2-PCV2d plasmid and the PCR product of a portion of the pHA-VT3-PCV2a plasmid, to construct the PCM4 2B plasmid (SEQ ID NO: 19) that expresses two proteins, respectively.

[0145] Afterwards, in order to proceed with cloning, the PCM4 2B plasmid was linearized by treating it with the Sacl restriction enzyme. In-fusion primers for the region including the T7 promoter, T_BLS gene, and T7 terminator gene in the pStrep-VT4-T_BLS plasmid were designed, and PCR was performed to obtain the PCR product of the region. Using the linearized PCM4 2B plasmid and the PCR product of a portion of the pStrep-VT4-T_BLS plasmid, in-fusion cloning was performed to construct the PCM4 3B plasmid (SEQ ID NO: 20) that expresses each of the three proteins.

[0146] Afterwards, to proceed with cloning, the PCM4 3B plasmid was linearized by treating it with Spel restriction enzyme. Using the in-fusion primer design tool, an in-fusion primer for the region including the T7 promoter, p65 gene, and T7 terminator gene in the pFLAG-VT1-p65 plasmid was designed, and PCR was performed with the primer to obtain a PCR product of the region. Using the linearized PCM4 3B and the PCR product of a portion of the pFLAG-VT1-p65 plasmid, in-fusion cloning was performed to construct a PCM4 4B plasmid (SEQ ID NO: 21) that expresses each of the four proteins.

[0147] The above production method is briefly illustrated in Fig. 2, and the base sequences of sequence numbers 19 to 21 are shown in Table 6 below.

[0148]

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156]

[0157]

[0158]

[0159]

[0160] [Example 1]

[0161] Production of 4-protein expression cells using PCM4 4B plasmid and confirmation of recombinant antigen expression

[0162]

[0163] The PCM4 4B plasmid for expression of the above four antigens was transformed into Escherichia coli BL21 (DE3) competent cells, plated on solid LB-amp medium, and cultured at 37°C for 16 hours. Colonies were then collected from the medium, inoculated into LB-amp liquid medium, and cultured with shaking at 37°C and 200 rpm for 14 hours. The cultured cells were mixed 1:1 with 50% glycerol, aliquoted into 500 μl each, and stored at -80°C.

[0164] The strains that were being frozen were taken out and thawed at room temperature, and then inoculated into LB-amp medium at 0.1% (v / v) and cultured at 37°C and 200 rpm for approximately 12 hours. After adding 100 μg / mL of ampicillin to sterilized TY medium (24 g / L yeast extract, 12 g / L bacto tryptone, 5 g / L glycerol, 0.005% Antifoam 204), the cultured bacteria were inoculated into the medium at 5% (v / v) and cultured at 37°C and 350 rpm. When the fermentor was operating, the pH condition was adjusted to 6.9-7.3, and the aeration condition was maintained at 1.6 vvm (air volume / medium volume / minute) of compressed air inflow to culture the bacteria. From 4 hours of incubation in the fermentor, the degree of bacterial growth was aseptically collected at 30-minute intervals and the absorbance was measured at 600 nm. When the measured value was approximately 4.2–4.5, the fermentor incubation temperature was changed to 27°C. When the culture temperature reached 27°C, IPTG was added to a final concentration of 0.1 mM, and the culture was further incubated at 27°C and 350 rpm for approximately 7 hours.

[0165] The cultured E. coli solution was centrifuged at 8,000×g for 10 minutes, the supernatant was removed, and the solution was diluted with PBS to an OD of 50 at 600 nm. The suspension was pressure-crushed four times using a high-pressure crusher at 1,250 bar (approximately 18,000 psi) to extract antigens from E. coli. After completion of the crushing, the solution was centrifuged at 10,000×g for 15 minutes, and only the supernatant was recovered, filtered through a 0.8 μm pore size depth filter, and finally filtered through a 0.45 μm pore size filter. Western blot was performed on the four expressed proteins, and the results are shown in Fig. 3.

[0166] As shown in Fig. 3, A) is a photograph of the results of a western blot performed using PCV2 polyclonal antibody (VMRD, PCV2 polyclonal antibody, Cat. No. PAB-PCV2), in which PCV2d protein was confirmed in a band of approximately 28 kDa and PCV2a protein was confirmed in a band of approximately 28.5 kDa.

[0167] Western blot was performed using 6xHis monoclonal antibody (TaKaRa, anti-6xHis monoclonal antibody, Cat. No. 631212), and PCV2d protein was confirmed as shown in B) of Fig. 3.

[0168] Western blot was performed using HA monoclonal antibody (Abcam, anti-HA monoclonal antibody, Cat. No. ab18181), and PCV2a protein was confirmed as shown in Fig. 3C).

[0169] Western blot was performed using Strep monoclonal antibody (IBA Lifescience, StrepMAB classic, Cat. No. 2-1507-001), and T_BLS protein (approximately 33.7 kDa) was confirmed as shown in D) of Fig. 3.

[0170] Western blot was performed using Mhp p65 monoclonal antibody (ID Screen® Mycoplasma hyopneumoniae Competition kit, “Ready to use conjugate 1X”, ID Vet, Cat. No. 5112-009), and p65 protein (approximately 78 kDa) was confirmed as shown in E) of Fig. 3.

[0171] Western blot was performed using FLAG monoclonal antibody (Abcam, anti-DDDDK tag antibody, Cat. No. ab125243). As shown in Fig. 3F), p65 protein (approximately 78 kDa) was confirmed at the same location as Fig. 3F.

[0172] Through the results of the above Test Example 1, it was confirmed that the PCM 4B plasmid of the present invention can stably express four types of antigens.

[0173]

[0174] [Example 2]

[0175] Immunogenicity assessment of PCV2d, PCV2a, PCM4 2B (PCV2d+PCV2a), T_BLS, and p65 recombinant antigens

[0176]

[0177] pHis-VT2-PCV2d, pHA-VT3-PCV2a, pStrep-VT4-T_BLS, pFLAG-VT1-p65, and PCM4 2B plasmids were each transformed into Escherichia coli BL21 (DE3) competent cells, plated on solid LB-amp medium, and cultured at 37°C for 16 hours. Colonies were then collected from the medium, inoculated into LB-amp liquid medium, and cultured with shaking at 37°C and 200 rpm for 14 hours. The cultured cells were mixed 1:1 with 50% glycerol, aliquoted into 500 μl each, and stored at -80°C.

[0178] Five different BL21(DE3) frozen strains were inoculated into LB-amp liquid medium at 0.1% (v / v) and cultured at 37°C and 200 rpm for approximately 12 hours. Each cultured strain was inoculated into LB-amp medium at 0.1% (v / v) and cultured at 37°C and 200 rpm for approximately 12 hours. After sterilization, 100 ㎍ / mL of ampicillin was added to sterilized TY medium (24 g / L yeast extract, 12 g / L bacto tryptone, 5 g / L glycerol, 0.005% Antifoam 204), and the cultured strains were inoculated into 5% (v / v) and cultured at 37°C and 350 rpm. During fermentor operation, the pH condition was adjusted to 6.9-7.3, and the aeration condition was maintained at 1.6 vvm (air volume / medium volume / minute) of compressed air inflow to culture the bacteria. After 4 hours of culture in the fermentor, the degree of bacterial growth was aseptically collected at 30-minute intervals and the absorbance was measured at 600 nm. When the measured value was approximately 4.2-4.5, the temperature of the fermentor culture was changed to 27℃, and when the culture temperature reached 27℃, IPTG was added to a final concentration of 0.1 mM, and then cultured for approximately 7 hours at 27℃ and 350 rpm.

[0179] Each E. coli culture was centrifuged at 8,000 × g for 10 minutes, the supernatant was removed, and the mixture was diluted with PBS to an OD of 50 at 600 nm. The suspension was pressure-crushed four times using a high-pressure crusher at 1,250 bar (approximately 18,000 psi) to extract antigens from five types of E. coli. After completion of the crushing, the homogenate was centrifuged at 10,000 × g for 15 minutes, and only the supernatant was recovered and filtered through a 0.8 μm pore size depth filter, and then finally through a 0.45 μm pore size filter to extract each antigen from five different bacterial strains. A vaccine (2 ml / head) was prepared by including 12% of an immune adjuvant (MVP adjuvant, Emulcigen-BCL) for the five different extracted antigen compositions as shown in Table 7.

[0180]

[0181]

[0182] As shown in Table 7, 2.0 mL of the five vaccines were administered intramuscularly to 3-4 piglets aged 3-4 weeks, and 2.0 mL of PBS was administered intramuscularly to 3-4 week-old piglets in the negative control group. Blood samples were collected before vaccination and 3 and 5 weeks after vaccination, and serum was separated and tested for ELISA antibody titers. In groups 3 and 4, peripheral blood mononuclear cells (PBMCs) were isolated from the blood to evaluate the effect of improving cell-mediated immunity. PCV2 ELISA antibody titers were tested for PCV2 antibodies using a PCV2 (Porcine Circovirus type 2 Antibody Test, BioChek BV, Reeuwijk, Holland) kit, and ELISA antibodies to the p65 antigen of Mhp were tested for PCV2 antibodies using the ID Screen® Mycoplasma hyopneumoniae Competition ELISA (ID.Vet, ELISA judgment criteria; positive: The presence of antibodies was confirmed using a kit (0.50, negative: >0.50; unit: S / N ratio).

[0183]

[0184] The results of measuring antibody titers using the PCV2 antibody diagnostic kit are shown in Figure 4 and Table 8.

[0185]

[0186]

[0187]

[0188] As shown in Table 8 above, positive antibody titers were observed from the third week after vaccination in groups 1 to 4, and it was confirmed that the antibody titers increased until the fifth week after vaccination.

[0189] In addition, antibody titers were compared using a PCV2 antibody diagnostic kit for different antigens in Group 1 (PCV2d vaccine) and Group 2 (PCV2a vaccine), but no relative significance in the superiority of antibody formation between the two groups could be observed.

[0190] PCV2 antibody titers were examined among the monovalent (Groups 1 and 2), bivalent (Group 3), and trivalent (Group 4) antigen vaccine groups. The polyvalent antigen vaccine groups (Groups 3 and 4) showed similar antibody formation to PCV2 as the monovalent antigen vaccine groups. However, no antibody formation to PCV2 was observed in the p65 (Group 5) and control (Group 6) groups.

[0191] Therefore, it can be seen that even if the types of recombinant antigens in the vaccine increase, the immunogenicity against PCV2 is not significantly affected.

[0192]

[0193] The results of measuring antibody titers using a commercial antibody diagnostic kit (ID Vet) for the p65 antigen of Mhp to prevent swine epidemic pneumonia are shown in Fig. 5. As shown in Fig. 5, in the group vaccinated with the Mhp-p65 vaccine (group 5), immunogenicity against Mhp was observed from 3 weeks after vaccination compared to the other groups (S / N ratio, 33.15±3.97), and an additional increase in antibody titers was observed at 5 weeks after vaccination (S / N ratio, 25.71±2.47).

[0194] Therefore, it was demonstrated that p65, a recombinant Mhp surface antigen protein, has value as a vaccine because it can form antibodies against Mhp in preventing epidemic pneumonia.

[0195] To analyze the results of cellular immunity against PCV2, blood was collected from groups 3, 4, and 6 in Table 8 above, and peripheral blood mononuclear cells (PBMCs) were separated from the blood using a lymphocyte separation reagent, and then the cellular immune function was evaluated in the cells. First, 5.0 × 10 PBMCs were isolated. 6.0 After preparing at a concentration of 10 cells / mL, the number of IFN-γ secreting cells was measured according to the protocol of the Porcine IFN-gamma ELISpot Kit (R&D Systems, Inc.). The stimulators for each PBMC cell were orf1 and orf3 T-cell epitope peptides (20 μg / mL for each peptide), and PBMCs were stimulated with the stimulators.

[0196] For groups 3 and 4, cellular immune stimulation was observed from 3 weeks after vaccination, but 5 weeks after vaccination, the number of interferon gamma-secreting cells in group 4 containing the T_BLS antigen (36.38±10.47) was slightly higher than that in group 3 (21.88±9.30) with a p-value of 0.08, which is slightly higher than the significance level of 5%. As shown in Fig. 6 and Table 9, the average cellular immune stimulation was confirmed to be enhanced by approximately 1.7 times in the individuals containing the T_BLS antigen. Based on these results, the T_BLS antigen was included in the vaccine formulation to improve the cellular immune function of the PCV2 vaccine.

[0197]

[0198]

[0199]

[0200] [Example 3]

[0201] Efficacy evaluation of the manufactured PCM4 vaccine

[0202]

[0203] Using the above PCM 4B plasmid, four recombinant antigens and two mycoplasma isolate antigens (Mhp and Mhr) were used to prepare a vaccine with the composition shown in Table 10, and 2.0 mL was inoculated intramuscularly into the musculature of 3-week-old piglets. 2.0 mL of PBS was inoculated into the positive control group (challenge after non-vaccination) and the negative control group (non-vaccination with vaccine and pathogenic strain).

[0204]

[0205]

[0206] * pp of Mhp and Mhr are protein particle measurements measured using Sartorius virus counter equipment.

[0207]

[0208] Three weeks after vaccination, each group was challenged with field pathogenic strains of PCV2d virus (group 1), PCV2a virus (group 2), Mycoplasma hyopneumoniae (group 3), and Mycoplasma hyorhinis and PCV2d virus (group 4). Antibody titers, clinical symptoms, and histopathological lesions according to the challenge vaccination were compared and evaluated from vaccination to three weeks after the challenge vaccination.

[0209] PCV2d (strain SNUVR202003: d type, GenBank no. MZ440695) and PCV2a (strain SNUVR000032: a type, GenBank no. KF871067) for challenge vaccination were 1.2×10 5.0 TCID 50 / mL was prepared and 3.0 mL was administered intranasally at 6 weeks of age, 3 weeks after vaccination. Mhp was 1×10 7.0 Intratracheal inoculation was performed with 7.0 mL of color changing units (CCU) / mL. Mhr was 1.0×10 9.0 4.0 mL was inoculated intraperitoneally and 2.0 mL intratracheally at a concentration of color changing units (CCU) / mL.

[0210] Clinical symptoms were measured using a clinical index ranging from 0 to 6, referring to the literature of Jeong et al (Vet Microbiol 177: 43-52, 2015). Specific antibodies to PCV2 using the collected blood were tested using a commercial ELISA kit (Porcine Circovirus type 2 Antibody Test, BioCheck, BB, Reeuwijk, Holland), and specific neutralizing antibodies to PCV2 in blood were tested according to the literature of Seo et al (Vaccine 30:6671-6677, 2012). The PCV2- and mycoplasma-specific INF-γ secreting cell measurement method was performed according to the literature of Seo et al (Vet Res 45:13, 2014). Specific antibodies to Mhp in the collected blood were measured using the commercial ID Screen® Mycoplasma hyopneumoniae Competition ELISA (ID.Vet). Specific antibodies to M. hyorhinis in the collected blood were measured using an in-house ELISA kit manufactured by Careside Co., Ltd. Real-time PCR for PCV2d was performed according to Jeong et al. (Vet Microbiol 177: 43-52, 2015). Real-time PCR for PCV2a was performed according to Gagnon et al. (J Vet Diagn Invest 20: 545-558, 2008). Real-time PCR for Mhp was performed according to Marois et al. (J Appl Microbiol 108: 1523-1533, 2010). Real-time PCR for Mhr was performed according to Clavijo et al. (J Vet Diagn Invest 26: 755-760, 2014).

[0211] In the case of PCV2-related histopathological examination, the degree of lymphoid depletion and granulomatous inflammation observed in the inguinal lymph nodes was measured by dividing them into grades 0 to 5 according to Kim et al (J Comp Pathol 131, 121-126, 2004).

[0212] The gross pneumonia lesions due to MHP were scored by dividing them into the volume ratio of each lung lobe and the ventral and dorsal parts of the lung, and assigning points to each part as follows: ventral Lt-anterior was 5 points, ventral Rt-anterior was 5 points, ventral Lt-middle was 5 points, ventral Rt-middle was 5 points, ventral Lt-causal was 12.5 points, ventral Rt-causal was 12.5 points, ventral accessory was 5 points, dorsal Lt-anterior was 5 points, dorsal Rt-anterior was 5 points, dorsal Lt-middle was 5 points, dorsal Rt-middle was 5 points, dorsal Lt-causal was 15 points, and dorsal Rt-causal was 15 points, and the score of the lesion was measured in each part.

[0213] Histological lesions of pneumonia caused by MHP were classified into grades 0 to 6 according to the severity of the lesions. The grading was based on the degree of thickening of the alveolar wall, the degree of inflammation of the alveolar cavity, and the hypertrophy of lymphocytes in the bronchioles and peribronchioles, and was indicated as 'score 0' for normal without lesions, 'score 1' for mild multifocal, 'score 2' for mild diffuse, 'score 3' for moderate multifocal, 'score 4' for moderate diffuse, 'score 5' for moderate multifocal, and 'score 6' for severe diffuse.

[0214] The gross lesion scoring for fibrinous serositis observed in the serosa of the liver in Mhr infection was measured and graded from 0 to 3 according to the method of Chen et al (Vet Microbiol. 182: 123-130, 2016).

[0215] Fibrinous hepatitis observed in the liver by MHR was graded from 0 to 6 according to the degree of histopathological lesions. The grade was determined based on the fibrinous inflammation as 'score 0' for normal without lesions, 'score 1' for mild multifocal, 'score 2' for mild diffuse, 'score 3' for moderate multifocal, 'score 4' for moderate diffuse, 'score 5' for moderate multifocal, and 'score 6' for severe diffuse.

[0216] The Shapiro-Wilk test was used to test for normal distribution. If the statistical data were normally distributed, an ANOVA test was performed to analyze the significance between groups. If the ANOVA test revealed significant differences between groups, the Tukey adjustment was used to analyze the significant differences between groups. If the statistical data were not normally distributed, the Kruskal-Wallis test was performed to analyze the significance between groups. If the Kruskal-Wallis test revealed significant differences between groups, the Mann-Whitney test was used to analyze the significant differences between groups. A p value of 0.05 or less was considered significant, and statistical significance indices a, b, and c were used to indicate the significance between groups.

[0217] The evaluation items for the PCM4 vaccine were clinical symptoms before and after challenge vaccination, immune response to PCV2 (IgG to PCV2, neutralizing antibodies to PCV2a and PCV2d, IFN-γ secreting cells), immune response to Mhp (IgG and IFN-γ secreting cells), immune response to Mhr (IgG and IFN-γ secreting cells), and microbiological analysis comparing and evaluating the antigen levels of PCV2d and PCV2a in blood and tissues, and Mhp and Mhr in nasal and tissues.

[0218] 3.1 Comparison of clinical symptoms according to the attack vaccination

[0219] After PCV2d challenge vaccination, the clinical symptoms of the vaccine group and the positive control group were observed. As a result, the clinical symptom index of the vaccine group was 0.6±0.5 and 1.0±0.7, respectively, and the positive control group was 1.8±0.4 and 2.4±0.9, respectively, at 2 and 3 weeks after challenge vaccination, as shown in Table 11 below. The clinical symptom index of the vaccine group was significantly lower at both 2 and 3 weeks after challenge vaccination. As a result of observing the clinical symptoms of the vaccine group and the positive control group after PCV2a challenge vaccination, the clinical symptom index of the vaccine group was 0.4±0.9 and 0.8±0.8, respectively, at 2 and 3 weeks after challenge vaccination, and the positive control group was 1.8±0.4 and 2.4±0.9, respectively, as shown in Table 11 below. The clinical symptom index of the vaccine group was significantly lower at both 2 and 3 weeks after challenge vaccination. After Mhp challenge vaccination, the clinical symptom indexes of the vaccine group and the positive control group were 0.6±0.5 and 1.6±0.5, respectively, and the positive control group was 1.8±0.4 and 3.2±1.1, respectively, at 2 and 3 weeks after challenge vaccination. As shown in Table 11 below, the clinical symptom indexes of the vaccine group were significantly lower at both 2 and 3 weeks after challenge vaccination. After Mhr and PCV2d combined challenge vaccination, the clinical symptom indexes of the vaccine group and the positive control group were 1.8±0.4 and 2.8±1.1, respectively, and the positive control group was 3.2±1.1 and 4.8±1.1, respectively, at 2 and 3 weeks after challenge vaccination. As shown in Table 11 below, the clinical symptom indexes of the vaccine group were significantly lower at both 2 and 3 weeks after challenge vaccination.

[0220]

[0221]

[0222] *a, b, c indicate statistical significance (p< 0.05)

[0223]

[0224] 3.2 Comparison of body weight (kg) by age according to the attack vaccination

[0225] After PCV2d, PCV2a, Mhp, or Mhr challenge vaccination, the body weights of the vaccine group and the positive control group were measured at different ages. As shown in Table 12 below, in the PCV2d challenge vaccination group, when body weights (kg) were compared between each group 3 weeks after challenge vaccination, the average body weight in the vaccine group (20.4±1.6) was higher than that in the positive control group (19.1±2.8), but no statistical significance was observed. In addition, in the PCV2a challenge vaccination group, when body weights (kg) were compared between each group 3 weeks after challenge vaccination, the average body weight in the vaccine group (20.5±0.7) was higher than that in the positive control group (19.2±2.9), but no statistical significance was observed. However, in the case of the Mhp challenge vaccination group, when comparing body weight (kg) between each group 3 weeks after the challenge vaccination, the average body weight of the vaccine group (20.9±1.4) was significantly higher than that of the positive control group (17.1±1.5), and the body weight of the vaccine group was measured at the level of the negative control group (21.1±14). In addition, when comparing body weight (kg) between each group 3 weeks after the combined challenge vaccination with Mhr and PCV2d viruses, the average body weight of the vaccine group (14.9±0.6) was significantly higher than that of the positive control group (11.9±0.8), but was measured significantly lower than that of the negative control group (21.1±1.4). This can be seen as proof of the effect of the PCM4 vaccine due to the high pathogenicity of the simultaneous challenge vaccination with PCV2d and Mhr, and the more severe weight loss in the non-vaccinated group (positive control group).

[0226]

[0227]

[0228] *a, b, c indicate statistical significance (p< 0.05)

[0229]

[0230] 3.3 Immunological evaluation

[0231] In the PCV2d challenge vaccination group, the vaccinated group had significantly higher PCV2 IgG antibody levels than the positive control group at 3 weeks after vaccination and 1, 2, and 3 weeks after challenge vaccination, as shown in Table 13 below.

[0232]

[0233]

[0234] *a, b, c indicate statistical significance (p< 0.05)

[0235]

[0236] In addition, as a result of evaluating the level of PCV2d virus neutralizing antibodies in the blood, as shown in Table 14 below, the level of PCV2d neutralizing antibodies in the sera of the vaccine group was significantly higher than that in the sera of the positive control group at 3 weeks after vaccination and 1, 2, and 3 weeks after challenge vaccination.

[0237]

[0238]

[0239] *a, b, c indicate statistical significance (p< 0.05)

[0240]

[0241] In the PCV2a challenge vaccination group, the PCV2 IgG antibody level of the vaccine group was significantly higher than that of the positive control group at 3 weeks after vaccination and 1, 2, and 3 weeks after challenge vaccination, as shown in Table 15 below.

[0242]

[0243]

[0244] *a, b, c indicate statistical significance (p< 0.05)

[0245]

[0246] In addition, as a result of evaluating the PCV2a neutralizing antibody level in the blood, as shown in Table 16 below, the PCV2a neutralizing antibody level in the sera of the vaccine group was significantly higher than that in the positive control group at 3 weeks after vaccination and 1, 2, and 3 weeks after challenge vaccination.

[0247]

[0248]

[0249] *a, b, c indicate statistical significance (p< 0.05)

[0250]

[0251] In the Mhp challenge vaccination group, the Mhp IgG antibody levels of the vaccine group and the positive control group were compared, and as shown in Table 17 below, the Mhp IgG antibody levels were significantly higher than those of the positive control group at 3 weeks after vaccination and 1, 2, and 3 weeks after challenge vaccination.

[0252]

[0253]

[0254]

[0255]

[0256] *a, b, c indicate statistical significance (p< 0.05)

[0257]

[0258]

[0259] *a, b, c indicate statistical significance (p< 0.05)

[0260] We also evaluated the levels of neutralizing antibodies to PCV2d virus in the blood. As shown in Table 20 below, the PCV2d neutralizing antibody levels in the sera of the vaccine group were significantly higher than those in the positive control group at 3 weeks post-vaccination and 1, 2, and 3 weeks post-challenge.

[0261]

[0262]

[0263]

[0264] *a, b, c indicate statistical significance (p< 0.05)

[0265]

[0266] 3.4 Measurement of IFN-γ secreting cells related to cellular immunity

[0267] In the PCV2d challenge vaccination group, the levels of PCV2d-related IFN-γ-secreting cells (PCV2d IFN-γ-SC) in the vaccine group and the positive control group were compared, and as shown in Table 21 below, the levels of PCV2d-related IFN-γ-secreting cells (PCV2d IFN-γ-SC) in the vaccine group were significantly higher than those in the positive control group at 3 weeks after vaccination and 1, 2, and 3 weeks after challenge vaccination.

[0268]

[0269]

[0270] *a, b, c indicate statistical significance (p< 0.05)

[0271]

[0272] In the PCV2a challenge vaccination group, the PCV2a-related IFN-γ-secreting cell (PCV2a IFN-γ-SC) levels of the vaccine group and the positive control group were compared, and as shown in Table 22 below, the PCV2a-related IFN-γ-secreting cell (PCV2a IFN-γ-SC) levels of the vaccine group were significantly higher than those of the positive control group at 3 weeks after vaccination and 1, 2, and 3 weeks after challenge vaccination.

[0273]

[0274]

[0275] *a, b, c indicate statistical significance (p< 0.05)

[0276]

[0277] In the Mhp challenge vaccination group, the Mhp-associated IFN-γ-secreting cell (Mhp IFN-γ-SC) levels of the vaccine group and the positive control group were compared, and as shown in Table 23 below, the Mhp-associated IFN-γ-secreting cell (Mhp IFN-γ-SC) levels of the vaccine group were significantly higher than those of the positive control group at 3 weeks after vaccination and 1, 2, and 3 weeks after challenge vaccination.

[0278]

[0279]

[0280] Finally, in the Mhr and PCV2d mixed challenge vaccination group, the levels of PCV2d-associated IFN-γ-secreting cells (PCV2d IFN-γ-SC) and Mhr-associated IFN-γ-secreting cells (Mhr IFN-γ-SC) in the vaccine group and the positive control group were compared. As shown in Table 24 below, the levels of PCV2d-associated IFN-γ-secreting cells in the vaccine group were significantly higher than those in the positive control group at 3 weeks after vaccination and 1, 2, and 3 weeks after challenge vaccination. The levels of Mhr IFN-γ-secreting cells in the vaccine group were also significantly higher than those in the positive control group at 3 weeks after vaccination and 1, 2, and 3 weeks after challenge vaccination, as shown in Table 25 below.

[0281]

[0282]

[0283] *a, b, c indicate statistical significance (p< 0.05)

[0284]

[0285]

[0286] *a, b, c indicate statistical significance (p< 0.05)

[0287]

[0288] 3.5 Evaluation of PCV2 antigen in blood and mycoplasma antigen in nasal cavity

[0289] Blood samples were collected from the vaccine, positive control, and negative control groups, and PCV2a or PCV2d were quantitatively analyzed using real-time PCR. Furthermore, nasal swabs were used to collect nasal samples, and Mhp and Mhr were quantitatively analyzed using real-time PCR.

[0290] In the PCV2d challenge vaccination group, the blood PCV2d amount of the vaccine group and the positive control group was compared, and as shown in Table 26 below, the PCV2d content of the vaccine group was significantly lower than that of the positive control group from 1 week after the challenge vaccination, and the blood PCV2d antigen amount of the vaccine group was continuously significantly lower than that of the positive control group until 2 and 3 weeks after the challenge vaccination.

[0291]

[0292]

[0293] In the PCV2a challenge vaccination group, the blood PCV2a content of the vaccine group and the positive control group was compared, and as shown in Table 27 below, the antigen content of the vaccine group was significantly lower than that of the positive control group from 1 week after the challenge vaccination, and the blood PCV2a antigen of the vaccine group was significantly lower than that of the positive control group continuously until 2 and 3 weeks after the challenge vaccination.

[0294]

[0295]

[0296] *a, b, c indicate statistical significance (p< 0.05)

[0297]

[0298] In the MHP challenge group, intranasal MHP antigen was observed from the second week after intratracheal MHP challenge. As shown in Table 28 below, nasal MHP antigen levels in the vaccine group were significantly lower than in the positive control group at both two and three weeks after challenge.

[0299]

[0300]

[0301] *a, b, c indicate statistical significance (p< 0.05)

[0302]

[0303]

[0304] Finally, in the Mhr and PCV2d mixed challenge vaccination group, the blood PCV2d content of the vaccine group and the positive control group was compared, and as shown in Table 29 below, the antigen content of the vaccine group was significantly lower than that of the positive control group from 1 week after the challenge vaccination, and the blood PCV2d antigen of the vaccine group was significantly lower than that of the positive control group continuously until 2 and 3 weeks after the challenge vaccination.

[0305]

[0306]

[0307] *a, b, c indicate statistical significance (p< 0.05)

[0308]

[0309] And we checked the Mhr antigen levels in the nasal cavity. Unlike Mhp, Mhr antigen was observed in the nasal cavity starting one week after the challenge vaccination. One week after the challenge vaccination, there was no significant difference in the Mhr antigen levels between the vaccine group and the positive control group. However, as shown in Table 30 below, the nasal Mhr antigen levels in the vaccine group were significantly lower than in the positive control group at two and three weeks after the Mhr challenge vaccination.

[0310]

[0311]

[0312] *a, b, c indicate statistical significance (p< 0.05)

[0313]

[0314]

[0315] 7.6 Histopathological evaluation

[0316] As a result of checking the lymph node lesions 3 weeks after PCV2d challenge vaccination, as shown in Table 31 below, the lymph node lesion index of the vaccine group was significantly lower than that of the positive control group.

[0317]

[0318]

[0319] *a, b, c indicate statistical significance (p< 0.05)

[0320]

[0321] In addition, as a result of evaluating the antigen level in the lymph nodes, the PCV2d antigen level in the vaccine group was significantly lower than that in the positive control group, as shown in Table 32 below.

[0322]

[0323]

[0324] *a, b, c indicate statistical significance (p< 0.05)

[0325]

[0326] As a result of examining lymph node lesions 3 weeks after PCV2a challenge vaccination, the lymph node lesion index of the vaccine group was significantly lower than that of the positive control group, as shown in Table 33 below.

[0327]

[0328]

[0329] *a, b, c indicate statistical significance (p< 0.05)

[0330]

[0331] And as a result of evaluating the antigen level in the lymph nodes, the PCV2a antigen level in the vaccine group was significantly lower than that in the positive control group (Table 34).

[0332]

[0333]

[0334] *a, b, c indicate statistical significance (p< 0.05)

[0335]

[0336] As a result of observing changes in macroscopic pneumonia lesions after Mhp challenge vaccination, as shown in Table 35 below, the macroscopic pneumonia lesion index of the vaccine group was significantly lower than that of the positive control group.

[0337]

[0338]

[0339] *a, b, c indicate statistical significance (p< 0.05)

[0340]

[0341] In addition, as shown in Table 36 below in the histopathological examination of the lungs, the histopathological pneumonia lesion scores of the vaccine group were significantly lower than those of the positive control group.

[0342]

[0343]

[0344] *a, b, c indicate statistical significance (p< 0.05)

[0345]

[0346] As a result of examining lymph node lesions 3 weeks after the combined challenge vaccination with Mhr and PCV2d, the lymph node lesion index of the vaccine group was significantly lower than that of the positive control group, as shown in Table 37 below.

[0347]

[0348]

[0349] In addition, as a result of evaluating the antigen level in the lymph nodes 3 weeks after the above-mentioned mixed challenge vaccination, the PCV2d antigen level in the lymph nodes of the vaccine group was significantly lower than that of the positive control group, as shown in Table 38 below.

[0350]

[0351]

[0352] *a, b, c indicate statistical significance (p< 0.05)

[0353]

[0354] As a result of comparing the changes in macroscopic lesions related to Mhr by the above-mentioned mixed challenge vaccination with the serosal lesions of the liver, the degree of macroscopic hepatic serositis lesions of the vaccine group was significantly lower than that of the positive control group, as shown in Table 39 below.

[0355]

[0356]

[0357] *a, b, c indicate statistical significance (p< 0.05)

[0358] In addition, as a result of examining the histopathological lesions of Mhr-related hepatic serositis, the degree of hepatic serositis lesions in the vaccine group was significantly lower than that in the positive control group, as shown in Table 40 below.

[0359]

[0360]

[0361] *a, b, c indicate statistical significance (p< 0.05)

Claims

1. A method for producing a vector, characterized by comprising the step of inserting a T7 terminator having a base sequence of sequence number 22 and a multiple cloning site (MCS) having a base sequence of sequence number 23 into a vector having a base sequence of sequence number 1.

2. A method for producing a vector, characterized in that the vector in the first paragraph has a base sequence of sequence number 2.

3. A method for producing a vector, characterized by including a step of converting a His tag in a vector having a base sequence of sequence number 2 into at least one selected from the group consisting of an HA tag, a Strep tag, and a FLAG tag.

4. A method for producing a vector, characterized in that in the third paragraph, the His tag has a base sequence of sequence number 24.

5. A method for producing a vector, characterized in that in the third paragraph, the HA tag, Strep tag, and FLAG tag each have a base sequence of sequence numbers 3 to 5.

6. A method for producing a vector, characterized in that the vector in the third paragraph has one of the base sequences of SEQ ID NOs: 6 to 8.

7. A method for producing a vector, characterized by comprising the step of introducing at least one selected from the group consisting of base sequences of sequence numbers 9 to 13 into a vector selected from the group consisting of base sequences of sequence numbers 2 and 6 to 8.

8. A method for producing a vector, characterized in that it comprises a step of introducing a base sequence of sequence number 9 into a vector having a base sequence of sequence number 2 in the 7th paragraph.

9. A method for producing a vector, characterized in that in paragraph 8, the vector has a base sequence of sequence number 14.

10. A method for producing a vector, characterized in that it comprises a step of introducing a base sequence of sequence number 10 into a vector having a base sequence of sequence number 6 in the 7th paragraph.

11. A method for producing a vector, characterized in that the vector in claim 10 has a base sequence of sequence number 15.

12. A method for producing a vector, characterized in that it comprises a step of introducing a base sequence of sequence number 11 into a vector having a base sequence of sequence number 7 in the 7th paragraph.

13. A method for producing a vector, characterized in that the vector in claim 12 has a base sequence of sequence number 16.

14. A method for producing a vector, characterized in that it further comprises a step of introducing a base sequence of sequence number 12 into a vector having a base sequence of sequence number 16 in the 13th paragraph.

15. A method for producing a vector, characterized in that the vector in claim 14 has a base sequence of sequence number 17.

16. A method for producing a vector, characterized in that it comprises a step of introducing a base sequence of sequence number 13 into a vector having a base sequence of sequence number 8 in the 7th paragraph.

17. A method for producing a vector, characterized in that the vector in claim 16 has a base sequence of sequence number 18.

18. A method for producing a vector, characterized in that the vector according to claims 1 to 17 is for producing a recombinant antigen related to swine wasting disease.

19. A step of obtaining a PCV2a base sequence from a vector having a base sequence of sequence number 15; and A method for producing a vector, characterized by comprising a step of introducing the PCV2a base sequence into a vector having the base sequence of sequence number 14.

20. A method for producing a vector, characterized in that in claim 19, the vector has a base sequence of sequence number 19 and is capable of obtaining PCV2a and PCV2d.

21. A step of obtaining a T_ BLS base sequence from a vector having a base sequence of sequence number 17; and A method for producing a vector, characterized by comprising a step of introducing the T_ BLS base sequence into a vector having the base sequence of sequence number 19.

22. A method for producing a vector, characterized in that in claim 21, the vector has a base sequence of sequence number 20 and is capable of obtaining PCV2a, PCV2d, and T_ BLS.

23. A step of obtaining a p65 base sequence from a vector having a base sequence of sequence number 18; and A method for producing a vector, characterized by comprising a step of introducing the p65 base sequence into a vector having the base sequence of sequence number 20.

24. A method for producing a vector, characterized in that in the 23rd paragraph, the vector has a base sequence of sequence number 21 and is capable of obtaining PCV2a, PCV2d, T_ BLS and p65.

25. A vector for producing a recombinant antigen related to swine wasting disease, comprising any one of the base sequences of SEQ ID NOs: 2, 6 to 8, and 14 to 21.

26. A vector for producing a recombinant antigen related to swine wasting disease, characterized in that it comprises any one of the base sequences of SEQ ID NOs: 19 to 21 in the 25th paragraph.

27. A vector for producing a swine wasting disease, characterized in that the vector in claim 26 is capable of producing PCV2a and PCV2d.

28. A vector for producing a recombinant antigen related to swine wasting disease, characterized in that it comprises the base sequence of sequence number 21 in claim 25.

29. A vector for producing a swine wasting disease-related product, characterized in that the vector in claim 28 is capable of producing PCV2a, PCV2d, T_ BLS, and p65.

30. A peptide manufactured using the vector of Article 25.

31. A multi-balanced peptide according to claim 30, characterized in that the peptide is at least two selected from the group consisting of PCV2a, PCV2d, T_ BLS and p65.

32. A vaccine composition for preventing or treating a swine wasting disease comprising the multi-balanced peptide of Article 31.

33. A vaccine composition for preventing or treating swine wasting disease, characterized in that it further comprises at least one of Mycoplasma hyopneumoniae (Mhp) cells and inactivated Mycoplasma hyorhinis (Mhr) cells in accordance with claim 32.

34. A vaccine composition according to claim 32, characterized in that the vaccine composition may additionally contain an immunostimulant.

35. A vaccine composition according to claim 32, characterized in that the vaccine composition can prevent or treat porcine circovirus type 2 infection, mycoplasma pneumonia, or mycoplasma peritonitis.

36. E. coli transformed with the vector of Article 25.

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