KR102133632B1 - Recombinant Porcine circovirus and vaccine composition for preventing or treating Porcine circovirus- infected disease comprising the same - Google Patents

Abstract

The present invention relates to a vaccine composition for preventing a porcine circovirus infection disease including the recombinant porcine circovirus and a variety of applications thereof. The PCV2 recombinant virus of the present invention overcomes the disadvantage that the conventional single-stranded DNA virus, PCV2, proliferates extremely little in cells, and is recombined in the form of double-stranded DNA, thereby increasing the efficiency of production. In addition, since the effect of inducing antibody formation is excellent, it can be usefully used in related industries.

Description

Recombinant Porcine circovirus and vaccine composition for preventing or treating Porcine circovirus-infected disease comprising the same}

The present invention relates to a vaccine composition for preventing a porcine circovirus infection disease including the recombinant porcine circovirus and a variety of applications thereof.

PCV is a single-stranded DNA virus. PCV is a small genome-sized virus that self-replicates in cells. The virus is characterized by self-replicating in the nucleus using a host polymerase. Porcine serotypes of PCV are classified into type 1 (PCV1) and type 2 (PCV2), and type 1 is non-pathogenic, but type 2 is known as a pathogenic virus in pigs and is characterized by significant resistance to disinfectants. PCV2 is further divided into subtypes of PCV2a, PCV2b, PCV2c, and PCV2d.

The PCV2 is known as a causative agent of various diseases related to swine, and in particular, is a causative agent most problematic for postweaning multisystemic wasting syndrome (PMWS). The systemic wasting syndrome (PMWS) occurs between 6 and 16 weeks of age, when the parental antibody falls in piglets, especially in pigs of 8 to 12 weeks of age, and infected pigs are chronically atrophic and have skin It is characterized by pale and enlarged lymph nodes. In some infected pigs, respiratory symptoms such as dyspnea, chronic pneumonia, and digestive symptoms such as diarrhea and stomach ulcers may be present. Another important feature is the slow progression of the disease, which can be confused with other diseases by slow onset. Infection rates in weaned pigs vary and mortality rates are high (5-50%).

Currently, vaccines that have inactivated the virus as a vaccine against the PCV2a type have been commercialized in Korea. However, it has been known that the protective ability of the vaccine is very weak for the PCV2b type that has recently occurred in pig farms, and accordingly, the need to develop a new vaccine for the PCV2b type has emerged.

An object of the present invention can provide a PCV2 (Porcine circovirus type 2) recombinant virus.

In addition, an object of the present invention can provide a vaccine composition for preventing PCV2 infection disease.

It is also an object of the present invention to provide a diagnostic kit for PCV2.

It is also an object of the present invention to provide a PCV2 detection method.

In addition, an object of the present invention can provide a method for providing information for the prevention of PCV2 infection disease.

An object of the present invention can provide a PCV2 (Porcine circovirus type 2) recombinant virus.

In addition, an object of the present invention can provide a vaccine composition for preventing PCV2 infection disease.

It is also an object of the present invention to provide a diagnostic kit for PCV2.

It is also an object of the present invention to provide a PCV2 detection method.

In addition, an object of the present invention can provide a method for providing information for the prevention of PCV2 infection disease.

The PCV2 recombinant virus of the present invention overcomes the disadvantage that the conventional single-stranded DNA virus, PCV2, proliferates extremely little in cells, and is recombined in the form of double-stranded DNA, thereby increasing the efficiency of production. In addition, since the effect of inducing antibody formation is excellent, it can be usefully used in related industries.

1 is a diagram schematically showing recombinant PCV2b C5 separation and recombinant PCV2b C5 production of the present invention.
2 is a diagram analyzing the PCV2b C5 phylogenetic tree of the present invention.
3 is a view confirming the results of immunohistochemical staining in the group treated with the recombinant PCV2b C5 virus.
FIG. 4 is a diagram in which the present invention compares and evaluates the induction effect of antibody production in vivo for vaccination of PCV2b C5.

The present invention provides a PCV2 (Porcine circovirus type 2) recombinant virus comprising a nucleotide sequence represented by SEQ ID NO: 1 or a nucleotide sequence represented by SEQ ID NO: 2.

The PCV2 is one or more subtypes selected from the group consisting of PCV2a, PCV2b, PCV2c, and PCV2d, and is preferably not limited to the PCV2b subtype.

In addition, the present invention provides a recombinant vector comprising a nucleotide sequence represented by SEQ ID NO: 1 or a nucleotide sequence represented by SEQ ID NO: 1.

According to a preferred embodiment of the present invention, the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2 in the vector of the present invention is operatively linked to a promoter.

In the present invention, the term "operably linked" refers to functional binding between a nucleic acid expression control sequence (eg, an array of promoters, signal sequences, or transcriptional regulatory factor binding sites) and other nucleic acid sequences, thereby The regulatory sequence controls the transcription and/or translation of the other nucleic acid sequence.

The vector system of the present invention can be constructed through various methods known in the art, and specific methods thereof are disclosed in Sambrook et al. (2001), Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, This document is incorporated herein by reference.

Vectors of the invention can typically be constructed as vectors for cloning or vectors for expression. In addition, the vector of the present invention can be constructed using prokaryotic or eukaryotic cells as hosts. When the vector of the present invention is an expression vector, and a prokaryotic cell is a host, a strong promoter capable of progressing transcription (eg, tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter and T7 promoter, etc.), and a ribosome binding site for initiation of translation and a transcription/translation termination sequence are common. When E. coli (eg, HB101, BL21, DH5α, etc.) is used as a host cell, the promoter and operator site of the E. coli tryptophan biosynthetic pathway (Yanofsky, C. (1984), J. Bacteriol., 158:1018- 1024) and the phage λ leftward promoter (pLλ promoter, Herskowitz, I. and Hagen, D. (1980), Ann. Rev. Genet., 14:399-445) can be used as a regulatory site.

On the other hand, when the vector of the present invention is an expression vector and a eukaryotic cell is a host, a promoter derived from the genome of a mammalian cell (eg, a metallothionine promoter) or a promoter derived from a mammalian virus (eg, adeno) Late virus promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter and HSV's four promoters) can be used and generally have a polyadenylation sequence as a transcription termination sequence.

The vector of the present invention may be fused with other sequences as necessary to facilitate purification of the protein, and the fused sequences may be, for example, glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA ), FLAG (IBI, USA) and 6x His (hexahistidine; Quiagen, USA) may be used, but are not limited thereto. In addition, the expression vector of the present invention, as a selection marker, may include an antibiotic resistance gene commonly used in the art, for example, ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin , There are genes for resistance to neomycin and tetracycline.

In addition, the recombinant vector comprising the nucleotide sequence of SEQ ID NO: 2 of the present invention is introduced into prokaryotic host cells or eukaryotic host cells in various ways, CaCl ₂ method (Cohen, SN et al. (1973), Proc. Natl. Acac. Sci. USA, 9:2110-2114), one method (Cohen, SN et al. (1973), Proc. Natl. Acac. Sci. USA, 9:2110-2114; and Hanahan, D. (1983), J. Mol. Biol., 166:557-580) and electroporation methods (Dower, WJ et al. (1988), Nucleic. Acids Res., 16:6127-6145).

The PCV2 recombinant virus may be represented by the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and variants of the nucleotide sequence are included within the scope of the present invention. The nucleic acid molecule that can be used as the gene of the present invention is a functional equivalent of the nucleic acid molecule constituting it, for example, some base sequences of the nucleic acid molecule have been modified by deletion, substitution, or insertion. , It is a concept that includes variants that can function functionally the same as a nucleic acid molecule. Specifically, the gene has a sequence homology of 70% or more, more preferably 80% or more, even more preferably 90% or more, and most preferably 95% or more with the base sequence of SEQ ID NO: 1 or SEQ ID NO: 2, respectively. Branches can include nucleotide sequences. “% of sequence homology” to a polynucleotide is identified by comparing two optimally aligned sequences with a comparison region, and a portion of the polynucleotide sequence in the comparison region is a reference sequence (addition or deletion) for the optimal alignment of the two sequences. (Not including), but may include addition or deletion (ie, gap).

In addition, the present invention provides a recombinant cell in which a recombinant viral vector comprising a nucleotide sequence represented by SEQ ID NO: 1 or a nucleotide sequence represented by SEQ ID NO: 2 is introduced.

The recombinant cells may be cultured if necessary, and in the present invention, the term "cultivation" means to grow microorganisms/cells under artificially controlled environmental conditions.

Recombinant cells of the present invention can be grown in a normal medium, and for the purpose of culturing a specific microorganism/cell, a culture target, that is, a nutrient that is required by a microorganism/cell that becomes a culture medium, contains a substance for a special purpose, and is additionally added. And may be mixed. The medium is also referred to as a culture medium or a culture medium, and is a concept including both natural medium, synthetic medium, or selective medium.

The medium used for cultivation must satisfy the requirements of a specific strain in an appropriate manner while controlling temperature, pH, etc. in a normal medium containing a suitable carbon source, nitrogen source, amino acid, vitamin, and the like. As a carbon source that can be used, mixed sugars of glucose and xylose are used as the main carbon source. In addition, sugars and carbohydrates such as sucrose, lactose, fructose, maltose, starch, and cellulose, soybean oil, sunflower oil, castor oil, and coconut Oils and fats such as oil, fatty acids such as palmitic acid, stearic acid and linoleic acid, alcohols such as glycerol and ethanol, and organic acids such as acetic acid. These materials can be used individually or as a mixture. Examples of nitrogen sources that can be used include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, anmonium carbonate, and ammonium nitrate; Amino acids such as glutamic acid, methionine and glutamine, and organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or its degradation products, skim soy cake or degradation products thereof Can. These nitrogen sources may be used alone or in combination. The medium may include potassium phosphate, potassium phosphate, and the corresponding sodium-containing salt as personnel. Personnel that can be used include potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salt. In addition, as the inorganic compound, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate and calcium carbonate may be used. Finally, in addition to the above materials, essential growth materials such as amino acids and vitamins can be used.

In addition, precursors suitable for the culture medium can be used. The above-mentioned raw materials may be added in a batchwise, fed-batch, or continuous manner in an appropriate manner to the culture in the culture process, but are not particularly limited thereto. The pH of the culture can be adjusted by using a basic compound such as sodium hydroxide, potassium hydroxide or ammonia or an acid compound such as phosphoric acid or sulfuric acid in an appropriate manner.

In addition, the present invention provides a vaccine composition for preventing a PCV2 infection disease, comprising a PCV2 recombinant virus comprising a base sequence represented by SEQ ID NO: 1 or a base sequence represented by SEQ ID NO: 2 as an active ingredient.

The PCV2 infection disease is Post-weaning Multisystemic Wasting Syndrome (PMWS), Pseudodorabies, Porcine Reproductive and Respiratory Syndrome (PRRS), Glasser's disease, Streptococcus One or more selected from the group consisting of streptococcal meningitis, salmonellosis, postweaning colibacillosis, dietic hepatosis, and suppurative bronchopneumonia.

The vaccine composition may further include one or more selected from the group consisting of a solvent, an adjuvant, and an excipient.

The vaccine composition may be at least one selected from the group consisting of live vaccines, attenuated vaccines, and dead vaccines.

In the present invention, the term, "immunity enhancer (adjuvant)" is a material that can be used in the development of vaccines by increasing the antigenicity or by enhancing the non-specific immune response to the antigen, such as treatment and prevention. Immuno-adjuvants are used when preparing vaccines because they act to maintain the immune response against antigens quickly, powerfully, and for a long time when the amount of antigen is low. It is possible to control or to control the type, subtype, etc. of the antibody against the antigen. The use of adjuvants can increase the efficacy of the vaccine and protect it from a wide range of viral infections. For the purposes of the present invention, it is a virus that causes PCV2 infectious diseases, but is not limited thereto.

The term "vaccine" in the present invention is a biological agent containing an antigenic substance that immunizes a living body, and refers to an immunogen that immunizes a living body by injecting or injecting it into a living body for the prevention of foot and mouth disease.

The vaccine of the present invention may be for animals, preferably for animals of cattle, and more preferably for cattle or pigs. Most preferably for pigs. However, the type of animal is not limited thereto.

The present invention may include a pharmaceutical composition in addition to the vaccine composition, and may further include an adjuvant in addition to the dextran polymerized nanoparticles. The adjuvant may be used without limitation as long as it is known in the art, but for example, Freund's complete adjuvant or incomplete adjuvant may be further included to increase its effect.

The vaccine composition or pharmaceutical composition according to the present invention may be prepared in the form of an active ingredient incorporated into a pharmaceutically acceptable carrier. Here, pharmaceutically acceptable carriers include carriers, excipients and diluents commonly used in the pharmaceutical field. Pharmaceutically acceptable carriers that can be used in the pharmaceutical compositions of the present invention are not limited to these, but are lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, Calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The vaccine composition or pharmaceutical composition of the present invention is formulated in the form of an oral dosage form such as a powder, a granule, a tablet, a capsule, a suspension, an emulsion, a syrup, an aerosol, an external preparation, a suppository, or a sterile injection solution, according to a conventional method. Can be used.

In the case of formulation, it may be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc., which are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient in an active ingredient such as starch, calcium carbonate, sucrose, lactose, and gelatin. It can be prepared by mixing the back. In addition, lubricants such as magnesium stearate and talc may be used in addition to simple excipients. Liquid preparations for oral administration include suspending agents, intravenous solutions, emulsions, syrups, etc. In addition to commonly used diluents such as water and liquid paraffin, various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, are included. Can. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories. Non-aqueous solvents, suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As a base for suppositories, witepsol, tween 61, cacao butter, laurin butter, and glycerogelatin may be used.

The vaccine composition or pharmaceutical composition according to the present invention can be administered to a subject by various routes. Any mode of administration can be envisaged, for example, intramuscular, subcutaneous, intraperitoneal, intravenous, oral, dermal, intraocular and brain administration.

The dosage of the vaccine composition or pharmaceutical composition according to the present invention is selected in consideration of the age, weight, sex, and physical condition of the individual. It is obvious that the concentration of the dextran polymerized nanoparticles contained in the vaccine composition or pharmaceutical composition can be variously selected depending on the subject, and is preferably included in the pharmaceutical composition at a concentration of 0.01 to 5,000 μg/ml. If the concentration is less than 0.01 μg/ml, the pharmaceutical activity may not appear, and when it exceeds 5,000 μg/ml, it may exhibit toxicity to the human body.

In addition, the present invention provides a diagnostic kit for PCV2, comprising a PCV2 recombinant virus comprising a nucleotide sequence represented by SEQ ID NO: 1 or a nucleotide sequence represented by SEQ ID NO: 2.

The diagnostic kit can be prepared using methods commonly used in the art.

Such a diagnostic kit includes a PCV2 recombinant virus or antigen containing at least one of the nucleotide sequences of SEQ ID NO: 1 or SEQ ID NO: 2, as well as tools, reagents, and the like commonly used in the art for immunological analysis. do. Such tools/reagents include, but are not limited to, suitable carriers, labeling substances capable of generating detectable signals, solubilizers, detergents, buffers, stabilizers, and the like. When the labeling substance is an enzyme, a substrate capable of measuring enzyme activity and a reaction stopper may be included. Suitable carriers include, but are not limited to, soluble carriers, such as physiologically acceptable buffers known in the art, such as PBS, insoluble carriers, such as polystyrene, polyethylene, polypropylene, polyester, Polymers such as polyacrylonitrile, fluorine resin, crosslinked dextran, polysaccharide, magnetic fine particles plated with metal in latex, other paper, glass, metal, agarose, and combinations thereof.

In addition, the present invention provides a PCV2 detection method comprising the step of detecting a PCV2 recombinant virus comprising a nucleotide sequence represented by SEQ ID NO: 1 or a nucleotide sequence represented by SEQ ID NO: 2.

The virus may further include its antigen, and may detect PCV2 in infected or infected cells through an antigen-antibody reaction. The antigen-antibody reaction includes tissue immunostaining, radioimmunoassay (RIA), enzyme immunoassay (ELISA), western blotting, immunoprecipitation assay, immunodiffusion assay, and complement fixation assay (Complement Fixation Assay), FACS (Fluorescence-activated cell sorter) and one or more methods selected from the group consisting of protein chip (protein chip) analysis method can be used.

In addition, the present invention provides a method for providing information for the prevention of PCV2 infection disease, comprising the step of administering the vaccine composition to mammals other than humans.

The mammal is at least one selected from the group consisting of pig, cow, bat, red fox, skunk, raccoon, badger, dog, wolf, mongoose, coyote, ferret, dog and cat, preferably pig, but limited thereto Does not work.

The present invention will be described in more detail through the following examples. However, the following examples are only intended to materialize the contents of the present invention, and the present invention is not limited thereby.

<Preparation Example 1> PCV2b C5 virus production

<1-1> Recombinant PCV2b C5 construction

PCV2 C5 was isolated from Porcine circovirus (PCV) 2 (FIG. 1A). Primers were prepared using the conserved sequence of PCV2, and PCR amplification was performed. Cloning was performed using T-blunt, and the full-length genome was analyzed through sequencing, followed by Clone C5.

In order to manufacture circular PCV2b C5, the base sequence of Replicase was partially modified to make Sal Isite and amplified by PCR. The amplified DNA was cloned using T-blunt. Thereafter, after cutting with Sal I, recombinant PCV2b C5 in a circular form was produced through self ligation (B in FIG. 1 ).

<1-2> Transfection of Recombinant PCV2b C5

PK-15 cells were prepared in a 96-well plate at 5X10 ⁴ Cells/ml. Prototype recombinant PCV2b C5 prepared by self-ligation was transfected with lipofectamine 3000 in a PK-15 cell line. After 6 hours of transfection, the medium was changed to new media, and 2 days later, the recombinant PCV2 C5 was stained.

<Preparation Example 2> Immunohistochemistry of recombinant PCV2b C5

Two days after transfection, PK-15 was fixed with 10% Formalin. After removing 10% Formalin using PBS, permeabilization is performed using 0.2% TritonX100. After washing with PBS again, the PCV2 antibody was treated and stored at 4°C overnight. The next day, after removing PCV2 antibody using PBS, Goat anti mouse (secondary antibody) is treated at room temperature for 1 hour. And DAB staining was performed.

<Preparation Example 3> Efficacy evaluation of PCV2b C5 inactivated vaccine

Guinea pigs were inoculated subcutaneously with 1 ml of the PCV2b C5 inactivated vaccine (105.5 TCID50/ml) prepared to evaluate the efficacy of the fluorinated vaccine. To compare and evaluate the efficacy of the vaccine, commercially available vaccines and antibody production were compared by ELISA method. The antibody production amount was Sandwich indirect ELISA method, and 10 μg/ml of PCV2 mAb antibody was coated on an ELISA plate at 37° C. for 2 hours, and then washed 5 times with 300 μl of PBST (Tween20 0.05%). To minimize non-specific binding, 200 μl of 3% skim milik was used to react at 37° C. for 2 hours. After washing 5 times with 300 μl of PBST, the PCV2b C5 antigen of the present invention was diluted 1/10 and coated with 100ul aliquots (1/10dose/ml, 37° C., 2 hours). After washing 5 times with 300 µl PBST, the guinea pig serum was diluted 1:50, and the final 100 µl serum was reacted at 37° C. for 2 hours. The non-specific reaction was washed 5 times with 300 μl of PBST, and then diluted with anti-Guinea pig-IgG HRP to 1:2000, reacted at 37° C. for 1 hour, and absorbance was measured at 450 nm to compare antibody production.

turn group Inoculation Inoculation method One Control (PBS) 1 ml SC 2 PCV2b C5 1 ml SC 3 A company 0.5ml SC 4 B company 0.5ml SC 5 C company 0.5ml SC 6 D company 1 ml SC 7 E company 1 ml SC 8 F company 1 ml SC

<Example 1> Full-length genome sequence analysis of PCV2b C5 clone

PCV2 C5 of the present invention isolated from wild-type PCV2 was identified by the full-length genome through sequencing, and the sequencing thereof was shown as SEQ ID NO: 1. Whole genome digestion for Clone C5 and amino acid sequence analysis of the capsid protein of the virus were performed. Specifically, amino acid sequence analysis was performed using the Megalign clustal W comparison method of the Lagergene program (Dnastar Inc. WI, USA). To confirm the subtype type of PCV2 C5, genome and capsid sequences of PCV2 subtypes identified in the GenBank database were used as a comparison group. Finally, phylogenetic analysis was performed using phylogenetic analysis of Megalign program based on the comparative sequence shown in Megalign clustal W.

As a result, it was confirmed that the isolated PCV2 C5 was a PCV2b type as a result of whole genome analysis and capsid protein amino acid sequence analysis.

<Example 2> Recombinant PCV2b C5 genomic sequence analysis

Recombinant PCV2b C5 clone was produced by partially changing the base sequence of Replicase of PCV2. The Sal I enzyme site was inserted into the replicase of PCV2 through PCR. Then, after cutting with Sal I, recombinant PCV2b C5 was produced through self-ligation, and base sequence analysis thereof was performed.

As a result, the recombinant PCV2b C5 of the present invention was identified as the nucleotide sequence represented by SEQ ID NO: 2.

<Example 3> PCV2b C5 recombinant virus production confirmation of the present invention

The recombinant PCV2b C5 clone of the present invention was introduced into PK-15 cells, and it was confirmed whether the virus generation of the recombinant PCV2b C5 clone was correct. Specifically, as a control group, PK-15 cell alone group, wild-type PCV2b virus infection group, and the recombinant PCV2b C5 clone transfection group of the present invention, each group using PCV2b antibody immunohistochemical staining as in Preparation Example 2 ( Staining by Immunohistochemistry) method.

As shown in FIG. 3, it was confirmed that the PK-15 cells transfected with the recombinant PCV2b C5 virus of the present invention of the present invention were stained brown, in the same manner as the cells infected with the wild-type PCV2b C5 virus used as a positive control. Therefore, it was confirmed that the recombinant PCV2b C5 virus of the present invention was produced in PK-15 cells.

Therefore, it was confirmed that the PCV2b C5 recombinant virus of the present invention was produced with high efficiency in cells. Therefore, it suggests that the conventional PCV2 viral genome replicates a rolling-circle replication mechanism, proliferating extremely little in cells and overcoming the disadvantage of low productivity.

<Example 4> Vaccine effect using the PCV2b C5 recombinant virus of the present invention

In order to verify the vaccine effect of the PCV2b C5 recombinant virus of the present invention, the guinea pig was inoculated with the PCV2b C5 inactivated vaccine, and the negative control group was divided into PBS and the conventional products produced by six companies as a comparison group, and vaccinated by group. Proceeded. Blood was collected 3 weeks after vaccination, and the antibody titer was confirmed using a sandwich indirect ELISA method.

As shown in FIG. 4, as a result of comparing and evaluating the induction effect of antibody production in vivo, PCV2b C5 was relatively high compared to the comparison group as a result of confirming the antibody titer for the PCV2b C5 virus of the present invention compared to the conventionally produced product. It was confirmed that the antibody production effect.

Therefore, the PCV2b C5 recombinant virus of the present invention has a higher antibody production effect than the conventional product, suggesting that the antibody formation-inducing effect against PCV2b is excellent.

<110> The Industry & Academic Cooperation in Chungnam National University (IAC) <120> Recombinant Porcine circovirus and vaccine composition for preventing or treating Porcine circovirus- infected disease comprising the same <130> PN1803-099 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 1767 <212> DNA <213> Artificial Sequence <220> <223> Clone C5 <400> 1 atgcccagca agaagaatgg aagaagcgga ccccaacccc ataaaaggtg ggtgttcact 60 ctgaataatc cttccgaaga cgagcgcaag aaaatacggg atcttccaat atccctattt 120 gattatttta ttgttggcga ggagggtaat gaggaaggac gaacacctca cctccagggg 180 ttcgctaatt ttgtgaagaa gcagactttt aataaagtga agtggtattt gggtgcccgc 240 tgccacatcg agaaagccaa aggaacagat cagcagaata aagaatactg cagtaaagaa 300 ggcaacttac tgattgagtg tggagctcct agatctcagg gacaacggag tgacctgtct 360 actgctgtga gtaccttgtt ggagagcggg agtctggtga ccgttgcaga gcagtaccct 420 gtaacgtttg tcagaaattt ccgcgggctg gctgaacttt tgaaagtgag cgggaaaatg 480 cagaagcgtg attggaagac taatgtacac gtcattgtgg ggccacctgg gtgtggtaaa 540 agcaaatggg ctgctaattt tgcagacccg gaaaccacat actggaaacc acctagaaac 600 aagtggtggg atggttacca tggtgaagaa gtggttgtta ttgatgactt ttatggctgg 660 ctgccctggg atgatctact gagactgtgt gatcgatatc cattgactgt agagactaaa 720 ggtggaactg tacctttttt ggcccgcagt attttgatta ccagcaatca gaccccgttg 780 gaatggtact cctcaactgc tgtcccagct gtagaagctc tttatcggag gattacttcc 840 ttggtatttt ggaagaatgc tacagaacaa tccacggagg aagggggcca gttcgtcacc 900 ctttcccccc catgccctga atttccatat gaaataaatt actgagtctt ttttatcact 960 tcgtaatggt ttttattatt cattaagggt taagtggggg gtctttaaga ttaaattctc 1020 tgaattgtac atacatggtt acacggatat tgtattcctg gtcgtttata ctgttttcga 1080 acgcagtgcc gaggcctacg tggtctacat ttccagcagt ttgtagtctc agccacagct 1140 ggtttctttt gttgtttggt tggaagtaat caatagtgga atctaggaca ggtttggggg 1200 taaagtagcg ggagtggtag gagaagggct gggttatggt atggcgggag gagtagttta 1260 cataggggtc ataggtgagg gctgtggcct ttgttacaaa gttatcatct agaataacag 1320 cactggagcc cactcccctg tcaccctggg tgatcgggga gcagggccag aattcgacct 1380 taacctttct tattctgtag tattcaaagg gcacagagcg ggggtttgag ccccctcctg 1440 ggggaagaaa gtcattaata ttgaatctca tcatgtccac cgcccaggag ggcgttttga 1500 ctgtggttcg cttgatagta tatccgaagg tgcgggatag gcgggtgttg aagatgccat 1560 ttttccttct ccagcggtaa cggtggcggg ggtggacgag ccaggggcgg cggcggagga 1620 tctggccaag atggctgcgg gggcggtgtc ttcttctccg gtaacgcctc cttggatacg 1680 tcatatctga aaacgaaaga agtgcgctgt aagtattacc agcgcacttc ggcagcggca 1740 gcacctcggc agcacctcag cagcaac 1767 <210> 2 <211> 1767 <212> DNA <213> Artificial Sequence <220> <223> PCV2b C5 <400> 2 atgcccagca agaagaatgg aagaagcgga ccccaacccc ataaaaggtg ggtgttcact 60 ctgaataatc cttccgaaga cgagcgcaag aaaatacggg atcttccaat atccctattt 120 gattatttta ttgttggcga ggagggtaat gaggaaggac gaacacctca cctccagggg 180 ttcgctaatt ttgtgaagaa gcagactttt aataaagtga agtggtattt gggtgcccgc 240 tgccacatcg agaaagccaa aggaacagat cagcagaata aagaatactg cagtaaagaa 300 ggcaacttac tgattgagtg tggagctcct agatctcagg gacaacggag tgacctgtct 360 actgctgtga gtaccttgtt ggagagcggg agtctggtga ccgttgcaga gcagtaccct 420 gtaacgtttg tcagaaattt ccgcgggctg gctgaacttt tgaaagtgag cgggaaaatg 480 cagaagcgtg attggaagac taatgtacac gtcattgtgg ggccacctgg gtgtggtaaa 540 agcaaatggg ctgctaattt tgcagacccg gaaaccacat actggaaacc acctagaaac 600 aagtggtggg atggttacca tggtgaagaa gtggttgtta ttgatgactt ttatggctgg 660 ctgccctggg atgatctact gagactgtgt gatcgatatc cattgactgt agagactaaa 720 ggtggaactg tacctttttt ggcccgcagt attttgatta ccagcaatca gaccccgttg 780 gaatggtact cctcaactgc tgtcccagct gtagaagctc tttatcggag gattacttcc 840 ttggtatttt ggaagaatgc tacagaacag tcgacggagg aagggggcca gttcgtcacc 900 ctttcccccc catgccctga atttccatat gaaataaatt actgagtctt ttttatcact 960 tcgtaatggt ttttattatt cattaagggt taagtggggg gtctttaaga ttaaattctc 1020 tgaattgtac atacatggtt acacggatat tgtattcctg gtcgtttata ctgttttcga 1080 acgcagtgcc gaggcctacg tggtctacat ttccagcagt ttgtagtctc agccacagct 1140 ggtttctttt gttgtttggt tggaagtaat caatagtgga atctaggaca ggtttggggg 1200 taaagtagcg ggagtggtag gagaagggct gggttatggt atggcgggag gagtagttta 1260 cataggggtc ataggtgagg gctgtggcct ttgttacaaa gttatcatct agaataacag 1320 cactggagcc cactcccctg tcaccctggg tgatcgggga gcagggccag aattcgacct 1380 taacctttct tattctgtag tattcaaagg gcacagagcg ggggtttgag ccccctcctg 1440 ggggaagaaa gtcattaata ttgaatctca tcatgtccac cgcccaggag ggcgttttga 1500 ctgtggttcg cttgatagta tatccgaagg tgcgggatag gcgggtgttg aagatgccat 1560 ttttccttct ccagcggtaa cggtggcggg ggtggacgag ccaggggcgg cggcggagga 1620 tctggccaag atggctgcgg gggcggtgtc ttcttctccg gtaacgcctc cttggatacg 1680 tcatatctga aaacgaaaga agtgcgctgt aagtattacc agcgcacttc ggcagcggca 1740 gcacctcggc agcacctcag cagcaac 1767

Claims (10)

PCV2b (Porcine circovirus type 2) recombinant virus consisting of the nucleotide sequence represented by SEQ ID NO: 2. delete A vaccine composition for preventing a PCV2b infection disease, comprising a PCV2b recombinant virus comprising a nucleotide sequence represented by SEQ ID NO: 2 as an active ingredient. The method of claim 3, wherein the PCV2b infection disease is Post-weaning Multisystemic Wasting Syndrome (PMWS), Pseudodorabies, Porcine Reproductive and Respiratory Syndrome (PRRS), Glaser's disease. disease), streptococcal meningitis, salmonellosis, postweaning colibacillosis, dietary hepatosis, and suppurative bronchopneumonia The vaccine composition. The vaccine composition of claim 3, wherein the vaccine composition further comprises one or more selected from the group consisting of a solvent, an adjuvant and an excipient. The vaccine composition according to claim 3, wherein the vaccine composition is at least one selected from the group consisting of live vaccines, attenuated vaccines, and dead vaccines. The vaccine composition according to claim 3, wherein the vaccine composition is administered through one or more administration routes selected from the group consisting of muscle, subcutaneous, intraperitoneal, intravenous, oral, dermal, ocular and brain. A diagnostic kit for PCV2b, comprising a PCV2b recombinant virus consisting of the nucleotide sequence represented by SEQ ID NO: 2. Comprising the step of detecting a PCV2b recombinant virus consisting of the nucleotide sequence represented by SEQ ID NO: 2, PCV2b detection method. A method of providing information for the prevention of PCV2b infectious diseases, comprising administering the vaccine composition of claim 3 to a mammal other than humans.

Images