KR101527832B1 - ORF2 recombinant gene of porcine circovirus type 2, transfer vector, recombinant vaculovirus, recombinant capsid protein of porcine circovirus type 2 and manufacturing method thereof - Google Patents

Abstract

The present invention relates to a recombinant vector encoding a porcine circovirus type 2 (PCV2) outdoor segregation capsid protein, which comprises an ORF2 recombinant gene encoding the ORF2 gene comprising SEQ ID NO: 1 codon-optimized, a transfer vector comprising the ORF2 recombinant gene, (PCV2) recombinant capsid protein expressed by recombinant baculovirus and a method for producing the recombinant capsid protein.

Description

(ORF2 recombinant gene of porcine circovirus type 2 (PCV2), a transfer vector, a recombinant baculovirus, a porcine circovirus type 2 (PCV2) recombinant capsid protein and a production method thereof vaculovirus, recombinant capsid protein of porcine circovirus type 2 and manufacturing method thereof}

The present invention relates to an ORF2 recombinant gene, a transfer vector, a recombinant baculovirus, a porcine circovirus type 2 (PCV2) recombinant capsid protein of a porcine circovirus type 2 (PCV2) and a production method thereof, and more particularly to a porcine circovirus Type 2 (PCV2) outdoor isolation CASSID protein, an ORF2 recombinant gene in which the ORF2 gene consisting of SEQ ID NO: 1 is codon-optimized, a transfer vector comprising the ORF2 recombinant gene, a recombinant baculovirus transfected with a transfer vector, (PCV2) recombinant capsid protein expressed by recombinant baculovirus and a method for producing the recombinant capsid protein.

Pancreatic virus type 2 (PCV2) is known to be the cause of postweaning multisystemic wasting syndrome (PMWS) in the first place. However, not only PMWS but also porcine respiratory disease complex (PRDC) (PDVD), reproductive disorders, and digestive diseases, all of the diseases associated with this virus are called porcine circovirus diseases (PCVD). It is named.

The main symptom of PCVD is PMWS, which causes various symptoms such as cough, fever, pale skin and discoloration, respiratory disorder, feed intake and growth rate decrease, atrophy and diarrhea, Dysfunction is the most common symptom, and symptoms are worsened by multiple infections with other respiratory diseases such as porcine reproductive tract respiratory syndrome virus (PRRSV), pig parvovirus, and mycoplasma, rather than a single infection of PCV2.

PCV2 is a very small envelope-free virus with a size of about 17 nm and is resistant to external environments and disinfectants. Therefore, PCV2-infected farms are susceptible to recurrent infections and are difficult to eradicate because they are on top of most farms.

The DNA of PCV2 consists of ORF1, a gene related to viral replication, and ORF2, which encodes a capsid protein, which is recognized as a serologic marker of infection, in a circular form of 1.76 kb. ORF2 of PCV2 Genotypes such as PCV2a, PCV2b-1A, PCV2b-1B and PCV2b-1C can be categorized according to the genotype and severe PMWS occurrence after 2003 is reported to be highly related to PCV2b.

However, the virulence of these genotypes is still unclear, and conclusions can not be drawn on the pathogenicity of the results of artificial infections. In order to reduce the infectious damage of PCV2, farm hygiene and protection should be given priority, but in recent years, various kinds of vaccines have been developed and used to prevent such infections.

PCV2 vaccine development research areas include inactivated vaccine using PCV2 isolate, subunit vaccine using capsid protein, DNA vaccine, pseudorabies virus with recombinant ORF1 and OFR2 gene inserted, ORF2 gene inserted Adenovirus and a chimeric inactivated vaccine in which PCV1 and PCV2 are mixed.

The most successful of these vaccines is a vaccine that induces an immune response to the capsid protein of PCV2. It is also true that the use of these vaccines has reduced the incidence and the incidence of PCVD, including PMWS. For example, inactivated vaccines using chimeric viruses in which ORF1 of PCV1 and ORF2 of PCV2 are combined and vaccines containing recombinant capsid proteins using baculovirus are effective in the field.

PCV2a is a source virus of the above-mentioned commercialized vaccine antigen. Although the protective immunity induced by PCV2a may be inhibited by amino acid changes in the PCV2b capsid protein in theory, it has been shown that cross protection is achieved through experimental results. The development of PCVD vaccine using PCV2b has been successful, This vaccine is used in farms that suffer a lot of damage, and it has direct effects such as reduction of dead money,

However, when the vaccine is discontinued or when the immunity level of the vaccine is lowered, PCV2 re-infections may be caused by viruses excreted from swine-infected pigs or exogenous viruses.

Therefore, farms that suffer from a wasting disease should pay more attention to on-farm hygiene and specification management, as well as continuous PCV2 vaccination, especially in the management of pathogens that cause multiple infections.

In order to solve the above problems, the present invention provides a recombinant protein having a high expression rate by using a codon-optimized gene of PCV2, which is a major cause of swine-wasting disease in pigs, The technical problem is to provide.

It is another object of the present invention to provide a method for producing the recombinant protein which can inhibit infection more economically and effectively by inducing an immune response to PCV2.

In order to solve the above-mentioned problems, the present invention provides an ORF2 recombinant gene encoding a porcine circovirus type 2 (PCV2) outdoor segregation capsid protein and codon-optimized the ORF2 gene of SEQ ID NO: 1.

The present invention also provides a transfer vector comprising an ORF2 recombinant gene.

The present invention also provides a recombinant baculovirus transduced with said delivery vector.

The present invention also provides a porcine circovirus type 2 (PCV2) recombinant capsid protein expressed by said recombinant baculovirus.

The present invention also provides a method for producing a porcine circovirus type 2 (PCV2) recombinant capsid protein comprising culturing the recombinant baculovirus.

The highly immunogenic vaccine against PCV2 is determined by the content of the capsid protein, which is the main immunity inducible protein including the protective antibody, so that the ORF2 gene of PCV2 contains the codon-optimized ORF2 recombinant gene of the present invention to be effectively translated in insect cells The recombinant capsid protein expressed by the recombinant baculovirus transduced with the transduction vector of the present invention and the production method thereof can produce an immunogenic antigen in the same condition, And it is possible to prevent the PCV2 from being managed more efficiently by using it.

1 is shown as the restriction enzyme cleavage picture after the cloning of the PCV2 ORF2 gene outdoor isolates, M is a size marker, 1, and 2 of pGEMTPCV2vORF2 Bgl II And the lower band represents the PCV2vORF2 gene.
FIG. 2 shows the nucleotide sequence and amino acid sequence of the synthetic ORF2 gene (sORF2) by the ORF2 gene (vORF2) and the codon optimization of PCV2 outdoor isolation.
Figure 3 shows a map of the transfer vector (pBacPAK8PCV2vORF2) in which the ORF2 gene (vORF2) of PCV2 outdoor isolation was inserted.
Fig. 4 shows a map of the transfer vector (pBacPAK8PCV2sORF2) into which the synthetic ORF2 gene (sORF2) was inserted by codon optimization.
FIG. 5 is a fluorescence image of baculovirus (reBacPCV2sORF2, right) infected insect cells in which the ORF2 gene insertion baculovirus (reBacPCV2vORF2, left) and the synthetic ORF2 gene inserted by codon optimization are inserted into PCV2 outdoor isolation.
FIG. 6 shows a PCR-confirmed photograph of baculovirus (reBacPCV2vORF2, left) inserted with ORF2 gene of PCV2 outdoor isolation and baculovirus (reBacPCV2sORF2, right) inserted with synthetic ORF2 gene by codon optimization, wherein reBacPCV2vORF2 The amplification product is 722 bp, and the amplification product of reBacPCV2sORF2 is 538 bp.
Figure 7 shows the specific reactivity of the recombinant capsid protein (vCap, left) and the recombinant capsid protein (sCap, intermediate) using the ORF2 gene in the recombinant baculovirus-infected cells to the synthetic ORF2 gene by codon optimization Control cells, right).
FIG. 8 shows the expression levels (by infectious dose and infection time) of recombinant capsid protein (vCap, gray) and recombinant capsid protein (sCap, black) using the codon-optimized synthetic ORF2 gene using ORF2 gene for PCV2 outdoor isolation.
9 shows the expression levels (20 MOI infection, 3 days culture) of recombinant capsid protein (sCap, left) using the codon-optimized synthetic ORF2 gene and recombinant capsid protein (vCap, right) using the PCF2 outdoor ORF2 gene .
FIG. 10 shows the results of comparison of recombinant capsid proteins (vCap, gray) using PCF2 outdoor isolation ORF2 gene and recombinant capsid proteins (sCap, black) using a codon-optimized synthetic ORF2 gene to chicken .

The present invention relates to an ORF2 recombinant gene encoding a porcine circovirus type 2 (PCV2) outdoor isolation caascade protein and codon-optimized the ORF2 gene comprising SEQ ID NO: 1.

In the present invention, the recombinant gene may be an ORF2 recombinant gene comprising the nucleotide sequence of SEQ ID NO: 4.

In the present invention, the codon optimization can be performed by equilibrating the GC ratio of the entire ORF2 gene sequence of SEQ ID NO: 1, and reducing the number and length of nucleotide sequences of the helix structure.

The present invention also relates to a transfer vector comprising an ORF2 recombinant gene.

In the present invention, the transfer vector may be a transfer vector pBacPAK8PCV2sORF2 having a cleavage map as disclosed in FIG.

The present invention also relates to a recombinant baculovirus transduced with said delivery vector.

The present invention also relates to a recombinant capsid protein of porcine circovirus type 2 (PCV2) expressed by said recombinant baculovirus.

In the present invention, the expression ratio of the recombinant capsid protein expressed by the recombinant baculovirus is expressed by a recombinant baculovirus transfected with a transfer vector comprising the porcine circovirus type 2 (PCV2) outdoor sorting ORF2 gene The expression level of the recombinant capsid protein may be 1.5 to 2 times higher than that of the recombinant capsid protein.

In the present invention, the antibody-forming ability of the recombinant capsid protein is determined by measuring the antibody-forming ability of the recombinant capsid protein expressed by the recombinant baculovirus transduced with the transfer vector containing the ORF2 gene of the porcine circovirus type 2 (PCV2) 1.5 to 2 times higher.

In addition, the present invention relates to a method for producing recombinant capsid protein of porcine circovirus type 2 (PCV2) comprising culturing the recombinant baculovirus.

Hereinafter, the present invention will be described in detail.

In the present invention, a codon usage gene was synthesized and used to enhance the expression of PCV2 recombinant capsid protein using baculovirus. Namely, the ORF2 gene encoding the capsid protein was amplified from the outdoor PCV2 isolate, and the nucleotide sequence thereof was analyzed.

The optimized nucleotide sequence of each codon was modified so that the codon of the analyzed ORF2 gene could be translated most efficiently in insect cells. The codon optimized gene was artificially synthesized, and recombinant baculovirus was prepared by cloning and transfection.

In addition, recombinant baculovirus using the original isolate ORF2 gene was also produced, and the expression efficiencies of the recombinant capsid proteins were compared.

In the insect cells infected with the recombinant baculovirus having the same titer, the recombinant capsid protein (sCap) using the codon-optimized ORF2 gene was 1.5 to 2 times higher in expression efficiency than the recombinant capsid protein (vCap) using the PCF2 outdoor ORF2 gene , Which was confirmed by enzyme immunoassay (ELISA).

In addition, it was confirmed that the expression was enhanced by western blotting. As a result of inoculating the chicken by dissolving and ultrasonically disrupting the cells infected with the same reverse transfection, the sCap-infected individual was found to have antibody formation Was found to be 1,5 to 21 times higher than that of the control group.

Hereinafter, the present invention will be described in more detail by way of examples, but the scope of the present invention is not limited to the examples.

< Example  1> PCV2 of ORF2  gene Cloning

Virus DNA was extracted from the lymph node of piglets showing symptoms of porcine PMWS using DNA Mini kit (Qiagen). To clone the entire ORF2 gene (SEQ ID NO: 1) of PCV2, restriction enzyme Bgl II (5'-ga agatct tcATGacgtatccaaggag-3 ') of SEQ ID NO: 2 and a start codon (upper case) containing an initiation codon (upper case) Primer (5'ga agatct tcTTAgggtttaagtgggg-3 ') was prepared.

ORF2 gene amplification was performed by mixing 5 μl of extracted DNA, 1.5 mM MgCl ₂ , 1 × PCR buffer, 0.2 mM dNTP, 0.4 μM of each primer and 2.5 U of Taq DNA polymerase (Qiagen), filling the final 25 μl with sterile distilled water, Respectively. At this time, the temperature condition was repeatedly amplified 35 times at 94 ° C. for 5 minutes, at 94 ° C. for 30 seconds, at 55 ° C. for 45 seconds, at 72 ° C. for 45 seconds, and the reaction was continued at 72 ° C. for 10 minutes.

The 722 bp gene amplified by PCR was confirmed by electrophoresis on 1.5% agarose gel. The amplified gene on the agarose gel was purified using a gene cleaning kit (Q-biogen), cloned into pGEM-easy Vector (Promega), and sequenced. The corresponding wild-type sequence (vORF2) is shown in Fig.

< Example  2> PCV2 of ORF2  Gene codon optimization and synthesis

Based on the ORF2 gene sequence information of the outdoor PCV2 used in the present invention, the ORF2 codon was optimized in order to increase the translation efficiency of genes in animal and insect cells and to harvest more recombinant proteins.

That is, there is no change in the 233 amino acid sequences encoded by the outdoor ORF2 gene. However, the codon ratios most suitable for the desired insect cells are adjusted for each amino acid, and the equilibrium of the CG ratio of the entire nucleotide sequence and the base of the helix structure The final optimized nucleotide sequence was newly created through shortening the sequence number and the length. The optimized nucleotide sequence (sORF2) of SEQ ID NO: 4 is shown in FIG.

The optimized restriction endonuclease Bgl II &lt; RTI ID = 0.0 &gt; Site and initiation codon were added to synthesize a 722 bp nucleotide.

< Example  3> PCV2 of vORF2  And sORF2  Insertion of gene transfer vector

For PCV2 capsid protein expression using viral expression systems in baculovirus and vORF2 sORF2 gene of the Bgl pBacPAK8 (Clontech) inserted into the vector and transfer vector II .

Only vORF2 and sORF2 genes were extracted and purified on the agarose gel, and the truncated transfer vector was treated with Alkaline phosphatase. The vORF2 and sORF2 genes and T4 ligase were added to the transfer vector and reacted overnight at room temperature.

The conjugated plasmid was transformed into E. coli (DH5a), and the vector inserted in the forward direction was selected through restriction enzyme treatment. FIG. 3 shows a map of the transfer vector cloned with the vORF2 gene, and FIG. 4 shows a map of the transfer vector cloned with the sORF2 gene.

< Example  4> PCV2 of vORF2  And sORF2  Transduction of the gene

To prepare recombinant baculovirus, insect cells (Spodoptera frugiperda, Sf9) cultured in serum-free Sf-900 II medium (Invitrogen) were suspended at 4x10 ⁶ cells / ml and 5 ml each was added to 60 mm cell culture petri dish 27 C &lt; / RTI &gt;

After 1 hour, the medium was discarded and washed twice with fresh medium to remove cells that did not adhere and had poor cell activity. For transfection, the transfer vectors (pBacPAK8PCV2vORF2 and pBacPAK8PCV2sORF2) in which the vORF2 and sORF2 genes of PCV2 were inserted were purified in large quantities to a final concentration of 0.5 μg / μl.

Baculovirus DNA, into which vORF2 and sORF2 genes of PCV2 are inserted, can be easily identified by the presence of heat shock protein 40 (Hsp40) and heat shock congnate 70 protein (Hsc70), which are thermal shock proteins that help protein folding , Profold C1 (AB vector) with green fluorescence protein (GFP) gene inserted was used.

10 μl each of the transfer vectors was dispensed into separate tubes, 250 μl of the antibiotic and serum-free medium and 0.5 μg of Profold C1 were added and mixed lightly. 30 쨉 l of cellfectin (Invitrogen) and 250 쨉 l of the above medium were added to a separate tube, mixed gently, and allowed to stand at room temperature for 30 minutes.

The cellfectin-containing medium was added to a tube containing the baculovirus transfer vector and baculovirus DNA, mixed gently, allowed to stand at room temperature for about 20 minutes, and then carefully dispensed into the cells one drop at a time.

The transgenic Petri dishes were incubated at 27 DEG C for 4 hours, and then the supernatant was discarded and replaced with a medium supplemented with antibiotics and serum for 6 days. During the incubation period, the proliferation of recombinant baculovirus was confirmed by fluorescence microscopy.

After collecting the final culture medium, the fluorescence and cytopathic effect (CPE) were confirmed by re-inoculation with new insect cells. The collected virus culture was subdivided and stored at -70 ° C and 4 ° C, respectively.

FIG. 5 is a fluorescence image of baculovirus (reBacPCV2sORF2, right) infected insect cells in which the ORF2 gene insertion baculovirus (reBacPCV2vORF2, left) and the synthetic ORF2 gene inserted by codon optimization are inserted into PCV2 outdoor isolation.

< Example  5> Recombination Baculovirus  refine

The recombinant baculovirus was purified by a virus plaque assay in the first virus culture solution.

First, 4 ml of 0.5 × 10 ⁶ cells / ml Sf9 cells were inoculated into 8 60 mm cell culturing petri dishes and cultured for 2 hours. After washing twice, cell activity was observed through microscopic examination. Each virus culture solution was decanted by using a medium, and 10 ml of 10 ^-2 , 10 ^-3 , 10 ^-4 , and 10 ^-5 diluted virus culture solutions were added to each petri dish in an amount of 1 ml each.

A medium containing 1.25% SeaPalaque agarose preheated at 37 캜 was prepared during the slow rotation of the inoculated petri dish for 1 hour.

After removing the inoculum from each petridish that had been cultured for one hour, carefully dilute the medium containing agarose with 4 ml each, add petri dish to a box filled with water-moistened paper, and incubate at 27 ° C for 7 days . During the incubation period, CPE and several plaques showing fluorescence were collected and cultured according to the above method.

< Example  6> PCV2  Recombination Baculovirus  Confirm

DNA was extracted from each of the virus cultures that were cultured through a plaque assay. PCR was performed using DNA extracted to identify recombinant baculovirus (reBacPCV2vORF2) inserted with PCV2 vORF2 gene and recombinant baculovirus (reBacPCV2sORF2) inserted with PCV2 sORF2 gene.

(5'-gaagatcttcatgacgacgtatccaaggag-3 ') of SEQ ID NO: 5 and the reverse primer (5'-gaagatcttcttagggtttaagtgggg-3') of SEQ ID NO: 6 were used to confirm reBacPCV2vORF2 and SEQ ID NO: (5'-cagatggaggcgtaagaac-3 ') of SEQ ID NO: 8 and a reverse primer (5'-aaccctgatgttgtag tcctg-3'

As shown in FIG. 6, amplification of reBacPCV2vORF2 was confirmed by PCR using a primer for outdoor field, and amplification of reBacPCV2sORF2 was confirmed by PCR using a primer for a codon optimized synthetic gene. The final confirmed viral clones were mass-grown and stored at -70 ° C and 4 ° C, respectively.

< Example  7> PCV2  Recombination Capsid  Identification of proteins

To confirm the expression of the PCV2 recombinant capsid protein, the finally selected recombinant baculoviruses reBacPCV2vORF2 and reBacPCV2sORF2 were inoculated into Sf9 cells attached to 24 wells.

On the third day of culture, the supernatant was discarded, Sf9 cells were dried, and then cold 80% acetone was added to fix the cells for 10 minutes. The immobilized infected cells were washed once with PBS, and reacted with a monoclonal antibody (12C48, Median diagnostic) specifically reacting with the capsid protein of PCV2 for 1 hour at 37 ° C.

After washing three times, biotinylated anti-mouse IgG (biotinylated anti-mouse IgG, Vector Lab) was reacted at 37 ° C for 1 hour. After washing three times, avidin-bound peroxidase (avidin-HRPO) was reacted at 37 ° C for 1 hour.

After 3 washes, fresh diaminobenzidine (DAB) was added, the cells were stained, washed with distilled water, and then light microscopically examined.

Fig. 7 shows the result of staining PCV2 capsid protein expressed in the infected Sf9 cytoplasm.

< Example  8> PCV2 vCap and sCap of Incidence  compare

To compare the intracellular expression efficiency of vCAP and sCap of PCV2, the reactivated reBacPCV2vORF2 and reBacPCV2sORF2 viruses were inoculated with 2MOI and 20MOI, respectively, in flasks incubated in the same number of cells.

The infected cells were harvested from the 2nd to the 4th day of the inoculation, and the insect cell lysis buffer was added in the same manner, followed by sonication, and the supernatant was collected. The collected supernatant was diluted 100-fold with a coating buffer for enzyme immunoassay, and 100 μl was added to a plate for enzyme immunoassay (Nunc). The supernatant was fixed at 4 overnight.

The plate with the recombinant antigen immobilized was washed three times with a washing solution, and 150 μl of 3% skim milk was dispensed into each well, followed by reaction at 37 for 1 hour. After washing three times, 100 ㎕ of specific monoclonal antibody (12C48, Median diagnostic) against 0.5 / / ml of PCV2 capsid protein was dispensed and reacted at 37 캜 for 1 hour.

After washing 5 times, 100 쨉 l of 1 쨉 g / ml anti-mouse IgG (anti-mouse IgG, KPL) was dispensed and reacted at 37 째 C for 1 hour. After washing 5 times, 100 .mu.l of TMB (tetramethyl benzidine substrate solution, KPL) was added to each well and reacted at room temperature for 20 minutes. After completion of the reaction, 4N H2SO4 was added in an amount of 50 .mu.l each.

Plate absorbance was measured at 450 nm and vCap and sCap expression efficiencies were compared based on wells with the highest optical density (OD) values. As shown in FIG. 8, when the viruses with 2MOI and 20 MOI titers were inoculated, the expression rate of sCap through reBacPCV2sORF2 inoculation was about twice as high as that of vCap through the reBacPCV2vORF2 inoculation.

In order to compare the intracellular expression efficiency of vCap of PCV2 with sCap, reBacPCV2vORF2 and reBacPCV2sORF2 were inoculated with 20MOI, respectively, and cultured for 3 days, and then compared by western blotting using collected cell lysate As a result, it was confirmed that the color intensity of sCap was higher than that of vCap as shown in Fig.

< Example  9> PCV2 vCap and sCap of Immunity  compare

In order to compare the immunity induction of vCAP of PCV2 with sCap, reBacPCV2vORF2 and reBacPCV2sORF2 of 20 MOI were inoculated and cultured for 3 days, respectively. The collected cell lysate was diluted 10 times with PBS, and adjuvant (ISA70) Lt; / RTI &gt;

PCV2 vCap and sCap were inoculated with 1 ml of muscle in 5 laying hens and inoculated with the same dose in 3 weeks. After 3 weeks of the first inoculation, 4 weeks after the second inoculation and 8 weeks after the second inoculation in the inoculated laying hens, blood was collected from the brachial veins and the serum was separated.

To determine the level of antibodies against the PCV2 capsid protein present in each serum, measurements were made using a commercial broaching enzyme immunoassay (Blocking ELISA, Synbiotics). Each serum was diluted 1,000-fold and then added to each test well and tested according to manufacturer's instructions.

Serum titers were calculated according to the procedure (1-test serum OD / negative control OD) after measuring the OD values of each well at the final 450 nm. As shown in FIG. 10, it can be confirmed that the antibody formed in the sCap-inoculated chicken was higher than the vCap of PCV2.

When an antigen is produced by a recombinant capsid protein expressed by a recombinant baculovirus transfected with a transfer vector containing the ORF2 recombinant gene of the present invention which is codon-optimized so that the ORF2 gene of PCV2 is effectively translated in insect cells and a method for producing the recombinant capsid protein, , The production efficiency of the immunoantigen can be improved under the same conditions. Therefore, it is possible to produce an economical and effective recombinant vaccine of PCV2, and it is possible to more effectively prevent the PCV2 from being managed.

<110> Animal, Plant and Fisheries Quarantine and Inspection Agency <120> ORF2 recombinant gene of porcine circovirus type 2, transfer          vector, recombinant vaculovirus, recombinant capsid protein of          porcine circovirus type 2 and manufacturing method thereof <160> 8 <170> Kopatentin 1.71 <210> 1 <211> 702 <212> DNA <213> porcine circovirus type 2 <220> <221> gene <222> (1) <223> ORF2 gene <400> 1 atgacgtatc caaggaggcg ttaccgcaga agaagacacc gtccccgcag ccatcttggc 60 cagatcctcc gccgccgccc ctggctcgtc cacccccgcc accgctaccg ttggagaagg 120 aaaaatggca tcttcaacac ccgcctctcc cgcaccttcg gatatactgt caagcgtacc 180 acagtcacaa cgccctcctg ggcggtggac atgatgagat ttaagcttga cgactttgtt 240 cccccgggag gggggaccaa caaaatctct ataccctttg aatactacag aataagaaag 300 gttaaggttg aattctggcc ctgctccccc atcacccagg gtgatagggg agtgggctcc 360 actgctgtta ttctagatga taactttgta ccaaaggcca cagccctaac ctatgaccca 420 tatgtaaact actcctcccg ccatacaatc ccccaaccct tctcctacca ctcccgttac 480 ttcacaccca aacctgttct ggactccact attgattact tccaaccaaa taacaaaagg 540 aatcagcttt ggctgaggct acaaacctct agaaatgtgg accacgtagg cctcggcact 600 gcgttcgaaa acagtaaata cgaccaggac tacaatatcc gtgtaaccat gtatgtacaa 660 ttcagagaat ttaatcttaa agacccccca cttaaaccct aa 702 <210> 2 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> sense primer <400> 2 gaagatcttc atgacgtatc caaggag 27 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> antisense primer <400> 3 gaagatcttc ttagggttta agtgggg 27 <210> 4 <211> 702 <212> DNA <213> porcine circovirus type 2 <220> <221> gene <222> (1) <223> recombinant ORF2 gene <400> 4 atgacctatc cgagacgaag gtacagacga aggagacacc gtccacgtag tcatctgggc 60 caaatcctga ggaggcgacc atggttggtt catcccagac acaggtacag atggaggcgt 120 aagaacggca tcttcaacac gcgtctgtca aggaccttcg gatacacggt taagcgcacc 180 acagtgacta ccccttcttg ggcagtcgac atgatgcggt tcaagctgga cgactttgtg 240 ccacctggcg gcgggactaa caagatcagc atccctttcg agtactaccg catccgaaag 300 gtgaaggtgg aattctggcc ttgctctccc ataacgcagg gtgatagggg tgttggcagt 360 actgctgtca tcctggacga caacttcgtg ccgaaagcaa ctgcgctcac atacgaccca 420 tacgtgaact acagctctag gcacactatc cctcagccat ttagctacca ctccaggtac 480 tttaccccta aaccggtcct tgatagcacc atcgactact tccagcccaa caacaagcgg 540 aatcagcttt ggcttcgtct gcaaacctca cgtaacgtgg atcacgtggg cctcggaacg 600 gctttcgaga actccaagta cgaccaggac tacaacatca gggttacgat gtacgtgcag 660 ttcagggagt tcaacctgaa ggatccgcca cttaagccct aa 702 <210> 5 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 5 gaagatcttc atgacgtatc caaggag 27 <210> 6 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 6 gaagatcttc ttagggttta agtgggg 27 <210> 7 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 7 cagatggagg cgtaagaac 19 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 8 aaccctgatg ttgtagtcct g 21

Claims (10)

As a recombinant gene of ORF2 encoding a porcine circovirus type 2 (PCV2) outdoor isolating capsid protein and codon-optimized ORF2 gene of SEQ ID NO: 1,
Wherein the recombinant gene comprises the nucleotide sequence of SEQ ID NO: 4. delete delete A delivery vector comprising the ORF2 recombinant gene of claim 1. 5. The transfer vector of claim 4, wherein the transfer vector is a transfer vector pBacPAK8PCV2sORF2 having a cleavage map as disclosed in FIG. 4. Recombinant baculovirus transduced with the delivery vector of claim 4. A method for producing a porcine circovirus type 2 (PCV2) recombinant capsid protein comprising culturing the recombinant baculovirus of claim 6. 8. The method according to claim 7, wherein the expression ratio of the recombinant capsid protein expressed by said recombinant baculovirus is determined by recombinant baculovirus transfected with a transfer vector comprising the porcine circovirus type 2 (PCV2) (PCV2) recombinant capsid protein, wherein the expression level of the recombinant capsid protein is 1.5 to 2 times higher than that of the expressed recombinant capsid protein. The recombinant capsid protein according to claim 7, wherein the antibody-forming ability of the recombinant capsid protein is selected from the group consisting of an antibody of a recombinant capsid protein expressed by a recombinant baculovirus transfected with a transfer vector comprising the porcine circovirus type 2 (PCV2) (PCV2) recombinant capsid protein, which is characterized in that it is 1.5 to 2 times higher than that of the recombinant capsid protein of porcine circovirus type 2 (PCV2). delete

Images