# Patent

## Title
Porcine circovirus type-2 (PCV2) subunit vaccine

## Abstract

A porcine circovirus type-2 (PCV2) immunogenic composition includes an antigenic peptide. The antigenic peptide is a non-arginine-rich peptide of a PCV2 open reading frame 2 (ORF2) and/or a recombinant fusion protein having the non-arginine-rich peptide of the PCV2 ORF2, a PE peptide, and a KDEL signal peptide. The number of arginines of the non-arginine-rich peptide of the PCV2 ORF2 is not greater than half of the number of arginines of the arginine-rich domain of the N terminal of the PCV2 ORF2.

## Background
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention is related to a porcine circovirus type 2 (PCV2) subunit vaccine, particularly to a PCV2 subunit vaccine having a peptide of PCV2 open reading frame (ORF2) that can be abundantly expressed as an antigen and an additionally proper carrier or adjuvant.
2. Description of the Prior Art
It is known that porcine circovirus type 2 (PCV2) is related to postweaning multisystemic wasting syndrome (PMWS) and porcine dermatitis and nephropathy syndrome (PDNS). PMWS was first found in pigs in Canada in 1991 and has been reported subsequently around the world. The syndrome has caused a huge loss in swine production industry worldwide. Main symptoms of PMWS include progressive weight loss, tachypnea, dyspnea, jaundice, et cetera. Visibly pathological changes in tissue include lymphocytic and granulomatous infiltrate, lymphadenopathy, lymphocytic and granulomatous hepatitis, and nephritis.
Porcine circovirus (PCV) was first recognized in a pig kidney cell line (PK-15, ATCC CCL33) in 1982. Although the porcine circovirus can continuously contaminate PK-15 cells, the virus does not cause cytopathic effect (CPE) in the contaminated PK-15 cells. Even though the porcine circovirus can infect pigs, it does not cause lesions in the infected pigs. The virus is named porcine circovirus type 1 (PCV1). PCV1 is an icosahedron, single-stranded DNA virus with a circular genome of 1,759 bp. The PK-15-derived PCV was classified in the Circoviridae family in 1995.
The PK-15-derived PCV1 is considered apathogenic. In contrast, the virus mutation isolated from pigs with PMWS in 1997 is pathogenic and is named porcine circovirus type 2 (PCV2). Postweaning Multisystemic Wasting Syndrome (PMWS) is a highly contagious pig disease. It mainly infects pregnant sows and their piglets and seriously affects health of pigs.
PCV2 is a single-stranded, circular DNA virus with a diameter of 17 nm, and its genome size is 1.76 kb. Genomic analysis with software shows a total of 11 open reading frames (ORFs) transcribed in the clockwise and counterclockwise directions. Among the 11 ORFs, ORF1 and ORF2 are probably the most important genes. ORF1 gene encodes Rep and Rep′ proteins, which are related to virus replication. It is known that ORF2 gene encodes immunogenic structure capsid protein of PCV2, which is used to induce immune response in organisms.
Inactivated PCV2 vaccine is the most common commercially available PCV2 vaccine. However, developing inactivated vaccine requires cell lines to be free of contaminant, and the possibility of incomplete inactivation of the virus by chemicals is the most significant disadvantage of inactivated vaccine. Another disadvantage is that the antigenic structures of the virus may be altered by chemical treatment, leading to failure to induce sufficient immune response to eliminate the virus and failure to protect pigs from infection of the disease. Therefore, developing inactivated vaccine can be difficult and costly, and vaccine safety may be a concern.
Unlike inactivated vaccine, in which the whole virus is the vaccine antigen, subunit vaccine uses a part of proteins from the pathogen as antigen protein, and the antigen protein is inoculated into animals or humans to induce immunity. Subunit vaccine can be prepared by cloning genes encoding antigen proteins from pathogens and then producing large amounts of the antigen proteins by genetic engineering. Safety is the most significant advantage of subunit vaccine because it uses parts of a pathogen, instead of a whole pathogen, to inoculate pigs without the issue of incomplete inactivation. Conventional PCV2 subunit vaccine uses PCV2 ORF2 protein as the antigen protein; however, the protein expression level of full-length PCV2 ORF2 protein in prokaryotic expression systems is quite low and does not meet the requirements of vaccine production. Therefore, developing antigen fragments of PCV2 ORF2 that can be highly expressed in biological expression systems is helpful to commercial application of PCV2 subunit vaccine.
SUMMARY OF THE INVENTION
The disclosure provides DNA sequences encoding protein fragments of PCV2 ORF2, and the protein fragments of PCV2 ORF2 can be highly expressed in biological expression systems.
The disclosure also provides PCV2 subunit vaccine in which protein fragments of PCV2 ORF2 that can be highly expressed in biological expression systems are used as antigen proteins to be inoculated into animals to induce sufficient immunity against PCV2 infection in the animals.
The disclosure further provides subunit vaccine developed by genetic engineering to produce low-cost, high-purity, and good safety PCV2 subunit vaccine with simple production process.
Due to the fact that protein expression level of full-length PCV2 ORF2 protein in biological expression systems is quite low, genetic engineering was used to achieve the objects above. DNA sequences (such as SEQ ID NO: 1) that encode full-length PCV2 ORF2 proteins were cut into fragments of different sizes, and the DNA fragments were inserted into expression vectors and then expressed in hosts. The levels of the expressed proteins were evaluated to determine which DNA fragments can produce high levels of proteins in protein expression systems.
Results of protein sequence analysis and protein expression tests show that there are about 30 arginines in a full-length PCV2 ORF2 protein, in which at least two thirds of the arginines locate at the N terminus of the PCV2 ORF2 protein. The more arginines at the N terminus being deleted, the higher level the ORF2 protein fragment being expressed. Further, after the first 234 nucleotides at the 5′ end of a full-length DNA sequence of PCV2 ORF2 is deleted, the protein fragment encoded by the remaining DNA fragment (i.e. from nucleotide 235 at the 5′ end to a stop codon) can be highly expressed.
Therefore, the PCV2 subunit vaccine provided in the disclosure comprises an antigenic peptide of PCV2 with proper carrier or adjuvant. The antigenic peptide can be highly expressed in expression systems and is a non-arginine-rich peptide of the PCV2 ORF2. The number of arginines of the non-arginine-rich peptide of the PCV2 ORF2 are not more than half (1⁄2) of the number of arginines of an arginine-rich domain at the N terminus of the full-length PCV2 ORF2. In an embodiment, when the number of arginines of the arginine-rich domain at the N terminus of a full-length PCV2 ORF2 is 20, the number of arginines of the non-arginine-rich peptide of the PCV2 ORF2 is one of the integers between 0 and 10. In another embodiment, when the number of arginines of the arginine-rich domain at the N terminus of a full-length PCV2 ORF2 is 21, the number of arginines of the non-arginine-rich peptide of the PCV2 ORF2 is one of the integers between 0 and 10. In yet another embodiment, when the number of arginines of the arginine-rich domain at the N terminus of a full-length PCV2 ORF2 is 22, the number of arginines of the non-arginine-rich peptide of the PCV2 ORF2 is one of the integers between 0 and 11.
In an embodiment, the arginine-rich domain has residues 1-78 at the N terminus of a full-length PCV2 ORF2 (which are encoded by nucleotides 1-234 at the 5′ end of a full-length DNA sequence of the PCV2 ORF2). In an embodiment, the non-arginine-rich peptide has the peptide sequence from residue 79 to the last amino acid residue at the C terminus of a full-length PCV2 ORF2 (which is encoded by the DNA sequence from nucleotide 235 at the 5′ end to a stop codon at the 3′ end of a full-length DNA sequence of the PCV2 ORF2).
The PCV2 ORF2 disclosed herein has a full-length peptide sequence having at least 80%, preferably 85%, more preferably 90%, even more preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology to one of SEQ ID NO: 2, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 51, and SEQ ID NO: 53. In a preferable embodiment, the PCV2 ORF2 disclosed herein has a full-length peptide sequence of one of SEQ ID NO: 2, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 51, and SEQ ID NO: 53.
In addition, the disclosure provides a PCV2 ORF2 fusion protein as an antigenic peptide by using the functions of the receptor binding domain I and the transmembrane targeting domain II of Pseudomonas aeruginosa exotoxin A (i.e. the “PE protein”) and an endoplasmic reticulum (ER) retention signal (i.e. the “KDEL signal peptide”).
The PE protein of Pseudomonas aeruginosa can be used as a guide of a target protein to its target location by the following procedure. First, the receptor binding domain of Pseudomonas aeruginosa exotoxin A is responsible for binding to receptors on the membrane of the target cells (CD8+ T cells), and then the ligand-receptor complexes enter the endosomes of the target cells through endocytosis. After enzymatic cleavage by a protease in the endosomes, truncated protein fragments (containing the transmembrane targeting domain and the linked target protein) are delivered into the Golgi bodies and the endoplasmic reticulum (ER) and further translocated into the cytoplasm of the target cells by the transmembrane targeting domain.
Furthermore, when a KDEL signal peptide is linked at the C terminus of a target protein, the target protein will be transported to the ER by the KDEL signal peptide and then interact in the ER.
Therefore, the disclosure provides a PCV2 ORF2 fusion protein as an antigenic peptide by using the functions of the PE protein and the KDEL signal peptide. PE protein and KDEL signal peptide were fused with a PCV2 ORF2 protein fragment at the N and C terminuses, respectively, to produce the fusion protein (PE-ORF2 fragment-KDEL) to be inoculated into animals to induce sufficient immunity against PCV2 infection in the animals.
In some embodiments, the antigenic peptides used herein include, but not limited to, PCV2 ORF2 fragments. The ORF2 fragments have a peptide sequence having at least 80%, preferably 85%, more preferably 90%, even more preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology to one of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 55, and SEQ ID NO: 57. In a preferable embodiment, the PCV2 ORF2 fragments has a peptide sequence of one of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 55, and SEQ ID NO: 57, and were obtained by genetic engineering. DNA sequences (SEQ ID NOs: 5, 7, 9, 11, 17, 21, 54, 56) encoding the PCV2 ORF2 fragments were cloned into expression vectors to form plasmids containing a DNA fragment encoding an antigenic peptide. The plasmids were then transformed into host cells to express the antigenic peptides.
The fusion proteins (PE-ORF2 fragment-KDEL) disclosed herein were obtained by genetic engineering. DNA sequences (SEQ ID NOs: 5, 7, 9, 11, 17, 21, 54, 56) encoding the PCV2 ORF2 fragments, DNA sequence (SEQ ID NO: 34) encoding the PE protein, and the DNA sequence (SEQ ID NO: 30) encoding the KDEL signal peptide were cloned into expression vectors to form plasmids containing a DNA fragment encoding a fusion protein. The plasmids were then transformed into host cells to express the fusion protein.
The expression vectors include but not limited to pET vectors and pGEX vectors. In an embodiment, the expression vector is pET24a. The expression systems (host cells) include but not limited to prokaryotic expression systems (such as Escherichia coli (E. coli.)), eukaryotic expression systems (such as animal cells (for example, CHO cell) and plant cells). In an embodiment, the expression system is E. coli. 
The adjuvant includes, but not limited to, aqueous adjuvant (such as aluminum hydroxide), potassium alum, Freund's Incomplete Adjuvant, oil adjuvant, water-soluble adjuvant, or water-in-oil-in-water (W/O/W) emulsion adjuvant. In an embodiment, the adjuvant is oil adjuvant.
The immunogenic composition disclosed herein further comprises antigenic peptides of other PCV2 ORFs. The other PCV2 ORFs include, but not limited to, ORF1 and ORF3. In addition, the immunogenic composition disclosed herein further comprises a pathogen antigen. The pathogen antigen is selected from the group consisting of antigen of Swine influenza virus (SIV), antigen of porcine reproductive and respiratory syndrome virus (PRRSV), antigen of mycoplasma, antigen of porcine parvovirus (PPV), antigen of erysipelas, and antigen of pseudorabies (Aujeszky's disease) virus.
In addition, the immunogenic composition disclosed herein further comprises one or more than one of the followings: vehicles, solvent, emulsifier, suspending agent, decomposer, binding agent, excipient, stabilizing agent, chelating agent, diluent, gelling agent, preservative, lubricant, surfactant, adjuvant, and biological carriers.
The meaning of the technical and scientific terms as described herein can be clearly understood by a person of ordinary skill in the art.
The present invention is described in more detail in the following illustrative examples. Although the examples may represent only selected embodiments of the invention, it should be understood that the following examples are illustrative and not limiting.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 provides phylogenetic analysis of PCV2 genome sequences.
FIG. 2 illustrates a schematic diagram of the PCV2 ORF2 fragments, in which pF1, pF2, pF3, pF4, pR1, pR2, and pR3 are PCR primers.
FIG. 3 shows the results of SDS-PAGE analysis of recombinant proteins of different PCV2 2a-ORF2 fragments expressed in E. coli. Total proteins of E. coli were collected at 6 hours after IPTG induction and then resolved by a 15% SDS-PAGE, in which lanes 1-9 show molecular weight ladders, empty pET24a vector (as negative control), PCV2 ORF2 2a-F1 fragment (12.7 KDa), PCV2 ORF2 2a-F2 fragment (11.6 KDa), PCV2 ORF2 2a-F3 fragment (12.1 KDa), PCV2 ORF2 2a-F4 fragment (16.5 KDa), PCV2 ORF2 2a-F5 fragment (21.4 KDa), PCV2 ORF2 2a-F6 fragment (20.8 KDa), and full-length PCV2 2a-ORF2 (27.5 KDa), respectively.
FIG. 4 provides the results of western bolt of recombinant proteins of different PCV2 2a-ORF2 fragments. Lanes 1-9 show molecular weight ladders, empty pET24a vector (as negative control), PCV2 ORF2 2a-F1 fragment (12.7 KDa), PCV2 ORF2 2a-F2 fragment (11.6 KDa), PCV2 ORF2 2a-F3 fragment (12.1 KDa), PCV2 ORF2 2a-F4 fragment (16.5 KDa), PCV2 ORF2 2a-F5 fragment (21.4 KDa), PCV2 ORF2 2a-F6 fragment (20.8 KDa), and full-length PCV2 2a-ORF2 (27.5 KDa), respectively.
FIG. 5 provides the results of PCV2 ELISA of serum samples collected at different time points from rats vaccinated with recombinant proteins of different PCV2 2a-ORF2 fragments, respectively. *, p<0.05. (PI: post-immunization; PB: post-booster)
FIG. 6 illustrates the results of PCV2 ELISA of serum samples collected at different time points from mice vaccinated with recombinant proteins of the 2a-F2 fragment and recombinant proteins of the PE-2a-F2-KDEL fragment, respectively. *, p<0.05; **, p<0.01; ***, p<0.001; #, p<0.05; ##, p<0.01; ###, p<0.001. (PI: post-immunization; PB: post-booster)
FIG. 7 shows the results of PCV2 ELISA of serum samples collected at different time points from mice vaccinated with recombinant proteins of the PE-2a-F2-KDEL fragment and PCV2 whole virus vaccine, respectively. *, p<0.05; **, p<0.01; ***, p<0.001; #, p<0.05; ##, p<0.01; ###, p<0.001. (PI: post-immunization; PB: post-booster)



DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
Example 1
Construction and Determination of Antigenic Peptides of PCV2 Subunit Vaccine
Due to the fact that protein expression level of full-length PCV2 ORF2 protein in biological expression systems is quite low, DNA sequences that encode full-length PCV2 ORF2 proteins are cut into fragments of different sizes, and the DNA fragments are inserted into expression vectors and then expressed in hosts. The levels of the expressed proteins are evaluated to determine which DNA fragments can produce high levels of proteins in protein expression systems.
A commonly used prokaryotic expression system (Escherichia coli) is used in the following examples for analysis and illustration.
1. Amplification of PCV2 ORF2 Gene Fragments of Different Sizes
The full-length PCV2 ORF2 gene used in this example has a sequence of SEQ ID NO: 1. PCV2 ORF2 gene sequences provided by Wang et al. (Wang et al., Virus Research 2009, Genetic variation analysis of Chinese strains of porcine circovirus type 2.) are used as standard sequences for phylogenetic analysis of SEQ ID NO: 1. The result shows that the PCV2 ORF2 sequence (SEQ ID NO: 1) belongs to PCV2 2a subgroup (as shown in FIG. 1). Primers (as shown in Table 1) were designed to amplify different sizes of fragments of the PCV2 2a ORF2 gene (2a-ORF2) by polymerase chain reaction (PCR).
As shown in FIG. 2, the full-length 2a-ORF2 gene was amplified with specific primers pF1 (forward primer) and pR1 (reverse primer) by PCR. The full-length 2a-ORF2 gene (2a-ORF2) has the DNA sequence of SEQ ID NO: 1, and the different sizes of fragments of the PCV2 2a-ORF2 are named 2a-F1, 2a-F2, 2a-F3, 2a-F4, 2a-F5, and 2a-F6, respectively. The 2a-F1 fragment has the DNA sequence of SEQ ID NO: 3, which is nucleotides 1-234 at the 5′ end of 2a-ORF2. The 2a-F2 fragment has the DNA sequence of SEQ ID NO: 5, which is nucleotides 235-468 at the 5′ end of 2a-ORF2. The 2a-F3 fragment has the DNA sequence of SEQ ID NO: 7, which is nucleotides 469-699 at the 5′ end of 2a-ORF2. The 2a-F4 fragment has the DNA sequence of SEQ ID NO: 9, which is nucleotides 349-699 at the 5′ end of 2a-ORF2. The 2a-F5 fragment has the DNA sequence of SEQ ID NO: 11, which is nucleotides 235-699 at the 5′ end of 2a-ORF2. The 2a-F6 fragment has the DNA sequence of SEQ ID NO: 13, which is nucleotides 1-468 at the 5′ end of 2a-ORF2. All forward primers contain a Hind III restriction site, and all reverse primers contain an Xho I restriction site.







TABLE 1







Sequence listing of primers for amplication of


different sizes of fragments of PCV2 ORF2 gene











Frag-






ment
Primer

SequenceNote





ORF2
pF1
Forward
CCCAAGCTTGCATGACGT
SEQ ID




primer
ATCCAAGGAGGCG
NO: 23



pR1
Reverse
CCG   GGGTTT
SEQ ID




primer
AAGTGGGGGGTCTTTA
NO: 24


 


F1
pF1
Forward 
CCCAAGCTTGCATGACGT
SEQ ID




primer
ATCCAAGGAGGCG
NO: 23



pR3
Reverse
CCG   GTCGTT 
SEQ ID




primer
AATATTAAATCTCATC
NO: 28


 


F2
pF2
Forward 
CCCAAGCTTGCTTTGTTC
SEQ ID




primer
CCCCGGGAGGGGG
NO: 25



pR2
Reverse
CCG   GTAGGA
SEQ ID




primer
GAAGGGTTGGGGGATT
NO: 26


 


F3
pF3
Forward
CCCAAGCTTGCCACTCCC
SEQ ID




primer
GGTACTTTACCCC
NO: 27



pR1
Reverse
CCG   GGGTTT
SEQ ID




primer
AAGTGGGGGGTCTTTA
NO: 24


 


F4
pF4
Forward
CCCAAGCTTGCGGAGTGG
SEQ ID




primer
GCTCCACTGCTGT
NO: 29



pR1
Reverse
CCG   GGGTTT
SEQ ID




primer
AAGTGGGGGGTCTTTA
NO: 24


 


F5
pF2
Forward
CCCAAGCTTGCTTTGTTC
SEQ ID




primer
CCCCGGGAGGGGG
NO: 25



pR1
Reverse
CCG   GGGTTT
SEQ ID




primer
AAGTGGGGGGTCTTTA
NO: 24


 


F6
pF1
Forward
CCCAAGCTTGCATGACGT
SEQ ID




primer
ATCCAAGGAGGCG
NO: 23



pR2
Reverse
CCG   GTAGGA
SEQ ID




primer
GAAGGGTTGGGGGATT
NO: 26





Note:


Sequences marked with “____” underneath are the sequences of Hind III restriction site (AAGCTT), and sequences marked with “ ” underneath are the sequences of Xho I restriction site (CTCGAG).






Conditions for PCR reaction comprises: 95° C. for 5 minutes, 25 cycles of 95° C. for 30 seconds, 55° C. for 30 seconds, and 72° C. for 30 seconds, and 72° C. for 5 minutes for elongation. Sizes of the PCR products 2a-ORF2, 2a-F1, 2a-F2, 2a-F3, 2a-F4, 2a-F5, and 2a-F6 are 699 bp, 234 bp, 234 bp, 231 bp, 351 bp, 465 bp, and 468 bp, respectively, and confirmed by 2% agarose electrophoresis. After confirmation, the PCR products were purified with PCR-M purification kit (Viogene).
2. Construction of pET24a Plasmids Containing Different Sizes of Fragments of PCV2 ORF2
One (1) μg of the purified PCR products and 1 μg of pET24a expression vector (Novagen) were digested with two restriction enzymes (New England Biolabs), 1 μl of Hind III and 1 μl of Xho I, respectively, for 8 hours at 37° C. After restriction enzyme cleavage reaction, the digested PCR products and pET24a expression vector were purified with PCR-M Clean up System (Viogene) respectively. The purified PCR products were ligated with the purified pET24a expression vector to form plasmids pET24a-2a-F1, pET24a-2a-F2, pET24a-2a-F3, pET24a-2a-F4, pET24a-2a-F5, pET24a-2a-F6, and pET24a-2a-ORF2, and then the plasmids were transformed into host cells (E. coli). Transformants containing the PCR products were selected, and DNA sequences were confirmed by DNA sequencing. Bacteria containing the plasmids above were then obtained.
3. Protein Expression and Confirmation of Different Sizes of Fragments of PCV2 ORF2
Bacteria containing the plasmids above were incubated in 2 ml of LB medium at 37° C. for 16-18 hours and then inoculated at a ratio of 1:50 in LB medium containing 25 μg/ml kanamycin to be further incubated in a 37° C., 200 rpm incubator until the O.D. 600 nm is 0.6. After that, β-D-thiogalactoside (IPTG) was added to a final concentration of 1 mM, and the E. coli host cells were incubated in a 37° C., 200 rpm incubator for 6 more hours. Then, 1 ml of the E. coli host cells was centrifuged at 10,000×g, and the pellet was treated with B-PE® Bacterial Protein Extraction (purchased from PIERCE) to check whether the recombinant proteins are soluble proteins or in inclusion bodies by the following steps. The pellet was added in 40 μl of reagent, mixed with a vortex shaker for 1 minute, and centrifuged at 10,000×g to separate soluble proteins (upper part) from insoluble inclusion bodies (lower part). The soluble proteins were dissolved in 1×SDS-PAGE sample buffer, and the pellet was added in 2×SDS-PAGE sample buffer. The samples were placed in a heat block at 100° C. for 20 minutes and then centrifuged. The supernatant was analyzed by 15% SDS-PAGE to measure the expression of the recombinant proteins.
The recombinant proteins were produced by inducing bacterial culture with IPTG. The 2a-ORF2 recombinant protein has the amino acid sequence of SEQ ID NO: 2. The 2a-F1 recombinant protein has the amino acid sequence of SEQ ID NO: 4. The 2a-F2 recombinant protein has the amino acid sequence of SEQ ID NO: 6. The 2a-F3 recombinant protein has the amino acid sequence of SEQ ID NO: 8. The 2a-F4 recombinant protein has the amino acid sequence of SEQ ID NO: 10. The 2a-F5 recombinant protein has the amino acid sequence of SEQ ID NO: 12. The 2a-F6 recombinant protein has the amino acid sequence of SEQ ID NO: 14.
Expression of the recombinant proteins was analyzed by SDS-PAGE, and the results are shown in FIG. 3, where lane 1 shows molecular weight ladders, lane 2 shows the negative control (empty pET24a vector), lane 3 shows the 2a-F1 recombinant protein (12.7 KDa), lane 4 shows the 2a-F2 recombinant protein (11.6 KDa), lane 5 shows the 2a-F3 recombinant protein (12.1 KDa), lane 6 shows the 2a-F4 recombinant protein (16.5 KDa), lane 7 shows the 2a-F5 recombinant protein (21.4 KDa), lane 8 shows the 2a-F6 recombinant protein (20.8 KDa), and lane 9 shows full-length 2a-ORF2 recombinant protein (27.5 KDa). Among the 6 fragments of 2a-ORF2, fragments 2a-F2, 2a-F3, 2a-F4, and 2a-F5 were expressed in large quantities (FIG. 3, lanes 4-7). The estimated sizes of fragments 2a-F2, 2a-F3, 2a-F4, and 2a-F5 proteins match their actual sizes of 11.6 kDa, 12.1 kDa, 16.5 kDa, and 21.4 kDa, respectively.
Next, western blotting was deployed to check whether the expressed recombinant proteins are PCV2 2a-ORF2 fragments. Following electrophoresis, the SDS-PAGE was transferred onto a PVDF Nylon membrane. The resulting membrane was blocked with blocking buffer, which is TBST (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% Tween 20) containing 5% skim milk, for 1 h at room temperature to prevent non-specific binding of proteins. After that, the proteins were identified using alkaline phosphatase (AP)-conjugated mouse anti-6×his monoclonal antibodies against the 6×his-tag fusion proteins for 1 hour at room temperature, followed by 6 times of wash with TBST, 5 minutes for each. After wash, NBT/BCIP substrate (Bio-Rad) was added. After 10 minutes, the reaction was stopped by adding water.
The results of western blot are shown in FIG. 4, where lane 1 shows molecular weight ladders, lane 2 shows the negative control (empty pET24a vector), lane 3 shows the 2a-F1 recombinant protein (12.7 KDa), lane 4 shows the 2a-F2 recombinant protein (11.6 KDa), lane 5 shows the 2a-F3 recombinant protein (12.1 KDa), lane 6 shows the 2a-F4 recombinant protein (16.5 KDa), lane 7 shows the 2a-F5 recombinant protein (21.4 KDa), lane 8 shows the 2a-F6 recombinant protein (20.8 KDa), and lane 9 shows full-length 2a-ORF2 recombinant protein (27.5 KDa). The results of western blot match the results of SDS-PAGE analysis (as shown in FIG. 3). The full-length 2a-ORF2 recombinant protein cannot be expressed (FIG. 4, lane 9). Among the 6 fragments of 2a-ORF2, fragments 2a-F2, 2a-F3, 2a-F4, and 2a-F5 are expressed in large quantities (FIG. 4, lanes 4-7). The estimated sizes of fragments 2a-F2, 2a-F3, 2a-F4, and 2a-F5 proteins match their actual sizes of 11.6 kDa, 12.1 kDa, 16.5 kDa, and 21.4 kDa, respectively. However, there is no expression of the 2a-F1 recombinant protein or the 2a-F6 recombinant protein (FIG. 4, lanes 3 and 8). Since both of the 2a-F1 recombinant protein and the 2a-F6 recombinant protein contain nucleotides 1-234 at the 5′ end of the full-length DNA sequence of the PCV2 2a-ORF2 (see FIG. 2), some sequences in the nucleotides 1-234 may affect expression of PCV2 ORF2 protein. Analysis of amino acid sequences of the 2a-F1 fragment (nucleotides 1-234 at the 5′ end of PCV2 2a-ORF2, SEQ ID NO: 3) and the 2a-F2+2a-F3 fragment (nucleotides 235-699 (without the stop codon) at the 5′ end of PCV2 2a-ORF2, SEQ ID NO: 11) shows that the arginine numbers of the amino acid sequence (SEQ ID NO: 4) of the 2a-F1 fragment are two times or more than the arginine numbers of the amino acid sequence (SEQ ID NO: 12) of the 2a-F2+2a-F3 fragment (as shown in Table 2). Analysis of addition or deletion of the arginine shows that deletion of the excessive arginine can increase protein expression of PCV2 ORF2.







TABLE 2







Analysis of Arginine Numbers of the


PCV2 2a-ORF2 Amino Acid Sequence








2a-F1
2a-F2 + 2a-F3












Codon
Amino Acid
Amount
Codon
Amino Acid
Amount















AGA
Arginine
5
AGA
Arginine
3


AGG
Arginine
3
AGG
Arginine
3


CGA
Arginine
1
CGC
Arginine
1


CGC
Arginine
10
CGG
Arginine
1


CGT
Arginine
2
CGT
Arginine
1


Total

21
Total

9









Example 2
Immunogenicity Analysis of Antigenic Peptides of PCV2 Subunit Vaccine
1. Immunization of Rats
PCV2 subunit vaccine was made with the 2a-F2, 2a-F3, 2a-F4, and 2a-F5 recombinant proteins prepared in Example 1, respectively, and Freund's complete adjuvant. Rats were vaccinated with the PCV2 subunit vaccine to analyze immunogenicity of the antigenic peptides of the PCV2 subunit vaccine.
Five- to six-week-old healthy specific-pathogen-free (SPF) rats were randomly divided into 5 groups of 3 rats each. Enzyme-linked immunosorbent assay (ELISA) showed that all the 15 rats were negative for anti-PCV2 antibodies. Each rat in the 4 vaccine groups (Groups 1 to 4) was injected subcutaneously with 200 μg of recombinant protein, and the total volume of each injection was 300 μL with 1:1 (v/v) ratio of protein to adjuvant formulation. Rats in Group 5 were injected with 300 μL PBS and served as negative control. Two weeks after primary immunization (p.i.), the rats in the 4 vaccine groups were boosted with the same dose of the 4 different recombinant proteins, respectively. Serum samples were collected at weeks 0, 2, 4, and 8 post-primary immunization. All the serum samples were tested by ELISA to measure the titer of anti-PCV2 antibodies.
2. Detection of Anti-PCV2 Antibodies by ELISA
Ninety-six well plates containing PCV2 pathogen antigen (300 ng/well) were used as the ELISA plates in this example. The ELISA plates were washed 3 times with 50 mmol/L PBS (pH 7.2) containing 500 μl/L Tween-20 (i.e. PBST) for 3 to 5 minutes each time. To block the ELISA plates, 2004 of 0.15% BSA blocking solution was added to each well of the ELISA plates, and then the ELISA plates were incubated for 2 hours at 37° C. After that, the ELISA plates were washed with PBS. Rat serum samples were diluted fifty-fold (1:50) with PBS and then diluted two-fold serially. Each sample had 8 repeats. Diluted serum samples were added to the wells of the ELISA plates (100 μl/well), and the plates were incubated for 1 hour at 37° C. After incubation, the plates were washed with PBS. Anti-rat IgG antibody conjugated with alkaline phosphatase (AP) was then added to the wells. After incubating for 1 hour at 37° C., the plates were washed with PBS. For visualization of results, para-Nitrophenylphosphate (pNPP) was added to the wells. Following incubation, the reaction was stopped by adding 1M NaOH. The optical density of each well was read using optical density at 405 nm. Each sample was analyzed in duplicate, and the O.D. values of duplicates were averaged.
ELISA results are shown in FIG. 5. All the 2a-F2, 2a-F3, 2a-F4, and 2a-F5 recombinant proteins prepared in Example 1 are able to induce serum antibodies against PCV2 in the tested animals. Among the recombinant proteins, 2a-F2 recombinant protein (SEQ ID NO: 6) induced the highest level of serum antibody against PCV2. For statistical analysis, all groups were compared with the PBS negative control at different sampling points, and there are significant differences between the negative control and each vaccination group (p<0.05).
Furthermore, pigs were vaccinated with the 2a-F2, 2a-F3, 2a-F4, and 2a-F5 recombinant proteins, respectively, and all the recombinant proteins were able to induce serum antibodies against PCV2 in pigs. In addition, the vaccinated pigs were challenged with PCV2 virus to evaluate the efficacy of the recombinant proteins. First, the recombinant proteins were formulated as subunit vaccine and injected into pigs. Then, the pigs were challenged with PCV2 virus. The results show that the protection rates of immunization groups are higher then that of the negative control (no vaccination). The protection rates used herein include a decrease in viremia and alleviation of PCV2 symptoms. Therefore, the results show that the PCV2 subunit vaccine prepared with the recombinant proteins can effectively induce immunity in animals and increase survival rate of the animals.
Example 3
Construction and Expression of Antigenic Peptides of PCV2 2a Subunit Vaccine
The results in Example 2 show that among the recombinant proteins, the F2 peptide of PCV2 ORF2 induces the highest level of serum antibody against PCV2. In order to enhance the immunogenicity of PCV2 subunit vaccine, receptor binding domain I and transmembrane targeting domain II of Pseudomonas aeruginosa exotoxin A (i.e. PE protein) and ER retention signal—KDEL signal peptide were fused with the 2a-F2 peptide prepared in Example 1 at the N and C terminuses, respectively, to produce a fusion protein (PE-2a-F2-KDEL) to induce sufficient immunity against PCV2 infection in animals.
The recombinant protein (PE-2a-F2-KDEL) was prepared by genetic engineering in this Example. DNA sequences encoding the proteins of interest were cloned into an expression vector to construct pET24a-PE-2a-F2-KDEL plasmid. The plasmid was induced to express PE-2a-F2-KDEL recombinant protein. First, DNA sequence (SEQ ID NO: 30) encoding KDEL signal peptide was cloned into pET24a vector to form pET24a-KDEL plasmid. After that, DNA sequence (SEQ ID NO: 5) of the 2a-F2 fragment obtained in Example 1 was cloned into pET24a-KDEL plasmid to form pET24a-2a-F2-KDEL plasmid. Finally, DNA sequence (SEQ ID NO: 34) encoding PE protein was cloned into pET24a-2a-F2-KDEL plasmid to form pET24a-PE-2a-F2-KDEL plasmid.
1. Construction of pET24a-KEDL
The DNA sequence encoding KDEL signal peptide (SEQ ID NO: 31) is shown as SEQ ID NO: 30. The DNA sequence was amplified by PCR. The sequences of KEDL specificity primers are shown as follows.







Forward primer (with a Hind III restriction site):


(SEQ ID NO: 32)


5′-CCC   CTCAAAAAAGACGAACTGAGAGATG


 


AACTGAAAGA-3′


Hind III


 


Reverse primer (with a Xho I restriction site)


(SEQ ID NO: 33)


5′-GTG   CAGTTCGTCTTTCAGTTCATCT-3′


Xho I






Conditions for PCR reaction comprises: 94° C. for 3 minutes, 5 cycles of 95° C. for 1 minute, 55° C. for 1 minute, and 72° C. for 20 seconds, and 72° C. for 1 minutes for elongation. PCR product and pET24a vector were subjected to double restriction enzymes digestion with Hind III and Xho I. Thereafter, the digested PCR products and pET24a vector were purified, respectively, followed by ligation to clone the PCR product into pET24a to form pET24a-KDEL. Then, the construct pET24a-KEDL was transformed into host cells (E. coli) to carry out mass replication. The replicate PCR products were further confirmed by sequencing.

2. Construction of pET24a-2a-F2-KDEL

The 2a-F2 DNA sequence (SEQ ID NO: 5) obtained in Example 1 was amplified by PCR. The PCR primers are shown as follows.









Forward primer pF2-1



(with a Sac I restriction site):



(SEQ ID NO: 38)



5′-C   TTTGTTCCCCCGGGAGGGGGG-3′



Sac I



 



Reverse primer pR2-1



(with a Hind III restriction site):



(SEQ ID NO: 39)



5′-CCC   GTAGGAGAAGGGTTGGGGGATT-3′



Hind III






Conditions for PCR reaction comprises: 95° C. for 5 minutes, 25 cycles of 95° C. for 30 seconds, 55° C. for 30 seconds, and 72° C. for 30 seconds, and 72° C. for 5 minutes for elongation. PCR product and pET24a-KDEL plasmid were subjected to double restriction enzymes digestion with Sac I and Hind III. Thereafter, the digested PCR products and pET24a-KDEL plasmid were purified, respectively, followed by ligation to clone the PCR product into pET24a-KDEL to form pET24a-2a-F2-KDEL. Then, the construct was transformed into host cells (E. coli) to carry out mass replication. The replicate PCR products were further confirmed by sequencing.

3. Construction of pET24a-PE-2a-F2-KDEL

The DNA sequence encoding PE protein (SEQ ID NO: 35) is shown as SEQ ID NO: 34. The DNA sequence was amplified by PCR. The sequences of PE specificity primers are shown as follows.







Forward primer (with a BamH I restriction site):


(SEQ ID NO: 36)


5′-CG   GAAGAAGCGTTCGAC-3′


BamH I


 


Reverse primer


(with a EcoRI and a Sac I restriction sites)


(SEQ ID NO: 37)


5′-CGGAATTC   GCAGGTCAGGCTCACCAC-3′


EcoR I Sac I






Conditions for PCR reaction comprises: 94° C. for 5 minutes, 30 cycles of 95° C. for 1 minute, 55° C. for 1 minute, and 72° C. for 1.5 minutes, and 72° C. for 7 minutes for elongation. PCR product and pET24a-2a-F2-KDEL plasmid were subjected to double restriction enzymes digestion with BamH I and Sac I. Thereafter, the digested PCR products and pET24a-2a-F2-KDEL plasmid were purified, respectively, followed by ligation to clone the PCR product into pET24a-2a-F2-KDEL to form pET24a-PE-2a-F2-KDEL. Then, the construct pET24a-PE-2a-F2-KDEL was transformed into host cells (E. coli) to carry out mass replication. The replicate PCR products were further confirmed by sequencing. The antigenic fusion protein PE-2a-F2-KDEL has amino acid sequence of SEQ ID NO: 41, and the DNA sequence encoding the fusion protein is SEQ ID NO: 40.

4. PE-2a-F2-KDEL Protein Expression

E. coli containing pET24a-PE-2a-F2-KEDL plasmid was incubated in LB medium. The bacterial culture was induced by addition of IPTG to express the antigenic fusion protein PE-2a-F2-KEDL (SEQ ID NO: 41). Methods for protein expression and extraction are described in Example 1.
Example 4
Analysis of PCV2 ORF2 Sequences of Different Subgroups
In addition to subgroup 2a, there are other subgroups of PCV2 virus. Analyses of arginine numbers of amino acid sequences encoded by nucleotides 1-234 at the 5′ end of PCV2 ORF2 and amino acid sequences encoded by the DNA sequences from nucleotide 235 to the last nucleotide of PCV2 ORF2 of PCV2 subgroups 2b, 2c, 2d, and 2e are shown as follows, respectively.
PCV2 ORF2 gene sequences provided by Wang et al. (Wang et al., Virus Research 2009, Genetic variation analysis of Chinese strains of porcine circovirus type 2.) are used as standard sequences for phylogenetic analysis of SEQ ID NO: 15 and SEQ ID NO: 19. The result shows that PCV2 ORF2 sequences SEQ ID NO: 15 and SEQ ID NO: 19 belong to PCV2 2b subgroup and PCV2 2d subgroup, respectively (as shown in FIG. 1). In addition, PCV2 ORF2 sequences SEQ ID NO: 50 and SEQ ID NO: 52 serve as a standard strain of PCV2 2c subgroup and PCV2 2e subgroup, respectively.
Results of sequence analysis of PCV2 2b subgroup are shown in Table 3. The ORF2 fragment of PCV2 2b subgroup (2b-ORF2) has the nucleotide sequence of SEQ ID NO: 15 and the amino acid sequence of SEQ ID NO: 16. There are 21 arginine residues in the amino acid sequences encoded by nucleotides 1-234 at the 5′ end of 2b-ORF2, whereas there are 10 arginine residues in the amino acid sequences (SEQ ID NO: 18) encoded by nucleotides 235-699 (without the stop codon, SEQ ID NO: 17) of 2b-ORF2. Results of sequence analysis of PCV2 2b subgroup are consistent with that of PCV2 2a subgroup. The arginine numbers at the N terminus of both ORF2 proteins are two times or more than the arginine numbers of the rest part of the ORF2 proteins (as shown in Tables 2 and 3).







TABLE 3







Analysis of Arginine Numbers of the PCV2 2b-ORF2 Amino Acid Sequence








Subgroup PCV2-2b -
Subgroup PCV2-2b -


nucleotides 1-234
nucleotides 235-699












Codon
Amino Acid
Amount
Codon
Amino Acid
Amount















AGA
Arginine
5
AGA
Arginine
5


AGG
Arginine
3
AGG
Arginine
1


CGA
Arginine
1
CGA
Arginine
0


CGC
Arginine
9
CGC
Arginine
3


CGG
Arginine
1
CGG
Arginine
0


CGT
Arginine
2
CGT
Arginine
1


Total

21
Total

10









Results of sequence analysis of PCV2 2c subgroup are shown in Table 4. The ORF2 fragment of PCV2 2c subgroup (2c-ORF2) has the nucleotide sequence of SEQ ID NO: 50 and the amino acid sequence of SEQ ID NO: 51. There are 20 arginine residues in the amino acid sequences encoded by nucleotides 1-234 at the 5′ end of 2c-ORF2, whereas there are 10 arginine residues in the amino acid sequences (SEQ ID NO: 55) encoded by nucleotides 235-702 (SEQ ID NO: 54) of 2c-ORF2. Results of sequence analysis of PCV2 2c subgroup are consistent with that of PCV2 2a subgroup and PCV2 2b subgroup. The arginine numbers at the N terminus of all the three ORF2 proteins are two times or more than the arginine numbers of the rest part of the ORF2 proteins (as shown in Tables 2, 3 and 4).







TABLE 4







Analysis of Arginine Numbers of the PCV2 2c-ORF2 Amino Acid Sequence








Subgroup PCV2-2c -
Subgroup PCV2-2c -


nucleotides 1-234
nucleotides 235-702












Codon
Amino Acid
Amount
Codon
Amino Acid
Amount















AGA
Arginine
5
AGA
Arginine
6


AGG
Arginine
3
AGG
Arginine
1


CGA
Arginine
0
CGA
Arginine
0


CGC
Arginine
9
CGC
Arginine
2


CGG
Arginine
1
CGG
Arginine
0


CGT
Arginine
2
CGT
Arginine
1


Total

20
Total

10









Results of sequence analysis of PCV2 2d subgroup are shown in Table 5. The ORF2 fragment of PCV2 2d subgroup (2d-ORF2) has the nucleotide sequence of SEQ ID NO: 19 and the amino acid sequence of SEQ ID NO: 20. There are 21 arginine residues in the amino acid sequences encoded by nucleotides 1-234 at the 5′ end of 2d-ORF2, whereas there are 10 arginine residues in the amino acid sequences (SEQ ID NO: 21) encoded by nucleotides 235-702 (without the stop codon, SEQ ID NO: 22) of 2d-ORF2. Results of sequence analysis of PCV2 2d subgroup are consistent with that of PCV2 2a subgroup, PCV2 2b subgroup, and PCV2 2c subgroup. The arginine numbers at the N terminus of all the four ORF2 proteins are two times or more than the arginine numbers of the rest part of the ORF2 proteins (as shown in Tables 2, 3, 4, and 5).







TABLE 5







Analysis of Arginine Numbers of the PCV2 2d-ORF2 Amino Acid Sequence








Subgroup PCV2-2d - 
Subgroup PCV2-2d - 


nucleotides 1-234
nucleotides 235-702












Codon
Amino Acid
Amount
Codon
Amino Acid
Amount















AGA
Arginine
5
AGA
Arginine
4


AGG
Arginine
3
AGG
Arginine
3


CGA
Arginine
1
CGA
Arginine
0


CGC
Arginine
10
CGC
Arginine
1


CGG
Arginine
0
CGG
Arginine
1


CGT
Arginine
2
CGT
Arginine
1


Total

21
Total

10









Results of sequence analysis of PCV2 2e subgroup are shown in Table 6. The ORF2 fragment of PCV2 2e subgroup (2e-ORF2) has the nucleotide sequence of SEQ ID NO: 52 and the amino acid sequence of SEQ ID NO: 53. There are 20 arginine residues in the amino acid sequences encoded by nucleotides 1-234 at the 5′ end of 2e-ORF2, whereas there are 9 arginine residues in the amino acid sequences (SEQ ID NO: 57) encoded by nucleotides 235-699 (without the stop codon, SEQ ID NO: 56) of 2e-ORF2. Results of sequence analysis of PCV2 2e subgroup are consistent with that of PCV2 2a subgroup, PCV2 2b subgroup, PCV2 2c subgroup, and PCV2 2d subgroup. The arginine numbers at the N terminus of all the five ORF2 proteins are two times or more than the arginine numbers of the rest part of the ORF2 proteins (as shown in Tables 2, 3, 4, 5, and 6).







TABLE 6







Analysis of Arginine Numbers of the PCV2 2e-ORF2 Amino Acid Sequence








Subgroup PCV2-2e -
Subgroup PCV2-2e -


nucleotides 1-234
nucleotides 235-699












Codon
Amino Acid
Amount
Codon
Amino Acid
Amount















AGA
Arginine
5
AGA
Arginine
4


AGG
Arginine
3
AGG
Arginine
2


CGA
Arginine
0
CGA
Arginine
0


CGC
Arginine
9
CGC
Arginine
2


CGG
Arginine
1
CGG
Arginine
0


CGT
Arginine
2
CGT
Arginine
1


Total

20
Total

9









Protein expression levels of the arginine-rich domains and the non-arginine-rich domains of PCV2 2b subgroup, PCV2 2c subgroup, PCV2 2d subgroup, and PCV2 2e subgroup are also analyzed by the method described in Example 1. The results are consistent with the results in Example 1. Protein expression level of the arginine-rich domains at N terminus is low, whereas protein expression level of the non-arginine-rich domain is high. Furthermore, analysis of addition or deletion of the arginine shows that deletion of the excessive arginine can increase protein expression of PCV2 ORF2, which is also consistent with the results of PCV2 2a subgroup.
The arginine-rich domain has an amino acid sequence of about residues 1-78 at the N terminus of the full-length peptide of PCV2 ORF2, and the non-arginine-rich domain has an amino acid sequence about from residue 79 to the last residue at C terminus.
Example 5
Construction and Expression of Antigenic Peptides of PCV2 2b Subunit Vaccine
1. Construction and Expression of pET24a-2b-F2
In this example, ORF2 gene of PCV2 2b subgroup in Example 4 was used in construction of the antigenic peptide of PCV2 subunit vaccine. As described in Examples 1 and 3, F2 fragment (nucleotides 235-468 at the 5′ end of the full-length DNA sequence of PCV2 2b ORF2 gene, 2b-F2 fragment) is cloned into expression vector. The 2b-F2 fragment of PCV2 2b subgroup has the nucleotide sequence of SEQ ID NO: 17 and the amino acid sequence of SEQ ID NO: 18. The nucleotide sequence of the 2b-F2 fragment was amplified by PCR. The PCR primers are shown as follows.









Forward primer pF-2b



(with a Sac I restriction site):



(SEQ ID NO: 42)



5′-C   TTTCTTCCCCCAGGAGGGGGC-3′



Sac I



 



Reverse primer pR-2b



(with a Hind III restriction site):



(SEQ ID NO: 43)



5′-CCC   GTAGGAGAAGGGCTGGGTTAT-3′



Hind III






Conditions for PCR reaction comprises: 95° C. for 5 minutes, 25 cycles of 95° C. for 30 seconds, 55° C. for 30 seconds, and 72° C. for 30 seconds, and 72° C. for 5 minutes for elongation. PCR product and pET24a expression vector (Novagen) were subjected to double restriction enzymes digestion with Sac I and Hind III. Thereafter, the digested PCR products and pET24a expression vector were purified, respectively, followed by ligation to clone the PCR product into pET24a expression vector to form pET24a-2b-F2 plasmid. Then, the construct was transformed into host cells (E. coli) to carry out mass replication. The replicate PCR products were further confirmed by sequencing. E. coli containing pET24a-2b-F2 μlasmid was incubated in LB medium. The bacterial culture was induced by addition of IPTG to express the antigenic protein 2b-F2 (SEQ ID NO: 18). Methods for protein expression and extraction are described in Example 1.

2. Construction and Expression of pET24a-PE-2b-F2-KDEL

In addition to using 2b-F2 peptide as the antigenic peptide of PCV2 subunit vaccine, in this Example, PE protein and KDEL signal peptide were fused with the 2b-F2 peptide at the N and C terminuses, respectively, to produce PE-2b-F2-KDEL recombinant fusion protein to induce sufficient immunity against PCV2 infection in animals.
PE-2b-F2-KDEL recombinant fusion protein has a DNA sequence of SEQ ID NO: 44 and an amino acid sequence of SEQ ID NO: 45. The strategy for construction of plasmid expressing PE-2b-F2-KDEL recombinant fusion protein (pET24a-PE-2b-F2-KDEL) is the same as the strategy described in Example 3. First, DNA sequence (SEQ ID NO: 30) encoding KDEL signal peptide was cloned into pET24a vector to form pET24a-KDEL plasmid. Method for construction of pET24a-KDEL plasmid is described in Example 3. After that, DNA sequence (SEQ ID NO: 17) of the 2b-F2 fragment was cloned into pET24a-KDEL plasmid to form pET24a-2b-F2-KDEL plasmid. Finally, DNA sequence (SEQ ID NO: 34) encoding PE protein was cloned into pET24a-2b-F2-KDEL plasmid to form pET24a-PE-2b-F2-KDEL plasmid. Method for construction of pET24a-PE-2b-F2-KDEL plasmid is described in Example 3. Then, E. coli containing pET24a-PE-2b-F2-KDEL plasmid was incubated in LB medium. The bacterial culture was induced by addition of IPTG to express PE-2b-F2-KDEL recombinant protein (SEQ ID NO: 45). Methods for protein expression and extraction are described in Example 1.
Example 6
Construction and Expression of Antigenic Peptides of PCV2 2d Subunit Vaccine
1. Construction and Expression of pET24a-2d-F2
In this example, ORF2 gene of PCV2 2d subgroup in Example 4 was used in construction of the antigenic peptide of PCV2 subunit vaccine. As described in Examples 1 and 3, F2 fragment (nucleotides 235-468 at the 5′ end of the full-length DNA sequence of PCV2 2d ORF2 gene, 2d-F2 fragment) is cloned into expression vector. The 2d-F2 fragment of PCV2 2d subgroup has the nucleotide sequence of SEQ ID NO: 21 and the amino acid sequence of SEQ ID NO: 22. The nucleotide sequence of the 2d-F2 fragment was amplified by PCR. The PCR primers are shown as follows.









Forward primer pF-2d



(with a Sac I restriction site):



(SEQ ID NO: 46)



5′-C   TTTCTTCCCCCAGGAGGGGGC-3′



Sac I



 



Reverse primer pR-2d



(with a Hind III restriction site):



(SEQ ID NO: 47)



5′-CCC   GTAGGAGAAGGGCTGGGTTAT-3′



Hind III






Conditions for PCR reaction comprises: 95° C. for 5 minutes, 25 cycles of 95° C. for 30 seconds, 55° C. for 30 seconds, and 72° C. for 30 seconds, and 72° C. for 5 minutes for elongation. PCR product and pET24a expression vector (Novagen) were subjected to double restriction enzymes digestion with Sac I and Hind III. Thereafter, the digested PCR products and pET24a expression vector were purified, respectively, followed by ligation to clone the PCR product into pET24a expression vector to form pET24a-2d-F2 plasmid. Then, the construct was transformed into host cells (E. coli) to carry out mass replication. The replicate PCR products were further confirmed by sequencing. E. coli containing pET24a-2d-F2 μlasmid was incubated in LB medium. The bacterial culture was induced by addition of IPTG to express the antigenic protein 2d-F2 (SEQ ID NO: 22). Methods for protein expression and extraction are described in Example 1.

2. Construction and Expression of pET24a-PE-2d-F2-KDEL

In addition to using 2d-F2 peptide as the antigenic peptide of PCV2 subunit vaccine, in this Example, PE protein and KDEL signal peptide were fused with the 2d-F2 peptide at the N and C terminuses, respectively, to produce PE-2d-F2-KDEL recombinant fusion protein to induce sufficient immunity against PCV2 infection in animals.
PE-2d-F2-KDEL recombinant fusion protein has a DNA sequence of SEQ ID NO: 48 and an amino acid sequence of SEQ ID NO: 49. The strategy for construction of plasmid expressing PE-2d-F2-KDEL recombinant fusion protein (pET24a-PE-2d-F2-KDEL) is the same as the strategy described in Example 3. First, DNA sequence (SEQ ID NO: 30) encoding KDEL signal peptide was cloned into pET24a vector to form pET24a-KDEL plasmid. Method for construction of pET24a-KDEL plasmid is described in Example 3. After that, DNA sequence (SEQ ID NO: 21) of the 2d-F2 fragment was cloned into pET24a-KDEL plasmid to form pET24a-2d-F2-KDEL plasmid. Finally, DNA sequence (SEQ ID NO: 34) encoding PE protein was cloned into pET24a-2d-F2-KDEL plasmid to form pET24a-PE-2d-F2-KDEL plasmid. Method for construction of pET24a-PE-2d-F2-KDEL plasmid is described in Example 3. Then, E. coli containing pET24a-PE-2d-F2-KDEL plasmid was incubated in LB medium. The bacterial culture was induced by addition of IPTG to express PE-2d-F2-KDEL recombinant protein (SEQ ID NO: 49). Methods for protein expression and extraction are described in Example 1.
Example 7
Immunogenicity Analysis of Antigenic Peptides of PCV2 Subunit Vaccine-1
1. Immunization of Mice
PCV2 subunit vaccine was made with the 2a-F2 recombinant protein (SEQ ID NO: 6) prepared in Example 1 and the PE-2a-F2-KDEL recombinant protein (SEQ ID NO: 41) prepared in Example 3, respectively, and Freund's complete adjuvant. Mice were vaccinated with the PCV2 subunit vaccine to analyze immunogenicity of the antigenic peptides of the PCV2 subunit vaccine.
Five- to six-week-old healthy SPF mice were randomly divided into 3 groups of 3 mice each. Enzyme-linked immunosorbent assay (ELISA) showed that all the 9 mice were negative for anti-PCV2 antibodies. Each mouse in the 2 vaccine groups (Groups 1 and 2) was injected intraperitoneally with 100 μg of recombinant protein. Mice in Group 3 were injected with PBS and served as negative control. Two weeks after primary immunization (p.i.), the mice in the 2 vaccine groups were boosted with the same dose of the 2 different recombinant proteins, respectively. Serum samples were collected at weeks 2, 4, 5, and 8 post-primary immunization. All the serum samples were tested by ELISA to measure the titer of anti-PCV2 antibodies.
2. Detection of Anti-PCV2 Antibodies by ELISA
Ninety-six well plates containing PCV2 pathogen antigen (300 ng/well) were used as the ELISA plates in this example. The ELISA plates were washed 3 times with 50 mmol/L PBS (pH 7.2) containing 500 μl/L Tween-20 (i.e. PBST) for 3 to 5 minutes each time. To block the ELISA plates, 2004 of 0.15% BSA blocking solution was added to each well of the ELISA plates, and then the ELISA plates were incubated for 2 hours at 37° C. After that, the ELISA plates were washed with PBS. Mice serum samples were diluted fifty-fold (1:50) with PBS and then diluted two-fold serially. Each sample had 8 repeats. Diluted serum samples were added to the wells of the ELISA plates (100 μl/well), and the plates were incubated for 1 hour at 37° C. After incubation, the plates were washed with PBS. Anti-mouse IgG antibody conjugated with alkaline phosphatase (AP) was then added to the wells. After incubating for 1 hour at 37° C., the plates were washed with PBS. For visualization of results, para-Nitrophenylphosphate (pNPP) was added to the wells. Following incubation, the reaction was stopped by adding 1M NaOH. The optical density of each well was read using optical density at 405 nm. Each sample was analyzed in duplicate, and the O.D. values of duplicates were averaged.
ELISA results are shown in FIG. 6. The PE-2a-F2-KDEL recombinant protein (SEQ ID NO: 41) induces higher level of serum antibodies against PCV2 in tested animals than the 2a-F2 recombinant protein (SEQ ID NO: 6) does. There are significant differences between the levels of serum antibodies against PCV2 in Group 1 and Group 2 at week 4 post-primary immunization (p<0.05). The differences between the levels of serum antibodies against PCV2 in Group 1 and Group 2 are more significant at week 6 post-primary immunization (p<0.01). Additionally, compared to the antibody level of negative control, the antibody levels of Group 1 and Group 2 are significantly higher (p<0.01).
Example 8
Immunogenicity Analysis of Antigenic Peptides of PCV2 Subunit Vaccine-2
1. Immunization of Mice
In this Example, immunogenicity of PE-2a-F2-KDEL recombinant protein disclosed herein in tested animals is compared with immunogenicity of PCV2 whole virus in tested animals.
PCV2 subunit vaccine was made with the PE-2a-F2-KDEL recombinant protein (SEQ ID NO: 41) prepared in Example 3 and oil adjuvant Montanide ISA 206 (Seppic, France). In addition, PCV2 whole virus vaccine was made with in activated PCV2 whole virus (106 TCID50/ml) and oil adjuvant Montanide ISA 206 (Seppic, France). There are 100 μl inactivated PCV2 whole virus and 250 μl adjuvant in a single dose of PCV2 whole virus vaccine. The two vaccines were used to vaccinate mice.
Five- to six-week-old healthy SPF mice were randomly divided into 3 groups of 5 mice each. Enzyme-linked immunosorbent assay (ELISA) showed that all the 15 mice were negative for anti-PCV2 antibodies. Each mouse in Groups 1 was injected intraperitoneally with 100 μg of recombinant protein. Each mouse in Groups 2 was injected intraperitoneally with a single dose of PCV2 whole virus vaccine. Mice in Group 3 were injected with oil adjuvant Montanide ISA 206 and served as negative control. Three weeks after primary immunization (p.i.), the mice in the 2 vaccine groups were boosted with the same dose of the 2 different vaccines, respectively. Serum samples were collected at weeks 0, 1, 2, 3, 4, and 5 post-primary immunization. All the serum samples were tested by ELISA to measure the titer of anti-PCV2 antibodies.
2. Detection of Anti-PCV2 Antibodies by ELISA
ELISA method for detection of anti-PCV2 antibodies is described in Example 7. ELISA results are shown in FIG. 7. The PE-2a-F2-KDEL recombinant protein (SEQ ID NO: 41) induces higher level of serum antibodies against PCV2 in tested animals than the inactivated PCV2 whole virus antigen does. There are significant differences between the levels of serum antibodies against PCV2 in Group 1 and Group 2 at week 1 post-primary immunization (p<0.001). Additionally, compared to the antibody level of negative control, the antibody level of mice vaccinated with the PE-2a-F2-KDEL recombinant protein (Group 1) is significantly higher (p<0.001).
Furthermore, pigs were vaccinated with the 2a-F2 and PE-2a-F2-KDEL recombinant proteins, respectively, and both of the recombinant proteins were able to induce serum antibodies against PCV2 in pigs. In addition, the vaccinated pigs were challenged with PCV2 virus to evaluate the efficacy of the recombinant proteins. First, the recombinant proteins were formulated as subunit vaccine and injected into pigs. Then, the pigs were challenged with PCV2 virus. The results show that the protection rates of immunization groups are higher then that of the negative control (no vaccination). The protection rates used herein include a decrease in viremia and alleviation of PCV2 symptoms. Therefore, the results show that the PCV2 subunit vaccine prepared with the recombinant proteins can effectively induce immunity in animals and increase survival rate of the animals.
Additionally, ORF2 proteins or fragments thereof of PCV2 2b subgroup, PCV2 2c subgroup, PCV2 2d subgroup, and PCV2 2e subgroup were prepared by the methods described in Examples 1-3, respectively, and immunogenicity of the ORF2 proteins or fragments thereof were analyzed by the methods described in Examples 7 and 8. The results show that the ORF2 fragments of these PCV2 subgroups (for example, F2 fragment) and recombinant fusion proteins thereof (for example, PE-F2-KDEL) can induce immunity in animals (such as pigs) and prevent the animals from PCV2 infection.
The PCV2 subunit vaccine provided in the disclosure has the following advantages comparing with other conventional techniques.
The PCV2 subunit vaccine provided in the disclosure is prepared by genetic engineering, in which PCV2 ORF2 protein fragments that can be highly expressed in biological expression systems are cloned and used as antigenic peptides of the subunit vaccine. The subunit vaccine can induce sufficient immunity against PCV2 infection in animals, and the PCV2 ORF2 protein fragments can be mass-produced by genetic engineering to reduce cost of manufacturing the vaccine.
Another PCV2 subunit vaccine provided in the disclosure contains a PE-F2-KDEL antigenic fusion protein, which is a recombinant protein of F2 peptide of PCV2 ORF2, PE protein, and KDEL signal peptide. Animal trials show that this PCV2 subunit vaccine can efficiently induce higher immunity against PCV2 infection in animals.
The subunit vaccine provided in the disclosure is developed by genetic engineering, and the vaccine has the advantages of simple production process, low cost, high purity, high yield, and good safety.
Many changes and modifications in the above-described embodiment of the invention can, of course, be carried out without departing from the scope thereof. Accordingly, to promote the progress in science and the useful arts, the invention is disclosed and is intended to be limited only by the scope of the appended claims.

## Claims

1. A porcine circovirus type 2 (PCV2) immunogenic composition, comprising an antigenic peptide, wherein the antigenic peptide consists of at least one of:
(a) at least one recombinant peptide fragment of a PCV2 open reading frame 2 (ORF2) selected from the group consisting of a recombinant peptide consisting of amino acids 79-156 at the N-terminus of a PCV2 ORF2 of SEQ ID NO: 2, a recombinant peptide consisting of amino acids 157-233 at the N-terminus of the PCV2 ORF2 of SEQ ID NO: 2, a recombinant peptide consisting of amino acids 117-233 at the N-terminus of the PCV2 ORF2 of SEQ ID NO: 2, a recombinant peptide consisting of amino acids 79-233 at the N-terminus of the PCV2 ORF2 of SEQ ID NO: 2, a recombinant peptide consisting of amino acids 79-156 at the N-terminus of a PCV2 ORF2 of SEQ ID NO: 16, a recombinant peptide consisting of amino acids 157-233 at the N-terminus of the PCV2 ORF2 of SEQ ID NO: 16, a recombinant peptide consisting of amino acids 117-233 at the N-terminus of the PCV2 ORF2 of SEQ ID NO: 16, a recombinant peptide consisting of amino acids 79-233 at the N-terminus of the PCV2 ORF2 of SEQ ID NO: 16, a recombinant peptide consisting of amino acids 79-156 at the N-terminus of a PCV2 ORF2 of SEQ ID NO: 20, a recombinant peptide consisting of amino acids 157-233 at the N-terminus of the PCV2 ORF2 of SEQ ID NO: 20, a recombinant peptide consisting of amino acids 117-233 at the N-terminus of the PCV2 ORF2 of SEQ ID NO: 20, a recombinant peptide consisting of amino acids 79-233 at the N-terminus of the PCV2 ORF2 of SEQ ID NO: 20, a recombinant peptide consisting of amino acids 79-156 at the N-terminus of a PCV2 ORF2 of SEQ ID NO: 51, a recombinant peptide consisting of amino acids 157-233 at the N-terminus of the PCV2 ORF2 of SEQ ID NO: 51, a recombinant peptide consisting of amino acids 117-233 at the N-terminus of the PCV2 ORF2 of SEQ ID NO: 51, a recombinant peptide consisting of amino acids 79-233 at the N-terminus of the PCV2 ORF2 of SEQ ID NO: 51, a recombinant peptide consisting of amino acids 79-156 at the N-terminus of a PCV2 ORF2 of SEQ ID NO: 53, a recombinant peptide consisting of amino acids 157-233 at the N-terminus of the PCV2 ORF2 of SEQ ID NO: 53, a recombinant peptide consisting of amino acids 117-233 at the N-terminus of the PCV2 ORF2 of SEQ ID NO: 53, and a recombinant peptide consisting of amino acids 79-233 at the N-terminus of the PCV2 ORF2 of SEQ ID NO: 53; and
(b) a recombinant fusion protein comprising, from the amino-terminus to the carboxyl-terminus of the recombinant fusion protein: a Pseudomonas aeruginosa exotoxin A (PE) peptide having the sequence of SEQ ID NO: 35; a PCV2 ORF2 protein sequence consisting of at least one of the recombinant peptide fragments of the PCV2 ORF2 of (a); and a KDEL signal peptide having the sequence of SEQ ID NO: 31; and
wherein the immunogenic composition does not include a peptide fragment consisting of amino acids 1-78 at the N-terminus of the PCV2 ORF2.

2. The PCV2 immunogenic composition of claim 1, further comprising open reading frames (ORFs) other than ORF 2 of the PCV2, wherein the ORFs other than ORF2 comprise ORF1 and ORF3.

3. The PCV2 immunogenic composition of claim 1, further comprising at least one pathogen antigen selected from the group consisting of an antigen of swine influenza virus (SIV), an antigen of porcine reproductive and respiratory syndrome virus (PRRSV), an antigen of mycoplasma, antigen of porcine parvovirus (PPV), an antigen of erysipelas, and an antigen of pseudorabies virus.

4. The PCV2 immunogenic composition of claim 1, wherein the peptide fragment of the PCV2 ORF2 of (a) is selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 55, and SEQ ID NO: 57.

5. The PCV2 immunogenic composition of claim 1, further comprising at least one agent selected from the group consisting of vehicles, solvent, emulsifier, suspending agents, decomposer, binding agents, excipient, stabilizing agents, chelating agents, diluent, gelling agents, preservatives, lubricant, surfactant, adjuvant, and biological carriers.

6. The PCV2 immunogenic composition of claim 1, wherein the recombinant fusion protein of (b) is selected from the group consisting of SEQ ID NO: 41, SEQ ID NO: 45, and SEQ ID NO: 49.