WO2013083036A1 - 猪第二型环状病毒次单位疫苗 - Google Patents

猪第二型环状病毒次单位疫苗 Download PDF

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WO2013083036A1
WO2013083036A1 PCT/CN2012/085907 CN2012085907W WO2013083036A1 WO 2013083036 A1 WO2013083036 A1 WO 2013083036A1 CN 2012085907 W CN2012085907 W CN 2012085907W WO 2013083036 A1 WO2013083036 A1 WO 2013083036A1
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seq
pcv2
arginine
protein
peptide
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PCT/CN2012/085907
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French (fr)
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郭村勇
陈旭中
杨舒翔
陈裕森
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施怀哲维克生物科技股份有限公司
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First worldwide family litigation filed litigation Critical https://patents.darts-ip.com/?family=48573551&utm_source=google_patent&utm_medium=platform_link&utm_campaign=public_patent_search&patent=WO2013083036(A1) "Global patent litigation dataset” by Darts-ip is licensed under a Creative Commons Attribution 4.0 International License.
Priority to UAA201407508A priority Critical patent/UA113192C2/uk
Application filed by 施怀哲维克生物科技股份有限公司 filed Critical 施怀哲维克生物科技股份有限公司
Priority to US14/363,341 priority patent/US9657063B2/en
Priority to EA201491123A priority patent/EA029698B1/ru
Priority to CN201280059357.6A priority patent/CN104039814B/zh
Priority to EP12855059.7A priority patent/EP2789627B1/en
Priority to ES12855059.7T priority patent/ES2661026T3/es
Publication of WO2013083036A1 publication Critical patent/WO2013083036A1/zh

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    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • A61K2039/55544—Bacterial toxins
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/04—Fusion polypeptide containing a localisation/targetting motif containing an ER retention signal such as a C-terminal HDEL motif
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    • C12N2720/00011—Details
    • C12N2720/12011—Reoviridae
    • C12N2720/12111—Orbivirus, e.g. bluetongue virus
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    • C12N2720/12011—Reoviridae
    • C12N2720/12111—Orbivirus, e.g. bluetongue virus
    • C12N2720/12134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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    • C12N2750/00011—Details
    • C12N2750/10011—Circoviridae
    • C12N2750/10034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to a porcine circovirus type 2 (PCV2) subunit vaccine, in particular to a PCV2 0RF2 protein fragment which can be expressed in a large amount as an antigen protein, and an appropriate carrier or A porcine type 2 circovirus subunit vaccine formed by adjuvant.
  • PCV2 porcine circovirus type 2
  • the porcine type 2 circovirus is known to be associated with Postweaning Multisystemic Wasting Syndrome (PMWS) and Porcine Dermatitis And Nephropathy Syndrome (PDNS).
  • PMWS Postweaning Multisystemic Wasting Syndrome
  • PDNS Porcine Dermatitis And Nephropathy Syndrome
  • PMWS was first discovered in Canada in 1991 and has since been reported around the world. The disease causes serious damage to the global swine industry. The main symptoms are progressive weight loss, tachypnea, dyspnea, jaundice and the like.
  • the macroscopic and histological lesions are lymphocytic and granulomatous infiltrate, lymphodenopathy, lymphocytic and granulomatous hepatitis, and nephritis.
  • PCV Porcine circovirus
  • PK-15 porcine kidney cell line
  • CPE cytopathic effect
  • the virus strain is named PCV1.
  • the PCV1 virus is a circular, single-strand, 1759 bp icosahedron virus that was classified as a member of the circulatory family CCircoviridae in 1995.
  • PCV1 isolated from PK-15 cells was found to be non-pathogenic, and in pigs from Postweaning Multisystemic Wasting Syndrome (PMWS) in 1997, pigs were isolated. A pathogenic mutant porcine circovirus named PCV2.
  • Post-weaning Multisystemic Dissipative Syndrome is a highly contagious swine disease that primarily infects young pigs and pregnant sows and seriously affects pig health.
  • the PCV2 virus has a diameter of 17 nm and has a circular single-stranded DNA with a DNA size of 1.76 Kb. After sequence analysis by software, it was found that there were 11 open reading frames (ORFs) clockwise plus counterclockwise, of which ORF1 and ORF2 are the two most important genes. ORF1 transcribes Rep and Rep' proteins and is involved in the replication of this virus (Mankertz et al, 2001). It is known that ORF2 itself is an immunogenic structure capsid protein of PCV2 virus, which is used to induce an immune response in an organism.
  • PCV2 dead vaccine The most common commercially available PCV2 vaccine is the PCV2 dead vaccine.
  • the development of the dead vaccine needs to obtain a cell strain without other viral infections, and its biggest disadvantage is that it cannot guarantee whether the chemical is completely not in the process of preparing the dead vaccine.
  • Activating the virus strain another disadvantage is that killing the pathogen with a chemical agent is likely to change the antigenic structure of the pathogen, so that the induced immune response cannot neutralize the toxicity of the pathogen, thus causing the cytotoxic vaccine to protect the pig from being avoided. Infected with this disease; it can be seen that the development of the dead vaccine product is not easy, the cost is high, and its safety is considered.
  • the whole virus is used as the antigen of the vaccine, and the sub-unit vaccine takes a part of the protein of the pathogen as the antigen protein, and inoculates the antigen protein into the animal or human body, so that the inoculated animal or The human body can produce immunity. It is prepared by first selecting the gene for the antigenic protein in the pathogen, and then mass producing the antigen protein by genetic engineering techniques. Its greatest advantage is its high safety, because it is only a part of the pathogen, so it is not a complete pathogen that is injected into the pig, and there is no concern that the pathogen is not activated.
  • the conventional PCV2 subunit vaccine uses the ORF2 protein fragment as an antigenic protein; however, the full-length protein of ORF2 has a low expression level in the prokaryotic expression system, which does not meet the requirements for preparing a vaccine. Therefore, the development of a high-performance PCV2 ORF2 vaccine antigen fragment that can be expressed in a biological expression system will contribute to the commercial application of the PCV2 subunit vaccine. Summary of the invention
  • the present invention provides a DNA sequence of a PCV2 virus ORF2 protein fragment which exhibits a large amount of a PCV2 virus ORF2 protein fragment in a biological expression system.
  • the invention also provides a PCV2 subunit vaccine, which is an antigenic protein of a PCV2 virus ORF2 protein fragment which can be expressed in a large amount in a biological expression system, so that the vaccine can induce sufficient production of the animal body after being applied to the animal body. Resistant to the immunity of PCV2 virus infection.
  • the invention also provides a sub-unit vaccine developed by using genetic recombination technology, which has simple production process and low cost. Low, high purity, safe PCV 2 subunit vaccine.
  • the inventors of the present invention used the gene recombination technique to encode the full-length ORF2 protein (shown as SEQ ID NO: 2) in the PCV2 virus.
  • the DNA sequence (as shown in SEQ ID NO: 1) is cleavage of different lengths, and each of the formed DNA fragments is separately constructed into a protein expression vector, and the protein is expressed in the biological system to be determined in the biological system. A DNA fragment of a high amount of protein.
  • PCV2 virus ORF2 full-length protein has about 30 arginine, of which more than two-thirds of arginine is concentrated at the amino terminus of ORF2 protein. , N-terminal), the more the number of arginine at the N-terminus of the ORF2 protein is removed, the more the ORF2 protein fragment is expressed in the biological expression system; and the removal of the 5'-end 234 bp of the DNA sequence encoding the ORF2 protein The remaining DNA fragment ( ⁇ 5, a fragment between the 235 bp and the stop codon) can be expressed in large quantities in the biological expression system.
  • the PCV2 subunit vaccine provided by the present invention comprises a PCV2 antigen protein fragment, and a suitable carrier or adjuvant; the antigen protein fragment can be expressed in a large amount in the biological expression system, and is an O F2 non of the PCV2 virus.
  • the PCV2 ORF2 Enriched with non-Arginine-rich peptide, the PCV2 ORF2 is not rich in arginine peptide.
  • the amount of arginine is the N-terminal arginine-rich region of the PCV2 ORF2 full-length peptide.
  • (Arginine-rich domian) is less than one-half (1/2) of the amount of arginine; in one embodiment, when the amount of arginine in the N-terminal arginine-rich region of the PCV2 0RF2 is 20 And the amount of arginine which is not rich in arginine peptide in the PCV2 ORF2 is equal to a positive integer between 0 or 1 and 10; in another embodiment, when the N-terminus of the PCV2 ORF2 is rich in arginine When the amount of arginine in the region is 21, the amount of arginine which is not rich in arginine peptide in the PCV2 ORF2 is equal to a positive integer between 0 or 1 to 10; in yet another embodiment, when the PCV2 When the amount of arginine in the arginine-rich region at the N-terminus of ORF2 is 22, the PCV2 ORF2 is not rich.
  • the arginine-rich region is the region of the amino-terminal (N-terminal) 1-78 amino acid of the PCV2 O F2 full-length peptide (corresponding to the 5-terminal end of the DNA sequence) a region between 1-234 nucleotides; in one embodiment, the non-arginine-rich peptide is the amino-terminal (N-terminal) 79th amino acid of the full-length peptide of the PCV2 ORF2 The peptide between the last amino acid to the carboxy terminus (C-terminus) (corresponding to the 235th nucleotide to the 3' end of the 5' end of the DNA sequence The area between the coders).
  • the PCV2 O F2 full-length peptide has at least 80% of one of the sequences set forth in SEQ ID No: 2, SEQ ID No: 16, SEQ ID No: 20, SEQ ID No: 51, and SEQ ID No: 53 Sequence homology, preferably, has 85% sequence homology, more preferably, 90% sequence homology, even 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98%, 99% sequence homology.
  • the PCV2 ORF2 full-length peptide is represented by SEQ ID No: 2, SEQ ID No: 16, SEQ ID No: 20, SEQ ID No: 51, and SEQ ID No: 53 One of the sequences.
  • the present invention utilizes a protein of a receptor binding domain I and a transmembrane targeting domain II of exotoxin A, which is a protein of PE protein.
  • a protein of a receptor binding domain I and a transmembrane targeting domain II of exotoxin A which is a protein of PE protein.
  • the characteristics of the ER retention signal (KDEL protein signal peptide) provide a PCV2 ORF2 fusion protein antigen fragment.
  • the Pseudomonas aeruginosa PE protein system can be used as a guiding medium for the target protein.
  • the mode of action is as follows: First, the receptor binding domain protein of Pseudomonas aeruginosa exotoxin A is responsible for cell receptors on the cell membrane of target cells (CD8+ T cells).
  • the target protein will be recovered into the endoplasmic reticulum and then enter the endoplasmic reticulum under the action of the KDEL protein signal sequence.
  • the present invention further utilizes the properties of the PE protein and the KDEL protein signal sequence to provide a PCV2 ORF2 fusion protein antigen fragment, which is linked to the N-terminus of the PCV2 virus ORF2 protein fragment and the PE protein, and the PCV2 virus ORF2 protein fragment.
  • the C-terminal plus KDEL protein signal sequence (signal) is used to generate an antigen fusion protein (PE-ORF2 fragment-KDEL), which allows the vaccine to induce immunity against PCV2 infection after being administered to the animal. force.
  • the antigenic protein used in the present invention includes, but is not limited to, a PCV2 virus ORF2 protein sheet.
  • the ORF2 protein fragment is 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
  • one of the sequences set forth in SEQ ID No: 57 has at least 80% sequence homology, preferably, 85% sequence homology, more preferably 90% sequence homology, even 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence homology.
  • the PCV2 virus ORF2 protein fragment is SEQ ID No: 6, SEQ ID No: 8, SEQ ID No: 10, SEQ ID No: 12, SEQ ID No: 18, SEQ ID No. 22, one of the sequences shown in SEQ ID No: 55 and SEQ ID No: 57, wherein the protein fragment is obtained by gene transfer; the DNA sequence encoding the PCV2 virus ORF2 protein fragment (SEQ ID NO) : 5, 7, 9, 11 , 17, 21 , 54, 56) are selected into expression vectors, each forming a plastid containing a DNA sequence encoding an antigenic protein, and then transferring the plastid into a biological expression host, An antigenic protein obtained by protein expression.
  • the antigen fusion protein (PE-ORF2 fragment-KDEL) used in the present invention is obtained by gene transfer, and the above DNA partial fragment sequence encoding PCV2 O F2 (SEQ ID NO: 5, 7, 9, 11 , 17, 21, 54, 56), a DNA fragment sequence encoding the PE protein signal sequence (shown as SEQ No: 34), and a DNA fragment sequence encoding the KDEL protein signal sequence (shown as SEQ No: 30) are selected.
  • a plastid containing a DNA sequence encoding an antigen fusion protein is formed, and the plastid is transferred to an antigen fusion protein which is expressed in a host and expressed by a protein.
  • the expression vector system includes, but is not limited to, a pET vector system and a pGEX vector system, etc.; in one embodiment, the expression vector is pET24a; the biological expression system (host) includes but is not limited to: a prokaryotic expression system (eg, Escherichia coli) /0), eukaryotic expression system (such as: CHO cell, plant cell), in one embodiment, the expression system is E. coli.
  • a prokaryotic expression system eg, Escherichia coli
  • eukaryotic expression system such as: CHO cell, plant cell
  • the expression system is E. coli.
  • the adjuvant includes, but is not limited to, aqueous aluminum hydroxide gel, alum, Freimd's incomplete adjuvant, oily adjuvant, water soluble adjuvant, or water-in-oil-in water-in-oil-in -water, W/0/W); In one embodiment, the adjuvant is an oleaginous adjuvant.
  • the immunological composition provided by the present invention may further comprise an antigenic protein of other open reading frames (ORFs) of PCV2, and the antigenic proteins of other ORFs of the PCV2 include, but are not limited to, ORF1 and ORF3.
  • the immunological composition may further comprise a pathogenic antigen selected from the group consisting of swine influenza virus (SIV) antigen, porcine reproductive and respiratory syndrome virus (PRRSV) antigen, porcine mold (Mycoplasma), Parvovirus (PPV), Erysipelas, and Aujeszky's disease
  • the immunological composition provided by the present invention may further comprise one or more selected from the group consisting of: a carrier, a solvent, an emulsifier, a suspending agent, a decomposing agent, a binder, an excipient, a stabilizer, a chelating agent, Diluents, gelling agents, preservatives, lubricants, surfactants, adjuvants, biotype carriers.
  • Figure 1 is a phylogenetic tree analysis of the porcine type 2 circovirus (PCV2) genomic sequence.
  • Figure 2 is a schematic diagram showing the segmentation of PCV2 0RF2, in which pFl, pF2, pF3, pF4, p K pR2, p 3 are PCR primers.
  • Figure 3 shows the results of SDS-PAGE analysis of recombinant protein of each fragment of PCV2 2a-O F2 in E. coli; after induction of E. coli by IPTG for 6 hours, whole protein was collected and analyzed by 15% SDS-PAGE;
  • Lane 1 is the molecular weight ladder;
  • lane 2 is the pET24a vector (negative control group);
  • lane 3 is PCV2 ORF2 2a-Fl fragment (12.7 KDa);
  • lane 4 is PCV2 ORF2 2a-F2 fragment (11.6 KDa);
  • lane 5 is PCV2 O F2 2a-F3 fragment (12.1 KDa);
  • lane 6 is PCV2 ORF2 2a-F4 fragment (16.5 KDa);
  • lane 7 is PCV2 O F2 2a-F5 fragment (21.4 KDa);
  • Lane 8 is the PCV2 ORF2 2a-F6 fragment (20.8 KDa);
  • lane 9 is the PCV2 2a
  • Figure 4 shows the results of detection of recombinant proteins of PCV2 2a-ORF2 fragments by Western blotting; the first lane is the molecular weight ladder; the second lane is the pET24a vector (negative control group); the third lane is PCV2 O F2 2a-Fl fragment (12.7 KDa); lane 4 is PCV2 ORF2 2a-F2 fragment (11.6 KDa); lane 5 is PCV2 O F2 2a-F3 fragment (12.1 KDa); lane 6 is PCV2 O F2 2a-F4 fragment (16.5 KDa); lane 7 is PCV2 O F2 2a-F5 fragment (21.4 KDa); lane 8 is PCV2 O F2 2a-F6 fragment (20.8 KDa); lane 9 is PCV2 2a-O F2 Full length fragment (27.5 KDa).
  • Figure 5 shows blood collected at different times after immunization with rat 2a-ORF2 recombinant proteins of different length fragments.
  • the liquid sample was assayed for antibody titer by PCV2 ELISA, and * indicates ⁇ ⁇ 0.05.
  • Figure 6 shows the results of PCV2 ELISA determination of antibody titers in mice after immunization with 2a-F2 recombinant protein and PE-2a-F2-KDEL recombinant protein, respectively, * indicates p ⁇ 0.05, ** indicates p ⁇ 0.01, *** indicates ⁇ ⁇ 0.001; # indicates ⁇ ⁇ 0.05, Ding indicates ⁇ ⁇ 0.01, and drain indicates ⁇ ⁇ 0.001.
  • Figure 7 shows the results of PCV2 ELISA for measuring the antibody titer of blood samples collected from PE-2a-F2-KDEL recombinant protein and PCV2 whole virus vaccine at different times, respectively, * indicates p ⁇ 0.05, ** indicates p ⁇
  • the inventors of the present invention divided the gene of the PCV2 O F2 protein into different DNA fragments, and constructed these different DNA fragments onto the vector in the biological expression system.
  • the protein encoded by each DNA fragment is expressed, and the protein yield of each DNA fragment in the biological expression system is analyzed to determine a preferred DNA fragment which can produce a high amount of protein in the biological expression system.
  • the full length of the PCV2 ORF2 gene sequence used in this example is shown in SEQ ID No: 1, according to Wang et al. (Wang et al., Virus Research 2009, Genetic variation analysis of Chinese strains of porcine circovirus type 2).
  • the sequence was analyzed as SEQ ID No: 1 as a standard sequence of the parental relationship tree; the results showed that the PCV2 ORF2 sequence (SEQ ID No: 1) belongs to a member of the PCV2 2a subgroup (as shown in Figure 1).
  • a primer for increasing the length of the 2a-O F2 gene fragment (as shown in Table 1) for the ORF2 (hereinafter referred to as 2a-ORF2) gene sequence of PCV2 2a was designed for polymerase chain reaction (polymerase chain reaction, PCR) amplification of each fragment.
  • the full-length 2a-ORF2 gene fragment was amplified by PCR with the specific primer pF1 (forward primer) and pR1 (reverse primer), and the DNA sequence of the full-length 2a-ORF2 gene fragment was obtained as SEQ ID. NO: 1, shown below as 2a-ORF2; and different lengths of PCV2 2a-ORF2 gene fragments are named 2a-Fl, 2a-F2, respectively. 2a-F3, 2a-F4, 2a-F5 and 2a-F6.
  • the 2a-Fl fragment is located at positions 1 to 234 nucleotides from the 5' end of PCV2 2a-ORF2, and the DNA sequence of the 2a-Fl fragment is shown in SEQ ID NO: 3.
  • the 2a-F2 fragment is located at positions 235 to 468 nucleotides from the 5' end of PCV2 ORF2, and the DNA sequence of the 2a-F2 fragment is shown in SEQ ID NO: 5.
  • the 2a-F3 fragment is located at positions 469 to 699 nucleotides from the 5' end of PCV2 O F2, and the DNA sequence of the 2a-F3 fragment is shown in SEQ ID NO: 7.
  • the 2a-F4 fragment is located at the position 349 to 699 nucleotides from the 5' end of PCV2 O F2, and the DNA sequence of the 2a-F4 fragment is shown in SEQ ID NO: 9.
  • the 2a-F5 fragment is located at the position 235 to 699 nucleotides from the 5' end of PCV2 O F2, and the DNA sequence of the 2a-F5 fragment is shown in SEQ ID NO: 11.
  • the 2a-F6 fragment is located at positions 1 to 468 nucleotides from the 5' end of PCV2 ORF2, and the DNA sequence of the 2a-F6 fragment is shown in SEQ ID NO: 13.
  • pR2 reverse primer CCGCTCGAGGTAGGAGAAGGGTTGGGGGATT SEQ ID NO: 26
  • pF3 forward primer CCCAAGCTTGCCACTCCCGGTACTTTACCCC SEQ ID NO: 27
  • pR2 reverse primer CCGCTCGAGGTAGGAGAAGGGTTGGGGGATT SEQ ID NO: 26 Note: The sequence below the sequence is the sequence of the enzyme cleavage H t III (AAGCTT), under the sequence The sequence shown is the enzyme cut-off Xho I sequence CCTCGAG).
  • the PCR reaction conditions were as follows: after reacting at 95 ° C for 5 minutes, performing 95 ° C for 30 seconds, 55 ° C for 30 seconds, and 72 ° C for 30 seconds for 25 cycles, and finally reacting at 72 ° C for 5 minutes to carry out the extension reaction (
  • the yield of the obtained PCR products are: 699 bp for 2a-ORF2, 234 bp for 2a-Fl, 234 bp for 2a-F2, 231 bp for 2a-F3, 351 bp for 2a-F4, and 2a-F5 for 2a-F5 465 bp and 2a-F6 are 468 bp.
  • the size of each PCR product fragment was identified by 2% agalose electrophoresis. After the identification, the PCR products were purified by PCR-M purification kit (Viogene).
  • Each PCR product was cloned into ET24a vector to obtain ET24a-2a-Fl, pET24a-2a-F2, pET24a-2a-F3, pET24a-2a-F4, pET24a-2a-F5, pET24a-2a-F6 and pET24a-2a.
  • - ORF2 plastid, and the above plastids are separately transferred into the expression host Escherichia coli (E. CO / ) by transformation, and the plastids carrying the PCR fragments in the strain are selected and selected.
  • the sequence of the PCR product confirmed to be propagated was confirmed to obtain a strain containing the above plastid.
  • the strain containing the above plastid is first cultured in 2 ml of LB medium at 37 ° C for 16-18 hours, and then the bacterial solution is inoculated into LB medium at a ratio of 1:50, and the medium contains 25 g / Ml kanamycin, cultured in an incubator at 37 ° C, 200 rpm until the concentration of the OD 600 nm is 0.6, adding ⁇ -D-thiogalactoside (IPTG) to a final concentration of 1 mM, then at 37 ° C.
  • IPTG ⁇ -D-thiogalactoside
  • the strain containing the above plastid is induced by IPTG to produce a recombinant protein, wherein the amino acid sequence of the 2a-ORF2 recombinant protein is as shown in SEQ ID No: 2; the amino acid sequence of the 2a-F1 recombinant protein is as shown in SEQ ID No: 4
  • the amino acid sequence of the 2a-F2 recombinant protein is shown in SEQ ID No: 6
  • the amino acid sequence of the 2a-F3 recombinant protein is shown in SEQ ID No: 8, the amino acid sequence of the 2a-F4 recombinant protein.
  • SEQ ID No: 10 the amino acid sequence of the 2a-F5 recombinant protein is shown in SEQ ID No: 12
  • the amino acid sequence of the 2a-F6 recombinant protein is shown in SEQ ID No: 14.
  • the results of confirming the expression of recombinant proteins by SDS-PAGE are shown in Fig. 3.
  • the first lane is the Molecular weight ladder
  • the second lane is the negative control group (pET24a)
  • the third lane is the 2a-Fl recombination.
  • lane 4 is 2a-F2 recombinant protein (11.6 kDa)
  • lane 5 is 2a-F3 recombinant protein (12.1 kDa)
  • lane 6 is 2a-F4 recombinant protein (16.5 kDa)
  • 7th The pathway is 2a-F5 recombinant protein (21.4 kDa)
  • lane 8 is 2a-F6 recombinant protein (20.8 kDa)
  • lane 9 is 2a-ORF2 full-length recombinant protein (27.5 kDa).
  • 2a-ORF2 can be expressed in a large amount (Fig. 3, lines 4, 5, 6, and 7), their molecular weight positions and predictions. 11.6kDa, 12.1kDa, 16.5kDa, 21.4kDa match.
  • the Western blot was then used to confirm whether the recombinant protein was a fragment of PCV2 2a-ORF2.
  • the colloid analyzed by SDS-PAGE electrophoresis was transferred onto a PVDF nylon membrane (Nylon membrane). After transfer, the nylon membrane was placed in a plugging solution (Blocking buffer: 5 % Skim milk, in TBST) at room temperature for 1 hour to remove unless specific reaction.
  • mouse monoclonal antibody against 6x histidine [labeled alkaline phosphatase (AP)] was allowed to act at room temperature for 1 hour, and then TBST solution (10 mM Tris-HCl H 8.0, 150 mM) NaCl, 0.1% Tween 20) Wash 6 times for 5 minutes each time. After washing, the substrate (NBT/BCIP, Bio-Rad) was colored for about 10 minutes, and then washed with water to terminate the color reaction.
  • the results of Western ink stain analysis are shown in Figure 4.
  • the first lane is the Molecular weight ladder
  • the second lane is the negative control group (pET24a)
  • the third lane is the 2a-Fl recombinant protein (12.7 kDa).
  • Lane 4 is 2a-F2 recombinant protein (11.6 kDa)
  • lane 5 is 2a-F3 recombinant protein (12.1 kDa)
  • lane 6 is 2a-F4 recombinant protein (16.5 kDa)
  • lane 7 is 2a-F5 recombination.
  • lane 8 is 2a-F6 recombinant protein (20.8 kDa)
  • ninth The lane is 2a-ORF2 full-length recombinant protein (27.5 kDa).
  • Western ink stain analysis results are the same as SDS-PAGE analysis results (shown in Figure 3). 2a-ORF2 full-length recombinant protein (Fig.
  • the 2a-F3 fragment amino acid sequence (SEQ ID No: 12) is more than 2 times (as shown in Table 2); the inventors further increase or delete the arginine, and then analyze the protein expression, and find that Excessive arginine reduction can indeed increase the protein expression of PCV2 O F2.
  • Codon amino acid amount codon amino acid amount
  • the 2a-F2, 2a-F3, 2a-F4, 2a-F5 recombinant protein obtained in Example 1 was prepared as Freund's complete adjuvant as various PCV 2 subunit vaccines, and rats were administered (rats ) immunoassay to analyze each Initiation of recombinant proteins
  • the test animals produced immunogenicity against the PCV2 antibody.
  • a 96-well antigen disk containing PCV2 virus antigen (300 ng/well) was used as a test kit.
  • the antigen plate was washed 3 times with 50 mmol/L PBS (pH 7.2) (ie PBST) for 3 to 5 minutes each time; then 0.15% BSA filling solution was added to the antigen plate (blocking solution) (20 (L/well), to fill the antigen disk, and after washing for 2 hours at 37 ° C, wash with PBS.
  • the rat serum to be tested was diluted 1:50 in PBS buffer, Then double-diluted; 8 replicates of each sample, ⁇ diluted rat serum per well, washed at 37 ° C for 1 hour, washed with PBS; then added alkaline phosphatase (AP) calibration
  • AP alkaline phosphatase
  • the anti-mouse IgG antibody was washed with PBS at 37 ° C for 1 hour, then added with a solution of para-Nitrophenylphosphate (pNPP), and finally terminated with 1 M NaOH.
  • pNPP para-Nitrophenylphosphate
  • the results of the ELISA analysis are shown in Figure 5.
  • the recombinant proteins 2a-F2, 2a-F3, 2a-F4, 2a-F5 obtained in Example 1 can induce serum antibodies against PCV2 in the test animals; wherein the 2a-F2 recombinant protein fragment (SEQ ID No: 6)
  • the test animals were allowed to produce the highest amount of anti-PCV2 serum antibody, and the antibody production was significantly different ⁇ 0.05 compared to the negative control group injected with PBS.
  • the inventors of the present invention further immunized the 2a-F2, 2a-F3, 2a-F4, and 2a-F5 recombinant proteins into pigs, and found that the recombinant proteins all induced the production of anti-PCV2 serum antibodies in pigs.
  • the immunoprotective effects of these recombinant proteins were also evaluated by the PCV2 challenge test: After the recombinant proteins were formulated into subunit vaccines, the pigs were immunized and then challenged with the PCV2 strain. Afterwards, the protection rate of the immunized group was higher than that of the control group (no vaccine); the increase in protection rate referred to here included: a decrease in viremia and a slowing of PCV2 symptoms. therefore, It was confirmed that the PCV2 subunit vaccine prepared by the recombinant proteins can effectively provide immunity to the animal to improve the survival rate of the animal.
  • Example 3 Construction and expression of PCV2 2a subunit vaccine antigen protein fragment
  • the F2 protein fragment of PCV2 ORF2 can induce the highest amount of anti-PCV2 serum antibody in the test animal; in order to increase the immunogenicity of the PCV2 subunit vaccine, in the present embodiment, the first embodiment will be obtained.
  • the N-terminus of the 2a-F2 protein fragment plus the receptor binding domain I and the transmembrane targeting domain ⁇ of the exotoxin A of ftem owomw aerag ⁇ ora Protein (ie, PE protein), and the endoplasmic reticulum recovery signal (KDEL) is added to the C-terminus of the 2a-F2 protein fragment to generate an antigen fusion protein (PE-2a- F2-KDEL), in order to induce organisms to produce a better immune response.
  • the antigen fusion protein (PE-2a-F2-KDEL) transfects the DNA sequence encoding each protein into a expression vector by gene transfer technology to form an antigen fusion protein expression vector (pET24a-PE-2a-F2- KDEL), and induces the antigen fusion protein expression vector to express the antigen fusion protein (PE-2a-F2-KDEL).
  • the DNA sequence encoding the KDEL protein signal sequence (SEQ ID No: 30) was transferred to the pET24a plastid to form the pET24a-KDEL plastid; then, the 2a-F2 fragment DNA sequence obtained in Example 1 was obtained ( SEQ ID No: 5), transfected into the pET24a-KDEL plastid to form the pET24a-2a-F2-KDEL plastid; finally, the DNA sequence encoding the PE protein (SEQ ID No: 34) was transferred to The pET24a-2a-F2-KDEL plastid forms a pET24a-PE-2a-F2-KDEL plastid.
  • the DNA sequence encoding the KDEL protein signal sequence (SEQ ID No: 31) is shown in SEQ ID No: 30, amplified by PCR, and the sequence of KEDL specificity primers is as follows: Forward primer (; contains HincHU restriction enzyme digestion site):
  • Reverse primer (containing 1 ⁇ 2 I restriction enzyme digestion position):
  • the PCR reaction conditions were as follows: after reacting at 94 ° C for 3 minutes, performing 95 ° C for 1 minute, 55 ° C for 1 minute, and 72 ° C for 20 seconds for 5 cycles, and finally reacting at 72 ° C for 1 minute to carry out the extension reaction ( Elongation;
  • the PCR product was digested with pET24a vector (digested with Hind III and Xho I restriction enzymes), and the digested PC product and pET24a vector were purified and ligated.
  • the sequencing confirmed that the amplified PCR product sequence was correct.
  • the 2a-F2 DNA fragment (SEQ ID No: 5) obtained in Example 1 was propagated by PCR, and the PC primer sequence was as follows: Forward primer pF2-l (containing c l restriction enzyme cleavage position):
  • Reverse primer pR2-l (; contains H"i m restriction enzyme cleavage position):
  • the PCR reaction conditions were as follows: after reacting at 95 ° C for 5 minutes, performing 95 ° C for 30 seconds, 55 ° C for 30 seconds, and 72 ° C for 30 seconds for 25 cycles, and finally reacting at 72 ° C for 5 minutes to carry out the extension reaction ( Elongation;; respectively, the PCR product and the above pET24a-KEDL plastid were double-digested (the Sac I and Hind III restriction enzymes were digested), and the digested PCR product and pET24a-KEDL plastid Purification and conjugation are performed to select the PCR product to pET24a-KEDL plastid to form pET24a-2a-F2-KEDL plastid, and the plastid is transferred to host cell E. coli by transformation. In the CO / ), the PCR product sequence which was subjected to a large amount of proliferation and which was confirmed to be proliferated was confirmed to be correct.
  • the DNA sequence encoding the PE protein (SEQ ID No: 35) is amplified by PCR as shown in SEQ ID No: 34, and the sequence of PE specificity primers is as follows:
  • Reverse primer (containing EcoRI and Sac I restriction enzyme digestion sites):
  • the PC reaction conditions were as follows: after reacting at 94 ° C for 5 minutes, performing 95 ° C for 1 minute, 55 ° C for 1 minute, and 72 ° C for 1.5 minutes for 30 cycles, and finally reacting at 72 ° C for 7 minutes to carry out the extension reaction;
  • the PCR product was digested with the above pET24a-2a-F2-KEDL plastid (digested with BamH I and Sac I restriction enzymes), and the digested PCR product was pET24a-2a-F2.
  • -KEDL plastids were purified and ligated to select the PCR product into the pET24a-2a-F2-KDEL plastid to form the pET24a-PE-2a-F2-KEDL plastid, and pET24a-PE-2a-F2- The KEDL plastid is transferred into the expression host E. coli co/ using a transformation, and the sequence of the PCR product confirmed by sequencing is correct.
  • the DNA sequence of the PE-2a-F2-KEDL antigen fusion protein is shown in SEQ ID No: 40, and the amino acid sequence is shown in SEQ ID No: 41.
  • the porcine type 2 circovirus has other subgroups of virus strains, the following are for a 2b subgroup PCV2 strain, 2c subgroup PCV2 strain, 2d subgroup
  • the PCV2 ORF2 sequence of the PCV2 strain and the 2e subgroup PCV2 strain was analyzed for the arginine of the 1st to 234th nucleotide of the 5' end of the ORF2 and the fragment between the 235th and the last nucleotide. content.
  • the sequence analysis results of the PCV2 2b subgroup strain are shown in Table 3.
  • the 2b-ORF2 nucleotide sequence of the PCV2 2b subgroup strain is shown in SEQ ID No: 15, and the amino acid sequence is shown in SEQ ID No: 16.
  • the 1st to 234th nucleotide fragment of the 5' end of 2b-O F2 encodes 21 arginine
  • the 5' end of 2b-ORF2 is 235th to 699th nucleotide (without stop codon)
  • the sequence is shown in SEQ ID No: 17, the amino acid sequence is shown in SEQ ID No: 18, and the fragment encodes 10 arginine; the sequence analysis result of the PCV2 2b subgroup and the sequence of the PCV2 2a subgroup
  • the results of the analysis were consistent.
  • the arginine content of the N-terminal sequences of both ORF2 proteins was more than twice that of other parts of arginine (as shown in Table 2 and Table 3).
  • PCV2 2c subgroup strain 2c-ORF2 nucleotide sequence as SEQ ID No: 50
  • the amino acid sequence is shown in SEQ ID No: 51; wherein the 1st to 234th nucleotide fragment of the 5' end of 2c-ORF2 encodes 20 arginine, and the 5th end of 2C-ORF2 is 235th.
  • SEQ ID No: 54 Up to 702 nucleotide sequences are shown in SEQ ID No: 54, the amino acid sequence is shown in SEQ ID No: 55, and the fragment encodes 10 arginine; sequence analysis results of PCV2 2c subgroup and PCV2 The sequence analysis results of the 2a and 2b subgroups were consistent.
  • the arginine content of the N-terminal sequence of the three ORF2 proteins was more than twice that of other parts of arginine (as shown in Table 2, Table 3 and Table 4). .
  • the sequence analysis results of the PCV2 2d subgroup strain are shown in Table 5.
  • the 2d-ORF2 nucleotide sequence of the PCV2 2d subgroup strain is shown in SEQ ID No: 19, and the amino acid sequence is shown in SEQ ID No: 20.
  • the 1st to 234th nucleotide fragment of the 5' end of 2d-ORF2 encodes 21 arginine, and the 5th end of 2d-ORF2 is 235th to 702th nucleotide fragment (without stop codon)
  • the sequence is shown in SEQ ID No: 21, the amino acid sequence is shown in SEQ ID No: 22, and the fragment encodes 10 arginine; the sequence analysis results of the PCV2 2d subgroup are compared with PCV2 2a, 2b, and 2c. The sequence analysis results of the subgroups were consistent.
  • the arginine content of the N-terminal sequence of the four ORF2 proteins was more than twice that of other parts of arginine (as shown in Table 2, Table 3, Table 4 and Table 5). .
  • the 1st to 234th nucleotide fragment of the 5' end of 2e-ORF2 encodes 20 arginine
  • the 5th, 235th to 699th nucleotide fragment of 2e-ORF2 (without the stop codon)
  • the sequence is shown in SEQ ID No: 56
  • the amino acid sequence is shown in SEQ ID No: 57
  • the fragment encodes 9 arginine
  • the sequence analysis results of the PCV2 2e subgroup are compared with PCV2 2a, 2b, 2c and The sequence analysis results of the 2d subgroups were consistent.
  • the arginine content of the N-terminal sequence of the five ORF2 proteins was more than twice that of other parts of arginine (Table 2, Table 3, Table 4, Table 5 and Table). Six shows).
  • this case also analyzed the N-terminus of PCV2 2b subgroup, PCV2 2c subgroup, PCV2 2d subgroup and PCV2 2e subgroup.
  • the arginitie-rich domian and the non-Arginine-rich domain protein expression were found to be the same as in Example 1.
  • the N-terminal is rich in spermine.
  • the protein fragment compiled by the acid region is difficult to express; however, the protein fragment compiled by the non-arginine-rich region can be easily expressed in the biological expression system; and further, the arginine is added or deleted, and analyzed After protein expression, the results were also consistent with the PCV2 2a subgroup. When the amount of excess arginine decreased, it was found that the protein expression of PCV2 O F2 was indeed increased.
  • the arginine-rich region is between the N-terminal 1st to 78th amino acids of the PCV2 ORF2 full-length peptide
  • the region of the non-arginine-rich region is between the 79th amino acid at the N-terminus of PCV2 ORF2 and the last amino acid at the C-terminus.
  • the subunit vaccine antigen protein fragment was constructed for the ORF2 gene of the PCV2 2b subgroup strain of Example 4.
  • the F2 fragment (the 235-468 nucleotide of the 5' end of the ORF2 gene of PCV2 2b, hereinafter referred to as the 2b-F2 fragment) was used for construction.
  • the nucleotide sequence of the 2b-F2 fragment of the PCV2 2b subgroup strain is shown in SEQ ID No: 17, and the amino acid sequence thereof is shown in SEQ ID No: 18.
  • the 2b-F2 fragment nucleotide sequence was amplified by PCR, and the PCR primer sequence was as follows - forward primer pF-2b (; containing Sac I restriction enzyme cleavage position):
  • Reverse primer pR-2b (containing Himim restriction enzyme cleavage position):
  • the PCR reaction conditions were as follows: after reacting at 95 ° C for 5 minutes, performing 95 ° C for 30 seconds, 55 ° C for 30 seconds, and 72 ° C for 30 seconds for 25 cycles, and finally reacting at 72 ° C for 5 minutes to carry out the extension reaction ( Elongation;
  • the PCR product was digested with pET24a expression vector (Novagen) (digested with Sac I and Hind III restriction enzymes), and the digested PCR product and pET24a expression vector were purified. Engagement to clone the PCR product into the pET24a expression vector to form the pET24a-2b-F2 plastid, and transfer the plastid into the host cell E. coli (E.
  • the 2b-F2 amino acid fragment is used as an antigenic fragment of the PCV secondary unit vaccine
  • the PE protein and KDEL protein signal sequence were also added to the N-terminus and C-terminus of the 2b-F2 amino acid fragment to form the antigen fusion protein PE-2b-F2-KDEL, in order to induce the organism to produce a better immune response.
  • the DNA sequence of the antigen fusion protein PE-2b-F2-KDEL is shown in SEQ ID No: 44, and the amino acid sequence thereof is shown in SEQ ID No: 45.
  • the structuring strategy for expressing the plastid (pET24a-PE-2b-F2-KDEL) of the antigen fusion protein PE-2b-F2-KDEL was the same as in Example 3.
  • the DNA sequence encoding the KDEL protein signal sequence (SEQ ID No: 30) was transferred to the pET24a plastid to form the pET24a-KDEL plastid.
  • the pET24a-KDEL plastid was constructed as described in Example 3.
  • the PCR-proliferated 2b-F2 fragment DNA sequence (SEQ ID No: 17) was transferred to the pET24a-KDEL plastid to form a pET24a-2b-F2-KDEL plastid.
  • the DNA sequence encoding the PE protein (SEQ ID No: 34) was transferred into the pET24a-2b-F2-KDEL plastid to form the pET24a-PE-2b-F2-KDEL plastid, pET24a-PE-
  • the structure of the 2b-F2-KDEL plastid is also as described in the third embodiment. Next, the above E.
  • the subunit vaccine antigen protein fragment was constructed for the ORF2 gene of the PCV2 2d subgroup strain of Example 4.
  • the F2 fragment (the 235-468 nucleotide of the 5' end of the ORF2 gene of PCV2 2d, hereinafter referred to as the 2d-F2 fragment) was used for construction.
  • the nucleotide sequence of the 2d-F2 fragment of the PCV2 2d subgroup strain is shown in SEQ ID No: 21, and the amino acid sequence thereof is shown in SEQ ID No: 22.
  • the 2b-F2 fragment nucleotide sequence was amplified by PCR, and the PCR primer sequence was as follows:
  • Reverse primer pR-2d (containing Himi m restriction enzyme cleavage position):
  • the PCR reaction conditions were as follows: after reacting at 95 ° C for 5 minutes, performing 95 ° C for 30 seconds, 55 ° C for 30 seconds, and 72 ° C for 30 seconds for 25 cycles, and finally reacting at 72 ° C for 5 minutes to carry out the extension reaction ( Elongation;
  • the PCR product was digested with pET24a expression vector (Novagen) (digested with Sac I and Hind III restriction enzymes), and the digested PCR product and pET24a expression vector were purified. Engagement to clone the PCR product into the pET24a expression vector to form the pET24a-2d-F2 plastid, and transfer the plastid into the host cell E. coli (E.
  • the PE protein and the KDEL protein signal are also added to the N-terminus and the C-terminus of the 2d-F2 amino acid fragment, respectively.
  • the sequence forms the antigen fusion protein PE-2d-F2-KDEL, in order to induce an organism to produce a better immune response.
  • the DNA sequence of the antigen fusion protein PE-2d-F2-KDEL is shown in SEQ ID No: 48, and the amino acid sequence thereof is shown in SEQ ID No: 49.
  • the structuring strategy for expressing the plastid (pET24a-PE-2d-F2-KDEL) of the antigen fusion protein PE-2d-F2-KDEL was the same as in Example 3.
  • the DNA sequence encoding the KDEL protein signal sequence (SEQ ID No: 30) was transferred to the pET24a plastid to form the pET24a-KDEL plastid.
  • the pET24a-KDEL plastid was constructed as described in Example 3.
  • the PCR-proliferated 2d-F2 fragment DNA sequence (SEQ ID No: 21) was introduced and transferred into the pET24a-KDEL plastid to form a pET24a-2d-F2-KDEL plastid.
  • the DNA sequence encoding the PE protein (SEQ ID No: 34) was transferred to the pET24a-2d-F2-KDEL plastid to form the pET24a-PE-2d-F2-KDEL plastid, pET24a-PE-
  • the structure of the 2d-F2-KDEL plastid is also as described in the third embodiment. Next, the above E.
  • the PE-2a-F2-KDEL recombinant protein (SEQ ID No: 41) was prepared as a PCV 2 subunit vaccine with Freund's complete adjuvant, and a mouse immunoassay was performed to compare the two recombinant proteins to induce test animals. The immunogenicity of the anti-PCV2 antibody is produced.
  • mice Nine healthy female clean grade Balb/c mice of 5 to 6 weeks old were used as test animals, and all of the mice were negative for porcine type 2 circovirus (PCV2) ELISA antibody.
  • the 9 mice were randomly divided into 3 groups, 3 in each group.
  • groups 1 and 2 each mouse was injected intraperitoneally (intraperitoally, LP.) with 100 ⁇ g of recombinant protein 2a-F2 and PE-2a. -F2-KDEL.
  • PBS was injected as a negative control group. Two weeks later, the same immunization dose was used to boost the immunization once; each group was isolated and observed. Blood samples were collected at 2, 4, 5, and 6 weeks after the first immunization, and the antibody titer of anti-PCV2 in serum was determined by ELISA.
  • a 96-well antigen disk containing PCV2 virus antigen (300 ng/well) was used as a test kit.
  • the antigen plate was washed 3 times with 50 mmol/L PBS (pH 7.2) (ie PBST) for 3 to 5 minutes each time; then 0.15% BSA filling solution was added to the antigen plate (blocking solution) (20 (L/well), to fill the antigen disk, and after washing for 2 hours at 37 ° C, wash with PBS.
  • the serum of the mouse to be tested was diluted 1:50 in PBS buffer.
  • the PE-2a-F2-KDEL recombinant protein (SEQ ID No: 41) can elicit a higher amount of anti-PCV2 serum antibody in the test animal, and is produced in the first and second groups of mice after the fourth week of the first immunization. Serum antibody levels began to show significant differences ⁇ 0.05) ; by week 6 after the first immunization, the difference in serum antibody production between the two groups was greater ⁇ 0.001). In addition, the amount of antibody production in the first group and the second group was significantly different (p ⁇ 0.01) compared to the negative control group in which PBS was injected.
  • the immunogenicity of the anti-PCV2 antibody produced by the PE-2a-F2-KDEL recombinant protein subunit vaccine of the present invention and the PCV2 whole virus vaccine-primed test animal was compared.
  • the PE-2a-F2-KDEL recombinant protein (SEQ ID No: 41) obtained in Example 3 was prepared as a PCV 2 subunit vaccine with an oil adjuvant Montanide ISA206 (Seppic, France).
  • inactivated PCV2 whole virus (10 6 TCID 5Q /ml) was prepared as a PCV2 whole virus vaccine with the oil adjuvant Montanide ISA206 (Seppic, France).
  • Each dose of PCV2 whole virus vaccine contained 100 ⁇ inactivated.
  • PCV2 whole virus and 250 ⁇ adjuvant was performed.
  • mice Fifteen healthy female clean grade Balb/c mice of 5 to 6 weeks old were used as test animals, and all of the mice were negative for the porcine type 2 circovirus (PCV2) ELISA antibody.
  • the 15 mice were randomly divided into 3 groups of 5 each; each mouse in the first group was intraperitoneally injected (LP.) with 100 ⁇ g of recombinant protein.
  • Each mouse in Group 2 was injected intraperitoneally (I.P.) with a dose of PCV2 whole virus vaccine.
  • the oil adjuvant Montanide ISA206 was injected as a negative control group. Three weeks later, the same immunization dose was used to boost the immunization once; each group was isolated and observed.
  • the PE-2a-F2-KDEL recombinant protein SEQ ID No: 41
  • the PE-2a-F2-KDEL recombinant protein can induce a higher amount of anti-PCV2 serum antibody in the test animals, and after 1 week of the first immunization, the first group and The amount of serum antibody produced by the mice in Group 2 was significantly different ⁇ 0.001); and compared with the negative control group injected with oily adjuvant, the amount of antibody produced by the mice injected with PE-2a-F2-KDEL recombinant protein, There was a significant difference (p ⁇ 0.001) after 1 week of the first immunization.
  • the protection rate of the immunized group was higher than that of the control group (no vaccine); the increase in protection rate referred to here included: a decrease in viremia and a slowing of PCV2 symptoms. Therefore, confirm The PCV2 subunit vaccine prepared by the recombinant proteins can effectively provide immunity to the animal to improve the survival rate of the animal.
  • the PCV2 2b subgroup strain, the PCV2 2c subgroup strain, the PCV2 2d subgroup strain, and the PCV2 2e subgroup strain ORF2 were further constructed in the manners of Examples 1 to 3, and further, Examples 7 and 8 were used.
  • the immunogenicity analysis described was carried out, and it was found that the PCV2 subunit vaccine prepared by the ORF2 fragment of the subgroup (such as F2 fragment) and its fusion protein (such as: PE-F2-KDEL) can induce animals. (eg, pigs) produce anti-PCV2 antibodies that protect animals from PCV2 infection.
  • the PCV secondary unit vaccine provided by the present invention utilizes a gene recombination technique to select a fragment of a full-length protein expressed in a biological expression system from a full-length fragment of the ORF2 protein of the PCV2 virus, and a PCV2 subunit vaccine prepared by using the ORF2 protein fragment. Not only can the animal produce immunity against PCV2 virus, but the ORF2 protein fragment can be mass-produced by genetic recombination technology, and the cost of manufacturing the vaccine can also be reduced.
  • Another PCV secondary unit vaccine provided by the invention comprises the F2 protein fragment of the PCV2 virus ORF2 and the PE protein and the KDEL protein signal sequence to form a PE-F2-KDEL antigen fusion protein, and the test results show that the primary unit vaccine can be further Improve the immunity and efficiency of animals against PCV2 virus.
  • the PCV2 subunit vaccine provided by the invention is a sub-unit vaccine developed by using genetic recombination technology, and has the advantages of simple production, low cost, high purity, high yield and good safety.

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Abstract

本发明提供了一种抗猪第二型环状病毒（PCV2）免疫组合物，包含抗原肽，该抗原肽为PCV2第二开放阅读区（0RF2）非富含精氨酸肽（non-Arginine-richpeptide）和/或将该PCV20RF2非富含精氨酸肽进一步与PE肽和KDEL肽构建形成的重组融合蛋白。

Description

猪第二型环状病毒次单位疫苗
技术领域
本发明系关于一种猪第二型环状病毒 (Porcine circovirus type 2, PCV2)次单位疫苗， 特别是指一种可以被大量表现的 PCV2 0RF2蛋白质片段做为抗原蛋白质， 以及加入适 当的载剂或佐剂而形成的猪第二型环状病毒次单位疫苗。 背景技术
猪第二型环状病毒已知与离乳后多系统消耗性综合症 (Postweaning Multisystemic Wasting Syndrome, PMWS)、 猪皮肤炎肾病症候群 (Porcine Dermatitis And Nephropathy Syndrome, PDNS)有关。 PMWS最早于 1991在加拿大发现，且之后于全世界各地陆续有 被报导。 该病对于全球养猪业造成严重损失， 其主要病征为患猪体重渐进性丧失 (progressive weight loss)、急性呼吸症 (tachypnea)、呼吸困难 (dyspnea)、黄疸 (jaundice)等。 病理学上， 肉眼及组织病变有淋巴球性肉芽肿性浸润 (lymphocytic and granulomatous infiltrate)、 淋巴结病（lymphadenopathy)、 淋巴球性肉芽肿性肝炎（lymphocytic and granulomatous hepatitis)及肾炎 (nephritis)。
猪环狀病毒 (porcine circovirus, PCV)最早于 1982 年在猪肾细胞株 (PK-15, ATCC CCL31)中被发现，虽然会持续感染该细胞，但是并不产生细胞病变效应 (cytopathic effect, CPE), 另外该病毒虽会感染猪只， 但是并不造成任何病变， 该病毒株系命名为 PCV1。 PCV1病毒为一环狀单股、 全长 1759 bp的正二十面体 (icosahedron)病毒， 在 1995年被 归類为环狀病毒科 CCircoviridae)的一员。
自 PK-15细胞中分离出的 PCV1被认定是不具备病源性， 而于 1997年自罹患离乳 后多系统消耗性综合症 (Postweaning Multisystemic Wasting Syndrome, PMWS)猪只体内， 分离出对猪只具有病原性的突变猪环状病毒， 该病毒株命名为 PCV2。 离乳后多系统消 耗性综合症 (PMWS)是一种具有高度传染力的猪只疾病，该病主要感染幼猪及怀孕母猪， 严重影响猪只健康。
PCV2病毒直径大小为 17 nm， 具有环状单股 DNA， DNA大小为 1.76 Kb， 基因组 序列经软体分析后发现顺时针加上逆时针共有 11 个开放阅读区 (open reading frame, ORF)， 其中 ORFl、 ORF2是 2个最主要的基因。 ORF1可转录出 Rep及 Rep'蛋白， 和 该病毒的复制有关 (Mankertz et al, 2001)。已知 ORF2本身是 PCV2病毒的免疫性结构壳 体蛋白（immunogenic structure capsid protein), 用以诱导生物体产生免疫反应。
最常见的市售 PCV2疫苗为 PCV2死毒疫苗， 然而死毒疫苗研发上需取得无其他病 毒感染的细胞株， 且其最大的缺点在于无法确保在制备死毒疫苗过程中， 化学药剂是否 完全不活化病毒株； 而另一缺点在于以化学药剂来杀死病原菌很有可能会改变病原菌的 抗原结构， 使得所诱发出来的免疫反应无法中和病原菌的毒性， 因此造成该死毒疫苗无 法保护猪只避免感染此疾病； 由此可知， 死毒疫苗产品发展不易、 成本高， 且其安全性 堪虑。
相对于死毒疫苗是以整株病毒作为疫苗的抗原，次单位疫苗则是取病原菌中一部分 的蛋白质作为抗原蛋白质， 并将该抗原蛋白质作为疫苗接种到动物或人体中， 使接种后 的动物或人体可产生免疫力。 它的制备方式是先选殖病原菌中的抗原蛋白质的基因， 然 后以遗传工程的技术将此抗原蛋白质大量生产。 其最大的优点是安全性高， 因为它只是 病原菌的一个部分， 所以注射到猪只体内的并不是一个完整的病原菌， 不会有病原菌不 活化不完全的顾虑。 习用 PCV2次单位疫苗系使用 ORF2蛋白片段作为抗原蛋白质； 然 而 ORF2的全长蛋白质在原核生物表现系统中表现量很低，不符制备疫苗的需求。因此， 研发可以在生物表现系统中表现量高的 PCV2 ORF2疫苗抗原片段， 将有助于 PCV2次 单位疫苗的商业应用。 发明内容
本发明提供一种 PCV2病毒 ORF2蛋白片段的 DNA序列，该 DNA序列可于生物表 现系统中表现出大量的 PCV2病毒 ORF2蛋白片段。
本发明并提供一种 PCV2次单位疫苗， 系以可在生物表现系统中大量表现的 PCV2 病毒 ORF2蛋白片段作为疫苗的抗原蛋白， 使该疫苗在施打于动物体后， 可以诱导动物 体产生足以抵抗 PCV2病毒感染的免疫力。
本发明并提供一种利用基因重组技术所研发的次单位疫苗， 以生产制程简单、 成本 低、 纯度高、 安全性佳的 PCV2次单位疫苗。
由于 PCV2病毒 ORF2蛋白全长片段难以在生物系统中大量表现， 为达成上述发明 目的，本案发明人利用基因重组技术，将 PCV2病毒中编码 ORF2全长蛋白 (如 SEQ ID NO: 2所示)的 DNA序列 (如 SEQ ID NO: 1所示)， 进行不同长度的切割， 再将所形成的各 DNA 片段分别构筑至蛋白表达载体上， 于生物系统中表现蛋白质， 以决定于生物系统 中可产生高量蛋白质的 DNA片段。
经蛋白质序列分析以及蛋白表现量试验结果显示， PCV2病毒 ORF2全长蛋白共有 约 30个精胺酸 (Arginine)，其中三分之二以上的精胺酸集中于 ORF2蛋白的胺基端 (amino terminal, N端)， 当 ORF2蛋白 N端的精胺酸数目被去除的越多， ORF2蛋白片段在生 物表现系统中的表现量就越多； 而去除编码 ORF2蛋白的 DNA序列 5'端 234 bp后所剩 的 DNA片段 (δ卩 5，端 235 bp至终止密码子 (stop codon)之间的片段)， 可以在生物表现系 统中大量表现。
是以， 本发明所提供的 PCV2次单位疫苗， 包括一 PCV2抗原蛋白质片段， 以及适 当的载剂或佐剂； 该抗原蛋白质片段可于生物表现系统中大量表现， 系为 PCV2病毒的 O F2非富含精胺酸胜肽 (non-Arginine-rich peptide), 该 PCV2 ORF2非富含精胺酸胜肽 的精胺酸数量系为该 PCV2 ORF2全长胜肽的 N端富含精胺酸区域 (Arginine-rich domian) 的精胺酸数量的二分之一 （1/2) 以下； 于一实施例中， 当该 PCV2 0RF2的 N端富含精 胺酸区域的精胺酸数量为 20时，则于该 PCV2 ORF2非富含精胺酸胜肽的精胺酸数量等 于 0或 1至 10之间的正整数； 于另一实施例中， 当该 PCV2 ORF2的 N端富含精胺酸 区域的精胺酸数量为 21时， 则于该 PCV2 ORF2非富含精胺酸胜肽的精胺酸数量等于 0 或 1至 10之间的正整数； 于又一实施例中， 当该 PCV2 ORF2的 N端富含精胺酸区域 的精胺酸数量为 22时， 则于该 PCV2 ORF2非富含精胺酸胜肽的精胺酸数量等于 0或 1 至 11之间的正整数。
于一实施例中，该富含精胺酸区域为该 PCV2 O F2全长胜肽的胺基端 (N端)第 1-78 个胺基酸的区域 (；相当于 DNA序列的 5端第 1-234个核苷酸之间的区域);于一实施例中， 该非富含精胺酸胜肽为该 PCV2 ORF2全长胜肽的胺基端 (N端)第 79个胺基酸至羧基端 (C端)最后一个胺基酸间的胜肽 (相当于 DNA序列的 5'端第 235个核苷酸至 3'端终止密 码子之间的区域)。
该 PCV2 O F2全长胜肽与如 SEQ ID No: 2、 SEQ ID No: 16、 SEQ ID No: 20、 SEQ ID No: 51及 SEQ ID No: 53所示的序列其中之一具有至少 80%序列同源性 (homology), 较佳者，具有 85%序列同源性，更佳者，具有 90%序列同源性，甚至是 91%、 92%、 93%、 94%、 95%、 96%、 97%、 98%、 99%序列同源性。 于一较佳实施例中， 该 PCV2 ORF2 全长胜肽系为如 SEQ ID No: 2、 SEQ ID No: 16、 SEQ ID No: 20、 SEQ ID No: 51及 SEQ ID No: 53所示的序列其中之一。
此外，本发明并利用绿脓杆菌 CP^m owomw aerag ora)外毒素 A (exotoxin A)的受体 结合区 (receptor binding domain I)及运输结构区 (transmembrane targeting domain II)的蛋 白（即 PE蛋白）以及内质网回收讯号 (ER retention signal,即 KDEL蛋白质讯号序列 (signal peptide))的特性， 提供一种 PCV2 ORF2融合蛋白抗原片段。
绿脓杆菌 PE蛋白系可作为目标蛋白的引导媒介， 其作用的方式为： 首先， 绿脓杆 菌外毒素 A 的受体结合区蛋白负责与目标细胞 (CD8+ T 细胞)的细胞膜上的细胞受体 (receptor)结合， 经由细胞内噬作用 (endocytosis)进入细胞的内膜体 (endosome)中， 当绿脓 杆菌外毒素 A被内膜体中的蛋白酶切割后， 绿脓杆菌外毒素 A的运输结构区蛋白会将 剩余的蛋白片段 (含运输结构区蛋白及其后所连接的目标蛋白）由内膜体位移至细胞质 内， 与高基氏体 (Golgi body)及内质网 (endoplasmic reticulum, ER)结合， 进而将目标蛋白 引导至目标细胞中。
另外， 若一目标蛋白的 C端具有一 KDEL蛋白质讯号序列 (signal peptide), 则在该 KDEL蛋白质讯号序列的作用下， 目标蛋白会被回收至内质网， 进而进入内质网进行作 用。
因此，本发明进一步利用 PE蛋白及 KDEL蛋白质讯号序列的特性，提供一种 PCV2 ORF2融合蛋白抗原片段， 系于 PCV2病毒 ORF2蛋白片段的 N端加上 PE蛋白， 且于 该 PCV2病毒 ORF2蛋白片段的 C端加上 KDEL蛋白质讯号序列 (signal peptide), 以产 生抗原融合蛋白 (PE-ORF2片段 -KDEL), 使该疫苗在施打于动物体后， 可以诱导动物体 产生足以抵抗 PCV2病毒感染的免疫力。
于部分实施态样中， 本发明所用的抗原蛋白包含但不限于 PCV2病毒 ORF2蛋白片 段，该 ORF2蛋白片段系与如 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及 SEQ ID No: 57所示的序列其中 之一具有至少 80%序列同源性， 较佳者， 具有 85%序列同源性， 更佳者， 具有 90%序列 同源性， 甚至是 91%、 92%、 93%、 94%、 95%、 96%、 97%、 98%、 99%序列同源性。 于一较佳实施例中， 该 PCV2病毒 ORF2蛋白片段系为如 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及 SEQ ID No: 57所示的序列其中之一，该蛋白片段系藉由基因转殖方式而得;分别将编码 PCV2 病毒 ORF2蛋白片段的 DNA序列 (SEQ ID NO: 5, 7, 9, 11 , 17, 21 , 54, 56)选殖到表现载体 中， 各形成含有编码抗原蛋白的 DNA序列的质体， 再将该质体转殖到生物表现宿主中， 经蛋白质表现后而得到的抗原蛋白。
本发明所用的抗原融合蛋白 (PE-ORF2片段 -KDEL)系藉由基因转殖方式而得， 系将 上述编码 PCV2 O F2的 DNA部分片段序列 (SEQ ID NO: 5, 7, 9, 11 , 17, 21 , 54, 56)、 编 码 PE蛋白质讯号序列的 DNA片段序列 (如 SEQ No: 34所示)， 以及编码 KDEL蛋白质 讯号序列的 DNA片段序列 (如 SEQ No: 30所示)选殖到表现载体系统中， 形成含有编码 抗原融合蛋白的 DNA序列的质体， 再将该质体转殖到表现宿主中， 经蛋白质表现后而 得到的抗原融合蛋白。
该表现载体系统包含但不限于 pET载体系统以及 pGEX载体系统等;于一实施例中， 该表现载体为 pET24a; 该生物表现系统 (宿主)包含但不限于： 原核表现系统 (如： 大肠 杆菌 co/0)、 真核表现系统 (如： 动物细胞 (CHO cell)、 植物细胞)， 于一实施 例中， 该表现系统为大肠杆菌 (E. coli)。
该佐剂包含但不限于水性氢氧化铝胶、 明矾、 Freimd氏不完全佐剂、 油质佐剂、 水 溶性佐剂、 或水包油包水双相佐剂 (water-in-oil-in-water, W/0/W); 于一实施例中， 该佐 剂为油质佐剂。
本发明所提供的免疫组合物， 进一步可包含 PCV2 其他开放阅读区 (open reading frame, ORF)的抗原蛋白， 该 PCV2其他 ORF的抗原蛋白包含但不限于： ORFl、 ORF3。 此外， 该免疫组合物可进一步包含一种病原抗原， 该病原抗原系选自由下列群组所组成 者： 猪流感病毒 (SIV)抗原、 猪繁殖与呼吸症候群病毒 (PRRSV)抗原、 猪霉浆菌 (Mycoplasma)、 猪小病毒 (Parvovirus, PPV)、 猪丹毒 ( Erysipelas )， 以及伪狂犬病 (Aujeszky's disease)
另， 本发明所提供的免疫组合物可进一步包含一或多种选自于下者： 载剂、 溶剂、 乳化剂、 悬浮剂、 分解剂、 黏结剂、 赋形剂、 安定剂、 螯合剂、 稀释剂、 胶凝剂、 防腐 剂、 润滑剂、 界面活性剂、 佐剂、 生物型载体。
本说明书中所述的所有技术性及科学术语， 除非另外有所定义， 皆为该所属领域具 有通常技艺者可共同了解的意义。
本发明系以下面的实施例予以示范阐明， 但本发明不受下述实施例所限制。 附图说明
图 1为猪第二型环状病毒 (PCV2)基因组序列的亲缘树分析图。
图 2为 PCV2 0RF2分段表现示意图， 其中 pFl、 pF2、 pF3、 pF4、 p K pR2、 p 3 为 PCR引子。
图 3为以 SDS-PAGE分析以大肠杆菌分别表现 PCV2 2a-O F2各片段重组蛋白的结 果； 将大肠杆菌以 IPTG诱导 6小时后， 收集全菌蛋白， 以 15% SDS-PAGE分析； 其中 第 1道为标准分子量 (molecular weight ladder); 第 2道为 pET24a载体 (负对照组)； 第 3 道为 PCV2 ORF2 2a-Fl片段 (12.7 KDa); 第 4道为 PCV2 ORF2 2a-F2片段 (11.6 KDa); 第 5道为 PCV2 O F2 2a-F3片段 (12.1 KDa);第 6道为 PCV2 ORF2 2a-F4片段 (16.5 KDa); 第 7道为 PCV2 O F2 2a-F5片段 (21.4 KDa);第 8道为 PCV2 ORF2 2a-F6片段 (20.8 KDa); 第 9道为 PCV2 2a-O F2全长片段 (27.5 KDa)。
图 4为以西方墨渍法检测 PCV2 2a-ORF2各片段重组蛋白表现的结果； 其中第 1道 为标准分子量 (molecular weight ladder);第 2道为 pET24a载体 (负对照组)；第 3道为 PCV2 O F2 2a-Fl片段 (12.7 KDa);第 4道为 PCV2 ORF2 2a-F2片段 (11.6 KDa);第 5道为 PCV2 O F2 2a-F3片段 (12.1 KDa);第 6道为 PCV2 O F2 2a-F4片段 (16.5 KDa);第 7道为 PCV2 O F2 2a-F5片段 (21.4 KDa);第 8道为 PCV2 O F2 2a-F6片段 (20.8 KDa);第 9道为 PCV2 2a-O F2全长片段 (27.5 KDa)。
图 5为大鼠分别以各不同长度片段的 2a-ORF2重组蛋白免疫后，不同时间采集的血 液样本以 PCV2 ELISA测定抗体力价的结果， *表示 ρ < 0.05。
图 6为小鼠分别以 2a-F2重组蛋白以及 PE-2a-F2-KDEL重组蛋白免疫后，不同时间 采集的血液样本以 PCV2 ELISA测定抗体力价的结果， *表示 p < 0.05， **表示 p < 0.01， ***表示 ρ < 0.001 ; #表示 ρ < 0.05， 鼎表示 ρ < 0.01， 漏表示 ρ < 0.001。
图 7为小鼠分别以 PE-2a-F2-KDEL重组蛋白以及 PCV2全病毒疫苗免疫后，不同时 间采集的血液样本以 PCV2 ELISA测定抗体力价的结果， *表示 p < 0.05， **表示 p <
0.01， ***表示 ρ < 0.001 ; #表示 ρ < 0.05， 鼎表示 ρ < 0.01， 漏表示 ρ < 0.001。 具体实施方式
实施例一 PCV2次单位疫苗抗原蛋白片段的构筑及决定
由于 PCV2病毒 ORF2蛋白全长片段难以在生物表现系统中大量生产， 因此本案发 明人将 PCV2 O F2蛋白的基因分成不同 DNA片段， 并将这些不同的 DNA片段构筑至 载体上， 于生物表现系统中表现各 DNA片段编码的蛋白质， 并分析各 DNA片段在生 物表现系统中的蛋白质产量， 以决定于生物表现系统中可产生高量蛋白质的较佳 DNA 片段。
以下， 系以常用的原核生物表现系统 (大肠杆菌)用以分析及说明。
1. 增幅不同长度 PCV2 0RF2基因片段
本实施例中所使用的 PCV2 ORF2基因序列全长如 SEQ ID No: 1所示， 根据 Wang 等人 (Wang et al., Virus Research 2009, Genetic variation analysis of Chinese strains of porcine circovirus type 2)所提供的序列作为亲源关系树标准序列来分析 SEQ ID No: 1；结 果显示该 PCV2 ORF2序列 (SEQ ID No: 1)属于 PCV2 2a亚群 (subgroup)的一员 (如图 1所 示)。 针对上述 PCV2 2a 的 ORF2 (；以下称为 2a-ORF2)基因序列设计可增幅不同长度 2a-O F2 基因片段的引子 (；如表一所示）， 以进行聚合酶连锁反应 (polymerase chain reaction, PCR)增幅各片段。
如图 2所示， 全长的 2a-ORF2基因片段以专一性引子 pFl(正向引子)及 pRl(反向引 子)进行 PCR增幅， 取得全长 2a-ORF2基因片段的 DNA序列如 SEQ ID NO: 1所示， 以 下简称 2a-ORF2; 而不同长度的 PCV2 2a-ORF2基因片段则分别命名为 2a-Fl、 2a-F2、 2a-F3、 2a-F4、 2a-F5及 2a-F6。 2a-Fl片段位于 PCV2 2a-ORF2的 5'端第 1至 234个核 苷酸的位置， 2a-Fl片段的 DNA序列如 SEQ ID NO: 3所示。 2a-F2片段位于 PCV2 ORF2 的 5'端第 235至 468个核苷酸的位置， 2a-F2片段的 DNA序列如 SEQ ID NO: 5所示。 2a-F3片段位于 PCV2 O F2的 5'端第 469至 699个核苷酸的位置， 2a-F3片段的 DNA 序列如 SEQ ID NO: 7所示。 2a-F4片段位于 PCV2 O F2的 5'端第 349至 699个核苷酸 的位置， 2a-F4片段的 DNA序列如 SEQ ID NO: 9所示。 2a-F5片段位于 PCV2 O F2的 5'端第 235至 699个核苷酸的位置， 2a-F5片段的 DNA序列如 SEQ ID NO: 11所示。 2a-F6 片段位于 PCV2 ORF2的 5'端第 1至 468个核苷酸的位置， 2a-F6片段的 DNA序列如 SEQ ID NO: 13所示。 所有的正向引子 (forward primer)均设计具 H «t m的限制酶切位， 而反 向引子 (reverse primer)均设计具 Xho I的限制酶切位。 表一、 用以增幅不同长度的 PCV2 ORF2基因片段的引子序列表
片段 引子 序列 ft
pFl 正向引子 CCCAAGCTTGCATGACGTATCCAAGGAGGCG SEQ ID NO: 23
ORF2
pRl 反向引子 CCGCTCGAGGGGTTTAAGTGGGGGGTCTTTA SEQ ID NO: 24 pFl 正向引子 CCCAAGCTTGCATGACGTATCCAAGGAGGCG SEQ ID NO: 23
F1
pR3 反向引子 CCGC1CQAQGTCGTTAATATTAAATCTCATC SEQ ID NO: 28 pF2 正向引子 CCCAAGCTTGCTTTGTTCCCCCGGGAGGGGG SEQ ID NO: 25
F2
pR2 反向引子 CCGCTCGAGGTAGGAGAAGGGTTGGGGGATT SEQ ID NO: 26 pF3 正向引子 CCCAAGCTTGCCACTCCCGGTACTTTACCCC SEQ ID NO: 27
F3
pRl 反向引子 CCGCTCGAGGGGTTTAAGTGGGGGGTCTTTA SEQ ID NO: 24 pF4 正向引子 CCCAAGCTTGCGGAGTGGGCTCCACTGCTGT SEQ ID NO: 29
F4
pRl 反向引子 CCGCTCGAGGGGTTTAAGTGGGGGGTCTTTA SEQ ID NO: 24 pF2 正向引子 CCCAAGCTTGCTTTGTTCCCCCGGGAGGGGG SEQ ID NO: 25
F5
pRl 反向引子 CCGCTCGAGGGGTTTAAGTGGGGGGTCTTTA SEQ ID NO: 24 pFl 正向引子 CCCAAGCTTGCATGACGTATCCAAGGAGGCG SEQ ID NO: 23
F6
pR2 反向引子 CCGCTCGAGGTAGGAGAAGGGTTGGGGGATT SEQ ID NO: 26 注： 序列下方以 标示者为酵素切位 H t III的序列 (AAGCTT), 序列下方以 标 示者为酵素切位 Xho I的序列 CCTCGAG)。
PCR反应条件为： 95°C反应 5分钟后， 进行 95°C 30秒、 55°C 30秒、 72°C 30秒， 共 25个循环， 最后以 72°C反应 5分钟以进行延伸反应 (elongation); 所得的 PCR产物 大小分别为： 2a-ORF2为 699 bp、 2a-Fl为 234 bp、 2a-F2为 234 bp、 2a-F3为 231 bp、 2a-F4 为 351 bp、 2a-F5 为 465 bp、 2a-F6 为 468 bp。 并以 2 %洋菜胶电泳 (agarose electrophoresis)鉴定各 PCR产物片段大小。 鉴定无误后， 以 PCR-M纯化套组 (Viogene) 纯化各 PCR产物。
2. 将不同长度 PCV2 O F2基因片段构筑于 pET24a表现载体
分别将 1 g PCR产物与 1 的 pET24a表现载体 (Novagen), 以 1 l Hind m及 1 μΐ Xho I (New England Biolabs)两种限制酶进行限制酶切割反应（Restriction Enzyme Cleavage Reaction),于 37°C反应 8小时。再以 PCR-M Clean up System纯化套组 (Viogene) 纯化酶切后的 PCR产物及 pET24a表现载体，接着进行接合反应 (ligation)。将各 PCR产 物选殖到 ET24a 载体以取得 ET24a-2a-Fl、 pET24a-2a-F2、 pET24a-2a-F3、 pET24a-2a-F4、 pET24a-2a-F5、 pET24a-2a-F6及 pET24a-2a-ORF2质体， 并将上述质体 分别利用转殖作用 (transformation)送入表现宿主大肠杆菌 (E. CO/ )中， 经选殖挑选出菌株 中带有 PCR片段的质体后， 进行定序确认增殖的 PCR产物序列无误， 以得到含有上述 质体的菌株。
3. 不同长度 PCV2 O F2基因片段的蛋白表达及确认
先将含有上述质体的菌株以 2 ml的 LB培养基， 于 37°C培养 16-18小时， 再将菌 液以 1 : 50的比例接种至 LB培养基中， 培养基中含有 25 g/ml kanamycin， 置于 37°C、 200 rpm的培养箱中培养，直到菌液浓度达 O.D 600 nm为 0.6时，加入 β-D-thiogalactoside (IPTG)使其最终浓度为 1 mM， 再于 37°C、 200 rpm的培养箱中培养 6小时， 吸取 1 ml 菌液，经 10,000 xg离心后，取离心后的菌块以 B-PERTM细菌蛋白质萃取试剂 (B-PERTM Bacterial Protein Extraction, 购自 PIERCE)来确认重组蛋白为可溶性蛋白或是包涵体 (inclusion body)。 确认方法为于菌块中加入 40 μΐ的反应试剂， 剧烈震荡 (vortex) 1分钟 后， 10,000 xg离心， 离心后重组蛋白在上层部份为可溶性蛋白， 在下层部份则为包涵 体。 将可溶性蛋白溶于 SDS-PAGE 用的 lx sample buffer, 下层的菌块则加入 2x 的 SDS-PAGE sample buffer。 以干浴槽 100°C煮沸 20分钟后离心之， 吸取上层液体部份， 以 15 %的 SDS-PAGE确认重组蛋白表现的情况。
以 IPTG诱导含有上述质体的菌株产生重组蛋白， 其中 2a-ORF2重组蛋白的胺基酸 序列如 SEQ ID No: 2所示； 2a-Fl重组蛋白的胺基酸序列如 SEQ ID No: 4所示， 2a-F2 重组蛋白的胺基酸序列如 SEQ ID No: 6所示， 2a-F3重组蛋白的胺基酸序列如 SEQ ID No: 8所示， 2a-F4重组蛋白的胺基酸序列如 SEQ ID No: 10所示， 2a-F5重组蛋白的胺基酸 序列如 SEQ ID No: 12所示， 2a-F6重组蛋白的胺基酸序列如 SEQ ID No: 14所示。
以 SDS-PAGE确认各片段重组蛋白表现的结果如图 3所示，第 1道为标准分子量梯 度 (Molecular weight ladder),第 2道为负对照组 (pET24a)，第 3道为 2a-Fl重组蛋白（12.7 kDa)， 第 4道为 2a-F2重组蛋白（11.6 kDa)， 第 5道为 2a-F3重组蛋白（12.1 kDa)， 第 6 道为 2a-F4重组蛋白（16.5 kDa)，第 7道为 2a-F5重组蛋白 (21.4 kDa)， 第 8道为 2a-F6重 组蛋白 (20.8 kDa)， 第 9道为 2a-ORF2全长重组蛋白 (27.5 kDa)。于 2a-ORF2的 6个片段 中， 2a-F2、 2a-F3、 2a-F4、 2a-F5片段都能大量表现 (；图 3第 4、 5、 6、 7道)， 其分子量 位置与预测的 11.6kDa、 12.1kDa、 16.5kDa、 21.4kDa符合。
接着以西方墨渍分析法 (Western Blot)确认表达重组蛋白是否为 PCV2 2a-ORF2的片 段。 将 SDS-PAGE电泳分析后的胶体转印到 PVDF尼龙膜 (Nylon membrane)上。 转印后 将尼龙膜置于填塞液 (Blocking buffer: 5 % Skim milk, in TBST)于室温作用 1小时， 以去 除非特异性反应。 之后， 加入第一级抗体： 老鼠抗 6x组织胺酸的单株抗体 [标帜碱性磷 酸酶 (AP)]于室温作用 1小时，再以 TBST液 (10 mM Tris-HCl H 8.0, 150 mM NaCl, 0.1 % Tween 20)清洗 6次， 每次各 5分钟。 洗涤后， 加受质 (NBT/BCIP, Bio-Rad)呈色约 10分 钟后， 以清水水洗终止呈色反应。
西方墨渍分析法结果如图 4 所示， 第 1 道为标准分子量梯度 (Molecular weight ladder),第 2道为负对照组 (pET24a)，第 3道为 2a-Fl重组蛋白（12.7 kDa)，第 4道为 2a-F2 重组蛋白（11.6 kDa)，第 5道为 2a-F3重组蛋白（12.1 kDa)，第 6道为 2a-F4重组蛋白（16.5 kDa)， 第 7道为 2a-F5重组蛋白 (21.4 kDa)， 第 8道为 2a-F6重组蛋白 (20.8 kDa)， 第 9 道为 2a-ORF2全长重组蛋白 (27.5 kDa)。西方墨渍分析法结果与 SDS-PAGE分析结果 (如 图 3所示)相同。 2a-ORF2全长重组蛋白（图 4第 9道)无法表现； 2a-ORF2的 6个片段中， 2a-F2、 2a-F3、 2a-F4、 2a-F5片段的蛋白都能被大量表现 (图 4第 4、 5、 6、 7道)， 其分 子量位置与预测的 11.6kDa、 12.1kDa、 16.5kDa、 21.4kDa符合。 然而， 2a-Fl禾 P 2a-F6 片段并没有重组蛋白的表现 (图 4第 3、 8道)，由于 2a-Fl和 2a-F6片段都涵盖了从 PCV2 2a-ORF2的 5'端第 1至 234个核苷酸的基因片段 (请参阅图 2所示)， 因此， 造成 ORF2 基因无法大量表现的原因可能是该 2a-Fl片段内的某些序列， 故针对 2a-Fl片段 (PCV2 2a-O F2的 5'端第 1至 234个核苷酸， SEQ ID No: 3)及 2a-F2 + 2a-F3片段 (PCV2 2a-O F2 的 5'端第 235至 699个核苷酸 (不含终止密码子)， SEQ ID No: 11)的胺基酸序列分析后， 发现 2a-Fl片段胺基酸序列 (SEQ ID No: 4)的精胺酸 (Arginine)含量是 2a-F2 + 2a-F3片段 胺基酸序列 (SEQ ID No: 12)的 2倍以上 (如表二所示)； 本案发明人进一步将该些精胺酸 增加或删除后，去分析蛋白质表现，发现将该些过多的精胺酸减少后，确实可增加 PCV2 O F2的蛋白表现量。
表二、 PCV2 2a-ORF2胺基酸序列中精胺酸的数量分析
2a-Fl 2a-F2 + 2a-F3
密码子 胺基酸 数量 密码子 胺基酸 数量
AGA 精胺酸 5 AGA 精胺酸 3
AGG 精胺酸 3 AGG 精胺酸 3
CGA 精胺酸 1 CGC 精胺酸 1
CGC 精胺酸 10 CGG 精胺酸 1
CGT 精胺酸 2 CGT 精胺酸 1 总数 21 总数 9 实施例二 PCV2次单位疫苗抗原蛋白片段的免疫原性 (immimogenicity)的决定
1. 大鼠免疫试验
将实施例一所得到的 2a-F2、 2a-F3、 2a-F4、 2a-F5重组蛋白以弗氏完全佐剂 (Freund's complete adjuvant)制备为各种 PCV2次单位疫苗， 并进行大鼠 (rats)免疫试验， 以分析各 重组蛋白的引发试验动物产生抗 PCV2抗体的免疫原性。
取 5〜6周龄健康雌性清洁级大鼠 15只作为试验动物， 所有大鼠的猪第二型环状病 毒 (PCV2)酵素结合免疫吸附分析 (ELISA)抗体皆呈阴性。 将这 15只大鼠随机分为 5组， 每组 3只； 第 1~4组中， 每只大鼠分别以皮下注射 (subcutaneously, S.C.)注射 200 μ§重 组蛋白， 注射疫苗总体积为 300 μί， 其中重组蛋白与佐剂的体积比为 1 : 1。 第 5组则 注射 300 PBS作为负对照组。 两周后用相同免疫剂量加强免疫一次； 各组隔离饲养 观察，首次免疫后第 0、2、4、8周分别采血分离血清，以酵素连结免疫分析 (Enzyme-linked immunosorbent assay, ELISA)测定血清内抗猪第二型环状病毒 (PCV2)的抗体力价。
2. 血清抗体的判定 - 以 ELISA测定抗 PCV2抗体力价
以含有 PCV2病毒抗原 (300 ng/孔)的 96孔抗原盘作为检测套组。 以含有 500 μΙ/L Tween-20的 50 mmol/L PBS (pH 7.2) (即 PBST)洗涤抗原盘 3次，每次 3~5分钟； 再于抗 原盘中加入 0.15% BSA填充液 (blocking solution)(20( L/孔)， 以填充 (block)抗原盘， 并 于 37°C下作用 2小时后， 以 PBS洗涤。将待检测的大鼠血清以 PBS缓冲液以 1 : 50倍 数稀释， 然后再做倍比稀释； 每个样品 8重复， 每孔加 ΙΟΟμί稀释的大鼠血清， 于 37°C 下作用 1小时后， 以 PBS洗涤； 然后加入碱性磷酸酶 (alkaline phosphatase, AP)标定的 anti-mouse IgG抗体， 于 37°C下作用 1小时后， 以 PBS洗涤； 接着加入磷酸对硝基苯 酯 (para-Nitrophenylphosphate, pNPP)溶液显色， 最后用 1M的 NaOH终止反应后判定结 果。 读取各样本在 405 nm波长下的吸光值， 每个样本读取两次， 并取平均值进行分析。
ELISA分析结果如图 5所示。 在实施例一所得到的 2a-F2、 2a-F3、 2a-F4、 2a-F5重 组蛋白皆可诱发试验动物产生抗 PCV2的血清抗体； 其中又以 2a-F2重组蛋白片段 (SEQ ID No: 6)可以引发试验动物产生最高量之抗 PCV2血清抗体，且相较于注射 PBS的负对 照组， 抗体产生量皆具有显著差异 <0.05)。
本案发明人进一步亦将该 2a-F2、 2a-F3、 2a-F4、 2a-F5重组蛋白进行猪只免疫， 结 果发现该等重组蛋白皆可诱发猪只产生抗 PCV2血清抗体。 此外， 亦藉由 PCV2攻毒试 验评估该等重组蛋白的免疫保护效力： 将该等重组蛋白配制成次单位疫苗后， 进行猪只 免疫后， 再以 PCV2病毒株攻毒， 结果发现经攻毒后， 免疫组的保护率高于对照组 (未施 以疫苗者)； 此处所指的保护率提高包含： 病毒血症的降低、 PCV2病征的减缓。 因此， 证实该等重组蛋白所制备的 PCV2次单位疫苗可有效提供动物产生免疫力， 以提高动物 的存活率。 实施例三 PCV2 2a次单位疫苗抗原蛋白片段的构筑及表达
由实施例二的试验结果可知， PCV2 ORF2的 F2蛋白片段可引发试验动物产生最高 量之抗 PCV2血清抗体； 为了提高 PCV2次单位疫苗的免疫原性， 在本实施例中， 将实 施例一所得的 2a-F2 蛋白片段的 N 端加上绿脓杆菌 (ftem owomw aerag^ora)外毒素 A(exotoxin A)的受体结合区 (receptor binding domain I)及运输结构区 (transmembrane targeting domain Π)的蛋白（即 PE蛋白）， 且于该 2a-F2蛋白片段的 C端加上内质网回收 讯号 (ER retention signal) - KDEL蛋白质讯号序列 (signal peptide), 以产生抗原融合蛋白 (PE-2a-F2-KDEL), 以期诱导生物体产生更佳的免疫反应。
该抗原融合蛋白 (PE-2a-F2-KDEL)系透过基因转殖技术，将编码各蛋白的 DNA序列 转入表现载体中， 形成一抗原融合蛋白表现载体 (pET24a-PE-2a-F2-KDEL)， 并诱导该抗 原融合蛋白表现载体表现该抗原融合蛋白 (PE-2a-F2-KDEL)。 首先， 取编码 KDEL蛋白 质讯号序列的 DNA序列 (SEQ ID No: 30)，转殖到 pET24a质体中，以形成 pET24a-KDEL 质体； 然后， 取实施例一所得的 2a-F2 片段 DNA 序列 (SEQ ID No: 5)， 转殖到该 pET24a-KDEL质体中，以形成 pET24a-2a-F2-KDEL质体；最后，取编码 PE蛋白的 DNA 序列（SEQ ID No: 34)， 转殖到该 pET24a-2a-F2-KDEL 质体中 ， 以形成 pET24a-PE-2a-F2-KDEL质体。
1. pET24a-KEDL质体的构筑
编码 KDEL蛋白质讯号序列 (SEQ ID No: 31)的 DNA序列如 SEQ ID No: 30所示， 以 PCR进行扩增， KEDL专一性引子 (； KEDL specificity primers)的序列如下所示： 正向引子 (；含有 HincHU限制酶酶切位置)：
Hz III
反向引子 (含有 ½ I限制酶酶切位置)：
5 ' -GTG QSd(JCAGTTCGTCTTTCAGTTCATCT-3 ' (SEQ ID No: 33) Xho I
PCR反应条件为： 94°C反应 3分钟后， 进行 95°C 1分钟、 55°C 1分钟、 72°C 20秒， 共 5个循环， 最后以 72°C反应 1分钟以进行延伸反应 (elongation); 分别将 PCR产物与 pET24a载体进行双酶切 (以 Hind III及 Xho I两种限制酶进行酶切反应)， 再将酶切后的 PC 产物与 pET24a载体进行纯化以及接合 (ligation)作用，以将 PCR产物选殖到 pET24a 载体形成 pET24a-KEDL质体， 并将 pET24a-KEDL质体利用转殖作用 (transformation)送 入寄主细胞大肠杆菌 (E. 中， 以进行大量增殖， 并进行定序确认增殖的 PCR产物序 列无误。
2. pET24a-2a-F2-KDEL质体的构筑
以 PCR增殖实施例一所得的 2a-F2 DNA片段 (SEQ ID No: 5)， PC 引子序列如下： 正向引子 pF2-l (含有 c l限制酶酶切位置)：
5 '-CGAGCTCTTTGTTCCCCCGGGAGGGGGG-3 ' (SEQ ID No: 38)
Sac I
反向引子 pR2-l (；含有 H"i m限制酶酶切位置)：
5 ' -CCC AAGCTTGTAGGAGAAGGGTTGGGGGATT-3 ' (SEQ ID No: 39) Hind III
PCR反应条件为： 95°C反应 5分钟后， 进行 95°C 30秒、 55°C 30秒、 72°C 30秒， 共 25个循环， 最后以 72°C反应 5分钟以进行延伸反应 (elongation); 分别将 PCR产物与上 述 pET24a-KEDL质体进行双酶切 (；以 Sac I及 Hind III两种限制酶进行酶切反应)， 再将 酶切后的 PCR产物与 pET24a-KEDL质体进行纯化以及接合作用， 以将 PCR产物选殖 到 pET24a-KEDL 质体形成 pET24a-2a-F2-KEDL 质体， 并将该质体利用转殖作用 (transformation)送入寄主细胞大肠杆菌 (E. CO/ )中， 以进行大量增殖， 并进行定序确认增 殖的 PCR产物序列无误。
3. pET24a-PE-2a-F2-KDEL质体的构筑
编码 PE蛋白（SEQ ID No: 35)的 DNA序列如 SEQ ID No: 34所示，以 PCR进行扩增， PE专一性引子 (PE specificity primers)的序列如下所示：
正向引子 (；含有 SawH I限制酶酶切位置)： 5 '- CGGGATCCGAAGAAGCGTTCGAC -3 ' (SEQ ID No: 36)
Bamii I
反向引子 (含有 EcoRI及 Sac I限制酶酶切位置)：
5 '- CGGAATTCGAGCTCGCAGGTCAGGCTCACCAC-3 ' (SEQ ID No: 37)
EcoR I Sac I
PC 反应条件为： 94°C反应 5分钟后， 进行 95°C 1分钟、 55°C 1分钟、 72°C 1.5分钟， 共 30 个循环， 最后以 72°C 反应 7 分钟以进行延伸反应； 分别将 PCR产物与上述 pET24a-2a-F2-KEDL质体进行双酶切 (以 BamH I及 Sac I两种限制酶进行酶切反应)， 再 将酶切后的 PCR产物与 pET24a-2a-F2-KEDL质体进行纯化以及接合作用，以将 PCR产 物选殖到 pET24a-2a-F2-KDEL 质体， 形成 pET24a-PE-2a-F2-KEDL 质体， 并将 pET24a-PE-2a-F2-KEDL 质体利用转殖作用 (transformation)送入表现宿主大肠杆菌 co/ )中， 并进行定序确认增殖的 PCR产物序列无误。 PE-2a-F2-KEDL抗原融合蛋白的 DNA序列则如 SEQ ID No: 40所示， 而其胺基酸序列则如 SEQ ID No: 41所示。
4. PE-2a-F2-KDEL抗原蛋白的表达
将上述含有 pET24a-PE-2a-F2-KEDL质体的大肠杆菌 (E. co/ )以 LB培养基培养， 并 以 IPTG诱导 PE-2a-F2-KEDL抗原融合蛋白 (SEQ ID No: 41)表达， 蛋白表达及萃取方法 如实施例一所述。 实施例四 不同亚群 (subgroup)的 PCV2 ORF2序列的分析
除了 2a亚群 (subgroup)之外， 猪第二型环状病毒 (PCV2)还有其他亚群的病毒株， 以 下分别针对一 2b亚群 PCV2病毒株、 2c亚群 PCV2病毒株、 2d亚群 PCV2病毒株、 及 2e亚群 PCV2病毒株的 PCV2 ORF2序列， 分析其 ORF2的 5 '端第 1至 234个核苷酸以 及第 235至最后一个核苷酸之间片段的精胺酸 (Arginine)含量。
首先， 根据 Wang等人 (Wang et al.， Virus Research 2009, Genetic variation analysis of Chinese strains of porcine circovirus type 2)所提供的序列作为亲源关系树标准序列来分析 SEQ ID No: 15、 SEQ ID No: 19，结果显示该 SEQ ID No: 15属于 PCV2 2b亚群 (subgroup) 的一员；该 SEQ ID No: 19属于 PCV2 2d亚群 (subgroup)的一员（如图 1所示)；而 SEQ ID No:50及 SEQ ID No:52则分别为 PCV2 2c亚群及 PCV2 2e亚群的标准株。
PCV2 2b亚群株的序列分析结果如表三所示， 该 PCV2 2b亚群株的 2b-ORF2核苷 酸序列如 SEQ ID No: 15所示，胺基酸序列如 SEQ ID No: 16所示;其中， 2b-O F2的 5' 端第 1至 234个核苷酸片段编码 21个精胺酸， 而 2b-ORF2的 5'端第 235至 699个核苷 酸 (不含终止密码子)序列如 SEQ ID No: 17所示， 其胺基酸序列如 SEQ ID No: 18所示， 该片段编码 10个精胺酸； PCV2 2b亚群株的序列分析结果与 PCV2 2a亚群株的序列分 析结果一致，两者 ORF2蛋白 N端序列的精胺酸含量皆为其他部份精胺酸含量的 2倍以 上 (如表二及表三所示)。
表三、 PCV2 2b-ORF2胺基酸序列中精胺酸的数量分析
2b亚群株-第 1-234个核苷酸 2b亚群株-第 235-699个核苷酸 密码子 胺基酸 数量 密码子 胺基酸 数量
AGA 精胺酸 5 AGA 精胺酸 5
AGG 精胺酸 3 AGG 精胺酸 1
CGA 精胺酸 1 CGA 精胺酸 0
CGC 精胺酸 9 CGC 精胺酸 3
CGG 精胺酸 1 CGG 精胺酸 0
CGT 精胺酸 2 CGT 精胺酸 1 总数 21 总数 10 PCV2 2c亚群株的序列分析结果如表四所示，该 PCV2 2c亚群株的 2c-ORF2核苷酸 序列如 SEQ ID No: 50所示， 胺基酸序列如 SEQ ID No: 51所示； 其中， 2c-ORF2的 5' 端第 1至 234个核苷酸片段编码 20个精胺酸， 而 2C-ORF2的 5，端第 235至 702个核苷 酸序列如 SEQ ID No: 54所示， 其胺基酸序列如 SEQ ID No: 55所示， 该片段编码 10个 精胺酸； PCV2 2c亚群株的序列分析结果与 PCV2 2a及 2b亚群株的序列分析结果一致， 三者 ORF2蛋白 N端序列的精胺酸含量皆为其他部份精胺酸含量的 2倍以上 (如表二、 表三、 表四所示)。
表四、 PCV2 2C-ORF2胺基酸序列中精胺酸的数量分析 2c亚群株-第 1-234个核苷酸 2c亚群株-第 235-702个核苷酸 密码子 胺基酸 数量 密码子 胺基酸 数量
AGA 精胺酸 5 AGA 精胺酸 6
AGG 精胺酸 3 AGG 精胺酸 1
CGA 精胺酸 0 CGA 精胺酸 0
CGC 精胺酸 9 CGC 精胺酸 2
CGG 精胺酸 1 CGG 精胺酸 0
CGT 精胺酸 2 CGT 精胺酸 1 总数 20 总数 10
PCV2 2d亚群株的序列分析结果如表五所示， 该 PCV2 2d亚群株的 2d-ORF2核苷 酸序列如 SEQ ID No: 19所示，胺基酸序列如 SEQ ID No: 20所示；其中， 2d-ORF2的 5' 端第 1至 234个核苷酸片段编码 21个精胺酸， 而 2d-ORF2的 5'端第 235至 702个核苷 酸片段 (不含终止密码子)序列如 SEQ ID No: 21所示，其胺基酸序列如 SEQ ID No: 22所 示， 该片段编码 10个精胺酸； PCV2 2d亚群株的序列分析结果与 PCV2 2a、 2b、 及 2c 亚群株的序列分析结果一致，四者 ORF2蛋白 N端序列的精胺酸含量皆为其他部份精胺 酸含量的 2倍以上 (如表二、 表三、 表四及表五所示)。
表五、 PCV2 2d-ORF2胺基酸序列中精胺酸的数量分析
2d亚群株-第 1-234个核苷酸 2d亚群株-第 235-702个核苷酸 密码子 胺基酸 数量 密码子 胺基酸 数量
AGA 精胺酸 5 AGA 精胺酸 4
AGG 精胺酸 3 AGG 精胺酸 3
CGA 精胺酸 1 CGA 精胺酸 0
CGC 精胺酸 10 CGC 精胺酸 1
CGG 精胺酸 0 CGG 精胺酸 1
CGT 精胺酸 2 CGT 精胺酸 1 总数 21 总数 10 PCV2 2e亚群株的序列分析结果如表六所示，该 PCV2 2e亚群株的 2e-ORF2核苷酸 序列如 SEQ ID No: 52所示， 胺基酸序列如 SEQ ID No: 53所示； 其中， 2e-ORF2的 5' 端第 1至 234个核苷酸片段编码 20个精胺酸， 而 2e-ORF2的 5，端第 235至 699个核苷 酸片段 (不含终止密码子)序列如 SEQ ID No: 56所示，其胺基酸序列如 SEQ ID No: 57所 示， 该片段编码 9个精胺酸； PCV2 2e亚群株的序列分析结果与 PCV2 2a、 2b、 2c及 2d 亚群株的序列分析结果一致，五者 ORF2蛋白 N端序列的精胺酸含量皆为其他部份精胺 酸含量的 2倍以上 (如表二、 表三、 表四、 表五及表六所示)。
表六、 PCV2 2e-ORF2胺基酸序列中精胺酸的数量分析
2e亚群株-第 1-234个核苷酸 2e亚群株-第 235-699个核苷酸 密码子 胺基酸 数量 密码子 胺基酸 数量
AGA 精胺酸 5 AGA 精胺酸 4
AGG 精胺酸 3 AGG 精胺酸 2
CGA 精胺酸 0 CGA 精胺酸 0
CGC 精胺酸 9 CGC 精胺酸 2
CGG 精胺酸 1 CGG 精胺酸 0
CGT 精胺酸 2 CGT 精胺酸 1 总数 20 总数 9 与实施例一相同， 本案亦分析了 PCV2 2b亚群株、 PCV2 2c亚群株、 PCV2 2d亚群 株及 PCV2 2e亚群株的 N端富含精胺酸区域 (Arginitie-rich domian), 及非富含精胺酸区 ¾(non-Arginine-rich domain)的蛋白表现量， 发现与实施例一结果相同， 该 N端富含精 胺酸区域所编译的蛋白片段难以被表达； 然而， 该非富含精胺酸区域所编译的蛋白片段 可易于生物表现系统中被表达； 且进一步将该些精胺酸增加或删除后， 并分析蛋白质表 现后， 亦发现结果与 PCV2 2a亚群株一致， 当该些过多的精胺酸数量减少后， 发现确实 可增加 PCV2 O F2的蛋白表现量。
其中该富含精胺酸区域约在 PCV2 ORF2全长胜肽的 N端第 1至 78个胺基酸之间 的区域； 该非富含精胺酸区域约在 PCV2 ORF2的 N端第 79个胺基酸至 C端最后一个 胺基酸之间的区域。 实施例五 PCV2 2b次单位疫苗抗原蛋白片段的构筑及表达
1. pET24a-2b-F2质体的构筑及表达
本实施例针对实施例四的 PCV2 2b亚群株的 ORF2基因进行次单位疫苗抗原蛋白片 段的构筑。 同实施例一及三， 选用 F2片段 (PCV2 2b的 ORF2基因 5 '端第 235-468个核 苷酸， 以下称为 2b-F2片段)进行构筑。 该 PCV2 2b亚群株的 2b-F2片段的核苷酸序列 如 SEQ ID No: 17所示， 其胺基酸序列如 SEQ ID No: 18所示。 以 PCR扩增 2b-F2片段 核苷酸序列， PCR引子序列如下- 正向引子 pF-2b (；含有 Sac I限制酶酶切位置)：
5 '- CGAGCTCTTTCTTCCCCCAGGAGGGGGC -3 ' (SEQ ID No: 42)
Sac I
反向引子 pR-2b (含有 Himim限制酶酶切位置)：
5 '- CCCAAGCTTGTAGGAGAAGGGCTGGGTTAT-3 ' (SEQ ID No: 43) Hind III
PCR反应条件为： 95°C反应 5分钟后， 进行 95°C 30秒、 55°C 30秒、 72°C 30秒， 共 25个循环， 最后以 72°C反应 5分钟以进行延伸反应 (elongation); 分别将 PCR产物与 pET24a表现载体 (Novagen)进行双酶切 (以 Sac I及 Hind III两种限制酶进行酶切反应)， 再将酶切后的 PCR产物与 pET24a表现载体进行纯化以及接合作用， 以将 PCR产物选 殖到 pET24a表现载体形成 pET24a-2b-F2质体，并将该质体利用转殖作用 (transformation) 送入寄主细胞大肠杆菌 (E. 中， 以进行大量增殖， 并进行定序确认增殖的 PCR产物 序列无误。 接着将上述含有 pET24a-2b-F2质体的大肠杆菌 (E. CO/ )以 LB培养基培养， 并以 IPTG诱导 2b-F2抗原蛋白 (SEQ ID No: 18)表达， 蛋白表达及萃取方法如实施例一 所述。
2. pET24a-PE-2b-F2-KDEL质体的构筑及表达
在本实施例中， 除了以 2b-F2胺基酸片段作为 PCV2次单位疫苗的抗原片段之外， 也在 2b-F2胺基酸片段的 N端及 C端分别加上 PE蛋白及 KDEL蛋白质讯号序列，形成 抗原融合蛋白 PE-2b-F2-KDEL， 以期诱导生物体产生更佳的免疫反应。
抗原融合蛋白 PE-2b-F2-KDEL的 DNA序列如 SEQ ID No: 44所示， 而其胺基酸序 列如 SEQ ID No: 45 所示。 表达该抗原融合蛋白 PE-2b-F2-KDEL 的质体 (pET24a-PE-2b-F2-KDEL)的构筑策略同实施例三。首先，取编码 KDEL蛋白质讯号序列 的 DNA序列 (SEQ ID No: 30)， 转殖到 pET24a质体中， 以形成 pET24a-KDEL质体， pET24a-KDEL质体的构筑方式如实施例三所述。 然后， 取上述 PCR增殖的 2b-F2片段 DNA序列 (SEQ ID No: 17)，转殖到该 pET24a-KDEL质体中，以形成 pET24a-2b-F2-KDEL 质体。最后，取编码 PE蛋白的 DNA序列 (SEQ ID No: 34)，转殖到该 pET24a-2b-F2-KDEL 质体中， 以形成 pET24a-PE-2b-F2-KDEL质体， pET24a-PE-2b-F2-KDEL质体的构筑方 式亦如实施例三所述。接着将上述含有 pET24a-PE-2b-F2-KEDL质体的大肠杆菌 (E. coli) 以 LB培养基培养，并以 IPTG诱导 PE-2b-F2-KEDL抗原融合蛋白 (SEQ ID No: 45)表达， 蛋白表达及萃取方法如实施例一所述。 实施例六 PCV2 2d次单位疫苗抗原蛋白片段的构筑及表达
1. pET24a-2d-F2质体的构筑及表达
本实施例针对实施例四的 PCV2 2d亚群株的 ORF2基因进行次单位疫苗抗原蛋白片 段的构筑。 同实施例一及三， 选用 F2片段 (PCV2 2d的 ORF2基因 5 '端第 235-468个核 苷酸， 以下称为 2d-F2片段)进行构筑。 该 PCV2 2d亚群株的 2d-F2片段的核苷酸序列 如 SEQ ID No: 21所示， 其胺基酸序列如 SEQ ID No: 22所示。 以 PCR扩增 2b-F2片段 核苷酸序列， PCR引子序列如下：
正向引子 pF-2d (含有 Sac I限制酶酶切位置)：
5 '- CGAGCTCTTTCTTCCCCCAGGAGGGGGC -3 ' (SEQ ID No: 46)
Sac I
反向引子 pR-2d (含有 Himi m限制酶酶切位置)：
5 '- CCCAAGCUGTAGGAGAAGGGCTGGGTTAT -3 ' (SEQ ID No: 47)
Hind III PCR反应条件为： 95°C反应 5分钟后， 进行 95°C 30秒、 55°C 30秒、 72°C 30秒， 共 25个循环， 最后以 72°C反应 5分钟以进行延伸反应 (elongation); 分别将 PCR产物与 pET24a表现载体 (Novagen)进行双酶切 (以 Sac I及 Hind III两种限制酶进行酶切反应)， 再将酶切后的 PCR产物与 pET24a表现载体进行纯化以及接合作用， 以将 PCR产物选 殖到 pET24a表现载体形成 pET24a-2d-F2质体，并将该质体利用转殖作用 (transformation) 送入寄主细胞大肠杆菌 (E. 中， 以进行大量增殖， 并进行定序确认增殖的 PCR产物 序列无误。 接着将上述含有 pET24a-2d-F2质体的大肠杆菌 (E. CO/ )以 LB培养基培养， 并以 IPTG诱导 2d-F2抗原融合蛋白 (SEQ ID No: 22)表达， 蛋白表达及萃取方法如实施 例一所述。
2. pET24a-PE-2d-F2-KDEL质体的构筑及表达
在本实施例中， 除了以 2d-F2胺基酸片段作为 PCV2次单位疫苗的抗原片段之外， 也在 2d-F2胺基酸片段的 N端及 C端分别加上 PE蛋白及 KDEL蛋白质讯号序列，形成 抗原融合蛋白 PE-2d-F2-KDEL， 以期诱导生物体产生更佳的免疫反应。
抗原融合蛋白 PE-2d-F2-KDEL的 DNA序列如 SEQ ID No: 48所示， 而其胺基酸序 列如 SEQ ID No: 49 所示。 表达该抗原融合蛋白 PE-2d-F2-KDEL 的质体 (pET24a-PE-2d-F2-KDEL)的构筑策略同实施例三。首先，取编码 KDEL蛋白质讯号序列 的 DNA序列 (SEQ ID No: 30)， 转殖到 pET24a质体中， 以形成 pET24a-KDEL质体， pET24a-KDEL质体的构筑方式如实施例三所述。 然后， 取上述 PCR增殖的 2d-F2片段 DNA序列 (SEQ ID No: 21)，转殖到该 pET24a-KDEL质体中，以形成 pET24a-2d-F2-KDEL 质体。最后，取编码 PE蛋白的 DNA序列 (SEQ ID No: 34)，转殖到该 pET24a-2d-F2-KDEL 质体中， 以形成 pET24a-PE-2d-F2-KDEL质体， pET24a-PE-2d-F2-KDEL质体的构筑方 式亦如实施例三所述。接着将上述含有 pET24a-PE-2d-F2-KEDL质体的大肠杆菌 (E. coli) 以 LB培养基培养，并以 IPTG诱导 PE-2d-F2-KEDL抗原融合蛋白 (SEQ ID No: 49)表达， 蛋白表达及萃取方法如实施例一所述。 实施例七 PCV2次单位疫苗抗原蛋白片段的免疫原性 (immimogenicity)的分析 -1
1. 小鼠免疫试验 将实施例一所得到的 2a-F2 重组蛋白（SEQ ID No: 6)以及实施例三所得到的
PE-2a-F2-KDEL重组蛋白 (SEQ ID No: 41)分别以弗氏完全佐剂制备为 PCV2次单位疫 苗， 并进行小鼠 (mice)免疫试验， 以比较这两种重组蛋白引发试验动物产生抗 PCV2抗 体的免疫原性。
取 5〜6周龄健康雌性清洁级 Balb/c小鼠 9只作为试验动物，所有小鼠的猪第二型环 状病毒 (PCV2) ELISA抗体皆呈阴性。 将这 9只小鼠随机分为 3组， 每组 3只； 第 1、 2 组中， 每只小鼠分别以腹腔注射 (intraperitoneally, LP.)注射 100 μg 重组蛋白 2a-F2 及 PE-2a-F2-KDEL。第 3组则注射 PBS作为负对照组。两周后用相同免疫剂量加强免疫一 次； 各组隔离饲养观察， 首次免疫后第 2、 4、 5、 6周分别采血分离血清， 以 ELISA测 定血清内抗 PCV2的抗体力价。
2. 血清抗体的判定 - 以 ELISA测定抗 PCV2抗体力价
以含有 PCV2病毒抗原 (300 ng/孔)的 96孔抗原盘作为检测套组。 以含有 500 μΙ/L Tween-20的 50 mmol/L PBS (pH 7.2) (即 PBST)洗涤抗原盘 3次，每次 3~5分钟； 再于抗 原盘中加入 0.15% BSA填充液 (blocking solution)(20( L/孔)， 以填充 (block)抗原盘， 并 于 37°C下作用 2小时后， 以 PBS洗涤。将待检测的小鼠血清以 PBS缓冲液以 1 : 50倍 数稀释， 然后再做倍比稀释； 每个样品 8重复， 每孔加 ΙΟΟμί稀释的小鼠血清， 于 37°C 下作用 1小时后， 以 PBS洗涤； 然后加入碱性磷酸酶 (alkaline phosphatase, AP)标定的 anti-mouse IgG抗体， 于 37°C下作用 1小时后， 以 PBS洗涤； 接着加入磷酸对硝基苯 酯 (para-Nitrophenylphosphate, pNPP)溶液显色， 最后用 1M的 NaOH终止反应后判定结 果。 读取各样本在 405 nm波长下的吸光值， 每个样本读取两次， 并取平均值进行分析。
ELISA 分析结果如图 6 所示。 相较于 2a-F2 重组蛋白片段 (SEQ ID No: 6)，
PE-2a-F2-KDEL重组蛋白 (SEQ ID No: 41)可以引发试验动物产生较高量的抗 PCV2血清 抗体， 且于首次免疫第 4周后， 第 1组及第 2组小鼠产生的血清抗体量开始具有显著差 异 < 0.05); 到了首次免疫后第 6周， 两组产生的血清抗体量差异更大 < 0.001)。 另 夕卜， 相较于注射 PBS的负对照组， 第 1组及第 2组小鼠的抗体产生量皆具有显著差异 (p <0.01)。 实施例八 PCV2次单位疫苗抗原蛋白片段的免疫原性 (immimogenicity)的分析 -2
1. 小鼠免疫试验
在本实施例中，比较本发明 PE-2a-F2-KDEL重组蛋白次单位疫苗以及 PCV2全病毒 疫苗引发试验动物产生抗 PCV2抗体的免疫原性。
将实施例三所得到的 PE-2a-F2-KDEL 重组蛋白（SEQ ID No: 41)以油质佐剂 Montanide ISA206 (Seppic, France)制备为 PCV2次单位疫苗。 另外， 取不活化的 PCV2 全病毒 (106TCID5Q/ml)， 以油质佐剂 Montanide ISA206 (Seppic, France)制备为 PCV2全病 毒疫苗， 每一剂量的 PCV2全病毒疫苗含有 100 μΐ不活化的 PCV2全病毒以及 250 μΐ 佐剂。 接着进行小鼠免疫试验。
取 5〜6周龄健康雌性清洁级 Balb/c小鼠 15只作为试验动物， 所有小鼠的猪第二型 环状病毒 (PCV2) ELISA抗体皆呈阴性。 将这 15只小鼠随机分为 3组， 每组 5只； 第 1 组中的每只小鼠分别以腹腔注射 (LP.)注射 100 μg重组蛋白。第 2组中的每只小鼠分别以 腹腔注射 (I.P.)注射一剂量的 PCV2全病毒疫苗。第 3组则注射油质佐剂 Montanide ISA206 作为负对照组。 三周后用相同免疫剂量加强免疫一次； 各组隔离饲养观察， 首次免疫后 第 0、 1、 2、 3、 4、 5周分别采血分离血清， 以 ELISA测定血清内抗 PCV2的抗体力价。 2. 血清抗体的判定 - 以 ELISA测定抗 PCV2抗体力价
同实施例七所述的"以 ELISA测定抗 PCV2抗体力价"的方法。 ELISA分析结果如图 7所示。 相较于 PCV2全病毒疫苗， PE-2a-F2-KDEL重组蛋白 (SEQ ID No: 41)可以引发 试验动物产生较高量的抗 PCV2血清抗体， 且于首次免疫 1周后， 第 1组及第 2组小鼠 产生的血清抗体量便具有显著差异 < 0.001)； 且相较于注射油质佐剂的负对照组， 注 射 PE-2a-F2-KDEL重组蛋白的小鼠的抗体产生量，在首次免疫 1周后便具有显著差异 (p <0.001)。
此外， 将前述的 2a-F2重组蛋白、 PE-2a-F2-KDEL重组蛋白进行猪只免疫， 结果发 现该等重组蛋白皆可诱发猪只产生抗 PCV2血清抗体。 此外， 亦藉由 PCV2攻毒试验评 估该等重组蛋白的免疫保护效力： 将该等重组蛋白配制成次单位疫苗后， 进行猪只免疫 后， 再以 PCV2病毒株攻毒， 结果发现经攻毒后， 免疫组的保护率高于对照组 (未施以疫 苗者)； 此处所指的保护率提高包含： 病毒血症的降低、 PCV2病征的减缓。 因此， 证实 该等重组蛋白所制备的 PCV2次单位疫苗可有效提供动物产生免疫力， 以提高动物的存 活率。
相同地， 本案进一步将 PCV2 2b亚群株、 PCV2 2c亚群株、 PCV2 2d亚群株、 及 PCV2 2e亚群株的 ORF2以实施例一至三的构筑方式构筑， 进而以实施例七、 八所述的 免疫原性分析进行分析， 结果发现， 该等亚群株的 ORF2片段 (如： F2片段)及其融合蛋 白 (如： PE-F2-KDEL)所制备的 PCV2次单位疫苗皆可诱发动物 (如： 猪只)产生抗 PCV2 抗体， 进而保护动物免于被 PCV2病毒感染。
本发明所提供的 PCV2次单位疫苗， 与其他习用技术相互比较时， 更具有下列的优 点：
本发明所提供的 PCV2次单位疫苗系利用基因重组技术，从 PCV2病毒 ORF2蛋白 全长片段中选殖出于生物表现系统中可表现大量蛋白质的片段， 利用该 ORF2蛋白片段 所制备的 PCV2次单位疫苗，不但可以引发动物产生抗 PCV2病毒的免疫力，且该 ORF2 蛋白片段可以基因重组技术大量生产， 亦可降低制造疫苗的成本。
本发明所提供的另一 PCV2次单位疫苗， 系以 PCV2病毒 ORF2的 F2蛋白片段与 PE蛋白及 KDEL蛋白质讯号序列形成 PE-F2-KDEL抗原融合蛋白， 经试验结果显示， 此一次单位疫苗更可提高动物对抗 PCV2病毒的免疫性及效率。
本发明所提供的 PCV2次单位疫苗系利用基因重组技术所研发的次单位疫苗， 其具 备有生产制备简单、 成本低、 纯度高、 产量高及安全性佳等优点。
上列详细说明系针对本发明的一可行实施例的具体说明，惟该实施例并非用以限制 本发明的专利范围， 凡未脱离本发明技艺精神所为的等效实施或变更， 均应包含于本案 的专利范围中。

Claims

1.一种抗猪第二型环状病毒免疫组合物， 包含一抗原胜肽， 该抗原胜肽选自由 及 (b)所组成的群组中至少一者-
(a) PCV2 第二开放阅读区非富含精胺酸胜肽，该 PCV2 0RF2非富含精胺酸胜肽 的精胺酸数量为该 PCV2 ORF2全长胜肽的 N端富含精胺酸区域的精胺酸数量 的 0至二分之一倍； 及
(b) 一重组融合蛋白， 该重组融合蛋白自胺基端到羧基端包含：
一 PE胜肽， 其具有如 SEQ ID No: 35的序列；
一 (a)项的 PCV2 0RF2非富含精胺酸胜肽； 以及
一 KDEL胜肽， 其具有如 SEQ ID No: 31的序列。
2.如权利要求 1所述的免疫组合物， 其中该 PCV2 ORF2全长胜肽具有一胺基酸序 列，该胺基酸序列选自由 SEQ ID No: 2、 SEQ ID No: 16、 SEQ ID No: 20、 SEQ ID No: 51、 及 SEQ ID No: 53所组成的群组中至少一者。
3.如权利要求 1所述的免疫组合物， 其中该 PCV2 0RF2全长胜肽具有一胺基酸序 列，该胺基酸序列与选自 SEQ ID No: 2、 SEQ ID No: 16、 SEQ ID No: 20、 SEQ ID No: 51、 及 SEQ ID No: 53的序列的一具有至少 80%序列同源性。
4.如权利要求 1所述的免疫组合物， 其中该富含精胺酸区域为该 PCV2 0 F2全长 胜肽的胺基端第 1-78个胺基酸。
5.如权利要求 1所述的免疫组合物，进一步包含 PCV2其他开放阅读区 (open reading frame, 0RF)的抗原蛋白， 该 PCV2其他 0RF的抗原蛋白包含 0RF1及 0RF3。
6.如权利要求 1所述的免疫组合物， 进一步包含至少一种病原抗原， 该病原抗原选 自由下列群组所组成者： 猪流感病毒抗原、 猪繁殖与呼吸症候群病毒抗原、 猪霉浆菌、 猪小病毒、 猪丹毒， 以及伪狂犬病。
7.如权利要求 1所述的免疫组合物， 其中该第 (a)项所述的 PCV2 ORF2非富含精胺 酸胜肽， 选自由下列群组所组成者： 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及 SEQ ID No: 57。
8.如权利要求 1所述的免疫组合物， 可进一步包含一或多种选自于下者： 载剂、 溶 剂、 乳化剂、 悬浮剂、 分解剂、 黏结剂、 赋形剂、 安定剂、 螯合剂、 稀释剂、 胶凝剂、 防腐剂、 润滑剂、 界面活性剂、 佐剂、 生物型载体。
9.一种抗猪第二型环状病毒免疫组合物， 包含一抗原胜肽， 该抗原胜肽选自由 (a)及
(b)所组成群组中至少一者- (a) — PCV2 O F2 F2胜肽，为 PCV2 ORF2全长胜肽的 N端第 79- 156个胺基酸； 以及
(b) 一重组融合蛋白， 该重组融合蛋白自胺基端到羧基端包含： 一 PE胜肽， 其具 有如 SEQ ID No: 35的序列； 一 PCV2 O F2 F2胜肽； 以及一 KDEL胜肽， 其 具有如 SEQ ID No: 31的序列。
10.如权利要求 9所述的免疫组合物，其中该 PCV2 0RF2 F2胜肽具有一胺基酸序列， 该胺基酸序列选自由 SEQ ID No: 6、 SEQ ID No: 18、 SEQ ID No: 22、 SEQ ID No: 55及 SEQ ID No: 57所组成的群组中至少一者。
11.如权利要求 9所述的免疫组合物，其中该 PCV2 ORF2 F2胜肽具有一胺基酸序列， 该胺基酸序列与选自 SEQ ID No: 6、 SEQ ID No: 18、 SEQ ID No: 22^ SEQ ID No: 55及 SEQ ID No: 57的序列的一具有至少 80%序列同源性。
12.如权利要求 9所述的免疫组合物，其中该第 (b)项的重组融合蛋白具有选自由 SEQ ID No: 41、 SEQ ID No: 45及 SEQ ID No: 49所组成的群组中至少一者。
13.—种制备猪第二型环状病毒 (PCV2)抗原片段的方法， 包含：
去除 PCV2 O F2 DNA序列中编码 N端富含精胺酸区域的 DNA序列， 以得到一编 码一 PCV2 0RF2非富含精胺酸区域的 DNA序列， 该非富含精胺酸区域的精胺酸数量 为该富含精胺酸区域的精胺酸数量的 0至二分之一倍； 以及
将该编码一 PCV2 0RF2非富含精胺酸区域的 DNA序列置于一生物表现系统中表 现， 得到抗猪第二型环状病毒的免疫组合物的抗原片段。
14.如权利要求 13所述的方法，其中该 PCV2 O F2 DNA序列为选自由 SEQ ID No: 1、 SEQ ID No: 15、 SEQ ID No: 19、 SEQ ID No: 50、 及 SEQ ID No: 52所组成的群组中 至少一者。
15.如权利要求 13所述的方法， 其中该富含精胺酸区域为该 PCV2 ORF2全长 DNA 序列的 5'端第 1-234个核苷酸之间的区域。
16.如权利要求 13所述的方法，其中该非富含精胺酸区域为该 PCV2 ORF2全长 DNA 序列的 5'端第 235个核苷酸至 3'端终止密码子之间的区域。
17.如权利要求 13所述的方法，其中该编码一 PCV2 ORF2非富含精胺酸区域的 DNA 序列， 选自由下列群组所组成者： SEQ ID No: 5、 SEQ ID No: 7、 SEQ ID No: 9, SEQ ID No: 11、 SEQ ID No: 17、 SEQ ID No: 21、 SEQ ID No: 54及 SEQ ID No: 56。
PCT/CN2012/085907 2011-12-06 2012-12-05 猪第二型环状病毒次单位疫苗 WO2013083036A1 (zh)

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CN104039814B (zh) 2017-09-01
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TW201323437A (zh) 2013-06-16
US20140348864A1 (en) 2014-11-27
EP2789627A4 (en) 2016-03-09
UA113192C2 (xx) 2016-12-26
EP2789627B1 (en) 2017-09-27
EA029698B1 (ru) 2018-05-31
EA201491123A1 (ru) 2014-12-30
US9657063B2 (en) 2017-05-23
EP2789627A1 (en) 2014-10-15
CN104039814A (zh) 2014-09-10

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