TW201428100A - Production of the porcine circovirus type 2 (PCV2) virus-like particles (VLPs) by pseudorabies virus - Google Patents

Abstract

The present invention relates to construct a gE gene-deleted (gE-) PRV recombinant viruses expressing PCV2 capsid protein (Cap), which could assembly to form virus-like particles (VLPs).

Description

Production of porcine circovirus type 2 virus particles by pseudorabies virus

The present invention relates to a method for producing a porcine circovirus type 2 virus particle by using pseudorabies virus (PRV), and a PVC unit virus particle obtained thereby for use in controlling a porcine circovirus infection subunit vaccine composition. More particularly, the present invention relates to the construction of a recombinant PRV strain having a gene fragment of at least gE gene and comprising a PCV2 capsid protein (Cap), and further exhibiting a virus particle of porcine circovirus type 2 .

Porcine circovirus type 2 (PCV2) is one of the important pathogens causing serious economic losses in the pig industry. PCV2 is a member of the circoviridae family and has a virus particle diameter of approximately 17 ± 1.3 nm. PCV2 has a single-stranded circular DNA of 1768 bp, consisting of an origin of replication (Ori) and two open transcribed regions (ORFs). After transcription, ORF1 is translated into a replication protein (Rep). ORF2 is a post-transcriptional translation of Capsid protein (Cap), which is the main structural protein of PCV2. PCV2 disease control, in addition to relying on good health monitoring and control, in addition to the use of vaccines to slow down the detoxification of pigs. Currently marketed PCV2 vaccine comprises inactivated whole virus type (e.g. Circovac®), insect cell expression systems subunit PCV2 vaccines of Cap protein production, such as Ingelvac® CircoFLEX ^TM (Boehringer Ingelheim) with Circumvent ^TM PCV vaccine (of Intervet) And non-activated PCV1-2 chimeric viruses such as Fostera® PCV (Fort Dodge) vaccine.

Pseudorabies (PR) is an important infectious disease in pigs caused by pseudorabib virus (PRV). At present, most of the PRV vaccine strains used in various countries are gE-deficient PRV viruses. If combined with serum monitoring, the advantages can be differentiated from vaccinated animals (DIVA) (Jacobs et al., The Journal of general virology 71 (Pt 4), 881-887, 1990). Therefore, in countries where pseudorabies (PR) clearance programs are carried out, marker vaccines are preferred. The glycoprotein gE of pseudorabies virus is a neuroinvasive and neurovirulent function of the host, and is a factor that causes PRV to cause neuroinvasion and neurotoxicity to the host, and the gE gene deficient virus is also confirmed to affect PRV in nerve cells. Advance transmission, and reduce the spread of the virus across synapses (Card et al., Journal of virology 66 , 3032-3041, 1992), but does not affect the replication capacity and immunogenicity of the virus itself (Mettenleiter et al., Journal of virology 61 , 4030-4032, 1987), and it has been demonstrated through field animal experiments that the application of this gene-deficient virus also has the advantage of serological differential diagnosis (van Oirschot et al., Veterinary microbiology 23 , 85-101, 1990). . Vaccine immunization strategies have been shown to be effective in preventing PRV infection, and PRV-labeled vaccines with glycoprotein E (gE) gene defects have been widely used throughout the world.

In addition, thymidine kinase (tk) of PRV is associated with viral pathogenesis and recurrence from latency, but is not required for viral replication. The PRV strain obtained from the defect of the tk gene shows that it is not pathogenic and does not detoxify (Efstathiou et al., The Journal of general virology 70 (Pt 4), 869-879, 1989), and can be developed into a safe and effective one. live vaccine (Azmi et al, Journal of Microbiology 4 0, 1-10,2002.), can also be used as a further security viral vectors carrying foreign genes (Li et al, vaccine 26, 2714-2722,2008;. Yuan Et al., Vaccine 26 , 1314-1321, 2008).

PRV has a large genome, which is suitable for embedding foreign genes in its genome and has the characteristics of stably expressing foreign genes. The pseudorabies virus is used as a carrier to carry foreign genes, which can stably express foreign genes, and has been confirmed by animal experiments to have a dual immunoprotective effect, thereby retaining the immunogenicity of pseudorabies virus and recombinant expression of foreign genes (Hooft). Van Iddekinge et al., Vaccine 14 , 6-12, 1996; van Zijl et al., Journal of virology 65 , 2761-2765, 1991). Multivalent vaccines have many advantages, such as reducing the urgency of multiple immunizations and the labor costs required to inject vaccines. The PRV and PCV2 bivalent inactivated recombinant vaccine developed in this experiment can be used to immunize pigs of all ages including pregnant sows, while live attenuated bivalent vaccines can be applied to empty sows, which are expected to be produced by sows. The high-valence transition antibody provides protection for piglets and provides dual protection against group immunity in pigs.

Therefore, the object of the present invention is to construct a PRV recombinant virus strain containing a cap gene of a PCV2 coat protein (Cap) by using pseudorabies virus (PRV), and further develop a porcine circovirus 2 Subunit vaccine of type virus particles. Song et al. published a cap protein expressing PCV2 in recombinant pseudorabies virus in 2007 (Song, Y. et al., Veterinary Microbiology 119 , 97-104, 2007), but its purpose is to develop a bivalent vaccine, and This document does not disclose that porcine circovirus type 2 virus particles have been obtained using pseudorabies virus as a expression vector.

Compared with the currently marketed PCV2 vaccine, the subunit vaccine of the present invention (i.e., the PCV2 Cap protein expressed by recombinant pseudorabies virus) has the characteristics of self-assembly into viroid-like particles. The viroid-like particles are mainly composed of one or two kinds of viral coat protein units, which are similar to the original virus but do not contain the genome. Because of the identification of the host innate immune response, the viroid particles are considered to be self-adjuvants. The efficacy, while reducing the dose required for immunization, is also a popular new vaccine development (Vicente et al., Journal of invertebrate pathology 107 Suppl, S42-48, 2011). If the PCV2 virus particles having antigenicity are expressed via a PRV vector, the development value thereof is bound to increase.

Accordingly, one aspect of the invention relates to a method for producing a porcine circovirus type 2 virus particle using pseudorabies virus (PRV), comprising: preparing a gene fragment comprising a PCV2 capsid protein (Cap) Recombinant transfer plastid; select a pro-viral strain whose viral DNA has at least gE gene defect, and co-transfect its viral DNA with the recombinant transfer plastid in a host cell to produce at least gE gene defect and include PCV2 a recombinant PRV strain of a gene fragment of capsid protein (Cap); and the recombinant PRV strain is propagated to produce an antigenic porcine circovirus type 2 virus particle (PCV2 VLP). In a specific embodiment of the invention, the PCV2 coat protein has the amino acid sequence of SEQ ID NO.

In some embodiments of the invention, the viral strain of the pro-viral strain has a gE gene defect (only the 3' end sequence is retained). In another embodiment of the present invention, the viral DNA of the pro-viral strain has a gE gene defect (retaining the 3' end sequence) and a tk gene defect (retaining the 5' end and the 3' end sequence).

In a specific embodiment of the invention, the pro-viral strain further comprises a gene fragment encoding a reporter protein that can be screened. In another embodiment of the invention, the reporter protein is a fluorescent protein.

In another aspect, the present invention provides a unit vaccine for controlling a porcine circovirus type 2 disease comprising a porcine circovirus type 2 virus particle produced by recombinant pseudorabies virus (PRV).

Figure 1 shows the construction process of recombinant transfer plastid pUC/gIPCV2CapgE3' (A) and recombinant gE ^- /PCV2 cap ⁺ PRV strain (B).

Figure 2 shows the construction of the recombinant transfer plastid pUCUL24PCV2Cap(A) UL23(A) and recombinant gE ^- tk ^- /PCV2 cap ⁺ PRV strain (B).

Fig. 3 is a result of analyzing the characteristics of the PCV2 Cap protein expressed by the recombinant virus of the present invention. (A) Analysis of non-viral-infected PK15 cells (lane 1), cells infected with wild-type virus strain (TNL) (lane 2), recombinant gE ^- /PCV2 cap ⁺ PRV virus-infected cells (lane) by SDS-PAGE 3), and the recombinant gE ^- tk ^- / PCV2 cap ⁺ virus PRV infected cells (lane 4) the collected liquid of the cell lysate results. (B) The results of Western blotting using a specific anti-PCV2 Cap2 monoclonal antibody. (C) Infected with PK15 cells for 18 hours with recombinant gE ^- /PCV2 cap ⁺ PRV virus (a, b, c, d) and recombinant gE ^- tk ^- /PCV2 cap ⁺ PRV virus (e, f, g, h) Then, the results of IFA determination were performed with anti-PCV2 Cap monoclonal antibody (a, e) and anti-PRV pig high serum (b, f), where c is the image of a and b combined, and g is e and The images of f are combined, and d and h are images of visible light observation.

Figure 4 shows that the method of the invention can be used to express PCV2 VLPs using recombinant viruses. (A) is a VLP exhibited by a recombinant gE ^- /PCV2 cap ⁺ PRV virus observed by a transmission electron microscope with a particle size of about 17-25 nm (as indicated by the arrow). (B) and (C) are VLPs expressed by anti-PCV2 Cap monoclonal antibody (Ingenasa) against recombinant gE ^- /PCV2 cap ⁺ PRV virus, and recombinant gE ^- tk ^- /PCV2 cap ⁺ PRV virus VLPs were obtained as a result of immunogold staining analysis.

Figure 5 shows the changes in antibodies against PRV in serum after immunization of mice with unactivated recombinant gE ^- /PCV2 cap ⁺ PRV virus. Mice immunized with 1.2× ^{10 8} pfu of non-activated recombinant virus or PBS (control group) were tested for anti-PRV antibodies in serum by indirect ELISA before and after primary immunization and booster immunization, respectively. The antibody reaction is expressed as the absorbance at the wavelength of 405 nm (OD ₄₀₅ ). (** indicates P<0.05)

Figure 6 shows the changes in antibodies against the PCV2 Cap protein in the serum after immunization of mice with VLPs expressed by the inactive recombinant gE ^- /PCV2 cap ⁺ PRV virus. Mice immunized with 50 μg of VLPs (immunized group) or PBS (control group) expressed by non-activated recombinant virus were assayed for anti-PCV2 Cap protein antibodies in serum by indirect ELISA before and after primary immunization and two booster immunizations, respectively. . The antibody reaction is expressed as the absorbance at the wavelength of 405 nm (OD ₄₀₅ ).

The other features and advantages of the present invention are further exemplified and illustrated in the following examples, which are intended to be illustrative only and not to limit the scope of the invention. In view of the various embodiments of the present invention, the various instruments, devices, methods, and related results described below are not intended to be limited to the scope of the present invention.

Example 1: Construction and Characterization of Recombinant Virus

Preparation of recombinant virus strains expressing PCV2 Cap protein

(1) Preparation by recombinant transfer plastid pUC/gIPCV2CapgE3'

First, using a specific primer pair (Capf-NheI: 5'-GCTAGCATGACGTATCCAAGG-3' and Capr-SalI: 5'-GTCGACTTAGGGTTTAAGTGG-3', SEQ ID NOS. 2 and 3), the recombinant plasmid pGapZ/ was amplified by PCR reaction. The PCV2 ORF2 gene (702 bp) fragment contained in PCV2 ORF2 was cloned into the vector pCRII-TOPO to construct the recombinant plasmid pTOPO/PCV2Cap. Then, using the restriction enzymes Nhe I and Sal I, the PCV2 Cap gene fragment contained in pTOPO/PCV2Cap was spliced, and the recombinant plasmid pUC/gIGFPgE3' was further ligated to generate recombinant transfer plastid pUC/gIPCV2CapgE3' (Fig. A), the gene sequence comprises the gI gene of the PRV Ka strain, the defective gE gene (retaining the 3' end sequence) and the CMV promoter, and the Cap protein of the PCV2 SC strain can be transcribed and translated.

A pro-virus strain (gE ^- GFP ⁺ PRV) was selected, and the gE gene of the viral DNA was deleted (only the 3' end sequence was retained), and the CMV promoter was transcribed and the green fluorescent protein (GFP) was transduced. The DNA of the parental strain (gE ^- GFP ⁺ PRV) was co-transfected into PK15 cells with the transfer plastid pUC/gIPCV2CapgE3' obtained as described above to obtain recombinant gE ^- /PCV2 cap ⁺ PRV strain (Fig. 1 B). The preparation and analysis methods are as follows: PK15 cells were cultured in a 24-well cell culture tray until the cells were cultured overnight until the formation of Bacheng was completed. First prepared containing 50 μl opti-MEM ^® I medium recombinant transfer plasmid is 0.8 μg of (Invitrogen), additionally are simultaneously containing 2 μl Lipofectamine ^TM 2000 (Invitrogen) in 50 μl opti-MEM ^® I medium the preparation and placed in the chamber After 5 minutes of warming, the two were gently mixed and allowed to stand at room temperature for 20 minutes. Before adding the mixture to the cells for transfection, the cell culture medium was replaced with 200 μl of opti-MEM ^® I medium, and the mixture was added to the cells and allowed to act in the incubator for 6 hours, after which the cells were removed and replaced with 500 cells. The cell culture medium of μl was analyzed by indirect immunofluorescence staining for 18 hours after transfection.

(2) Recombinant plastid pUC/UL24PCV2Cap(A)UL23

Using the specific primer pair (Capf-NheI: 5'-GCTAGCATGACGTATCCAAGG-3' and Capr-SalI: 5'-GTCGACTTAGGGTTTAAGTGG-3', SEQ ID NOS. 2 and 3), the recombinant plasmid pUC/gIPCV2CapgE3' was obtained by PCR amplification. The PCV2 Cap gene (702 bp) contained in the vector was cloned in the vector pJET2.1 to construct the recombinant plasmid pJET2.1/PCV2Cap. Then, the PCV2 Cap gene fragment of pJET2.1/PCV2Cap was cleaved by restriction enzymes Nhe I and Blp I, and further ligated with recombinant plastid pUC/UL24GFP(A)UL23 to generate recombinant transfer plastid pUC/UL24PCV2Cap ( A) UL23 (Fig. 2A), whose gene sequence includes the UL24 gene of PRV Ka strain, the defective UL23 gene (tk gene) (retaining the 5' end, 3' end sequence but central deletion of the sequence) and the CMV promoter can be transcribed and translated. The Cap protein of the PCV2 SC strain was obtained.

Select the pro-virus strain (gE ^- tk ^- GFP ⁺ PRV), the gE gene of the viral DNA is deleted (retaining the 3' end sequence), the tk gene is defective (retaining the 5' end and the 3' end sequence), and the CMV is activated. The child can transcribe and translate green fluorescent protein (GFP). The viral DNA of the parental strain (gE ^- tk ^- GFP ⁺ PRV) was co-transfected into PK15 cells together with the transfer plastid pUCUL24PCV2Cap(A) UL23 obtained to produce recombinant gE ^- tk ^- /PCV2 cap ⁺ PRV Virus strain (Figure 2B). The preparation and analysis methods are as follows: PK15 cells were cultured in a 24-well cell culture tray until the cells were cultured overnight until the formation of Bacheng was completed. First prepared containing 50 μl opti-MEM ^® I medium recombinant transfer plasmid is 0.8 μg of (Invitrogen), additionally are simultaneously containing 2 μl Lipofectamine ^TM 2000 (Invitrogen) in 50 μl opti-MEM ^® I medium the preparation and placed in the chamber After 5 minutes of warming, the two were gently mixed and allowed to stand at room temperature for 20 minutes. Before adding the mixture to the cells for transfection, the cell culture medium was replaced with 200 μl of opti-MEM ^® I medium, and the mixture was added to the cells and allowed to act in the incubator for 6 hours, after which the cells were removed and replaced with 500 cells. The cell culture medium of μl was analyzed by indirect immunofluorescence staining for 18 hours after transfection.

Characterization of PCV2 Cap Protein Expressed by Recombinant Virus

In order to confirm the presence of the PCV2 Cap protein in the recombinant virus, the infective cells were stained with indirect immunofluorescence, and the protein expression was measured and analyzed by Western blotting. The two recombinant viruses (gE ^- /PCV2 cap ⁺ PRV and gE ^- tk ^- /PCV2 cap ⁺ PRV) obtained above were collected, and the cell lysate was collected after the infected cells were overnight, and the PCV2 was detected by Western blotting. The performance of Cap protein (28 kDa), while the solution of wild virus strain (TNL) and PK15 cells did not detect PCV2 Cap protein expression (Fig. 3B). By indirect immunofluorescence staining analysis, both recombinant viruses were able to detect the expression of PCV2 Cap protein (Fig. 3C a, e) and PRV viral proteins after infection (Fig. 3C, b, f ). From the above results, it was revealed that the two recombinant viruses obtained by the present invention can produce antigenic PRV and PCV2 Cap proteins during viral replication.

To understand whether the PCV2 Cap protein expressed by the recombinant virus is capable of self-assembling into a viroid-like particle (VLP) during viral replication, two recombinant viruses (gE ^- /PCV2 cap ⁺ PRV and gE ^- tk ^- /PCV2 cap, respectively) ⁺ PRV) Cell lysate of infected cells, PCV2 VLPs were further purified by ultracentrifugation with 40% sucrose cushion and observed by transmission electron microscopy (TEM). The results showed that the two recombinant viruses produced VLPs during virus replication and their size was about 17-25 nm (Fig. 4A), which showed similarity to the original PCV2 virus particles. Further, the specific monoclonal antibody MAb α-PCV2 Cap was used for identification, and the PCV2 VLPs produced by the two recombinant viruses were all calibrated by gold particles (Fig. 4B, C). Based on the above results, the recombinant virus produced by the present invention can produce antigenic PCV2 VLPs during viral replication.

Example 2: Analysis of vaccine efficacy of recombinant virus strain

Virus strain (gE ^- /PCV2 cap ⁺  PRV) Vaccine efficacy in immunized mice

First, an anti-PRV-specific antibody in mouse serum was present using an indirect ELISA assay. Nine 6-week-old BALB/c mice were randomly divided into two groups. The immunized mice (6) were immunized with PCV2 VLPs (1.2 x ^{10 8} pfu) expressed by 50 μg of recombinant virus, and the control group (3) were immunized with 500 μl of PBS. Each mouse was immunized with 500 μl of 250 μl of antigen and 250 μl of ISA201 adjuvant (SEPPIC) and immunized intraperitoneally at 6 weeks, 8 weeks and 10 weeks of age, respectively.

Specific antibodies against PRV in mouse serum were determined by indirect ELISA. The PRV soluble antigen was antigen-coated on the ELISA assay disk in advance, and the antigen was diluted with a coating buffer (150 mM Na ₂ CO ₃ , 348 mM NaHCO ₃ , pH 9.6), and allowed to act overnight at 4 ° C. After washing once with PBST, 100 μl of a PBS solution containing 1% BSA (blocking solution) was added to each well and allowed to act at 37 ° C for two hours. The cells were washed twice with PBST, and 50 μl of the test mouse serum diluted 100-fold with blocking solution was added to each well, and allowed to act at 37 ° C for one hour. After washing three times with PBST, 50 μl of HRP-conjugated goat anti-mouse antibody (diluted 4000 times with blocking solution) was added to each well, followed by one hour at 37 °C. Wash three times with PBST, wash twice with PBS, and finally add 50 μl of color reagent (ABTS) to each well. The experimental results were performed using an ELISA analyzer to perform absorbance reading at a wavelength of 405 nm.

It was found that mice immunized with the non-activated recombinant virus strain (gE ^- /PCV2 cap ⁺ PRV) produced anti-PRV specific antibodies two weeks after the first immunization and one week after the booster immunization, and produced obvious after the booster immunization. The antibody was ascending (P<0.05). In the mice immunized with PBS, specific antibodies against PRV were not detected during the experiment (Fig. 5).

The challenge virus infection test was further carried out using a 10-fold LD ₅₀ PRV wild virus strain (TNL) to evaluate the protective efficacy of the vaccine. Nine 6-week-old BALB/c mice were randomly divided into two groups. The immunized mice (6) were immunized with 1.2 x ^{10 8} pfu of inactivated recombinant virus, while the control group (3) immunized with 500 μl of PBS. Each mouse was given an immunization dose of 500 μl containing 250 μl of antigen and 250 μl of ISA563 adjuvant (SEPPIC), and immunized intraperitoneally at 6 and 8 weeks of age, respectively, at 11 weeks of age. PRV challenge virus infection was carried out in 500 μl DMEM containing 10 times LD ₅₀ of PRV wild virus strain (TNL). As a result, it was found that the mice in the immunized group did not develop clinical symptoms and all survived, while the experimental control group died in all. From the above results, it was revealed that the immuno-inactivated recombinant virus strain (gE ^- /PCV2 cap ⁺ PRV) can induce anti-PRV neutralizing antibodies in mice and has good protective efficacy.

Non-activated recombinant virus strain (gE ^- /PCV2 cap ⁺  PRV) The efficacy of vaccines in mice immunized with VLPs

First, prepare the non-activated recombinant virus: prepare 0.1 M BEI (1.025 g 2-bromoethylamine dissolved in 50 ml 0.2 N NaOH, shake at 150 rpm for 1 hour at 37 ° C, filter with 0.22 μm filter membrane, and store at 4 ° C) and 1 M sodium thiosulphate. 0.1 M BEI was first added to the virus solution for inactivation, to a final concentration of 3 mM, and shaken at 150 rpm for 24 hours at 37 °C. Then, 1 ml of virus solution was taken, and 10 μl of iced 1 M sodium thiosulphate was added for a small amount of cell infection test to determine an inactivated virus solution which was not infected with cells. Finally, the non-activated virus solution was added to 1 M sodium thiosulphate to terminate the action of BEI.

Nine 6-week-old BALB/c mice were randomly divided into two groups. The immunized mice (6) were immunized with PCV2 VLPs expressed by 50 μg of recombinant virus, and the control group (3) were immunized with 500 μl of PBS. Each mouse was immunized at a dose of 500 μl containing 250 μl of antigen and 250 μl of ISA201 adjuvant (SEPPIC) and immunized intraperitoneally at 6 weeks, 8 weeks and 10 weeks of age, respectively.

Specific antibodies against the PCV2 Cap protein in mouse serum were determined by indirect ELISA. The PCV2 Cap protein expressed in E. coli was subjected to antigen coating on the ELISA assay disk, and the antigen was diluted in a coating buffer (150 mM Na ₂ CO ₃ , 348 mM NaHCO ₃ , pH 9.6) at 4 ° C. Function overnight. After washing once with PBST, 100 μl of a PBS solution containing 1% BSA (blocking solution) was added to each well and allowed to act at 37 ° C for two hours. The cells were washed twice with PBST, and 50 μl of the test mouse serum diluted 100-fold with blocking solution was added to each well, and allowed to act at 37 ° C for one hour. After washing three times with PBST, 50 μl of HRP-conjugated goat anti-mouse antibody (diluted 4000 times with blocking solution) was added to each well, followed by one hour at 37 °C. Wash three times with PBST, wash twice with PBS, and finally add 50 μl of color reagent (ABTS) to each well. The experimental results were performed using an ELISA analyzer to perform absorbance reading at a wavelength of 405 nm.

From the results of Figure 6, it was shown that mice immunized with VLPs expressed by the non-activated recombinant virus strain (gE ^- /PCV2 cap ⁺ PRV) produced anti-PCV2 two weeks after one week and two times of booster immunization after booster immunization. The specific antibody of Cap protein, and after the second immunization, the amount of antibody produced by the immunized group was significantly higher than that of the control group. In the immunized PBS group, specific antibodies against the PCV2 Cap protein were not detected during the experiment. From the above results, it was revealed that mice immunized with VLPs expressed by recombinant virus strain (gE ^- /PCV2 cap ⁺ PRV) can induce specific antibodies against anti-PCV2 Cap protein in mice.

Recombinant virus strain (gE-tk-/PCV2 cap+ PRV) was determined in mouse LD50

To understand the safety of recombinant gE ^- tk ^- /PCV2 cap ⁺ PRV strain, 10 6-week-old BALB/cJNarl mice were randomly divided into two groups, 5 mice in each group, inoculated with 500 intraperitoneal injections. Ll contains 7x10 ⁶ and 7x10 ⁵ pfu of recombinant virus solution. After the mice were inoculated with the virus solution, the health of the mice was observed daily. The results of the experiment showed that the mice were vaccinated at a dose of ⁷ × ^{10 6} or 7× ^{10 5} pfu, and no clinical symptoms were observed and no mice died. Therefore, the LD of the recombinant gE ^- tk ^- /PCV2 cap ⁺ PRV strain in mice was confirmed. ₅₀ is greater than 7x10 ⁶ pfu and has completely attenuated virulence.

Other specific aspects

All of the features disclosed in this specification can be combined in any combination. Thus, the individual features disclosed in this specification can be replaced by alternative features that are the same, equivalent, or similar. Therefore, the various features disclosed are merely examples of a series of equivalents or similar features, unless otherwise clearly indicated.

From the foregoing description, those skilled in the art can readily determine the essential features of the invention, and various changes and modifications of the invention can be made to adapt to various different uses and conditions without departing from the scope thereof. Therefore, other specific aspects are included in the scope of patent application.

<110> National Chung Hsing University

<120> Production of porcine circovirus type 2 virus particles using pseudorabies virus

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<170> Patent In Version 3.3

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<211> 233

<212> PRT

<213> Porcine circovirus type 2

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<212> DNA

<213> Artificial sequence

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<223> Forward introduction Capf-NheI

<400> 1

<210> 3

<211> 21

<212> DNA

<213> Artificial sequence

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<223> Reverse introduction Capr-SalI

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Claims (10)

A method for producing porcine circovirus type 2 virus particles by using pseudorabies virus (PRV), comprising: preparing a recombinant transfer plastid comprising a gene fragment encoding a PCV2 capsid protein (Cap); The viral DNA has a pro-viral strain having at least gE gene defect, and the viral DNA and the recombinant transfer plastid are co-transfected into the host cell to produce a defect having at least gE gene and comprising a PCV2 capsid protein (Cap). a recombinant PRV strain of the gene fragment; and the recombinant PRV strain is propagated to produce a viral particle (PCV2 VLP) such as the antigenic porcine circovirus type 2. The method of claim 1, wherein the PCV2 coat protein has the amino acid sequence of SEQ ID NO. The method of claim 1, wherein the viral DNA of the pro-virus strain has a gE gene defect (only the 3' end sequence is retained). The method of claim 1, wherein the viral DNA of the pro-virus strain has a gE gene deletion (retaining a 3'-end sequence) and a tk gene defect (retaining a 5'-end and a 3'-end sequence). The method of claim 1, wherein the pro-viral strain further comprises a gene fragment encoding a reporter protein that can be screened. The method of claim 5, wherein the reporter protein is a fluorescent protein. A subunit vaccine for preventing and treating porcine circovirus type 2 disease, characterized by comprising a recombinant pseudorabies virus (PRV) expressing a PCV2 coat protein The porcine circovirus type 2 virus particle produced, wherein the porcine circovirus type 2 virus particle has a particle size of 17-25 nm. The subunit vaccine according to claim 7, wherein the recombinant PRV strain has at least a gE gene defect and comprises a gene fragment of a PCV2 capsid protein (Cap). The subunit vaccine according to claim 7, wherein the recombinant PRV strain has a gE gene defect and a tk gene defect, and comprises a gene fragment of a PCV2 capsid protein (Cap). A subunit vaccine as described in claim 7 or 8, wherein the PCV2 coat protein self-assembles into a viroid-like particle (VLP) during replication of the virus.