TWI425088B - Dna vaccine for waterfowl parvovirus - Google Patents

Description

DNA vaccine for waterfowl small virus

The present invention relates to a DNA vaccine, and more particularly to a DNA vaccine for a waterfowl small virus.

Waterfowl parvovirus causes viral enteritis in waterfowl. The infection route is contact infection. In addition to causing high mortality of young birds, it also slows the growth of adult birds and causes serious losses to the operators.

At present, the prevention of waterfowl small viruses on the market is mainly based on attenuated vaccines and non-activated vaccines for duck embryo proliferation, but the non-activated vaccines cannot cause cell type immune responses, and have poor protection against viral diseases. While the attenuated waterfowl vaccine can induce a cell type immune response, its residual virulence is still toxic to ducklings and goslings, and the side effects after inoculation are large.

Since the waterfowl small virus is not easy to culture in vitro , the current vaccine preparation method still uses the traditional duck, goose embryo inoculation method, or the duck and goose embryonic fibroblast cell proliferation method, thereby obtaining the attenuation. Vaccines and non-activated vaccines are the mainstays.

However, the preparation method of the conventional vaccine has the following problems. First, the duck and goose embryo source that meets the SPF specification is insufficient, so there is still a disadvantage of weak antigenicity and insufficient protection regardless of the use of the non-activated or domesticated live vaccine. . Secondly, the waterfowl small virus is further divided into goose parvovirus (GPV) and duck parvovirus (also known as Muscovy duck parvovirus; MDPV), and the gene sequence is compared with goose virus (GPV) and After the duck virus (MDPV) results, the sequence difference between the two is quite large. Therefore, it is difficult to achieve cross-protection through the vaccine prepared by the traditional duck and goose embryo inoculation method. Furthermore, the inactivated vaccine does not cause a cell type immune response, and there is a poor protection against viral diseases. Although the attenuated waterfowl small vaccine can induce a cell type immune response, its residual virulence is still toxic to ducklings and goslings, and the side effects after inoculation are large.

In addition, the main adjuvants used in animal vaccines are aluminum gel adjuvants and oily adjuvants, but the disadvantage is that both are chemical adjuvants, which cannot enhance the immune response of specific Th1 cells, especially for inactivated vaccines. Live attenuated vaccines, unable to produce adequate protective humoral and cellular immune responses.

In view of this, it is urgent to propose a DNA vaccine for waterfowl small viruses to provide vaccines for waterfowls and to improve the various vaccines and adjuvants.

Accordingly, one aspect of the present invention provides a DNA vaccine composition for a waterfowl small virus, wherein the DNA vaccine composition comprises at least a recombinant plasmid having a first polynucleotide having a sequence of Sequence Identification No. 1, And the recombinant system is uncoated to promote at least one immunized animal against at least one waterfowl virus. In this way, the DNA vaccine composition can be mass-produced by using the prokaryotic expression system, and the problem of insufficient SPF specifications of duck and goose embryo eggs required by the conventional vaccine can be eliminated.

Secondly, another aspect of the present invention provides a DNA vaccine composition for a waterfowl small virus, which is composed of a first recombinant plastid and a DNA adjuvant, wherein the first recombinant plastid has a sequence identification number of The first polynucleotide of the sequence, the DNA adjuvant is a second polynucleotide of the sequence as shown in SEQ ID NO: 2 or a second recombinant plastid comprising the same, and the first recombinant plastid, DNA adjuvant and / or the second recombinant system is uncoated to promote at least one immunized animal against a plurality of waterfowl small viruses at the same time, and overcome the disadvantages of conventional adjuvant immunoreaction.

According to the above aspect of the invention, a DNA vaccine composition of a waterfowl small virus is proposed. In one embodiment, the DNA vaccine composition comprises at least one recombinant plastid, wherein the recombinant plastid has a first polynucleotide having a sequence as shown in SEQ ID NO: 1, and the recombinant system is uncoated. To promote at least one immunized animal against at least one waterfowl small virus.

According to other aspects of the invention, a DNA vaccine for a waterfowl small virus is presented. In one embodiment, the DNA vaccine composition can be composed of a first recombinant plastid having a first polynucleotide having a sequence as shown in SEQ ID NO: 1, a DNA adjuvant, and a DNA adjuvant. Is a second polynucleotide or a second recombinant substance comprising the sequence of sequence identification number 2, and the first recombinant plasmid, the DNA adjuvant and/or the second recombinant system are uncoated, Promote at least one immunized animal against multiple waterfowl small viruses.

According to an embodiment of the invention, the at least one waterfowl small virus is goose virus and/or duck virus.

The DNA vaccine composition of the waterfowl small virus of the present invention is an uncoated recombinant plastid as a vaccine, which can be mass-produced not only by the prokaryotic expression system, but also without the SPF specifications of ducks and geese required for the conventional vaccine. Problems such as insufficient embryonic egg source. When the DNA vaccine composition is administered to at least one immunized animal (e.g., waterfowl), at least one immunized animal can be promoted against at least one waterfowl small virus.

As described above, the present invention provides a DNA vaccine composition for a waterfowl small virus, which utilizes an uncoated recombinant plastid as a vaccine, and can be mass-produced using a prokaryotic expression system, eliminating the SPF specifications required for a conventional vaccine. The duck and goose embryos have insufficient source of eggs.

DNA vaccine composition

First, the first recombinant plastid

In one embodiment, the "DNA vaccine composition" described herein comprises at least a first polynucleotide having the sequence shown in SEQ ID NO: 1, a first recombinant plastid containing the same, a transformed strain, or any of the above. Combine to induce both humoral and cellular immune responses. Therefore, the DNA vaccine composition of the present invention excludes animal vaccines prepared by conventional conventional duck and goose embryo inoculation methods, such as non-DNA vaccines such as inactivated vaccines and attenuated vaccines.

Many studies have shown that DNA vaccines promote the proliferation of immune cells and the performance of MHC class I and MHC class II-peptides complexes. At this point, professional antigen-presenting cells provide a complete signal to activate surrounding CD4+ T cells, which in turn activate B cells and promote the production and secretion of antibodies. At the same time, CD8+ T cells are also activated to eliminate cells that are infected with the pathogen. Therefore, DNA vaccines can induce both humoral and cellular immune responses.

The viral single-strand linear DNA of goose parvovirus (GPV) is divided into two major open reading frames (ORFs), in which the right ORF encodes three capsid proteins VP1 to VP3. Therefore, in one embodiment, the first recombinant system contained in the first recombinant plastid comprises a gene sequence of 2433 to 4640 nucleotides of a full-length gene of goose virus (GPV), about 2200 base pairs. (base pair; bp), the sequence of which is shown in sequence identification number 1.

The aforementioned first recombinant plastid can be mass produced using a prokaryotic expression system. In one embodiment, first, a primer pair of GPV gene DNA and a full-length gene of GPV is subjected to polymerase chain reaction (PCR) to obtain a large number of PCR products of the first polynucleotide, wherein the full-length gene of GPV The primer pair is designed according to the GPN sequence of the GenBank accession number U25749 in the National Center for Biotechnology Information (NCBI) website, as shown in Sequence Identification No. 3 and Sequence Identification No. 4 of Table 1. The upstream and downstream primers:

After the above PCR product is confirmed by DNA sequencing, the first polynucleotide and the appropriate conventional vector are cleaved by the same restriction enzyme and then ligated into the first recombinant plasmid.

In an exemplary embodiment, the restriction enzyme cleavage site at the 5' end of the first polynucleotide can be, for example, EcoRI (the cleavage sequence is 5'-G↓AATTC-3', ↓ indicates the enzyme cleavage point), and the 3' end thereof The restriction enzyme cleavage site can be, for example, BamHI (the cleavage sequence is 5'-G↓GATCC-3', and ↓ indicates the enzyme cleavage site). It should be noted that the sequence of the restriction enzyme cleavage site varies depending on the vector to be ligated, and thus is not limited thereto.

The first recombinant plastid containing the first polynucleotide obtained above can be further transformed into a host cell, such as Escherichia coli , for mass production, thus eliminating the need for a conventional vaccine The SPF specification of duck and goose embryo source is insufficient. Since the steps of transformation and mass culture of the bacteria are carried out using conventional techniques, it is well known to those of ordinary skill in the art to which the present invention pertains, and therefore will not be described one by one.

Second, DNA adjuvant

In one embodiment, the DNA vaccine composition of the waterfowl small virus may further comprise a DNA adjuvant in addition to the first polynucleotide, the first recombinant plasmid, the transformed strain or any combination thereof. Thereby, the peripheral blood cell proliferation of at least one immunized animal is effectively promoted, and the vaccine efficacy of the first recombinant plastid is enhanced.

A "DNA adjuvant" as used herein may include, but is not limited to, a second polynucleotide, a second recombinant plastid comprising the same, a transformant strain, or any combination thereof, wherein the second polynucleotide has a waterfowl specific Multiple CpG module sequences that are connected end to end.

The above-mentioned waterfowl-specific immunostimulatory oligonucleotide may comprise 6 to 24 sets of CpG modules having waterfowl specificity, wherein each CpG module sequence is from the 5' end to the 3' end. The sequence can be, for example, "GACGTT". In one example, the waterfowl-specific immunostimulatory oligonucleotide can comprise a second polynucleotide having the sequence set forth in SEQ ID NO: 2, having 12 CpG modules and having a length of about 112 bp. The preparation of the DNA adjuvant or the second recombinant plastid containing the same may be carried out by mass production using a conventional prokaryotic expression system, or by reference to the content of the Patent Application No. 100100846 of the Republic of China, which is hereby incorporated by reference. .

Briefly, the second polynucleotide can be prepared by the following method. First, PCR can be performed using the designed primer pair to obtain a nucleic acid fragment containing three sets of CpG modules, and then ligated with a suitable vector to form a recombinant plastid (n=3) P1 containing three sets of CpG modules. Next, based on the recombinant plastid (n=3) P1, a fragment N1 was obtained by cutting a recombinant plastid (n=3) P1 with a set of restriction enzymes, and cleavage by another set of restriction enzymes to obtain a fragment N2, such that the fragment was obtained. The 5' end of N1 has three sets of CpG modules, and the other part N2 has three sets of CpG modules at the 3' end. Then, three sets of CpG modules at the 3' end of the fragment N2 are bonded to three sets of CpG modules at the 5' end of the fragment N1 by ligase to form a recombinant body having a head-to-tail connection and six sets of CpG modules ( n=6) P2. Then, referring to the above strategy, using the above-mentioned recombinant plastid (n=6) P2 containing six sets of CpG modules, 12 sets of CpG modules were obtained by repeating the cutting, conjugation and nucleic acid selection techniques of specific restriction enzymes. The recombinant plastid (n=12) P3. In this example, after confirming the sequence by DNA sequencing, the sequence containing 12 sets of CpG modules is the second polynucleotide of the sequence as shown in SEQ ID NO: 2, and the second polynucleotide or the like The second recombinant plastid can be used as a DNA adjuvant.

The second recombinant plastid obtained above can also be mass-produced by a prokaryotic expression system, thereby eliminating the cumbersome steps that conventional DNA adjuvants must utilize chemical modification (for example, phosphorus vulcanization treatment). Since the steps of transformation and mass culture of the bacteria are carried out using conventional techniques, it is well known to those of ordinary skill in the art to which the present invention pertains, and therefore will not be described one by one.

When the DNA vaccine composition of the aforementioned waterfowl small virus is applied to at least one immunized animal (for example, a waterfowl goose or duck), the ratio of the concentration of the first recombinant plasmid to the DNA adjuvant (for example, μg/mL) may be, for example, 0.5:2 to 2:0.5, but the concentration of both (eg μg/mL) is better than 1:1 to induce both humoral and cellular immune responses, thereby promoting at least one immunized animal against at least one waterfowl Small virus. Furthermore, the composition of the waterfowl or other animal vaccine thus obtained is confirmed by a cell model experiment of peripheral blood mononuclear cells (PBMC), respectively, and the waterfowl small virus obtained by the present invention. The DNA vaccine composition does allow at least one immunized animal (e.g., goose or duck) to simultaneously combat at least one waterfowl small virus, such as goose virus and/or duck parvovirus, and has a cross-protective effect.

It should be noted that the DNA vaccine composition of the waterfowl small virus of the present invention can not only reduce the use amount and cost of the conventional chemical adjuvant, but also overcome the conventional chemical adjuvant when it is selectively added with the DNA adjuvant. The shortcomings of the immune reaction are insufficient, and the blood cell proliferation of the peripheral blood of at least one immunized animal can be effectively promoted, and the vaccine efficacy of the first recombinant plastid is enhanced. It is worth mentioning that the animal immunized by the DNA vaccine composition of the waterfowl small virus of the present invention can maintain an antibody titer of at least 5 months or more.

The following examples are used to illustrate the application of the present invention, and are not intended to limit the present invention. Those skilled in the art can make various changes without departing from the spirit and scope of the present invention. Retouching.

Example 1: Preparation of DNA vaccine

1. Constructing the first recombinant plastid

In this example, first, each reaction reagent was uniformly mixed according to the second table, and then placed in a temperature cycle controller (Thermocycler; TaKaRa, Shiga, Japan) to carry out a polymerase chain reaction (PCR).

The upstream primer and the downstream primer of the second table were commissioned by Mingxin Biotechnology Co., Ltd. (Taipei, Taiwan) for synthesis. The reaction reagent is reacted in the temperature cycle controller at 94 ° C for 1 minute, at 48 ° C for 1 minute, and at 72 ° C for 1 minute, for a cycle reaction, after a total of 30 cycles of reaction, and then at 72 ° C for 5 minutes. By reason.

After the above PCR product was confirmed by DNA sequencing to be the first polynucleotide, the first polynucleotide and the pTCY vector were cleaved by EcoRI / BamHI [derived from the vector pEGFP-N1 (Clontech, Palo Alto, CA, USA). ], the two are joined as a first recombinant plastid (pTCY/GPV). Here, it is explained that the promoter of the vector pTCY has removed the coding region of the original enhanced green fluorescent protein of the vector pEGFP-N1, and the vector pEGFP-N1 was originally The CMV immediate early promoter is replaced by the rat β-actin promoter.

Thereafter, the first recombinant plastid (pTCY/GPV) can be further transformed into a suitable host, such as a competent cell of an E. coli BL21 (DE3) strain, as the first recombinant plastid. The host of reproduction and preservation. Then, using antibiotic screening method, for example, the bacterial liquid is evenly spread on a LB plate containing 50 μg/mL Ampicillin, placed in a 37 ° C incubator, and after 14 hours of culture, a single colony that has been successfully transformed is selected, and the restriction enzyme EcoRI is selected. / BamHI cutting and / or sequencing to determine the sequence of the correct construction of the bacteria behind, you can carry out a large number of cultures. However, the above-mentioned construction of recombinant plastids, transformation into host cells, screening of antibiotics, mass culture, extraction of plastid DNA, optical density (OD 260) values, establishment of standard curves, PCR and DNA sequencing are the technical fields. Anyone with ordinary knowledge is well known, so it will not be repeated here.

1 is a diagram showing agaric electrophoresis analysis of a first recombinant plastid by a restriction enzyme according to an embodiment of the present invention, wherein the first lane represents a DNA ladder mark with a 100 base pair progression ( DNA ladder, Bertec, Taipei, Taiwan), and lane 2 represents the nucleic acid fragment of the first polynucleotide, as indicated by arrow 101. From the results of the second lane of Fig. 1, it can be seen that after the first recombinant plasmid is subjected to the restriction enzyme, the nucleic acid fragment of the full-length gene of GPV (about 2200 bp; as indicated by arrow 101) can be specifically cut out. In addition, after the first recombinant plastid obtained above was subjected to DNA sequencing, the sequence was confirmed to be correct (not shown).

Example 2: Evaluation of the performance of the first recombinant plasmid in a cell line

1. Preparation of a large amount of recombinant plastid DNA for cell transfection

After the large number of cultured transformants of the first recombinant plasmid obtained in Example 1 were cultured, a large amount of plastid DNA was extracted by a commercially available product or a conventional method, and no bacterial endotoxin was contained, thereby facilitating subsequent transfection of the cell strain. However, the extraction of large amounts of plastid DNA should be well known to anyone of ordinary skill in the art and is not limited here.

2. Transfection of cell lines

The African green monkey kidney epithelial cell line Vero cells [for example, purchased from the Hsinchu Food Industry Research and Development Institute of Taiwan, registered vero cell line numbered BCRC 60013, or American Type Culture Collection (ATCC) Hosting the vero cell line numbered ATCC CCL-81], cultured in minimum essential medium (MEM; minimum essential medium; MEM; BRL, Grand Island, NY, USA) medium, plus 2 mM L-glutamine, Earle's balanced salt solution (Earle's BSS), 1.5 g/L Sodium bicarbonate, 0.1 mM non-essential amino acids, 1.0 mM sodium pyruvate and 10 vol% fetal calf serum (FBA; GIBCO/BRL, Rockville, MD USA) , or other commercially available products with similar functions, were placed in a 37 ° C, 5% CO ₂ incubator for cultivation.

The cells of 1 × 10 ⁶ /mL were seeded in a petri dish having a diameter of 6 cm, and cultured at 37 ° C in a 5% CO ₂ incubator for 16 hours to 18 hours to allow the cells to reach about 7 to 8 minutes. Next, the fresh medium was replaced and cultured for 3 hours at 37 ° C in a 5% CO ₂ incubator. Then, about 5 μg of the first recombinant plastid (pTCY/GPV) DNA and about 10 μL of the transfection reagent, such as TurboFect in vitro Transfection Reagent (Fermentas, Canada), first with about 250 μL, 150 mM NaCl, respectively. After the solution is uniformly mixed, the two are uniformly mixed to form a transfection reaction solution, and allowed to stand at room temperature (for example, about 15 ° C to 35 ° C) for 15 minutes to 30 minutes. At this time, the cells to be transfected should be replaced with a culture medium containing no serum and antibiotics to increase the success rate of transfection. Thereafter, the transfection reaction solution was added to the cells to be transfected, and cultured at 37 ° C in a 5% CO ₂ incubator for about 5 hours. After the reaction was completed, the fresh medium (10% FBS MEM) was replaced, and after 7 days of incubation in a 37 ° C, 5% CO ₂ incubator, the supernatant was collected for subsequent Western blot analysis (Western Immunoblot). Analysis).

2. Evaluation of the expression of recombinant protein of VP1-3 full-length gene in cell lines

This example uses the Western blot method to evaluate the expression of recombinant proteins of the VP1-3 full-length gene in the above stable cell lines.

The supernatant obtained above was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for about 2 hours at 120 volts. Thereafter, it was equilibrated with a transfer buffer containing 192 mM glycine (Biotechnology Grade, aM ReSCO, U.S.A.) and 25 mM Tris-HCl solution (J.T. Baker, U.S.A.). The preparation of the above SDS-PAGE and related drugs and related devices should be well known to those of ordinary skill in the art to which the present invention pertains, and will not be further described herein.

The aforementioned SDS-PAGE electrophoresis gel can be subjected to Western blotting analysis. In this embodiment, for example, a wet transfer tank (for example, Bio-Rad Scientific Instruments Transfer Unit; Amersham Pharmacia biotech, USA) is used at 250 mA for about 75 minutes to transfer the protein of the aforementioned SDS-PAGE gel. To the transfer film, wherein the transfer film may be, for example, a polyvinylidene difluoride membrane (PVDF membrane; Immobilon TM-P Transfer Membrane; Millipore, Ireland). Thereafter, the transfer film can be washed with 1×PBST (1×PBS containing 0.05% (v/v) Tween-20 (Showa, Japan)) for about 10 minutes, and then added to Blocking solution (20 mM Tris-Base, 0.1%). Sodium azide, 1% BSA (Sigma, USA), 125 mM NaCl, 0.2% Tween-20 (Showa, Japan), pH 7.4) was shaken at 4 ° C for about 1 hour.

Thereafter, the Blocking solution was removed, and a primary antibody (antibody: 1 × PBST = 1:3500 dilution) was added to react with the transfer membrane, and reacted at 4 ° C until overnight (about 14 hours to 16 hours). Then, the transfer film was washed 6 times with 1×PBST for 10 minutes, and then the secondary antibody was added to react with the transfer membrane (antibody: Blocking solution = 1:15000 dilution), and shaken at 4 ° C for about 50 minutes. Then, the transfer film was washed 6 times with 1 × PBST for 10 minutes each time. Subsequently, a cold-light color former such as Enhanced Chemi Luminescence (ECL) Plus Western blotting detection reagent (GE Healthcare, UK) is added in the dark, and the reaction is carried out for about 1 minute, using a negative film (for example, Kodak). ,USA). The above Western blotting method uses β-actin as a house keeping gene.

The primary antibody used as described above may be, for example, a rabbit anti-GST-GPV polyclonal antibody; Abcam, USA, and a mouse anti-β-actin monoclonal antibody (Chemicon) ,USA). The secondary antibody used as described above may be, for example, a goat anti-rabbit IgG-HRP (Zymed, USA) that binds to wasabi peroxidase, and a goat anti-mouse that binds to wasabi peroxidase (goat anti-mouse). IgG-HRP; Chemicon, USA). However, the above-mentioned use of cold light color development, film development and fixing, etc. can be carried out according to the manual provided by the manufacturer, and is well known to those of ordinary skill in the art, and will not be further described herein.

Please refer to FIG. 2, which shows the results of Western blot analysis of the first recombinant plasmid expressing VP1-3 recombinant protein in a cell line according to an embodiment of the present invention, wherein the Mth line represents a protein marker, and the first lane represents The Vero cell line expressing the VP1-3 recombinant protein, while the second lane represents the Vero cell control group, and the left side indicates the relative position of the protein marker.

As can be seen from the results of lane 1 of Figure 2, the specific antibody did detect the expression of the recombinant protein of VP1-3 full-length gene (about 80 kDa), as indicated by arrow 201. From the results of the second lane of Fig. 2, it was found that the specific antibody did not detect the recombinant protein of the VP1-3 full-length gene in the cell control group (lane 2), and the Vero cell strain representing the above example was indeed intracellular. A recombinant protein stably expressing the full length VP1-3 gene.

Example 2: Evaluation of the immune promoting effect of DNA vaccine composition of waterfowl small virus in animals

ELISA price

In this example, a 9-day-old duck was subjected to an immunoassay in which ducks were randomly divided into 3 groups of 4 each. The experimental group used 100 μg of the first recombinant plastid (pTCY/GPV) DNA of Example 1 with or without 100 μg of the aforementioned DNA adjuvant (including the second polynucleotide, the second recombinant containing the same) The body, the transformed strain, or any combination of the above) was inoculated into the duck by intramuscular injection (total amount 0.2 mL/dose/only). Two weeks after the inoculation, intramuscular injection into the hind legs for reinforced immunization once. The control group was administered with 0.2 mL of physiological saline. The whole blood of the ducks taken before immunization, 7 days, 14 days, 21 days 28 days, and 5 months after the first immunization, and the antibody titer in the serum was detected by enzyme-linked immunosorbent assay (ELISA). The ELISA value (OD _{450 nm} ) at a wavelength of 450 nm was detected by an ELISA reader (Anthos 2020, Cambridge, Austria) to evaluate the ELISA valency of the DNA vaccine composition of the waterfowl small virus in vivo, and the results are shown in Fig. 3. Shown.

Please refer to FIG. 3, which is a bar graph showing the absorbance (OD _450nm ) of duck serum immunized with DNA vaccine composition of waterfowl small virus according to an embodiment of the present invention at a wavelength of 450 nm, wherein the horizontal axis represents immunity. Time, the vertical axis represents the absorbance (OD _450nm ), the right oblique line column represents the experimental group that only applies the first recombinant plastid (pTCY/GPV) DNA, and the cross-hatched column represents the simultaneous application of the first recombinant The experimental group of the body (pTCY/GPV) DNA and the DNA adjuvant, and the white column column represents the control group administered with physiological saline. The average values of the figure numbers a and b shown in Fig. 3 are a>b in order. The same figure number (eg, between a and a) indicates a non-significant difference between the groups. Conversely, differences between different numbers (eg, a and b) indicate significant differences between groups ( p < 0.05).

From the results of Fig. 3, it was found that there was no significant change in the ELISA power price of the control group. However, the experimental group that only applied the first recombinant plastid (pTCY/GPV) DNA and the experimental group that simultaneously applied the first recombinant plastid (pTCY/GPV) DNA and DNA adjuvant, with the increase of the immunization time, the ELISA The price of power has also increased. Moreover, compared with the control group, only the experimental group that applied the first recombinant plastid (pTCY/GPV) DNA and the experimental group that simultaneously applied the first recombinant plastid (pTCY/GPV) DNA and the DNA adjuvant had statistics. Significant difference ( p <0.05). Furthermore, the DNA vaccine composition of the waterfowl small virus of the present invention can cause the antibody titer in the duck to be maintained for at least 5 months.

2. Cellular immune response

2.1 Stimulating the original stimulus

In this example, peripheral blood mononuclear cells (PBMC) of ducks 14 days and 28 days after immunization were extracted, and the effect of the DNA vaccine composition of the waterfowl small virus on the cellular immune response to the living body was evaluated.

First, the whole blood of the ducks of 14 days and 28 days after the above immunization was added, and EDTA (0.2%) anticoagulant was added, and then centrifuged at 1300 × g for about 30 minutes at 4 ° C to collect a buffy coat. . The aforementioned leukocyte layer is mixed with an equal volume of 1×PBS (pH 7.2, 37 ° C) and then added to an equal volume density gradient solution, such as Ficoll-Paque (GE Healthcare, Stgiles, Sweden) at about 4 ° C. Centrifuge at 200 xg for 25 minutes.

Next, the cell layer was aspirated, washed once with 1×PBS, centrifuged at 200×g for 10 minutes at 4° C., and then cultured with RPMI-1640 (for example, HEPES buffer containing 2.05 mM L-glutamine, 25 mM). 2 g of sodium bicarbonate) (GIBCO, NY, USA) was washed twice to obtain purified lymphocytes. After counting the cells, adjust the cell concentration using a cell culture medium such as RPMI-1640 (GIBCO, NY, USA) containing 10% fetal bovine serum, penicillin (40 μg/mL), and 5×10 ^-5 M β-Mercaptoethanol. After 1 × 10 ⁷ cells/mL, 100 μL of the cell liquid (1 × 10 ⁶ cells/well) was added to a 96-well cell culture plate.

In the experimental group, 1.25 μg of recombinant protein of GPV/VP1-3 full-length gene (hereinafter referred to as rVP1-3 recombinant protein) was added to each well. The positive control group was 1.25 μg of recombinant protein of GPV/VP1-3 full-length gene (hereinafter referred to as rVP1-3 recombinant protein) and T cell mitogen (such as Concanavalin A; Con). A; Sigma, MO, USA)]. The cells of the negative control group were not added with antigen and ConA. All of the above cells had 3 replicates and were incubated at 37 ° C, 5% CO ₂ for 72 hours.

2.2 MTT test

In each of the above cells, add 20 μL of MTS reagent per well, such as CellTiter 3-(4,5-dimethyloxo-2)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazole in the kit reagent [3- (4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; MTS tetrazolium; 5 mg/mL; Promega, WI, USA] Thereafter, the reaction was carried out at 37 ° C, 5% CO ₂ for 4 hours, and an absorbance (OD _{492 nm} ) at a wavelength of 492 nm was detected by an ELISA reader (Anthos 2020, Cambridge, Austria).

2.3 Calculation of cell proliferation index (SI)

The calculation of the cell proliferation index (SI) can be calculated according to the following formula (II):

In formula (II), the above background value is the OD _{492 nm} value detected after the cells were only added with RPMI-1640 medium and reacted with 20 μL of MTS. The result is shown in Fig. 4.

Please refer to FIG. 4, which is a bar graph showing the cellular immune response of a duck immunized with a DNA vaccine composition of waterfowl virus according to an embodiment of the present invention, wherein the horizontal axis represents antigen and the vertical axis represents cell proliferation. The index (SI), the right oblique line column represents the experimental group that only applied the first recombinant plastid (pTCY/GPV) DNA, and the cross-hatched column represents the simultaneous application of the first recombinant plastid (pTCY/GPV) DNA. In the experimental group with DNA adjuvant, the white column column represents the control group administered with physiological saline, and the black column column represents the positive control group (ConA). The average values of the numbers a and b in Fig. 4 are a>b. The same figure number (eg, between a and a) indicates a non-significant difference between the groups. Conversely, different numbers between different numbers (eg, a and b) indicate significant differences between groups.

From the results of Fig. 4, it was found that there was no statistically significant difference in the effect of the control group and the experimental group administered only with the first recombinant plastid (pTCY/GPV) DNA on the cell proliferation of duck PBMC, representing implementation. The first recombinant plastid (pTCY/GPV) DNA of Example 1 is non-toxic to duck PBMC. Secondly, the rVP1-3 recombinant protein could not effectively induce cell proliferation of the duck PBMC of the experimental group and the experimental group in which only the first recombinant plastid (pTCY/GPV) DNA was administered. However, the rVP1-3 recombinant protein effectively caused the proliferation of duck PBMC cells in the experimental group in which the first recombinant plastid (pTCY/GPV) DNA and DNA adjuvant were simultaneously administered, and only the first recombinant plastid was administered with the control group. There were significant differences in the experimental groups of (pTCY/GPV) DNA. Therefore, the results of Fig. 4 show that the first recombinant plastid (pTCY/GPV) DNA of Example 1 is non-toxic to duck PBMC, and has the effect of promoting the immunological activity of waterfowl animals after simultaneous application of the DNA adjuvant. effect.

3. Evaluation of cytokines

In this example, the peripheral blood mononuclear cells (PBMC) of the ducks 28 days after the above-mentioned immunization were taken, and the DNA vaccine composition of the waterfowl small virus was evaluated. The effect on the amount of cytokine expression in living organisms.

First, peripheral blood mononuclear cells (PBMC) of the ducks 28 days after the above-mentioned immunization were extracted in the same manner as above, and 1 mL of the cell liquid (1 × 10 ⁷ cells/mL) was added to the 24-well cell culture plates, respectively. The positive control group was added with 1.25 μg of rVP1-3 recombinant protein per well, and the cells of the negative control group were not added with antigen. All of the above cells had 3 replicates, and were cultured at 37 ° C, 5% CO ₂ for 3 hours, and then taken out, and the supernatant was removed by centrifugation.

After each well was uniformly dissolved in 1 mL of Trizol reagent (Invitrogen, CA, USA), RNA was extracted and subjected to reverse transcription polymerase chain reaction (RT-PCR) under the reaction conditions of 25 ° C for 10 minutes, 37 The reaction was carried out at ° C for 120 minutes, at 85 ° C for 5 minutes, and at 4 ° C for 10 minutes. Then, real-time polymerase chain reaction (real-time PCR) was used to analyze the expression of GAPDH, IFN-α, IFN-γ, IL-6 and IL-12 cytokine gene expression (mRNA). The results are shown in Figures 5A to 5D.

Please refer to FIG. 5A to FIG. 5D, which are bar graphs showing changes in cytokines of ducks immunized with DNA vaccine composition of waterfowl small virus according to an embodiment of the present invention, wherein FIG. 5A to FIG. 5D are diagrams. The horizontal axis represents time (day), the vertical axis represents a multiple of cytokine changes, and the right oblique line column represents the experimental group in which only the first recombinant plastid (pTCY/GPV) DNA is applied, and the cross-sectional line column represents both An experimental group that applied the first recombinant plastid (pTCY/GPV) DNA and DNA adjuvant. The average values of the numbers a and b of the 5A to 5D drawings are a>b. The same figure number (eg, between a and a) indicates a non-significant difference between the groups. Conversely, different maps Inter-numbers (eg, a and b) indicate significant differences between groups.

From the results of Fig. 5A to Fig. 5D, it was found that PBMC of each group before stimulation (day 0) was stimulated with rVP1-3 for 3 hours in vitro, IFN-α (Fig. 5A), IFN-γ (5B). Fig.), IL-6 (Fig. 5C) and IL-12 (Fig. 5D) have no increase in mRNA gene expression fold. However, on the 14th day after the booster immunization (the 28th day after the first immunization), the cytokine IFN-α (the first) was compared to the experimental group in which only the first recombinant plastid (pTCY/GPV) DNA was administered. 5A), IFN-γ (Fig. 5B), IL-6 (Fig. 5C), and IL-12 (Fig. 5D) mRNA expression levels, simultaneously applying the first recombinant plastid (pTCY/GPV) DNA and The experimental group of DNA adjuvants was significantly different ( p < 0.05). Therefore, the results of Figures 5A to 5D show that the first recombinant plastid (pTCY/GPV) DNA of Example 1 has no toxic effect on duck PBMC, and promotes waterfowl after simultaneous application of DNA adjuvant. The effect of cytokines in animals.

The data obtained in the above experimental examples are expressed as mean ± standard error of mean, and analyzed by SAS version 9.1, and the Tukey-type multiple comparison test is used. Studentized comparisons were used to compare the significance of differences between the groups. The average values of the numbers a and b are a>b. The same figure number (eg, between a and a) indicates a non-significant difference between the groups. Conversely, differences between different numbers (eg, a and b) indicate significant differences between groups ( p < 0.05).

It should be noted that the present invention describes the DNA vaccine composition of the waterfowl small virus of the present invention, although specific plastids, expression systems, reaction conditions, immunized animals, analytical methods, induction conditions or specific instruments are exemplified. It is to be understood by those of ordinary skill in the art that the present invention is not limited thereto, and that the DNA vaccine composition of the waterfowl small virus of the present invention may use other plastids, expression systems, etc., without departing from the spirit and scope of the present invention. The reaction conditions, the immunized animal, the analytical method, the induction conditions or the instrument are carried out.

For example, the DNA vaccine composition of the waterfowl small virus of the present invention may use a first polynucleotide such as the sequence of Sequence Identification No. 1, a first recombinant plasmid containing the same, a transformed strain, or any combination thereof. To induce both humoral and cellular immune responses. Secondly, the present invention further adds a "DNA adjuvant" to the DNA vaccine composition, which may comprise a second polynucleotide, a second recombinant substance containing the same, a transformed strain or any combination thereof, and a second The number of sets of CpG modules for waterfowl specificity contained in the polynucleotide can range from 6 to 24 sets. Furthermore, the DNA vaccine of goose virus is originally used for immunizing goose. The present invention uses the DNA vaccine of goose virus to be used for immunizing ducks, and can also cause a good immune response, and has confirmed the effect of cross protection.

It can be seen from the above embodiments of the present invention that the DNA vaccine composition of the waterfowl small virus of the present invention has the advantages that the uncoated recombinant plastid is used as a vaccine, and the prokaryotic expression system can be mass-produced without the conventional vaccine. The required SPF specifications for duck and goose embryos are insufficient. When the DNA vaccine composition is administered to at least one immunized animal (eg, waterfowl), at least one immunized animal can be promoted against at least one waterfowl small virus, and the antibody titer in the body can be maintained for at least 5 months or more. .

While the invention has been described above in terms of several embodiments, it is not intended to limit the scope of the invention, and the invention may be practiced in various embodiments without departing from the spirit and scope of the invention. The scope of protection of the present invention is defined by the scope of the appended claims.

<110> National Pingtung University of Science and Technology

<120> DNA vaccine composition of waterfowl small virus

<130> None

<160> 4

<210> 1

<211> 2208

<212> DNA

<213> Goose parvovirus (GPV)

<220>

<223> Amplified GPV VP1-3 nucleic acid fragment

<400> 1

<210> 2

<211> 112

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleic acid fragments containing twelve CpG modules

<400> 2

<210> 3

<211> 22

<212> DNA

<213> Artificial sequence

<220> primer

<230> PCR forward primer

<400> 3

<210> 4

<211> 18

<212> DNA

<213> Artificial sequence

<220> primer

<230> PCR reverse primer

<400> 4

101/201. . . arrow

The above and other objects, features, advantages and embodiments of the present invention will become more apparent and understood.

Fig. 1 is a diagram showing the agarose electrophoresis analysis of the first recombinant plastid by a restriction enzyme according to an embodiment of the present invention.

Figure 2 is a graph showing the results of Western blot analysis of the first recombinant plasmid expressing VP1-3 recombinant protein in a cell line according to an embodiment of the present invention.

Figure 3 is a bar graph showing the absorbance (OD _{450 nm} ) of duck serum immunized with a DNA vaccine composition of waterfowl small virus at a wavelength of 450 nm according to an embodiment of the present invention.

Figure 4 is a bar graph showing the cellular immune response of ducks immunized with a DNA vaccine composition of waterfowl virus according to an embodiment of the present invention.

5A to 5D are bar graphs showing changes in cytokines of ducks immunized with a DNA vaccine composition of waterfowl small virus according to an embodiment of the present invention.

Claims (3)

A DNA vaccine composition for a waterfowl small virus, which is composed of a first recombinant plastid having a sequence of the first recombinant nucleoside and a DNA adjuvant, wherein the first recombinant plastid has a sequence as shown in SEQ ID NO: 1. Acid, the DNA adjuvant is a second polynucleotide comprising one of the sequences shown in SEQ ID NO: 2 or a second recombinant plastid comprising the first recombinant plastid, the DNA adjuvant and/or the The second recombinant system is uncoated, the DNA adjuvant is not subjected to phosphorus vulcanization, and the ratio of the concentration of the first recombinant plasmid to the DNA adjuvant (μg/mL) is 1:1 to promote at least one The animals are immunized against both goose virus and duck virus. The DNA vaccine composition of the waterfowl small virus according to claim 1, wherein the at least one immunized animal is a waterfowl. A DNA vaccine composition for a waterfowl small virus according to claim 2, wherein the waterfowl is a goose or a duck.