TW201229238A - DNA adjuvant for waterfowl and livestock vaccines - Google Patents

Abstract

A DNA adjuvant for waterfowl and livestock vaccines is disclosed, which is characterized that the DNA adjuvant includes multi-copy waterfowlspecific CpG motifs or a recombinant plasmid including thereof, and the DNA adjuvant excludes any phosphorothioate (PTO) modification. Moreover, the DNA adjuvant and vaccines including thereof can enhance peripheral blood mononuclear cell (PBMC) growth of at least one immunized animal, for example, waterfowl, bovine or swine animals.

Description

201229238 VI. Description of the Invention: [Technical Field of the Invention] The present invention relates to a vaccine adjuvant, and more particularly to a DNA adjuvant for waterfowl and veterinary disease. [Prior Art] At present, the main adjuvant used in animal vaccines is aluminum gel adjuvant and oily adjuvant', but the disadvantage is that both are chemical adjuvants, which cannot improve specificity. • Thl cell immune response' especially Activated vaccines and live attenuated vaccines cannot produce adequate humoral and cellular immune responses.

Krieg A. M. et al. first tested the unmethylated cytosine guanine dinucleotide module (CpG motif) in a mouse model in nature in 1995. Deoxyribonucleic acid (DNA) has the function of promoting the activation of various immune cells, inducing the production of various cytokines, and being easily absorbed by tissues. After being confirmed in 1998, it was confirmed to be more effective in 2003. Vaccine adjuvant utility. Oligodeoxynucleotides (ODN) containing CpG modules can stimulate the activity of many large-scale livestock animals including lymphocytes and antigens of cattle, pigs, sheep, etc., and increase dendritic cells (Dendritic Cell; DC). The antigen exhibits activation and maturation, and promotes the immune system's response to specific antigens toward Thl cells. For example, the inventors of the present invention have proposed the use of phosphorylthioate-modified (PTO-modified) oligonucleotides containing Cp(} modules and plastids containing different sets of cpG modules for species specificity in waterfowl. It has immunostimulating activity against waterfowl. 201229238 v Oligonucleotides containing CpG modules can transmit the following mechanisms: (1) increase the activation, maturation and antigen presentation of dendritic cells; (2) dendritic cells Increased migration; (3) Significantly increased cell markers of mouse and human dendritic cells (eg, PCT 11 (:-11, 〇040, €080, €086, and 11^12); (4) increased Unstimulated dendritic cells (priming DC) respond to Thl cells of specific antigens; (5) increase the activity of CD8+ T cells, induce cytotoxicity (CTL), etc. The use of oligonucleotides containing CpG modules produces a protective immune response against infects of viruses, bacteria and extracellular parasites in the innate immune response, and activates B cells when combined with vaccines. Produce resistance Activated antigen-presenting cells (APC) secrete cytokines such as interferon-7 (interfer〇n_γ; ΐρΝ_r) to promote the immune response of the vaccine, so the oligonucleotide containing the CpG module can be replaced. As an adjuvant, in general, CpG modules containing oligonucleotides of CpG modules are mostly unpurified CpG modules, and conventional techniques are chemically synthesized to synthesize phosphorus-modified oligonucleotides containing CpG modules. Glycosyl acid. However, artificially synthesized oligonucleotides containing the spring C p G module must use chemically modified phosphodiester bonds between nucleic acids to replace phosphorus with sulfur (ie, the aforementioned phosphorus vulcanization modification) to reduce deoxyribose Nuclease (deoxyrib〇nudease; DNase) decomposes the rate of oligonucleotides containing CpG modules, which is not only time-consuming, incapable of mass production, but also expensive, and may lose the effect of continuously eliciting an immune response. In addition, CpG modules are included. The oligonucleic acid has different specificity, and the structure of the CpG module with better immune promoting effect is also different. The CpG module sequence which has immunomodulatory effects on humans and mice has been confirmed 2012292 The 38 columns are not the same, so the different substances are confirmed by the experiment. The study containing the effective CpG module sequence still needs to be carried out in the industry. The research starts in 2〇〇2 years. The group (4) acid uses the oligonucleoside in the poultry group. The evaluation of the chemically synthesized CPG-containing VZ.VO in the chemical synthesis of the acid is also limited to the evaluation of the anti-CD 0). In vivo, there is a real humoral immunity in the case of prion-infected cells in vivo. , 'the cellular immune response does not go deep into the animal's immune irritant to provide a suitable for animal use DNA adjuvant must use chemical repair

In view of this, it is urgent to propose (immunostimulatory) oligonucleotide CpG DNA adjuvant and improve the cumbersome steps of conventional decoration. SUMMARY OF THE INVENTION Accordingly, one aspect of the present invention provides a DNA adjuvant for a waterfowl and a livestock vaccine, wherein the DNA adjuvant is a multi-set (muUi_c〇py) waterfowl specific CpG module and has not been Phosphorylation-treated immunostimulatory oligonucleotide or recombinant plastid containing the same. This DNA adjuvant can be mass produced using a prokaryotic expression system, and the cumbersome steps of conventional DNA adjuvants that must utilize chemical modification are eliminated. Secondly, another aspect of the present invention provides a vaccine composition comprising an antigen and a DNA adjuvant, wherein the DNA adjuvant is a waterfowl-specific immunostimulatory oligonucleotide or a recombinant plastid comprising the same, Moreover, the immunostimulatory oligonucleotic acid contains multiple sets of waterfowl-specific CpG modules and is not treated with thiosulfide. When the vaccine composition is administered to at least one immunized animal (eg, waterfowl, cow or pig), thereby promoting the proliferation of peripheral immune cells of at least one immunized 201229238 animal, not only reducing the amount of conventional adjuvant used and The cost can also increase the efficacy of vaccines with less antigenic immunity. According to the above aspect of the invention, a DNA adjuvant for waterfowl and livestock vaccine is proposed. In one embodiment, the DNA adjuvant is characterized by a waterfowl-specific immunostimulatory oligonucleotide or a recombinant plastid containing the same, and is not subjected to phosphorus vulcanization. The above waterfowl immunostimulatory oligonucleotide may include, but is not limited to, a first polynucleotide such as the sequence of SEQ ID NO: 1, a second polynucleotide such as the sequence of SEQ ID NO: 2, or The third polynucleotide of the sequence shown in Figure 3. When the above DNA adjuvant is administered to at least one immunized animal (e.g., waterfowl, cow or pig), it promotes proliferation of peripheral immune cells of at least one immunized animal. According to other aspects of the invention, a vaccine composition is presented. In one embodiment, the vaccine composition may comprise an antigen and a DNA adjuvant, wherein the DNA adjuvant is an immunostimulatory oligonucleotide comprising a plurality of sets of waterfowl-specific CpG modules and not subjected to phosphorus vulcanization treatment. Or recombinant plastids containing this. In an exemplary embodiment, the waterfowl specific immunostimulatory oligonucleotide may include, but is not limited to, a first polynucleotide having a sequence as shown in SEQ ID NO: 1, and a second concentrating sequence as shown in SEQ ID NO: 2 A nucleotide or a third polynucleotide of the sequence as shown in SEQ ID NO: 3. When the aforementioned vaccine composition is administered to at least one immunized animal (e.g., waterfowl, cow or pig), peripheral immune cell proliferation of at least one immunized animal can be promoted. According to an embodiment of the invention, the vaccine composition described above may include, but is not limited to, a recombinant protein, a live vaccine or an inactivated vaccine. In an exemplary embodiment, the recombinant protein described above may, for example, be a polypeptide of the sequence of SEQ ID NO: 4. A DNA adjuvant for use in a waterfowl and livestock vaccine of the present invention, which utilizes an immunostimulatory neonatal nucleotide or a recombinant substance containing the same in 201229238, a waterfowl-specific CpG module and which has not been subjected to phosphorus vulcanization treatment. The vaccine adjuvant eliminates the cumbersome steps that conventional DNA adjuvants must utilize chemical modification. When the vaccine composition containing the DNA adjuvant and the antigen is administered to at least one immunized animal (for example, waterfowl, cow or pig), it not only promotes peripheral immune cell growth in the animal, but also reduces the amount of the conventional adjuvant used. The cost can also increase the efficacy of vaccines with less antigenic immunity. [Embodiment] As described in the above, the present invention provides a DNA adjuvant for a waterfowl and a livestock vaccine, and the DNA_ is an immunostimulatory oligo which contains a multi-water_specific CpG module and is not subjected to phosphorus vulcanization treatment. Nucleotide or recombinant plasmid containing this (_mbinant plasmid). This dNA adjuvant can be produced using the prokaryotic expression system, which eliminates the cumbersome steps that conventional DNA adjuvants must utilize chemical modification. φ Preparation of Adjuvants In one embodiment, the plurality of "waterfowl specific Cp modules" as described herein refers to a plurality of "single waterfowl specific types; groups" which are connected end to end. In the example, the "single set of waterfowl-specific CpG modules" is a single-CPG module sequence. From the 5th end to the 3rd end, the sequence can be, for example, a poultry-specific cpG module, and the tail is connected (4). P is linked to a 3rd (muln_c〇py) waterfowl class - immunostimulatory oligonucleic acid. P '°' ^ In an example, the aforementioned waterfowl-specific immunostimulatory oligo (4) 201229238 • Acid Z c includes 6 sets to 24 sets of waterfowl-specific CpG modules. In another example, the waterfowl-specific immunostimulatory oligonucleotide may include, but is not limited to, the first-polynuclear acid of the sequence shown in column identification number 1, such as sequence identification, and the sequence of the sequence indicated by the suffix 2. A dinucleotide or a third polynucleotide of the sequence as shown in SEQ ID NO: 3. In another example, the recombinant plastid containing one or more sets of waterfowl-specific CpG modules can be firstly obtained by repeating several specific restriction enzyme cleavage, binding specific sequences, and nucleic acid selection techniques. Recombinant plastid of the above immunostimulatory oligonucleotide. _ 凊 1A to 1C are partial flow charts showing a method of producing a DNA adjuvant according to several embodiments of the present invention. In Figure 1A, a recombinant plastid (η = 3) Ρ 1 containing three sets of cpG modules is first provided. In an example, the three sets of CpG modules (n=3) contained in the recombinant plastid can be polymerase-chain-locked by using the primer pair shown in Table 1 (Mingxin Biotech Co., Ltd. for synthesis of 'Taipei, Taiwan). Obtained by (PCR). The upstream primer of the above primer pair can be sequenced as shown in sequence identification number 4, and the downstream primer can be sequenced as sequence identification number 5: • The first table is identified by the sequence number 5'-GATCTTCGkC GTT G ACGTT TT G ACGTT G -35 4 ~ ~~^ ' .. __BgUI \CpG ^MM\ \CpG 1 \CpG Module_ 5'-GATCC\kACGTC\ AA jAACGTCl |AACGTC| GA -3 5 _ ~T _

BamHI \CpG module]1 \cpG MM \CPg _ 1 :linking base 201229238 Using the primer pair of the first table to perform the bonding reaction, the obtained kernel_segment (n=3) contains three sets of CpG modules, such as a sequence. Identification of 6 households =, a sequence of the fourth polynucleotide, wherein the 5' end to the 3' end may include a positive = enzyme A cleavage, three sets of CpG modules and a restriction enzyme B cleavage. In the exemplification, the three sets of CpG modules of the aforementioned fourth polynucleotide (n=3) may include, 'the base (as shown in the first table number *), to ensure subsequent colonization. The sequence in 2 is correct. In an example, the 5th end of the fourth polynucleotide (η = 3) can be, for example, 5g/// (the cleavage sequence is 5'-Α | GATCT 3, < I Indicates the enzyme cut point), as indicated by the italicized text in Table 1, G^rCT. Next, the restriction enzyme cleavage site at the 3' end of the nucleic acid fragment (n=3) can be, for example, (the cleavage sequence is 5, -G i GATCC-V , J , I ± shows the enzyme cleavage point)' as in the first table. In the sequence indicated by the italicized text, in addition, the ρΕΤ-3Μ vector (about WO kb) is taken as an example, and the restriction enzyme c cleavage is carried out on the carrier, for example, 八/(the cleavage sequence is G-3', 丨Indicates the enzyme cutting point) to facilitate subsequent bonding of more A|

CpG module. However, it should be noted that the above restriction enzyme a is cleavage,

The cleavage site and the sequence of the restriction enzyme C cleavage site are opt-connected, and the chymase B is not limited to the above, and the fourth smear acid is optionally cultured into any commercially available carrier, such as a recombinant substance. The body (shown as P1.) In the present invention - the embodiment, the DNA_ may be a recombinant plastid containing six sets of CPG modules (n = 6). Please refer to Figure 1A again. Restriction enzyme A can be used to cleave recombinant plastid (n=3) P1 (for example, about 5885_, and obtain fragment I and N1 (about 4547 bp); and use restriction enzyme B and restriction enzyme c to cut 201229238. The segment N2 (about 1366 bp) is obtained. The 5' end of the segment N1 has three CpG modules (indicated by internal blanks), and the other segment N2 has three CpG modules at the 3' end (to fill up). Gray, for example. Then, for example, a bonding reaction can be performed using a ligase, and three sets of CpG modules (indicated by filling gray) at the 3' end of the fragment N2 are bonded to three sets of CpG modules at the 5' end of the fragment N1 ( The internal blank indicates), and a recombinant body (n=6) P2 (about 5913 kb) having six sets of CpG modules is formed, as shown in Fig. 1A. In an example, this includes 6 The immunostimulatory oligonucleotide of the CpG module is the first polynucleotide of the sequence _ as shown in SEQ ID NO: 1, and contains 6 sets of CpG modules or recombinant plastids containing the same (n = 6) Ρ 2 It may be a DNA adjuvant. In another embodiment of the invention, the DNA adjuvant may comprise twelve sets of CpG modules or recombinant bodies containing the same (n = 12). See Figure 1B, Referring to the same strategy as in Figure 1A, using the above-described recombinant plastid (n = 6) P2 containing six sets of CpG modules, the cleavage of the specific restriction enzyme, the splicing fragment Ν3 and the fragment Ν4, and the nucleic acid selection technique were repeated. A recombinant plasmid (n=12) Ρ3 (about 5969 kb) containing twelve sets of CpG modules was obtained. In one example, an immunostimulatory oligonucleotide containing 12 sets of CpG modules was identified as a sequence identification number. The second polynucleotide of the sequence shown in Fig. 2. In another embodiment of the present invention, the DNA adjuvant may also be a CpG module containing 廿 or a recombinant plastid containing the same (n = 24). 1C, referring to the same strategy as FIG. 1A or FIG. 1B, the above-mentioned obtained recombinant plastid (n=12) P3 containing twelve sets of CpG modules can be used to repeatedly cleave and ligate specific restriction enzymes. N5 and fragment N6 and nucleic acid selection techniques, resulting in a recombinant plastid containing four sets of CpG modules (η=24) P4 (about S] 10 201229238 . 6081 kb). In one example, there are 24 sets of CpG modules. The immunostimulatory oligonucleotide of the group is the third polynucleotide of the sequence as shown in Sequence Identification No. 3. &quot; The resulting recombinant plastid containing multiple sets of CpG modules, such as recombinant plastid (n=6) P2, recombinant plastid (n=12) P3, recombinant plastid (n=24) P4, can be further transformed into host cells. For example, Escherichia coli (heart c/zeWc/zk co//) is used for mass production, so that the cumbersome steps of the conventional DNA adjuvant to utilize chemical modification (for example, structuring treatment) can be eliminated. Since the steps of transformation and mass culture of 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 repeated here. Vaccine Composition The term "vaccine composition" as used herein is mainly meant to include a conventional animal vaccine and the above DNA adjuvant, wherein a suitable DNA adjuvant comprises an immunostimulatory oligo comprising a plurality of sets of waterfowl-specific CpG modules. Nucleotide, recombinant plasmid comprising the immunostimulatory oligonucleotide, transformed strain, or any combination thereof. When the aforementioned vaccine composition is administered to at least one immunized animal (e.g., avian, bovine or porcine), peripheral immune cell proliferation of at least one immunized animal can be promoted. Furthermore, the composition of the waterfowl or other animal vaccine obtained thereby is confirmed by the cell model experiment of the peripheral blood mononuclear cells of the animal respectively, and the reconstitution of the multi-set waterfowl-specific CpG module obtained by the present invention is obtained. The plastids are indeed effective in inducing peripheral blood cell proliferation in at least one immunized animal, such as waterfowl and livestock. However, it should be noted that the DNA used in the waterfowl and livestock vaccine of the present invention 201229238 is an insufficient amount of vaccine and cost 'improving the antigen immunity compared with the production of the CPG module: the effect is far beyond the set, Two or three sets of forces can be used to improve the efficacy of vaccines for waterfowls or other animal vaccines. It is mentioned that the waterfowl and livestock, cattle or pigs of the present invention can cause at least one immunity. The antibody price in animals (eg, waterfowl-pig) is maintained for at least $month. </ RTI> </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> With retouching. Example 1: Preparation of DNA adjuvant 1· Construction of recombinant plastid containing three sets of waterfowl-specific CpG modules In this example, first, polymerase chain reaction (PCR) was performed using the primer pair shown in Table 1. And a fourth polynucleotide (n = 3) having the sequence shown in SEQ ID NO: 6 was obtained. Place 10 μί of the upstream primer and the downstream primer of the primer of the first table into a 200 μί microcentrifuge tube, place it in a temperature cycle controller (Thermocycler; TaKaRa, Shiga, Japan), and heat to about 55 °C. It takes about 5 minutes. Then, the reaction was allowed to stand at room temperature for 5 hours to naturally bind to form a fourth polynucleotide (η = 3; about 28 kb). The pET-32a plastid (empty plastid; Novagen, Darmatatt, Germany) was cleaved with restriction enzyme A and restriction enzyme B, and the purified plastid DNA was cleaved, and the fourth polynucleoside was sequenced as shown in SEQ ID NO: 6. The acid (n = 3; about 28 bp) was subjected to a ligation reaction to form a recombinant plastid containing three sets of CpG module 201229238 (n=3; about 5885 bp). Thereafter, the plastids that have been joined are transformed into host cells by heat shock, such as the competent cells of E. coli (5.co//) DH5α, as the transfer and preservation of the above recombinant plasmids. Host. Then, using antibiotic screening method, for example, sputum is evenly applied to LB plate containing 50 pg/mL Ampicillin, placed in a 37 ° C incubator, and after 14 hours of culture, a single colony with successful transformation is selected and sequenced. A large number of cultures can be carried out by determining that the sequence of the constructed sequence is backward. However, the above-mentioned construction of recombinant plastids, transformation into host cells, screening of antibiotics, mass culture, and extraction of plastid DNA are well known to those of ordinary skill in the art, and therefore will not be further described herein. Next, three sets of CpG modules of recombinant plastids (pET-32a) will be added. Reconstituted to pGEM-T easy plastid (empty vector; pGEM-T Easy Vector system I®; Promega, wi, USA) to facilitate subsequent preparation of recombinant plastids containing multiple sets of waterfowl-specific CpG modules (n= 6~24). It is stated that the recombinant plastid (n=3) containing three sets of CpG modules constructed above can be used to cleave the fourth polynucleotide via restriction enzyme a and restriction enzyme b, and is then constructed for pGEM for subsequent convenience. In the T Easy plastid, Taq polymerase (polymerase) is used to add a prominent A (3'-A overhand) to the 3' end of the fourth polynucleotide, and the reagents thereof are as shown in Table 2: __ 2 Table__ Reagent__ Volume (μ!&gt;) Fourth polynucleotide (5 mM) 7.7 10x PCR buffer (containing, for example, 200 mM Tris-Hcl, pH 1 8.4, 500 mM KC1, Mg2+ free; TaKaRa , Shiga,

Japan) 201229238 • dNTPs (10 mM; TaKaRa, Shiga, Japan) 〇 2

Taq DNA polymerase (5 U/pL, Invitrogen, U.S.A.) 〇 j

MgCl2 (50 mM) \ Total ___ ι〇 The above reagents were reacted in the above temperature cycle controller for 25 minutes at 72 ° C and then at 4 ° C for 10 minutes. Then, the 3' end was added with the protruding fourth polynucleotide of A, and the pGEM-Teasy plastid (empty vector; pGEM-T Easy Vector system I®; Promega, WI, USA) DNA was directly subjected to the ligation reaction. A recombinant plastid containing three sets of CpG modules (n==3; about 5885 bp) was formed, wherein the pGEM-T Easy empty vector did not need to be cleaved by any restriction enzyme. Thereafter, the recombinant plastid can be further transformed into a suitable host, such as a competent cell of the E. coli (5.m/z·) BL21 (DE3) strain, as a transfer of the above-described recombinant expression plastid. Colonization and preservation of the host. After that, blue-white screening was performed in an LB dish containing 100 mg/mL φ Ampicillin, and 5-bromo-4-chloro-3-indolyl-bD-galactoside (5-bromo-4-chloro-) was used. 3-indolyl-bD-galactopyranoside; X-gal) A large number of cultures can be carried out by allowing the colonies of successful transformation to develop color and confirming the sequence of the constructed bacteria by PCR and DNA sequencing. The plastid DNA extracted from the successfully transplanted host cells (n=3) was cleaved by restriction enzyme A and restriction enzyme C, or restriction enzyme b and restriction enzyme C, respectively, and subjected to DNA electrophoresis analysis to confirm that three sets of CpG were contained. The fourth polynucleotide (n=3) of the ODN module is embedded in the constructed plastid, and the recombinant plastid (n=3) containing three sets of CpG modules containing [S] 14 201229238 is correct. The formula, wherein the types of the restriction enzyme A, the restriction enzyme B, and the restriction enzyme C are as described above, are not described herein. Next, the fragment of the above fourth polynucleotide (n = 3) was confirmed by DNA sequencing, and the results are shown in Annex 1. Please refer to Annex 1, which shows a DNA sequence diagram of an immunostimulatory oligonucleotide containing multiple sets of (n=3) waterfowl specific CpG modules according to an embodiment of the present invention, which is entrusted by an upstream primer. Mingxin Biotechnology Co., Ltd. (Taipei, Taiwan) performs DNA sequencing using a nucleic acid sequence automatic sequencer. As is apparent from the results of Annex 1, the nucleic acid fragments containing multiple sets of (η = 3) waterfowl-specific CpG modules according to an embodiment of the present invention were confirmed by DNA sequencing, and the above sequences were confirmed to be correct. 2. Constructing recombinant plastids containing multiple sets of waterfowl-specific CpG modules. This embodiment utilizes the above-described constructed recombinant plastids containing three sets of CpG modules (n=3; approximately 5885 kb, hereinafter referred to as P1). Using the methods exemplified in FIGS. 1A to 1C, the cleavage of a specific restriction enzyme, the splicing of a specific sequence, and the nucleic acid selection technique are repeated, and 6 sets, 12 sets, and 24 sets of CpG modules are respectively obtained ( n= 6, 12, 24; about 5913, 5969, 6081 kb) recombinant plastids (hereinafter referred to as P2, P3, P4, respectively). The process is as described above and is not awkward here. The plastid DNA (P2, P3, P4) extracted from the successfully transplanted host cells was cleaved by restriction enzyme A and restriction enzyme C, or restriction enzyme B and restriction enzyme C, respectively, and subjected to DNA electrophoresis analysis to confirm the first Polynucleotides (n=6; about 56 bp), second polynucleotide (n=12; about 112 bp), and third polynucleotide (n=24; about 224 bp) were embedded in the constructed In the plastid 201229238., the formation of the 6,2, 2QCpC} module (n=6, 12, 24), the recombinant plastid is correct (not shown in green), which includes restriction enzyme A, restriction enzyme B and restriction enzyme The type of C is also as mentioned in the previous example. It is not like here. Next, the above first polynucleotide (n=6), second polynucleotide (η=12), second polynucleotide (η=24), and fourth polynucleotide (η=3) After the fragment is confirmed by DNA sequencing, the results are shown in Annex 2 to Annex 4. Please refer to Annex 2 to Annex 4, which respectively show DNA sequencing diagrams of immunostimulatory nucleotides containing a set of 12, 12, 24) waterfowl-specific CpG modules according to the present invention. The upstream primer was commissioned by Mingxin Biotechnology Co., Ltd. (Taipei, Taiwan) to perform DNA sequencing using a nucleic acid sequence automatic sequencer. From the results of the attachments 2 to 4, it is understood that the nucleic acid fragments of the multiple waterfowl-specific CpG modules of one embodiment of the present invention are confirmed by DNA sequencing, and the above sequences are confirmed to be correct. Example 2··Evaluation of the immune-promoting effect of recombinant plastids containing multiple sets of waterfowl-specific CpG modules in cell mode 1. Extraction of peripheral blood mononuclear cells This example is a healthy experimental animal (duck, Peripheral blood mononuclear cell (PBMC) of cattle and pigs. First, whole blood of healthy ducks, cattle, and pigs was taken, and EDTA (0.2%) anticoagulant was added, and then centrifuged at 1300 x g for about 30 minutes at 4 ° C to collect a buffy coat. The above-mentioned white blood cell layer is mixed with an equal volume of 'lxPBS (pH 7 '2 ' 37 ° C), and then added to an equal volume density ladder 201229238 degree solution, such as Ficoll-Paque (GE Healthcare, Stgiles, Sweden), Centrifuge at 200 xg for 25 minutes at about 4 °C. Next, the cell layer was aspirated, washed once in 1X PBS, centrifuged at 20 〇xg for 1 〇 at 4 ° C, and then RPMI-1640 broth (for example, HEPES buffer containing 2.05 mM L_glutamine '25 mM, 2 The sodium bicarbonate of g (GIBCO, NY, USA) was washed twice to obtain purified lymphocytes. After counting the cells, adjust the cell concentration to ΙχΙΟ7 using a cell culture medium such as RPMI-1640 (GIBCO, NY, USA) containing 10% fetal bovine serum, penicillin (40 pg/mL), and 5xl CT5 Μ β-Mercaptoethanol. After the cells/mL, add 1 〇〇 0 ^ 6 cells/1111 于 to the 96-well cell culture plate. 2. Recombinant plastids containing multiple sets of waterfowl-specific CpG modules Evaluation of immunostimulatory activity in duck spleen cells 2.1 Stimulation of stimuli In this example, peripheral blood mononuclear cells (PBMC) of experimental animals (duck, cow, pig) were divided into 4 groups of experimental groups and 3 groups of control groups. 'Evaluate the recombinant plastids containing multiple sets of waterfowl-specific CpG modules as the stimulating effect of the original stimulating effect. The experimental group added 1 〇pg of recombinant substance containing multiple sets of waterfowl-specific CpG modules per well. The sequence of the body (P1, p2, p3, P4), the empty vector, and the two sets of CpG modules for artificially synthesizing phosphorus sulfide (hereinafter referred to as CpG ODN). The positive control group is added with 2 gg of mitogen, for example Concanavalin a (Concanavalin a; c〇na; Sigma, MO, USA). Negative The cells in the group were not added with antigen and c〇nA. All of the above cells had 3 replicates and were cultured at 37 ° C, 51⁄4 C02 for 201229238 72 hours. The above groups of samples had significant differences (p &lt; 〇〇 5) 2.2 MTT assay In each of the above cells, 2 μM of MTS reagent was added to each well, for example, 3_(4,5-dimercaptozol-2)-5-(3-refenyl) in the CellTiter 96® kit reagent. 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2 -(4-sulfophenyl) -2H-tetrazolium; MTS tetrazolium; 5 mg/mL; Promega, WI, USA] After mixing evenly, avoiding light at 37 °C, 50/〇C02 for 4 hours, using Elisa reader (Anthos 2020, Cambridge,

Austria), detected at a wavelength of 492 nm (〇D492nm). 2.3 Cell proliferation index (8丨丨11111丨化0|1丨11(16\;81) calculation The calculation of the cell proliferation index (SI) can be obtained according to the following formula (II): Cell proliferation index ( §丨)=test group OD492nm-background value〇D492nm negative control group OD492nm-background value〇D492nm (II) • In formula (II), the above background value is the only reaction of cells with RPMI-1640 medium and 2〇hl MTS reaction After that, the detected value of 492d492 nm is shown in Fig. 2. Referring to Fig. 2, it is shown that the DNA adjuvant of one embodiment of the present invention is subjected to a cellular immunoreactivity test result. Figure, in which the horizontal axis represents the vertical axis of the antigen represents the cell proliferation index (SI), the blank column represents the empty vector 4 test group, the left oblique dense diagonal column represents the CpG 〇 DN test group, and the loose and diagonal column Represented the recombinant plastid (P1) test group, the loose dot column represents the recombinant plastid (P2) test group, the dense dot column represents the recombinant 201229238 plastid (P3) test group, and the large checkerboard column represents the recombinant plastid ( P4) Test group, black column indicates the positive control group (ConA). Figure 2 The statistical analysis was performed using SAS version 9.0, and the Tukey-type multiple comparison test was used to compare the significance of the differences between the groups by the Tuized-type multiple comparison test. Figure numbers a, b, c The average value of d is in the order of a&gt;b&gt;c&gt;d. The same figure number (for example, between a and a) indicates that there is no significant difference between the groups. Conversely, between different figure numbers (for example, b and c) There is a significant difference between the groups. As for the figure be, this value is not significantly different from b and c. From the results of Fig. 2, it can be seen that the empty vector DNA can not effectively cause cell proliferation of duck PBMC. There was no statistically significant difference in the effect of the recombinant plastid (pi) test group and the synthetic CpG ODN test group on the cell proliferation of duck PBMC. However, compared with the recombinant plastid (P1) test group or The synthetic CpG ODN test group had significant differences in recombinant plastids (P2, P3, P4), representing the non-toxic effects of the recombinant plastids (P2, P3, P4) of Example 1 on duck PBMC. Body (P4) causes good cell proliferation of duck PBMC However, there was no significant difference from the positive control group ConA. Therefore, the results of Fig. 2 show that the recombinant plastids (P2, P3, P4) of Example 1 have no toxic effect on duck PBMC, and have the effect of promoting waterfowl animals. The effect of immunological activity, but this effect does not have a corresponding difference as the number of CpG modules increases. 3. The recombinant immunogenic CpG module containing multiple sets of waterfowls is evaluated for the immunostimulatory activity of bovine blood mononuclear cells. This example divides the peripheral blood mononuclear cells (PBMC) of cattle into 4 groups. Group and 3 groups of control groups, evaluation of multiple sets of waterfowl specificity 201229238

The recombinant plastid of the CpG module acts as an immunostimulatory effect to stimulate the original. The stimulation of the original thorn, the calculation of the MTT test and the SI, etc. are as described above, and there is no further rumor here. FIG. 3 is a diagram showing the results of an immunological reaction test for immunizing a DNA according to an embodiment of the present invention in a bovine, wherein the horizontal axis of the towel represents the original and the vertical axis represents the cell proliferation index (SI). The blank column represents the empty carrier test group, the left oblique dense diagonal column represents the CpG ODN test group, the cross-diagonal column represents the recombinant plastid (ρι) test group, and the loose mesh B column represents the recombinant substance. In the body (P2) test group, the dense dot column represents the recombinant plastid (P3) test group, the large checkerboard column represents the recombinant plastid (P4) test group, and the black matrix column represents the positive control group (c〇nA). The statistical analysis in Figure 3 was performed using SAS version 9.0, and the Tukey-type multiple comparison test was used to compare the significance of the differences between the groups. The average values of the figure numbers a, b, c, and d are a>b&gt;c&gt;d&gt;e. The same figure number (for example, between a and a) indicates a non-significant difference between the groups. Conversely, different numbers between different numbers (eg b and c) indicate significant differences between the groups. As for the figure number be, it means that there is no significant difference between this value and b and c. From the results of Fig. 3, it was found that the empty vector DNA was not effective in causing cell proliferation of bovine PBMC. Secondly, there was no statistically significant difference in the effect of the recombinant plastid (pi) test group and the synthetic CpG ODN test group on the cell proliferation of bovine PBMC. Compared with the synthetic CpG ODN test group, the recombinant plastids (P2, P3) had significant differences. Furthermore, compared with the recombinant plastid (P1) test group or the synthetic CpG ODN test group, recombinant plastids (P4) had a significant difference, but there was no statistically significant difference from the positive control 201229238 • Group ConA. Therefore, the results of Fig. 3 show that the recombinant plastids (P2, P3, p4) of Example 1 are non-toxic to bovine pBMC and have an effect of promoting the immunological activity of other animals, but this effect does not follow CpG. There is a corresponding difference in the number of modules. 4. Evaluation of the immunostimulatory activity of recombinant plastids containing multiple sets of waterfowl-specific CPG modules in pig blood mononuclear cells. This example is to treat peripheral blood mononuclear cells (pBMC) of pigs into 4 The experimental group and the three control groups were evaluated for the immunostimulatory effect of the recombinant plastid containing multiple sets of waterfowl specific φ CPG modules. The stimulation of the stimuli, the calculation of the MTT test and the SI are also discussed here, and are not described here. Referring to Fig. 4, there is shown a graph showing the results of a cellular immunological test for immunizing a DNA adjuvant according to an embodiment of the present invention, wherein the horizontal axis represents antigen and the vertical axis represents cell proliferation index (SI), blank column. The representative of the empty vector test group's left oblique dense diagonal column represents the CpG ODN test group, the cross-loose diagonal column represents the recombinant plastid (P1) test group, and the loose mesh point φ column represents the recombinant plastid (P2) In the experimental group, the dense dot column represents the recombinant plastid (P3) test group. The large checkerboard column represents the recombinant plastid (P4) test group, and the black matrix column represents the positive control group (C〇nA). The statistical analysis in Fig. 4 was analyzed using SAS version 9.0, and the difference significance between the groups was compared by the Tukey type multiple comparison test (Tukey_type multiple comparison test). The average values of the figure numbers a, b, c, and d are sequentially a&gt;b&gt;c&gt;d. The same figure number (for example, between &amp; and 3) indicates a non-significant difference between the groups. Conversely, different numbers between different numbers (eg, b and C) indicate significant differences between groups. As for the figure number be, it means [S] 21 201229238 This value is not significantly different from b and c. From the results of Fig. 4, it was found that empty vector DNA could not effectively cause cell proliferation of pig PBMC. Secondly, there was no statistically significant difference in the effect of the recombinant plastid (P1) test group and the synthetic CpG ODN test group on the cell proliferation of pig PBMC. Compared with the recombinant plastid (P1) test group or the synthetic CpGODN test group, the recombinant plastids (P2, P3) had significant differences. Furthermore, compared with the recombinant plastid (PI, P2, P3) test group or the synthetic CpGODN test group, the recombinant plastid (P4) had a significant difference, but it was statistically insignificant with the positive control group ConA. The difference. Therefore, the results of Fig. 4 show that the recombinant plastids (P2, P3, P4) of Example 1 are non-toxic to pig PBMC and have an effect of promoting the immunological activity of other animals, but this effect does not follow the CpG mode. There is a corresponding difference in the number of groups. It should be noted that the present invention describes DNA adjuvants for waterfowl and livestock vaccines of the present invention, exemplified by specific plastids, expression systems, reaction conditions, immunized animals, analytical methods, induction conditions or specific instruments. It is to be understood by those skilled in the art that the present invention is not limited thereto, and other plastids may be used for the DNA adjuvants for waterfowl and livestock vaccines of the present invention without departing from the spirit and scope of the present invention. The system, reaction conditions, immunized animals, analytical methods, induction conditions or instruments are performed. Secondly, the DNA adjuvant for waterfowl and livestock vaccine of the present invention can effectively induce cell proliferation of duck PBMC, and can effectively induce cell proliferation of bovine PBMC and pig PBMC without cell cytotoxicity. Has the potential to be used in DNA adjuvants for waterfowl and livestock vaccines. 22 201229238 Furthermore, it can be seen from the results of the cell model experiment of the second embodiment that the number of CpG modules contained in the DNA adjuvant of the present invention is not proportional to the degree of immunostimulating activity against the vaccine. The relationship does not have a corresponding difference as the number of CpG modules increases. In addition, it is to be noted that the present invention is exemplified by a specific plastid, an immunostimulatory oligonucleotide containing a specific set of CpG modules, a specific host cell, a waterfowl test animal, a cell type or a vaccine species. The present invention is not limited thereto, and the waterfowl of the present invention is not limited thereto, and the present invention is not limited thereto, and the waterfowl of the present invention is not departing from the spirit and scope of the present invention. DNA adjuvants for livestock vaccines can be carried out using other plastids, host cells, recombinant plasmids, transformed strains, other test animals, cell types or vaccine types. For example, recombinant plastids containing an even number of sets of waterfowl-specific CpG modules other than 6, 12, and 24 sets can be used to generate additional sets in the host cell (eg, 8, 10, 14, 16, 18, 20). 22 sets of immunostimulatory oligonucleotides for waterfowl-specific CpG modules, used as DNA adjuvants for waterfowl and livestock vaccines. In addition, the DNA adjuvant obtained by the present invention, for example, an immunostimulatory oligonucleotide containing a plurality of sets of waterfowl-specific CpG modules, containing the immunostimulatory oligo, may also be included without departing from the spirit and scope of the present invention. The recombinant plastid, transformed strain, or any combination of the above, is applied to other animals (such as mammals) or other animal vaccine types to enhance the immune stimulating effect of the vaccine against other animals. It is worth mentioning that the DNA adjuvant for waterfowl and livestock vaccines of the present invention can cause antibody titers in at least one immunized animal (e.g., waterfowl, cow or pig) to be maintained for at least 5 months. 23 201229238 It can be seen from the above embodiments of the present invention that the DNA adjuvant of the waterfowl and livestock of the present invention has the advantage that the DNA adjuvant is a multi-set waterfowl-specific immunostimulatory oligonucleotide or a recombinant containing the same The body, and does not need to be treated by phosphorus vulcanization, can eliminate the cumbersome steps that conventional DNA adjuvants must utilize chemical modification. When the vaccine composition containing the DNA adjuvant and the antigen is administered to at least one immunized animal (for example, waterfowl, cow or pig), it not only promotes peripheral immune cell proliferation in the animal, but also reduces the amount of the conventional adjuvant used. The cost can also increase the efficacy of vaccines with less antigenic immunity. 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. BRIEF DESCRIPTION OF THE DRAWINGS The above and other objects, features, advantages and embodiments of the present invention will become more <RTIgt; A partial flow diagram of a method of making a DNA adjuvant of an embodiment. Fig. 2 is a graph showing the results of a cellular immunoreactivity test for immunizing a DNA adjuvant according to an embodiment of the present invention in ducks. Fig. 3 is a graph showing the results of a cellular immunological test for immunizing a DNA adjuvant according to an embodiment of the present invention in cattle. Figure 4 is a graph showing the results of a cellular immunoreactivity test for immunizing a DNA adjuvant according to an embodiment of the present invention in pig 24 201229238. Annexes 1 to 4 show DNA sequencing diagrams of immunostimulatory oligonucleotides containing multiple sets (n=6, 12, 24) of waterfowl-specific CpG modules according to an embodiment of the present invention. [Description of main component symbols] P1: Contains three sets of waterfowl specific P3: Recombinant plastids of recombinant plastid CpG modules containing 12 sets of waterfowl-specific CpG modules φ P2: Contains six sets of waterfowl-specific P4 : Recombinant plastids of recombinant plastid CpG modules containing four sets of waterfowl-specific CpG modules

[s] 25 201229238 Sequence Listing &lt;11〇&gt;National Pingtung University of Science &Technology&lt;120&gt;DNA adjuvant for waterfowl and livestock vaccine &lt;130&gt; No &lt;160&gt; 6

&lt;210&gt; 1 &lt;211&gt; 56 &lt;212&gt; DNA &lt;213&gt;Artificial sequence &lt;400&gt; 1 56 gatcttcgac gttgacgttt tgacgttgga tcttcgacgt tgacgttttg acgttg &lt;210&gt; 2 &lt;211&gt; 112 &lt;212&gt; DNA &lt;213&gt; Artificial sequence

&lt;400&gt; 2 60 112 gatcttcgac gttgacgttt tgacgttgga tcttcgacgt tgacgttttg acgttggatc ttcgacgttg acgttttgac gttggatctt cgacgttgac gttttgacgt tg &lt;210&gt; 3 &lt;211> 224 &lt;212&gt; DNA &lt;213&gt; Artificial sequence [si 201229238 &lt;400&gt; 3 gatcttcgac gttgacgttt Tgacgttgga tcttcgacgt tgacgttttg acgttggatc 60 ttcgacgttg acgttttgac gttggatctt cgacgttgac gttttgacgt tggatcttcg 120 acgttgacgt tttgacgttg gatcttcgac gttgacgttt tgacgttgga tcttcgacgt 180 tgacgttttg acgttggatc ttcgacgttg acgttttgac gttg 224

&lt;210&gt; 4 &lt;211&gt; 28 &lt;212&gt; DNA &lt;213>Artificial sequence &lt;400&gt; 4 gatcttcgac gttgacgttt tgacgttg 28 &lt;210&gt; 5 &lt;211&gt; 27 &lt;212&gt; DNA &lt;213&gt; Sequence &lt;400&gt; 5 gatccaacgt caaaacgtca acgtcga 27 &lt;210&gt; 6 &lt;211&gt; 32 &lt;212&gt; DNA &lt;213&gt; artificial sequence 2 201229238 &lt;400&gt; 6 32 gatcttcgac gttgacgttt tgacgttgga tc

[SI 3

Claims (1)

201229238 VII. Patent application scope: 1. A DNA adjuvant for waterfowl and livestock vaccine, characterized in that the DNA adjuvant is a water-fowl-specific immunostimulatory oligonucleotide or the like which is not subjected to sulfurization treatment. Recombinant plastid, the waterfowl immunostimulatory oligonucleotide is selected from the group consisting of a first polynucleotide such as the sequence number shown in SEQ ID NO: 1, and a second nucleoside as shown in SEQ ID NO: 2 An acid and a group consisting of a third polynucleotide consisting of one of the sequences identified by SEQ ID NO: 3. 2. The DNA adjuvant for waterfowl and livestock vaccine according to claim 1, wherein the DNA adjuvant is administered to at least one immunized animal, the at least one immunized animal is a waterfowl, a cow or a pig. And the DNA adjuvant promotes proliferation of peripheral immune cells of the at least one immunized animal. A vaccine composition comprising: an antigen; and a DNA adjuvant, wherein the DNA adjuvant is a non-phosphorus sulfided treatment and is a waterfowl-specific immunostimulatory oligonucleotide or a recombinant plastid comprising the same, The immunostimulatory oligonucleotide is selected from the group consisting of a first polynucleotide such as sequence identification number 1, a second polynucleotide such as the sequence number shown in SEQ ID NO: 2, and a sequence recognition a group consisting of a third polynucleotide comprising a sequence of number 3, and wherein the vaccine composition is administered to at least one immunized animal to promote peripheral immune cell proliferation of the at least one immunized animal, wherein the at least An immunized animal is a waterfowl, cow or pig. 1 201229238 4. The vaccine composition according to claim 3, wherein the antigen is a recombinant protein, a live vaccine or an inactivated vaccine. i S] 2