TWI425091B - Dna adjuvant for waterfowl and livestock vaccines - Google Patents

Description

DNA adjuvant for waterfowl and livestock vaccines

This invention relates to a vaccine adjuvant, and more particularly to a DNA adjuvant for waterfowl and livestock vaccines.

At present, the main adjuvants for 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 and live poisons. Attenuated vaccines do not produce adequate humoral and cellular immune responses.

Krieg AM et al. first tested the demethylation of the unmethylated cytosine guanine dinucleotide module (CpG motif) in a mouse model in nature in 1995. Deoxyribonucleic acid (DNA) has the effects of promoting the activation of various immune cells, inducing the production of various cytokines, and being easily absorbed by tissues. It was confirmed in 1998 and its effectiveness was confirmed. In 2003, it was confirmed to have the effect of vaccine adjuvant. . Oligodeoxynucleotides (ODN) containing CpG modules can stimulate the activity of various large-scale livestock animals, including lymphocytes and antigens of bovine, pig, and sheep, and increase the activity of dendritic cells (Dendritic Cell; DC). The antigen exhibits activation and maturation, and promotes the immune system's response to specific antigens toward Th1 cells. For example, the inventors of the present invention have proposed that the use of a phosphorylthioate-modified (PTO-modified) CpG-containing oligonucleotide and a plastid containing a different set of CpG modules have water species for waterfowl. Immune promoting activity.

Oligonucleotides containing CpG modules can transmit the following mechanisms: (1) increase the activation, maturation and antigen presentation of dendritic cells; (2) increase the migration of dendritic cells; (3) significantly increase mice And the expression of cell markers of human dendritic cells (eg, MHC-II, CD40, CD80, CD86, and IL-12); (4) increase the Th1 cell response of unstimulated dendritic cells (priming DC) to specific antigens (5) Increasing the activity of CD8 ⁺ T cells can induce cytotoxicity (CTL) against specific viruses or tumors, and enhance immunity. The use of oligonucleotides containing CpG modules produces a protective immune response against viral, bacterial and extracellular parasites in the innate immune response, and activates B cells to produce antibodies and activates when combined with vaccines. Antigen-presenting cells (APCs) secrete cytokines such as interferon-γ (IFN-γ) to promote the immune response of vaccines, so oligonucleotides containing CpG modules can be used instead. Adjuvant.

In general, CpG modules containing oligonucleotides of CpG modules are mostly unmethylated CpG modules, whereas conventional techniques chemically synthesize phosphorous-modified oligonucleotides containing CpG modules. However, artificially synthesized oligonucleotides containing CpG modules must use chemically modified phosphodiester bonds between nucleic acids to replace phosphorus with sulfur (ie, the aforementioned phosphorus sulfide modification), and reduce deoxyribonuclease (DNase). The rate at which oligonucleotides containing CpG modules are decomposed 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, oligonucleotides containing CpG modules have species specificity, and the structure of CpG modules with better immunostimulatory effects among different species is also different. It has been confirmed that the sequences of CpG modules which have immunomodulatory effects on humans and mice are not the same, so the effective CpG module sequences of different species still need to be further confirmed by experiments. The use of CpG-modulated oligonucleotides in the poultry industry began in 2002, but the in vitro evaluation of in vivo reactions using chemically synthesized oligonucleotides containing CpG modules ( in vivo) The assessment is also limited to humoral immunity against antibody production, and there is no in-depth discussion of the truly effective cellular immune response to viral infection of cells.

In view of this, it is urgent to propose an animal immunostimulatory oligonucleotide to provide a CpG DNA adjuvant suitable for use in animal vaccines and to improve the cumbersome steps in which conventional DNA adjuvants must utilize chemical modification.

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-copy waterfowl-specific CpG module and is not subjected to phosphorus vulcanization. An immunostimulatory oligonucleotide or a recombinant plastid containing the same. This DNA adjuvant can be mass-produced using a prokaryotic expression system, and the cumbersome steps that conventional DNA adjuvants 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 oligonucleotide contains multiple sets of waterfowl-specific CpG modules and is not subjected to phosphorus vulcanization treatment. When the vaccine composition is administered to at least one immunized animal (eg, waterfowl, cow or pig), thereby promoting proliferation of peripheral immune cells of at least one immunized animal, not only reducing the amount and cost of conventional adjuvants It can also improve 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 growth 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, recognize the 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 is a vaccine using an immunostimulatory oligonucleotide containing a plurality of sets of waterfowl-specific CpG modules and not subjected to phosphorus vulcanization, or a recombinant substance containing the same Adjuvants eliminate the need for conventional DNA adjuvants to take advantage of the cumbersome steps of 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 usage and cost of the conventional adjuvant. It can also improve the efficacy of vaccines with less antigenic immunity.

As described above, the present invention provides a DNA adjuvant for waterfowl and livestock vaccine, which is an immunostimulatory oligonucleotide containing multiple sets of waterfowl-specific CpG modules and which has not been subjected to phosphorus vulcanization treatment or contains Recombinant plasmid. This DNA adjuvant can be mass-produced using a prokaryotic expression system, and the cumbersome steps that conventional DNA adjuvants must utilize chemical modification are eliminated.

Preparation of DNA adjuvant

In one embodiment, the plurality of "waterfowl-specific CpG modules" described herein refers to a plurality of "single waterfowl-specific CpG modules" that are connected end to end. In an example, a "single set of waterfowl-specific CpG modules" is a single CpG module sequence, and the sequence from the 5' end to the 3' end can be, for example, "GACGTT". The aforementioned "single set of waterfowl-specific CpG modules" is connected end-to-end, that is, an immunostimulatory oligonucleotide linked to a multi-copy waterfowl-specific CpG module.

In one example, the aforementioned waterfowl-specific immunostimulatory oligonucleotides may comprise from 6 to 24 sets of waterfowl-specific CpG modules. In another illustration, the waterfowl specific immunostimulatory oligonucleotide may include, but is not limited to, a first polynucleotide such as the sequence of sequence number 1 or a second sequence of sequence number 2 A polynucleotide or a third polynucleotide as in the sequence identified by the sequence numbering 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.

Please refer to FIGS. 1A to 1C, which are partial flow charts showing a method of manufacturing a DNA adjuvant according to several embodiments of the present invention.

In Figure 1A, a recombinant plastid (n = 3) P1 containing three sets of CpG modules is first provided. In one example, the three sets of CpG modules (n=3) contained in the recombinant plastid can be polymerase-chain-locked using the primer pair shown in Table 1 (Mingxin Biotech Co., Ltd., Taipei, Taiwan). Obtained by (PCR). The upstream primer of the above primer pair can be the sequence of sequence identification number 4, and the downstream primer can be the sequence of sequence identification number 5:

Using the primer pair of the first table, the obtained nucleic acid fragment (n=3) contains three sets of CpG modules, such as the fourth polynucleotide of the sequence of sequence identification number 6, wherein the 5' end Sequences to the 3' end may include restriction enzyme A cleavage, three sets of CpG modules, and restriction enzyme B cleavage. In an exemplary embodiment, a linker (eg, shown in Figure 1) can be included between the three sets of CpG modules of the aforementioned fourth polynucleotide (n=3) to ensure subsequent colonization. The sequence is correct.

In an exemplary embodiment, the restriction enzyme A cleavage at the 5' end of the fourth polynucleotide (n=3) may be, for example, BglII (the cleavage sequence is 5'-A↓GATCT-3', and ↓ indicates the enzyme cleavage point. ), as indicated by the italicized text in Table 1, GATCT . Next, the restriction enzyme B cleavage at the 3' end of the aforementioned nucleic acid fragment (n=3) may be, for example, BamHI (the cleavage sequence is 5'-G↓GATCC-3', and ↓ indicates the enzyme cleavage point), as in Table 1. The sequence GATCC indicated in italics . In addition, taking the pET-32a vector (about 5900 kb) as an example, a restriction enzyme C cleavage is also designed on the vector, for example, PstI (the cleavage sequence is 5'-CTGCA↓G-3', and ↓ indicates the enzyme cleavage point). In order to facilitate the subsequent integration of more sets of CpG modules. It should be noted that the restriction enzyme A cleavage site, the restriction enzyme B cleavage site, and the restriction end of the restriction enzyme C cleavage site are different depending on the vector to be ligated, and thus are not limited thereto. Next, the fourth polynucleotide (n=3) obtained above can be optionally cultured into any commercially available vector, as shown by recombinant plastid (n=3) P1.

In one embodiment of the invention, the DNA adjuvant can be a recombinant plastid containing six sets of CpG modules (n=6). Please refer to Figure 1A again. Restriction enzyme A and restriction enzyme C can be used to cleave recombinant plastid (n=3) P1 (for example, about 5885 bp) to obtain fragment N1 (about 4547 bp); and restriction enzyme B is cleaved with restriction enzyme C to obtain fragment N2. (about 1366 bp). The 5' end of the segment N1 has three sets of CpG modules (indicated by internal blanks), while the 3' end of the other segment N2 has three sets of CpG modules (indicated by filled gray).

Then, for example, a bonding reaction can be performed using a ligase, and three sets of CpG modules (shown in 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 (indicated by internal blanks). A recombinant plastid (n=6) P2 (about 5913 kb) having a head-to-tail connection and containing six sets of CpG modules was formed, as shown in Fig. 1A. In an exemplary embodiment, the immunostimulatory oligonucleotide comprising 6 sets of CpG modules is the first polynucleotide of the sequence as shown in SEQ ID NO: 1, and comprises 6 sets of CpG modules or recombinants containing the same Body (n=6) P2 can 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). Referring to Figure 1B, the same strategy as in Figure 1A can be used to join the fragment N3 and fragments by repeating the cleavage of a specific restriction enzyme by using the above-described recombinant protein (n=6) P2 containing six sets of CpG modules. N4 and nucleic acid selection techniques resulted in recombinant plastids (n=12) P3 (approximately 5969 kb) containing twelve CpG modules. In one example, an immunostimulatory oligonucleotide comprising 12 sets of CpG modules is a second polynucleotide of the sequence set forth in SEQ ID NO: 2.

In still another embodiment of the present invention, the DNA adjuvant may also be a set of four CpG-containing modules or a recombinant body containing the same (n=24). Please refer to Figure 1C. Refer to the same strategy as Figure 1A or Figure 1B. The above-mentioned obtained recombinant plastid (n=12) P3 containing 12 sets of CpG modules can be used to repeatedly cut specific restriction enzymes. The fragment N5 and the fragment N6 and the nucleic acid selection technique were used to obtain a recombinant plastid (n=24) P4 (about 6081 kb) containing four sets of CpG modules. In one example, an immunostimulatory oligonucleotide comprising 24 sets of CpG modules is a third polynucleotide of the sequence set forth in Sequence ID #3.

The above-mentioned recombinant plastids 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, such as Escherichia coli , are produced in large quantities, so that the cumbersome steps of conventional DNA adjuvants that must utilize chemical modification (e.g., phosphorus vulcanization) can be eliminated. 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.

Vaccine composition

The term "vaccine composition" as used herein mainly refers to a vaccine for a conventional animal and the above-mentioned DNA adjuvant, wherein a suitable DNA adjuvant comprises an immunostimulatory oligonucleotide comprising a plurality of sets of waterfowl-specific CpG modules. A recombinant plastid, a transformed strain, or any combination thereof, comprising the immunostimulatory oligonucleotide.

When the aforementioned vaccine composition is administered to at least one immunized animal (e.g., avian, bovine or porcine), peripheral immune cell growth 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.

It should be noted that the DNA adjuvant for the waterfowl and livestock vaccine of the present invention can not only reduce the amount and cost of the adjuvant, but also enhance the efficacy of the vaccine with insufficient antigen immunity, and the effect is far more than one set and two. The yield of one or three sets of CpG modules will increase the predictability of the effectiveness of waterfowl or other animal vaccines. 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.

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 adjuvant

1. Construct a recombinant plastid containing three sets of waterfowl-specific CpG modules

In this example, first, a polymerase chain reaction (PCR) was carried out 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.

Put 10 μL of the upstream primer and the downstream primer of the primer of the first table into a 200 μL microcentrifuge tube, place it in a temperature cycle controller (Thermocycler; TaKaRa, Shiga, Japan), and heat to about 55 °C. About 5 minutes. Then, the reaction was allowed to stand at room temperature for 5 hours to allow it to naturally bind to form a fourth polynucleotide (n = 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 modules (n=3; about 5885 bp). Thereafter, the plastids that have been joined are transformed into a host cell, such as a competent cell of E. coli DH5α, in a heat shock manner as a host for the transfer and preservation of the above recombinant plastid. Then, using antibiotic screening method, for example, the bacterial liquid is evenly applied to the 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 sequenced. A large number of cultures can be carried out by constructing the sequence of the bacteria that are not correct. 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. Reconstructed to pGEM-T easy mass (empty carrier; pGEM-T Easy Vector system Promega, WI, USA) to facilitate the subsequent preparation of recombinant plastids containing multiple sets of waterfowl-specific CpG modules (n=6-24).

In other words, the recombinant plasmid (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 then constructed into pGEM- for subsequent convenience. In the T Easy plastid, a prominent A(3'-A overhand) was added to the 3' end of the fourth polynucleotide by Taq polymerase (polymerase), and the reagents thereof are as shown in Table 2:

The above reagent was reacted in the above temperature cycle controller at 72 ° C for 25 minutes and then at 4 ° C for 10 minutes. Then, the 3' end is added with the protruding fourth nucleotide of A, and the pGEM-T easy plastid (empty vector; pGEM-T Easy Vector system ;Promega, WI, USA) DNA, directly performing the ligation reaction to form a recombinant plastid containing three sets of CpG modules (n=3; about 5885 bp), wherein the pGEM-T Easy empty vector does not require any restriction enzymes. Cutting.

Thereafter, the recombinant plastid can be further transformed into a suitable host, such as a competent cell of E. coli BL21 (DE3) strain, for transfer and preservation of the recombinant plastid. Host. After that, blue-white screening was performed in an LB culture dish containing 100 mg/mL Ampicillin, and 5-bromo-4-chloro-3-indolyl-bD-galactoside (5-bromo-4-chloro-3) was utilized. -indolyl-bD-galactopyranoside; X-gal) allows the colonies of successful transformation to develop color, and the PCR- and DNA sequencing determines that the sequence of the constructed bacteria is backward, and a large number of cultures can be performed.

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, and then 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 is formed without error (not shown). The types of the enzyme A, the restriction enzyme B, and the restriction enzyme C are as described above, and 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 Fig. 5. Please refer to FIG. 5, which is a diagram showing the 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 utilizes an upstream primer. Entrusted Mingxin Biotechnology Co., Ltd. (Taipei, Taiwan) to perform DNA sequencing using a nucleic acid sequence automatic sequencer.

As is apparent from the results of Fig. 5, the nucleic acid fragment containing a plurality of sets (n=3) of waterfowl-specific CpG modules according to an embodiment of the present invention was confirmed by DNA sequencing, and the above sequence was confirmed to be correct.

2. Construct a recombinant plastid containing multiple sets of waterfowl-specific CpG modules

This embodiment uses the above-described constructed recombinant body containing three sets of CpG modules (n=3; about 5885 kb, hereinafter abbreviated as P1), and repeats several times using the methods exemplified in FIGS. 1A to 1C. Recombination of 6 sets, 12 sets, and 24 sets of CpG modules (n=6, 12, 24; about 5913, 5969, 6081 kb) were obtained by cleavage of specific restriction enzymes, binding of specific sequences, and nucleic acid selection techniques. Body (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 Polynucleotide (n=6; about 56 bp), second polynucleotide (n=12; about 112 bp), and third polynucleotide (n=24; about 224 bp) were respectively embedded in the constructed plastid In the middle, the recombinant plastids containing 6, 12, and 24 sets of CpG modules (n=6, 12, 24) are correct (not shown), and the types of restriction enzyme A, restriction enzyme B, and restriction enzyme C are also As the previous example, here is not awkward.

Second, the above first polynucleotide (n=6), second polynucleotide (n=12), third polynucleotide (n=24), and fourth polynucleotide (n=3) After the fragments were confirmed by DNA sequencing, the results are shown in Figures 6 to 8.

Please refer to FIG. 6 to FIG. 8 , which respectively show DNAs of immunostimulatory oligonucleotides containing multiple sets of (n=6, 12, 24) waterfowl specific CpG modules according to an embodiment of the present invention. The sequence diagram, which uses the upstream primer to commission Mingxin Biotechnology Co., Ltd. (Taipei, Taiwan), performs DNA sequencing with a nucleic acid sequence automatic sequencer. From the results of Fig. 6 to Fig. 8, it is understood that the nucleic acid fragments containing multiple sets of waterfowl-specific CpG modules according to an embodiment of the present invention are confirmed by DNA sequencing, and the above sequences are confirmed to be correct.

Example 2: Evaluating 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 to extract peripheral blood mononuclear cells (PBMC) from healthy experimental animals (duck, cow, pig).

First, whole blood of healthy ducks, cattle, and pigs was taken, 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/mL) was added to a 96-well cell culture plate.

2. Evaluation of immunostimulatory activity of recombinant plastids containing multiple sets of waterfowl-specific CpG modules in duck spleen cells

2.1 Stimulating the original stimulus

In this example, peripheral blood mononuclear cells (PBMC) of experimental animals (duck, cow, pig) were divided into four groups of test groups and three groups of control groups to evaluate recombinant plastids containing multiple sets of waterfowl-specific CpG modules. As an irritating immune stimulating effect.

The experimental group was supplemented with 10 μg of recombinant plastids (P1, P2, P3, P4) containing multiple sets of waterfowl-specific CpG modules, empty vectors and two sets of CpG modules for synthetic phosphorous vulcanization. Hereinafter referred to as CpG ODN). The positive control group was supplemented with 2 μg of 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. Each of the above groups of samples had a significant difference (p < 0.05).

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. 2.

Please refer to FIG. 2, which is a diagram showing the results of a cellular immunoreactivity 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 empty vector test group, the 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 column represents the recombinant plastid (P2) test. The 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 (ConA). The statistical analysis of Fig. 2 was analyzed using SAS version 9.0, and the difference significance between the groups was compared by Tukey-type multiple comparison test with t-saturated residual. The average values of the figure numbers a, b, c, and d are a>b>c>d. The same figure number (eg, 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 groups. As for the figure number bc, it means that there is no significant difference between this value and b and c.

From the results of Fig. 2, it was found that the empty vector DNA could not effectively cause cell proliferation of duck 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 duck PBMC. However, compared with the recombinant plastid (P1) test group or the synthetic CpG ODN test group, the recombinant plastids (P2, P3, P4) have significant differences, representing the recombinant plastids of Example 1 (P2, P3, P4) Non-toxic effect on duck PBMC. In particular, recombinant plastid (P4) produced a good effect on cell proliferation of duck PBMC, but there was no significant difference from ConA in the positive control group. Therefore, the results of Fig. 2 show that the recombinant plastids (P2, P3, P4) of Example 1 are non-toxic to duck PBMC and have an effect of promoting the immunological activity of waterfowl animals, but this effect does not follow CpG. There is a corresponding difference in the number of modules.

3. Evaluation of the immunostimulatory activity of recombinant plastids containing multiple sets of waterfowl-specific CpG modules in bovine blood mononuclear cells

In this embodiment, the peripheral blood mononuclear cells (PBMC) of the cows were divided into four groups of test groups and three groups of control groups, and the recombinant plastids containing multiple sets of waterfowl-specific CpG modules were evaluated as immunostimulating effects of the stimulating original. . The stimulation of the original stimulus, the calculation of the MTT test and the SI, etc. are as described above, and there is no further ambiguity here.

Please refer to FIG. 3, which is a diagram 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 empty vector test group, the 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 column represents the recombinant plastid (P2) test. The 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 (ConA). The statistical analysis of Fig. 3 was analyzed using SAS version 9.0, and the difference significance between the groups was compared by Tukey-type multiple comparison test with t-saturated residual. The average values of the figure numbers a, b, c, and d are a>b>c>d>e. The same figure number (eg, 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 groups. As for the figure number bc, 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 (P1) test group and the synthetic CpG ODN test group on the cell proliferation of bovine PBMC. Recombinant plastids (P2, P3) were significantly different compared to the synthetic CpG ODN test group. Furthermore, the recombinant plastid (P4) had a significant difference compared to the recombinant plastid (P1) test group or the synthetic CpG ODN test group, but there was no statistically significant difference from the positive control 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 the CpG mode. There is a corresponding difference in the number of groups.

4. Evaluation of the immunostimulatory activity of recombinant plastids containing multiple sets of waterfowl-specific CpG modules in pig blood mononuclear cells

In this example, peripheral blood mononuclear cells (PBMC) of pigs were divided into four groups of test groups and three groups of control groups, and the recombinant plastids containing multiple sets of waterfowl-specific CpG modules were evaluated as immunostimulating effects of stimulating substances. . The stimulation of the original stimulus, the calculation of the MTT test and the SI are also discussed in the previous section and will not be repeated here.

Please refer to FIG. 4, which is a diagram showing the results of a cellular immunoreactivity test for immunizing a DNA adjuvant in a pig according to an embodiment of the present invention, wherein the horizontal axis represents antigen and the vertical axis represents cell proliferation index (SI), and a blank column is shown. The empty vector test group, the 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 column represents the recombinant plastid (P2) test. The 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 (ConA). The statistical analysis of Fig. 4 was analyzed using SAS version 9.0, and the difference significance between the groups was compared by Tukey-type multiple comparison test with t-saturated residual. The average values of the figure numbers a, b, c, and d are a>b>c>d. The same figure number (eg, 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 groups. As for the figure number bc, it means that there is no significant difference between this value and 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 CpG ODN test group, the recombinant plastids (P2, P3) had significant differences. Furthermore, compared with the recombinant plastid (P1, P2, P3) test group or the synthetic CpG ODN test group, the recombinant plastid (P4) had a significant difference, but it was statistically insignificant with the positive control group ConA. Sexual differences. 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.

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 does not exhibit a proportional relationship with the degree of immunostimulating activity against the vaccine. At the same time, there is no corresponding difference with the increase in the number of CpG modules.

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.

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 comprises a plurality of sets of waterfowl-specific immunostimulatory oligonucleotides or recombinant bodies containing the same. Moreover, it is not necessary to be subjected to phosphorus vulcanization, which 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 proliferation in the animal, but also reduces the use amount and cost of the conventional adjuvant. It can also improve 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.

<110> National Pingtung University of Science and Technology

<120> DNA adjuvant for waterfowl and livestock vaccines

<130> None

<160> 6

<210> 1

<211> 56

<212> DNA

<213> Artificial sequence

<400> 1

<210> 2

<211> 112

<212> DNA

<213> Artificial sequence

<400> 2

<210> 3

<211> 224

<212> DNA

<213> Artificial sequence

<400> 3

<210> 4

<211> 28

<212> DNA

<213> Artificial sequence

<400> 4

<210> 5

<211> 27

<212> DNA

<213> Artificial sequence

<400> 5

<210> 6

<211> 32

<212> DNA

<213> Artificial sequence

<400> 6

P1‧‧‧Recombinant plastids containing three sets of waterfowl-specific CpG modules

P2‧‧‧Recombinant plastids containing six sets of waterfowl-specific CpG modules

P3‧‧‧Recombinant plastids containing twelve sets of waterfowl-specific CpG modules

P4‧‧‧Recombinant plastids containing four sets of waterfowl-specific CpG modules

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

1A to 1C are partial flow charts showing a method of manufacturing a DNA adjuvant according to an embodiment of the present invention.

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 a duck.

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.

Fig. 4 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 pigs.

5 to 8 are DNA sequence diagrams showing immunostimulatory oligonucleotides containing multiple sets (n=6, 12, 24) of waterfowl-specific CpG modules according to an embodiment of the present invention.

P3. . . Recombinant plastids containing twelve sets of waterfowl-specific CpG modules

P4. . . Recombinant plastids of four sets of waterfowl-specific CpG modules

Claims (4)

A DNA adjuvant for waterfowl and livestock vaccine, characterized in that the DNA adjuvant is a recombinant plastid which is not subjected to phosphorus vulcanization treatment and is an aqueous poultry-specific immunostimulatory oligonucleotide, and the waterfowl immunostimulating oligo The nucleotide is selected from the group consisting of a first polynucleotide such as the sequence number shown in Sequence Identification No. 1, a second polynucleotide such as the sequence of Sequence Identification No. 2, and a sequence as shown in SEQ ID NO: 3 A group consisting of a third polynucleotide. 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 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 according to claim 1 of the patent application, 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 one immunized animal is a waterfowl, cow or pig. The vaccine composition according to claim 3, wherein the antigen is a recombinant protein, a live vaccine or an inactivated vaccine.