TWI378144B - Recombinant plasmic containing multicopy cpg motifs and transformant thereof for applying on dna adjuvant in avian vaccines - Google Patents

Description

1378144 • VI. Description of the invention: • [Technical field to which the invention pertains] The present invention relates to a vaccine adjuvant, and more particularly to an oligonucleotide containing a plurality of CpG modules for use in an animal vaccine. Agent. [Prior Art] At present, the main adjuvant used in animal vaccines is aluminum gel adjuvant and oily zoru-agent. However, the disadvantage is that both are chemical adjuvants, which cannot enhance the immune response of specific Th1 cells, especially Activated vaccines and live attenuated vaccines cannot produce adequate humoral and cellular immune responses.

In 1995, Krieg AM et al. first confirmed the inclusion of unmethylated (unmethyiate) Oxyribonucleic acid (DNA) has the functions of promoting the activation of various immune cells, inducing the production of various cytokines, and being easily absorbed by tissues. It was confirmed to be effective in φ in 1998, and it was confirmed to have a vaccine in 2003. The oligodeoxynucleotides (ODN) containing CpG modules can stimulate the activity of various large-scale livestock animals, including lymphocytes and antigens of cattle, pigs, sheep, etc., and increase dendritic cells (Dendritic Cell; The antigen of DC) exhibits activation and maturation, and promotes the immune system's reaction to specific antigens toward Thl cells. For example, the inventors of the present invention have proposed to use phosphorothioate-modified (PTO_modified) for pig specificity. Oligonucleotides containing CpG modules and plastids containing different sets of CpG modules for pigs 4 1378144 revised March 31, 2010 It has immunostimulatory activity. • Nucleotide containing CpG modules can pass through the following mechanisms: (1) increase • dendritic cell activation, maturation and antigen presentation; (2) dendritic fine • cell migration Increased effect; (3) significantly increased the expression of cell markers (eg, MHC-II, CD40, CD80, CD86, and IL-12) in mouse and human dendritic cells; (4) increased dendritic appearance of presensible treatment Cells (Priming DC) respond to Thl cells of specific antigens; (5) Increase the activity of CD8 + T cells' can induce cytotoxicity (cyt〇toxicity; CTL) of specific viruses or tumors. An oligonucleotide containing a CpG module produces a protective immune response against infection by viruses, bacteria, and extracellular parasites in an innate immune response', and activates B cells when combined with a vaccine. Producing antibodies, antigen-presenting cells (APCs) secrete cytokines, such as interferon-7 (interferon-γ; IFN-γ), to promote the immune response of vaccines, and thus contain oligonucleotides of CpG modules. Acid instead as an adjuvant. In general, the CpG-containing oligonucleotide modules mostly CpG module

• Unpurified CpG modules, while conventional techniques chemically synthesize phosphorothioated oligonucleotides containing CpG modules. However, synthetic oligonucleotides containing CpG modules must use chemically modified phosphodiester bonds between nucleic acids to replace phosphorus with sulfur (ie, phosphorothioated as described above) to reduce deoxyribonuclease ( Deoxyribonuclease; Dnase) Decomposes the rate of oligonucleotides containing CpG • modules', which is not only time consuming, incapable of mass production, but also expensive. • In addition, 'the oligonucleotides containing CpG modules are species-specific, and the structure of CpG modules with better immune-promoting effects between species is also poor. 5 1378144 foio year 3_month 31曰correct replacement page' different. It has been confirmed that the sequence of C:pC5 modules that have immunomodulatory effects on humans and mice is not the same, so the sequence of effective CpG modules for different species still needs to be further confirmed by experiments. The use of oligonucleotides containing CpG modules in the poultry industry began in 2002, but the chemical synthesis of oligonucleotides containing CpG modules was used for in vitro (ζ·„ν^0) evaluation, in vivo. Internal (/wWv〇) • The assessment is also limited to the humoral immunity against antibody production, and there is no in-depth discussion of the truly effective cellular immune response when the virus infects cells. In view of this, it is urgent to propose an animal immune irritant. (immunostimulatory) oligonucleoside acid (ODN), which provides a cpG DNA adjuvant suitable for animal vaccines and improves the cumbersome steps that conventional DNA adjuvants must utilize chemical modification. [Invention] One aspect of the invention is to provide a recombinant plastid containing a plurality of sets of CpG modules, which embed an immunostimulatory oligonucleotide comprising a plurality of sets of CpG modules into a vector to form a recombinant plastid, wherein the foregoing An immunostimulatory oligonucleotide, a recombinant plastid, or any combination of the above can be used as an adjuvant for avian vaccine, thus eliminating the cumbersome steps that conventional DNA adjuvants must utilize chemical modification. Another aspect of the present invention provides a transformed strain comprising a plurality of sets of CpG modules, wherein the recombinant plasmid is transformed into a host cell by using a plurality of sets of CpG modules, wherein These CpG modules are avian specific sequences. The above host cells can stably, rapidly and abundantly express the above-mentioned immunostimulatory oligonucleic acid, and the resulting immunostimulatory nucleus is acid, recombinant plastid, transformed strain, or Any combination of the above can be applied to an adjuvant for avian vaccines, thus eliminating the cumbersome steps of the prior art. March 31 DNA adjuvant must be chemically modified, and another aspect of the present invention is a right-handed product. Use immunostimulatory oligonuclear 2 for: =" group of recombinant acid, recombinant strain, or: lb immunostimulatory oligo ^Tianzhi #1, due to the aforementioned immune money transfer to nuclear pure, recombinant plastid When the transformant strain, or any combination of the above, has multiple sets of adjuvants for poultry vaccines, it can not only reduce, but also can be used for antigen immunization; and * and cost 'the above r' Set of cpG modules; Γ trection t: two The recombinant plastid of the CpG module can be embedded in the vector, and the immunostimulatory oligo(tetra) acid can be embedded in the carrier, wherein the immunostimulatory oligo surface can comprise multiple sets of CPG molds, and the immunostimulatory oligosaccharide Can be packaged with pure money in the order of the rhyme number 2 in the order - the first oligo-nucleus m as the phase identification (four) 3 sequence - the second oligo thief, such as the serial identification number 4 one of the sequence of the third oligo Any combination of the above-mentioned immunoglobulin oligo (IV) obtained from the recombinant plastid containing multiple sets of CpG modules, the recombinant plastid containing the immunostimulatory oligonucleotide, or any combination thereof, can be used as Adjuvant for poultry vaccine. According to the above aspect of the invention, a transformant strain comprising a plurality of sets of CpG modules and recombinant bodies is proposed. In one embodiment, the transformed strain of the recombinant plasmid comprising the plurality of sets of CpG modules can include, but is not limited to, a host cell, and a recombinant plasmid comprising a plurality of sets of CpG modules is transformed into the host cell. In one embodiment, the sputum primary cell can be a prokaryotic expression system, such as the large intestine rod 1378144, March 31, 2010, corrected replacement of the bacterium (where co/z·). The recombinant body comprising the plurality of sets of CpG modules may comprise a vector, and an immunostimulatory oligonucleotide is embedded in the vector, wherein the immunostimulatory oligonucleotide may comprise a plurality of CPG modules, and the immunization The stimulatory oligonucleotide may include, but is not limited to, a first oligonucleotide such as one of the sequences shown in SEQ ID NO: 2, a second oligonucleotide such as the sequence of SEQ ID NO: 3, such as SEQ ID NO: 4 a third oligonucleotide of the sequence shown or any combination thereof, wherein the host cell exhibits an immunostimulatory oligonucleotide comprising a plurality of sets of recombinant plasmids of a CpG module, comprising the immunostimulatory oligonucleoside The acid recombinant plastid, the transformed strain, or any combination of the above is used as an adjuvant for poultry vaccines. In the preferred embodiment of the invention, the above-mentioned CpG modules are avian. According to another aspect of the present invention, a composition of a poultry vaccine is provided, and the composition of the poultry vaccine may include - poultry An immunostimulatory oligo (IV), a recombinant plastid containing the immunostimulation, a transformed strain, or any combination of the above: an adjuvant: wherein the immunostimulatory oligonucleotide may comprise a plurality of sets of two: However, it is not limited to any combination of the second oligo thief or the domain such as sequence identification = second _ acid, sequence identification. ^ Sequence ~ In the chicken hair C example, the above-mentioned poultry vaccine may include #不质=, avian influenza inactivated vaccine, poultry respiratory c live pan-field, or knotted city disease, material u 1378144 March 31 Corrected a replacement trivalent unactivated oily vaccine for bronchitis. The transformant strain of the recombinant plastid containing the plurality of sets of CpG modules of the present invention is inserted into the plastids containing the immunostimulatory oligonucleotides containing multiple sets of CpG modules, wherein the CpG modules are avian specific Sexual sequence. After the resulting recombinant plasmid is transformed into a host cell, the immunostimulatory oligonucleotide having multiple sets of CpG modules can be stably, rapidly and abundantly expressed. When the immunostimulatory oligonucleotide, the recombinant plastid containing the immunostimulatory oligonucleotide, the transformed strain, or any combination thereof is used as an adjuvant for poultry or other animal vaccines, Multiple sets of CpG modules not only reduce the amount and cost of adjuvants, but also improve the efficacy of vaccines with insufficient antigenic immunity, and eliminate the cumbersome steps that conventional DNA adjuvants must utilize chemical modification. [Embodiment] The present invention provides a recombinant plasmid containing multiple sets of CpG modules and a transformed strain containing the recombinant plasmid, which utilizes a recombinant plastid transformation containing multiple sets of CpG modules. (transf〇rm) to a host cell's immunostimulatory oligonucleotide with multiple sets of CpG modules in a stable, rapid and abundant manner. The above-mentioned recombinant system containing multiple sets of CpG modules refers to inserting an immunostimulatory oligonucleotide into a vector (vect〇r), wherein the immunostimulatory oligonucleotide may comprise multiple sets. The CpG module and its related functional sequences are described below for simplicity. The CpG modules include CpG modules and their associated functional sequences. The term "multiple sets of CpG modules" as used herein refers to a plurality of sets of CpG modules that are connected end to end and their associated functional sequences. Take a single set of CpG modules and 1378144 March 31, 2010 revised replacement page's related functional sequence as an example, the 5' end to the 3' end may include 5, the end - a plurality of TCG repeats, a single CpG module, adjacent avian specific sequence on both sides of the CpG module, and 3, 4 to 6 guanine sequences, wherein a plurality of TCG repeats at the -5' end can promote cellular immunity, cp ( } The module itself is an immune-promoting sequence for poultry, avian-specific sequence

There is a specificity, and 3, 4 to 6 guanine sequences can form a polyG structure to increase the uptake of cells by this CpG DNA adjuvant. However, by using the immunostimulatory oligonucleotide containing multiple sets of CpG modules designed by the present invention, the recombinant plastid obtained can be stably, rapidly and abundantly expressed in the host cell, and the obtained immunostimulatory oligonucleotide is also obtained. Because it contains multiple sets of cp (3 modules, it can reduce the dosage and cost of adjuvant, and can improve the efficacy of vaccines with insufficient antigen immunity, the effect far exceeds the single set of CpG modules and their related functional sequences. The extent to which production 4 and its efficacy in boosting poultry or other animal vaccines can be predicted. _ In the examples, recombinant plastids containing a single set of CpG modules can be used to replicate several specific restriction enzymes by repeating Recombinant plastids of the above-mentioned immunostimulatory oligo-ceuic acid obtained by binding a specific sequence to the nucleus and nuclear cytogenetic selection technique, and the plurality of sets of CpG modules described in the middle are referred to as multiple sets of c-modes sequentially connected. Groups and their associated functional sequences. In detail, please refer to FIG. 1A to FIG. 1C for a partial flow chart of a method for manufacturing a set of CpG modules according to the present invention. In Figure 1A, first provide 3 sets of CpG modules. Recombinant plastid (n = 1) 1〇〇, where a single set • CpOHM is the sequence shown in Sequence Identification No. 1 of the first table: Table 1 1378144 March 31, 2010 Revision Replacement Page No. 5, - CTAGTiyC^C ^AAlGTCGTTlTT.SOGGOOiT -3' ' _Spel fCG Repeat ~~~~| * CpG Module* [ poly G Ixbal * : Avian Specific Sequence • · • In the first table 'This single CpG module and its related functions Sexual sequence, from 5' to 3', may include restriction enzyme A cleavage (eg CTAGT shown in bold at the 5' end), and multiple TCG repeats at the 5' end (eg gray text φ word) Shown), a single CPG module (as indicated by the boxed text), avian specific sequence on both sides of the adjacent CpG module (eg shown in Figure *), 3, 4 to 6 guanine sequences (eg P〇ly G) as shown in the gray text, and restriction enzyme B cleavage (for example, T shown in bold text at the 3' end) 'The restriction enzyme a cleavage at the 5' end and the restriction enzyme B cleavage at the 3' end The sequences are different, but the sequence with the same binding end's binding cannot be cleaved with restriction enzyme A and restriction enzyme B. For example, the 3' end of DNA fragment A is sticky. The end of the DNA fragment B can be joined to the 5' end of the DNA fragment B (iigati〇n) to form a sequence of head-to-tail phase φ. It should be noted that the present invention is intended to produce a plurality of CpG modules. Recombinant plastids, and the resulting recombinant plastids are transformed into host cells, such as E. coli, for mass production. Therefore, restriction enzyme A cleavage and 3 are designed respectively at the 5' and 3' ends of immunostimulatory oligonucleotides. The restriction enzyme B is restricted, and the restriction enzyme C cleavage is designed on the vector. In one embodiment of the present invention, the immunostimulatory oligonucleotide 5, the restriction of the end of the enzyme A can be, for example, e/( The cleavage sequence is 5, -A丨CTAGT-3,, 丨 indicates the enzyme cleavage point), as indicated by the bold text in the first table, CTAGT; in addition, the restriction enzyme B cleavage at the 3' end can be, for example ( The cut position 1378144 is 5'-Τ I CTAGA-3', where j is the enzyme cut point), as indicated by the bold text in the i-th table. In addition, there are other restrictions on the vector c • cleavage, such as heart c / (cut sequence is 5, _GAGGCT | C-3, j represents • enzyme cut point). As the above-mentioned applicable vector, any commercially available vector such as pBluescript® vector, pBiueseriptKS vector or the like can be used, and the above restriction enzyme A cleavage, restriction enzyme B cleavage, and restriction enzyme C cleavage are contiguous. The carrier varies, and is not limited to the ones presented herein. Please refer to Figure 1A again. Next, restriction enzyme A and restriction enzyme c can be used to cleave the recombinant plastid containing a single set of CpG modules to obtain fragment N1, and restriction enzyme B and restriction enzyme C are used to cleave the recombinant containing a single set of Cp G modules. quality

The segment N2 is obtained by the body 100, wherein the 5th end of the segment N1 has a single set of CpG modules and related functional sequences (indicated by internal blanks), and the 3' end of the segment N2 has a single set of CpG modules and related functions. Sexual sequence (indicated by filling a slash). Then, for example, a bonding reaction can be performed using a ligase, and a single CpG module (shown by filling a diagonal line) at the 3' end of the segment N2 is bonded to a single CpG module at the 5' end of the segment N1 engine (indicated by an internal space) ), formed with a head

The tail is connected to a recombinant body (η=2)1ι〇 containing two sets of CpG modules, as shown in Figure 1A. Similarly, the recombinant plastids containing the two sets of CpG modules can be used to obtain a recombinant plastid containing four sets of CpG modules by repeating the cleavage of specific restriction enzymes, ligating specific sequences, and nucleic acid selection techniques. • 120. Please refer to Figure 1B. Firstly, the recombinant substance (n=2)ll〇 containing two sets of CpG modules is provided. Next, the restriction enzyme a and the restriction enzyme c are also used to cleave the recombinant plastid 110 containing the two sets of CpG modules to obtain the fragment N3, and the M and the tanning enzyme C are cleaved by the m 12 and the enzyme C to cut the CpG module. Recombinant plastid 110 White = 'The 5' end of its + fragment N3 has a dual set of CPG modules ("The 3' end of the fill line has a dual set (10) module (for example, ligase can be used to join the above Ν3 Γ) Two = 3' end of the dual CPG module (to fill, =: into 4: - as the = = four sets of cpG module reorganization called group: after several times, 'available with 16 sets (10) The weight of the module is 3_0, + shows) or the recombinant body containing 32 sets of coffee

11 , I will not repeat them here. In an example, 16 sets of CpG 3 nuclei are included: the excito 2=2 acid is a sequence as shown in Sequence Identification No. 2 ===, acid. (n=32) After l5〇, the same 3 for the recombinant body set CPG module containing 32 sets of CpG modules, ^== enzyme A and rhyme C cut contain 32 enzyme B spot to limit the plastid 150 Obtain the fragment N11, and use the restriction to obtain the fragment ν12,:;=2^ CpG module of the recombinant plastid 15〇 and the internal teaching white ▲ - 5中 5' end has 32 sets of CpG modules (to fill the slash table 2 The 3rd end of the fragment has 32 sets of CPG modules (for example, conjugated enzyme can be used, for example, the above-mentioned fragment Nil and fragment N12' can be used to make 32 sets of CpG modules of the 3' end of the piece N12 (to fill the slash) Indicates that the 5 sets of CpG modules bonded to the 5th end of the segment Nil (indicated by the internal blank 1378144, March 31, 2010 revised replacement page) form a head-to-tail connection with 64 sets of cpG modules. The recombinant plastid (pHCLS-64) (n:=64) 160, as shown in Figure 1C. In one case, the immunostimulatory oligonucleotide containing 64 sets of CpG modules is as in Sequence Identification No. 4 One of the oligonucleotides shown above may be further transformed into a primary cell such as Escherichia coli (five (3)") for mass production, as When the conventional DNA adjuvant can be dispensed with, it is necessary to utilize the complicated steps of chemical modification. Since the transformation of the bacterial body and the large-scale cultivation and the like are carried out by using conventional techniques, it is well known to any person having ordinary knowledge in the technical field to which the present invention pertains. Therefore, it will not be described one by one. The immunostimulatory oligonucleotide containing multiple sets of CpG modules, the recombinant plastid containing the immunostimulatory oligonucleotide, the transformed strain, and the like, are produced and purified in large quantities. Or any combination of the above, which may further be used as an adjuvant for poultry or other animal vaccines, and the composition of the vaccine for poultry or other animal use thus obtained is separately associated with peripheral blood mononuclear cells and spleen cells of the animal. In vitro culture and in vivo immunization experiments confirmed that the immunostimulatory nutrient phytic acid obtained by the present invention can promote interferon (interfer〇n_T; IFN~r) and steroid-like receptor 3 (toll-like Gene expression of receptor 3; TLR3) and TLR7. The following examples are used to illustrate the application of the present invention, but are not intended to limit the present invention, and those having ordinary knowledge in the technical field of the present invention, Various modifications and refinements can be made without departing from the spirit and scope of the invention. Embodiment 1: Construction of recombinant plastids containing a single set of CpG modules In this embodiment, first, with restriction enzyme A and restriction Enzyme B cleavage 1378144 Modified on March 31, 2010, the replacement page pGEM-T easy plastid (empty vector), after cleavage, the purified plastid DNA is ligated with the nucleic acid sequence as shown in SEQ ID NO: 1, and A recombinant plastid containing a single set of CpG modules is formed. 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//) DH5 α, as the recombinant cells are transferred and preserved. Host. After that, blue-white screening was performed, that is, using isopropyl-wan-D-thiosemicarbazone (iS0pr0pyi_beta-D-

Thiogalactopyranoside; IPTG) induces the lactose operator of the recombinant plastid (/ac operon) 'and utilizes 5_bromo-4_qi_3-吲哚_b_D_galactoside (5-bromo-4-chloro-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 sequencing to determine the sequence of the bacteria that are not correct. However, the above-mentioned construction of recombinant plastids, transfer of host cells, blue-and-white screening, mass culture, and extraction of plastid DNA are described. The sputum domain + any one with common knowledge is well known, so the plastid DNA extracted from the successful host cells is not separately exemplified by _electrophoresis 2 =, 6 and restriction enzyme C cleavage, functional The sequence is embedded in the constitutive hua body, and the CPG0 simple module and its related recombination f are formed, and a single set of eP is formed, which is exemplified as described above. The types of restriction enzyme 3 and restriction enzyme c 0青 Refer to FIG. 2A and 篦2B H1 for a single set of CPG molds, which are according to an embodiment C of the present invention (Fig. 2A), respectively, by restriction enzyme A and restriction _ Lipid electrophoresis analyst limit = Fig.) The cut ocean and 遒 indicate the DNA ladder mark (DN/ 15 1378144 March 3, 2010 factory ladder), and the “1.5 kb” and “2.0 kb” on the left side of the second channel indicate The 1500 base pair size and the DNA ladder mark indicating the size of 2000 base pairs. At the first lane of Figure 1A, the DNA fragment containing the recombinant plastid containing a single set of CpG modules was cleaved by restriction enzyme A and restriction enzyme C. (approximately 1.1 kb, as indicated by arrow 201), the second lane of Figure 1B indicates that the recombinant enzyme containing a single set of CpG modules is cleaved by restriction enzyme B and restriction domain c DNA fragment (about 1.8 kb, as indicated by arrow 203). It can be seen from Fig. 2A and Fig. 2B that a restriction fragment A can be used to cleave a recombinant plastid containing a single set of CpG modules to obtain a 1.1 kb fragment (corresponding to fragment N1 of Fig. 1A), and the restriction is utilized. Enzyme B and restriction enzyme c cleave a recombinant plastid containing a single set of CpG modules to obtain a 1.8 kb fragment (handle is fragment N2 of Figure 1A), wherein the 1.5 kb fragment has a single set of CpG modules at the 5' end. Its related functional sequence, while the 3' end of the 1.8 kb fragment has a single set of CpG modules and their associated functional sequences. Example 2: Construction of recombinant plastids containing multiple sets of CpG modules This example uses a recombinant plastid containing a single set of CpG modules constructed in the first embodiment to perform a specific restriction enzyme cleavage and joint specificity by repeating several times. The sequence and the nucleic acid selection technique respectively obtained recombinant bodies containing 2, 4, 8, 16, 32, and 64 sets of CpG modules. The process is as described above and illustrated in Figures 1A through ic, and is not awkward here. Please refer to FIG. 3 , which is a diagram of agarose agar electrophoresis analysis of recombinant plastids containing 4, 8, 16, 32, and 64 sets of CpG modules by restriction enzyme A and restriction enzyme restriction enzyme B according to an embodiment of the present invention. In the figure, the μth channel represents the ladder mark of the DNA, and the “3〇kb” and “〇5” on the left side of the centromere are divided into 1378144 and 2012. The screening indicates the size of 3000 base pairs and the representation of 1500 base pairs. The size of the dna ladder mark, the first lane indicates that the restriction enzyme A and the restriction enzyme b cleave the DNA fragment containing 4 sets of CpG modules and their related functional sequences (as indicated by arrow 301), and the second lane indicates Restriction enzyme a and restriction enzyme B cleave a DNA fragment containing 8 sets of CpG modules and their related functional sequences (respective bp, as indicated by arrow 303), and lane 3 indicates that 16 is cleaved by restriction enzyme A and restriction enzyme b A set of CpG modules and related functional sequences of DNA fragments (approx. 8 bp, as indicated by arrow 305). Lane 4 indicates that 32 sets of CpG modules and related functions are cleaved by restriction enzyme A and restriction enzyme B. The DNA sequence of the sequence (about 896 bp, as indicated by arrow 307), the fifth channel shows that 64 sets of CpG modules are cleaved by restriction enzyme A and restriction enzyme B. The DNA fragment of the relevant functional sequence (about i.8 kb, as indicated by the arrow 3〇9), the above fragment was confirmed by the sequence, and the results are shown in Fig. 13 and Fig. 14. Figure 13 and Figure 14 are diagrams showing DNA sequencing of immunostimulatory oligonucleotides containing 64 sets of CpG modules according to the present invention, wherein Figures 13 and 14 respectively utilize upstream The primer (Fig. 13) and the downstream primer (Fig. 14) perform DNA sequencing, while the numbers 1 to 0 represent 1 to 64 sets of CpG modules, respectively. As can be seen from the results of the πth and 14th, the present invention In one embodiment, the DNA fragments containing 64 sets of CpG modules and their related functional b-sequences are confirmed by DNA sequencing. The second embodiment of the recombinant plastids of the CpG module is in vitro (~v //? Evaluation of immune-promoting activity 1. Evaluation of immunostimulatory activity of recombinant plastids containing multiple sets of CpG modules in chicken spleen cells 1378144 March 31, 2010 Revision replacement page. In this example, 'First 'Using aseptic technique to take chicken gaZ/ws' spleen, make spleen cell suspension, adjust with RPMI 1640 complete medium The cell concentration was about 1×1〇7 cells/mL, and the cells were cultured in a 24-well cell culture dish with a cell volume of about 0.5 mL per well (about 5×106 cells). Next, Example 1 and implementation were respectively carried out. In the second example, the recombinant plastids (10 pg/50 μΙ〇 and empty vector (control group) containing 1, 2, 4, 8, 16, 32, and 64 sets of CpG modules were added to the chicken spleen cell fluid ( In lxl07cells/mL), it was placed in an incubator with 5% CO 2 at 41 °C. After culturing for about 6 hours of Φ, the cells were centrifuged at 1000 rpm for 10 minutes with the culture solution, and the resulting cell pellet was extracted with total RNA using Trizol® reagent, and combined into a cDNA using a reverse transcription set. Real-time PCR was used to detect the gene expression of IFN-γ, TLR 3 and TLR 7 in cells, and for example, actin-actin (dot-actin) was used as a housekeeping gene ( The house keeping gene) was used to evaluate the immunostimulatory activity of recombinant plastids containing 64 CpG modules. The conditions for the above-mentioned instant quantitative polymerase chain reaction are based on the method of the product • Lightcycle-faststart DNA master SYBR green I (Roche Applied Science, Germany), which contains Lightcycle-faststart enzyme, Lightcycle-faststart enzyme reaction mix , 25 mM MgCl2. Briefly, first, 5.8 pL of PCR-Grade Water, 1.2 pL of 25 mM MgCl2, 0.5 pL of primer pair, 1 pL of SYBR green I, 1 pL of cDNA were sequentially added and placed in Roche Molecular Biochemicals Lightcycle. The reaction was carried out in Software (Roche Applied Science, Germany), and the reaction was completed. After 'analysis of the gene expression levels of IFN-γ, TLR 3 and TLR 7, the results are as shown in 18 1378144 March 31, 2010. Replacement page. Figure to Figure 6 shows. Please refer to FIG. 4 to FIG. 6 , which respectively illustrate recombinant plastids containing single or multiple sets of CpG modules and empty carriers in vitro (/« Wiro ) stimulation according to the first embodiment and the second embodiment of the present invention. Histograms of IFN-γ, TLR 3 and TLR 7 genes in chicken spleen cells, in which the horizontal axis of Figures 4 to 6 indicates 1, 2, 4, 8, 16, 32, 64 sets of CpG modules The recombinant plastids (pHCLS-64 refers to recombinant plastids containing 64 sets of CpG modules) or empty vectors, and the vertical axes of Figures 4 to 6 represent the IFN-Y, TLR3 and TLR7 genes. Mean (Log2 (2_"et), each set of samples has at least three replicates. Here is the geometric mean of the IFN-γ, TLR 3 and TLR7 gene expressions in Figures 4 to 6 (2· -Δ ACt in ΔΔα) represents the fluorescein expression of IFN-γ, TLR3 and TLR7 genes in spleen cells stimulated by recombinant plastid or empty vector containing single or multiple sets of CpG modules of Example 1 and Example 2. Intensity change (△ (: 〇, minus the fluorescein intensity of IFN-γ, TLR 3 and TLR 7 genes of spleen cells stimulated by the recombinant plastid or empty vector of Example 1) (ACt), and the figure "*" indicates the degree of stimulation of IFN-γ, TLR 3 and TLR 7 genes by recombinant plastids containing multiple sets of CpG modules. As can be seen from the results of Figure 4, In the second example, the recombinant plastid-stimulated spleen cells containing 32 and 64 sets of CpG modules showed obvious IFN-γ gene, including 32 sets of CpG modules (ie 32x CpG) or 64 sets of CpG modules. The stimulating effect of the recombinant plastid (ie pHCLS-64) was significantly higher - in the control group of the phosphate buffer (Control) and in the other 16 sets or less than 16 sets of CpG modules. From the results of Fig. 5 to Fig. 6, it can be seen that the recombinant plastid-stimulated spleen cells of Example 丄 378144, 010, 3, 8, 16, 32, and 64 sets of CpG modules, • TLR 3, TLR 7 Genes have a more pronounced performance, in which the effect of stimulating with a recombinant plastid containing 64 CpG modules (ie pHCLS-64) is significantly higher than using recombinant plastids containing 8, 16, 32 CpG modules (ie 8X CpG, 16x CpG, 32x CpG) stimulating effect.

Furthermore, it can be seen from the results of the in vitro experiment of Example 3 that the recombinant plastids containing 8, 10, 32, and 64 sets of CpG modules have immunostimulatory activity, but the CpG module contained in the immunostimulatory oligonucleotide. The number should be at least higher than the 16-set CpG module', while the 32-set and 64-set CpG modules significantly increase the expression of IFN-γ, TLR3 and TLR7 genes, thus immunostimulatory oligonucleotides The number of C p G modules involved and the degree of their immunostimulating activity are not proportional to each other, so it is not possible to reasonably predict the resulting immunostimulatory oligonucleotides by the number of CpG modules. The extent of immune activity. Example 4: Preparation of a poultry vaccine composition containing multiple sets of CpG modules. 1. Preparation of chicken Newcastle disease virus antigen and vaccine First, 'Ishii strain of chicken Xincheng disease virus (Gaosheng Pharmaceutical Co., Ltd.) Company; original source: The Agricultural College of the Executive Yuan, Freshwater Animal Health Laboratory) was inoculated into BHK strained cells (BCRC No.: 60041; ATCC No.: CCL-10) at 37 °C containing 5〇/0 c〇2 The virus solution was harvested after 72 hours of proliferation in the incubator. The resulting virus solution was inactivated at a final concentration of 0.2% formalin (Sigma, USA). Then, the de-active virus solution was mixed with the gelatin adjuvant to prepare a Newcastle disease tissue culture vaccine (1378144^10), and the Newcastle Disease Tissue Culture (NDTC) was taken. The recombinant plastid containing 64 sets of CpG modules was added as a control vaccine. 2. Preparation of H5N2 Avian Influenza Virus Antigen and Vaccine First, the H5N2 Avian Influenza Virus (A/Duck/Taiwan/04, No. 3233, provided by the Laboratory of Professor Wang Jinhe, Department of Veterinary Medicine, National Taiwan University; Original Source: Executive Yuan Freshwater Animal Health Laboratory) was inoculated into a chicken skin urethral cavity of 9-11 days old specific pathogen-free (SPF), and virus solution was harvested after incubating for 72 hours in a 37 °C incubator. The resulting disease ® venom was deactivated at a final concentration of 22% of formalin (Sigma, USA). Then, the deactivated virus solution was mixed with an aluminum gel adjuvant to prepare an inactivated vaccine, and the vaccine was added to the recombinant plastid (pHCLS-64) containing 64 sets of CpG modules in the second embodiment to prepare a control vaccine. 3. Nobilis trivalent vaccine will treat Newcastle disease (ND), infectious bursal disease (IBD) or Gumboro disease (G) and infectious bronchitis (IB) trivalent vaccine, for example A trivalent unactivated oil vaccine produced by Lu Intervet/Schering-Plough Animal Health under the trade name Nobilis IB+G + ND (the strains are IB strain M41, Gumboro strain D78 and ND, respectively) Strain Clone 30). 4. Poultry respiratory mycoplasma vaccine poultry respiratory mycoplasmosis; MG) vaccine, for example, can be produced by Merial Animal Health (Ginesville, GA) poultry respiratory mycoplasma inactivated oily disease m * seeded (vaccine oil vaccine) (the source of the virus strain is S6 strain). 21 1378144 Revised replacement on March 31, 2010 |~| Example 5: Recombinant plastids containing 64 sets of CpG modules (pHCLS-64) Evaluation of immunostimulatory activity in vivo (1) 1. i Evaluation of the Ball Agglutination Inhibition Antibody Valence Test This example divides the experimental chickens into 4 groups of test groups and 1 group of control groups. Among them, the mother and the mother are each 5 week old specific-pathogen-free (SPF) chicken. Only 5, each group was given i dose, respectively the first group (negative pair

Group] muscle> ph〇Sphate buffer saline (PBS) 1 mL, the second group (experimental group) intramuscular injection 丨mL Example 4 chicken Newcastle disease tissue culture vaccine (vaccine), the first Groups 3 to 5 (experimental group) were intramuscularly injected with the Newcastle disease tissue culture vaccine (vaccine + vector) containing the empty vector of Example 1, and the recombinant plastid containing the single set of CpG modules of Example 1. Chicken Newcastle disease tissue culture vaccine (vaccine + containing a single set of plastids) and the weight of the 64 # CpG 1 containing Example 2

Chicken pH Newcastle disease tissue culture vaccine (pHCLS-64) (recombinant plastid (pHCLS-64) of vaccine + containing set of modules) & ! mL. All the test chickens were collected before immunization, 2 weeks after immunization and 3 weeks after immunization, and the blood contained in the blood contained the hemagglutination inhibitory antibody against chicken Newcastle disease virus (Hemagglutinin inhibiti〇n amib〇dy version s; 冚-; L〇g2), the results are shown in the figure. In this example, a hemagglutination inhibition antibody test was used, and the method of haemagglutination inhibition her body test; ΗΙ(4) was used to exemplify a v-type microplate, and each well was added with 0.85 % of physiological food ^^= For example, in the 8 wells of the first column, the pores are sequentially piped (pipet_) from the first (four) of each row, 25 hearts are diluted 2 times to the nth hole by quantitative drip, 22 1378144, the 12th hole is used as a control Then, in each well containing the serum sample, 4 blood unit (HAunits) of the virus antigen solution was added. After the chamber was placed for a few minutes, the body was added with 25 μg of 1% (V/V). Chicken red: The ball suspension is allowed to stand at room temperature for 3G minutes, and the blood cell agglutination of each sample is observed. = If the reciprocal of the highest dilution factor of red blood cell agglutination can be completely inhibited, it is the blood coagulation inhibition antibody strength. , the system (4) according to the present invention - the embodiment contains the =:= map, wherein the horizontal axis represents different immune vaccination vaccines = species of dystrophic new city disease tissue culture (four) lie carrier" represents the co-inoculation example - the carrier of the work Experimental group of cytopathic tissue culture vaccine, "vaccine + single set

The recombinant f body of the CpG module represents the experimental group of the chicken Newcastle disease tissue culture vaccine containing the recombinant plastid of a single set of Cp=modules, and the vaccine +PHCLS-64 represents the common inoculation example 2 The experimental group of 64 Newcastle disease groups containing 64 sets of P 4, ', heavy, and plastid (pHCLS64). · _ indicates that there is a titer of the titer of the Newcastle disease virus (hi titers ' Log 2) 2 weeks after immunization and 3 weeks after immunization (p<〇〇5). The figure "wood" indicates that the weight of the set of 64 sets, and the plastid (PHCLS-64) produces hemagglutination inhibition antibody. From the results of Figure 7A, the negative control group (PBS) did not detect the antibody price. . After 2 weeks of immunization (shown as gray bars) and 3 weeks after immunization (shown as black bars), co-inoculation of the tissue-culture of chicken Newcastle disease containing a recombinant set of CpG modules The experimental group of the vaccine (ie, the epidemic 23 1378144 ^〇ϊ〇~#Γ is replacing the seedlings + the recombinant plastid containing the CpG module), and the experimental group of the chicken new field disease tissue culture vaccine which is co-inoculated with the empty vector (The vaccine and the experimental group that only vaccinated (4) d cultivating the vaccine (ie, the antibody price of the three people of the vaccination did not differ significantly. However, the co-inoculation implementation of the chicken-new city containing 64 sets of CpG modules The experimental group of the disease tissue culture vaccine (ie vaccine + pHCLS_64) 'the test chickens in the household; the specific antibody strength produced, significantly higher than the above three experimental groups (second group to the fourth group).

2. Evaluation of stimulation index of cell proliferation As mentioned above, the immune status of all test chickens is as described in point 5 of Example 5, and 3 weeks after immunization, peripheral blood mononuclear cells (peripheral blood mononuclear cells) ; PBMC) Perform a cell lean test 'Assess the immune activity of the vaccine to promote cell proliferation, and the results are shown in Figure 7B. The method for using the cell proliferation assay (Cell Pr〇liferati〇n) in this example is exemplified as follows. First, take the chicken blood, for example

Histopaque® 1077 density gradient medium

The PBMC cells were isolated (the cell source was the same as that of the PBMC cells obtained in the first point of Example 5). After counting the cells, the cell concentration was adjusted to 2 X 1 〇 5 cells/100 pL/well and dispensed into 96-well plates. Next, add 1 gg/50pL of Concanavalin A (Con A) and the same volume of the culture solution to each well, and repeat them in 5% CO 2 and 37 ° C incubator for 48 hours. The cells were labeled with 10 pL/well BrdU reagent and cultured for another 24 hours. Thereafter, the mixture was centrifuged at 1000 rpm for 10 minutes, and after removing the supernatant, 200 pL /well FixDenat solution was placed at room temperature for 30 minutes. After removing the FixDenat solution, add 100 pL /well m 24 1378144 Modified the replacement page anti-BrdU-pod working solution on March 31, 2010, and leave it at room temperature for 90 minutes. Then, the anti-BrdU-pod working solution was removed, and the washing was repeated three times by adding 300 pL /well wash solution'. After washing s〇luti〇n, 100 pL/well of substrate solution was added and allowed to stand at room temperature for 5 minutes, and then the value was read at 370 nm using an ELISA reader. Please refer to FIG. 7B, which is a histogram showing the in vivo (9) wvo) i cell proliferation stimulation index (SI) of a recombinant plastid (pHCLS_64) containing 64 sets of CPG modules according to an embodiment of the present invention. Wherein the horizontal axis indicates different immunization methods, for example, pBS represents a negative control group, "vaccine" represents an experimental group inoculated only with a Newcastle disease tissue culture vaccine, and "vaccine + vector" represents a Newcastle disease in which the empty vector of Example 1 is co-inoculated. The experimental group of the tissue culture vaccine, "vaccine + recombinant plastid containing a single set of CpG modules" represents an experimental group of the chicken Newcastle disease tissue culture vaccine containing the recombinant plastid of the single set of CpG modules of Example 1. "Vaccine + pHCLS_64" represents an experimental group of chicken Newcastle disease tissue culture vaccine containing the recombinant plastid (pHCLS-64) containing 64 sets of CpG modules of Example 2. The vertical axis indicates that the PBMC proliferation index (SI) of the vaccinated animals was detected 3 weeks after immunization, and the number of stimulating ticks was not shown by the empty vector, including a single set or 64 sets of CpG molds, and the weight and quality The Brdu fluorescence value measured by pbmc of the body (pHCLS-64) was compared with the bBMc measured by pBMc obtained by the PBS control group, and the relative value (4). Figure No. "*" indicates the use of recombinant plastids containing 64 modules (pHCLS_64) to produce a cell proliferation stimulation index 25 1378144

曰Revise the recombinant plastids of the CPG module of the Newcastle disease tissue culture vaccine that replaces the recombinant plastid of page I), and the experimental group that co-inoculates the W disease (four) core (ie, the experimental group only (P epidemic S), although the cell proliferation index between the three is slightly different, the degree is not large. However, the chicken of 64 ΐ ϋ (PHCLS-64) in the second example is co-inoculated. The new domain disease tissue culture epidemic H seedling + PHCLS_64) was significantly higher than the above three experimental groups (second group to fourth group) for the proliferation of peripheral blood mononuclear cells. It is thus apparent that the recombinant plastid (pHCLS_64) containing 64 sets of CpG modules is a slant of the chicken Newcastle disease tissue culture vaccine, and can effectively promote the immune promoting activity in vivo. Example 6 · Recombinant plastid containing 64 sets of CpG modules (pHCLS_64) 免疫 In vivo evaluation of the immune-promoting activity in vivo (ί> ι WVi?) (1) 1. Evaluation of the blood cell agglutination inhibition antibody valence test The experimental chickens were divided into four groups: the experimental group and the sputum control group, each of which was 5 weeks old non-specific pathogen (SPF) chickens, each group was given 1 dose, respectively, the first group (negative In the control group) intramuscular injection of phosphate buffered saline (PBS) 1 mL, the second group (real, group) intramuscularly injected with the H5N2 avian influenza inactivated vaccine (vaccine) of the fourth example, the third group to the fifth The group (experimental group) was intramuscularly injected with the H5N2 avian influenza inactivated vaccine (vaccine + vector) containing the empty vector of Example 1, and the H5N2 avian influenza containing the recombinant plastid containing the single set of CpG modules of Example 1.

I SI 26 1378144 Revised replacement page on March 31, 2010 - Activated vaccine (vaccine + recombinant plastid containing a single set of CpG modules) and H5N2 avian influenza containing recombinant plastids containing 64 sets of CpG modules of Example 2 The vaccine was not activated (vaccine + recombinant plasmid containing 64 sets of CpG modules (pHCLS-64)) 1 mL each. All the test chickens were vaccinated with 1 dose as the second immunization 2 weeks after the first immunization. Thereafter, all the test chickens were collected before the immunization, 2 weeks after the first immunization, and 2 weeks after the second immunization, and blood samples were collected for blood cell agglutination inhibition antibody containing H5N2 avian influenza virus; ) 'The result is not shown in Figure 8A. The method for using the hemagglutination inhibition antibody test (HI test) in this example is as described in the first point of the fifth embodiment, and is not limited here. Please refer to FIG. 8A, which is a bar graph of a recombinant plastid containing 64 sets of CpG modules in vivo (ζ·„wvc〇 blood cell agglutination inhibition antibody titer according to an embodiment of the present invention, wherein The axis indicates different immunization methods, for example, PBS represents a negative control group, "vaccine" represents an experimental group inoculated only with H5N2 avian influenza inactivated vaccine, and "vaccine + vector" represents a non-activated H5N2 avian influenza inoculated with the empty vector of Example 1 The vaccine experimental group '"vaccine + recombinant plastid containing a single set of CpG modules" represents the experimental group of the H5N2 avian influenza inactivated vaccine containing the recombinant plastid of the single set of CpG modules of the first example. +pHCLS-64" represents the experimental group of the H5N2 avian influenza inactivated vaccine containing the recombinant plastid (pHCLS-64) containing 64 sets of CpG modules of Example 2, and the vertical axis indicates 2 weeks after the first immunization. And 2 weeks after the second 'immunization test, the blood contains a blood clot agglutination inhibitor antibody against H5N2 avian influenza virus (111 out of 6; [(^2)(^<0.05). and the figure "*" Indicates the use of recombinant plastids (pHCLS-64) containing 64 CpG modules 27 1378144 March 31, 2010 Corrected the degree of hemoglobin agglutination antibody titer on the replacement page. From the results of Figure 8A, the negative control group (pbs) did not detect the antibody price. After the first immunization 2 Weeks (shown as gray bars) and 2 weeks after the second immunization (shown as black bars), co-inoculated with the recombinant plastid H5N2 avian influenza inactivated vaccine containing a single set of CpG modules of Example 1. The experimental group (ie vaccine + recombinant plastid containing a single set of CpG modules), the experimental group (ie vaccine + vector) that co-inoculated the H5N2 avian influenza inactivated vaccine of the empty vector of Example 1 and the inoculation only H5N2 avian influenza were not activated. The experimental group (ie vaccine) of the vaccine, although the antibody price difference between the three is slightly different, but the degree of difference is not large. However, the recombinant plastid containing 64 sets of CpG modules (pHCLS) of the second embodiment was co-inoculated. -64) The experimental group of H5N2 avian influenza inactivated vaccine (ie vaccine + pHCLS-64), the specific antibody strength produced in the test chicken body was significantly higher than the above three experimental groups (the second group to Group 4) 2. Evaluation of the stimulation index of cell proliferation As mentioned above, the immune status of all the tested chickens was the same as in the first point of Example 6 and 2 weeks after the second immunization, and the peripheral blood mononuclear cells (PBMC) were taken for cell proliferation test to evaluate the vaccine. The immunological activity of promoting cell proliferation is shown in Fig. 8B. The method for using the cell proliferation test in this example is also as described in the second point of the fifth embodiment, and is not described herein. The present invention provides a histogram of cell proliferation (SI) of a recombinant plastid containing 64 sets of CpG modules in vivo (9) viv〇 according to an embodiment of the present invention, wherein the horizontal axis represents different immune modalities, for example. PBS represents a negative control group, and "vaccine" represents an experimental group in which only H5N2 avian influenza inactivated vaccine is inoculated. 'Vaccine + vector' stands for co-inoculation Example 1 28 1378144 [October 31, 2010 ^^γ~| The experimental group of H5N2 avian influenza inactivated vaccine, "vaccine + recombinant plastid containing a single set of CpG modules" represents the H5N2 avian influenza inactivated vaccine co-inoculated with the recombinant plastid containing the single CPG module of Example 1. Experimental group + PHCLS-64 "representatives to inoculate Example Two sets of 64 modules of CpG recombinant plasmid (pHCLS-64) implementation of the H5N2 avian influenza inactivated vaccine • experimental group. The vertical axis indicates that the PBMC hyperplasia index (SI) of the vaccinated animals was detected 2 weeks after the second immunization, wherein the stimulation index indicates that the recombinant plasmid (pHCLS-64) was empty vector, containing a single set or 64 sets of CpG modules. • The BrdU fluorescence value measured by the immunized PBMC, and the BrdU fluorescence value measured by the PBMC obtained by the PBS control group, the relative ratio of the two is 0.05). The figure "*" indicates the degree of cell proliferation index generated by recombinant plastids containing multiple sets of CpG modules. From the results of Fig. 8B, it was found that the cell proliferation index was not detected in the negative control group (pBS). Two weeks after the second immunization, the experimental group of the H5N2 avian influenza inactivated vaccine containing the recombinant plastid of the single set of CpG modules of Example 1 was co-inoculated (ie, vaccine + recombinant plastid containing a single set of CpG modules). ), an experimental group (ie vaccine + vector) that co-inoculates the H5N2 avian influenza inactivated vaccine of the empty vector of Example 1 and an experimental group (ie vaccine) inoculated only with the H5N2 avian influenza inactivated vaccine, cells between the three There was no significant difference in the proliferative stimulation index. However, the experimental group (ie vaccine + PHCLS-64) of the H5N2 avian influenza inactivated vaccine containing the recombinant plastid (pHCLS-64) containing 64 sets of CpG modules of Example 2 was co-inoculated to stimulate peripheral blood mononuclear cells. The effect of cell proliferation was significantly higher than the above three experimental groups (second group to group four). It is obvious that the recombinant plastid containing 64 sets of CpG modules (pHCLS_64) 29 1378144 Modified on March 31, 2010 替泰ΐ As an adjuvant for Η5Ν2 avian influenza inactivated vaccine, it can effectively promote immune promotion in vivo. active. Example 7: Recombinant plasmid containing 64 sets of CpG modules (pIICLS_64) Evaluation of immunostimulatory activity in vivo (ί·#ι Wv<?) (3) 1. Evaluation of hemagglutination inhibition antibody titer test This implementation The experimental chickens were divided into two groups: one test group and one control group, each of which was 5 weeks old non-specific pathogen (SPF) chickens, each group was given 1 dose, respectively, the first group (negative The control group was intramuscularly injected with phosphate buffered saline (PBS) 1 mL, the second group (experimental group) was intramuscularly injected with the H5N2 avian influenza inactivated vaccine (vaccine) of the fourth example, and the third group (experimental group) was administered intramuscularly. Example 2 H5N2 avian influenza inactivated vaccine (vaccine + PHCLS 64) mL containing recombinant CpG modules (pHCLS-64). All the test chickens were incubated with the sputum dose as the second immunization 2 weeks after the first immunization.

After that, all the test chickens were collected before the immunization, 2 weeks after the first immunization and 2 weeks after the second immunization, and blood samples were collected to detect the hemagglutination inhibition antibody titer of the H5N2 avian influenza virus (hi L〇). G2) 'The result is shown in Figure 9A. The method for using the hemagglutination-suppressing antibody test in this embodiment (the method of ΗΙ_ is as described in the fifth point of the fifth embodiment, in the reference picture (4), which shows a recombinant plastid of 64 sets of CPG modules according to an embodiment of the present invention. A histogram of the strength of the antibody in the living system, in which the horizontal axis is dry and the PBS is negative, and the PBS is negative for Zhao Zhi. "The vaccine is not immune, such as,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, 30 1378144 March 31, 2010 revised the experimental group of the replacement page activated vaccine, and "vaccine + PHCLS-64" represents the H5N2 co-inoculated with the recombinant plastid (pHCLS-64) containing 64 sets of CpG modules of Example 1. The experimental group of avian influenza inactivated vaccine. The vertical axis indicates that the blood contains a hemagglutination inhibitory antibody against H5N2 avian influenza virus 2 weeks after the first immunization and 2 weeks after the second immunization (mtiters; L〇 G2) 0 < 0.05). The figure numbers "a, b, and e" represent the average value of the blood coagulation-inhibiting antibody titers obtained by statistical analysis of each group at 2 weeks after the first immunization, and the figure numbers "X, y, z" represent respectively. The mean value of the A-ball agglutination-inhibiting antibody titer obtained by statistical analysis of each group 2 weeks after the second immunization. From the results of Fig. 9A, it was found that the antibody strength was not detected in the negative control group (PBS). However, the experimental group (ie, vaccine + PHCLS-64) of the H5N2 avian influenza inactivated vaccine containing 64 sets of recombinant plastids (pHCLS-64) of Example 2 was co-inoculated, and the test chickens were produced in vivo. The specific antibody strength was significantly higher than that of the experimental group (vaccine) inoculated only with the H5N2 avian influenza inactivated vaccine. 2. Evaluation of the stimulation index of cell proliferation As described above, all the test chickens were immunized as described in point VII of Example VII and 2 weeks after the second immunization, peripheral blood mononuclear cells were taken. (PBMC) A cell proliferation assay was performed to evaluate the immunological activity of the vaccine to promote cell proliferation, and the results are shown in Fig. 9B. The method for using the cell culture test in this example is also as described in the second point of Example 5, and is not limited here. As for the figure numbers "a, b, c", respectively, the average value of the PBMC hyperplasia stimulation index of the animals was measured after 2 weeks after the second immunization. - 结果9B results show that the negative control group (pBS) did not detect. PBMC proliferation stimulation index 1 and co-inoculation of the second embodiment of the material cover 31 1378144

Corrected replacement page I on March 31, 2010

The experimental group of H5N2 avian influenza inactivated vaccine (vaccine + PHCLS_64) of recombinant plastid (pHCLS-64) of CpG module has a significantly higher effect on stimulating cell proliferation of peripheral blood mononuclear cells than H5N2 avian influenza alone. The experimental group (vaccine) that does not activate the vaccine. 3. Evaluation of vaccine protection rate As mentioned above, all test chickens were infected with H5N1 avian influenza virus virulent strain of 108 5EID50 (50% embryo infective dose) by intramuscular injection 2 weeks after the second immunization. /Duck/China/E319-2/03, H5N1 Golden Gate strain; Source: Freshwater Animal Health Laboratory of the Agricultural Research Council of the Executive Yuan). Then all chickens were observed for another 2 weeks to observe the clinical symptoms of avian influenza and calculate the survival rate. Please refer to FIG. 10, which is a histogram of the immunoprotection rate of a recombinant plastid containing 64 sets of CpG modules in vivo (z > 7 Wv〇) according to an embodiment of the present invention, wherein the horizontal axis represents Different immunization methods, for example, PBS represents a negative control group, "vaccine" represents an experimental group inoculated only with H5N2 avian influenza inactivated vaccine, and "vaccine + pHCLS_64" represents a recombinant plastid containing 64 sets of CpG modules in co-inoculation of Example 2. (1) 11 (: 1^-64) experimental group of H5N2 avian influenza inactivated vaccine. The vertical axis indicates the immunoprotection rate (%) against the H5N1 avian influenza virus virulent strain (H5N1 Golden Gate strain) after 2 weeks of the second immunization; each group of samples has at least five replicates (/? < 0.05). From the results of Fig. 10, the immunoprotection rate of the negative control group was zero. The experimental group of the H5N2 avian influenza inactivated vaccine containing the recombinant plastid (pHCLS-64) containing 64 sets of CpG modules of Example 2 was co-inoculated, and the immunity against the virulent strain of h5N1 avian influenza virus was 2 weeks after the second immunization. The protection rate is as high as about 100% 'the protection effect is better than the inoculation only H5N2 avian influenza does not live m 32 ^/8144 The experimental group of the modified paged vaccine was revised on March 31, 2010 (immunization protection rate is about 80%). It is apparent that the recombinant plastid (pHCLS-64) containing 64 sets of CpG modules can be used as an adjuvant for the H5N2 avian influenza inactivated vaccine, which not only can effectively promote the immune promoting activity in vivo, but also is highly virulent to the H5N1 avian influenza virus. The strain also provides cross-immunoprotective effects. Example 8: Evaluation of immunostimulatory activity of recombinant plastids (phcLS-64) containing 64 sets of CpG modules in vivo (iVi νί·νο) (4) In this example, 40 experimental chickens were not mixed. Feeding to 3 weeks old's feed uses the special grade chicken feed produced by Fushou Industrial Co., Ltd. (containing about 22.5% crude protein (CP), about 22.5% fat), drinking water using RO After the system is processed, it can be eaten freely by the drinker. The experimental chickens were divided into 4 groups at the age of 3 weeks, and each group was 10, numbered one by one and immunized with different immunological reagents according to the second table. The feed was changed to the late feed of the broiler produced by Fushou Industrial Co., Ltd. ( It contains about 19.0% crude protein, about 5.5% fat.) Drinking water is treated with the r〇 system and then fed freely through the drinker. Experimental group, Table 2, immunological reagent composition, first group PBS, second group, fourth example, Nobilis trivalent vaccine, third group, fourth example, Nobilis trivalent vaccine + empty vector (5 〇β g), fourth group of fourth embodiment Nobilis Trivalent Vaccine + Example 2 contains _____ 64 sets of CpG module recombinant plastids (pHCLS-64; 50 #g) 33 13/8144 Letter ^"¥3月31日修换苜~Μ After the immunization, the blood collection method was performed at the peri-week, peri-week, 2 weeks, and 3 weeks. The specific antibody titer was detected by the knife. The detection method of the terry antibody in the blood can be exemplified by the following examples. Hemagglutination test money serum enzyme linked immunosorbent assay.

(1) Hemagglutination inhibition antibody test (HI test): This example can be tested according to the current present invention of the hematopoietic blood cell repression test in Taiwan, and the test is carried out by the method of preparing an antigen of 8 coagulation units (HAUNs; HAU). First, in a 96-well V-type microplate, 85% of physiological saline 25 is added to each well, and for example, 25 μM of the serum to be tested is sequentially added to the 8 wells of the first column to quantify the burette from each row. The first well was diluted 2 times to the nth well, and the 12th well was used as a control group. Next, to each well containing the serum sample, 4 blood unit (HA units) of the virus antigen solution 25 was added. After standing at room temperature for 15 minutes, add 5 〇 9% (ν/ν) to each well.

The chicken red blood cell suspension was allowed to stand at room temperature for an hour, and the blood cell agglutination inhibition state of each sample was observed. If the reciprocal of the highest dilution factor of red blood cell agglutination was completely suppressed, it was the hemagglutination inhibition antibody titer. (2) Serum enzyme-linked immunosorbent assay (ELISA test): This example can be used to determine infectious bursal disease (IBD or G) and avian infectious bronchitis (IB) using a special ELISA kit produced by KPL. The specific antibody strength in the blood. Please refer to FIG. 11A to FIG. 11C, which illustrate the blood coagulation inhibitory antibody titer of the recombinant plastid (pHCLS-64) containing 64 sets of CpG modules in vivo Wv<3) according to an embodiment of the present invention. The histogram, in which the horizontal axis represents the number of weeks of the first immunization, and the vertical axis represents the peri-implantation week, 1 week, 2 34 1378144, March 31, 2010, corrected replacement page 'week and 3 weeks test blood Contains the titer of hemagglutination inhibition for chicken Newcastle disease virus (Fig. ha), for infectious bursal disease in chickens (Fig. 11B), and for infectious bronchitis (article graph) of chickens (hi titers; Log2) b<0.05). In the 11A to lie diagrams, the twill strips (PBS) represent the negative control group. The blank strip (vaccine) represents the experimental group in which only the Nobilis trivalent vaccine was inoculated. The gray strip (vaccine + vector) represents the co-inoculation implementation. In the experimental group of the Nobilis trivalent vaccine of the empty vector of Example 1, the black strip (vaccine + pHCLS-64) represents Nobilis III co-inoculated with the recombinant plastid (PHCLS-64) containing 64 sets of cpG modules of Example 2. Experimental group of valency vaccines. The figure "*" indicates the degree of hemagglutination inhibition antibody titer produced by recombinant plastids (pHCLS-64) containing 64 sets of CpG modules. From the results of Fig. 11A to Fig. 11C, it was found that there was no significant difference in the antibody titer between the groups after immunization and one week. However, after 2 weeks and 3 weeks of immunization, the experimental group of N〇bilis trivalent field containing the recombinant plastids of 64 sets of CpG modules (pHCLS_64) of Example 2 was co-inoculated (ie vaccine) compared to the negative control group. +pHCLS_64), the specific antibody titer produced by the chickens in the tested chickens for chicken Luxinxin virus (Fig. 11A) and chicken infectious bursal disease (Fig. 11B) was significantly higher than that of the co-inoculation example one. The experimental group of the Nobilis trivalent vaccine (ie vaccine + vector) of the empty vector and the experimental group (ie vaccine) inoculated only with the Nobilis trivalent vaccine. As for the experimental group of the N〇blllS trivalent vaccine containing the recombinant plastid (pHCLS-64) containing 64 sets of CpG modules (ie, vaccine + PHCLS-64), the test chickens were inoculated in the first place. The specific antibody-forced mussels produced by avian infectious bronchitis (figure lie) and the N〇bUis trivalent epidemic vaccine + finely vaccinated with the empty vector of Example 1 were higher than those of NGbilis only. Price epidemic m. 35 1378144 March 31, 2010 Revised the experimental group (ie vaccine) for replacing the seedlings. It is thus apparent that the recombinant plastid (pHCLS-64) containing 64 sets of CpG modules can also effectively promote the immunopromoting activity in vivo when used as an adjuvant for the Nobilis trivalent vaccine. Example 9: Evaluation of immunostimulatory activity of recombinant plastids containing 64 sets of CpG modules in vivo (/η vivo) (5) In this example, 40 experimental chickens were raised in a non-grouped manner to 3 weeks. Age, the feed method is as described in Example 8. The experimental chickens were divided into 4 groups at the age of 3 weeks, and each group was 10, numbered one by one and immunized with different immunological reagents according to the third table, and the feed method was also described in Example 8. _第3表_ Experimental group Immunoreagent components

The first group of PBS, the second group of the fourth group of poultry respiratory mycoplasmosis (MG) vaccine, the third group of the fourth group of MG vaccine + empty vector (50 / zg) The fourth group of example IV MG vaccine + implementation Example 2 Recombinant plastids containing 64 sets of CpG modules (pHCLS-64; 50# g) All the above chickens were collected at 0, 1, 2, and 3 weeks after immunization, and serum was separated for specificity. The antibody valence, wherein the blood antibody specific antibody is detected by a serum enzyme-linked immunosorbent assay as described in Example 8, to determine poultry respiratory mycoplasmosis (MG) in the blood t!jj 36 1378144

The 10-year 3^31曰 correction replaces page I • the specific antibody price. - Please refer to Fig. 12, which shows a recombinant plastid (phCLS-64) containing 64 sets of CpG modules in vivo (with bamboo > 0) according to an embodiment of the present invention. A histogram of the inhibition of antibody valence, wherein the horizontal axis represents the number of weeks of the first immunization, and the vertical axis indicates that the blood in the test contains the respiratory mold for poultry after week, week, week, and week after immunization. The hemagglutination inhibition antibody titer of the disease (MG) vaccine (HI titers; LogO (p< 0.05). In Fig. 12, the twill strip (PBS) represents the negative control group, and the blank strip (vaccine) represents • inoculation only The experimental group of the MG vaccine, the gray strip (vaccine + vector) represents the experimental group of the MG vaccine co-inoculated with the empty vector of Example 1, and the black strip (vaccine + PHCLS-64) represents 64 sets of the co-inoculation Example 2. The experimental group of the MG vaccine of the recombinant plastid (pHCLS-64) of the CpG module, and the figure "*" indicates that the recombinant plastid (pHCLS-64) containing 64 sets of CpG modules produces hemagglutination inhibition antibody. From the results of Fig. 12, it can be seen that the antibody price between the groups was not significant after the immunization week and week after immunization. Difference. However, after 2 weeks and 3 weeks of immunization, the experimental group of the MG vaccine containing the recombinant plastids (pHCLS-64) containing 64 sets of CpG modules of Example 2 was co-inoculated (ie, the vaccine). +' pHCLS-64), the specific antibody titer produced by the test chickens for poultry respiratory mycoplasma was significantly higher than the experimental group of the mg vaccine co-inoculated with the empty vector of Example 1 (ie vaccine + The vector) and the experimental group (ie vaccine) that only inoculated the seedlings. It is thus apparent that the recombinant plastid (PHCLS-64) containing 64 sets of CpG modules is used as a vaccine for the poultry respiratory plaque disease (MG) vaccine. At the same time, it can also effectively promote the immune promoting activity in vivo. - Secondly, the results of the in vivo experiment from Example 5 to Example 9 can be found in the number of CpG modules contained in the immunostimulatory oligonucleotide. There is no proportional relationship between the two and the degree of immunostimulating activity of the vaccine, so the degree of immunological activity of the immunostimulatory oligonucleotide obtained by the number of CpG modules cannot be reasonably predicted. .

Furthermore, it should be noted that the present invention exemplifies a specific plastid, an immunostimulatory oligonucleotide containing a specific set of CpG modules, a specific host cell, a poultry test animal, a cell type or a vaccine species. It is to be noted that the recombinant plastosome containing multiple sets of CpG modules of the present invention is applied to an adjuvant for avian vaccine, but it is known to those having ordinary knowledge in the art to which the present invention pertains, and the present invention is not limited thereto. Within the spirit and scope of the invention, the recombinant plastids containing multiple sets of CpG modules of the present invention are carried out using other plastids, host cells, recombinant plastids, transformed strains, other test animals, cell types or vaccine types. For example, a recombinant plastid containing 16, 32, and 64 coats of CpG modules can be used to generate other sets of host cells (eg, 17 to 31 sets, or 33 to 63 sets) of CpG modules. The stimulatory oligonucleotide ' acts as an adjuvant for avian vaccines. In addition, the resulting immunostimulatory oligonucleotide containing multiple sets of CpG modules, recombinant plastids containing the immunostimulatory oligonucleotides, transformed strains, and the like, may be used without departing from the spirit and scope of the present invention. Or any combination of the above, applied to other animal (such as mammals) or other animal vaccine types to enhance the immune stimulating effect of the vaccine on other animals. According to the preferred embodiment of the present invention, the transformant strain of the recombinant plasmid containing multiple sets of CpG modules of the present invention has the advantages of embedding an immunostimulatory oligonucleotide containing multiple sets of CpG modules into the plastid, Among them, these CpG modules are 38 1378144 revised on March 31, 2010 to replace the page specific sequence of poultry. After the resulting recombinant plasmid is transformed into a host cell, the immunostimulatory oligonucleotides having multiple sets of CpG modules can be stably, rapidly and abundantly expressed. When the immunostimulatory oligonucleotide, the recombinant plastid containing the immunostimulatory oligonucleotide, the transformed strain, or any combination thereof is used as an adjuvant for poultry or other animal vaccines, The CpG module not only reduces the amount and cost of adjuvants, but also enhances the efficacy of vaccines with insufficient antigenic immunity, and eliminates the cumbersome steps that conventional DNA adjuvants must utilize chemical modification. 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 chart of a method of manufacturing a recombinant plastid containing 64 sets of CpG modules in one embodiment. Fig. 2A is a diagram showing the electrophoresis analysis of a recombinant agar containing a single set of CpG modules by restriction enzyme A and restriction enzyme C according to an embodiment of the present invention. Fig. 2B is a diagram showing the modification of the recombinant plasmid containing a single set of CpG modules according to an embodiment of the present invention by restriction enzyme B and restriction enzyme C cleavage of agar extract agar 1 39144. Fig. 3 is a diagram showing the electrophoresis analysis of agar extracts of the recombinant plastids containing 4, 8, 16, 32, and 64 sets of CpG modules, which were cleaved by restriction enzyme A and restriction enzyme restriction enzyme B, according to an embodiment of the present invention. Figure 4 is a diagram showing the expression of IFN-γ gene in chicken spleen cells stimulated by recombinant plastids containing one or more sets of CpG modules according to Examples 1 and 2 of the present invention and empty vector in vitro vzYro) Figure. Fig. 5 is a bar graph showing the expression of TLR3 gene in a chicken spleen cell stimulated in vitro by recombinant plastids containing one or more sets of CpG modules according to Examples 1 and 2 of the present invention and an empty vector. Fig. 6 is a bar graph showing the expression of TLR7 gene in a chicken spleen cell stimulated in vitro by recombinant plastids containing one or more sets of CpG modules according to Examples 1 and 2 of the present invention and an empty vector. 7A, 8A, 9A, 11A to 11C, and 12 are diagrams showing recombinant bodies containing 64 sets of CpG modules in vivo according to several embodiments of the present invention (/« A histogram of the blood cell agglutination inhibition antibody titer of v/vo). Fig. 7B, Fig. 8B and Fig. 9B are bar graphs showing the cell growth stimulation index of recombinant plastids containing 64 sets of CpG modules in vivo according to several embodiments of the present invention. Figure 10 is a bar graph showing the immunoprotection rate of recombinant plastids containing 64 sets of CpG modules in vivo according to an embodiment of the present invention. 11A to 11C are diagrams showing a recombinant plastid (pHCLS-64) containing 64 sets of CpG modules in vivo (ζ·« νζ·νο) 1378144 July 10, 2012 according to an embodiment of the present invention. A daily histogram of the replacement of the blood cell agglutination inhibition antibody. Figure 12 is a bar graph showing the blood coagulation inhibitory antibody titer of a recombinant plastid (pHCLS-64) containing 64 sets of CpG modules in vivo (ζ&gt;7 νζ·νο) according to an embodiment of the present invention. . Fig. 13 and Fig. 14 are diagrams showing the DNA sequencing of immunostimulatory oligonucleotides containing 64 sets of CpG modules according to an embodiment of the present invention. Φ [Main component symbol description] 100: Recombination 150 with single CpG module: Heavy mass group with 32 sets of CpG modules 110: Recombination 160 with dual CpG modules: 64 sets of CpG modules The weight of the plastid body (pHCLS-64) 120: Recombination of four sets of CpG modules 201/203/301/303/305/307/309 plastid: arrow 4] 1378144 revised March 31, 2010 Replacement page sequence table &lt;110&gt;National Pingtung University of Science &Technology&lt;120&gt; Recombinant plasmid containing multiple sets of CpG modules for avian vaccine adjuvant and its transformed strain &lt;130&gt; No &lt;160&gt; 2

&lt;210&gt; 1 &lt;211&gt; 28 • &lt;212&gt; DNA &lt;213&gt;Artificial sequence &lt;400&gt; 1 ctagttcgtc gaagtcgttt tggggggt &lt;210&gt; 2 &lt;211&gt; 448 &lt;212&gt; DNA &lt;213&gt; sequence

&Lt; 400 &gt; 2 ctagttcgtc gaagtcgttt tggggggtct agttcgtcga agtcgttttg gggggtctag 60 ttcgtcgaag tcgttttggg gggtctagtt cgtcgaagtc gttttggggg gtctagttcg 120 tcgaagtcgt tttggggggt ctagttcgtc gaagtcgttt tggggggtct agttcgtcga 180 agtcgttttg gggggtctag ttcgtcgaag tcgttttggg gggtctagtt cgtcgaagtc 240 gttttggggg gtctagttcg tcgaagtcgt tttggggggt ctagttcgtc gaagtcgttt 300 tggggggtct agttcgtcga agtcgttttg gggggtctag ttcgtcgaag tcgttttggg 360 gggtctagtt cgtcgaagtc gttttggggg Gtctagttcg tcgaagtcgt tttggggggt 420 March 31, 2010 Correction replacement page ctagttcgtc gaagtcgttt tggggggt 448 &lt;210&gt; 3 &lt;211&gt; 896 &lt;212&gt; DNA &lt;213&gt;Artificial sequence&lt;400&gt; 3 ctagttcgtc gaagtcgttt tggggggtct agttcgtcga agtcgttttg gggggtctag 60 ttcgtcgaag tcgttttggg gggtctagtt cgtcgaagtc gttttggggg gtctagttcg 120 tcgaagtcgt tttggggggt ctagttcgtc gaagtcgttt tggggggtct agttcgtcga 180 agtcgttttg gggggtctag ttcgtcgaag tcgttttggg gggtctagtt cgtcgaagtc 240 gttttggggg gtctagttcg tcgaagtcgt tttggggggt ctagttcgtc gaagtcgt tt 300 tggggggtct agttcgtcga agtcgttttg gggggtctag ttcgtcgaag tcgttttggg 360 gggtctagtt cgtcgaagtc gttttggggg gtctagttcg tcgaagtcgt tttggggggt 420 ctagttcgtc gaagtcgttt tggggggtct agttcgtcga agtcgttttg gggggtctag 480 ttcgtcgaag tcgttttggg gggtctagtt cgtcgaagtc gttttggggg gtctagttcg 540 tcgaagtcgt tttggggggt ctagttcgtc gaagtcgttt tggggggtct agttcgtcga 600 agtcgttttg gggggtctag ttcgtcgaag tcgttttggg gggtctagtt cgtcgaagtc 660 gttttggggg gtctagttcg tcgaagtcgt tttggggggt ctagttcgtc gaagtcgttt 720 tggggggtct agttcgtcga agtcgttttg gggggtctag ttcgtcgaag tcgttttggg 780 gggtctagtt cgtcgaagtc gttttggggg gtctagttcg tcgaagtcgt tttggggggt 840 ctagttcgtc gaagtcgttt tggggggtct agttcgtcga agtcgttttg gggggt 896

&lt;210&gt; 4 &lt;211&gt; 1792 &lt;212&gt; DNA 1378144 Corrected replacement page on March 31, 2010 &lt;213&gt; Artificial sequence &lt;400&gt;

ctagttcgtc ttcgtcgaag tcgaagtcgt agtcgttttg gttttggggg tggggggtct gggtctagtt ctagttcgtc ttcgtcgaag tcgaagtcgt agtcgttttg gttttggggg tggggggtct gggtctagtt ctagttcgtc ttcgtcgaag tcgaagtcgt agtcgttttg gttttggggg tggggggtct gggtctagtt ctagttcgtc ttcgtcgaag tcgaagtcgt gaagtcgttt tcgttttggg tttggggggt gggggtctag gtctagttcg agttcgtcga cgtcgaagtc gaagtcgttt tcgttttggg tttggggggt gggggtctag gtctagttcg agttcgtcga cgtcgaagtc gaagtcgttt tcgttttggg tttggggggt gggggtctag gtctagttcg agttcgtcga cgtcgaagtc gaagtcgttt tcgttttggg tttggggggt tggggggtct gggtctagtt ctagttcgtc ttcgtcgaag tcgaagtcgt agtcgttttg gttttggggg tggggggtct gggtctagtt ctagttcgtc ttcgtcgaag tcgaagtcgt agtcgttttg gttttggggg tggggggtct gggtctagtt ctagttcgtc ttcgtcgaag tcgaagtcgt agtcgttttg gttttggggg tggggggtct gggtctagtt ctagttcgtc agttcgtcga cgtcgaagtc gaagtcgttt tcgttttggg tttggggggt gggggtctag gtctagttcg agttcgtcga cgtcgaagtc gaagtcgttt tcgttttggg tttggggggt gggggtctag gtctagttcg agttcgtcga cgtcgaagtc gaagtcgttt tcgttttggg tttggggggt gggggtctag gtctagttcg agttcgtcga cgtcgaagtc gaagtcgttt agtcgttttg gttttggggg tggggggtct gggtctagtt ctagttcgtc ttcgtcgaag tcgaagtcgt agtcgttttg gttttggggg tggggggtct gggtctagtt ctagttcgtc ttcgtcgaag tcgaagtcgt agtcgttttg gttttggggg tggggggtct gggtctagtt ctagttcgtc ttcgtcgaag tcgaagtcgt agtcgttttg gttttggggg tggggggtct gggggtctag gtctagttcg agttcgtcga cgtcgaagtc gaagtcgttt tcgttttggg tttggggggt gggggtctag gtctagttcg agttcgtcga cgtcgaagtc gaagtcgttt tcgttttggg tttggggggt gggggtctag gtctagttcg agttcgtcga cgtcgaagtc gaagtcgttt tcgttttggg tttggggggt Gggggtctag gtctagttcg agttcgtcga 60 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 3 1378144 [March 31, 2010 曰agtcgttttg gggggtctag ttcgtcgaag tcgttttggg gggtctagtt cgtcgaagtc l5〇〇gttttggggg gtctagttcg tcgaagtcgt tttggggggt ctagttcgtc Gaagtcgttt lbe〇tggggggtct agttcgtcga agtcgttttg gggggtctag ttcgtcgaag tcgttttggg 1620 gggtctagtt cgtcgaagtc gttttggggg gtctagttcg tcgaagtcgt tttggg Gggt 1680 ctagttcgtc gaagtcgttt tggggggtct agttcgtcga agtcgttttg gggggtctag 11 AO ttcgtcgaag tcgttttggg gggtctagtt cgtcgaagtc gttttggggg gt 1192

Claims (1)

1378144 @2年July 10曰Amendment VII. Patent application scope: The recombinant plastid of the 3rd set CpG module comprises at least: a carrier; and a oligo-oligonucleotide embedded in the carrier, wherein the immunospin „At least 32 to 64 sets of CPG modules are included, and the immune oligonuclear acid is selected from the group consisting of a nucleotide sequence such as the sequence identification number 3 and a sequence identification number nucleotide. One of the ethnic groups, jin yi, one of the recombinant plastids containing multiple sets of CPG modules or the immunostimulatory oligo (4) obtained from the recombinant plastid containing the plurality of (4) ^, and the recombinant plastid is a poultry plague , = adjuvant ', where the vaccine for reading is a non-activated vaccine for poultry respiratory molds>, or a trivalent inactivated oily vaccine that combines Newcastle disease, infectious bursal disease, and infectious bronchitis. Recombinant plastid containing multiple sets of CpG modules according to the scope of the patent application, wherein the carrier is a pGEM_T carrier, a teacher (10) cript Η carrier or a pBluescript KS carrier. 3. According to the scope of the patent application 帛 1 Containing multiple sets of CpG modules ^recombined plastids It also includes - chicken Xincheng disease live poisoning disease -, - chicken Newcastle disease attenuated vaccine 'one chicken Newcastle disease * live flu not activated vaccine. Tian 4 get 4. - a transformation of recombinant plastids containing multiple sets of CpG modules The strain, at least 1378144 comprises: ^Ξΐ2Ζΐ〇ΖΐΐΈ55ΖΖ1 a host cell; and ... = the recombinant plastid of the CPG module is transformed into the host cell, wherein the recombinant plastid containing the plurality of sets of CPG modules comprises at least: a vector; And an immunostimulatory oligo (4) embedded in the vector, and (4) stimulating oligonucleotides comprising at least "set to 64 sets of CpG modulo" immunization, sexual oligonuclear lines, selected from, for example, sequence identification number 3 "column, H-nucleus (4) and a group of second oligonucleotides comprising one of the sequences shown in SEQ ID NO: 4, wherein the transformed bacterium comprising a plurality of sets of CpG modules is transformed by the &lt; The modified strain of the recombinant plastid of the CPG module obtains the multi-set plastid or the Qin nucleoside acid obtained from the recombinant plastid containing the plurality of CPG modules is an adjuvant for a poultry vaccine. Where the poultry is broad: 豕 豕 avian respiratory mycoplasma does not activate oily vaccine, Or a combination of Newcastle disease, Pneumatic bursal disease, and trivalent non-activated oily vaccine for infectious bronchitis. 5. Transformation of recombinant shells containing multiple sets of CpG modules as described in item 4 of the patent application scope. a strain, wherein the host cell is Escherichia coli 〇6', according to the fourth aspect of the patent application, a recombinant strain containing a plurality of sets of CpG modules, and the carrier is a pGEM-T easy vector. , 1378144 pBluescript 载体 carrier or pBiuescrjptKs P丨 2 July 7th revised carrier 7 · According to the scope of claim 4, the transitional strain containing multiple sets (10) of the module body, wherein the avian vaccine further contains - Chicken New Field Disease „ - Chicken Newcastle Disease Attenuated Vaccine, One Chicken Newcastle Disease Inactivated Vaccine or One Avian Influenza Inactivated Vaccine. 8* A composition of a poultry vaccine comprising at least: 壬儿丄ί用疫田' wherein the poultry vaccine is a poultry respiratory bacterium that does not combine Newcastle disease, infectious bursal disease, and chicken transmission The trivalent unactivated oily vaccine of the tuberculosis; and the silk core (4), as one of the riding seedlings, the smear oligonuclear nucleus (4) contains at least 32 sets to 64 sets (10). The bitter acid is selected from the group consisting of a second oligo (4), such as the sequence identification number 3, and a sequence of the second oligonucleotide, as indicated by the sequence identification number 4. The composition of the poultry vaccine described in item 8 of the patent application. The city disease is reduced to the live vaccine of chicken Newcastle disease, and the new chicken vaccine. Chicken Newcastle disease inactivated vaccine or avian influenza inactivated substance, == According to the composition of the poultry vaccine according to item 8 of the patent scope, &quot;immunosimgenic oligonuclei (4) is as claimed in the patent scope ^ 1378144 On July 10, 2012, the recombinant plastid containing multiple sets of CpG modules described in the alternative page was modified or obtained from a transformed strain containing recombinant plastids containing multiple sets of CpG modules as described in claim 4 of the patent application. 1378144 1378144 1378144 Corrected replacement page on March 31, 2010 IV. Designated representative map: (1) The representative representative of the case is: (1C). (2) A brief description of the symbol of the representative figure: 150: Weight of 32 sets of CpG modules: Recombination of plastids with 64 sets of CpG modules (pHCLS-64) 5. If there is a chemical formula in this case, please reveal the chemical formula that best shows the characteristics of the invention: