DE102011000036B4 - Recombinant plasmid-containing multiple copy CpG motifs and their transformants for use as DNA adjuvants in avian vaccines - Google Patents

Abstract

A transformant of a recombinant plasmid containing multi-copy CpG motifs is disclosed. The recombinant plasmid containing an immunostimulatory polynucleotide having a sequence of the continuous multiple copy CpG motifs is transformed into a host cell to form the transformant such that the multi-copy CpG motifs of the immunostimulatory polynucleotide are stable and fast Mass production are expressed. The immunostimulatory polynucleotide, the recombinant plasmid containing the immunostimulatory polynucleotide, the transformant containing the recombinant plasmid, or any combination thereof may serve as an adjunct to avian vaccines. Such an adjuvant can significantly reduce the burden and cost of the adjuvant and specifically improve the avian vaccines having low immunization strength and effectively solve the problem of complicated chemical modification of the prior art DNA adjuvant.

Description

RELATED APPLICATIONS

This application claims the priority of the application filed on 09 February 2010 Taiwan Application No. 99 104 026 , which has been incorporated by reference into the present application.

STATE OF THE ART

Field of the invention

The present invention relates to a vaccine adjuvant. In particular, the present invention relates to a polynucleotide adjuvant which continuously contains multi-copy CpG motifs for animal vaccines.

Description of the Prior Art

The currently used animal vaccines have certain disadvantages. These are in the conventional adjuvant such as the aluminum hydroxide gel adjuvant and the oil adjuvant. For example, the above two adjuvants are chemical adjuvants that can not improve the specific immune response of the Th1 cell. In particular, the conventional adjuvant can not adequately stimulate innate and adaptive immune responses to infections.

A.M. Krieg et al. have found that non-methylated dinucleotide CpG motifs in bacterial deoxyribonucleic acid (DNA) are beneficial in that they stimulate some immune cells to secrete cytokines to enhance innate and adaptive immunity. See A.M. Krieg et al., Nature 374: 546-549, 1995. The potency of DNA with CpG motifs has been confirmed in 1998. In 2003, DNA with CpG motifs was used as a vaccine adjuvant. In addition, in some large domestic animals such as cattle, swine, sheep, etc., those olidodeoxynucleotides (ODN) containing CpG motifs may increase the activity of lymphocytes and antigen-containing cells (APC), the maturation of dendritic cells (DC) and the Triggering the function of antigen presentation and directing immune systems towards the immune response of the Th-1 cell to specific antigens. For example, one of the inventors of the present invention has presented an ODN containing a porcine specific and phosphorothioate (PTO) modified CpG motif and an immunostimulatory plasmid containing various sets of CpG motifs specific for pigs.

In general, the mechanisms involved in improving immune responses by the ODN containing CpG motif (CpG-ODN) may be the following mechanisms. First, the CpG ODN enhances DC activation, maturation, and antigen presentation function. Second, the CpG ODN increases DC migration. Third, the CpG ODN significantly increases the expression of DC cell markers such as MHC-II, CD40, CD80, CD86 and IL-12 in mice and humans. Fourth, the CpG ODN induces a more sensitive start of DC over antigen-specific Th1 cells. Fifth, CpG ODN increases the cytotoxic activity (CTL) of CD8 ⁺ T cells against specific viruses or tumor cells. Using CpG ODN, the CpG ODN induces an innate immune response to confer a protective immune response to infection by viruses, bacteria, and extracellular parasites, and B cells are activated by the CpG ODN and vaccines thereto to activate the APC to eliminate cytokines such as interferon-γ (IFN-γ), thus improving the immune response to vaccines.

Typically, CpG ODN contains unmethylated CpG motif in most cases, but without a phosphorus atom of a phosphodiester bond in the PTO-modified CpG ODN being whisked by a sulfur atom, causing the phosphothioate to degrade the deoxyribonuclease (DNase) -cleaved PTO-modified CpG However, the prior art ODN synthesized by chemical processes containing the DNA with the PTO-modified CpG motif is time-consuming, expensive, and can not be produced in large quantities.

In addition, the sequence of CpG ODN is species specific; the CpG structures between different species are different in immunostimulatory activity. It is well known that the sequence of the most effective immunoregulatory CpG motif is different from mouse and human, and therefore, specific CpG motifs which are effective in different species are necessarily confirmed by experiments. The CpG ODN has been used since 2002 to increase the immunity of poultry. Related research on the efficacy of CpG ODN in poultry in most cases involves evaluating chemically synthesized CpG ODN in vitro or focusing on the antibody-related humoral immune response in vivo. Research on the effective supporting effect of CpG ODN on the cell-mediated immune response against viral infection in poultry is very limited.

Therefore, it is necessary to prepare an effective poultry-based immunostimulatory CpG ODN as a DNA adjuvant in poultry vaccines, thereby overcoming the disadvantages of the prior art complicatedly chemically modified DNA adjuvant.

PRESENTATION OF THE INVENTION

There is provided a recombinant plasmid containing continuous multi-copy CpG motifs. This recombinant plasmid contains an immunostimulatory polynucleotide inserted into a vector, this immunostimulatory polynucleotide having a sequence of continuous multi-copy CpG motifs. The immunostimulatory polynucleotide, the recombinant plasmid containing the immunostimulatory polynucleotide, or any combination thereof, may serve as adjunct of a bird vaccine to specifically provide the immunized bird with the expression of the genes interferon-γ (IFN-γ), Toll -like to increase receptor 3 (TLR3) or TLR7. Therefore, it is not necessary to subject the adjuvant to chemical modification, as is the case with the adjuvants of the prior art.

In addition, a transformant is provided which contains the aforementioned recombinant plasmid. The recombinant plasmid containing, as mentioned above, the immunostimulatory polynucleotide is transformed into a host cell to form a transformant. The immunostimulatory polynucleotide has a bird-specific sequence of the continuous multiple copy CpG motif. The immunostimulatory polynucleotide, the recombinant plasmid containing the immunostimulatory polynucleotide, the transformant containing the recombinant plasmid, or any combination thereof, are an adjunct of a bird vaccine. Such an adjuvant is beneficial in that it increases the expression of IFN-γ, TLR3 or TLR7 genes in an immunized bird, and thus can be administered without undergoing chemical modification, as in the DNA adjuvants According to the prior art is the case.

Further, a bird seed is provided. This avian vaccine comprises a DNA adjuvant contained in a bird's vaccine, said DNA adjuvant, as mentioned above, an immunostimulatory polynucleotide, a recombinant plasmid with the immunostimulatory polynucleotide, the transformant containing the recombinant plasmid contains, or is any combination of them. When such an avian vaccine is administered to an immunized bird, it is beneficial in that it specifically increases expression of the IFN-γ, TLR3 or TLR7 genes in the immunized bird, without chemical modification or higher dosage the DNA adjuvants of the prior art is required.

In addition, a method is provided for immunizing a bird against a pathogen. This method involves administering a vaccine with a DNA adjuvant to a bird, and this DNA adjuvant is an immunostimulatory polynucleotide containing a sequence of the continuous multipiece CpG motif, a recombinant plasmid containing the immunostimulatory polynucleotide, the transformant containing the recombinant plasmid or any combination thereof. The sequence may include, but is not limited to, a third polynucleotide having SEQ ID NO. 2, a first polynucleotide with SEQ ID NO. 3 and a second polynucleotide with Seq.id.No. 4 included. The DNA adjuvant is beneficial in that it is effective in the immunized bird specifically increases the expression of the genes IFN-γ, TLR3 or TLR7, and requires less effort and lower cost than the DNA adjuvants of the prior art.

Thus, the invention provides a recombinant plasmid containing continuous multi-copy CpG motifs. In one embodiment, the recombinant plasmid may contain an immunostimulatory polynucleotide incorporated into a vector. The immunostimulatory polynucleotide has a sequence of continuous multiple copy CpG motifs and these continuous multiple copy CPG motifs are continuous copies 16 to 24 of the bird specific CpG motifs. The immunostimulatory polynucleotide, the recombinant plasmid containing the immunostimulatory polynucleotide, or any combination thereof, may serve as adjunct of a bird vaccine such that expression of the genes interferon-γ, Toll-like receptor 3, is specific to an immunized bird (TLR3) or TLR7 is increased.

In addition, the invention provides a transformant having the aforementioned recombinant plasmid. This transformant comprises a recombinant plasmid which, as mentioned above, contains the immunostimulatory polynucleotide and has been transformed into a host cell. In one embodiment, the host cell is a prokaryotic expression system such as Escherichia coli. The immunostimulatory polynucleotide has a bird-specific sequence of the continuous multiple copy CpG motifs, and this bird-specific sequence may include, but is not limited to, a third polynucleotide having SEQ ID NO. 2, a first polynucleotide with SEQ ID NO. 3 or a second polynucleotide with Seq.id. 4 included. The immunostimulatory polynucleotide expressed by the host cell from the recombinant plasmid, the recombinant plasmid, the transformant containing the recombinant plasmid, or any combination thereof are adjuvants of a bird vaccine for specifically expressing the genes of an immunized bird Increase IFN-γ, TLR3 or TLR7.

In addition, the invention provides a bird vaccine. This bird vaccine contains a DNA adjuvant which is added to a bird's vaccine. In one embodiment, this DNA adjuvant is an immunostimulatory polynucleotide having a bird-specific sequence of continuous multiple copy CpG motifs, which may include, but are not limited to, a bird-specific sequence: a third polynucleotide having SEQ ID NO. 2, a first polynucleotide with SEQ ID NO. 3 and a second polynucleotide with Seq.id.No. 4, a recombinant plasmid containing the immunostimulatory polynucleotide, a transformant containing the recombinant plasmid, or any combination thereof, such that in a bird to which the avian inoculum has been administered, expression of the genes IFN -γ, TLR3 or TLR7 is increased.

According to one embodiment of the invention, the aforementioned avian vaccine is a live virus against Newcastle disease virus, a Newcastle disease virus-based live attenuated virus, an inactivated vaccine against Newcastle disease, an inactivated bird flu vaccine , an oil kill vaccine against avian mycoplasmosis (Mycoplasma gallisepticum; MG) or a trivalent oil dead vaccine against Nobilis (ie, combined with IB strain M41, Gumbaro strain D78, and ND strain Clone 30).

In addition, the invention provides a method of immunizing a bird against a pathogen. This method involves the administration of a vaccine with a DNA adjuvant to the bird. In one embodiment, this DNA adjuvant is an immunostimulatory polynucleotide comprising a sequence of the continuous multiprotocol CpG motif, a recombinant plasmid containing the immunostimulatory polynucleotide, a transformant containing the recombinant plasmid, or any combination thereof. The immunostimulatory polynucleotide contains a sequence of the continuous multiple copy CpG motif, and this sequence may include, but is not limited to: a third polynucleotide having SEQ ID NO. 2, a first polynucleotide with SEQ ID NO. 3 and a second polynucleotide with Seq.id.No. 4, a recombinant plasmid containing the immunostimulatory polynucleotide or any combination thereof so as to specifically increase expression of the genes IFN-γ, TLR3 or TLR7 in the bird.

In the application of the recombinant plasmid containing continuous multi-copy CpG motifs, the immunostimulatory polynucleotide having a bird-specific sequence of the continuous multiple copy CpG motifs is introduced into the vector. The recombinant plasmid is transformed into the host cell to achieve stable and rapid mass production of the immunostimulatory polynucleotide. The above-mentioned immunostimulatory polynucleotide, a recombinant plasmid containing the immunostimulatory polynucleotide, the transformant containing the recombinant plasmid, or any combination thereof may be a bird-seed adjuvant. When such an avian vaccine is administered to an immunized bird, it has the beneficial effect of increasing the expression of the IFN-γ, TLR3 or TLR7 genes in the immunized bird in a specific manner, and of the expense and cost over the adjuvants can be lowered according to the prior art and complicated chemical modifications with their higher cost, as required in the DNA adjuvants according to the prior art, omitted.

It should be understood that both the foregoing general description and the following detailed description are exemplary in nature and are intended to serve to the purpose of further elucidating the invention with its claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention can be better understood by reading the following description of the embodiments with reference to the accompanying drawings.

1A to 1C Figure 15 are partial flow diagrams of a method for producing a recombinant plasmid with continuous multiple copy CpG motifs according to the present invention;

2A and 2 B show the results of agarose electrophoresis of the recombinant plasmid (n = 1), which is represented by RE A and C ( 2A ) or by the RE B and C ( 2 B ) has been split;

3 shows a result of agarose electrophoresis of the recombinant plasmid (n = 4, 8, 16, 32 and 64) which has been split by RE A and B;

4 to 6 Figure 12 shows bar graphs of the expression of the chick splenocyte IFN-γ, TLR3 or TLR7 genes generated by the recombinant plasmids with the single copy or multiple copy CpG motif (s) and the empty vector of Examples 1 and 2 in vitro, respectively has been stimulated;

7A Figure 12 shows a bar graph of the HI titer of SPF chicks fed with the cell-cultured ND vaccine (NDTC) and / or the recombinant plasmid DNA with the 64-copy CpG motifs (pHCLS-64) according to a preferred embodiment of the present invention Invention have been vaccinated;

7B Figure 12 shows a stimulus index (SI) bar graph of SPF chicks vaccinated with the NDTC vaccine and / or the recombinant plasmid DNA with the 64-copy CpG motifs (pHCLS-64) according to a preferred embodiment of the present invention are;

8A Figure 12 shows a bar graph of the HI titer of SPF chickens infected with the inactivated H5N2 avian influenza vaccine and / or the recombinant plasmid DNA with the 64-copy CpG motifs (pHCLS-64) according to a preferred embodiment of the present invention have been vaccinated;

8B Figure 12 shows a bar graph of the SI value of SPF chicks infected with the inactivated H5N2 avian influenza vaccine and / or the recombinant plasmid DNA with the 64-copy CpG motifs (pHCLS-64) according to a preferred embodiment of the present invention have been vaccinated;

9A Figure 12 shows a bar graph of the HI titer of SPF chickens infected with the inactivated H5N2 avian influenza vaccine and / or the recombinant plasmid DNA with the 64-copy CpG motifs (pHCLS-64) according to a preferred embodiment of the present invention have been vaccinated;

9B Figure 12 shows a bar graph of the SI value of SPF chicks infected with the inactivated H5N2 avian influenza vaccine and / or the recombinant plasmid DNA with the 64-copy CpG motifs (pHCLS-64) according to a preferred embodiment of the present invention have been vaccinated;

10 Figure 12 shows a bar graph of the protection rate of SPF chicks vaccinated with the inactivated H5N2 avian influenza vaccine and / or the recombinant plasmid DNA with the 64-copy CpG motifs (pHCLS-64) according to a preferred embodiment of the present invention are;

11A Figure 12 shows a bar graph of the ND-HI titer in vivo of commercial chicks raised with the trivalent vaccine Nobilis ND-IB-IBD and / or the recombinant plasmid DNA with the 64-copy CpG motifs (pHCLS-64) according to a preferred embodiment of the present invention have been vaccinated;

11B Figure 12 shows a bar graph of the IBD-Ab titer in vivo of commercial chicks bound with the trivalent vaccine Nobilis ND-IB-IBD and / or the recombinant plasmid DNA with the 64-copy CpG motifs (pHCLS-64) according to a preferred embodiment of the present invention have been vaccinated;

11C Figure 12 shows a bar graph of the IBD-Ab titer in vivo of commercial chicks bound with the trivalent vaccine Nobilis ND-IB-IBD and / or the recombinant plasmid DNA with the 64-copy CpG motifs (pHCLS-64) according to a preferred embodiment of the present invention have been vaccinated;

12 Figure 12 shows a bar graph of MG-Ab titer in vivo of commercial chicks bound with the commercial MG vaccine and / or the recombinant plasmid DNA with the 64-copy CpG motifs (pHCLS-64) according to a preferred embodiment of the present invention Invention have been vaccinated; and

13 and 14 are DNA sequence diagrams of the immunostimulatory polynucleotide containing 64-copy CpG motifs according to an embodiment of the present invention, wherein an upstream-type initiator ( 13 ) and a downstream starter ( 14 ) can be used to perform the DNA sequence analysis.

DETAILED DESCRIPTION

Reference will now be made in detail to the present embodiments of the invention, examples of which are illustrated in the accompanying drawings. Wherever possible, the same reference numbers will be used in the drawings and the description to refer to the same or similar parts.

DNA adjuvant

As already mentioned above, the invention relates to a DNA adjuvant comprising a recombinant plasmid containing continuous multi-copy CpG motifs and a transformant containing the aforementioned recombinant plasmid. This recombinant plasmid, which contains an immunostimulatory polynucleotide inserted into a vector, is transformed into a host cell to achieve stable and rapid mass production of the immunostimulatory polynucleotide containing continuous multi-copy CpG motifs.

In particular, the recombinant plasmid described herein relates to an immunostimulatory polynucleotide inserted into a vector, which immunostimulatory polynucleotide may contain a sequence of continuous multi-copy CpG motifs and related functional sequences. In one example, the continuous multiple copy CpG motifs may be continuous copies 16-64 of bird specific CpG motifs, and the continuous multiple copy CpG motifs may have a sequence which may include, but is not limited to, a third polynucleotide with the Seq.id.-Nr. 2, a first polynucleotide with SEQ ID NO. 3 and a second polynucleotide with Seq.id.No. 4, a recombinant plasmid containing the immunostimulatory polynucleotide, or any combination thereof. In another example, the recombinant containing the third polynucleotide with SEQ ID NO. 4, deposited at the China Center for the Collection of Cultural Types of the University of Wuhan in China under the accession number CCTCC M 2010302.

Before proceeding, it is convenient to refer to the term "single-copy CpG motif", as discussed below, for an abbreviation of a sequence of the "copy-in CpG motif" that is linked by its associated functional sequence , to acquire.

The "continuous multiple-copy CpG motifs" as discussed below refer to a sequence of head-to-tail binding of multi-copy CpG motifs (which also contain related functional sequences, such as discussed above). For example, a "single-copy CpG motif" is typically intended to include a sequence of a certain number of TCG tandem repeats at the 5 'end, a "copy-in CpG" is intended to include bird-specific flanking sequences at two ends of the CpG motif, and 4 to Contains 6 guanines at a 3 'end, from the 5' end to the 3 'end of the "single copy CpG motif" in sequential form. In one example, the single-copy CpG motif may have a sequence with SEQ ID NO. 1 have. Also, "the single-copy CpG motif" is referred to an unmethylated dinucleotide sequence containing a cytosine, followed by guanine and linked by a phosphodiester bond. The TCG tandem repeats at a 5 'end can specifically increase the cell-mediated immunity of birds, the single CpG motif itself is the immunostimulatory sequence of birds, the bird-specific flanking sequences at two ends of the CpG motif provide the species specificity and the poly-G structure consisting of 4 to 6 guaninan at a 3 'end facilitates cellular uptake of the CpG DNA adjuvant. However, the immunostimulatory polynucleotide containing the "continuous multiple copy CpG motifs provided by the present invention can be constructed in a recombinant plasmid and then transformed into a host cell for stable and rapid mass production of the immunostimulatory polynucleotide, which is the continuous multiple copy CPG motives to achieve. The resulting immunostimulatory polynucleotide containing the continuous multi-copy CpG motifs can be less costly and lower Costs are as the DNA adjuvants of the prior art, and it can specifically improve those bird vaccines that have a low immunization strength. In addition, the immunostimulatory polynucleotide of the present invention containing the continuous multiple copy CpG motifs has resulted in unexpected or unaccumulated production and has an immunostimulatory effect of various avian vaccines far beyond the predictable results of the "single copy CpG motif" as will be recognized by one skilled in the art.

Process for the preparation of the DNA adjuvant

*: vogelspezifische FlankiersequenzIn one embodiment, the recombinant plasmid containing the aforementioned immunostimulatory polynucleotide with the continuous multicopy CpG motifs can be obtained by multiple enzymatic cleavage and ligations of a recombinant plasmid containing the single copy CpG motif, followed by gene cloning techniques. The resulting continuous multi-copy CpG motifs contain a sequence of head-to-tail binding of multi-copy CpG motifs (and also contain related functional sequences as discussed above). In detail, it should now open 1A to 1C Referring now to Figure 1, partial flowcharts of a method for producing a recombinant plasmid with continuous multi-copy CpG motifs according to the present invention are shown. In 1A becomes a recombinant plasmid 100 with the single-copy CpG motif (n = 1), and the single-copy CpG motif (n = 1) is shown in Table 1 as a sequence with SEQ ID NO. 1 listed. Tab. 1

*: bird-specific flanking sequence

In Table 1, the "single-copy CpG motif" typically includes, for example, from the 5 'end to the 3' end sequentially a restriction enzyme (RE) site A (eg, the italicised text "CTAGT" at 5 '). End), a certain number of TCG tandem repeats at the 5 'end (eg the bold text on the gray background), a copy CpG (eg the text enclosed in square brackets), bird specific Flanking sequences at two ends of the CpG motif (eg the text above the symbol "*"), 4 to 6 guanines at a 3 'end (eg the bold and italicized text on the gray background) and another place RE B (eg the italic text "T" at the 3 'end). The RE A site at the 5 'end of the single-copy CpG motif may have a sequence that is different from the RE B site at the 3' end, but both have the same cohesive end. The same two cohesive ends can be melted but not split by RE-A and / or RE-B splitting. For example, the cohesive 3 'end of the DNA fragment A can be ligated to the cohesive 5' end of the DNA fragment B such that a sequence of head-to-tail bonds is formed. It should be clarified here that for the purpose of mass production of the recombinant plasmid with the immunostimulatory polynucleotide, the sites RE A and RE B are provided at the 5 'end and at the 3' end of the immunostimulatory polynucleotide, respectively, and a site RE C on the vector provided that the recombinant plasmid is transformed with the continuous multi-copy CpG motifs into a host cell such as Escherichia coli.

For example, in one example, the RE A site at the 5 'end of the immunostimulatory polynucleotide may be SpeI as the italicized CTAGT sequence (ie, Spel recognizes and cuts the 6 bp sequence of 5'-ACTAGT-3', supra that five overhanging remnants CTAGT remain). For example, the RE B site at the 3 'end of the immunostimulatory ODN may be XbaI site like the italicized T sequence (ie XbaI recognizes and cuts the 6bp sequence of 5'-TCTAGA-3') Remaining piece T remains). In addition, the site RE C on the vector may, for example, be the site SacI, as does the sequence GAGGCT (ie SacI recognizes and cuts the 7 bp sequence of 5'-GAGGCTC). 3 'so that six overhanging remnants remain GAGGCT). The suitable vector may be other commercially available vectors such as the vector pBluescript II, the vector pBluescriptKS or the like. The sequences of the site RE A, the site RE B and the site RE C are more dependent on the desired vector and are less limiting to the aforementioned sequences.

It should now again on 1A Be referred. Thus, REs A and C are used to produce the recombinant plasmid 100 to recognize and cut to obtain the fragment N1, and the fragment N1 has at its 5 'end a single-copy CpG motif (ie, copy CpG motif and its associated functional sequence, represented as an empty block). The RE B and C are used to construct the recombinant plasmid 100 to recognize and cut to obtain fragment N2, and fragment N2 has at its 3 'end another single-copy CpG motif (represented as a block filled with diagonal pattern).

Then, the continuous single-copy CpG motif (represented as a block filled with a diagonal pattern) at the 3 'end of fragment N2 may be overlaid with another continuous single-copy CpG motif (represented as an empty block) at the 5' end of the fragment N1 ligated, creating a recombinant plasmid 110 (n = 2) is formed with a head-to-tail binding of two continuous single-copy CpG motifs, such as those in 1A is shown.

Similarly, the recombinant plasmid then becomes 110 (n = 2) were subjected to restriction enzyme digestion, ligation and gene cloning procedures in a similar manner as mentioned above, yielding a recombinant plasmid 110 (n = 4) is formed with head-to-tail binding of four continuous single-copy CpG motifs.

It should be up now 1B Be referred. First, the recombinant plasmid 110 (n = 2) containing two continuous single-copy CpG motifs (n = 2). Subsequently, REs A and C are also used to construct the recombinant plasmid 110 (n = 2) and to cut to obtain the fragment N3, and the fragment N3 has two single-copy CpG motifs at its 5 'end. The RE B and C are also used to produce the recombinant plasmid 110 (n = 2) and to cut to obtain fragment N4, and fragment N4 has another two single-copy CpG motifs at its 3 'end (represented as blocks filled with diagonal patterns).

Next, the two single copy CpG motifs (represented as blocks filled with diagonal patterns) at the 3 'end of fragment N4 may be overlaid with another single copy CpG motif (represented as empty blocks) at the 5' end of the fragment N3, resulting in a recombinant plasmid 120 (n = 4) is formed with head-to-tail binding of four single-copy CpG motifs, such as those in 1B is shown.

Then, a recombinant plasmid (n = 16, not shown) and a recombinant plasmid 150 (n = 32) by repeating the above steps several times without the need for unnecessary detail. In one example, the immunostimulatory polynucleotide containing sixteen copy CpG motifs (n = 16) is shown as a sequence with SEQ ID NO. 2 and the immunostimulatory polynucleotide containing thirty-two copy CpG motifs (n = 32) as a sequence with Seq.id.No. 3 listed.

It should be up now 1C Be referred. Here is the recombinant plasmid 150 (n = 32) containing thirty-two copy CpG motifs (n = 32). Subsequently, REs A and C are also used to construct the recombinant plasmid 150 (n = 32) and to obtain the fragment N11, and this fragment N11 has thirty-two-copy CpG motifs at its 5 'end. The RE B and C are also used to produce the recombinant plasmid 150 (n = 32) and to obtain the fragment N12, and this fragment N12 has another thirty-two-copy CpG motifs at its 3 'end (represented as blocks filled with diagonal patterns).

Next, the thirty-two-copy CpG motifs (represented as blocks filled with diagonal pattern) at the 3 'end of fragment N12 may be overlaid with another thirty-two copy CpG motif (represented as blank blocks) at the 5' end. End of the fragment N11, whereby a recombinant plasmid 160 (n = 64, pHCLS-64) is formed with head-to-tail binding of sixty-four copy CpG motifs as described in U.S. Pat 1C is shown. In one example, the immunostimulatory polynucleotide containing sixty-four copy CpG motifs (n = 64) is shown as a sequence with SEQ ID NO. 4 listed.

The aforementioned recombinant plasmid containing continuous multi-copy CpG motifs can be further transformed into a host cell such as Escherichia coli for the purpose of mass production. Thus, the present invention obviates the need for the complicated chemical modification of the prior art DNA adjuvant. Since bacterial transformation and large-scale production are subject to conventional techniques known to those skilled in the art, it is not necessary to provide details thereof.

hydrate seed

The invention additionally provides a avian vaccine containing a DNA adjuvant and a bird vaccine in which the DNA adjuvant comprises the above-mentioned immunostimulatory polynucleotide having the continuous multiple copy CpG motifs, the recombinant plasmid having the immunostimulatory polynucleotide which is transformant with the recombinant plasmid or any combination thereof. In one example, the aforementioned bird vaccine is a live vaccine against Newcastle disease, a live attenuated Newcastle disease vaccine, an inactivated Newcastle disease virus vaccine, an avian killed bird oil vaccine vaccine against avian mycoplasmosis ( Mycoplasma gallisepticum; MG) or the trivalent oil Nobilis dead vaccine (which is combined with the IB strain M41, the Bumboro strain D78 and the ND strain Clone 30).

In addition, the invention further provides a method for immunizing a bird against a pathogen, which comprises administering to a bird a bird vaccine containing such a DNA adjuvant and preparing this DNA adjuvant, as exemplified above is. In one embodiment, the method may include administering a vaccine with the aforementioned DNA adjuvant to the bird such that expression of the genes IFN-γ, TLR3 or TLR7 is increased in the bird.

Next, several applications of the DNA adjuvant to bird vaccines will be described in more detail, with reference being made below to several exemplary, non-limiting embodiments. Thus, one skilled in the art can appreciate the essential characteristics of the present invention and make various changes and modifications to the invention to adapt it to a variety of applications and conditions, without departing from the spirit and scope of the invention.

EXAMPLE 1

Construction of the recombinant plasmid containing the single-copy CpG motif

In this example, a polynucleotide having a sequence of SEQ ID NO. into a pGEM-T light vector which is cleaved by the RE A and B and then purified to form a recombinant plasmid containing a single-copy CpG motif. Next, this recombinant plasmid is subjected to a heat shock treatment to be transformed into a host cell such as a competent cell of E. coli DH5a for transformation and storage. Subsequently, the successfully transformed colonies are selected according to the blue-white screening method. The competent cells are cultured in the presence of isopropyl-β-D-1-thiogalactopyranoside (IPTG) and 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside (X-gal) to select the successfully transformed colonies. If the ligation was successful, the lacZ operon of the recombinant plasmid can be induced in the successfully transformed colonies by IPTG to express the β-galactosidase for cleavage of the X-gal and generation of an insoluble blue product. The DNA sequence of the successfully transformed colonies is accurate by DNA sequence confirmation and is mass-produced. However, construction of recombinant plasmids, transformation of host cells, blue-white screening, mass production, purification of plasmid DNA and the like are known to those skilled in the art, so that it is not necessary to refer to the corresponding Details to enter.

The plasmid DNA can be separated from the successfully transformed colonies, split by the RE A and C or the RE B and C, and then analyzed by DNA electrophoresis to check whether the single copy CpG motif is indeed recombinant Plasmid (n = 1) has been constructed. The RE A, B and C have already been described above, so that there is no need to specify unnecessary details here.

It should be up now 2A and 2 B Be referred. These are the results of agarose electrophoresis from the recombinant plasmid (n = 1), expressed by RE A and B ( 1A ) or the RE B and C ( 2 B ) has been split. In 2A and 1B the lane M refers to the DNA markers of the DNA ladders, and the indications "1.5 kb" and "2.0 kb" on the lefthand side of the lane M refer to a place of 1500 bp DNA ladders or 2000 bp. In 2A Lane 1 refers to the DNA fragment of the recombinant single-copy CpG motif plasmid that has been cleaved by the RE A and C and has a molecular weight of approximately 1.1 kb as indicated by the arrow 201 specified. In 2 B Lane 2 refers to the DNA fragment of the recombinant single copy CpG motif plasmid which has been cleaved by the RE B and C and has a molecular weight of approximately 1.8 kb as indicated by the arrow 203 specified.

According to the results of 2A and 2 B For example, the recombinant plasmid containing the single-copy CpG motif is cleaved from the RE A and C to obtain 1.1 kb of the DNA fragment (corresponding to fragment N1 in FIG 1A ) having a single-copy CpG motif at its 5 'end. The recombinant plasmids are cleaved by the RE B and C to obtain 1.8 kb of the DNA fragment (corresponding to fragment N2 in FIG 1A ) having another single-copy CpG motif at its 3 'end.

EXAMPLE 2

Construction of recombinant plasmids containing multi-copy CpG motifs

In this example, the recombinant plasmid (n = 1) of Example 1 is subjected to restriction enzyme cleavage, ligation, and gene cloning techniques in a manner similar to that of 1A to 1C whereby the recombinant plasmids are formed with multi-copy CpG motifs (n = 2, 4, 8, 16, 32, 64) with head-to-tail bonds. It is not necessary here to go into the details.

It should be up now 3 Be referred. This shows a result of agarose electrophoresis of the recombinant plasmid (n = 4, 8, 16, 32, 64) which has been split by RE A and B. In 3 the lane M refers to the DNA ladder DNA marker, and the "3.0 kb" and "0.5 kb" on the left side of the lane M refers to a place of the 3000 bp DNA ladder or 500 bp. Lane 1 refers to a four-copy CpG motif DNA fragment which has been cleaved by RE A and B and has a molecular weight of approximately 112 bp as indicated by arrow 301 specified. Lane 2 refers to an eight-copy CpG motif DNA fragment which has been cleaved by RE A and B and has a molecular weight of approximately 224 bp as indicated by arrow 303 specified. Lane 3 refers to a sixteen copy CpG motif DNA fragment which has been cleaved by RE A and B and has a molecular weight of approximately 448 bp as indicated by arrow 305 specified. Lane 4 refers to a thirty-two-copy CpG motif DNA fragment which has been cleaved by RE A and B and has a molecular weight of approximately 896 bp as indicated by arrow 307 specified. Lane 5 refers to a sixty-four-copy CpG motif DNA fragment which has been cleaved by RE A and B and has a molecular weight of approximately 1.8 kp, as indicated by arrow 309 specified. The above-mentioned DNA fragments are accurate by DNA sequence confirmation as in 13 and 14 shown.

It should now be on the 13 and 14 Which contain the DNA sequence diagrams of the immunostimulatory polynucleotide with 64-copy CpG motifs according to an embodiment of the present invention in which an upstream starter ( 13 ) and a downstream starter ( 14 ) can be used to perform the DNA sequence analysis. The texts o, 1 to o, 64 marked with a circle o respectively refer to the 1 to 64 copy CpG motifs. According to the results of 13 and 14 For example, the DNA fragments with the 64-copy CpG motifs are exact according to DNA sequence confirmation.

EXAMPLE 3

Evaluation of immunostimulatory activity in vitro using recombinant plasmids with multi-copy CpG motifs

1. Apply immunostimulatory activity in vitro to chick cells using recombinant plasmids with multi-copy CpG motifs

In this example, the spleen of chicks (Gallus gallus) is aseptically collected and gently homogenized to obtain a splenocyte suspension. The splenocytes are in a complete Medium RPMI-1640 (Sigma-Aldrich Co., MO) was resuspended at a cell density of approximately 1 x 10 ⁷ cells / ml, and 0.5 ml (approximately 5 x 10 ⁶ cells) of the cells are grown in each well a cell culture plate with 24 wells cultivated.

Subsequently, 50 μl of the vector (as control vector 10 μg / 50 ml) and the recombinant plasmid DNA disclosed in Examples 1 and 2 (n = 1, 2, 4, 8, 16, 32 and 64, 10 μg / 50 ml ) each added to the chick splenocytes. This is followed by incubation in the incubator at 41 ° C. for 6 hours. The culture of chick splenocytes is then centrifuged at 1000 U / min for 10 minutes and the cell pellet is subjected to the total RNA extraction by using the Trizol reagent ^® (Invitrogen Corp., Carlsbad, California). The total RNA of the splenocytes is subjected to PCR reverse transcription (RT-PCR) to obtain cDNA. Next, the expression of splenocyte IFN-γ, TLR3 or TLR7 genes is detected by real-time PCR and then normalized by expression of the β-actin gene. Here, the β-actin gene serves as an internal standard in RT-PCR because the β-actin gene is a routine gene that is constitutively expressed and participates in basic functions required for the maintenance of the cell. Thereby, the immunostimulatory activity of the recombinant plasmid containing the 64-copy CpG motifs can be evaluated on chick lymphocytes in vitro.

Real-time PCR can be performed under the conditions suggested in the manufacturer's operating manual, such as Lightcycle Faststart DNA Master SYBR Green I (Roche Applied Science, Germany). This commercial product may contain the Lightcycle Fast-Start Enzyme, the Lightcycle Fast-Start Enzyme Reaction Reaction, and 25 mM MgCl ₂ . Briefly, 5.8 μl of PCR-grade water, 1.2 μl of the 25 mM MgCl ₂ , 0.5 μl of the starter pair, 1 μl of SYBR Green I and 1 μl of cDNA are mixed well, and then the reaction mixture is allowed in Roche Molecular Biochemicals Lightcycle Software (Roche Applied Science, Germany). After the reaction is complete, the expression of the genes IFN-γ, TLR3 or TLR7 is analyzed; the result is in 4 to 6 shown.

It should be up now 4 to 6 Reference is made to where in the form of bar graphs the expression of the chick splenocyte IFN-γ, TLR3 or TLR7 genes is represented by the recombinant plasmids with the single or multiple copy CpG motif (s) Examples 1 and 2 have been stimulated in vitro. In 4 to 6 For example, the horizontal axis shows the recombinant plasmids containing the single or multiple copy CpG motifs (n = 1, 2, 4, 8, 16, 32 and 64) and pHCLS-64 refers to the recombinant plasmids containing the 64-copy CpG motifs) or the empty vector, and on the vertical axis the expression of the genes IFN-γ, TLR3 or TLR7 is shown, calculated as geometric mean (Log ₂ (2 ^-ΔΔCt )) and each group is analyzed in triplicate. It should be noted here that the geometric mean exponent "-ΔΔCt" (Log ₂ (2 ^-ΔΔCt ) refers to the fact that the change in fluorescence intensity (ΔCt) of the expression of the genes IFN-γ, TLR3 or TLR7, the from the recombinant plasmids with the continuous multiple copy CpG motifs or from the empty vector of Example 2, the change in fluorescence intensity (ΔCt) is subtracted from that gene expression which has not been stimulated by the recombinant plasmids or empty vector of Example 2 The symbol "*" refers to the significantly increased expression of the genes IFN-γ, TLR3 or TLR7 in cells stimulated by the recombinant plasmids with the continuous multi-copy CpG motifs in the Comparison with those in the control studies (P <0.05).

According to the result of 4 the expression of the IFN-γ gene stimulated by the recombinant plasmids with the 32- or 64-copy CpG motifs (32 × CpG or 64 × CpG) is more significant than the expression of the IFN-γ gene; that by the control group (PBS) and by the recombinant plasmids with the 16-copy CpG motifs (16x CpG) or the lower than the 16-copy CpG motifs (8x, 4x, 2x or 1x) CpG) has been stimulated.

In addition, according to the results of the 5 and 6 the expression of the TLR3 and TLR7 genes stimulated by the recombinant plasmids with 64-copy CpG motifs (64 x CpG), more significant than the expression of the TLR3 and TLR7 genes produced by the control group (PBS) and the recombinant Plasmids with the 32-copy CpG motifs (32 × CpG) or lower than the 32-copy CpG motifs (16 ×, 8 ×, 4 ×, 2 × or 1 × CpG) has been stimulated.

In addition, the results of the in vitro experiments of Example 3 show that the recombinant plasmid with the 8, 16, 32 and 64 copy CpG motifs has immunostimulatory activity. Therefore, the immunostimulatory polynucleotide should have at least higher than 16-copy CpG motifs and the immunostimulatory polynucleotide with the 32- and 64-copy CpG motifs can Expression of the genes considerably increase IFN-γ, TLR3 or TLR7. However, the degree of immunostimulatory activity is not proportional to the copy number of CpG motifs in the immunostimulatory polynucleotide. In other words, the degree of immunostimulatory activity may not be plausibly and accurately expected from the copy number of CpG motifs, especially for 16-copy CpG motifs or above.

EXAMPLE 4

Representation of avian vaccine containing recombinant plasmids with multi-copy CpG motifs

1. Presentation of the viral antigen and the vaccine against Newcastle Disease (ND) of chicks

Initially, the immortalized cell line (accession number: ATCC CCL-10, deposited at the American Collection Center for Culture Types, USA) or accession number: BCRC 60041, deposited with the Research and Development Institute for the Food Industry (FIRDI), Collection and Research Center for Bioresources (BCRC ), Hsinchu, Taiwan) with the chicken ND virus strain Ishii (Kaohsiung Biological Product Co. Ltd., Taiwan, original source: Animal Health Research Institute, Agriculture Council, Yuan Branch, Taiwan). The inoculated cultures are incubated for 72 hours in a humidified atmosphere with 5% CO ₂ at 37 ° C before the virus fluid is collected. The collected virus fluid is inactivated by formalin (Sigma, USA), which has 0.2% of the final concentration. Next, the Newcastle Disease Tissue Culture Vaccine (NDTC vaccine) is mixed with the inactivated virus and the aluminum adjuvant, and test vaccines are mixed with the DNA adjuvant, such as the recombinant plasmid DNA with the individual (s) or 64-copy CpG motif (s) of Example 2 or the vector DNA of Example 1 (NDTC-1 × CpG or NDTC-64 × CpG or NDTC vector, respectively).

2. Presentation of the viral antigen and vaccine against H5N2 avian influenza virus

First, allantoic fluid from 9 to 11 day old specific pathogen-free eggs (SPF eggs) is inoculated with the H5N2 avian influenza virus (A / Duck / Taiwan / 3233/04 (H5N2) provided by Professor Ching-Ho Wang, University Veterinary School Taiwan) (V52). The inoculated eggs are incubated at 37 ° C for 72 hours before the virus fluid is collected. The collected virus fluid is inactivated by formalin (Sigma, USA), which has 0.2% of the final concentration. Next, the inactivated vaccine V52 is mixed with the inactivated virus fluid and the aluminum adjuvant, and test vaccines are mixed with a DNA adjuvant, such as the recombinant plasmid DNA with the single or 64-copy CpG motif of Example 2 or the vector DNA of Example 1 (V52-1 × CpG or V52-64 × CpG or V52 vector, respectively).

3. Presentation of the trivalent vaccine Nobilis-ND-IB-IBD

The trivalent vaccine Nobilis-ND-IB-IBD can be a commercially available product, such as, for example, the dead oil vaccine Nobilis-IB (inactivated infectious bronchitis strain M41) + G (inactivated Gumboro strain D78) or so-called infectious bursal disease ( IBD) + ND (inactivated ND clone 30) manufactured by Intervet / Schering-Plow Animal Health (Netherlands).

4. Vaccine against avian mycoplasma gallisepticum (MG vaccine)

The avian mycoplasma gallisepticum (MG vaccine) vaccine may be a commercially available product such as the MG vaccine (inactivated MG strain S6) as an oil death vaccine (manufactured by Merial Animal Health (Gainesville, GA).

EXAMPLE 5

Evaluation of immunostimulatory activity in vivo using recombinant plasmids with 64-copy CpG motifs (pHCLS-64) - 1

1. Evaluation of hemagglutination inhibitor titre (HI titer)

In this example, the five week old SPF chicks are divided into four test groups and one control group, with each group having 5 chicks each. These receive intramuscular immunization by application of a dose of phosphate buffered saline (PBS, first group, negative control group) 1 ml, NDTC vaccine of Example 4 (second group, test group) 1 ml, NDTC vector vaccine of Example 4 (third group, test group, vector of Example 1) 1 ml, NDTC-1 x CPG vaccine of Example 4 (fourth group, test group, the recombinant plasmid with the single-copy CpG motif of Example 2) 1 ml and NDTC -pHCLS-64 vaccine of Example 4 (fifth group, test group; the recombinant plasmid with the 64-copy CpG motif (pHCLS-64) of Example 2) 1 ml.

Prior to immunization, two weeks and three weeks after primary immunization (PI-2wk or PI-3wk), total chick blood is collected to analyze the hemagglutination inhibition antibody titers (HI titer; Log 2) against the ND virus; the results are in 7A shown.

A typical HI test is exemplified as follows. First, 25 μl of a solvent (0.85% normal saline, pH 7.2) is added to each V-well well of a 96-well microplate. Next, 25 μl of the test sera from each column from the first well to the eleventh well are diluted sequentially using the Pipetman pipette, and each last row column (eg, the twelfth well) is left for the control group ( control well). Then 4 HA units of viral antigen fluid per 25 μl are added to each well containing the test sera. After 30 minutes rest at room temperature, 25 μl of 1% (v / v) chick red blood cell suspension is added to each well including the control wells in the last row. After another 30 minute rest period at room temperature, the settling patterns for each serum sample are determined and the end point determined at which complete inhibition of hemagglutination occurs, taking the control well as a benchmark. The reciprocal of the endpoint value is the HI titer.

Now reference should be made 7A be taken. Shown here is a bar graph of HI titre of chicks raised with the inactivated ND vaccine (NDTC) obtained in cell culture and / or the plasmid DNA with the 64-copy CpG motifs (pHCLS-64) according to the preferred Embodiment of the present invention have been inoculated. The horizontal axis of the 7A represents the different ways of immunization. For example, "PBS" means immunization with the phosphate buffer saline solution (PBS), "NDTC" means mere immunization with the inactivated NDTC vaccine, "NDTC vector" the common immunization with NDTC Vaccine and the Vector DNA of Example 1, "NDTC-1 × CpG", the co-immunization with NDTC vaccine and the recombinant plasmid DNA with the single-copy CpG motif of Example 2 and finally "NDTC-pHCLS-64" co-immunization with NDTC vaccine and recombinant plasmid DNA with the 64-copy CpG motifs (pHCLS-64) of Example 2. On the vertical axis of 7A For example, the HI titers (Log 2) to the ND virus are plotted in the test sera for two weeks and three weeks post-immunization (p <0.05). The symbol "*" refers to the significantly increased HI titers in chicks co-administered with the NDTC-pHCLS-64 vaccine (containing pHCLS-64 of Example 2) compared to those in the control groups (P <0 , 05).

According to the result of 7A can not be detected in the negative group of HI titers. HI titers in chicks co-administered with the NDTC vaccine, the NDTC vector vaccine and the NDTC-1 x CPG vaccine are significantly increased over those of the control group, but have no significant differences between the three test groups , neither two weeks (empty bar) nor three weeks (with bars filled) after immunization. However, the HI titer in chicks co-administered with the NDTC-pHCLS-64 vaccine (containing pHCLS-64 of Example 2) is significantly increased over those in the above three test groups (test groups two, three and four) ( P <0.05).

2. Evaluation of the stimulation index (SI)

In this example, all the chicks are immunized as in the aforementioned first evaluation of Example 5. Three weeks after the immunization, the peripheral blood mononuclear cells (PBMC) are collected to perform the SI test so that vaccine stimulated immune activity can be assessed; the result is in 7B shown.

In this embodiment, the peripheral blood of the chicks is collected. Next, a density gradient such as a Ficoll-Hypaque will ^® solution (Sigma-Aldrich Co., MO, USA) was added to lymphocyte separation into a tube, followed by a careful recovery of the PBMC. Subsequently, the PBMC are counted and resuspended in a complete RPMI1640 medium (Sigma-Aldrich Co., MO, USA) at a cell density of approximately 4 × 10 ⁶ cells / ml, and 2 × 10 ⁵ cells / 50 μl of the PBMC cultured in each well of a 96-well microplate. Subsequently, the same volume of Concanavalin A (Con A; 1 μg / 50 μl) and the culture medium are added to each well, each group being one Duplicate and incubated for 48 hours in a humidified atmosphere with 5% CO ₂ at 37 ° C. Later, 10 μm / well BrDU reagent (bromodeoxyuridine, analog of thymidine) is added to each well and incubated for 24 hours to label the cells. After centrifuging the microplate at 1000 rpm for 10 minutes to remove the supernatant, add 200 μl / well of FixDenat solution to each well, incubate at room temperature for 30 minutes, and discard the fix-denate solution. Then, 100 μl / well of anti-BrdU-pod working solution is added to each well and incubated for 90 minutes at room temperature. After removing the anti-BrdU-pod working solution, add 300 μl / well of wash solution to each well and rinse three times. The wash solution is then removed and 100 μl / well substrate solution added to each well, incubated at room temperature for 5 minutes and stained. Then the absorbance is measured with the ELISA reader at 370 nm with a reference wavelength of 492 nm.

It should be up now 7B Referring to Figure 1, a bar graph illustrates chick stimulation index (SI) associated with the NDTC vaccine and / or the recombinant plasmid DNA with the 64-copy CpG motifs (pHCLS-64) according to a preferred embodiment of the present invention vaccinated according to the present invention. The horizontal axis of the 7B represents the different ways of immunization. For example, "PBS" means immunization with the phosphate buffer saline solution (PBS), "NDTC" the mere immunization with inactivated NDTC vaccine, "NDTC vector" the joint immunization with the NDTC vaccine and of the vector DNA of Example 1, "NDTC-1 × CpG", the co-immunization with the NDTC vaccine and the recombinant plasmid DNA with the single-copy CpG motif of Example 2 and finally "NDTC-pHCLS-64" the common Immunization with the NDTC vaccine and the recombinant plasmid DNA with the 64-copy CpG motifs (pHCLS-64) of Example 2. On the vertical axis of 7B the PBMC-SI value of the immunized animals is plotted during the three weeks after immunization. The SI value also refers to the ratio between the measured value of the BrdU chemiluminescence of the PBMC of the test groups (ie the immunized animals containing the vector, the NDTC vaccine, the NDTC vector vaccine and the NDTC-1 x CpG Vaccine co-administered) relative to that in the control groups, referred to as "1-fold increment," and the measured value of BrdU chemiluminescence of the control PBMC (PBS) (p ☐ 0.05). The symbol "*" refers to the significantly increased PBMC-SI in chicks coadministered with the NDTC-pHCLS-64 vaccine (containing pHCLS-64 of Example 2) compared to those in the control groups (P < 0.05).

According to the result of 7B For example, the SI values at which the NDTC vaccine, the NDTC vector vaccine and the NDTC-1 × CpG vaccine were co-administered are significantly increased over those of the control group, but show no significant differences between the three test groups for the three weeks after immunization. However, the SI value in chicks administered NDTC-pHCLS-64 vaccine (containing pHCLS-64 of Example 2) is significantly increased over those in the above three test groups (test groups two, three and four) (P < 0.05).

It is apparent that the recombinant plasmid DNA containing the 64-copy CpG motifs (pHCLS-64) can serve as an adjunct of the NDTC vaccine and indeed promotes immunostimulatory activity in vivo.

EXAMPLE 6

Evaluation of immunostimulatory activity in vivo using recombinant plasmids with 64-copy CpG motifs (pHCLS-64) - 2

1. Evaluation of the HI titer

In this example, the five week old SPF chicks are divided into four test groups and one control group, with each group having 5 chicks each. These receive intramuscular immunization by application of a dose of phosphate buffered saline (PBS, first group, negative control group) 1 ml, vaccine V52 of example 4 (second group, test group) 1 ml, V52 vector vaccine of example 4 (third group, Test Group; Vector of Example 1) 1 ml, V52-1x CPG vaccine of Example 4 (fourth group, test group; the recombinant plasmid with the single copy CpG motif of Example 2) 1 ml and V52-pHCLS-64 vaccine of Example 4 (Fifth Group, Test Group; the recombinant plasmid with the 64-copy CpG motif (pHCLS-64) of Example 2) 1 ml. All experimental chicks are second immunized with 1 dose of the appropriate vaccine two weeks after the primary immunization been refreshed (BI).

Prior to immunization, two weeks after primary immunization (PI-2wk) and 2 weeks after secondary immunization (BI-2wk), total chick blood is collected to analyze HI titer (Log 2) against H5N2 avian influenza virus; the result is in 8A shown. The HI test is carried out according to the method of the above-mentioned HI test of Example 5, first item, so that it is unnecessary to specify unnecessary details.

It should be up now 8A Be referred. This represents a bar graph of the HI titer of chicks infected with the inactivated H5N2 avian influenza vaccine (vaccine V52) and / or the recombinant plasmid DNA with the 64-copy CpG motifs (pHCLS-64) according to a preferred Embodiment of the present invention have been vaccinated. The horizontal axis of the 8A represents the different ways of immunization. Thus, "PBS" designates the immunization with the phosphate buffer saline solution (PBS), "V52" the mere immunization with the inactivated H5N2 avian influenza vaccine (vaccine V52), "V52 vector" the common Immunization with vaccine V52 and the DNA vector of Example 1, "V52-1 × CpG", the co-immunization with vaccine V52 and the recombinant plasmid with the single-copy CpG motif of Example 2 and finally "V52-pHCLS-64 "Co-immunization with vaccine V52 and recombinant plasmid DNA with the 64-copy CpG motifs (pHCLS-64) of Example 2. On the vertical axis of 8A For example, the HI titers (Log 2) against the V52 virus are plotted in the test sera two weeks after the primary immunization and two weeks after the secondary immunization (PI-2wk and Bi-2wk respectively) (p <0.05). The symbol "*" refers to the significantly increased HI titers in chicks co-administered with the V52 pHCLS-64 vaccine (containing pHCLS-64 of Example 2) compared to those in the control groups (P < 0.05).

According to the result of 8A can not be detected in the negative group (PBS) of the HI titers. HI titers in chicks coadministered with vaccine V52, V52 vector vaccine and vaccine V52-1x CPG are more significantly elevated than those of the negative group, but show no significant differences between the three test groups two weeks after the primary immunization (empty bar) two weeks after the secondary immunization (gray bar). However, the specific HI titer in chicks co-administered with V52-pHCLS-64 vaccine (containing pHCLS-64 of Example 2) is significantly more elevated than those in the above three test groups (test groups two, three and four) (P <0.05).

2. Evaluation of the stimulation index (SI)

In this example, all experimental chicks are immunized as in the first evaluation of Example 6. Two weeks after the secondary immunization (BI-2wk), the PBMC are collected to perform the SI test to evaluate vaccine-stimulated immune activity, such as 8B shows. Chick PBMC are collected using the second-guess method of Example 5, thus eliminating the need for detail.

It should be up now 8B In which a bar graph depicts the stimulation index (SI) of chicks infected with the inactivated H5N2 avian influenza vaccine and / or the recombinant plasmid DNA with the 64-copy CpG motifs (pHCLS-64) according to a preferred embodiment of the present invention have been vaccinated. The horizontal axis of the 8B represents the different ways of immunization. For example, "PBS" refers to immunization with PBS, "V52" refers to mere immunization with inactivated H5N2 avian flu vaccine, "V52-vectors" common immunization with vaccine V52 and the DNA vector of Example 1, "V52-1 × CpG", co-immunization with vaccine V52 and the recombinant plasmid DNA with the single-copy CpG motif of Example 2, and "V52-pHCLS-64" finally refers to co-immunization vaccine V52 and recombinant plasmid DNA with the 64-copy CpG motifs (pHCLS-64) of Example 2. On the vertical axis of 8B the PBMC-SI value of the immunized animals is plotted during the two weeks following the secondary immunization. The SI value also refers to the ratio of the measured value of the BrdU chemiluminescence of the PBMC of the test groups (ie, the immunized animals receiving the vector, the V52 vaccine, the V52 vector vaccine and the V52-1x CpG vaccine co-administered) relative to the measured value of BrdU chemiluminescence of the control PBMC (PBS) (p <0.05). The symbol "*" refers to the significantly increased PBMC-SI in chicks co-administered with the V52-pHCLS-64 vaccine (including pHCLS-64 of Example 2) compared to those in the control groups (P <0.05).

According to the result of 8B For example, PBMC-SI levels are more significant in chicks coadministered with V52 vaccine, V52 vector vaccine and V52-1x CpG vaccine increased compared to those of the control group, but showed no significant differences between the three test groups for the two weeks after the secondary immunization. However, the PBMC-SI value in chicks administered V52-pHCLS-64 vaccine (including pHCLS-64 of Example 2) is significantly increased over those in the above three test groups (test groups two, three and four) ( P <0.05).

It is apparent that the recombinant plasmid DNA containing the 64-copy CpG motifs (pHCLS-64) can serve as an adjunct of the vaccine V52 and indeed promotes immunostimulatory activity in vivo.

EXAMPLE 7

Evaluation of immunostimulatory activity in vivo using recombinant plasmids with 64-copy CpG motifs (pHCLS-64) - 3

1. Evaluation of the HI titer

In this example, the five-week-old SPF chicks are divided into two test groups and one control group, with each group having 5 chicks each. These receive intramuscular immunization by the application of a dose of phosphate buffered saline (PBS, first group, negative control group) 1 ml, vaccine V52 of example 4 (second group, test group) 1 ml and V52-pHCLS-64 vaccine of example 4 (third Group, Test Group; the recombinant plasmid with the 64-copy CpG motif (pHCLS-64) of Example 2) 1 ml. All experimental chicks were boosted (BI) with 1 dose of the appropriate vaccine two weeks after the primary immunization as a second immunization ,

Prior to immunization, two weeks after primary vaccination and two weeks after secondary immunization, total chick blood is collected to analyze HI titers (Log 2) against H5N2 avian influenza virus; the result is in 9A shown. The HI test is carried out according to the method of the above-mentioned HI test of Example 5, first item, so that the explanation of unnecessary details is unnecessary.

It should be up now 9A Be referred. This represents a bar graph of the HI titer of chicks infected with the inactivated H5N2 avian influenza vaccine and / or the recombinant plasmid DNA with the 64-copy CpG motifs (pHCLS-64) according to a preferred embodiment of the present invention have been vaccinated. The horizontal axis of the 9A illustrates the different ways of immunization. For example, "PBS" refer to immunization with phosphate buffer saline (PBS), "V52" to mere immunization with inactivated H5N2 bird flu vaccine (V52 vaccine) and "V52-pHCLS-64". co-immunization with vaccine V52 and recombinant plasmid DNA with the 64-copy CpG motifs (pHCLS-64) of Example 2. On the vertical axis of 9A For example, the HI titers (Log 2) against the V52 virus are plotted in the test sera two weeks after the primary immunization (PI-2wk) and two weeks after the secondary immunization (Bi-2wk) (p <0.05). The data "a, b and c" indicate that the averages with different letters are significantly different two weeks after the primary immunization (P <0.05) and the indications "x, y and z" indicate that the Means with different letters two weeks after the secondary immunization are significantly different (P <0.05).

According to the result of 9A the HI titer in the negative group (PBS) can not be detected. The specific HI titer in chicks co-administered with vaccine V52-pHCLS-64 (containing pHCLS-64 of Example 2) is significantly increased over that of the control and test group (vaccine V52) (P <0.05). ,

2. Evaluation of the stimulation index (SI)

In this example, all experimental chicks are immunized as in the first evaluation of Example 7. Two weeks after the secondary immunization, the chicks' PBMC are collected to perform the SI test to evaluate vaccine-stimulated immune activity, such as 9B shows. Chick PBMC are collected using the second-guess method of Example 5, thus eliminating the need for detail. The data "a, b and c" indicate that the mean values with different letters are significantly different two weeks after the primary immunization (P <0.05).

According to the result of 9B the SI value in chicks co-administered with vaccine V52-pHCLS-64 (containing pHCLS-64 of Example 2) is significantly increased over that in the control and test groups (V52 vaccine) (P <0, 05).

3. Evaluation of the protection rate of vaccines

As mentioned earlier, two weeks after secondary immunization, all test-pups are further challenged for intramuscular injection by the application of 10 ^8.5 EID ₅₀ (50% embryonic infectious dose) of the highly virulent avian influenza H5N1 virus, subtype (A / Duck / China / E319). This sub-type was isolated from a smuggled end on the Kinmen Kinmen Island (source: Research Institute for Animal Health, Agriculture Council, Yuan Branch, Taiwan). Later, the clinical syndromes of all test chicks are observed for two weeks and the survival rate is calculated for all birds.

It should be up now 10 In which a bar graph represents the protection rate of chicks infected with the inactivated H5N2 avian influenza vaccine and / or the recombinant plasmid DNA with the 64-copy CpG motifs (pHCLS-64) according to a preferred embodiment of the present invention vaccinated according to the present invention. The horizontal axis of the 10 illustrates the different ways of immunization. For example, "PBS" refer to immunization with phosphate buffer saline (PBS), "V52" to mere immunization with inactivated H5N2 bird flu vaccine (V52 vaccine) and "V52-pHCLS-64". co-immunization with vaccine V52 and recombinant plasmid DNA with the 64-copy CpG motifs (pHCLS-64) of Example 2. On the vertical axis of 10 the protection rate (%) against the H5N1 virus is shown in the test sera two weeks after the primary immunization, with each group being analyzed with at least five replications (p <0.05).

According to the result of 10 the protection rate of the control group is 0%. However, the chick protection rate co-administered with vaccine V52-pHCLS-64 (containing about 100%) (containing pHCLS-64 of Example 2) is higher than the test groups two weeks after the secondary immunization (V52 vaccine, protection rate 80%).

It is apparent that the recombinant plasmid DNA containing the 64-copy CpG motifs (pHCLS-64) can serve as an adjunct of the V52 vaccine and indeed promotes immunostimulatory activity in vivo and strongly cross-protective virulent subtype H5N1 of avian influenza virus.

EXAMPLE 8

Evaluation of immunostimulatory activity in vivo using recombinant plasmids with 64-copy CpG motifs (pHCLS-64) - 4

In this example, forty chicks are fed until they are three weeks old without being grouped. The younger chicks are given chick starter feed (containing 22.5% crude protein (CP) and at least 25% fat, Fwusow Industry Co., Ltd.) ad libitum, reverse osmosis water is also available ad libitum.

Then these three week old chicks are divided into 4 groups of 10 chicks per group. These are given an intramuscular immunization according to Table 2. The chicks are given ad libitum chick growth supplement (containing 19.0% CP and more than 5.5% fat, Fwusow Industry Co., Ltd.), and reverse osmosis water is also available ad libitum available. Tab. 2 group hydrate seed 1 2 3 4 PBS Trivalent vaccine Nobilis ND-IB-IBD of Example 4 Vaccine Nobilis ND-IB-IBD + Vector of Example 4 (50 μg of the vector DNA) Vaccine Nobilis ND-IB-IBD-pHCLS-64 of Example 4 (50 μg of the pHCLS-64-DNA)

• (1) HI-Test: 4-HA-Einheiten (HAU) des Antigens können beim HI-Test nach den derzeit auf Taiwan üblichen Methoden, die nachfolgend exemplarisch dargestellt werden, benutzt werden. Zunächst erhält jede Vertiefung mit V-Boden einer Mikroplatte mit 96 Vertiefungen 25 μl Lösungsmittel (0,85% normale Kochsalzlösung, pH 7,2). Als Nächstes werden 25 μl der Testseren aus jeder Spalte von der ersten Vertiefung bis zur elften Vertiefung unter Verwendung einer Poipetman-Pipetten nacheinander verdünnt, und die Spalte der jeweils letzten Reihe (z. B. die zwölfte Vertiefung) wird für die Kontrollgruppe gelassen (Kontrollvertiefung). Dann werden 4 HA-Einheiten der Virus-Antigen-Flüssigkeit pro 25 μl in jede Vertiefung, welche die Testseren enthält, gegeben. Nach einer 15-minütigen Ruhezeit bei Zimmertemperatur werden 50 μl 0,9% (v/v) Suspension der roten Blutzellen der Küken jeder Vertiefung einschl. der Kontrollvertiefung in der letzten Reihe gegeben. Nach einer weiteren Ruhezeit über eine Stunde bei Zimmertemperatur werden die Absetzmuster für jede Serumprobe ermittelt und der Endpunkt festgelegt, wo vollständige Hemmung der Hämagglutination erfolgt, wobei das Kontrollvertiefung als Vergleichspunkt genommen wird. Der Kehrwert des Endpunktes ist der HI-Titer.
• (2) ELISA-Test: Bei diesem Beispiel kann der ELISA-Kit (Kirkegaard & Perry Laboratories, Inc.; KPL) benutzt werden, um den spezifischen IBD- oder IB-Antikörpertiter im Kükenblut nachzuweisen.

The total blood of the test chicks is collected at 0, 1, 2 and 3 weeks after the primary immunization to analyze the specific antibody titer in the sera using the following exemplary methods of the HI test or the ELISA test.

• (1) HI test: 4-HA units (HAU) of the antigen can be used in the HI test according to the methods currently used in Taiwan, which are exemplified below. First, each V-bottom well of a 96-well microplate receives 25 μl of solvent (0.85% normal saline, pH 7.2). Next, 25 μl of the test sera from each column from the first well to the eleventh well are successively diluted using a Poipetman pipette, and the last-row column (eg, the twelfth well) is left for the control group (control well ). Then 4 HA units of viral antigen fluid per 25 μl are added to each well containing the test sera. After a 15 minute rest period at room temperature, 50 μl of 0.9% (v / v) suspension of chick red blood cells are added to each well including the control well in the last row. After a further one hour rest period at room temperature, the settling patterns for each serum sample are determined and the end point determined where complete inhibition of hemagglutination occurs taking the control well as a benchmark. The reciprocal of the endpoint is the HI titer.
• (2) ELISA Assay: In this example, the ELISA kit (Kirkegaard & Perry Laboratories, Inc., KPL) can be used to detect the specific IBD or IB antibody titer in chick blood.

It should be up now 11A to 11C Figures in which bar graphs depict the in vivo HI titers of chicks raised with the trivalent Nobilis ND IB IBD vaccine and / or the recombinant plasmid DNA with the 64-copy CpG motifs (pHCLS). 64) according to a preferred embodiment of the present invention have been vaccinated. On the horizontal axes of the 11A to 11C are the weeks after the primary immunization shown. On the vertical axes of the 11A to 11C are the ND HI titers (Log 2; 11A ), the antibody titer (Ab titer) to IBD (Log 2; 11B ) or the Ab titers to IB (Log 2; 11C ) in the test sera at weeks 0 to 3 after the primary immunization (p <0.05). In the 11A to 11C "PBS" refers to the immunization with PBS (control, the column with the diagonal strips from left to right), "ND-IB-IBD" the mere immunization with the trivalent vaccine Nobilis-ND-IB-IBD (the test group, column with the Diagonal bars from right to left), "ND-IB-IBD + vector" co-administration of the trivalent vaccine Nobilis-ND-IB-IBD and the vector DNA of Example 1 (test group, empty column) and "ND-IB-IBD + pHCLS-64 "coadministration of the trivalent vaccine Nobilis-ND-IB-IBD and the recombinant plasmid DNA with the 64-copy CpG motifs (pHCLS-64) of Example 2 (test group, dotted column). The symbol "*" refers to the significantly increased HI titer in chicks co-administered with the trivalent vaccine Nobilis ND-IB-IBD and the recombinant plasmid DNA with the 64-copy CpG motifs (pHCLS-64) compared with those in control groups (P <0.05).

After the result of 11A to 11C In the control and test groups, the Ab titers in the weeks zero and one after the primary vaccination do not differ significantly. However, two and three weeks after the secondary immunization, the specific ND-Ab titers in chicks containing "ND-IB-IBD + pHCLS-64" (containing the trivalent vaccine Nobilis-ND-IB-IBD and pHCLS-64 of Example 2 ) in 11A significantly higher than those in the other test groups (trivalent vaccine Nobilis-ND-IB-IBD) and the control group (PBS). In addition, three weeks after immunization, the specific IBD-Ab titers, coadministration "ND-IB-IBD + pHCLS-64" (containing the trivalent vaccine Nobilis-ND-IB-IBD and pHCLS-64 of Example 2) and " ND-IB-IBD + vector "(trivalent vaccine Nobilis-ND-IB-IBD and vector DNA) in 11B higher than those of the other test groups (only trivalent vaccine Nobilis-ND-IB-IBD).

It is apparent that the recombinant plasmid DNA containing the 64-copy CpG motifs (pHCLS-64) can serve as adjuvant of the trivalent vaccine Nobilis ND-IB-IBD and indeed promotes immunostimulatory activity in vivo and provides immunological protection against the ND virus and the IBD virus.

EXAMPLE 9

Evaluation of immunostimulatory activity in vivo using recombinant plasmids with 64-copy CpG motifs (pHCLS-64) - 5

In this example, forty chicks are fed as described in Example 8 until three weeks old without being grouped. Then these three week old chicks are divided into 4 groups to 10 chicks per group. These are numbered and subjected to an intramuscular immunization according to Tab. The chicks are also fed as in Example 8. Tab. 3 group hydrate seed 1 2 3 4 PBS MG vaccine of Example 4 MG vaccine of Example 4 (50 μg of vector DNA) MG vaccine of Example 4 (50 μg of pHCLS-64 DNA)

The total blood of the test chicks is collected at 0, 1, 2, and 3 weeks after the primary vaccination to analyze the MG-specific antibody titer in the sera using the following exemplary methods of the ELISA assay, as exemplified in Example 8.

It should be up now 12 In which a bar graph depicts the MG-Ab titer in vivo of chicks raised with the MG avian influenza vaccine and / or the recombinant plasmid DNA with the 64-copy CpG motifs (pHCLS-64). have been vaccinated according to a preferred embodiment of the present invention. On the horizontal axis of the 12 the individual weeks are shown after the primary vaccination. On the vertical axis of the 12 the MG-Ab titer against MG (log 2) is shown in the test sera at weeks 0 to 3 after primary vaccination (P <0.05). In the 12 "PBS" refers to the immunization with PBS (control, the column with the diagonal strips from left to right), "MG" the mere immunization with the MG vaccine (the test group, column with the diagonal bars from right to left), "MG + Vector "the co-administration of the MG vaccine and the vector DNA of Example 1 (test group, empty column) and" MG + pHCLS-64 "the co-administration of the MG vaccine and the recombinant plasmid DNA with the 64-copy CpG motifs (pHCLS-64 of Example 2 (test group, dotted column). The symbol "*" refers to the significantly increased Ab titre in chicks containing the MG vaccine and the recombinant plasmid DNA with the 64-copy -CpG motifs (pHCLS-64) were co-administered compared with those in control groups (P <0.05).

After the result of 12 In the three groups at week zero and one after primary vaccination, ab titers did not significantly differ. However, two and three weeks after the primary immunization, the specific Ab titers in chicks co-administered with "MG + pHCLS-64" (containing the MG, vaccine and pHCLS-64 of Example 2) are significantly higher than in the chicks labeled with "MG + Vector" (MG vaccine and vector DNA) in 12 or those in the test groups (MG vaccine only).

It is apparent that the recombinant plasmid DNA containing the 64-copy CpG motifs (pHCLS-64) can serve as an adjunct of the MG vaccine and indeed promotes immunostimulatory activity in vivo.

Moreover, according to the in vivo embodiments of Examples 5 to 9, the degree of immunostimulatory activity is not proportional to the copy number of CpG motifs in the immunostimulatory polynucleotide. In other words, the degree of immunostimulatory activity can not be reasonably and accurately predicted according to the copy number of the CpG motif, especially for 16 × copy CpG motifs or more.

In addition, it is to be understood that specific vectors, immunostimulatory polynucleotides having specific copy numbers of CpG motifs, specific host cells, specific birds (such as chickens), specific cell types, or specific types of vaccines are exemplified in the present invention to evaluate the applications of the recombinant plasmid with the multi-copy CpG motifs of the present invention as adjuncts to avian vaccines. As is known to those skilled in the art, the copy-numbering of the CpG motifs of the present invention can be prepared as, for example, copies 17 to 31 or 33 to 63 and can serve as an adjunct for avian vaccines. In addition, the recombinant plasmid and its transformant, or any combination thereof, can also be applied to other types of animals (mammals) or other types of animal vaccines to promote the immunostimulatory effects without limiting the aforementioned examples.

Preferably, according to embodiments of the present invention, the transformant contains the immunostimulatory polynucleotide having a bird-specific sequence of the continuous multiple copy CpG motifs. The immunostimulatory polynucleotide is introduced into the vector to form the recombinant plasmid with the continuous multiple copy CpG motifs. The recombinant plasmid is transformed into the host cell to obtain stable and rapid mass production of the immunostimulatory polynucleotide. The aforementioned immunostimulatory polynucleotide, a recombinant plasmid containing the immunostimulatory polynucleotide, the transformant containing the recombinant plasmid, or any combination thereof, may serve as an adjunct of a bird vaccine. If such a bird seed is administered to an immunized bird, this has the beneficial effect of increasing the expression of the genes IFN-γ, TLR3 or TLR7 in the immunized bird, as well as reducing the effort and reducing the costs compared to the DNA. Adjuvants of the prior art with their complicated chemical modifications or greater effort

As will be apparent to those skilled in the art, the foregoing preferred embodiments are illustrative and not restrictive of the present invention. In view of the above, various modifications and similar arrangements consistent with the spirit of the invention are intended to be within the scope of the appended claims to be covered. Thus, the scope of the claims is to be accorded the broadest interpretation so as to cover all such modifications and similar arrangements.

This is followed by a sequence protocol according to WIPO St. 25. This can be downloaded from the official publication platform of the DPMA.

Claims (5)

Continuous multi-copy CpG motifs containing recombinant plasmid containing an immunostimulatory polynucleotide of a first polynucleotide with the Seq.Id.-No. 3 or a second polynucleotide with the Seq.Id.-No. 4 and wherein the recombinant plasmid containing the immunostimulatory polynucleotide or the immunostimulatory polynucleotide obtained from the recombinant plasmid is an adjuvant of a bird's vaccine, the avian vaccine being an oil killed vaccine against avian mycoplasmosis ( Mycoplasma gallisepticum; MG), a trivalent oil death vaccine against Nobilis ND-IB-IBD (ie, combined with IB strain M41, Gumboro strain D78, and ND strain Clone 30), a live vaccine against Newcastle disease , an attenuated live vaccine against Newcastle disease, an inactivated vaccine against Newcastle disease or an inactivated avian influenza vaccine and wherein the level of immunostimulatory activity of the avian vaccine has a non-linear correlation with the copy number of the CpG motif. A recombinant plasmid according to claim 1, wherein the recombinant carrying the second polynucleotide with the Seq.Id.-No. 4, deposited at the China Center for the Collection of Cultural Types of Wuhan University in China under the accession number CCTCC M 2010302. A transformant containing a recombinant plasmid with continuous multi-copy CpG motifs and comprising a recombinant plasmid containing an immunostimulatory polynucleotide transformed into a host cell, wherein the immunostimulatory polynucleotide comprises a first polynucleotide having the Seq. Id. 3 or a second polynucleotide with the Seq.Id.-No. 4, and the transformant carrying the second polynucleotide with Seq. Id. 4, deposited at the China Center for the Collection of Cultural Types of the University of Wuhan in China under the accession number CCTCC M 2010302, and wherein the recombinant plasmid containing the immunostimulatory polynucleotide or the immunostimulatory polynucleotide obtained from the recombinant plasmid, or the transformant containing the recombinant plasmid is an adjuvant of a bird vaccine, wherein the transformant is not a human embryonic stem cell obtained by destruction of embryos, the avian vaccine being an oil killed vaccine against avian mycoplasmosis ( Mycoplasma gallisepticum; MG), a trivalent oil death vaccine against Nobilis ND-IB-IBD (ie, combined with IB strain M41, Gumboro strain D78, and ND strain Clone 30), a live vaccine against Newcastle disease , a live attenuated vaccine against Newcastle disease, an inactivated Newcastle disease vaccine, or e is in inactivated avian influenza vaccine and wherein the level of immunostimulatory activity of the avian vaccine has a non-linear correlation with the copy number of CpG motifs. The transformant of claim 3, wherein the host cell is Escherichia coli. A bird vaccine containing a DNA adjuvant contained in a bird's vaccine, said DNA adjuvant comprising an immunostimulatory polynucleotide of a first polynucleotide having the Seq. Id. 3 or a second polynucleotide with the Seq.Id.-No. 4, a recombinant plasmid containing the immunostimulatory polynucleotide, a transformant containing the recombinant plasmid, or any combination thereof, and the recombinant containing the third polynucleotide with Seq. Id. 4, deposited at the China Center for the Collection of Cultural Types of Wuhan University in China under accession number CCTCC M 2010302, wherein the transformant is not a human embryonic stem cell obtained by destroying embryos, the avian vaccine being an oil Anti-avian mycoplasmosis (Mycoplasma gallisepticum; MG), a trivalent oil death vaccine against Nobilis ND-IB-IBD (ie, combined with IB strain M41, Gumbaro strain D78, and ND strain Clone 30). , a live vaccine against Newcastle disease, a live attenuated vaccine against Newcastle disease, an inactivated Newcastle disease vaccine, or an inactivated avian influenza vaccine, and wherein the degree of immunostimulatory activity of the avian vaccine is a non-linear correlation with the copy number of CpG creatives.

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