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CpG-ODN having specific immuno-stimulatory effect on pigs and application thereof

An immune-stimulating and specific technology, applied in the field of CpG-ODN, can solve the problems of inappropriateness and high cost, and achieve the effects of less toxic and side effects of the body, lower production costs, and reduced morbidity

Active Publication Date: 2019-07-23
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Although the reported CpG-ODN has a certain immune stimulating effect, there is no CpG-ODN that can effectively improve the innate and acquired immunity of pigs at the same time to report
In addition, most of the reported CpG-ODNs need thiolation in order to prevent their easy degradation, and the cost of synthesis is very high, which is not suitable for the existing pig breeding

Method used

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  • CpG-ODN having specific immuno-stimulatory effect on pigs and application thereof
  • CpG-ODN having specific immuno-stimulatory effect on pigs and application thereof
  • CpG-ODN having specific immuno-stimulatory effect on pigs and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0032] The screening of embodiment 1 specificity CpG-ODN

[0033] 1. This example uses existing genetic engineering or chemical methods to design and synthesize more than 50 CpG motifs (parts are shown in Table 1), and the rapid determination of IFN-r and SI in peripheral blood mononuclear cells is efficient and reliable. The directional screening mode of the method was used to screen out the best porcine CpG motif (results are shown in Table 2).

[0034] Table 1 Structural features of porcine-specific CpG ODN screened in vitro

[0035]

[0036]

[0037] Note: CpG motifs are underlined, where lowercase letters indicate modified with thio, and uppercase letters indicate not modified with thio.

[0038] Table 2 Stimulation effect of different pig-specific CpG ODNs on pig PBMC in vitro

[0039]

[0040]

[0041] Note: The above data is the average value of 5 individuals; the same letter means no significant difference p>0.05, different letters mean significant differe...

Embodiment 2

[0050] The nucleotide sequence of CpG-ODN (SEQ ID NO: 1) can be synthesized by existing genetic engineering or chemical methods. After synthesis, construct the pFAR4-CpG recombinant plasmid, and connect the nucleotide sequence (SEQ ID NO: 1) of the target gene CpG to the pFAR4 fragment (such as Figure 2-4 shown), the recombinant plasmid was obtained and stored at -15°C for future use.

[0051] A preparation for treating and / or preventing porcine diseases, said preparation is a sterile PBS solution containing 30 μg / mL of the above recombinant plasmid (ie pFAR4-CpG preparation).

[0052] Described aseptic PBS solution (1L) also contains each component of following percentage by weight: 0.6%NaCl, 0.01%KCl, 0.0124%Na 2 HPO 4 , 0.014% KH 2 PO 4 , the balance deionized water.

[0053] The preparation method of the sterile PBS solution is as follows: After mixing the components uniformly in proportion, they are sterilized at 115° C. for 20 minutes by a damp heat autoclaving met...

Embodiment 3

[0057] The nucleotide sequence of CpG-ODN (SEQ ID NO: 1) can be synthesized by existing genetic engineering or chemical methods. After synthesis, construct the pFAR4-CpG recombinant plasmid, and connect the nucleotide sequence (SEQ ID NO: 1) of the target gene CpG to the pFAR4 fragment (such as Figure 2-4 shown), the recombinant plasmid was obtained and stored at -15°C for future use.

[0058] A preparation for treating and / or preventing porcine diseases, said preparation is a sterile PBS solution containing 30 μg / mL of the above recombinant plasmid (ie pFAR4-CpG preparation).

[0059] Described aseptic PBS solution (1L) also contains each component of following percentage by weight: 0.7%NaCl, 0.02%KCl, 0.0134%Na 2 HPO 4 , 0.024% KH 2 PO 4 , the balance deionized water.

[0060] The preparation method of the sterile PBS solution is as follows: After mixing the components uniformly in proportion, they are sterilized at 117° C. for 21 minutes by a damp heat autoclaving met...

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Abstract

The present invention discloses a CpG-ODN having a specific immuno-stimulatory effect on pigs and an application thereof. A nucleotide sequence of the CpG-ODN is shown in SEQ ID NO:1. The CpG-ODN canimprove induction of innate immunity and specific humoral and cell-mediated immune responses of the pigs, and can be transferred into a plasmid vector pFAR4 to construct a recombinant plasmid pFAR4-CpG to prepare a preparation containing the recombinant plasmid pFAR4-CpG. The preparation can effectively activate natural immune functions of the pigs, also successfully replaces or partially replaces antibiotics as an immune activator, can also effectively activate acquired immune functions of the pigs, especially cellular immunity, can be used as an immune adjuvant to improve a protective effect of vaccines, also has little toxic and side effects on body, has great potential, can be widely used in live pig breeding industry, and is conducive to reducing breeding cost, reducing incidence ofthe pigs, and improving survival rate and breeding efficiency.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a CpG-ODN with a specific immune stimulating effect on pigs and an application thereof. Background technique [0002] CpG motifs (CpG motifs) refer to a specific structure composed of unmethylated cytosine guanine dinucleotide (CpG) as the core sequence. A large number of studies have shown that the CpG motif is a powerful non-specific immunostimulatory DNA sequence that can directly or indirectly activate T cells, B cells, dendritic cells, natural killer cells (Natural killer cells, NK) and macrophages and other immune cells. The DNA containing the CpG motif is called CpG-DNA; the artificially synthesized oligodeoxyribonucleotide (Oligodeoxynucleotide, ODN) containing the unmethylated CpG motif is called CpG-ODN. Because CpG-ODN can specifically increase the immunogenicity of antigens, activate various immune effector cells, produce certain cytokines, and make the auxil...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/85A61K31/711A61P31/04A61P31/14
CPCC12N15/113C12N15/85A61K31/711A61P31/04A61P31/14Y02A50/30
Inventor 张玲华贾军皓马苗鹏
Owner SOUTH CHINA AGRI UNIV
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