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Chicken specificity immune activator CpG-ODN and application thereof

A specific and activator technology, applied in the field of genetic engineering, can solve the problems of high cost and unsuitable for chicken breeding, and achieve the effects of low production cost, improved vaccine protection effect, and reduced morbidity.

Active Publication Date: 2019-09-10
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The currently reported CpGs are mainly used as chicken vaccine adjuvants, and chicken-specific CpG preparations with both innate immunity enhancement and vaccine adjuvant (acquired immunity) functions have not been reported yet.
In addition, most of the reported CpG-ODNs require thiolation in order to prevent their easy degradation, and the cost of synthesis is very high, which is not suitable for the existing chicken breeding

Method used

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  • Chicken specificity immune activator CpG-ODN and application thereof
  • Chicken specificity immune activator CpG-ODN and application thereof
  • Chicken specificity immune activator CpG-ODN and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] The screening of embodiment 1 specificity CpG-ODN

[0035] 1. In this example, more than 35 CpG motifs (as shown in Table 1) were designed and synthesized by adopting existing genetic engineering or chemical methods, and an efficient and reliable method for rapid determination of IFN-r and SI in peripheral blood mononuclear cells was established. Oriented screening mode, screen out the best chicken CpG motif (results are shown in Table 2).

[0036] Table 1 Structural features of chicken-specific CpG ODN screened in vitro

[0037]

[0038]

[0039] Note: CpG motifs are underlined, where lowercase letters indicate modified with thio, and uppercase letters indicate not modified with thio.

[0040] Table 2 Stimulation effect of different chicken-specific CpG ODNs on chicken PBMCs in vitro

[0041]

[0042]

[0043] Note: The above data is the average value of 5 individuals; the same letter means no significant difference p>0.05, different letters mean signific...

Embodiment 2

[0052] The nucleotide sequence of CpG-ODN (SEQ ID NO: 1) can be synthesized by existing genetic engineering or chemical methods. After synthesis, the pVAX1-CpG recombinant plasmid is constructed, and the nucleotide sequence (SEQ ID NO: 1) of the target gene CpG is connected with the pVAX1 fragment (such as Figure 1~3 shown), the recombinant plasmid was obtained and stored at -15°C for future use.

[0053] A preparation for treating and / or preventing chicken diseases, said preparation is a sterile PBS solution containing 30 μg / mL of the above recombinant plasmid (ie pVAX1-CpG preparation).

[0054] 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.

[0055] 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 aut...

Embodiment 3

[0059] The nucleotide sequence of CpG-ODN (SEQ ID NO: 1) can be synthesized by existing genetic engineering or chemical methods. After synthesis, the pVAX1-CpG recombinant plasmid is constructed, and the nucleotide sequence (SEQ ID NO: 1) of the target gene CpG is connected with the pVAX1 fragment (such as Figure 1~3 shown), the recombinant plasmid was obtained and stored at -15°C for future use.

[0060] A preparation for treating and / or preventing chicken diseases, said preparation is a sterile PBS solution containing 30 μg / mL of the above recombinant plasmid (ie pVAX1-CpG preparation).

[0061] 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.

[0062] 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 aut...

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Abstract

The invention discloses a chicken specificity immune activator CpG-ODN and an application thereof, and a nucleotide sequence of the CpG-ODN is shown as SEQ ID NO: 1. The CpG-ODN can enhance an induction effect of innate immunity and specific humoral and cell-mediated immune responses of chickens, and can be transferred into a plasmid vector pVAX1 to construct a recombinant plasmid pVAX1-CpG, and apreparation containing the recombinant plasmid pVAX1-CpG is prepared. The preparation can effectively activate a natural immune function of the chickens, and also successfully replace or partially replace antibiotics as an immune activator; the preparation can also effectively activate an acquired immune function of the chickens, especially cellular immunity, and can be used as an immune adjuvantto enhance a protective effect of vaccines; and the preparation has little toxic and side effects on a body, has great potential, can be widely applied to a breeding industry of raw chickens, is beneficial to reduction of breeding cost, enables incidence of a disease of the raw chickens to be reduced, and improves the survival rate and the breeding efficiency of the chickens.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a chicken-specific immune activator CpG-ODN and an application thereof. Background technique [0002] For a long time, people have always thought that the role of DNA is only to control the expression of traits, and the immunological role of DNA has not been recognized. As early as the 19th century, it was discovered that cancer patients infected with bacteria were more likely to remove tumors. Later, a surgeon discovered that injecting bacterial extracts into cancer patients could significantly reduce the disease (Coley, 1991), and believed that the active ingredients that worked might be is the lipopolysaccharide in the extract. Later, Braw et al. immunized mice with oligodeoxyribonucleic acid and sheep red blood cells in 1965, and found that compared with the immunization group of red blood cells alone, the mice could produce a stronger level of immune response, s...

Claims

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Application Information

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IPC IPC(8): C12N15/117C12N15/63A61K39/39A61P37/04A61P31/04
CPCC12N15/117C12N15/63A61K39/39A61P37/04A61P31/04C12N2310/17A61K2039/55561Y02A50/30
Inventor 张玲华马苗鹏贾军皓
Owner SOUTH CHINA AGRI UNIV
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