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Sika deer specific CpG oligodeoxynucleotide and application thereof

A technology of deoxynucleotide and sika deer, applied in veterinary vaccines, allergic diseases, medical preparations containing active ingredients, etc., can solve the problems of economic loss, secondary infection, doe abortion and other problems in the deer industry, and achieve Improving the effect of immune protection, enhancing disease resistance, and improving the effect of antibody levels

Active Publication Date: 2019-01-18
JILIN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Deer infected with BVDV can cause miscarriage in doe, stillbirth, weak fetus, deformed fetus, viremia offspring, intractable diarrhea and mucosal disease in deer, resulting in obstruction of feeding and nutrient absorption, and secondary infection, which seriously affects The development of young deer and the production performance of adult deer may even cause a large number of deer to die, causing serious economic losses to the deer industry

Method used

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  • Sika deer specific CpG oligodeoxynucleotide and application thereof
  • Sika deer specific CpG oligodeoxynucleotide and application thereof
  • Sika deer specific CpG oligodeoxynucleotide and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Design and synthesis of CpG-ODN sequence

[0028] Using the solid-phase phosphoramidite triester method, multiple CpG-ODNs were designed with the CpG motif GTCGTT that can activate the lymphocyte proliferation of various livestock and poultry including humans, cattle, sheep, pigs and chickens. The sequence involves the length of the sequence in the CpG-ODN sequence, the backbone modification, the CpG base sequence and the number of positions of the motif, etc. The designed and synthesized CpG-ODN sequence (SEQID NO.3) is shown in Table 1, wherein the phosphodiester bonds are all thio-modified.

[0029] Table 1 CpG-ODN sequence

[0030] Numbering

Embodiment 2

[0031] Example 2 Separation of Peripheral Blood Mononuclear Cells from Sika Deer

[0032] Sika deer adopts jugular vein blood collection method: 3 normal healthy sika deer for blood collection; peripheral blood mononuclear cell isolation kits from various animals were purchased from Tianjin Haoyang Biological Products Technology Co., Ltd.; isolated peripheral blood mononuclear cells What used for the cultivation was the IMDM nutrient solution containing 10% fetal bovine serum.

[0033] method:

[0034] (1) Baoding the sika deer, shearing the neck, wiping and disinfecting alcohol cotton balls along the jugular vein back and forth, using a disposable blood collection device, piercing from the bottom to the top at an angle of 45 degrees to the skin, and using a disposable sterile heparin anticoagulant to collect blood The blood vessels are used to collect blood, and 10ml of blood is collected from each sika deer. After the blood collection is completed, the blood collection devi...

Embodiment 3

[0040] Example 3 Stimulation of CpG-ODN on peripheral blood mononuclear cells of sika deer

[0041] The peripheral blood mononuclear cells of the sika deer are counted, and diluted with the IMDM nutrient solution containing 10% fetal bovine serum, so that the final concentration of the peripheral blood mononuclear cells of the sika deer is 5 × 10 6 cells / ml, the cells were seeded in a 96-well cell culture plate, 0.1ml per well. Artificially synthesized CpG-ODN was added to the cell culture wells at a final concentration of 5-50 μg / ml, and three replicate wells were set up for each group. Place at 37°C, 5% CO 2 Cultivate in the incubator for 32 hours, take out the cell culture plate, add 20 μl MTS solution to each well, and continue to store at 37°C, 5% CO 2 Incubator for 4 hours. After culturing, the cell culture plate was placed on a microplate reader to measure the OD value of each well at a wavelength of 490 nm. Set CpG-2006 (numbering SEQ ID NO.1) as positive control, ...

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Abstract

The invention provides a sika deer specific CpG oligodeoxynucleotide and application thereof, and relates to the field of immunopotentiator for deer and vaccine adjuvant for deer. To boost the sika deer's immunity, the immune efficacy of sika deer vaccine and the antibody level of deer-derived bovine viral diarrhea / mucosal disease inactivated vaccine were increased, A CpG oligomeric deoxynucleotide specific to sika deer, As shown in SEQ ID NO. 3, A Chinese medicinal preparation is use for preparing immunopotentiator for sika deer, immunizing adjuvant for sika deer vaccine, and preparation of antiviral preparation against deer-derived bovine viral diarrhea / mucosal disease virus, and preparation of induce cells to produce antiviral substance against deer-derived bovine viral diarrhea / mucosaldisease virus, so as to improve antibody level of deer-derived bovine viral diarrhea / mucosal disease inactivated vaccine. As prove by experiments, that artificially synthesize sika deer specific CpGoligonucleotide can be used for sika deer peripheral blood mononuclear cells to exhibit antiviral activity, and at the same time, the deer-derived bovine viral diarrhea / mucosal disease inactivated vaccine immunity can be enhance. The sika deer specific CpG oligonucleotide can be used for sika deer peripheral blood mononuclear cells to show antiviral activity.

Description

technical field [0001] The invention relates to the technical field of deer immune enhancer and deer vaccine adjuvant, in particular to a sika deer-specific CpG oligodeoxynucleotide and its application. Background technique [0002] CpG ODN is a kind of oligodeoxynucleotide chain containing cytosine-guanine dinucleotide, which is in the form of single-stranded DNA and contains one or more unmethylated CpG motifs. It is often used as an adjuvant in clinical research because of its effect on the immune response. Based on differences in the structure of CpG ODNs and the properties of the immune responses they induce, four distinct classes of CpG ODNs have been identified in humans, although each class of CpG ODNs contains at least one central unmethylated CG dinucleotide plus binding domains composed of motifs, but they differ in structure and immunological activity: Type B ODNs typically contain 1 to 5 CpG motifs within a phosphorothioate backbone that enhances their Resista...

Claims

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Application Information

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IPC IPC(8): C12N15/117A61K39/39A61K39/12A61P31/14A61P37/04
CPCA61K39/12A61K39/39A61K2039/5252A61K2039/552A61K2039/55561A61P31/14A61P37/04C12N15/117C12N2310/17C12N2770/24334
Inventor 冷雪杜锐李健明时坤孙志博徐宁郜艳雪栾美慧刘艺宫庆龙葛桂阳李东丽
Owner JILIN AGRICULTURAL UNIV
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