Swine-specific CpG oligodeoxynucleotide and application thereof

A deoxynucleotide and specific technology, applied in nucleic acid vectors, veterinary vaccines, allergic diseases, etc., can solve problems such as increased losses, harm to the pig industry, and aggravation of diseased pigs, and achieve good immunity. , immune-enhancing effect

Pending Publication Date: 2020-04-24
JILIN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The virus not only caused serious harm to my country's pig industry, but also co-infected with porcine parvovirus or porcine reproductive and respiratory synd

Method used

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  • Swine-specific CpG oligodeoxynucleotide and application thereof

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Experimental program
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Effect test

Embodiment 1

[0023] The design of CpG-ODN sequence, according to the CpG motifs ATCGAT, GTCGTT and GACGTT which have good stimulating activity to pigs and humans, designed and synthesized a sequence containing CpG units, among which, GC-ODN is CpG motif-free The negative sequence, all the nucleic acid backbones of CpG-ODN were modified by sulfo modification, which was synthesized by Sangon Bioengineering (Shanghai) Co., Ltd., as shown in Table 1.

[0024] Table 1 CpG-ODN sequence

[0025]

Embodiment 2

[0027] Isolation and culture of porcine peripheral blood mononuclear cells (PBMC): The skin of the porcine neck was disinfected with 75% alcohol, and blood was aseptically collected from the anterior vena cava after fixation, and the blood was collected with a sterile vacuum blood collection tube containing heparin. According to the instructions of the porcine peripheral blood mononuclear cell isolation kit, the porcine PBMCs were isolated. Resuspend the cells in 1 mL of IMDM nutrient solution containing 10% fetal bovine serum, take 90 μL of the resuspended cells, add 10 μL of trypan blue staining solution, mix gently, and stain for 3 min; absorb a small amount of stained cells , observe and count the number of cells under an inverted microscope for later use.

Embodiment 3

[0029] Detection of pig PBMC proliferation ability stimulated by CpG-ODN sequence: Use IMDM nutrient solution containing 10% fetal bovine serum to adjust the concentration of pig PBMC to 1×10 6 cells / mL, seeded in 96-well cell culture plate, 100 μL per well. The negative control GC-ODN and the designed and synthesized CpG-ODN sequence were added respectively, and the final concentration of CpG-ODN was 5 μg / mL; the positive control well was added with ConA solution, and the final concentration was 5 μg / mL; the blank group was added with CpG- ODN equal volume of PBS, 3 replicate wells for each group. Place the 96-well cell culture plate at 37 °C, 5% CO 2 Cultivate in the incubator for 32 h, add 20 μL of MTS solution to each well 4 h before the end of the culture, detect the OD value of each well at a wavelength of 490 nm with a microplate reader immediately after the end of the culture, and calculate the SI value (stimulation index), SI The value calculation formula is as foll...

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Abstract

The invention belongs to the technical field of biology, and provides a swine-specific CpG oligodeoxynucleotide. The swine-specific CpG oligodeoxynucleotide comprises a CpG-08 sequence, and the CpG-08sequence is represented by SEQ ID NO.1. The invention also provides a plasmid containing the repetitive sequence of the porcine-specific CpG oligodeoxynucleotide. The pCpG-08 recombinant plasmid constructed in the invention has a good enhancement effect on the immunity of swine fever live vaccines and porcine circovirus inactivated vaccines, and can be put into the market as a new vaccine adjuvant.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a pig-specific CpG oligodeoxynucleotide and its application. Background technique [0002] CpG-ODN (Oligodeoxynucleotides containing CpG motifs) is a synthetic oligodeoxynucleotide containing unmethylated CpG motifs, which can mimic the immunostimulatory activity of bacterial DNA, as a pathogen-associated molecular pattern (Pathogen associated molecular patterns, PAMP) binds to the pattern recognition receptors (Pattern recognition receptors, PRR) TLR-9 in many immune cells in the animal body, stimulates the body to generate an innate immune response, and effectively enhances dendritic cells, B cells, monocytes, and macrophages. The activity of immune cells such as phagocytes promotes the secretion of various cytokines, thereby non-specifically enhancing the strength of the adaptive immune response, and playing the role of an immune enhancer in the body's resistance to pa...

Claims

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Application Information

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IPC IPC(8): C12N15/117C12N15/85A61K39/39A61K39/187A61K39/12A61K31/7088A61P37/04A61P31/14A61P31/20
CPCC12N15/117C12N15/85A61K39/39A61K39/12A61K31/7088A61P37/04A61P31/14A61P31/20C12N2310/17C12N2800/107A61K2039/55561A61K2039/5252A61K2039/552C12N2770/36034C12N2750/10034
Inventor 冷雪周宇杨艳文杜锐张进进伭婷盛陈艳麻宝艺夏辉马青霞刘雅馨高沙沙
Owner JILIN AGRICULTURAL UNIV
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