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Preparation method of CpG ODN with pig immune enhancement activity

An immune enhancement and activity technology, applied in the direction of medical preparations containing active ingredients, pharmaceutical formulas, allergic diseases, etc., to achieve the effects of improving survival rate, good immune effect, and improving specific and non-specific immune response ability

Inactive Publication Date: 2017-03-15
NANNING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, major animal diseases such as highly pathogenic porcine blue-ear disease, highly pathogenic avian influenza, foot-and-mouth disease, and swine fever have been threatening the healthy development of animal husbandry in Suining District. The current research on CpG ODN focuses on its use as an immune adjuvant In terms of enhancing the immune enhancement effect of vaccine strains, there are few reports on the application of CpG ODN as a feed additive. This study will further promote the application of the new type of immune enhancer CpG ODN in the immune prevention and control of swine diseases, and provide new methods for the prevention and treatment of swine diseases. methods and ideas

Method used

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  • Preparation method of CpG ODN with pig immune enhancement activity

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Comparison scheme
Effect test

Embodiment 1

[0021] A kind of preparation method of the CpG ODN that pig has immunoenhancing activity, comprises the following steps:

[0022] (1) Mass production of CpG ODN, including the following steps:

[0023] a. Fermentation: transfer the CpG ODN plasmid into the E. coli DH5a strain, culture at 35° C. with shaking at 250 rpm for 2 h to obtain the E. coli liquid, inoculate it in the fermentation medium at a volume ratio of 1:70, and ferment for 15 h.

[0024] b. Extraction: Shake and stir the fermented bacterial solution in step a, then add 50 μg of lysozyme per ml of bacterial solution, stir evenly, stir for 8 minutes in a constant temperature water bath at 35.2°C, and then add 50 μg of lysozyme per ml of bacterial solution Add 0.2ml of LiCl to the liquid, centrifuge at 10000r / min for 6min, and take the supernatant.

[0025] c. Heating: shake and stir the supernatant in step b for 15 minutes, heat in a boiling water bath for 20 minutes, cool to 20° C., add isopropanol of the same qu...

Embodiment 2

[0034] A kind of preparation method of the CpG ODN that pig has immunoenhancing activity, comprises the following steps:

[0035] (1) Mass production of CpG ODN, including the following steps:

[0036] a. Fermentation: transfer the CpS ODN plasmid into the E. coli DH5α strain, culture at 36°C and 280 rpm for 2.5 hours with shaking to obtain the E. coli liquid, inoculate it in the fermentation medium at a volume ratio of 1:80, and ferment for 20 hours .

[0037] b. Extraction: Shake and stir the fermented bacterial solution in step a, then add 60 μg of lysozyme per ml of bacterial solution, stir evenly, stir for 12 minutes in a constant temperature water bath at 35.8°C, and then add 60 μg of lysozyme per ml of bacterial solution Add 0.3ml of LiCl to the liquid, centrifuge at 10000r / min for 8min, and take the supernatant.

[0038] c. Heating: shake and stir the supernatant in step b for 20 minutes, heat in a boiling water bath for 25 minutes, cool to 25° C., add isopropanol of...

Embodiment 3

[0047] A kind of preparation method of the CpG ODN that pig has immunoenhancing activity, comprises the following steps:

[0048] (1) Mass production of CpG ODN, including the following steps:

[0049] a. Fermentation: transfer the CpG ODN plasmid into the E. coli DH5α strain, culture at 35.5°C and 270 rpm for 2.3 hours with shaking to obtain the E. coli bacterial liquid, inoculate it in the fermentation medium at a volume ratio of 1:75, and ferment for 18 hours .

[0050] b. Extraction: Shake and stir the fermented bacterial solution in step a, then add 55 μg of lysozyme per ml of bacterial solution, stir evenly, stir for 10 minutes in a constant temperature water bath at 35.5°C, and then add 55 μg of lysozyme per ml of bacterial solution Add 0.25ml of LiCl to the liquid, centrifuge at 10000r / min for 7min, and take the supernatant.

[0051] c. Heating: shake and stir the supernatant in step b for 18 minutes, place in a boiling water bath and heat for 23 minutes, cool to 23°...

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Abstract

The invention relates to the technical field of veterinarian biological products, in particular to a preparation method of CpG ODN with the pig immune enhancement activity. The method comprises the following steps of 1 volume production of CpG ODN, 2 envelope additive preparation and 3 ingredient preparation. The body is induced to generate various immunology effects and promoted to secrete various cell factors, the specific and non-specific immunity reaction capability of the body are improved, the pig immunity can be effectively improved when the CpG ODN is added into feed, and the good immunity effect on great animal epidemic diseases such as the high pathogenicity pig ear disease, the high pathogenicity bird flu, foot-and-mouth diseases and pig fevers is achieved, the pig survival rate is effectively increased, diseases are reduced, and the breeding cost is lowered.

Description

technical field [0001] The invention relates to the technical field of veterinary biological products, in particular to a preparation method of CpGODN which has immune enhancing activity on pigs. Background technique [0002] Cytosine-guanine oligodeoxynucleotides (CpGoligodeoxymieleotides, CpG ODN), also known as immunostimulatory DNA sequences, is a specific nuclear The nucleotide sequence can induce the body to produce various immunological effects (such as activation of immune cells), promote its secretion of various cytokines, and improve the body's specific and non-specific immune response capabilities. Functional studies have found that CpG ODN can activate immune cells such as B cells, NK cells, monocytes / macrophages, dendritic cells, and T cells, and induce the production of various cytokines such as IL-12, TNF-d, and INF-γ , Promote the expression of MHC class II molecules and co-stimulatory factors CD40, CD80, and CD86. More importantly, it can enhance the proce...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A23K50/30A23K20/153C12P19/34A61K39/39A61P37/04
CPCC12P19/34A61K39/39A61K2039/55561
Inventor 张艳雯郑一民张宁兰宗宝
Owner NANNING UNIV
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