CN110684768A - Duck natural immunopotentiator based on pattern recognition receptor ligand - Google Patents
Duck natural immunopotentiator based on pattern recognition receptor ligand Download PDFInfo
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Abstract
The invention discloses a duck natural immunopotentiator based on a pattern recognition receptor ligand, which has the sequence as follows: 5'-GATATGCGACCGATT-3' are provided. The duck natural immunopotentiator based on the pattern recognition receptor ligand can obviously increase the levels of duck serum cytokines IL-1 beta/12 p40, IL-1 beta, IFN-alpha and IFN-beta, up-regulate the tissue expression levels of natural immunity related genes RIG-I, TLR3, TLR7, IFN-alpha, IFN-beta, IL-1 beta and TNF-alpha in duck immune organs, inflammation related genes COX2 and iNOS and apoptosis related gene Caspase3 in liver, spleen, thymus and bursa of Fabricius, and enhance the natural immune response and disease resistance of ducks.
Description
Technical Field
The invention belongs to the technical field of agriculture, and relates to a duck natural immunopotentiator based on a Pattern Recognition Receptor (PRRs) ligand.
Background
With the rapid development of animal husbandry in China, particularly the popularization and promotion of large-scale and intensive breeding modes, animal diseases are diversified, animal infectious diseases, particularly viral infectious diseases, are increasingly damaged, not only are animal production seriously affected, but also human health is possibly threatened.
The natural immune system is the first checkpoint of body defense, and can recognize pathogenic microorganisms, trigger signal pathways between cells and prevent pathogen infection. The host embryonic line coded Pattern Recognition Receptors (PRRs) are important components of the natural immune system, and can release cell factors (such as IFN-beta and the like) with the pathogenic microorganism resisting effect by recognizing pathogen related molecular patterns (PAMPs), such as microbial flagella, peptidoglycan, lipopolysaccharide, CpG motifs, single-stranded and double-stranded RNAs and the like, thereby inducing the organism to generate the pathogenic microorganism natural immune response and playing an important role in controlling pathogenic microorganism infection and epidemic disease occurrence. Since the discovery of PRRs, the research on PRRs immunomodulators for regulating natural immunity and adaptive immunity and preventing and treating human diseases has led to great adverse reaction and extensive research in the medical field, and lays the foundation for the research and development of novel immunopotentiators and targeted therapeutic drugs by targeting PRRs. At present, the safety and efficacy of various agonists of PRRs as immunopotentiators and therapeutic agents for human vaccines have been validated and widely used throughout the world. However, the research on the animal PRRs immunomodulator is relatively late, and although researches show that some livestock PRRs ligands have potential immunoregulation effect, can induce DC activation to generate a large amount of IL-12 and I-type interferon, and play an important role in natural immune response of organisms, the detailed mechanism of the effect is not clear, and the application in livestock production is rarely reported.
Disclosure of Invention
The invention aims to provide a duck natural immunopotentiator based on a pattern recognition receptor ligand.
The specific technical scheme is as follows:
a duck natural immunopotentiator based on pattern recognition receptor ligand has the sequence as follows: 5'-GATATGCGACCGATT-3' are provided.
The invention relates to a preparation method of a duck natural immunopotentiator based on a pattern recognition receptor ligand, which comprises the following steps:
step 1, synthesizing a duck natural immunopotentiator
The duck natural immunopotentiator is synthesized by a chemical synthesis method.
Step 2, purifying the duck natural immunopotentiator
The duck natural immunopotentiator is purified and synthesized by adopting a denaturing polyacrylamide gel electrophoresis (PAGE) method.
Step 3, calculating the concentration of the duck natural immunopotentiator
Measuring the light absorption value of the duck natural immunopotentiator solution at 260nm by using an ultraviolet spectrophotometer according to a formula: and (3) calculating the concentration of the duck natural immunopotentiator by A260 multiplied by the transformation coefficient multiplied by the dilution rate.
Compared with the prior art, the invention has the beneficial effects that:
the duck natural immunopotentiator based on the pattern recognition receptor ligand can obviously increase the levels of duck serum cytokines IL-1 beta/12 p40, IL-1 beta, IFN-alpha and IFN-beta, up-regulate the tissue expression levels of natural immunity related genes RIG-I, TLR3, TLR7, IFN-alpha, IFN-beta, IL-1 beta and TNF-alpha in duck immune organs, inflammation related genes COX2 and iNOS and apoptosis related gene Caspase3 in liver, spleen, thymus and bursa of Fabricius, and enhance the natural immune response and disease resistance of ducks.
Detailed Description
The technical solution of the present invention will be described in further detail with reference to specific embodiments.
1. Synthesis of duck natural immunopotentiator
According to the ligand molecular structure of animal PRRs family member TLR9, duck natural immunopotentiator is designed and synthesized.
2. Grouping and handling of test animals
Selecting 1 day old ducklings which are taken out of shells in the same batch, adaptively feeding the ducklings for 7d, and then randomly dividing the ducklings into a test group and a control group, wherein each group comprises 20 ducklings. Wherein the duckling of the test group is injected with synthetic duck natural immunopotentiator (5 mug/feather) subcutaneously, and the duckling of the control group is injected with normal saline with the same volume subcutaneously for 3 days continuously. During the experiment, each group of ducklings can eat and drink water freely.
3. Sample collection
On day 18 after the start of the test, blood was collected from the jugular vein, and serum was separated at room temperature and stored frozen at-20 ℃; the test duck is killed, and tissues and organs such as liver, spleen, thymus, bursa of Fabricius and the like are collected and immediately put into liquid nitrogen for preservation.
4. Detection of duck natural immune response indexes
(1) Detection of serum cytokines and immunoglobulin levels
Serum cytokine IL-1 beta/12 p40, IFN-alpha and IFN-beta and immunoglobulin IgG, IgA and IgM levels were determined by ELISA.
(2) Detection of native immune-related genes in immune tissue organs
The mRNA expression levels of the natural immune related genes RIG-I, TLR3, TLR7, IFN-alpha, IFN-beta, IL-1 beta and TNF-alpha in liver, spleen, thymus and bursa of Fabricius tissues are detected by a qRT-PCR method.
(3) Detection of inflammation-associated genes in immune tissue organs
The mRNA expression levels of inflammation related genes COX2 and iNOS in liver, spleen, thymus and bursal disease tissues are detected by adopting a qRT-PCR method; the levels of COX2 and iNOS proteins were measured using the Western Blot method.
(4) Detection of apoptosis-related genes in immune tissue and organs
The mRNA expression levels of apoptosis related genes Bcl2 and Caspase3 in tissues of liver, spleen, thymus and bursa of Fabricius are detected by adopting a qRT-PCR method; the levels of Bcl2 and Caspase3 proteins were measured using the Western Blot method.
5. Test results
(1) Detection of serum cytokines and immunoglobulin levels
The duck serum cytokines IL-1 beta/12 p40, IL-1 beta, IFN-alpha and IFN-beta and the levels of immunoglobulins IgG, IgA and IgM in the test and control groups are shown in tables 1 and 2.
TABLE 1 serum cytokine levels
TABLE 2 serum immunoglobulin levels
As can be seen from tables 1 and 2, the duck serum cytokines IL-1 beta/12 p40, IL-1 beta, IFN-alpha and IFN-beta in the test group are all significantly higher than those in the control group, while the serum immunoglobulins IgA, IgG and IgM are not significantly different. The synthetic duck natural immunopotentiator is shown to be capable of remarkably increasing the levels of duck serum cytokines IL-1 beta/12 p40, IL-1 beta, IFN-alpha and IFN-beta, and has no remarkable influence on the levels of serum immunoglobulin IgA, IgG and IgM.
(2) Detection of native immune-related genes in immune tissue organs
The mRNA expression levels of the natural immune related genes RIG-I, TLR3, TLR7, IFN-alpha, IFN-beta, IL-1 beta and TNF-alpha in the tissues of the duck liver, spleen, thymus and bursa of Fabricius in the test group and the control group are shown in tables 3-6.
TABLE 3 expression levels of innate immunity-related genes in liver tissue
TABLE 4 expression levels of innate immunity-related genes in spleen tissue
TABLE 5 expression levels of innate immunity-related genes in thymus tissue
TABLE 6 expression levels of native immune-related genes in bursal tissue
As can be seen from tables 3 to 6, the mRNA expression levels of the natural immunity related genes RIG-I, TLR3, TLR7, IFN-alpha, IFN-beta, IL-1 beta and TNF-alpha in the liver, spleen, thymus and bursa of Fabricius tissues of the duck in the test group are higher or remarkably higher than those of the control group, which indicates that the synthesized natural duck immunity enhancer can up-regulate or remarkably up-regulate the expression level of the natural immunity related genes in the immune organ tissues of the duck.
(3) Detection of inflammation-associated genes in immune tissue organs
The expression levels of inflammation-related genes COX2 and iNOS mRNA and proteins in the liver, spleen, thymus and bursa of Fabricius tissues of the duck in the test group and the control group are shown in tables 7-10.
TABLE 7 expression levels of mRNA and protein of inflammation-related genes COX2 and iNOS in liver tissue
TABLE 8 expression levels of mRNA and protein of inflammation-related genes COX2 and iNOS in spleen tissue
TABLE 9 expression levels of mRNA and protein of COX2 and iNOS genes, genes involved in inflammation, in thymus tissue
TABLE 10 expression levels of inflammation-associated genes COX2 and iNOS mRNA and protein in bursal tissue of Fabricius
As can be seen from tables 7 to 10, the expression levels of inflammation-related genes COX2 and iNOS mRNA and protein in liver, spleen, thymus and bursa of Fabricius tissues of the experimental group ducks are almost all higher or remarkably higher than those of the control group except COX2mRNA in spleen, which indicates that the synthesized duck natural immunopotentiator can up-regulate or remarkably up-regulate the expression levels of inflammation-related genes COX2 and iNOS mRNA and protein in immune organ tissues of the ducks.
(4) Detection of apoptosis-related genes in tissue and organ
The expression levels of apoptosis-related genes Bcl2 and Caspase3mRNA and protein in the liver, spleen, thymus and bursa of Fabricius tissues of the duck in the test group and the control group are shown in tables 11-14.
TABLE 11 mRNA and protein expression levels of apoptosis-related genes Bcl2 and Caspase3 in liver tissue
TABLE 12 expression levels of mRNA and protein of apoptosis-related genes Bcl2 and Caspase3 in spleen tissue
mRNA and protein expression levels of apoptosis-related genes Bcl2 and Caspase3 in thymus tissue of TABLE 13
TABLE 14 expression levels of mRNA and protein of apoptosis-related genes Bcl2 and Caspase3 in bursal tissue
As can be seen from tables 11 to 14, the expression levels of Caspase3 gene mRNA and protein in liver, thymus and bursa of Fabricius of the duck in the test group and Caspase3 gene mRNA in spleen of the duck are all obviously higher than those of the control group, and the difference between the expression levels of Bcl2 gene mRNA and protein of the duck in the test group and the control group is not obvious. The synthetic duck natural immunopotentiator is shown to be capable of up-regulating the expression level of Caspase3 gene mRNA and protein related to apoptosis in duck immune organ tissues, and has no significant influence on the expression level of Bcl2 gene mRNA and protein.
The above description is only a preferred embodiment of the present invention, and the scope of the present invention is not limited thereto, and any simple modifications or equivalent substitutions of the technical solutions that can be obviously obtained by those skilled in the art within the technical scope of the present invention are within the scope of the present invention.
Sequence listing
<110> Zhejiang province academy of agricultural sciences
<120> a duck natural immunopotentiator based on pattern recognition receptor ligand
<160>1
<170>SIPOSequenceListing 1.0
<210>1
<211>15
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>1
gatatgcgac cgatt 15
Claims (2)
1. A duck natural immunopotentiator based on pattern recognition receptor ligand is characterized by comprising the following sequences: 5'-GATATGCGACCGATT-3' are provided.
2. The duck natural immunopotentiator based on pattern recognition receptor ligand according to claim 1, which is characterized by:
the method comprises the following steps:
step 1, synthesizing a duck natural immunopotentiator
Synthesizing a duck natural immunopotentiator by adopting a chemical synthesis method;
step 2, purifying the duck natural immunopotentiator
Purifying the synthesized duck natural immunopotentiator by adopting a modified polyacrylamide gel electrophoresis PAGE (PAGE electrophoresis) method;
step 3, calculating the concentration of the duck natural immunopotentiator
Measuring the light absorption value of the duck natural immunopotentiator solution at 260nm by using an ultraviolet spectrophotometer according to a formula: and (3) calculating the concentration of the duck natural immunopotentiator by A260 multiplied by the transformation coefficient multiplied by the dilution rate.
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Citations (4)
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| US20080152665A1 (en) * | 2006-09-01 | 2008-06-26 | Claude Leclerc | Therapy of cancer based on targeting adaptive, innate and/or regulatory component of the immune response |
| CN101304759A (en) * | 2003-07-14 | 2008-11-12 | 巨瓦日斯生物治疗股份有限公司 | Vaccines using pattern recognition receptor-ligand:lipid complexes |
| WO2018060514A1 (en) * | 2016-09-30 | 2018-04-05 | Galderma Research & Development | Methods and compositions combining at least one pattern recognition receptor (prr) agonist with an anti-il10 receptor antibody |
| CN111182925A (en) * | 2017-05-30 | 2020-05-19 | 纽约市哥伦比亚大学理事会 | Cationic nucleic acid scavengers and uses thereof |
-
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- 2019-10-12 CN CN201910968634.9A patent/CN110684768B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101304759A (en) * | 2003-07-14 | 2008-11-12 | 巨瓦日斯生物治疗股份有限公司 | Vaccines using pattern recognition receptor-ligand:lipid complexes |
| US20080152665A1 (en) * | 2006-09-01 | 2008-06-26 | Claude Leclerc | Therapy of cancer based on targeting adaptive, innate and/or regulatory component of the immune response |
| WO2018060514A1 (en) * | 2016-09-30 | 2018-04-05 | Galderma Research & Development | Methods and compositions combining at least one pattern recognition receptor (prr) agonist with an anti-il10 receptor antibody |
| CN111182925A (en) * | 2017-05-30 | 2020-05-19 | 纽约市哥伦比亚大学理事会 | Cationic nucleic acid scavengers and uses thereof |
Non-Patent Citations (2)
| Title |
|---|
| TIANTIAN GU: "Effects of immunopotentiators on biochemical parameters,proinflammatory cytokine, and nonspecific immune responses in Shaoxing ducklings", 《POULTRY SCIENCE》 * |
| 汪雪峰: "TLR7_8和TLR9配体对日本血吸虫DNA疫苗免疫效应的影响", 《全国临床微生物与感染免疫学术研讨会》 * |
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