KR102497647B1 - A composition for modulating the innate immune response of chickens comprising gga-miR-200a-3p, mimics thereof or inhibitors of gga-miR-200a-3p - Google Patents

Abstract

The present invention relates to a composition for regulating the innate immune response of chickens, comprising gga-miR-200a-3p, a mimic thereof or an inhibitor of the expression or activity of gga-miR-200a-3p as an active ingredient, and the present invention The composition for suppressing or enhancing the innate immune response of chickens according to the MAPK signaling pathway involved in the innate immune response, in particular ZAK , TGFβ2 and MAP2K4, and by regulating the expression of ERK1/2 and p32 pro-inflammatory cytokines Expression can be indirectly regulated, and through this, the innate immune response of chickens to protozoal or bacterial diseases can be controlled. In addition, disease-resistant chickens can be selected by comparing the expression level of gga-miR-200a-3p.

Description

A composition for modulating the innate immune response of chickens comprising gga-miR-200a-3p, a mimic thereof or a gga-miR-200a-3p inhibitor {A composition for modulating the innate immune response of chickens comprising gga-miR-200a-3p, mimics thereof or inhibitors of gga-miR-200a-3p}

The present invention relates to a composition for regulating the innate immune response of chickens, comprising gga-miR-200a-3p, a mimic thereof, or an inhibitor of the expression or activity of gga-miR-200a-3p as an active ingredient.

Necrotic enteritis (NE), which mainly occurs in avian species, is caused by high levels of Clostridium perfringens (CP ) infection. CP is a gram-positive, spore-forming anaerobic microorganism (anaerobe) found in generally low abundance (<10 ⁴ cfu) in the gastrointestinal tract (GIT) of most birds. However, an excessive number of CPs , particularly in the small intestine, leads to the development of necrotizing enterocolitis. CP produces toxins and the intestinal mucosa can be covered with a fibrino-necrotic layer. This damage to the intestinal epithelium is often associated with coccidiosis caused by the coccidian genus, Eimeria . Necrotizing enteritis usually occurs in 17- to 18-day-old broiler chickens, and affected birds show symptoms such as huddling, ruffled feathers, anorexia, reduced growth rate, low feed efficiency, and diarrhea. , resulting in a high mortality rate.

For the treatment of necrotizing enteritis, antimicrobial agents such as antibiotic growth promoters and other therapeutic agents were initially used to effectively reduce necrotizing enteritis, and these treatment methods have been used worldwide. However, in response to emerging concerns about antibiotic resistance, the EU has banned the use of antimicrobials in poultry production since 2006 and Korea since 2012. Therefore, there is a need for an effective and novel method for controlling necrotizing enteritis, which causes serious economic loss and affects animal welfare. Moreover, studies examining the immunological and pathological responses of birds to CP and Eimeria spp. are limited.

On the other hand, microRNA (miRNA) is an endogenous non-coding RNA of about 21-24 nucleotides in length. Animal miRNAs mainly regulate target gene expression at both the post-transcriptional and transcriptional level through imperfect matching between the seed region (2-8 nucleotides of the miRNA) and the 3' untranslated region (UTR) of the target mRNA. Regulation by this matching plays an important role not only in animal development, health and disease, but also in various other biological pathways. Accordingly, studies of miRNAs in humans and many animal models have been well performed, but identification of the total miRNA complement in many animals remains a challenge.

Several functional studies of miR-200a have been performed in vertebrates. In humans and mice, MALAT1 has been reported to affect proliferation, migration, invasion and apoptosis during the progression of hypoxic hepatocellular carcinoma by sponging miR-200a. In addition, miR-200a mediates the proliferation of hepatic stellate cells and the development of fibrosis by targeting the 3'-UTR of SIRT1 through the SIRT1/Notch signaling pathway, and reduces cardiac microvasculature after hypoxia/reoxidative injury through the NRF2 antioxidant pathway. It is involved in protecting thymosin β-4 in endothelial cells. Furthermore, the expression of miR-200a was reported to be down-regulated in fibrostenosing Crohn's disease, HBV-induced hepatocellular carcinoma and human glioma, functioning as a suppressor of many diseases. In chicken, gga-miR-200a is known to regulate the differentiation and proliferation of breast muscle cells by targeting the 3'-UTR of GRB2. In addition, gga-miR-200a-3p was abundantly expressed between 14 and 22 weeks of age, and was reported to target TGFB3 involved in the TGF-β signaling pathway and MAPK signaling pathway in abdominal adipose tissue during postnatal development. . However, there is no study on the regulatory mechanism of gga-miR-200a-3p in the immune response.

Keren Jiang, Meng Zhang, Fang Li, Donghua Li, Guirong Sun, Xiaojun Liu, Hong Li, Ruili Han, Ruirui Jiang, Zhuanjian Li, Xiangtao Kang & Fengbin Yan (2017) Study on the role of gga-miRNA-200a in regulating cell differentiation and proliferation of chicken breast muscle by targeting Grb2, Animal Cells and Systems, 21:6, 365-373.

The present inventors have completed the present invention by confirming that gga-miR-200a-3p plays an important role in regulating the immune response in chickens, and that the immune response can be suppressed or enhanced when the expression level of gga-miR-200a-3p is artificially controlled in the body.

Accordingly, an object of the present invention is to provide a composition for suppressing the innate immune response of chickens, comprising gga-miR-200a-3p or a mimic thereof as an active ingredient.

Another object of the present invention is to provide a composition for enhancing the innate immune response of chickens, comprising an inhibitor of the expression or activity of gga-miR-200a-3p as an active ingredient.

Another object of the present invention is to provide a composition or kit for screening protozoal or bacterial disease resistant chickens, including an agent for measuring the expression or activity level of gga-miR-200a-3p.

Another object of the present invention is to provide a method for screening protozoal or bacterial disease resistant chickens.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned can be clearly understood by those skilled in the art from the description below. will be.

In order to achieve the object of the present invention as described above, the present invention provides a composition for suppressing the innate immune response of chickens, comprising gga-miR-200a-3p or a mimic thereof as an active ingredient.

The present inventors selected gga-miR-200a-3p, which is one of the candidate miRNAs related to immunity in the chicken intestine, and artificially changed its expression to reveal the biological and immunological functions of gga-miR-200a-3p. , Differential expression patterns of gga-miR-200a-3p in the intestinal mucosal layer between necrotizing enteritis-induced chickens and control chickens, and gga-miR-200a-3p directly targeting genes that play a critical role in the innate immune response of chickens It was confirmed that the present invention was completed.

In one embodiment of the present invention, the gga-miR-200a-3p may consist of the nucleotide sequence represented by SEQ ID NO: 2, but is not limited thereto.

In another embodiment of the present invention, the gga-miR-200a-3p or a mimetic thereof may be provided in a form included in a vector or introduced into a cell, but is not limited thereto.

In another embodiment of the present invention, the composition is one selected from the group consisting of ZAK (sterile alpha motif and leucine zipper containing kinase), TGFβ2 (transforming growth factor beta 2) and MAP2K4 (mitogen-activated protein kinase kinase 4) Expression of the above genes can be reduced.

In another embodiment of the present invention, the composition can reduce the expression of one or more protein kinases selected from the group consisting of ERK1/2 (Extracellular signal-regulated protein kinases 1 and 2) and p38.

In another embodiment of the present invention, the composition can reduce the expression of proinflammatory cytokines (proinflammatory cytokine).

In another embodiment of the present invention, the proinflammatory cytokines are IL-1β (Interleukin 1 beta), IFN-γ (Interferon gamma), IL-12p40 (Interleukin-12 subunit p40), IL-17A (Interleukin 17A ) and LITAF (Lipopolysaccharide-induced tumor necrosis factor-alpha factor), but may be one or more selected from the group consisting of, but is not limited thereto.

In another embodiment of the present invention, the innate immune response may be an immune response caused by necrotizing enteritis, but is not limited thereto.

In addition, the present invention provides a composition for enhancing the innate immune response of chickens, comprising an inhibitor of the expression or activity of gga-miR-200a-3p as an active ingredient.

In one embodiment of the present invention, the inhibitor may be a nucleic acid molecule capable of binding to all or part of gga-miR-200a-3p consisting of the nucleotide sequence represented by SEQ ID NO: 2, but is not limited thereto.

In another embodiment of the present invention, the nucleic acid molecule is RNA, DNA, antagomir, antisense molecule, siRNA, shRNA, 2'-O-modified oligonucleotide, phosphorothioate-backbone deoxyribonucleotide, phosphothioate-backbone deoxyribonucleotide, It may be a porothioate-backbone ribonucleotide, a decoy oligonucleotide, a peptide nucleic acid (PNA) oligonucleotide, or a locked nucleic acid (LNA) oligonucleotide, but is not limited thereto.

In another embodiment of the present invention, the composition can increase the expression of one or more genes selected from the group consisting of ZAK , TGFβ2 and MAP2K4 .

In another embodiment of the present invention, the composition can increase the expression of one or more protein kinases selected from the group consisting of ERK1/2 and p38.

In another embodiment of the present invention, the composition can increase the expression of pro-inflammatory cytokines.

In another embodiment of the present invention, the pro-inflammatory cytokine may be one or more selected from the group consisting of IL-1β, IFN-γ, IL-12p40, IL-17A and LITAF, but is not limited thereto.

In another embodiment of the present invention, the innate immune response may be an immune response caused by necrotizing enteritis, but is not limited thereto.

In addition, the present invention provides a composition or kit for screening chickens resistant to protozoal or bacterial diseases, including an agent for measuring the expression or activity level of gga-miR-200a-3p consisting of the nucleotide sequence represented by SEQ ID NO: 2 do.

In one embodiment of the present invention, the agent may include one or more selected from the group consisting of a primer pair, a probe, an antisense oligonucleotide, and an antibody that specifically bind to gga-miR-200a-3p. It is not limited.

In another embodiment of the present invention, the protozoal disease is Eimeria acervulina , Eimeria brunette , Eimeria maxima , Eimeria necatrix ) And Eimeria tenella ( Eimeria tenella ) It may be a disease caused by one or more infections selected from the group consisting of, but is not limited thereto.

In another embodiment of the present invention, the bacterial disease may be a disease caused by infection of Clostridium perfringens ( Clostridium perfringens ), but is not limited thereto.

In another embodiment of the present invention, the chicken may be a strain susceptible to Marek's disease, but is not limited thereto.

In addition, the present invention screens protozoal or bacterial disease-resistant chickens, comprising the step of measuring the expression or activity level of gga-miR-200a-3p consisting of the nucleotide sequence represented by SEQ ID NO: 2 in a biological sample of the subject. provides a way

In one embodiment of the present invention, the method is the expression level of gga-miR-200a-3p consisting of the nucleotide sequence represented by SEQ ID NO: 2 in a state infected with a protozoal disease or a bacterial disease compared to the normal state If it is reduced or unchanged, it can be determined that there is resistance to the disease.

In another embodiment of the present invention, the protozoan is at least one selected from the group consisting of Aimeria acerbulina, Aimeria bruneti, Aimeria maxima, Aimeria necatrix, and Aimeria tenella, and the bacteria are It may be Clostridium perfringens.

In another embodiment of the present invention, the expression level may be measured using a primer pair, probe, antisense oligonucleotide or antibody that specifically binds to gga-miR-200a-3p, but is not limited thereto. .

In another embodiment of the present invention, the expression level is reverse transcription polymerase chain reaction (Reverse Transcription Polymerase Chain Reaction; RT-PCR), competitive reverse transcription polymerase chain reaction (Competitive RT-PCR), real-time reverse transcription polymerase chain reaction (Real-time RT-PCR), RNase protection assay (protection assay), northern blot (northern blot), or may be measured using a DNA chip, but is not limited thereto.

According to the present invention, gga-miR-200a-3p or a mimic thereof inhibits the innate immune response of chickens, and conversely, inhibitors of the expression or activity of gga-miR-200a-3p enhance the innate immune response of chickens. has the effect of More specifically, the composition for suppressing or enhancing the innate immune response of chickens according to the present invention contains TGFβ2 , MAP2K4 , and ZAK , which are genes related to the MAPK (mitogen-activated protein kinase) signaling pathway in response to various external stresses. By regulating the expression, it is possible to indirectly regulate the expression of pro-inflammatory cytokines, which has the effect of regulating the innate immune response of chickens. In addition, by comparing the expression level of gga-miR-200a-3p, chickens resistant to protozoal or bacterial diseases can be selected.

1 is a diagram showing the stem-loop structure of pre-gga-miR-200a and the sequence of mature gga-miR-200a-3p (red font: seed sequence).
Figure 2 is a diagram showing the predicted binding structure and minimum free energy (mfe) between gga-miR-200a-3p and the 3'-UTR of the candidate target gene using the RNA-hybrid algorithm. Green lines and letters represent mature miRNA sequences, and red lines and letters represent part of the 3'-UTR sequence of the target gene.
3a and 3b are diagrams showing the expression profiles of gga-miR-200a-3p (Fig. 3a) and its candidate target genes (Fig. 3b) in the intestinal mucosa of strains 6.3 and 7.2 chickens (* p <0.05; ** p < 0.01).
Figures 4a to 4d show that miR-200a-3p mimics in chicken inhibit the expression of immune genes related to the MAPK signaling pathway, and gga-miR-200a-3p mimics and/or LPS were used in a chicken macrophage cell line. Expression profiles of gga-miR-200a-3p (FIG. 4a) and its candidate target genes (FIG. 4b) and expression levels of signaling molecules in the MAPK signaling pathway (FIG. 4c) and the levels of the signaling molecules after treatment with HD11 Western blot results (FIG. 4d) are shown.
Figure 5 shows gga-miR-200a-3p mimics and cytokine genes involved in the MAPK signaling pathway in the HD11 chicken macrophage cell line treated with LPS ( IL-1β , IFN -γ, IL-20p40 , IL-17A and LITAF) ) It is a diagram showing the expression profile of.
6a to 6c show a vector constructed for verification of the target gene of gga-miR-200a-3p and the result of luciferase reporter analysis using the same, and FIG. 6a shows DsRed and TGFβ2 having gga-miR-200a-3p. It is a schematic diagram showing expression vectors for eGFP having a 3'-UTR part of and luciferase having a 3'-UTR part of a selected gene (selected genes: TGFβ2 , MAP2K4 , or ZAK ), and FIG. 6B is miR200a/DsRed and TGFβ2 Fluorescence microscopy image (scale bar, 400 μm) of the dual fluorescence reporter assay after co-transfection of /EGFP into HD11 chicken macrophages. It is a graph showing the results of analysis by injection or transfection of miR200a/DsRed and luciferase vector as a control.

miRNAs are involved in both the adaptive and innate immune systems of host animals and play important roles in many immune-related pathways. In one embodiment of the present invention, the systemic biological role of gga-miR-200a-3p on the immune response was investigated in Marek's disease (MD) resistant (line 6.3) and susceptible strains (line 6.3), which induced necrotizing enterocolitis (NE). line 7.2). We investigated the expression pattern of gga-miR-200a-3p in the intestinal mucosal layer of chickens when necrotizing enteritis was induced, and identified target genes related to the host immune response to pathogens. As a result, it was found that the expression of gga-miR-200a-3p in the intestinal mucosal layer of Marek's disease-susceptible chicken strain 7.2 was significantly up-regulated during necrotizing enteritis (see Example 2), which is a chicken strain without resistance to the disease. It was suggested that the miRNA is expressed abnormally high in infection situations such as bacteria or protozoa.

Therefore, in another embodiment of the present invention, by confirming the expression change of the candidate target gene according to gga-miR-200a-3p overexpression and/or LPS treatment in chicken macrophage cell line HD11, the gga-miR-200a-3p It was found that 200a-3p down-regulated the expression levels of TGFβ2 , ZAK and MAP2K4 (see Example 3). This regulation of expression was confirmed through luciferase reporter analysis and dual fluorescence analysis that gga-miR-200a-3p directly binds to the 3'-UTR region of the above genes to inhibit expression (see Example 4).

In another embodiment of the present invention, the expression of ERK (Extracellular signal-regulated protein kinase) and p38 protein kinase, which are major MAP kinases that greatly contribute to the innate immune response, are inhibited by gga-miR-200a-3p mimics. It was confirmed that it was significantly inhibited by the transfection of (see Example 5). The above results suggest that gga-miR-200a-3p plays an important role as a regulator of the MAPK signaling pathway, thereby regulating the innate immune response.

Therefore, the present invention can provide a composition capable of controlling the innate immune response of chickens by artificially controlling the level of gga-miR-200a-3p in chickens.

More specifically, the present invention can provide a composition for suppressing the innate immune response of chickens, comprising gga-miR-200a-3p or a mimic thereof as an active ingredient.

As another aspect of the present invention, the present invention may provide a composition for enhancing the innate immune response of chickens, comprising an inhibitor of the expression or activity of gga-miR-200a-3p as an active ingredient.

As used herein, the term “miR” or “miRNA” refers to post-transcriptional regulation of gene expression by binding to the 3' UTR (untranslated region) of target RNA and promoting its degradation or inhibiting their translation. 21 to 23 non-coding RNAs. The mature sequences of the miRNAs used herein can be obtained from the miRNA database (http://www.mirbase.org). As of August 2012, according to the miRNA database (19th edition, miRBase), 25,141 mature miRNAs from 193 species have been registered. In general, microRNAs are transcribed into precursors of about 70-80 nt (nucleotides) in length having a hairpin structure called pre-miRNAs, and then cleaved by Dicer, an RNAse III enzyme, to be produced in a mature form. MicroRNAs form ribonucleocomplexes called miRNPs to cleave target genes or inhibit translation through complementary binding to target sites. More than 30% of human miRNAs exist in clusters, and after being transcribed as one precursor, final mature miRNAs are formed through a cleavage process.

In the present invention, gga-miR-200a-3p is up-expressed when necrotizing enteritis is induced, binds to the 3'-UTR of genes involved in immune response, ZAK , TGFβ2 and/or MAP2K4 , and eventually pro-inflammatory cytokine expression is to suppress

In the present invention, the gga-miR-200a-3p may consist of the nucleotide sequence represented by SEQ ID NO: 2.

As used herein, the term “mimic” refers to a miRNA having the same or similar sequence as the corresponding miRNA molecule, and exhibits the same or extremely similar phenotype. The mimic of gga-miR-200a-3p according to the present invention includes all or part of the gga-miR-200a precursor sequence (SEQ ID NO: 1) capable of performing functions equivalent to gga-miR-200a in the body of a chicken; or all or part of the mature gga-miR-200a-3p sequence (SEQ ID NO: 2); or an oligonucleotide containing the seed sequence of gga-miR-200a-3p.

The precursor sequence of gga-miR-200a (SEQ ID NO: 1) and the mature gga-miR-200a-3p sequence (SEQ ID NO: 2) are as follows:

pre-gga-miR-200a, 5′-GGUCCUCUGUGGGCAUCUUACUAGACAGUGCUGGAUUUCUUGGAUCUAUUCUAACACUGUCUGGUAACGAUGUUUAAAGGGUGAACC-3′ (SEQ ID NO: 1);

gga-miR-200a-3p, 5'-UAACACUGUCUGGUAACGAUGU-3' (SEQ ID NO: 2).

The gga-miR-200a-3p directly binds to the 3'-UTR region of the ZAK , TGFβ2 and/or MAP2K4 genes to suppress the expression of the genes (see Example 4). In addition, as the above MAPK signaling pathway is inhibited by gga-miR-200a-3p, the expression of ERK and p38 protein kinase, which contribute to the innate immune response, is significantly downregulated, and these series of signals The expression of pro-inflammatory cytokines is suppressed by the change in the delivery route, and as a result, the immune response is suppressed. In this case, the pro-inflammatory cytokine whose expression is suppressed may be one or more selected from the group consisting of IL-1β, IFN-γ, IL-12p40, IL-17A and LITAF, but is not limited thereto.

In the present invention, in order to increase the expression of gga-miR-200a-3p, a composition comprising the nucleic acid sequence of gga-miR-200a-3p or gga-miR-200a-3p may be provided. In addition, the gga-miR-200a-3p itself may be included, but it may also include a fragment functionally equivalent thereto, and the fragment of gga-miR-200a-3p contains the seed sequence of gga-miR-200a-3p. It may be an oligonucleotide containing The seed sequence refers to a nucleotide sequence of a part of the miRNA that binds with perfect complementarity when the miRNA recognizes the target gene, and is an essential part for the miRNA to bind to the target. In addition, the gga-miR-200a-3p may be gga-miR-200a in a precursor form.

As used herein, the term "innate immune response" includes various innate resistance mechanisms by which cells or organisms recognize the presence of pathogens and respond. As used herein, “innate immune response” includes intracellular and intercellular events and responses that occur when cells recognize molecular patterns or signals associated with pathogens. The pivotal function of MAPKs in the innate immune response signaling network is well established in both plants and animals. For example, microbial-derived molecules are known to induce MAPK signaling.

When gga-miR-200a-3p or its mimetic is administered to a living body, gga-miR-200a is activated in cells and participates in the functioning pathway, increasing the expression level of gga-miR-200a-3p or reducing its activity. can increase In addition, it directly interacts with endogenous gga-miR-200a and/or gga-miR-200a-3p or indirectly interacts with upstream regulators of gga-miR-200a and/or gga-miR-200a-3p. act to increase the expression of gga-miR-200a and/or gga-miR-200a-3p at the transcriptional level, or to decrease the degradation of expressed gga-miR-200a and/or gga-miR-200a-3p , By increasing the activity, the expression level or activity of gga-miR-200a-3p can be increased.

In the present invention, gga-miR-200a-3p or a mimic thereof may be provided in a form included in a vector or introduced into a cell. In one embodiment, the gga-miR-200a-3p or its mimic may be provided in an expression vector for intracellular delivery. In the present invention, miRNA nucleic acid molecules can be introduced into cells using various transformation techniques, such as a complex of nucleic acid and DEAE-dextran, a complex of nucleic acid and nuclear protein, and a complex of nucleic acid and lipid. It can be provided in a form contained within a delivery system that enables the efficient introduction of Preferably, the delivery vehicle is a vector, and both viral vectors and non-viral vectors can be used. As a viral vector, for example, lentivirus, retrovirus, adenovirus, herpes virus, and avipox virus vectors can be used, It is not limited thereto.

In another embodiment, in the present invention, gga-miR-200a-3p or a mimic thereof may be provided in a form introduced into cells. These cells can express miRNA nucleic acid molecules at high levels, and by transplanting these cells into chickens, the innate immune response can be suppressed.

As a method of introducing into cells, G-fectin, Mirus TrasIT-TKO lipophilic reagent, lipofectin, lipofectamine, cellfectin, cationic phospholipid nanoparticles, cationic polymers, cationic micelles, cationic Intracellular uptake may be increased by introducing into cells together with delivery reagents including emulsions or liposomes, or by conjugating biocompatible polymers such as polyethylene glycol.

In addition, in the present invention, the inhibitor may be a nucleic acid molecule capable of binding to all or part of gga-miR-200a-3p consisting of the nucleotide sequence represented by SEQ ID NO: 2, but is not limited thereto.

As described above, “gga-miR-200a-3p” means the mature sequence of gga-miR-200a consisting of the nucleotide sequence represented by SEQ ID NO: 2. The inhibitor according to the present invention reduces the inhibitory action of gga-miR-200a-3p on ZAK , TGFβ2 and/or MAP2K4 mRNA expression by specifically binding to all or part of gga-miR-200a-3p, and thus The expression of pro-inflammatory cytokines is promoted and, as a result, the immune response is enhanced. At this time, the pro-inflammatory cytokine whose expression is promoted may be one or more selected from the group consisting of IL-1β, IFN-γ, IL-12p40, IL-17A and LITAF, but is not limited thereto.

In the present invention, the inhibitor includes those capable of inhibiting the expression of gga-miR-200a-3p, degrading it, or inhibiting its activity. Inhibiting the activity of gga-miR-200a-3p means inhibiting or interfering with the intracellular action or function of gga-miR-200a-3p, typically the binding of gga-miR-200a-3p to its target molecule. Including direct inhibition of the function of gga-miR-200a-3p using small molecule inhibitors, antibodies or antibody fragments, or indirect regulation using siRNA (small interfering RNA) molecules do. Furthermore, inhibition or inhibition of the activity of gga-miR-200a-3p includes directly or indirectly inhibiting the activity of the precursor sequence (SEQ ID NO: 1) and the mature sequence (SEQ ID NO: 2). In addition, suppressing the activity of gga-miR-200a-3p includes suppressing the transcription of gga-miR-200a itself to lower its intracellular concentration.

As used herein, the term "gga-miR-200a-3p expression or activity inhibitor" includes any substance capable of inhibiting its expression and/or activity. Such substances include, for example, antagomirs, antisense molecules, short hairpin RNA (shRNA) molecules, siRNA molecules, seed target LNA (Locked Nucleic Acid) oligonucleotides, decoy oligonucleotides, aptamers, ribozymes ( ribozyme), or an antibody recognizing a DNA:RNA hybrid.

As used herein, the term “antisense oligonucleotide” refers to a target miRNA, in particular, all or part of the miRNA seed sequence, or all or part of the miRNA including all or part of the miRNA seed sequence. It encompasses nucleic acid-based molecules that can form duplexes with miRNAs. In one embodiment it is an RNA-RNA dimer. Thus, as used herein, the term "antisense oligonucleotide" can be referred to as "complementary nucleic acid-based inhibitor".

The antisense oligonucleotide includes various molecules, such as ribonucleic acid (RNA), deoxyribonucleic acid (DNA), antagomir, 2'-O-modified oligonucleotide, phosphorothioate-backbone deoxyribonucleotide, phosphoro thioate-backbone ribonucleotides, peptide nucleic acid (PNA) oligonucleotides, or locked nucleic acid (LNA) oligonucleotides, but are not limited thereto.

The “LNA” is a modified ribonucleotide, which has a locked form by including an additional bridge between the 2′ to 4′ carbons of the ribose sugar site, so that oligonucleotides with LNA have improved thermal stability. do.

In addition, the “PNA (peptide nucleic acids)” include a peptide-based backbone instead of a sugar-phosphate backbone. As the 2'-O-modified oligonucleotide, for example, a 2'-O-alkyl oligonucleotide may be used.

The "antisense oligonucleotide" includes antisense oligonucleotides in a narrow sense, antagomirs and inhibitory RNA molecules described later.

As used herein, the term “antagomir” refers to a single-stranded, chemically modified oligonucleotide used for silencing endogenous microRNA. Antagomir contains a non-complementary sequence at the Ago2 (Arganoute-2) cleavage site, or the base is modified with, for example, a 2' methoxy group, a 3' cholesterol group, or phosphorothioate to inhibit Ago2 cleavage. , has a sequence complementary to the target sequence. Antagomirs according to the present invention are gga-miR-200a-3p, in particular a seed sequence of gga-miR-200a-3p, in particular a sequence at least partially or completely complementary to all or part of the gga-miR-200a-3p seed sequence. have In one embodiment, the antagomirs contain one or more modifications (eg, 2'-O-methyl-sugar modifications, or 3' cholesterol modifications). In other embodiments, the antagomirs contain one or more phosphorothioate linkages and will at least partially have a phosphorothioate backbone.

The length of antisense oligonucleotides or antagomirs suitable for inhibiting gga-miR-200a-3p according to the present invention may be 6-50 nucleotides, particularly 10-40 nucleotides, more particularly 15-30 nucleotides, but the length is limited It is not, and may include various lengths as long as it achieves the effect according to the present invention.

As used herein, the term "complementary" means that the antisense oligonucleotide is sufficiently complementary to hybridize selectively to the gga-miR-200a-3p target under predetermined hybridization or annealing conditions, preferably physiological conditions. It means that, partially or partially, it has a meaning encompassing both substantially complementary and completely complementary, and preferably means completely complementary. Substantially complementary, but not completely complementary, refers to a degree of complementarity that binds to a target sequence and produces an effect according to the present disclosure, that is, an effect sufficient to block the activity of gga-miR-200a-3p.

As used herein, the term “nucleic acid” includes polynucleotides, oligonucleotides, DNA, RNA, analogues and derivatives thereof, including, for example, peptide nucleic acids (PNA) or mixtures thereof. In addition, nucleic acids may be single or double stranded, and may encode molecules including polypeptides, mRNAs, microRNAs, or siRNAs.

In one embodiment according to the present invention, the expression or activity inhibitor of gga-miR-200a-3p complementarily binds to all or part of the progenitor and/or mature sequence of gga-miR-200a, particularly the seed sequence, thereby It is an antisense oligonucleotide capable of inhibiting activity. Inhibiting the expression or activity is to inhibit transcription of gga-miR-200a and/or binding of gga-miR-200a-3p to target mRNA, such as TGFβ2 , ZAK and/or MAP2K4 mRNA. The antisense oligonucleotide according to the present invention may or may not include one or more of the following modifications in the nucleotides constituting it or the backbone (backbone) linking the nucleotides. That is, the antisense oligonucleotide may include LNA in one or more nucleotides constituting it, 2'-O-methylation in the sugar of one or more nucleotides constituting it, or one or more phosphothioates in its backbone.

Also, in the present invention, the innate immune response may be an immune response induced by necrotizing enterocolitis. That is, in chickens infected with the causative agent of necrotizing enteritis, when an excessive immune response occurs or an immune response does not occur, the composition for regulating the immune response according to the present invention can be used to suppress the excessive immune response of chickens or induce an appropriate immune response. can

On the other hand, the composition for regulating the innate immune response of chickens according to the present invention, that is, the composition for inhibiting the innate immune response of chickens or the composition for enhancing the innate immune response of chickens, includes appropriate carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions. can include more. The excipient may be, for example, one or more selected from the group consisting of a diluent, a binder, a disintegrant, a lubricant, an adsorbent, a moisturizer, a film-coating material, and a controlled release additive.

The compositions according to the present invention are powders, granules, sustained-release granules, enteric granules, liquids, eye drops, elsiliks, emulsions, suspensions, spirits, troches, perfumes, rimonadese, tablets, respectively, according to conventional methods. , sustained-release tablets, enteric tablets, sublingual tablets, hard capsules, soft capsules, extended-release capsules, enteric capsules, pills, tinctures, soft extracts, dry extracts, fluid extracts, injections, capsules, perfusate, warnings , Lotions, pastas, sprays, inhalants, patches, sterile injection solutions, or external preparations such as aerosols may be formulated and used, and the external preparations may include creams, gels, patches, sprays, ointments, warning agents, and lotions. It may have formulations such as agent, liniment agent, pasta agent, or cataplasma agent.

Carriers, excipients and diluents that may be included in the composition according to the present invention include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginates, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Corn starch, potato starch, wheat starch, lactose, sucrose, glucose, fructose, di-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, phosphoric acid as additives for tablets, powders, granules, capsules, pills, and troches according to the present invention Calcium monohydrogen, calcium sulfate, sodium chloride, sodium bicarbonate, purified lanolin, microcrystalline cellulose, dextrin, sodium alginate, methylcellulose, sodium carboxymethylcellulose, kaolin, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropylmethyl excipients such as cellulose, 1928, 2208, 2906, 2910, propylene glycol, casein, calcium lactate, and Primogel; Gelatin, gum arabic, ethanol, agar powder, cellulose phthalate acetate, carboxymethyl cellulose, calcium carboxymethyl cellulose, glucose, purified water, sodium caseinate, glycerin, stearic acid, sodium carboxymethyl cellulose, sodium methyl cellulose, methyl cellulose, microcrystalline cellulose, dextrin Binders such as hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, purified shellac, starch arc, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinyl alcohol, and polyvinylpyrrolidone may be used, Hydroxypropyl Methyl Cellulose, Corn Starch, Agar Powder, Methyl Cellulose, Bentonite, Hydroxypropyl Starch, Sodium Carboxymethyl Cellulose, Sodium Alginate, Calcium Carboxymethyl Cellulose, Calcium Citrate, Sodium Lauryl Sulfate, Silicic Anhydride, 1-Hydroxy Propyl cellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gum, sodium bicarbonate, polyvinylpyrrolidone, calcium phosphate, gelled starch, gum arabic, disintegrants such as amylopectin, pectin, sodium polyphosphate, ethyl cellulose, white sugar, magnesium aluminum silicate, di-sorbitol solution, and light anhydrous silicic acid; Calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopod, kaolin, vaseline, sodium stearate, cacao butter, sodium salicylate, magnesium salicylate, polyethylene glycol 4000, 6000, liquid paraffin, hydrogenated soybean oil (Lubri wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, macrogol, synthetic aluminum silicate, silicic anhydride, higher fatty acid, higher alcohol, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, starch, sodium chloride, A lubricant such as sodium acetate, sodium oleate, dl-leucine, light anhydrous silicic acid; may be used.

Additives for the liquid formulation according to the present invention include water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, sucrose monostearate, polyoxyethylene sorbitol fatty acid esters (tween esters), polyoxyethylene monoalkyl ethers, lanolin ethers, Lanolin esters, acetic acid, hydrochloric acid, aqueous ammonia, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethyl cellulose, sodium carboxymethyl cellulose, and the like may be used.

In the syrup according to the present invention, a solution of white sugar, other sugars, or a sweetener may be used, and aromatics, coloring agents, preservatives, stabilizers, suspending agents, emulsifiers, thickeners, etc. may be used as necessary.

Purified water may be used in the emulsion according to the present invention, and emulsifiers, preservatives, stabilizers, fragrances, etc. may be used as needed.

Acacia, tragacantha, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose, 1828, 2906, 2910, etc. may be used as the suspending agent according to the present invention. Surfactants, preservatives, stabilizers, colorants, and fragrances may be used as needed.

Injections according to the present invention include distilled water for injection, 0.9% sodium chloride injection, IV injection, dextrose injection, dextrose + sodium chloride injection, PEG, lactated IV injection, ethanol, propylene glycol, non-volatile oil-sesame oil , solvents such as cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, and benzene benzoate; solubilizing agents such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene glycol, twins, nijuntinamide, hexamine, and dimethylacetamide; buffers such as weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, albumin, peptone, and gums; tonicity agents such as sodium chloride; Stabilizers such as sodium bisulfite (NaHSO ₃ ) carbon dioxide gas, sodium metabisulfite (Na ₂ S ₂ O ₃ ), sodium sulfite (Na ₂ SO ₃ ), nitrogen gas (N ₂ ), ethylenediaminetetraacetic acid; Sulfating agents such as sodium bisulfide 0.1%, sodium formaldehyde sulfoxylate, thiourea, ethylenediamine disodium tetraacetate, acetone sodium bisulfite; analgesics such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; Suspending agents such as Siemesis sodium, sodium alginate, Tween 80, aluminum monostearate may be included.

The suppository according to the present invention includes cacao butter, lanolin, witapsol, polyethylene glycol, glycerogelatin, methylcellulose, carboxymethylcellulose, a mixture of stearic acid and oleic acid, subanal, cottonseed oil, peanut oil, palm oil, cacao butter + Cholesterol, Lecithin, Lannet Wax, Glycerol Monostearate, Tween or Span, Imhausen, Monolen (Propylene Glycol Monostearate), Glycerin, Adeps Solidus, Buytyrum Tego-G -G), Cebes Pharma 16, Hexalide Base 95, Cotomar, Hydroxycote SP, S-70-XXA, S-70-XX75 (S-70-XX95), Hyde Hydrokote 25, Hydrokote 711, Idropostal, Massa estrarium (A, AS, B, C, D, E, I, T), Massa-MF, Masupol, Masupol-15, Neosupostal-N, Paramound-B, Suposiro (OSI, OSIX, A, B, C, D, H, L), suppository type IV (AB, B, A, BC, BBG, E, BGF, C, D, 299), Supostal (N, Es), Wecobi (W, R, S, M, Fs), testosterone triglyceride base (TG-95, MA, 57) and The same mechanism can be used.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations contain at least one excipient, for example, starch, calcium carbonate, sucrose, etc. ) or by mixing lactose and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

Liquid preparations for oral administration include suspensions, solutions for oral administration, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. there is. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents.

The composition of the present invention can be administered to a subject by various routes. For example, it may be administered by oral administration, intranasal administration, transbronchial administration, arterial injection, intravenous injection, subcutaneous injection, intramuscular injection, or intraperitoneal injection. The daily dose may be divided and administered once or several times a day.

As another aspect of the present invention, the present invention comprises a preparation for measuring the expression or activity level of gga-miR-200a-3p consisting of the nucleotide sequence represented by SEQ ID NO: 2, screening protozoal or bacterial disease resistant chickens It is possible to provide a composition for protozoal disease or a kit for screening chickens resistant to bacterial diseases.

In the present invention, the agent may include one or more selected from the group consisting of a primer pair, a probe, an antisense oligonucleotide, and an antibody specifically binding to gga-miR-200a-3p, but is not limited thereto. .

In addition to the above components, the kit of the present invention may additionally include other components. For example, when the kit of the present invention is applied to a PCR amplification process, the kit of the present invention optionally contains reagents necessary for PCR amplification, such as buffers, DNA polymerases [eg, Thermus aquaticus (Taq), Thermus thermophilus (Tth), a thermostable DNA polymerase obtained from Thermus filiformis, Thermis flavus, Thermococcus literalis or Pyrococcus furiosus (Pfu)], DNA polymerase cofactors and dNTPs. The kit of the present invention may be manufactured in a number of separate packaging or compartments containing the reagent components described above. According to one embodiment of the present invention, the kit of the present invention may be a microarray. According to another embodiment of the present invention, the kit of the present invention is a gene amplification kit.

In the present invention, the protozoal diseases are Eimeria acervulina , Eimeria brunette , Eimeria maxima , Eimeria necatrix and Eimeria Tenella ( Eimeria tenella ) It may be a disease caused by one or more infections selected from the group consisting of, the bacterial disease may be a disease caused by infection of Clostridium perfringens ( Clostridium perfringens ).

In addition, in the present invention, the chicken may be a strain susceptible to Marek's disease.

In this study, the expression level of gga-miR-200a-3p significantly increased during the induction of necrotizing enteritis in Marek's disease-susceptible chickens, whereas gga-miR-200a-3p was observed before and after the induction of necrotizing enteritis in Marek's disease-resistant chickens. No change in expression was observed (see Example 2). These experimental results show that chickens resistant to protozoal or bacterial diseases do not significantly increase the expression level of miR-200a-3p, which properly regulates the immune response when diseases occur due to infiltration of protozoa or bacteria. This means that an appropriate level of activation of the immune response is achieved.

In another aspect, the present invention is a protozoal disease or bacterial disease resistant chicken comprising the step of measuring the expression or activity level of gga-miR-200a-3p consisting of the nucleotide sequence represented by SEQ ID NO: 2 in a biological sample of a subject. A selection method can be provided.

In the present invention, the method is performed when the expression level of gga-miR-200a-3p consisting of the nucleotide sequence represented by SEQ ID NO: 2 is reduced or unchanged compared to the normal state when the subject is infected with protozoa or bacteria. It can be identified as being resistant to disease.

In addition, in the present invention, the type of protozoa or bacteria is as described above, and the biological sample of the subject in which the expression level of gga-miR-200a-3p is measured includes tissue, cell, whole blood, serum, plasma, saliva, sputum, spinal fluid, urine, and the like, but is not limited thereto. For example, when the disease is necrotic enteritis, the expression level of gga-miR-200a-3p can be measured in the intestinal mucosa of the subject.

In addition, in the present invention, the expression level of gga-miR-200a-3p can be measured using a primer pair, probe, antisense oligonucleotide or antibody that specifically binds to gga-miR-200a-3p, and the measurement method Examples include Reverse Transcription Polymerase Chain Reaction (RT-PCR), Competitive RT-PCR, Real-time RT-PCR, and RNase protection assay Methods known in the art including protection assay, northern blot, or DNA chip may be used.

After the reaction such as the reverse transcription polymerase chain reaction, the product is subjected to electrophoresis to check the pattern and thickness of the band, thereby confirming the expression of gga-miR-200a-3p and the degree of expression. ·By comparing with the after, it is possible to easily determine chicken individuals or openings with resistance to disease. On the other hand, the DNA chip uses a DNA chip in which nucleic acids corresponding to the gga-miR-200a-3p or a fragment thereof are attached to a glass-like substrate at high density. A cDNA probe labeled at the end or inside with a fluorescent material is prepared, hybridized to a DNA chip, and then whether or not the individual has resistance to a disease can be read.

The terms or words used in this specification and claims should not be construed as being limited to ordinary or dictionary meanings, and the inventors may appropriately define the concept of terms in order to explain their invention in the best way. It should be interpreted as a meaning and concept consistent with the technical idea of the present invention based on the principle that there is.

Hereinafter, a preferred embodiment is presented to aid understanding of the present invention. However, the following examples are provided to more easily understand the present invention, and the content of the present invention is not limited by the following examples.

[Example]

experimental example. Experimental Materials and Methods

1. Necrotizing Enteritis Disease Model Animals

Two lines of White Leghorn chickens (6.3 and 7.2), highly bred and maintained since 1931, are resistant or susceptible to avian leucosis virus (ALV) and Marek's disease virus (MDV), respectively. showed To induce necrotizing enteritis, Eimeria maxima (EM ) strain 41A (1.0 × 10 ⁴ oocysts/birds) was infected by oral gavage at 14 days after hatching, During the next 2 days (4 days after EM infection) Clostridium perfringens (CP ) strain Del (1.0 x 10 ⁹ cfu/birds) was infected by oral gavage. Infection experiments were conducted for a total of 6 days, and intestinal mucosal layers (IMLs) were collected from 5 chickens per group after induction of necrotizing enteritis. Additional intestinal mucosal samples were provided and used by the Animal Life Sciences and Biotechnology Laboratory of the USDA-Agricultural Research Service. All animal experiments were performed under the approval of the Institutional Animal Care and Use Committee of Beltsville Agricultural Research Center (protocol #09-019). The intestinal mucosal layer was frozen in liquid nitrogen and carefully homogenized, and total RNA was extracted using TRIzol (Invitrogen, USA).

2. Target gene prediction of gga-miR-200a-3p

Target gene prediction of gga-miR-200a-3p was performed through miRDB (v6.0), which includes chicken miRNA and mRNA data and provides a custom prediction mode based on mature miRNA sequences. Genes with a target score of 80 or higher were further functionally analyzed using the DAVID Bioinformatic Resources and KEGG PATHWAY databases, through which the MAPK signaling pathway, TGF-β pathway and/or toll- Mapping of genes associated with immune-related pathways, such as toll-like receptor signaling pathways, was induced. Next, potential targets were predicted based on whether they met certain criteria and whether there was a binding site for gga-miR-200a-3p in the 3'-UTR.

3. Cell culture, miRNA mimics and LPS stimulation

The HD11 chicken macrophage cell line was grown at 41° C. under humidified conditions in RPMI-1640 medium supplemented with L-glutamine (Gibco, USA), 10% heat-inactivated fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin. It was cultured under % CO ₂ environment.

The gga-miR-200a-3p mimetic oligonucleotide for overexpression of gga-miR-200a-3p in cells was commissioned by Bioneer (Bioneer, Korea) based on the gga-miR-200a precursor sequence obtained from the miRbase catalog. It was synthesized, and the base sequence is as follows:

gga-miR-200a-3p mimic, 5'-UAACACUGUCUGGUAACGAUGU-3', SEQ ID NO: 2.

The experimental group of the present invention consisted of four treatments as follows: (1) control, (2) Salmonella enteritis ( Lipopolysaccharide ) exposure, (3) miRNA mimic overexpression and (4) LPS exposure. upon miR-200a mimic overexpression. Each group consisted of 3 independent replicates. To overexpress the miRNA mimic, the synthesized gga-miR-200a-3p mimic oligonucleotide was diluted with Opti-MEM medium (Gibco) according to the manufacturer's instructions, using Lipofectamine 3000 (Invitrogen, USA), and finally HD11 chicken macrophage cell line seeded in a 12-well plate at a concentration of 50 nM was transiently transfected. After 20 hours of transient transfection, treated cells were stimulated with LPS (1.0 μg/mL; Sigma-Aldrich, USA) or normal medium for 4 hours. Controls were treated with similar amounts of Lipofectamine 3000 and Opti-MEM without oligonucleotides. Samples were collected from each group for further experiments.

4. Construction of gga-miR-200a-3p or candidate target gene 3'-UTR constructs

Oligonucleotides having the mature gga-miR-200a-3p sequence using NotI restriction enzymes (5' and 3') were converted to pDsRed2-N1 containing red fluorescent protein (RFP) expressed by the cytomegalovirus (CMV) promoter. It was cloned into a plasmid (Clontech, USA). This construct, named miR200a/DsRed, co-expressed a small fragment of the miRNA molecule and RFP to visualize the RNA molecule. The miR-200a binding site 3'-UTR flanking region predicted in the candidate target gene was amplified using pooled cDNA from ADOL chicken strains as a template.

For dual fluorescence analysis, the 3'-UTR portion of TGFβ2 was ligated into a pcDNA3 plasmid containing enhanced green fluorescence protein (eGFP) using NotI/XbaI restriction enzymes. The resulting construct was named TGFβ2/EGFP.

For the luciferase assay, the PCR product of the target gene was digested with SacI/HindIII and cloned into the pMIR-REPORT luciferase vector (Ambion, USA) to obtain “Gene” containing TGFβ2, ZAK and MAP2K4 independently. A /Luc recombinant construct was created. Competent E. coli (Invitrogen, USA) was transformed using the ligated construct, and the recombinant plasmid was identified by sequencing (Genotech, Korea).

5. Quantitative real-time PCR (qRT-PCR) for mRNA and miRNA quantification

Total RNA from chicken lines 6.3 and 7.2 and HD11 cells was isolated using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. For cDNA synthesis from mRNA, RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, USA) was used. More specifically, the reaction mixture (20 μL) contained RNA (2 μg), 5X reaction buffer (4 μL), 10 mM dNTP mix (2 μL), oligo (dT) ₁₈ primer (1 μL), RiboLock RNase inhibitor (1 μL). U) and RevertAid M-MuLV RT (10 U). The reaction was carried out under the following conditions: 42 °C for 60 min, followed by 70 °C for 5 min. In the case of miRNA cDNA synthesis, it was performed using the Ncode™ miRNA first-strand cDNA synthesis Kit (Invitrogen) according to the manufacturer's protocol. A diluted amount of cDNA was used as a template to perform qRT-PCR according to the manufacturer's instructions. A specific forward primer for miRNA was designed, and a universal primer was used as the reverse primer. Primers for mRNA were designed using Primer-BLAST. qRT-PCR was performed using 2X Power SYBR Green Master Mix (Roche Life Science, Germany) and the LightCycler ^® 96 system (Roche Life Science) according to the manufacturer's instructions. Ct (Threshold cycle) values were normalized to GAPDH (mRNA) or U1A micronuclear RNA (miRNA) values by the 2 ^-ΔΔCt method.

6. Western blotting

HD11 chicken macrophage cells were transfected with gga-miR-200a-3p mimics or gga-miR-200a-3p mimics in an environment stimulated with endotoxins, and protein expression was analyzed. Protein samples whose concentration was measured by BCA assay were subjected to electrophoresis on an SDS-PAGE gel containing 10% polyacrylamide and transferred to a polyvinylidene difluoride (PVDF) membrane (GE Healthcare, Australia). The PVDF membrane was blocked with 5% non-fat milk containing 0.05% Tween 20 in PBS (pH 7.4) (PBST) for 1 hour. Rabbit anti-chicken phosphor-p44/42 MAPK (Cell Signaling Technology, ERK1/2; Thr202/Tyr204, #4370, specific), rabbit anti-chicken phospho-p38 MAPK (Cell Signaling Technology, Thr180/Tyr182, #4631, Primary antibodies including mouse anti-chicken GAPDH antibody (Thermo Fisher Scientific, AM4300, specific) and mouse anti-chicken GAPDH antibody (specific) were prepared in PBST containing 2% non-fat milk based on the specific dilution ratio of each primary antibody. It was prepared by dilution, and the PVDF membrane was incubated overnight at 4°C. After washing with PBST, membranes were treated with horseradish peroxidase (HRP)-linked anti-rabbit or anti-mouse secondary antibodies (Thermo Fisher Scientific; based on primary antibodies) diluted in PBST containing 2% skimmed milk. and incubated for 2 hours at room temperature. Then, band signals were detected using Western Lightning ECL Plus substrate (Thermo Fisher Scientific) and exposed to Hyperfilm (GE Healthcare).

7. Dual fluorescence assay

HD11 chicken macrophages were seeded in a 12-well plate (Corning, USA) at 1.0 × 10 ⁶ cells/well and cultured. When confluency reached 80%, the cells were co-transfected with miR200a/DsRed or TGFβ2/EGFP, or both plasmids using Lipofectamine 3000 (Invitrogen) according to the manufacturer's instructions. . 3 hours after transfection, 50 μM of β-mercaptoethanol was added to each well. 48 hours after transfection, eGFP or DsRed was visualized using the EVOS ^® FL color imaging system (Life Technologies). The fluorescence microscope was set up as follows: green fluorescent protein (eGFP), 470 nm excitation filter and 525 nm emission filter; Red fluorescent protein (DsRed), 530 nm excitation filter and 593 nm emission filter.

8. Luciferase reporter assay

HD11 chicken macrophages were seeded in a 12-well plate (Corning) at 1.0 × 10 ⁶ cells/well and cultured. “Gene”/Luc (“Gene” is the TGFβ2, ZAK or MAP2K4 gene) was co-transfected with miR200a/DsRed into cells using Lipofectamine 3000 (Invitrogen) according to the manufacturer's instructions, and pMIR-REPORT β -gal control plasmid (Ambion) was also transfected to quantify transfection efficiency. Cells were collected after 24 hours and lysed with 1X luciferase cell culture lysis reagent (Promega, USA). After centrifugation at 20,000 × g for 1 minute, the obtained supernatant was analyzed using a luciferase assay system (Promega) and a β-galactose solution (o-nitrophenyl-β-D-galactopyranoside or OPNG), respectively, according to the manufacturer's instructions. was used to measure the activity of luciferase and β-galactosidase in a 96-well plate (Corning). For verification of the results, all experiments were independently repeated three times.

9. Statistical Analysis

All in vitro experiments were performed in triplicate, and differences between groups were analyzed using one-way ANOVA, followed by Duncan's multiple range test with IBM SPSS software (SPSS 25.0; USA). was performed. Data for each group ( N =3) were expressed as mean ± standard error of the mean (SEM) values. Statistical significance was set at p < 0.05.

Example 1. Structural analysis of gga-miR-200a-3p and prediction of candidate genes

The miRDB algorithm used to predict miRNA target genes mainly searched for target genes based on the sequence of gga-miR-200a-3p (Fig. 1), and as a result, a total of 555 candidate target genes were found. Next, the genes were classified based on the target score, and 102 candidate target genes were identified with a score of 90 or higher. Of these, as predicted by the DAVID and KEGG tools, 4 genes were involved in immune-related pathways, and 3 genes among them were involved in MAPK signaling pathways. These three genes, ZAK (sterile alpha motif and leucine zipper containing kinase), TGFβ2 (transforming growth factor beta 2), and MAP2K4 (mitogen-activated protein kinase kinase 4) were selected for further validation experiments. The binding site between the 3'-UTR of each candidate gene and gga-miR-200a-3p is RNA based on the seed match (2-8 nucleotides of miRNA and 3'-UTR of candidate gene) between the two molecules. -Predicted by hybrid software tools. The results are shown in Figure 2.

Example 2. Expression analysis of gga-miR-200a-3p and its target genes in chicken intestinal mucosa

In order to determine the expression of gga-miR-200a-3p and putative target genes associated with induction of necrotizing enteritis, intestinal mucosal layers obtained from necrotizing enteritis induction and control groups (both Marek's disease-resistant line 6.3 and Marek's disease-susceptible line 7.2) (IMLs) qRT-PCR was performed using cDNA from samples. On day 6 after induction of necrotizing enteritis, the expression level of gga-miR-200a-3p in the intestinal mucosal layer was significantly increased in line 7.2, but not in line 6.3 (Fig. 3a). In addition, expression of the three candidate target genes TGFβ2, ZAK and MAP2K4 was found to be significantly downregulated only in line 7.2 chickens (FIG. 3B). These results suggest that the expression of each candidate target gene in line 7.2 chickens was negatively correlated with the expression of gga-miR-200a-3p.

Example 3. Expression analysis of candidate target genes according to synthetic gga-miR-200a and LPS treatment in chicken macrophage cell line HD11

Next, to investigate whether the expression of candidate target genes could be altered by overexpressing gga-miR-200a-3p upon LPS stimulation (endotoxin stimulation), we investigated gga-miR-200a-3p mimics ( mimic) was transfected with HD11, and the cells were treated with LPS derived from SE . As a result, the expression of gga-miR-200a-3p was found to be significantly increased in both cells transfected with miRNA alone and cells treated with LPS after miRNA transfection, compared to the control group (see Fig. 4a). This indicates that gga-miR-200a-3p was successfully induced.

Among the three candidate target genes, the expression level of ZAK mRNA was significantly reduced in cells transfected with miRNA mimics alone (Fig. 4b middle graph), and in cells treated with LPS after miRNA transfection, TGFβ2 and ZAK and MAP2K4 mRNA expression levels were strongly decreased compared to cells stimulated only with LPS (FIG. 4b, p < 0.05). However, the expression of gga-miR-200a-3p and its target gene ZAK did not show any change in response to SE -derived LPS compared to the control group (middle graphs in Figs. 4a and 4b). On the other hand, significant increases (4-fold and 14-fold, respectively) were observed in the expression of TGFβ2 and MAP2K4 by LPS treatment (upper and lower graphs in Fig. 4b). The above results directly show that the overexpression of gga-miR-200a-3p can down-regulate the expression levels of TGFβ2 , ZAK and MAP2K4 .

Example 4. Verification of target gene of gga-miR-200a-3p in HD11 chicken macrophage cell line

To confirm that miR-200a inhibits the expression of candidate target genes by targeting the predicted seed region of the 3'-UTR region, a luciferase reporter assay and dual fluorescence reporter assay were performed in HD11 cells. Each target gene region complementary to gga-miR-200a-3p was predicted using an RNA-hybrid computational analysis program (see Fig. 2). In addition, for this purpose, an eGFP reporter vector “TGFβ2/EGFP” was constructed (Fig. 6a). After transfection of miR200a/DsRed and/or TGFβ2/EGFP vectors, expression of eGFP and DsRed proteins was analyzed by fluorescence microscopy.

As a result, as shown in FIG. 6B , in the cells co-transfected with the miR200a/DsRed and TGFβ2/EGFP constructs, it was confirmed that DsRed and GFP fluorescence were significantly reduced compared to the control group. These results suggest that gga-miR-200a-3p suppresses the expression of TGFβ2 by targeting the 3'-UTR of the gene.

In addition, a luciferase reporter vector containing TGFβ2 , MAP2K4 or ZAK (hereinafter “Gene”) was constructed, and the DsRed reporter vector was named miR200a/DsRed (see FIG. 6a). The miR200a/DsRed construct was co-transfected into the HD11 cell line either with the “Gene/Luc” vector or with an empty luciferase vector as a control.

As a result, as shown in FIG. 6, the expression pattern of the target gene showed a significant decrease in luciferase activity when miR200a/DsRed was co-transfected with TGFβ2/Luc, ZAK/Luc or MAP2K4/Luc compared to the control group. Confirmed. The above results indicate that gga-miR-200a-3p can directly target TGFβ2 , MAP2K4 and ZAK .

Example 5. Inhibitory effect of gga-miR-200a-3p on MAPK signaling pathway

Extracellular signal-regulated protein kinase (ERK) and p38 MAP kinase are two major MAP kinases that greatly contribute to the innate immune response. Accordingly, the present inventors investigated whether gga-miR-200a-3p affects the expression of molecules related to the MAPK signaling pathway.

As a result, in the group transfected with the miR-200a mimic, the levels of p38α and ERK1 mRNA were downregulated 5-fold compared to the control group. On the other hand, no difference in ERK2 expression was observed between the samples transfected with the miR-200a mimic and the control group (see Figs. 4c and 4d). However, when treated with LPS after miR-200a mimic transfection, it was confirmed that ERK1 and p38α mRNA levels were down-regulated by 2-fold and ERK2 mRNA by 70-fold (Fig. 4c). In addition, the protein levels of p38 and ERK1/2 were analyzed by Western blotting. As a result, as shown in Fig. 4d, the miR-200a mimic significantly suppressed the expression of p-p38 and pERK1/2 in the LPS-treated environment.

As a downstream component of the MAPK signaling pathway, pro-inflammatory cytokines including Th1 type cytokines IL-1β , IFN-γ and IL-12p40 ; The Th17 cytokine family IL-17A and the pro-inflammatory mediator LITAF were significantly down-regulated in the LPS-stimulated group transfected with miR-200a mimic compared to the LPS-only treated group (FIG. 5). In particular, it was confirmed that IFN-γ and IL-17A mRNA expressions, which were significantly suppressed when gga-miR-200a-3p was overexpressed, were strongly upregulated by about 60-fold and 100-fold, respectively, when treated with LPS alone (FIG. 5). These results indicate that the miR-200a mimic negatively regulates the MAP kinase-related pathway and can suppress the expression of pro-inflammatory cytokines in HD11 chicken macrophages.

Summarizing the results disclosed in the above examples, the present inventors have demonstrated the biological role of Gallus gallus miRNA-200a-3p using two genetically different chicken strains, 6.3 and 7.2, induced with necrotizing enteritis (NE). and a new mechanism of the basic signal transduction pathway in chickens. Expression of gga-miR-200a-3p in the intestinal mucosa of necrotizing enterocolitis-induced chickens was found to be upregulated during necrotizing enterocolitis infection in disease-susceptible chicken strain 7.2, identical to gga-miR-200a-3p. Overexpression analysis using chemically synthesized oligonucleotides, reporter gene analysis including luciferase reporter analysis, and dual fluorescence reporter analysis in chicken macrophage cell line HD11 were performed to confirm target genes. It was confirmed that it is a direct transcriptional repressor of ZAK , MAP2K4 , and TGFβ2 involved in the mitogen-activated protein kinase pathway, and targets the 3'-UTR of these transcripts. In addition, gga-miR-200a-3p can indirectly affect both the transcriptional and translational levels of protein kinases including p38 and ERK1/2 , indicating that the miRNA is an important regulator of the MAPK signaling pathway. It means that it can function as a ruler. Proinflammatory cytokines composed of IL-1β , IFN-γ , IL-12p40 , IL-17A and LITAF belonging to Th1 and Th17 type cytokines were upregulated by overexpression of gga-miR-200a-3p. These series of findings suggest that gga-miR-200a-3p mediates the immune response of chickens through regulation of the MAPK signaling pathway, and therefore, the immune response of chickens can be controlled by artificially regulating the level of the miRNA, and livestock is expected to serve as an important biomarker of disease.

The above description of the present invention is for illustrative purposes, and those skilled in the art can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, the embodiments described above should be understood as illustrative in all respects and not limiting.

<110> Chung-Ang University Industry-Academy Cooperation Foundation <120> A composition for modulating the innate immune response of chickens comprising gga-miR-200a-3p, mimics thereof or inhibitors of gga-miR-200a-3p <130> MP20-058 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 87 <212> RNA 213 <br> <400> 1 gguccucugu gggcaucuua cuagacagug cuggauuucu uggaucuauu cuaacacugu 60 cugguaacga uguuuaaagg gugaacc 87 <210> 2 <211> 22 <212> RNA 213 <br> <400> 2 uaacacuguc ugguaacgau gu 22

Claims (27)

In the composition for inhibiting the innate immune response of chickens,
It contains gga-miR-200a-3p consisting of the nucleotide sequence represented by SEQ ID NO: 2 as an active ingredient,
The innate immune response is characterized in that the immune response by necrotizing enteritis induced by Eimeria maxima and Rostridium perfringens ( Clostridium perfringens )
A composition for suppressing the innate immune response of chickens.
delete According to claim 1,
The gga-miR-200a-3p is contained in a vector or provided in a form introduced into a cell, characterized in that, the composition for suppressing the innate immune response of chickens.
According to claim 1,
The composition is characterized by reducing the expression of one or more genes selected from the group consisting of ZAK (sterile alpha motif and leucine zipper containing kinase), TGFβ2 (transforming growth factor beta 2) and MAP2K4 (mitogen-activated protein kinase kinase 4) To, a composition for inhibiting the innate immune response of chickens.
According to claim 1,
The composition is characterized by reducing the expression of one or more protein kinases selected from the group consisting of ERK1/2 (Extracellular signal-regulated protein kinases 1 and 2) and p38, a composition for inhibiting the innate immune response of chickens.
According to claim 1,
The composition is a composition for suppressing the innate immune response of chickens, characterized in that reducing the expression of pro-inflammatory cytokines.
According to claim 6,
The pro-inflammatory cytokine is characterized in that at least one selected from the group consisting of IL-1β, IFN-γ, IL-12p40, IL-17A and LITAF, composition for suppressing the innate immune response of chickens.
delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete

Images