Application of gga-miR-7b in relieving chicken eimeria tenella infection
By regulating the expression amount and detection methods of gga-miR-7b, the limitations and drug resistance of chemical drugs in coccidiosis infection in chicken tender Emmel is solved, effective inflammatory damage detection and proliferation vitality regulation are provided, and the resistance of chickens to coccidiosis infection is enhanced, and scientific basis for early diagnosis and prevention is provided.
Patent Information
- Application Number
- CN202510513328.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-23
- Publication Date
- 2025-08-22
AI Technical Summary
The prior art has problems with chemical drug residues, drug resistance and vaccine safety in preventing and treating chicken tender Emmel coccidiosis infection. It lacks effective miRNA regulation methods, making it difficult to effectively alleviate inflammatory damage and growth hysteresis caused by infection.
By regulating the expression amount of gga-miR-7b, using its overexpression and interfering reagents, combined with specific primers and fluorescence quantitative PCR detection methods, the inflammatory damage during coccidiosis infection is detected and regulated, and the proliferation vitality of host cells is promoted or inhibited.
It has achieved rapid and accurate judgment of the degree of inflammatory damage of coccidiosis infection, provided new molecular markers and targets, enhanced chicken body's resistance to coccidiosis infection, promoted early diagnosis and prevention, and provided scientific basis and molecular targeted treatment plans.
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Figure CN120514723A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of bioengineering, and particularly relates to application of gga-miR-7b in alleviating Eimeria tenella (E. tenella) infection in chickens. Background Art
[0002] Coccidiosis is a type of intestinal parasitic disease caused by Eimeria spp., of which Eimeria tenella is considered one of the most pathogenic coccidia. Symptoms include intestinal mucosal ulceration, bloody diarrhea, and hemorrhagic lesions of the intestinal wall, accompanied by systemic symptoms such as depression, decreased food intake, and impaired nutrient metabolism. These symptoms ultimately lead to growth retardation and poor feed utilization, causing significant economic losses to the global poultry industry. Currently, domestic and international approaches to coccidiosis prevention and control primarily rely on the use of chemically synthesized anticoccidial drugs and coccidial vaccines. However, with the expansion of intensive farming, the limitations and potential risks of their application have become increasingly prominent. These include public food safety risks caused by drug residues, the increasing prevalence of drug resistance, and complex issues such as the safety and preparation of coccidial vaccines, which severely restrict their effectiveness.
[0003] miRNAs are small, single-stranded, endogenous, highly conserved non-coding RNA molecules, approximately 18-25 nucleotides in length. They primarily regulate biological processes by specifically binding to the seed sequence in the 3′ UTR region of target gene mRNAs, inducing mRNA degradation and inhibiting protein translation. Numerous studies have demonstrated that miRNAs play important regulatory roles in pathogenic microbial infection and inflammatory responses. However, the identification and functional investigation of miRNAs that mitigate E. tenella infection in chickens are currently scarce. Therefore, systematic screening and identification of key miRNAs involved in chicken coccidia infection, as well as in-depth analysis of the molecular mechanisms and regulatory relationships that mediate host-pathogen interactions, provide a solid foundation for future applications in genetic diagnosis, molecular markers, and targeted therapies, and are currently a hot research topic.
[0004] RNA sequencing (RNA-seq), as a high-throughput technology, has been widely used to explore key miRNAs associated with important traits and to identify new miRNAs. The discovery of new miRNAs will help to elucidate the regulatory mechanism of miRNA in the chicken host immune response. Summary of the Invention
[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide a reagent for regulating the expression level of gga-miR-7b and its use in the preparation of a drug for preventing or treating diseases related to Eimeria tenella infection in chickens.
[0006] The technical problem that the present invention also aims to solve is to provide the application of the overexpression of gga-miR-7b and its interfering sequence reagent in verifying its ability to alleviate inflammatory damage caused by E. tenella infection.
[0007] The technical problem that the present invention also aims to solve is to provide specific primers for the expression of gga-miR-7b related to chicken E. tenella infection.
[0008] The technical problem that the present invention also aims to solve is to provide a kit and a detection method for detection.
[0009] The technical problem that the present invention also aims to solve is to provide the application of the gga-miR-7b overexpression and interference sequence reagent, the specific primer, the detection kit and the detection method in studying the regulatory effect on the host inflammatory response during E. tenella infection.
[0010] Technical solution: In order to solve the above technical problems, the present invention provides the use of a reagent for regulating the expression level of gga-miR-7b in the preparation of a drug for preventing or treating diseases related to chicken Eimeria tenella infection, wherein the gga-miR-7b nucleotide sequence is shown in SEQ ID NO.1.
[0011] Wherein, the regulation includes up-regulation or down-regulation. Preferably, the up-regulation includes using a gga-miR-7b overexpression reagent, and the down-regulation includes using a gga-miR-7b interference reagent.
[0012] Among them, the gga-miR-7b overexpression reagent includes the gga-miR-7b overexpression sequence shown in SEQ ID NO.6 with a positive chain and the antisense chain shown in SEQ ID NO.7, and the gga-miR-7b interference reagent includes the gga-miR-7b interference sequence shown in SEQ ID NO.8.
[0013] Among them, the diseases related to chicken Eimeria tenella infection include inflammatory damage caused by infected hosts.
[0014] The application includes upregulating the expression of gga-miR-7b to inhibit the mRNA and protein concentration levels of inflammatory factors in host cells and / or promote the proliferation activity of host cells.
[0015] The present invention also includes specific primers for detecting the expression level of gga-miR-7b, and the sequence of the specific primers is shown in SEQ ID NO.2.
[0016] The present invention also includes a detection kit, which contains the specific primers.
[0017] The detection kit further comprises a universal reverse primer as shown in SEQ ID NO.3 and a primer pair of internal reference U6, and the primer sequence of the internal reference U6 is shown in SEQ ID NO.4 and SEQ ID NO.5.
[0018] The detection kit is a fluorescence quantitative detection kit, which also includes 2×miRNA Universal SYBR qPCR Master Mix, a universal reverse primer and enzyme-free double-distilled water.
[0019] The present invention also includes the use of gga-miR-7b overexpression and interference reagents, the specific primers, and the detection kit in studying or detecting the degree of inflammatory damage in hosts infected with chicken coccidia. The gga-miR-7b overexpression reagent includes a gga-miR-7b overexpression sequence as shown in SEQ ID NO.6 for the positive chain and SEQ ID NO.7 for the antisense chain. The gga-miR-7b interference reagent includes a gga-miR-7b interference sequence as shown in SEQ ID NO.8.
[0020] The application is to detect the expression level of gga-miR-7b in the host by fluorescence quantitative PCR to verify the degree of inflammatory damage caused by chicken coccidiosis infection.
[0021] Preferably, the gga-miR-7b specific primers and the detection kit are used to detect the expression level of gga-miR-7b in chicken cecal tissue to verify the degree of inflammatory damage caused by coccidia infection in chickens.
[0022] The application is to detect the correlation between gga-miR-7b and the expression of inflammatory factors in chicken cecal tissue by using fluorescent quantitative PCR and the miRNA specific primers.
[0023] The present invention also includes the use of the gga-miR-7b overexpression and its interference sequence reagent, the specific primer pair and the fluorescent quantitative detection kit in studying or regulating the inflammatory damage regulation effect of chicken E. tenella infection in the host.
[0024] The present invention also includes a method for alleviating inflammatory damage caused by E. tenella infection in chickens and promoting the proliferation activity of chicken somatic cells, which includes increasing the expression level of gga-miR-7b.
[0025] The present invention also includes a method for promoting inflammatory damage caused by E. tenella infection in chickens and weakening the proliferation activity of chicken somatic cells, which includes inhibiting the expression level of gga-miR-7b.
[0026] Principle of the present invention: The present invention extracts total RNA from chicken cecal tissue and reverse transcribes it to obtain cDNA. A kit consisting of a specific upstream primer for amplifying gga-miR-7b, a reverse universal primer, and an internal reference U6 primer is used to perform fluorescence quantitative PCR. The PCR reaction system and amplification procedure are the same as conventional fluorescence quantitative PCR. -△△CT Methods The expression level of this miRNA in each individual was determined to determine its correlation with the inflammatory expression of coccidia infection. Then, the overexpression and interference reagent of gga-miR-7b were transfected into chicken embryo fibroblasts to observe their effects on the expression levels of inflammation and proliferation activity in the cells. According to the expression level of gga-miR-7b, it was judged that it could alleviate the inflammatory damage of coccidia infection and promote the proliferation and repair of the body. The application of this miRNA will help enhance the resistance and tolerance of chickens to coccidia infection and provide a new molecular marker target for the targeted treatment of coccidiosis, specifically by promoting the expression of gga-miR-7b.
[0027] Beneficial effects: Compared with the prior art, the present invention has the following advantages: the present invention is the first to study and obtain miRNA related to inflammatory damage caused by E. tenella infection in chickens, and based on the research on the miRNA, specific primers and detection kits for detecting the miRNA are obtained. The specific primers and detection kit provided by the present invention can be used to detect the expression level of gga-miR-7b in chicken cecal tissue, and the detection method is simple and quick. The gga-miR-7b provided by the present invention can be used as a key marker for detecting inflammatory damage during coccidia infection. By detecting the expression level of gga-miR-7b in chicken cecal tissue during coccidia infection, the degree of inflammatory damage caused by the infection can be quickly and accurately determined, and can be used to develop auxiliary selection markers for early diagnosis of coccidia infection in chickens. At the same time, the present invention verified by overexpression and interference reagents of gga-miR-7b in chicken embryo fibroblasts infected with coccidia that increasing the expression of gga-miR-7b can alleviate the inflammatory damage caused by coccidia infection and promote cell proliferation activity, while inhibiting the expression of gga-miR-7b can aggravate the degree of inflammatory damage caused by coccidia infection and weaken cell proliferation activity. Therefore, the present invention fully verifies that the expression level of gga-miR-7b can effectively regulate the resistance and tolerance of chickens to coccidiosis infection, which can provide a scientific basis and new molecular targets for the early diagnosis and prevention of chicken coccidiosis, and provide an important basis for molecular-assisted disease-resistant breeding. BRIEF DESCRIPTION OF THE DRAWINGS
[0028] Figure 1 Figure 2 is the anatomical lesion diagram of the cecum in the challenge group and the control group;
[0029] Figure 2This figure shows the effect of E. tenella infection on the concentration of inflammatory factor proteins in chicken blood;
[0030] Figure 3 Figure 2 is the result of RNA-seq and qPCR validation;
[0031] Figure 4 This is a correlation analysis diagram between inflammatory factors and differential miRNAs in chicken cecal tissues infected with E. tenella;
[0032] Figure 5 This is a diagram of the extraction process of E. tenella sporozoites;
[0033] Figure 6 This figure shows the effect of overexpression of gga-miR-7b on the mRNA expression of inflammatory cytokines detected by RT-qPCR;
[0034] Figure 7 This is a graph showing the effect of overexpression of gga-miR-7b on the protein concentration of inflammatory cytokines detected by ELISA;
[0035] Figure 8 This is a diagram showing the effect of interfering with gga-miR-7b on the mRNA expression of inflammatory cytokines detected by RT-qPCR;
[0036] Figure 9 This is a graph showing the effect of interfering with gga-miR-7b on the concentration of inflammatory cytokine proteins detected by ELISA.
[0037] Figure 10 This figure shows the effect of overexpression of gga-miR-7b on the proliferation activity of host cells infected with coccidia.
[0038] Figure 11 This figure shows the effect of interfering with gga-miR-7b on the proliferation activity of host cells infected with coccidia. DETAILED DESCRIPTION
[0039] The technical solution of the present invention is further described in detail below through specific embodiments.
[0040] Example 1: miRNA gga-miR-7b that can alleviate chicken Eimeria tenella infection based on combined RNA-seq and functional studies
[0041] 1. Preparation of E. tenella sporulated oocysts
[0042] The sporulated oocysts of E. tenella stored in our laboratory (Cui Xiaoxia. Screening of AMA1-acting proteins of Eimeria tenella and preliminary study of the functions of EtMIC2 and Et0440 genes [D]. Chinese Academy of Agricultural Sciences, 2017.) were orally inoculated into 25-day-old healthy chicks, with each chick receiving 3.5×10 4 Coccidian oocyst solution: Feces were collected for four consecutive days starting from the fifth day after inoculation. The feces were graded and filtered through 60-mesh and 260-mesh sieves, then allowed to stand and stratify. The precipitate was retained and incubated with 5% potassium dichromate solution (purchased from Shanghai Sinopharm Group) at 28°C for 48-72 hours under constant temperature and shaking. During the incubation process, regular stirring or shaking was required to achieve oxygenation and promote oocyst sporulation. The supernatant was then removed by centrifugation (3500 rpm, 6 min), and the oocyst suspension was collected by flotation with saturated salt water. This was repeated 2-3 times, and the supernatant was finally removed by centrifugation. 2.5% potassium dichromate solution was added to the oocyst precipitate and stored at 4°C for later use.
[0043] 2. Collection of sequencing samples
[0044] Thirty one-day-old female Jinghai yellow chickens (a new yellow-feathered broiler breed approved by the state) were selected from Jiangsu Jinghai Poultry Group Co., Ltd. as the test materials of the present invention. Each chicken was raised separately in a pathogen-free animal room and had free access to feed and drinking water that did not contain any anticoccidial drugs. When the chickens were raised to 30 days of age, the feces of the chickens were randomly selected and placed in saturated physiological saline. The supernatant was observed under a microscope to see if there were coccidian oocysts to ensure that there was no coccidian infection. The 30 female chicks were randomly divided into a challenge group (n=15) and a control group (n=15). Each chicken in the challenge group was orally administered 3.5×10 4 The E. tenella sporulated oocysts prepared in step 1 were injected into the control group, and the same amount of normal saline was administered orally. On the 5th day after infection (acute infection period), the growth performance data of the control group and the challenge group were collected, and blood samples were collected. Then, cecal tissue samples of 3 chicks were randomly selected from the control group and the challenge group (the pathological changes of cecal tissue are shown in Figure 1 ) for subsequent RNA-seq sequencing analysis. The sequencing was completed by Shanghai Ouyi Biotechnology Co., Ltd., and the specific steps included RNA extraction, RNA quality detection and library construction. The growth performance data of the challenge group and the control group are shown in Table 1, and the expression of blood inflammation-related factors is shown in Figure 2 .
[0045] Table 1 Effects of E. tenella infection on growth performance of chickens
[0046]
[0047] Note: P>0.05 indicates no significant difference, P<0.05 indicates significant difference, P<0.01 and P<0.001 indicate extremely significant difference.
[0048] 3. Screening of differentially expressed miRNAs and prediction of target genes
[0049] After quality control of the RNA-seq results, the miRNA expression levels between the control group and the challenge group were compared. When P ≤ 0.05 and Fold change ≥ 1.5 times, it was defined as differentially expressed miRNA. A total of 17 differentially expressed miRNAs were obtained, including 10 known miRNAs such as gga-miR-6604-5p, gga-miR-12286-5p, gga-miR-7b, gga-miR-6606-5p, gga-miR-1551-5p, gga-miR-2131-3p, and gga-miR-1458, and 7 new miRNAs such as novel241-mature, novel179-mature, novel39-mature, and novel295-mature. Among them, gga-miR-7b showed the greatest differential expression between the cecal tissues of the challenged and control groups. Specifically, compared with the control group, the expression level of gga-miR-7b in the cecal tissues of the E. tenella-challenged chickens was significantly downregulated by over 100-fold. The differentially expressed gga-miR-7b target genes were predicted using miRDB and TargetScan software, respectively. A total of 621 and 181 target genes were screened, respectively. KEGG pathway enrichment analysis was performed after the intersection of the obtained target genes. The enriched signaling pathways were found to be involved in host immune regulation. Table 2 lists several important signaling pathways enriched by gga-miR-7b target genes.
[0050] Table 2 Pathways related to host immunity that are significantly enriched in gga-miR-7b target genes
[0051]
[0052] 4. qPCR verification of gga-miR-7b expression level in cecal tissue
[0053] Total RNA was extracted from the cecal tissue of the control group and the challenge group, and reverse transcription was performed to obtain cDNA. The nucleotide sequence of gga-miR-7b was obtained according to high-throughput sequencing by Shanghai Ouyi Biotechnology Co., Ltd. (the sequence number of the miRbase database is: Accession: MIMAT0001192, species: chicken; mature sequence length: 22bp), namely SEQ ID NO.1, and a specific upstream primer SEQ ID NO.2 was designed. The primer sequence of the internal reference gene was designed based on the mRNA sequence of U6 in the NCBI database (accession number: NM_001006337.2). A fluorescent quantitative kit containing specific primers was used to measure the expression level of the miRNA according to the conventional reaction system and amplification procedure.
[0054] The kit consists of 2×miRNA Universal SYBR qPCR Master Mix, universal reverse primer and enzyme-free double-distilled water, wherein the sequence of the universal reverse primer is shown in SEQ ID NO.3; the internal reference gene is the U6 primer pair shown in SEQ ID NO.4 and SEQ ID NO.5; miRNA Universal SYBR qPCR Master Mix, universal reverse primer and enzyme-free double-distilled water are all from the fluorescent quantitative kit (miRNA Universal SYBR qPCR Master Mix) of Nanjing Novozymes Biotechnology Co., Ltd.
[0055] Table 3 Sequence information
[0056]
[0057] The fluorescence quantitative reaction system (20 μl) consisted of 10.0 μl of 2× miRNA Universal SYBR qPCR Master Mix, 0.4 μl of specific upstream primer, 0.4 μl of universal reverse primer, 2.0 μl of template (cDNA), and 7.2 μl of ddH2O. The fluorescence quantitative reaction protocol was as follows: initial denaturation at 95°C for 5 min; 40 cycles of 95°C for 10 sec, 60°C for 30 sec, and then melting curve acquisition at 95°C for 15 sec, 60°C for 60 sec, and 95°C for 15 sec.
[0058] Application 2 -△△CT Methods The relative expression of gga-miR-7b in cecal tissue of E. tenella-challenged chickens and normal control chickens was analyzed. Figure 3qPCR revealed that the expression level of this miRNA in the control group was approximately 10 times higher than that in the infected group, consistent with the RNA-seq results. These results suggest that E. tenella infection significantly alters the expression level of gga-miR-7b in chicken cecal tissue and preliminarily suggest that its expression level may directly affect the degree of inflammatory damage in infected chickens.
[0059] 5. gga-miR-7b showed a significant negative correlation with the expression of inflammatory cytokines in chicken cecal tissue
[0060] In order to further confirm that gga-miR-7b can be used as a key molecular marker for inflammatory damage during coccidia infection, the present invention extracted RNA from the cecal tissue of chickens in the challenge group, performed reverse transcription, and obtained cDNA. RT-qPCR was used to detect the expression level of inflammatory cytokines in the cecal tissue of chickens in the challenge group, and the Pearson correlation coefficient was used to calculate the correlation between the expression of inflammatory cytokines and the miRNA. The data results were visualized using Origin (2024) software. Figure 4 The results showed that other differentially expressed miRNAs had no significant correlation with the expression of inflammatory cytokines in cecal tissue, while gga-miR-7b showed a significant negative correlation with the expression of inflammatory cytokines (P<0.05). Therefore, these results indicate that gga-miR-7b is closely related to the expression of inflammatory cytokines in cecal tissue and confirm that gga-miR-7b can be used as a key marker for inflammatory expression.
[0061] Example 2
[0062] 1. Preparation of E.tenella sporozoites
[0063] The sporulated oocysts of E. tenella ( Figure 5 A, B) were placed in a 2 ml centrifuge tube containing magnetic beads and shaken at high speed to successfully break the E. tenella oocysts ( Figure 5 C black arrow), release of sporangium ( Figure 5 C blue arrow); then the sporangium was digested with 10% fresh chicken bile (purchased from Jinghai Yellow Chicken Resource Farm, Haimen, Jiangsu Province) and 0.5% trypsin (purchased from Beijing Solebaugh) in a water bath at 42°C for 1 hour. Figure 5 The sporangium indicated by the red arrow in D was successfully digested, releasing E. tenella sporozoites; finally, the digested E. tenella sporozoites were filtered using a G3 sand core funnel (purchased from Changchun Glass Instrument Factory) to remove the undigested sporangium ( Figure 5D blue arrow), purify and collect the obtained E.tenella sporozoites ( Figure 5 E).
[0064] 2. gga-miR-7b alleviates inflammatory damage in chicken coccidia infection
[0065] To verify whether gga-miR-7b can alleviate inflammatory damage induced by coccidia infection in hosts, the present invention constructed gga-miR-7b overexpression (mimic) and interference (inhibitor) sequence reagents. The gga-miR-7b overexpression sequence is shown in SEQ ID NO. 6 (sense chain) and SEQ ID NO. 7 (antisense chain), and the gga-miR-7b interference sequence is shown in SEQ ID NO. 8. The specific sequence information is shown in Table 4 below. After the overexpression and interference sequence reagents were synthesized, 250 μl of DEPC water was added to prepare a working solution with a concentration of 20 μM. The gga-miR-7b overexpression and interference sequence reagents were transfected into the chicken embryo fibroblast cell line (purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences) in vitro. 24 hours after transfection, coccidian sporozoites were injected and the cells were infected for an additional 6 hours. Then, RT-qPCR and ELISA experiments were used to detect the mRNA and protein concentration levels of inflammatory cytokines IL-6, IL-1β, IL-8, TNF-α and IL-12 in the cells and cell supernatants. Among them, miR-7b mimic: gga-miR-7b overexpression group; miR-7b-mimic-NC: gga-miR-7b overexpression negative control group; Spo-miR-7b-mimic: gga-miR-7b overexpression group infected with coccidian sporozoites (where Spo is the abbreviation of E. tenella sporozoite, the same below); Spo-miR-7b-mimic-NC: gga-miR-7b overexpression negative control group infected with coccidian sporozoites. Figure 6 and Figure 7 It can be seen that the results of RT-qPCR and ELISA tests found that overexpression of gga-miR-7b can significantly or extremely significantly inhibit the mRNA and protein concentration levels of inflammatory factors in normal cells and cells infected with coccidian sporozoites (P<0.05, P<0.01 or P<0.001). In addition, miR-7b-inhibitor: gga-miR-7b interference group; miR-7b-inhibitor-NC: gga-miR-7b interference negative control group (referring to the negative control group without interference sequence); Spo-miR-7b-inhibitor: gga-miR-7b interference group infected with coccidian sporozoites; Spo-miR-7b-inhibitor-NC: gga-miR-7b interference negative control group infected with coccidian sporozoites. Figure 8 and 9 Interference with gga-miR-7b significantly or extremely significantly increased the mRNA and protein levels of inflammatory factors in normal cells and cells infected with coccidian sporozoites (P < 0.05, P < 0.01, or P < 0.001). The results of the present invention confirm that increasing the expression of gga-miR-7b can alleviate the inflammatory damage caused by coccidian infection.
[0066] Table 4gga-miR-7b overexpression and interference sequence information
[0067]
[0068] 3. gga-miR-7b promotes the proliferation of coccidia-infected cells
[0069] E.tenella infection causes inflammatory damage to the chicken body, causing apoptosis of damaged tissues and cells. Therefore, repairing damaged tissues and cells plays a key role in the host's defense against coccidia infection. Therefore, the present invention further verified the effect of gga-miR-7b on the proliferation activity of host cells infected with coccidia. The above-mentioned synthesized gga-miR-7b overexpression and interference sequence reagents were transfected into chicken embryo fibroblasts, and coccidia sporozoites were injected into the cells to infect the cells. Then, RT-qPCR, Western Blot and CCK-8 experiments were used to verify the effect of gga-miR-7b on cell proliferation activity. The results are as follows. Figure 10 As shown in the results, overexpression of gga-miR-7b significantly or extremely significantly promoted the proliferation of normal cells and cells infected with coccidian sporozoites (P<0.05, P<0.01 or P<0.001); on the contrary, interference with gga-miR-7b significantly or extremely significantly inhibited the proliferation of normal cells and cells infected with coccidian sporozoites ( Figure 11 The above results indicate that gga-miR-7b can effectively promote the proliferation of host cells.
[0070] The results of the above examples fully demonstrate that gga-miR-7b can effectively regulate the resistance and tolerance of chickens to coccidiosis infection, and the method includes increasing the expression level of gga-miR-7b. Therefore, the kit provided by the present invention can be used to detect the expression level of gga-miR-7b in chicken cecal tissue. The detection method is simple and rapid. The gga-miR-7b provided by the present invention can be used as a key marker for alleviating coccidiosis infection in chickens, providing a scientific basis and new molecular targets for the early diagnosis and prevention of coccidiosis in chickens, and providing an important basis for molecular-assisted disease-resistant breeding.
Claims
1. Use of a reagent for regulating the expression level of gga-miR-7b in the preparation of a drug for preventing or treating diseases related to chicken Eimeria tenella infection, wherein the nucleotide sequence of the gga-miR-7b is shown in SEQ ID NO.
1.
2. The use according to claim 1, characterized in that The regulation includes up-regulation or down-regulation. Preferably, the up-regulation includes using a gga-miR-7b overexpression reagent, and the down-regulation includes using a gga-miR-7b interference reagent.
3. The use according to claim 2, characterized in that The gga-miR-7b overexpression reagent includes a gga-miR-7b overexpression sequence as shown in SEQ ID NO.6 as the positive chain and an antisense chain as shown in SEQ ID NO.7 as the antisense chain. The gga-miR-7b interference reagent includes a gga-miR-7b interference sequence as shown in SEQ ID NO.
8.
4. The use according to claim 1, characterized in that The diseases associated with Eimeria tenella infection in chickens include inflammatory damage caused by infected hosts.
5. The use according to claim 1, characterized in that The application includes upregulating the expression of gga-miR-7b to inhibit the mRNA and protein concentration levels of inflammatory factors in host cells and / or promote the proliferation activity of host cells.
6. Specific primers for detecting the expression level of gga-miR-7b, characterized in that: The specific primer sequence is shown as SEQ ID NO.
2.
7. A detection kit, characterized in that The kit comprises the specific primer according to claim 4.
8. The detection kit according to claim 7, characterized in that The detection kit further comprises a universal reverse primer as shown in SEQ ID NO.3 and a primer pair of internal reference U6, and the primer sequence of the internal reference U6 is shown in SEQ ID NO.4 and SEQ ID NO.
5.
9. Use of gga-miR-7b overexpression and interference reagents, the specific primers described in claim 6, and the detection kit described in claim 7 or 8 in studying or detecting the degree of inflammatory damage in hosts infected with chicken coccidia, the gga-miR-7b overexpression reagent comprising a gga-miR-7b overexpression sequence as shown in SEQ ID NO.6 for the positive chain and SEQ ID NO.7 for the antisense chain, and the gga-miR-7b interference reagent comprising a gga-miR-7b interference sequence as shown in SEQ ID NO.
8.
10. The use according to claim 9, characterized in that The application is to detect the expression level of gga-miR-7b in the host by fluorescence quantitative PCR to verify the degree of inflammatory damage caused by chicken coccidiosis infection.