Inflammation regulation sequence of dairy cow mastitis and application thereof

Abstract

The invention discloses an inflammation regulation sequence of dairy cow mastitis and application thereof, and belongs to the technical field of biology, the inflammation regulation sequence is a regulation sequence responding to a gram-negative bacterium inflammation signal, the regulation sequence is a promoter sequence of a CXCL2 gene and is shown as SEQ ID NO: 1, and the regulation sequence is a promoter sequence of a CXCL2 gene and is shown as SEQ ID NO: 2. A regulatory sequence responding to a gram-positive bacterium inflammation signal, which is a promoter partial sequence of the NLRP3 gene and is as shown in SEQ ID NO: 2; the regulatory sequence responding to a gram-positive bacterium inflammation signal is a promoter reverse complementary sequence of the TLR2 gene and is shown as SEQ ID NO: 3; the endogenous inflammation regulation sequence provided by the invention is composed of genome elements of the dairy cow, and no exogenous coding gene is introduced, so that potential risks caused by exogenous proteins are avoided.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically an inflammatory regulatory sequence for mastitis in dairy cows and its application. Background Technology

[0002] Mastitis is the most common disease in dairy cows, accounting for about one-third of common production diseases in dairy farms. Currently, it is mainly treated with antibiotics in clinical practice, but the resulting problems of drug residues, bacterial resistance, and environmental biosafety are becoming increasingly serious.

[0003] At the genetic breeding level, early anti-mastitis transgenic breeding involved knocking exogenous antimicrobial proteins (such as human lysozyme and lysostaphylococcal lysin) entirely into the genome to obtain transgenic animals. However, random insertion of exogenous genes may disrupt the function of endogenous genes or affect genome stability; their expression is susceptible to position effects, leading to uncontrollable expression levels or even gene silencing; in addition, the continuous expression of non-self proteins may disrupt host immune homeostasis and bring potential risks in terms of biosafety and ethics. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides an inflammatory regulatory sequence for bovine mastitis, selected from at least one of the following: The regulatory sequence responding to Gram-negative bacterial inflammatory signals is the promoter sequence of the CXCL2 gene, as shown in SEQ ID NO:1; the regulatory sequence responding to Gram-negative bacterial inflammatory signals is obtained based on LPS induction; The regulatory sequence responding to Gram-positive bacterial inflammatory signals is a partial promoter sequence of the NLRP3 gene, as shown in SEQ ID NO:2; the regulatory sequence responding to Gram-positive bacterial inflammatory signals was obtained based on Staphylococcus aureus induction. The regulatory sequence responding to Gram-positive bacterial inflammatory signals is the reverse complementary sequence of the TLR2 gene promoter, as shown in SEQ ID NO:3. The regulatory sequence responding to Gram-positive bacterial inflammatory signals is based on Staphylococcus aureus induction.

[0005] On the other hand, the present invention provides the application of the aforementioned inflammatory regulatory sequence in the preparation of a kit for detecting or diagnosing bovine mastitis, or in bovine gene editing, or in the preparation of a drug for treating bovine mastitis.

[0006] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. Traditional transgenic breeding relies on the random insertion and continuous expression of exogenous antibacterial genes, which poses risks such as genome damage, unstable expression, and immune interference. The endogenous inflammation regulation sequence provided by this invention is composed of elements of the dairy cow's own genome and does not introduce any exogenous coding genes, thereby avoiding the potential risks brought by exogenous proteins.

[0007] 2. The inflammatory regulatory sequence provided by this invention can specifically activate the cow's own anti-inflammatory / antibacterial genes (such as lactoferrin and antimicrobial peptide S100A9) when infected by Gram-positive or Gram-negative bacteria. It can achieve rapid and efficient upregulation of expression only when pathogens infect mammary tissue. In the healthy and uninfected state of the animal, its expression level is maintained at a very low physiological baseline level, which fundamentally avoids the biosafety and ethical issues of introducing foreign genes. Attached Figure Description

[0008] Figure 1 Image of primary fibroblasts; Figure 2 Image showing the purification of mammary epithelial cells; Figure 3 The effects of different LPS concentrations and treatment times on cell viability; Figure 4 To detect the effect of LPS treatment on the expression of inflammatory factors in cells using RT-qPCR; Figure 5 To detect the effect of LPS treatment on CXCL2 expression using RT-qPCR; Figure 6 The effect of LPS on luciferase activity expressed by CXCL2 promoter-related sequences; Figure 7 The effect of LPS on the regulation of luciferase activity by the CXCL2 promoter sequence in the expression of the antimicrobial peptide S100A9 promoter; Figure 8 To observe the growth of S. aureus colonies by plating the extracellular culture medium; Figure 9 The intracellular lysis buffer was plated to observe the growth of S. aureus colonies. Figure 10 To investigate the effect of RT-qPCR on the expression of cellular inflammatory factors IL-18 and IL-1β mRNA; Figure 11 To detect the effect of S. aureus treatment on NLRP3 mRNA expression by RT-qPCR; Figure 12 To detect the effect of S. aureus treatment on TLR2 mRNA expression by RT-qPCR; Figure 13The effect of S. aureus on luciferase activity expressed by NLRP3 promoter-related sequences; Figure 14 The effect of S. aureus on the regulation of lactoferrin promoter expression luciferase activity by the NLRP3 promoter sequence; Figure 15 The effect of S. aureus on luciferase activity expressed by TLR2 promoter-related sequences; Figure 16 The effect of S. aureus on the regulation of lactoferrin promoter expression luciferase activity by the TLR2 promoter sequence. Detailed Implementation

[0009] Example 1: Primary isolation and purification culture of bovine fibroblasts Bovine mammary gland tissue was collected from slaughterhouses, cleaned with 75% alcohol, and then soaked in DPBS. Hair was scraped off, and surface fat was removed. The tissue was cut into small pieces, placed at the bottom of culture dishes, and filled with DMEM medium containing 10% fetal bovine serum and 3% antibiotics. The culture was carried out at 37°C and 5% CO2 for 4-6 days, during which elongated primary fibroblasts were observed to emerge. Culture was continued for 15 days until 80% confluence was achieved. The cells were then digested and passaged. Results were as follows... Figure 1 As shown, in primary cultured bovine mammary gland tissue, cells migrated and grew outward from the edge of the tissue block, and fibroblasts with slender, spindle-shaped or fusiform morphology were successfully isolated. These primary fibroblasts were used as reserve cells for gene editing.

[0010] Example 2: Isolation and purification of bovine mammary epithelial cells Mammary epithelial cells can synthesize and secrete milk fat, participate in innate immunity, recognize pathogen-associated molecular patterns (PAMPs), secrete various cytokines, and exert antimicrobial infection functions. However, when a cow's mammary gland infection is severe, it usually damages mammary tissue and affects the function of mammary cells, ultimately leading to a decline in milk yield and quality, and even endangering the cow's life.

[0011] Escherichia coli and its LPS bacteria mostly cause severe clinical mastitis, with clinical symptoms ranging from severe and even fatal to mild. Staphylococcus aureus can cause subclinical or even chronic mastitis. Both are the main pathogens of mastitis in dairy cows. Escherichia coli typically colonizes mammary tissue and does not easily invade cells, primarily acting through LPS. Staphylococcus aureus can both colonize mammary tissue and invade and proliferate mammary cells. Therefore, using LPS and Staphylococcus aureus to stimulate mammary epithelial cells and induce an inflammatory environment is a reasonable model for in vitro research on bovine mastitis.

[0012] Bovine mammary gland tissue retrieved from the slaughterhouse was treated with 75% alcohol and physiological saline to clean it thoroughly. Using a sterile scalpel, the tissue was divided into small pieces along the mammary ducts and washed with DPBS in centrifuge tubes. In a laminar flow hood, the surface tissue was removed, and the tissue was washed again with DPBS, followed by washing with 75% alcohol. This process was repeated three times. The washed tissue pieces were then placed in inverted T25 culture dishes. After 5 hours, DMEM culture medium containing 10% fetal bovine serum and 1% penicillin-dextrose antibiotics was added, and the cells were cultured until cobblestone-like mammary epithelial cells emerged. When the cell confluence exceeded 80%, trypsin was added for digestion. Taking advantage of the different tolerances of mammary epithelial cells and fibroblasts to trypsin, the digestion was completed after 5 minutes. The digested fibroblasts were discarded, and the remaining cells were cultured repeatedly to obtain purified mammary epithelial cells. The results are as follows: Figure 2 As shown in the figure, the cells exhibit typical epithelial cell morphology, and the obtained cells are in good condition and have high purity, which meets the requirements for establishing an in vitro inflammatory cell model.

[0013] The purified mammary epithelial cells were passaged in DMEM medium containing 10% fetal bovine serum and 1% penicillin-dextrin antibodies. Once the cells were stable and proliferating actively, mammary epithelial cells in the logarithmic growth phase could be obtained for the subsequent establishment of an inflammation model.

[0014] Example 3: Establishment and Validation of an LPS-Induced Inflammation Model To construct an in vitro inflammation model of bovine mammary epithelial cells, LPS stimulation was used. Logarithmic growth phase mammary epithelial cells obtained in Example 2 were selected, and after discarding the old culture medium, they were washed with PBS and digested with trypsin. Once all cells became rounded, an equal volume of complete culture medium was added to terminate the digestion. The cells were gently pipetted to detach, and the cell suspension was centrifuged at 800 rpm at room temperature for 5 minutes. The supernatant was discarded, and the cells were resuspended in complete culture medium at 9 × 10⁻⁶ centrifuges. 4 Cells were seeded at a density of 100 cells / well in 12-well plates and cultured overnight in DMEM medium containing 10% fetal bovine serum and 1% penicillin-dextrose antibiotics.

[0015] The following day, cells were washed with PBS and then replaced with serum-free DMEM medium, followed by starvation treatment for 12 hours. Subsequently, five LPS concentration gradients (0, 10, 50, 100, and 200 μg / mL) were established, and cells were treated for 6 hours, 12 hours, and 24 hours, respectively. Cell viability after LPS treatment was assessed using the CCK-8 assay. The results showed that LPS treatment did not affect cell viability within the 6-24 hour range; therefore, the above treatment concentrations and times can be used for subsequent experiments. Figure 3 ).

[0016] Based on CCK-8 cell activity results, to verify the successful establishment of the LPS inflammation model, we selected IL-8 (CXCL8) and IL-1β, key pro-inflammatory cytokines rapidly and highly expressed in mammary epithelial cells in response to LPS stimulation, as biomarkers. After 12 h of starvation treatment, cells were randomly treated with 100 μg / mL LPS for 2 h, 4 h, 6 h, 8 h, and 12 h, respectively. Total RNA was extracted from the cells, reverse transcribed, and then qPCR was used to detect the expression levels of inflammatory factors. Primer sequences used: F-IL-1β: SEQ ID NO:10; R-IL-1β: SEQ ID NO:11; F-IL-8: SEQ ID NO:12; R-IL-8: SEQ ID NO:13; qPCR results showed ( Figure 4 The expression levels of two inflammatory factors, IL-8 and IL-1β, were significantly upregulated after treatment with 100 μg / mL LPS for 2–12 h. This result demonstrates that stimulation with 100 μg / mL LPS can successfully activate the classic inflammatory signaling pathway in bovine mammary epithelial cells, thus confirming the effectiveness and reliability of the LPS-based in vitro inflammation model constructed in this embodiment.

[0017] Example 4: Identification of the CXCL2 gene inflammatory response regulatory sequence Using the same cell treatment and qPCR methods as in Example 3, the mRNA expression level of CXCL2 at different time points was detected using CXCL2-specific primers (F-CXCL2: SEQ ID NO:4; R-CXCL2: SEQ ID NO:5). The results showed that the expression of CXCL2 was higher than that of the control group at all treatment time points. Figure 5 The mRNA expression level of CXCL2 was significantly upregulated after LPS stimulation, indicating that CXCL2 has a stable response under inflammatory conditions.

[0018] Furthermore, to identify whether the CXCL2 gene promoter region contains a specific regulatory sequence in response to LPS inflammatory signals, a reporter gene vector (pGL4.10-CXCL2) containing a 262bp regulatory sequence of the CXCL2 promoter was constructed using pGL4.10 as the donor vector, and its function was verified by the dual-luciferase reporter gene system described below.

[0019] 293T cells were fed at a dose of 2.3 × 10⁻⁶. 5Cells were seeded at a density of 100 cells / well in 12-well plates and cultured overnight to allow cell adhesion. Plasmid transfection was then performed, with a ratio of Renida luciferase vector to firefly luciferase vector of 1:5, strictly following the JetPRIME® in vitro DNA & siRNA transfection reagent instructions. Twenty-four hours after transfection, cell lysis buffer was prepared according to the instructions of the Dual Luciferase Reporter Gene Assay Kit (Coolaber, MH107). Firefly luciferase activity (FLUC value) and Renida luciferase activity (RLUC value) were measured using a multi-functional microplate reader, with Renida luciferase activity used as an internal control. The transcriptional activity of the CXCL2 promoter was assessed by calculating the FLUC / RLUC ratio. Figure 6 As shown, the reporter system containing the CXCL2 promoter regulatory sequence exhibited significantly increased luciferase activity, indicating that the CXCL2 promoter regulatory sequence can respond to LPS-induced inflammatory signals and also drive downstream gene expression. Therefore, the CXCL2 gene-related promoter region is identified as having a regulatory function in inflammatory response.

[0020] Example 5: CXCL2 promoter sequence regulates the expression of the antimicrobial peptide S100A9 gene. To screen for inflammatory regulatory sequences in the CXCL2 gene that respond to LPS induction and drive the expression of the antimicrobial peptide S100A9 gene promoter, a reporter gene vector (pGL4.10-S100A9) containing 259 bp upstream of the TSS region of the S100A9 core promoter was constructed using pGL4.10 as the donor vector. Simultaneously, a reporter gene vector containing 245 bp upstream of the S100A9 core promoter region and the complete CXCL2 promoter was constructed using pGL4.10 as the donor vector; that is, the 245 bp upstream region of the S100A9 core promoter region was inserted 1 bp into the 200 bp region of the complete CXCL2 promoter element (pGL4.10-CXCL2-S100A9-1). The transcriptional driving activity of these regulatory sequences was detected under LPS-induced conditions using a dual-luciferase reporter gene assay.

[0021] Experimental results showed that, compared with the uninduced group, the pGL4.10-CXCL2-S100A9 reporter gene vector exhibited significant upregulation of luciferase activity upon LPS induction (e.g., ...). Figure 7 This result indicates that the complete CXCL2 promoter element can specifically respond to LPS-triggered inflammatory signals and effectively drive the expression of downstream antimicrobial peptide genes. Therefore, the complete CXCL2 promoter element is identified as a gene editing inflammatory regulatory sequence element.

[0022] Example 6: Establishment and Validation of a Staphylococcus aureus-Induced Inflammation Model To establish a Staphylococcus aureus infection model, LB medium was first prepared (1g tryptone, 1g sodium chloride, and 0.5g yeast extract in 100 mL distilled water, sterilized at 121℃ for 20 min). 70µL of Staphylococcus aureus glycerol culture was added to 7 mL of LB medium, and the medium was incubated with shaking at 230 rpm for 3-5 h until the OD600 value reached 0.7-1.0. The medium was then stored at 4℃ for later use.

[0023] Simultaneously, bacterial quantification was performed using a serial dilution colony counting method: 8 sterile centrifuge tubes were taken and labeled as 10... -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 10 -8 Each tube was pre-filled with 900 μL of LB medium. 100 μL of the prepared bacterial culture was then added to 10... -1 Mix well in the tube, then take 100 μL from this tube and add 10 -2 In the tube, the series of dilutions were completed in sequence, resulting in 8 concentration gradients of bacterial suspensions, leaving 10 -6 10 -7 10 -8 Dilute the bacterial suspension and mix thoroughly by repeated pipetting. Take 10 of the above-mentioned amount. -6 10 -7 10 -8 Spread 100 μL of each dilution of bacterial suspension onto solid LB agar plates, with 3 replicates for each dilution. Incubate at 37°C for 24 h. When the colony morphology and size are appropriate, count the colonies (30-300 single colonies on the plate is ideal) and calculate the original bacterial suspension CFU / mL.

[0024] Before conducting the bacterial invasion experiment, after mixing the bacterial solution by pipetting and aspirating, 1 mL was transferred to a centrifuge tube and centrifuged at 4°C and 12000 rpm for 3 min. The supernatant was discarded, and the solution was resuspended in PBS and centrifuged again. This process was repeated three times. The final resuspended solution was obtained by adding 1 mL of PBS.

[0025] Mammary epithelial cells were infected with Staphylococcus aureus of varying multiplicity of infection (MOI) for 2 hours, followed by culturing in DMEM medium containing 100 μg / mL gentamicin and 1 mg / mL lysozyme for another 4 hours to eliminate extracellular bacteria. Finally, cells were lysed with 1% saponin, and the extracellular culture medium and intracellular lysis buffer were plated and cultured separately. The results are as follows: Figure 8 As shown, no colonies grew after plating extracellular culture media treated with different MOIs (12.5, 25, 50, 100); while... Figure 9As shown, the intracellular lysate plate showed obvious colonies, and the number of colonies increased with increasing MOI (for example, the number of colonies was low when MOI was 12.5, and significantly increased when MOI was 100), proving that Staphylococcus aureus can successfully invade mammary epithelial cells in a dose-dependent manner, and the invasion efficiency is positively correlated with MOI.

[0026] Further analysis of post-infection inflammatory factor expression changes was performed using qPCR. Cells treated with different MOIs (12.5, 25, 50, 100) were collected, RNA was extracted and reverse transcribed, and qPCR analysis was conducted using the following primers. Figure 10 As shown, compared with the untreated group, the mRNA expression levels of IL-18 and IL-1β increased with increasing MOI. Primer sequences used: F-IL-1β: SEQ ID NO:10; R-IL-1β: SEQ ID NO:11; F-IL-18: SEQ ID NO:14; R-IL-18: SEQ ID NO:15; In the constructed Staphylococcus aureus-induced inflammation model, the expression levels of pattern recognition receptor components NLRP3 and TLR2 were detected by qPCR using specific primers. The primers for detecting NLRP3 were SEQ ID NO:6 and SEQ ID NO:7, and the primers for detecting TLR2 were SEQ ID NO:8 and SEQ ID NO:9. The results are as follows: Figure 11 , Figure 12 As shown, under MOI=50 and MOI=100 treatment conditions, the mRNA expression levels of NLRP3 and TLR2 were relatively high, indicating that Staphylococcus aureus infection can specifically upregulate the expression of NLRP3 and TLR2 genes.

[0027] Furthermore, the NLRP3 inflammasome and TLR2 are involved in the mammary epithelial cell death pathway and are significantly upregulated in a variety of cell death-related inflammatory diseases, indicating that their promoters have the potential to act as inflammatory regulatory sequences in response to inflammatory responses.

[0028] Example 7 Identification of the NLRP3 gene inflammatory response regulatory sequence To verify the specific regulatory region in the NLRP3 gene promoter that responds to Staphylococcus aureus infection, this embodiment constructed reporter gene vectors containing 2000bp of the complete NLRP3 promoter (pGL4.10-2) and -2000 to -2500bp upstream of the NF-κB and NLRP3 promoter recognition core region TSS (pGL4.10-0.5) using pGL4.10 as the donor vector.

[0029] The transcriptional activity of the above-mentioned regulatory sequences was detected using a dual-luciferase reporter gene assay. The constructed reporter vector and internal control plasmid were co-transfected into 293T cells, and infected with Staphylococcus aureus at MOIs of 50 and 100, respectively. A control group with an empty vector (pGL4.10) was also included. After infection, cells were lysed, and the luciferase activities of firefly and kidney luciferase were measured. The relative luciferase activities were calculated after correction with the internal control luciferase activity.

[0030] The experimental results showed that, compared with the uninfected control group, when the multiplicity of infection (MOI) was 50 and 100, reporter vectors containing the complete NLRP3 promoter 2000 bp (pGL4.10-2) and the NF-κB and NLRP3 promoter recognition core region TSS upstream of -2000 to -2500 bp (pGL4.10-0.5) both showed significant upregulation of luciferase activity (e.g., ...). Figure 13 This indicates that both regulatory regions can specifically respond to inflammatory signals triggered by Staphylococcus aureus infection and effectively drive downstream gene expression. Furthermore, the data in the figure show that the region upstream of the TSS in the NF-κB and NLRP3 promoter recognition core region, where expression levels are high, serves as a gene-editing inflammatory regulatory sequence element.

[0031] Example 8: NLRP3 promoter sequence regulates lactoferrin gene expression To verify that specific regulatory regions in the NLRP3 gene promoter responding to Staphylococcus aureus infection affect lactoferrin gene promoter expression, this example constructed a reporter gene vector (pGL4.10-LTF) containing 500 bp upstream of the TSS region of the LTF core promoter region using pGL4.10 as the donor vector. Another reporter gene vector (pGL4.10-NLRP3-LTF-337) was constructed using pGL4.10 as the donor vector, containing 500 bp upstream of the TSS region of the LTF core promoter region and -2000 to -2500 bp upstream of the TSS region of the NF-κB and NLRP3 promoter recognition core region. Specifically, the NF-κB and NLRP3 promoter recognition core region was inserted at a position of 337 bp within the 500 bp upstream of the TSS region of the LTF core promoter region. The transcriptional drive activity of the above regulatory sequences was detected under Staphylococcus aureus infection conditions using a dual-luciferase reporter gene assay.

[0032] Experimental results show (e.g.) Figure 14Compared with the uninfected group, when the multiplicity of infection (MOI) was 50, the luciferase activity of the basic promoter vector pGL4.10-LTF did not change significantly; however, the vector containing the NLRP3 regulatory sequence specifically inserted at 337 bp of the LTF gene core promoter showed significant upregulation of luciferase activity. These results indicate that the NF-κB and NLRP3 promoter recognition core region (SEQ ID NO:2) can specifically respond to the inflammatory signals triggered by Staphylococcus aureus infection and effectively drive the expression of the downstream LTF gene. Therefore, identifying the region -2000 to -2500 bp upstream of the NF-κB and NLRP3 promoter recognition core region TSS as a gene editing inflammatory regulatory sequence element can effectively enhance lactoferrin gene expression under inflammatory conditions.

[0033] Example 9 Identification of the TLR2 gene inflammatory response regulatory sequence To verify the specific regulatory region in the TLR2 gene promoter that responds to Staphylococcus aureus infection, this embodiment uses pGL4.10 as the donor vector to construct a reporter gene vector containing the complete TLR2 promoter 200bp (named pGL4.10-TLR2).

[0034] The transcriptional activity of this regulatory sequence was detected using a dual-luciferase reporter gene assay. The pGL4.10-TLR2 reporter vector and internal control plasmid were co-transfected into 293T cells, and infected with Staphylococcus aureus at MOIs of 50 and 100, respectively. An empty vector (pGL4.10) control group and an uninfected control group were also included. Cells were lysed after infection, and the luciferase activities of *Firefly* and *Reniformis rubrum* were measured. Relative luciferase activities were calculated after internal control correction.

[0035] Experimental results showed that, compared with the uninfected control group, pGL4.10-TLR2 exhibited significant upregulation of luciferase activity at MOI values ​​of 50 and 100 (e.g., ...). Figure 15 Furthermore, the expression level was higher at an MOI of 50 than at an MOI of 100. This result indicates that the complete promoter reverse complement sequence of the TLR2 gene (SEQ ID NO:3) can specifically respond to the inflammatory signals triggered by Staphylococcus aureus infection and effectively drive the expression of downstream genes. Therefore, the 200bp reverse complement sequence of the complete TLR2 promoter is identified as a key sequence for inflammatory regulation in response to Staphylococcus aureus infection.

[0036] Example 10: TLR2 promoter sequence regulates lactoferrin gene expression To verify that a specific regulatory region in the TLR2 gene promoter responding to Staphylococcus aureus infection affects lactoferrin (LTF) gene promoter expression and to determine its optimal insertion position, this embodiment constructed a reporter gene vector (pGL4.10-LTF) containing 500 bp upstream of the TSS region of the LTF core promoter region using pGL4.10 as the donor vector. Another reporter gene vector (pGL4.10-TLR2-LTF) containing 500 bp upstream of the TSS region of the LTF core promoter region and 200 bp of the inverse complement sequence of the complete TLR2 promoter was constructed using pGL4.10 as the donor vector. Specifically, the 200 bp inverse complement sequence of the complete TLR2 promoter was specifically inserted into positions 37 bp, 337 bp, and 467 bp within the 500 bp upstream of the TSS region of the LTF gene core promoter region. The transcriptional activity of the above regulatory sequences was detected under Staphylococcus aureus infection conditions using a dual-luciferase reporter gene assay.

[0037] Experimental results show that ( Figure 16 When the multiplicity of infection (MOI) was 50, compared with the uninfected pGL4.10-TLR2-LTF-37 group, pGL4.10-TLR2-LTF-37 (i.e., the TLR2 promoter is inserted at 37 bp of the LTF promoter) showed a significant upregulation of luciferase activity; compared with the uninfected pGL4.10-TLR2-LTF-337 group, pGL4.10-TLR2-LTF-337 (i.e., the TLR2 promoter is inserted at 337 bp of the LTF promoter) showed no significant increase in luciferase activity; compared with the uninfected pGL4.10-TLR2-LTF-467 group, pGL4.10-TLR2-LTF-467 (i.e., the TLR2 promoter is inserted at 467 bp of the LTF promoter) showed a significant decrease in luciferase activity. Therefore, this result indicates that the inverse complementary sequence of the complete TLR2 promoter (SEQ ID NO:3) specifically responds to the inflammatory signal triggered by Staphylococcus aureus infection at a specific insertion site (37 bp of the LTF promoter) and effectively drives lactoferrin gene expression. Thus, the 200 bp inverse complementary sequence of the complete TLR2 promoter is identified as a key sequence for the inflammatory regulation of the lactoferrin gene in response to Staphylococcus aureus infection, and it can effectively enhance lactoferrin gene expression under inflammatory conditions.

Claims

1. An inflammation regulatory sequence for bovine mastitis, characterized by, selected from at least one of: a regulatory sequence responsive to gram-negative bacterial inflammatory signals, which is a promoter sequence of the CXCL2 gene, as shown in SEQ ID NO: 1 ; a regulatory sequence responsive to gram-positive bacterial inflammatory signals, which is a partial promoter sequence of the NLRP3 gene, as shown in SEQ ID NO: 2; a regulatory sequence responsive to gram-positive bacterial inflammatory signals, which is a reverse complement sequence of the TLR2 gene promoter, as shown in SEQ ID NO:

3.

2. The inflammation regulatory sequence of claim 1, wherein, The regulatory sequence responsive to gram-positive bacterial inflammatory signals is based on induction by S. aureus.

3. The inflammation regulatory sequence of claim 1, wherein, The regulatory sequence responsive to gram-negative bacterial inflammatory signals is based on induction by LPS.

4. Use of the regulatory sequence of inflammation according to any one of claims 1-3 in the preparation of a kit for detecting or diagnosing mastitis in dairy cows.

5. Use of the regulatory sequence of inflammation according to any one of claims 1-3 in the gene editing of dairy cows.

6. Use of the regulatory sequence of inflammation according to any one of claims 1-3 in the preparation of a medicament for the treatment of mastitis in dairy cows.

Images