Porcine beta-defensin-3 gene core promoter and construction method and application thereof

Abstract

The application discloses a pig beta-defensin-3 gene core promoter and a construction method and application thereof, and belongs to the technical field of genetic engineering. The sequence of the core promoter is shown as SEQ ID No. 1, the core promoter has significant promoter activity in pig small intestinal epithelial cells, and can be regulated by nutrients such as sodium butyrate and glutamine. The application also discloses a construction method for specifically amplifying the core promoter, which comprises the following steps: performing PCR amplification by taking pig small intestinal epithelial cell DNA as a template, taking the upstream primer and the downstream primer shown as SEQ No. 2-SEQ No. 3 as the primer, constructing a pMD-18T-pBD-3-P recombinant plasmid, and taking the recombinant plasmid as a template to amplify a core promoter fragment. The core promoter provides an excellent experimental system for studying the transcriptional regulation mechanism of the pig beta-defensin-3 gene, the nutrient response characteristics provide a new platform for studying the interaction between nutrition and immunity, and the core promoter has a wide application prospect in the field of healthy breeding of livestock and poultry.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and molecular biology, specifically relating to a core promoter of the porcine β-defensin-3 gene, its construction method, and its application. Background Technology

[0002] In nature's immune defense system, defensins, as a class of cationic antimicrobial peptides widely present in organisms, are indispensable key effector molecules of the innate immune system. With their unique molecular structure and mechanism of action, they exhibit broad-spectrum antimicrobial activity, effectively resisting the invasion of various pathogens such as bacteria, fungi, and viruses. They also play a crucial role in regulating the body's immune response and promoting the proliferation and differentiation of immune cells, serving as an important bridge connecting innate and adaptive immunity.

[0003] Porcine β-defensin-3 (pBD-3), an important member of the β-defensin family, is closely related to the health status of pigs. Studies have found that pBD-3 is specifically expressed mainly in the mucosal epithelial cells of the intestines, respiratory tract, and reproductive tract of pigs. These sites are the main gateways for pathogen invasion, therefore pBD-3 plays a crucial role in mucosal immune defense. It can not only directly kill invading pathogens, but also enhance the overall immune level of the body through chemotaxis of immune cells and strengthening of mucosal barrier function, which is of great significance for reducing the incidence of infectious diseases in pig herds.

[0004] In recent years, with the deepening of research in nutritional immunology, increasing evidence suggests that nutrients can influence the body's immune function by regulating the expression of immune-related genes. Studies on pBD-3 have found that short-chain fatty acids (such as butyrate) and functional amino acids (such as arginine and glutamine) can significantly upregulate the expression level of pBD-3 in porcine intestinal epithelial cells (Dou et al., 2019; Lan et al., 2020). This finding provides a new approach to enhancing porcine immunity through nutritional regulation, making pBD-3 an ideal target for research on nutritional immune regulation.

[0005] However, the molecular mechanisms by which nutrients regulate pBD-3 expression are not fully elucidated. In eukaryotic gene expression regulation, the promoter, as a DNA sequence located upstream of the transcription start site, is a key region for the binding of RNA polymerase and transcription factors. Its activity directly determines the initiation and intensity of gene transcription, making it a core element of gene expression regulation. Therefore, elucidating the structure and function of the pBD-3 gene promoter, and clarifying its core sequence and regulatory elements, is crucial for revealing the molecular mechanisms by which nutrients regulate pBD-3 expression.

[0006] Currently, although there are studies on the cloning and expression patterns of the pBD-3 gene, research on its promoter, especially the core promoter, identification, functional verification, and interaction mechanisms with nutrients remains scarce. The few existing studies only involve preliminary analysis of the upstream sequence of the pBD-3 gene, failing to identify the transcriptionally active core promoter region or explore in depth how nutrients regulate gene expression by acting on the promoter. This research status quo significantly limits our understanding of the pBD-3 gene transcriptional regulatory network and hinders the development of novel immune enhancement strategies and nutrient screening platforms based on pBD-3 regulation.

[0007] Therefore, cloning and identifying the core promoter of the pBD-3 gene, clarifying its activity in porcine small intestinal epithelial cells and its response characteristics to nutrients, has become a key issue that urgently needs to be addressed in current research in this field. This has important theoretical and practical significance for promoting research on nutritional immunity in pigs and disease-resistant breeding. Summary of the Invention

[0008] To address the above shortcomings, this invention provides a core promoter for the porcine β-defensin-3 gene, which solves the problem of unclear core promoters for the pBD-3 gene. This promoter has promoter activity in porcine small intestinal epithelial cells and is regulated by the nutrients sodium butyrate and glutamine.

[0009] The present invention adopts the following technical solution: a core promoter of the porcine β-defensin-3 gene, the sequence of which is shown in SEQ ID No.1.

[0010] Another object of the present invention is to provide a primer pair for amplifying the core promoter of the porcine β-defensin-3 gene as described above, the primer pair comprising: an upstream primer as shown in SEQ No. 2 for amplifying the core promoter sequence and a downstream primer as shown in SEQ No. 3.

[0011] Another objective of this invention is to provide a method for constructing the core promoter of the porcine β-defensin-3 gene as described above, comprising the following steps: first, using DNA extracted from porcine small intestinal epithelial cells as a template, amplification, ligation, and transformation are performed to obtain the pMD-18T-pBD-3-P recombinant plasmid; then, using the pMD-18T-pBD-3-P recombinant plasmid as a template, PCR amplification is performed using the primers described above to obtain the core promoter of the β-defensin-3 gene.

[0012] Another object of the present invention is to provide a recombinant plasmid comprising the porcine β-defensin-3 gene core promoter as described above.

[0013] Another objective of this invention is to provide an application of the porcine β-defensin-3 gene core promoter as described above, applied in the following aspects: (1) to initiate the expression of firefly luciferase in eukaryotic cells, wherein the eukaryotic cells are porcine small intestinal epithelial cells; (2) in an experimental system for studying the transcriptional regulation mechanism of the porcine β-defensin-3 gene; (3) in the study of porcine anti-infection immune mechanisms; and (4) in screening for nutrients with immune-enhancing functions, by detecting the activity changes of the core promoter under nutrient stimulation, and screening for nutrients that can enhance its activity.

[0014] The present invention has the following technical effects and advantages:

[0015] (1) Clear promoter activity and targeted regulatory ability: The core promoter of the porcine β-defensin-3 gene provided in this invention has been shown to have significant promoter activity in porcine small intestinal epithelial cells, and can directly drive the transcriptional expression of downstream genes (such as firefly luciferase). This characteristic makes it a molecular switch for precisely regulating the expression of the porcine β-defensin-3 gene, providing a reliable tool for studying the physiological function of this gene, and laying the foundation for targeted regulation of its expression.

[0016] Specific response to nutrients: The activity of this core promoter is significantly enhanced upon stimulation by nutrients such as sodium butyrate and glutamine. This characteristic not only reveals the molecular pathway by which nutrients regulate the expression of the porcine β-defensin-3 gene (i.e., by enhancing transcriptional activity through action on the core promoter), but also makes it a key molecular target connecting nutrient intake and immune function, providing direct evidence and experimental models for the study of the "nutrient-gene-immunity" regulatory network.

[0017] (2) Advancing research on transcriptional regulation mechanisms: The clear identification of the core promoter fills a gap in research on the transcriptional regulation of the porcine β-defensin-3 gene. Recombinant plasmids containing this promoter can serve as standardized experimental systems, helping researchers to explore the effects of transcription factor binding and epigenetic modifications on gene expression, analyze its regulatory patterns under different physiological or pathological conditions, and provide important support for improving the theoretical system of expression regulation of porcine immune-related genes.

[0018] (3) Facilitating the application of anti-infection immunity and nutritional screening: At the application level, this core promoter can be directly used to study the anti-infection immune mechanism of pigs. By regulating its activity, the resistance effect of changes in the expression of pig β-defensin-3 on pathogen invasion can be observed. At the same time, based on its response characteristics to nutrients, an efficient screening model can be established to quickly identify nutrients that can enhance immune function, providing a scientific basis for the development of new feed additives and the optimization of breeding formulas, and has significant industrial application value.

[0019] (4) Stability and reproducibility of the experimental system: By constructing recombinant plasmids containing this core promoter, a standardized experimental system can be formed, reducing experimental errors caused by unclear promoter sequences and improving the stability and reproducibility of research results. This advantage enables different laboratories to conduct research based on the same molecular tools, promotes the comparison and integration of academic achievements, and accelerates scientific research progress in related fields.

[0020] In summary, this invention not only theoretically elucidates the key molecular basis of porcine β-defensin-3 gene expression regulation, but also provides practical tools for immune research and nutritional screening, possessing both scientific value and industrial potential. Attached Figure Description

[0021] Figure 1 Comparative activity diagram of the core promoter of the porcine β-defensin-3 gene;

[0022] Figure 2 Comparative activity diagram of the core promoter of porcine β-defensin-3 gene after sodium butyrate stimulation;

[0023] Figure 3 A comparative activity diagram of the core promoter of the porcine β-defensin-3 gene after glutamine stimulation;

[0024] Figure 4 This is a graph showing the relative expression levels of porcine β-defensin-3 gene mRNA after glutamine stimulation. Detailed Implementation

[0025] The present invention will now be described in further detail with reference to the embodiments and accompanying drawings.

[0026] Example 1

[0027] Cloning of the core promoter of the porcine β-defensin-3 gene

[0028] Using genomic DNA extracted from the jejunum of piglets raised at the Acheng base of Northeast Agricultural University as a template, the 5' upstream 2kb sequence of the porcine β-defensin-3 gene was amplified by PCR. The gel-recovered product was ligated into the pMD-18T vector, and the ligation product was transformed into E. coli DH5α competent cells. Positive clones were screened by colony PCR and sequencing to obtain the recombinant plasmid pMD-18T-pBD-3-P. Using the successfully identified pMD-18T-pBD-3-P positive plasmid as a template, the core promoter fragment of the β-defensin-3 gene was amplified by PCR using primer pairs pBD-3-F (SEQ ID No. 2) and pBD-3-R (SEQ ID No. 3). The gel-recovered product and the dual-luciferase vector pGL3-Basic were double-digested and ligated with KpnⅠ and XhoⅠ. The ligation product was transformed into E. coli DH5α competent cells, and positive clones were screened by colony PCR and sequencing to finally obtain the recombinant plasmid pGL3-pBD-3.

[0029] Example 2

[0030] Analysis of the core promoter activity of the porcine β-defensin-3 gene

[0031] Porcine small intestinal epithelial cells (IPEC-J2) in good growth condition were seeded into 96-well plates. When the cells reached approximately 80% confluence, Lipofectamine was used to... TM The pGL3-pBD-3 plasmid and the internal control pRL-TK plasmid were co-transfected into cells at a ratio of 100:1 using 3000 transfection reagent. The negative control was transfected with the pGL3-Basic empty vector and the internal control pRL-TK plasmid (100:1). Forty-eight hours after transfection, firefly luciferase activity was detected using a Promega dual-luciferase reporter gene detection system, corrected for by Renida luciferase activity. The relative reporter gene activity is the ratio of firefly luciferase activity to Renida luciferase activity. The reporter gene activity analysis results showed that, as... Figure 1 As shown, the pGL3-pBD-3 luciferase activity was significantly increased in the experimental group transfected with the pGL3-pBD-3 recombinant plasmid compared with that in the control group transfected with the pGL3-Basic empty vector (P<0.001). This indicates that the core promoter of the porcine β-defensin-3 gene initiates the transcriptional expression of the downstream reporter gene and has promoter activity.

[0032] Example 3

[0033] Activation of the core promoter of the porcine β-defensin-3 gene by nutrients

[0034] Porcine small intestinal epithelial cells (IPEC-J2) in good growth condition were seeded into 96-well plates. When the cells reached approximately 80% confluence, they were treated with Lipofectamine. TM Recombinant plasmids pGL3-pBD-3 and pRL-TK were co-transfected into cells using a 3000 transfection reagent. Twenty-four hours after transfection, cells were stimulated with 4 mM sodium butyrate (NaB) or 1 mM glutamine (Gln), using cell culture medium as a control. After another 24 hours of culture, firefly luciferase activity was detected using a Promega dual-luciferase reporter gene detection system, corrected for by Renida luciferase activity. The relative reporter gene activity is the ratio of firefly luciferase activity to Renida luciferase activity. Figure 2-3 The results show that both sodium butyrate and glutamine treatment significantly enhanced the activity of the porcine β-defensin-3 core promoter, indicating that the porcine β-defensin-3 gene core promoter can serve as an experimental system for studying its transcriptional regulatory mechanisms.

[0035] Example 4

[0036] The regulatory effect of glutamine on porcine β-defensin-3 gene mRNA expression

[0037] Porcine small intestinal epithelial cells (IPEC-J2) in good growth condition were seeded into 12-well plates. When the cells reached approximately 80% confluence, they were treated with different concentrations of glutamine (0.5 mM, 1 mM, 2 mM, 4 mM, and 8 mM) for 24 hours. Cells were then collected, and total RNA was extracted for reverse transcription. The resulting cDNA was used as a template for quantitative real-time PCR detection. Figure 4 The results show that glutamine increases porcine β-defensin-3 mRNA expression in a concentration-dependent manner, with 1 mM glutamine showing the most significant effect. Compared with the control group, 1 mM glutamine treatment increased porcine β-defensin-3 mRNA expression by 3.2 times. These results indicate that the core promoter of the porcine β-defensin-3 gene is a key element in the regulation of porcine β-defensin-3 expression by glutamine.

Claims

1. A core promoter for the porcine β-defensin-3 gene, characterized in that, The sequence of the core promoter of the porcine β-defensin-3 gene is shown in SEQ ID No.

1.

2. A primer pair, characterized in that, For amplifying the porcine β-defensin-3 gene core promoter as described in claim 1, the primer pair comprises: an upstream primer as shown in SEQ ID No. 2 and a downstream primer as shown in SEQ ID No.

3.

3. A method for constructing a recombinant plasmid containing the porcine β-defensin-3 gene core promoter as described in claim 1, characterized in that, The method is as follows: First, DNA extracted from porcine small intestinal epithelial cells was used as a template to amplify the 5' upstream 2kb sequence of the porcine β-defensin-3 gene using PCR technology. The gel-recovered product was ligated into the pMD-18T vector, and the ligation product was transformed into E. coli DH5α competent cells. Positive clones were screened by colony PCR and sequencing to obtain the pMD-18T-pBD-3-P recombinant plasmid. Using the pMD-18T-pBD-3-P recombinant plasmid as a template, the core promoter fragment of the β-defensin-3 gene was amplified by PCR technology. The primer pair used included the upstream primer shown in SEQ ID No. 2 and the downstream primer shown in SEQ ID No.

3. The gel-recovered product and the dual-luciferase vector pGL3-Basic were double-digested and ligated with Kpn I and Xho I. The ligation product was transformed into E. coli DH5α competent cells, and positive clones were screened by colony PCR and sequencing to finally obtain the recombinant plasmid pGL3-pBD-3.

4. A recombinant plasmid, characterized in that, Including the porcine β-defensin-3 gene core promoter as described in claim 1.

5. An application of the porcine β-defensin-3 gene core promoter as described in claim 1, characterized in that, Including any of the following: (1) application to initiate the expression of firefly luciferase in eukaryotic cells, wherein the eukaryotic cells are porcine small intestinal epithelial cells; (2) application as a tool for studying the transcriptional regulation mechanism of porcine β-defensin-3 gene; (3) application in the study of porcine anti-infection immune mechanisms; (4) application in screening for nutrients with immune-enhancing functions, wherein the activity of the core promoter is detected by detecting changes in the activity of the core promoter under the stimulation of nutrients, and nutrients that can enhance its activity are screened out.

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