Use of recombinant intestinal alpha-defensin for the preparation of a medicament for the treatment of inflammatory bowel disease

Abstract

The application discloses application of recombinant intestinal alpha-defensin in preparation of a medicine for treating inflammatory bowel disease. The application constructs an expression vector of Defa20 defensin by optimizing a gene sequence (Defa20 gene) of mouse-derived alpha-defensin, adopts a eukaryotic expression system to optimize protein expression, so as to obtain sufficient yield of Defa20 recombinant protein, and finds that exogenous administration of alpha-defensin can regulate immune response of colitis and protect intestinal tight junction, thereby providing a train of thought and direction for preparation of the medicine for inflammatory bowel disease.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of recombinant intestinal α-defensin in the preparation of drugs for treating inflammatory bowel disease. Background Technology

[0002] Antimicrobial peptides are small polypeptides naturally found in almost all organisms. They are not only effective against various bacteria, fungi, protozoa, viruses, and cancer cells, but also an important component of the body's innate immune system. Alpha-defensins, as a key member of the antimicrobial peptide family, are expressed and secreted by Paneth cells in the gut. They participate in the host's immune defense and regulate the composition of the gut microbiota, thereby maintaining gut balance and health. The amphipathic structure of alpha-defensins allows them to embed into cell membranes, forming dimer or multimerous transmembrane ion channels. These channels alter membrane permeability, creating defects in the cell membrane that lead to the extravasation of essential ions and nutrients, ultimately resulting in bacterial death. Simultaneously, alpha-defensins also have the ability to regulate the function of immune cells. Immune cells such as monocytes, lymphocytes, and macrophages play crucial roles in the immune response. Alpha-defensins can influence the activity of these immune cells, regulating their proliferation, migration, and secretion of inflammatory factors. This regulatory effect helps balance the immune system's response and prevent excessive inflammatory responses. In summary, alpha-defensins maintain intestinal immune balance and health through both direct bactericidal action and regulation of immune cell function. Unlike traditional antibiotics, alpha-defensins are bioactive molecules produced by the body itself, thus less likely to induce bacterial resistance and possessing a regulatory effect on the body's symbiotic flora. Due to their ability to inhibit pathogenic microorganisms, defensins hold promise as alternatives to traditional bactericides and antibiotics, showing broad application prospects in the medical and food industries. Furthermore, sufficient quantities of defensin monomers are prerequisites for further structural analysis, physicochemical property analysis, and in-depth research into their mechanisms of action. Modern research indicates that the expression of alpha-defensins and the overall physiology of Paneth cells may play a crucial role in the development of colitis and other intestinal inflammatory or dysbiotic diseases. As researchers gain a better understanding of intestinal alpha-defensins, their potential as clinical intervention tools becomes increasingly apparent. Studies have shown reduced alpha-defensin expression in the ileal tissue of patients with ileal Crohn's disease; however, it remains unclear whether altered defensin expression is a cause of the pathogenesis of inflammatory bowel disease or a byproduct of disease progression.

[0003] Despite the broad application prospects and enormous development potential of defensins, their industrialization process and mechanism research have been relatively slow. The main production methods for defensins include natural extraction, chemical synthesis, and genetic engineering. However, the content of natural defensins in animals is low, and extraction from them has many drawbacks, such as low yield, long cycle time, complex process, low potency, and high price. Chemical synthesis of defensin peptides is costly, difficult, and struggles to control the disulfide bonds and spatial folding conformation of the peptides, thus affecting their activity. Therefore, developing and preparing next-generation defensins using genetic engineering methods can overcome these shortcomings and is of great significance. Summary of the Invention

[0004] One objective of this invention is to provide a recombinant intestinal α-defensin with the following amino acid sequence: SRDLICYCRKGGCNRGEQVYGTCSGRLLFCCRRRHRH.

[0005] The nucleotide sequence encoding the amino acid is as follows: GAAGAGCAGCCGGGACCTGATCTGCTACTGCAGAAAGGGCGGATGTAATAGAGGCGAGCAAGTGTACGGCACCTGTAGCGGCAGACTGCTCTTCTGCTGCCGGAGACGGCACAGACACCACCACCACCATCACCACTGA.

[0006] A second objective of this invention is to provide the application of the above-mentioned recombinant intestinal α-defensin in the preparation of a medicament for treating inflammatory bowel disease.

[0007] Genetic engineering methods, such as microbial fermentation and cell expression purification, allow for the large-scale production of active defensin peptides, which is currently the best approach for defensin production. Genetic engineering methods are mainly divided into two types: prokaryotic bacterial expression and eukaryotic cell expression. While prokaryotic cell expression can produce large quantities of peptides, it may lack post-translational modifications, affecting peptide function. In contrast, peptides produced by eukaryotic cell expression avoid this problem and yield active defensins similar to those naturally produced by the body.

[0008] This invention constructs an expression vector for Defa20 defensin by optimizing the gene sequence of murine α-defensin (now named the Defa20 gene). The constructed expression vector was introduced into prokaryotic host *Escherichia coli*, and the vector plasmid was amplified and extracted. Subsequently, the extracted plasmid was transfected into HEK293T cells for eukaryotic expression. The expressed recombinant protein was purified by NI column affinity chromatography, and its antibacterial activity was detected, showing significant antibacterial activity. Further investigation revealed that this recombinant protein significantly inhibited the expression of LPS-induced pro-inflammatory factors Il-1β and Il-6 in macrophages, exhibiting significant anti-inflammatory activity. Furthermore, in mice, it was found that by regulating pro-inflammatory factors, it could improve DSS-induced acute colitis.

[0009] In this invention, an α-defensin Defa20 plasmid was constructed, and protein expression was optimized using a eukaryotic expression system to obtain a sufficient yield of recombinant Defa20 protein. Furthermore, it was discovered that exogenous administration of α-defensin can regulate the immune response to colitis and protect intestinal tight junctions, providing ideas and directions for the preparation of drugs for inflammatory bowel disease. Attached Figure Description

[0010] Figure 1 A comparison of the optimized gene sequence for α-defensin with the original sequence.

[0011] Figure 2 This is a schematic diagram of the recombinant plasmid structure.

[0012] Figure 3 The results are from the PCR gel electrophoresis of the recombinant plasmid.

[0013] Figure 4 SDS-PAGE electrophoresis results of Defa20 eluted with different concentrations of imidazole.

[0014] Figure 5 The SDS-PAGE electrophoresis results for the Defa20 protein.

[0015] Figure 6 The results of the antibacterial experiment of Defa20 recombinant protein.

[0016] Figure 7 The anti-inflammatory experimental results of overexpressing Defa20.

[0017] Figure 8 The results of the anti-inflammatory experiment for the Defa20 recombinant protein.

[0018] Figure 9 Results of experiments demonstrating how Defa20 recombinant protein improves DSS-induced acute colitis in mice. Detailed Implementation

[0019] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0020] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0021] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. Example 1

[0022] First, based on codon optimization of the eukaryotic expression vector, the optimized sequence of the Defa20 target gene was obtained (completed by GenScript). In this process, an XbaⅠ restriction site and a Kozak sequence (GCCACC) were added sequentially before the nucleotide fragment, and a 6×His sequence, a stop codon, and an EcoRV restriction site were added after the nucleotide fragment.

[0023] Comparison of optimized gene sequences with original sequences, for example Figure 1 As shown.

[0024] The obtained nucleic acid fragment was inserted into the multiple cloning sites XbaⅠ and EcoRV of the pCDNA3.4 eukaryotic expression vector. After ligation with DNA ligase, the plasmid with complete insertion was verified by PCR and gene sequencing. The plasmid was then transformed into Top10 competent bacteria. Transformed bacteria were selected using ampicillin and preserved with glycerol.

[0025] Recombinant plasmid structure such as Figure 2 As shown, the PCR gel electrophoresis results of successful recombinant plasmid construction are as follows: Figure 3 As shown.

[0026] The glycerol bacteria transformed with the Defa20 plasmid were activated. The activated glycerol bacteria were added to ampicillin-resistant LB medium and shaken at 37°C for 12 hours. Then, the overnight bacteria were transferred to fresh ampicillin-resistant LB medium at a ratio of 1:10 and cultured at 37°C for another 6-12 hours.

[0027] Centrifuge the above bacterial culture at 5000g for 10 minutes to remove as much supernatant as possible. Extract the plasmid of Defa20 using an endotoxin-free plasmid extraction kit (Kangwei Century) and transform it into HEK 293T cells for eukaryotic expression. HEK293T cells were seeded in six 100mm dishes and transfected using lipo3000 and P3000 reagents. The plasmid mass per dish was 12μg, and the transfection time was 48 hours. The target protein was purified by Ni column affinity chromatography. The specific steps are as follows: (1) Each dish was lysed with 2ml of lysis buffer (20mM Tris-HCl, 150mM NaCl, 20mM imidazole, pH=8.0). The lysis buffer was placed on ice and sonicated for 10min at 50% intensity. The sonicated lysis buffer was centrifuged at 12000rpm for 20min at 4℃. The supernatant was collected and centrifuged once more to remove the precipitate and avoid column blockage. (2) The Ni column was rinsed with 5mL of lysis buffer (20mM Tris-HCl, 150mM NaCl, 20mM imidazole, pH=8.0). (3) The supernatant of the recombinant protein was added and mixed by inversion. (4) Place the Ni column vertically, and after the packing material settles to the bottom, drain the liquid from the column and collect the liquid after passing through the column; (5) Add 5 mL of washing buffer (20 mM Tris-HCl, 150 mM NaCl, 50 mM imidazole, pH=8) to the column, invert and mix well, let stand, and after the packing material settles, drain the liquid from the column to ensure that the impurities are fully eluted; (6) Add 1 mL of elution buffer (imidazole concentrations of 20 mM, 50 mM, 100 mM, and 300 mM) to the column according to the imidazole concentration from low to high, invert and mix well, let stand, and after the packing material settles, collect the liquid from the column, elute according to the imidazole concentration from low to high, and elute twice for each concentration, collect the elution liquid separately, and obtain the target protein. The SDS-PAGE electrophoresis results of Defa20 protein eluted with different concentrations of imidazole are as follows. Figure 4 As shown.

[0028] The purified protein was analyzed and identified using 15% SDS-PAGE to confirm that it was the desired protein. The SDS-PAGE electrophoresis results of successful Defa20 purification are shown below. Figure 5 As shown.

[0029] The antibacterial activity of Defa20 protein was determined using a microspectrophotometric method. First, Salmonella, Escherichia coli, and Bacillus licheniformis were cultured to the logarithmic growth phase (OD600 = 0.5). The bacterial suspensions were then diluted to 1 × 10⁻⁶. 6The concentration of CFU / mL was determined, and 50 μL of the diluted bacterial culture was added to each well of a 96-well plate. Different concentrations of purified Defa20 recombinant protein were added to each well, and LB broth was added to bring the total volume to 100 μL to achieve the target concentrations of Defa20 recombinant protein (10, 50, 100, 200, and 300 μg / mL). The control group received no Defa20 protein, only the corresponding volume of LB broth. After incubation at 37°C for 12 hours, the OD value of each well was measured at 600 nm using a microplate reader. The antibacterial activity of Defa20 protein was expressed as the growth inhibition rate by comparing the OD values. Each concentration of Defa20 protein and the control group were tested three times to ensure the reliability of the results.

[0030] The antibacterial results of the Defa20 recombinant protein are as follows: Figure 6 As shown.

[0031] RAW 264.7 cells were seeded in 12-well plates at a density of 300,000 cells per well. After 24 h, the cell density reached 60-70%. Defa20 plasmid was transfected at 1 μg per well. Twelve h after transfection, cells were stimulated with 1 μg / mL lipopolysaccharide for 12 h. The culture medium was discarded, and each well was washed with 1 mL of pre-chilled PBS to remove residual medium. Another 1 mL of pre-chilled PBS was added, and cells were scraped into new 1.5 mL centrifuge tubes. After centrifugation, the supernatant was discarded, and 1 mL of TRIZOL was added to the cell pellet to extract RNA. The expression levels of Il-6, Tnf-α, and Il-1β mRNA in the cells were detected by RT-qPCR. Figure 7 As shown, the expression of the three inflammatory factors mentioned above increased after LPS induction, while the expression of Tnf-α, Il-6, and Il-1β mRNA decreased in the group transfected with Defa20 plasmid; moreover, overexpression of Defa20 plasmid in mouse primary cells BMDM and human macrophage cell line THP-1 also reduced the expression of pro-inflammatory factors.

[0032] RAW 264.7 cells were seeded in 12-well plates at a density of 300,000 cells per well. After 24 h, the cell density reached 60-70%. 2 mg of Defa20 recombinant protein was added to each well. After 12 h, the cells were stimulated with 1 μg / mL lipopolysaccharide for 12 h. The culture medium was then discarded. Each well was washed with 1 mL of pre-chilled PBS to remove residual culture medium, and then another 1 mL of pre-chilled PBS was added. Cells were scraped into new 1.5 mL centrifuge tubes, centrifuged, and the supernatant was discarded. 1 mL of TRIZOL was added to the cell pellet to extract RNA. The expression levels of Il-6, Tnf-α, and Il-1β mRNA in the cells were detected by RT-qPCR. Figure 8 As shown, the Defa20 recombinant protein also exhibits anti-inflammatory function in the LPS-induced cellular inflammation model, and can significantly inhibit the expression of pro-inflammatory factors. Example 2

[0033] Fifteen wild-type male C57BL / 6J mice were introduced to the Animal Experiment Center of China Pharmaceutical University. After one week of acclimatization, they were randomly divided into three groups of five mice each based on their body weight: a control group (CTL, n=5), a model group (DSS, n=5), and a Defa20 administration group (Defa20, n=5). Two days before the DSS-induced colitis model was established, the administration group was administered 2 mg of purified Defa20 recombinant protein by gavage, while the control and model groups were administered an equal volume of physiological saline. Two days later, the model and administration groups were induced with experimental colitis by 3% DSS. On the third day of modeling, the administration group was administered Defa20 recombinant protein by gavage, while the control and model groups were administered an equal volume of physiological saline by gavage. The weight changes of the mice were recorded daily. After the model was established, the mice were euthanized by cervical dislocation, and colonic tissue was dissected to measure length changes. The colonic tissue near the cecum was harvested, the mesentery was removed, and 1 cm of colonic tissue was harvested. The intestinal contents were rinsed with PBS and then placed in 4% paraformaldehyde for HE staining and AB-PAS staining. RNA was extracted from colon tissue near the cecum of mice, and the expression levels of Il-6, Tnf-α, and Il-1β mRNA in the colon tissue were detected by RT-qPCR.

[0034] like Figure 9 As shown, the recombinant Defa20 protein improved the degree of DSS-induced weight loss and colon shortening in mice, and inhibited pro-inflammatory factors in colon tissue, while also improving the degree of colon tissue damage in mice.

Claims

1. The use of recombinant intestinal α-defensin, or biological vectors for expressing recombinant intestinal α-defensin, or cells expressing recombinant intestinal α-defensin, in the preparation of drugs for treating DSS-induced acute colitis; The recombinant intestinal α-defensin has the following amino acid sequence: SRDLICYCRKGGCNRGEQVYGTCSGRLLFCCRRRHRH.

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