A fusion protein comprising selenomethionine and its use in the treatment of acute pancreatitis

Abstract

The present application relates to a kind of fusion protein comprising selenomethionine and its application in the treatment of acute pancreatitis, belong to the field of biological medicine.The fusion protein is prepared by in vivo biological chelation method from TAT-14-PTD-p65 fusion protein and organic selenium (selected from selenomethionine), the fusion protein is designed by "Nrf2 activation-NF-κB inhibition-selenium donor" trinity, comprising Nrf2 activation element (TAT-14 peptide), NF-κB inhibition element (PTD-p65-P1) and selenium donor element (selenomethionine), is connected by (Gly4Ser) 2 connecting peptide to avoid spatial interference.Experiments show that the fusion protein and its selenium chelate can inhibit the activation of pancreatic tissue NF-κB inflammatory pathway, up-regulate pancreatic Nrf2 / HO-1 antioxidant pathway, significantly reduce the serum lipase and amylase levels of C57 mice with acute pancreatitis, inhibit inflammatory response and oxidative stress, relieve pancreatic tissue damage, and selenium chelate is better.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a fusion protein containing selenomethionine, its preparation method, and its application in the preparation of drugs for the treatment and / or prevention of acute pancreatitis. This fusion protein achieves synergistic anti-inflammatory and antioxidant effects by precisely regulating key signaling pathways in the pathogenesis of acute pancreatitis, providing a novel drug candidate for the treatment of this common acute abdominal condition. It can be widely applied in the research and development and production of drugs for the treatment of acute pancreatitis. Background Technology

[0002] Acute pancreatitis (AP) is an inflammatory syndrome caused by abnormal activation of pancreatic digestive enzymes, which can manifest as edema, hemorrhage, and even necrosis. It is one of the most common acute abdominal conditions in clinical practice. In recent years, with a deeper understanding of the pathogenesis of AP, the cross-regulation of the Nrf2 (nuclear factor erythroid 2-related factor 2) and NF-κB (nuclear factor κB) pathways has become a hot topic in inflammation and oxidative stress research.

[0003] In the pathogenesis of acute pancreatitis (AP), abnormal activation of digestive enzymes in pancreatic acinar cells leads to cell damage, which in turn triggers an inflammatory cascade. Studies have shown that the pathogenesis of AP involves multiple steps, including abnormal activation of trypsinogen, pathological calcium signaling, mitochondrial dysfunction, abnormal autophagy, endoplasmic reticulum stress, inflammation, and immune regulation. Among these, oxidative stress and inflammatory response are the core components of the AP pathological process, and the two promote each other, forming a vicious cycle.

[0004] Nrf2 is a key transcription factor in cellular antioxidant stress response, exerting a protective effect by regulating the expression of downstream antioxidant genes. Under normal conditions, Nrf2 binds to Keap1 (Kelch-like epichlorohydrin-associated protein 1) and is degraded by its ubiquitination. When cells are stimulated by oxidative stress, Nrf2 is released from Keap1 and enters the nucleus, activating the transcription of downstream antioxidant genes such as heme oxygenase-1 (HO-1) and NAD(P)Hquinone oxidoreductase 1 (NQO1).

[0005] NF-κB is a key regulator of the inflammatory response, and under normal conditions it is inactive due to its binding to the inhibitory protein IκBα. When cells are stimulated by inflammation, IκBα is phosphorylated and degraded, releasing NF-κB, which enters the nucleus and initiates the transcription of pro-inflammatory factors such as TNF-α, IL-6, and IL-1β. In acute prostatitis (AP), excessive activation of NF-κB is an important mechanism leading to the inflammatory cascade and tissue damage.

[0006] Recent studies have revealed a complex cross-regulatory relationship between the Nrf2 and NF-κB pathways. On the one hand, Nrf2 activation can inhibit NF-κB activity, reducing the production of inflammatory mediators through antioxidant effects; on the other hand, NF-κB activation can also inhibit Nrf2 transcriptional activity, forming a mutually restrictive regulatory network. This cross-regulatory mechanism plays a crucial role in the pathophysiology of acute pancreatitis (AP).

[0007] Selenium, an important trace element, exhibits unique advantages in simultaneously regulating the Nrf2 and NF-κB pathways. Studies have shown that selenium participates in the cellular antioxidant defense system through multiple mechanisms, including directly scavenging free radicals as a component of glutathione peroxidase (GPx) and enhancing cellular antioxidant capacity by regulating the Nrf2 / Keap1 signaling pathway. Simultaneously, selenium can also alleviate inflammatory responses by inhibiting the activation of the NF-κB signaling pathway. This dual regulatory effect makes selenium a potential candidate drug for the treatment of acute pancreatitis (AP).

[0008] However, traditional single-target therapy strategies have limited effectiveness in treating acute pancreatitis (AP). Therefore, developing selenomethionine-containing fusion protein therapy strategies that can simultaneously regulate the Nrf2 and NF-κB pathways is of great significance. Selenomethionine-containing fusion proteins, by linking proteins or peptides with different functions together, can achieve multiple therapeutic effects, improving the specificity and effectiveness of treatment. Summary of the Invention

[0009] To address the limited efficacy of traditional single-target treatments for acute pancreatitis, this invention provides a fusion protein containing selenomethionine, prepared by chelating the TAT-14-PTD-p65 fusion protein with an organoselenium.

[0010] In some embodiments, the TAT-14-PTD-p65 fusion protein is selected from the polypeptide shown in SEQ ID NO:3; the organic selenium is selected from selenomethionine (SeMet), and the fusion protein adopts a "Nrf2 activation-NF-κB inhibition-selenium donor" triad design, containing TAT-14 peptide (SEQ ID NO:1), PTD-p65-P1 (SEQ ID NO:2) and selenomethionine elements, linked by (Gly4Ser)2 linker peptides.

[0011] This invention also provides a method for preparing a targeted selenium-coupled anti-inflammatory fusion protein, the method comprising the following steps: 1. Designing and synthesizing the encoding nucleotide sequence according to the amino acid sequence shown in SEQ ID NO:3, constructing a recombinant expression vector and transforming it into *E. coli*, and after IPTG induction expression, obtaining the TAT-14-PTD-p65 fusion protein by Ni²⁺-NTA affinity chromatography; 2. Activating and culturing *E. coli* containing the target fusion protein gene in methionine-free medium until OD... 600 When the concentration reached 0.6-0.8, L-selenomethionine (final concentration 0.1-1 mmol / L) and IPTG (final concentration 0.5 mmol / L) were added for induction culture. Subsequently, the fusion protein selenium chelate was obtained by cell disruption, centrifugation, and Ni²⁺-NTA affinity chromatography purification.

[0012] Finally, this invention provides the application of the above-mentioned fusion protein, namely, its use in the preparation of medicaments for the treatment and / or prevention of acute pancreatitis.

[0013] In some embodiments, the fusion protein alleviates pancreatic tissue damage by inhibiting the activation of the NF-κB inflammatory pathway in pancreatic tissue, upregulating the Nrf2 / HO-1 antioxidant pathway, reducing serum lipase, amylase, and inflammatory factor levels in C57 mice with acute pancreatitis.

[0014] Compared with the prior art, the present invention has at least the following beneficial effects: This invention is the first to construct a fusion protein consisting of "Nrf2 activation-NF-κB inhibition-selenium donor", which achieves multi-target synergistic regulation of key pathways in the pathological process of acute pancreatitis, breaking through the bottleneck of limited efficacy of traditional single-target therapy and providing a new strategy for the treatment of acute pancreatitis.

[0015] The selenomethionine in the fusion protein of this invention not only acts as a selenium donor but also synergistically enhances the anti-inflammatory and antioxidant effects by working with the TAT-14-PTD-p65 fusion protein. Experiments have confirmed that the selenium chelate of the fusion protein is superior to the TAT-14-PTD-p65 fusion protein and dexamethasone alone in reducing serum lipase and amylase levels in C57 mice with acute pancreatitis, inhibiting inflammatory responses and oxidative stress, and alleviating pancreatic tissue damage, demonstrating greater therapeutic potential.

[0016] The method for preparing the fusion protein of this invention uses a prokaryotic expression system combined with Ni²⁺-NTA affinity chromatography purification technology. The operation is simple and low-cost, and it can obtain fusion proteins with high purity (greater than 97%). At the same time, the selenium chelation efficiency is as high as 87.5%, which is convenient for large-scale production and lays a solid foundation for its subsequent drug research and development and application. Attached Figure Description

[0017] Figure 1 SDS-PAGE assay of the TAT-14-PTD-p65 fusion protein.

[0018] Figure 2 Comparison of serum lipase and amylase levels in C57 mice with acute pancreatitis in different groups.

[0019] Figure 3 Comparison of serum MDA and SOD levels in C57 mice with acute pancreatitis in different groups.

[0020] Figure 4 Comparison of serum IL-1β, IL-6, and TNF-α levels in C57 mice with acute pancreatitis from different groups.

[0021] Figure 5 Comparison of Schmidt scores of pancreatic tissue from C57 mice with acute pancreatitis in different groups.

[0022] Figure 6 Gray-scale analysis of Western blot results for the expression of NF-κB, Nrf2, and HO-1 proteins in pancreatic tissues of C57 mice with acute pancreatitis in each group. Detailed Implementation

[0023] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0024] Example 1: Preparation of TAT-14-PTD-p65 fusion protein TAT-14 peptide is a 14-mer peptide that acts as an Nrf2 activator. TAT-14 peptide has no effect on Nrf2 mRNA expression, but it increases Nrf2 protein levels by targeting the Keap1 binding site on Nrf2. Its amino acid sequence is: YGRKKRRQRRRLQLDEETGEFLPIQ (SEQ ID NO:1).

[0025] PTD-p65-P1 is an inhibitor of the nuclear transcription factor NF-kappaB, consisting of an antennapedia-derived membrane transport peptide sequence (PTD) linked to p65-P1. It selectively inhibits NF-kappaB activity induced by various stimuli, and its amino acid sequence is: DRQIKIWFQNRRMKWKKQLRRPSDRELSE (SEQ ID NO:2). The fusion protein employs a "Nrf2 activation-NF-κB inhibition-selenium donor" triad design, with the overall sequence consisting of three functional elements: Nrf2 activating element: TAT-14 peptide (SEQ ID NO:1, YGRKKRRQRRRLQLDEETGEFLPIQ), which enhances Nrf2 protein levels by targeting Keap1; NF-κB inhibitory element: PTD-p65-P1 (SEQ ID NO:2, DRQIKIWFQNRRMKWKKQLRRPSDRELSE), inhibits NF-κB activity; Selenium donor element: Methionine in the sequence is replaced with Se-Met to ensure stable selenium integration; Linking peptides: (Gly4Ser)2 link TAT-14 peptide to Se-Met and Se-Met to PTD-p65-P1 respectively, avoiding spatial interference between components.

[0026] TAT-14-PTD-p65 fusion protein: YGRKKRRQRRRLQLDEETGEFLPIQGGGGSGGGGSDRQIKIWFQNRRMKWKKQLRRPSDRELSEGGGGSGGGGSYGRKKRRQRRRLQLDEETGEFLPIQGGGGSGGGGSDRQIKIWFQNRRMKWKKQLRRPSDRELSE (SEQ IDNO: 3); Nanjing Genscript Biotechnology Co., Ltd. was commissioned to design and synthesize the corresponding coding nucleotide sequence based on the amino acid sequence of the TAT-14-PTD-p65 fusion protein (SEQ ID NO:3: YGRKKRRQRRRLQLDEETGEFLPIQGGGGSGGGGSDRQIKIWFQNRRMKWKKQLRRPSDRELSEGGGGSGGGSYGRKKRRQRRRLQLDEETGEFLPIQGGGGSGGGGSDRQIKIWFQNRRMKWKKQLRRPSDRELSE), taking into account the codon bias of *E. coli*. Simultaneously, adapter sequences (NdeⅠ and XhoⅠ) matching the restriction enzyme sites of the expression vector were introduced at both ends of the sequence. The prokaryotic expression vector pET-28a(+) with a HIS tag was selected. This vector contains a T7 promoter, which allows for efficient initiation of the target gene expression under IPTG induction, and the HIS tag facilitates subsequent purification of the target protein. The synthesized coding nucleotide sequence was then used in conjunction with pET-28a... The (+) vector was double-digested with the same restriction endonucleases (NdeⅠ and XhoⅠ), incubated at 37℃ for 2-3 h, and then the digested target fragment and vector backbone were separated and recovered by agarose gel electrophoresis. Using T4 DNA ligase, the target fragment and vector backbone were ligated overnight at 16℃ to construct the recombinant expression vector pET-28a(+)-TAT-14-PTD-p65. The recombinant expression vector was transformed into *E. coli* DH5α competent cells and plated on LB solid medium containing kanamycin (50 μg / mL), and cultured at 37℃ for 12-16 h. Single colonies were picked and inoculated into LB liquid medium (containing 50 μg / mL kanamycin), cultured with shaking, and the plasmid was extracted. Double enzyme digestion and DNA sequencing were performed to verify that the target gene was correctly inserted into the vector and that the sequence was error-free. The correctly identified recombinant plasmid was transformed into *E. coli* BL21 (DE3) competent cells, and positive clones were screened. Positive clones were picked and inoculated into 5 mL of LB broth (containing 50 μg / mL kanamycin), and cultured overnight at 37°C with shaking at 220 rpm to activate the bacterial strain. The next day, the activated bacterial solution was inoculated into 500 mL of LB broth (containing 50 μg / mL kanamycin) at a ratio of 1:100, and cultured at 37°C with shaking at 220 rpm until OD (out of control) was reached. 600The pH value reached 0.6-0.8; IPTG was added to the culture system to a final concentration of 0.5 mmol / L, and the culture was continued at 30℃ and 220 rpm with shaking for 4-6 h to induce the expression of the target protein. After the induction culture, the bacterial culture was centrifuged at 4℃ and 8000 rpm for 10 min to collect the bacterial cells. The bacterial cells were washed twice with pre-cooled PBS buffer (pH 7.4) and resuspended in lysis buffer (20 mmol / L Tris-HCl, 500 mmol / L NaCl, 20 mmol / L imidazole, pH 8.0). The bacterial cells were disrupted using an ultrasonic disruptor (300W power, 3s working time, 5s interval, total time 20 min), centrifuged at 4℃ and 12000 rpm for 20 min, and the supernatant (containing soluble target protein) was collected. The supernatant was loaded onto a Ni²⁺-NTA affinity chromatography column pre-equilibrated with lysis buffer at a flow rate of 1 mL / min to allow the target protein to bind to Ni²⁺ via the HIS tag. Subsequently, impurities were eluted with washing buffer (20 mmol / L Tris-HCl, 500 mmol / L NaCl, 50 mmol / L imidazole, pH 8.0) until the absorbance of the eluent at 280 nm stabilized. Finally, the target protein was eluted with elution buffer (20 mmol / L Tris-HCl, 500 mmol / L NaCl, 500 mmol / L imidazole, pH 8.0), and the fraction corresponding to the elution peak was collected. The collected target protein fraction was placed in a dialysis bag and dialyzed overnight at 4°C in dialysis buffer (20 mmol / L Tris-HCl, 150 mmol / L NaCl, pH 7.4), changing the dialysis buffer 2-3 times to remove imidazole and excess salt, yielding purified TAT-14-PTD-p65 fusion protein. (See [link to dialysis buffer]). Figure 1 .

[0027] Figure 1 The results showed that the theoretical relative molecular mass of the TAT-14-PTD-p65 fusion protein was consistent with the band detected by SDS-PAGE (15.85 kDa), indicating that the purified protein was the target fusion protein with a purity greater than 97%.

[0028] Example 2: Preparation of TAT-14-PTD-p65 fusion protein selenium chelate via in vivo biosynthetic incorporation. The *E. coli* BL21(DE3) containing the target TAT-14-PTD-p65 fusion protein gene, preserved in a glycerol tube, was removed. Under aseptic conditions, a small amount of bacterial culture was inoculated using an inoculation loop into 5 mL of methionine-free LB broth containing 50 μg / mL kanamycin. The inoculated culture was then placed in a shaker at 37°C and 220 rpm overnight for bacterial activation.

[0029] The following day, the activated bacterial culture was inoculated into 500 mL of methionine-free LB broth containing 50 μg / mL kanamycin at a ratio of 1:100, and cultured at 37°C with shaking at 220 rpm. When the OD of the bacterial culture... 600 When the L-selenomethionine concentration reaches 0.6-0.8, indicating that the bacteria are in the mid-logarithmic growth phase, add an appropriate amount of L-selenomethionine stock solution to the culture medium to achieve a final concentration of 0.1-1 mmol / L. During this period, *E. coli* are metabolically active and have a strong ability to absorb nutrients. Adding L-selenomethionine at this time allows it to be more effectively absorbed by *E. coli* and participate in the synthesis of the fusion protein, thereby achieving efficient integration of selenium into the fusion protein.

[0030] After adding L-selenomethionine, IPTG was added to the culture system to a final concentration of 0.5 mmol / L, and the culture was continued at 30°C and 220 rpm with shaking for 4-6 hours. IPTG can bind to the lacI product, causing a conformational change that releases lacO, thereby activating transcription and initiating the expression of the target fusion protein. During the fusion protein expression process, due to the presence of L-selenomethionine in the culture medium, it will replace methionine in the synthesis of the fusion protein, thus forming a fusion protein containing selenium, i.e., achieving selenium chelation of the fusion protein.

[0031] After induction culture, the bacterial culture was transferred to centrifuge tubes and centrifuged at 4°C and 8000 rpm for 10 min to precipitate the bacteria. The supernatant was discarded, and the bacterial cells were collected. This step is to separate the bacterial cells containing the fusion protein selenium chelate from the culture medium for subsequent purification and other operations.

[0032] The collected bacterial cells were washed twice with pre-cooled PBS buffer (pH 7.4) to remove residual culture medium and impurities from the cell surface. The washed bacterial cells were then resuspended in lysis buffer (20 mmol / L Tris-HCl, 500 mmol / L NaCl, 20 mmol / L imidazole, pH 8.0) and the cells were lysed using an ultrasonic disruptor at 300W for 3 seconds followed by 5-second intervals, for a total of 20 minutes. The ultrasound caused cell rupture, releasing the intracellular fusion protein selenium chelate, which could then be processed in subsequent separation and purification steps.

[0033] The disrupted bacterial culture was centrifuged at 12,000 rpm for 20 min at 4 °C, and the supernatant was collected. The supernatant contained soluble selenium chelates of the fusion protein. The supernatant was loaded onto a Ni²⁺-NTA affinity chromatography column pre-equilibrated with lysis buffer at a flow rate of 1 mL / min, allowing the selenium chelates to specifically bind to Ni²⁺ via the HIS tag. Subsequently, impurities were eluted with washing buffer (20 mmol / L Tris-HCl, 500 mmol / L NaCl, 50 mmol / L imidazole, pH 8.0) until the absorbance of the eluent at 280 nm stabilized, indicating that impurities had been largely removed. Finally, the target selenium chelate of the fusion protein was eluted with elution buffer (20 mmol / L Tris-HCl, 500 mmol / L NaCl, 500 mmol / L imidazole, pH 8.0), and the fractions corresponding to the elution peaks were collected to obtain high-purity selenium chelates of the fusion protein.

[0034] Turn on the inductively coupled plasma mass spectrometry (ICP-MS) instrument and allow it to warm up to a stable operating state. Set the instrument's radio frequency power, typically 1100-1500W, to ensure stable plasma generation and sufficient sample ionization. Optimize the nebulizer flow rate, usually controlled at 0.8-1.2 L / min, to ensure uniform nebulization of the sample solution into the plasma. Select an appropriate sampling depth, typically 7-10 mm, to obtain the optimal ion signal. To eliminate interference, use helium collision mode, setting the helium flow rate to 4-6 mL / min to effectively eliminate Ar2⁺ interference. 78 Interference from Se. Select 7 ²Ge is used as an internal standard element to correct for matrix effects and instrument drift, ensuring the accuracy of the measurement results.

[0035] The sample was measured in parallel three times, and the selenium response value was recorded. Based on the fixed volume and the sample amount, the selenium chelation efficiency was calculated to be 87.5%, which indicates that the TAT-14-PTD-p65 fusion protein selenium chelate was successfully prepared.

[0036] Example 3: Experiments on TAT-14-PTD-p65 fusion protein and its selenium chelate in an acute pancreatitis C57 mouse model. Experimental animals: Sixty 8-week-old SPF-grade male C57 mice, weighing (20±0.5) g, were purchased from Shulaibao (Wuhan) Biotechnology Co., Ltd. (Wuhan, China). Normal mouse food was purchased from Jiangsu Xietong Biotechnology Co., Ltd. The animals were housed at the SPF-grade Experimental Animal Center of Hubei University of Medicine under an ambient temperature of 22±2℃ and a relative humidity of 55%±5%, with a 12-hour light / dark cycle. Experiments were conducted after one week of acclimatization. This experimental protocol was approved by the Animal Ethics Committee of Hubei University of Medicine, and the experimental procedures strictly followed the 3R principle.

[0037] Model establishment and grouping for drug administration: Fifty C57 mice were fasted for 12 hours and then anesthetized by intraperitoneal injection of 35 mg / kg sodium pentobarbital. Under sterile conditions, the abdomen was opened, and the bile and pancreatic ducts near the porta hepatis were clamped with arterial clamps. The ampulla of Vater was punctured and injected with 3.5% sodium taurocholate solution (1 mL / kg, 0.1 mL / min). Pancreatic edema and hemorrhage were observed after 5 minutes, indicating successful model establishment. The wound was then sutured. C57 mice that successfully developed the taurocholate model were randomly divided into 5 groups of 10 mice each: model group (intraperitoneal injection of an equal volume of saline), dexamethasone group (intraperitoneal injection of 2 mg / kg dexamethasone in saline), TAT-14-PTD-p65 fusion protein group (intraperitoneal injection of 2 mg / kg fusion protein), and TAT-14-PTD-p65 fusion protein selenium chelate group (intraperitoneal injection of 2 mg / kg selenium chelate). The remaining 10 C57 mice underwent sham surgery using saline instead of sodium taurocholate, serving as the sham surgery group, and received an intraperitoneal injection of an equal volume of saline. All groups received the medication once, and relevant indicators were measured 24 hours later.

[0038] Blood was collected from the abdominal aorta, and serum was separated by centrifugation at 3000 rpm for 15 min. Lipase and amylase levels were measured using an automated biochemical analyzer, and oxidative stress and inflammatory factor levels were detected using reagent kits. SOD, MDA, IL-1β, IL-6, and TNF-α ELISA kits were purchased from R&D Company, USA. SPSS 27.0 software was used. Quantitative data are expressed as mean ± standard deviation (x±s). One-way ANOVA combined with the SNK-q test was used. P < 0.05 was considered statistically significant. Figure 2-4 ; Figure 2 The results showed that the serum lipase and amylase levels of C57 mice in the model group were significantly higher than those in the sham-operated group (P < 0.05); the serum lipase and amylase levels of C57 mice in the dexamethasone group, fusion protein group, and fusion protein selenium chelate group were significantly lower than those in the model group (P < 0.01), and the reduction in the protein selenium chelate group was more significant compared with the other groups.

[0039] Figure 3The results showed that the serum MDA level in the model group of C57 mice was significantly higher and the SOD level was significantly lower than that in the sham-operated group (P < 0.05). The serum MDA level in the dexamethasone group, fusion protein group, and fusion protein selenium chelate group of C57 mice was significantly lower and the SOD level was significantly higher than that in the model group (P < 0.01); and the decrease in MDA and increase in SOD were more significant in the protein selenium chelate group compared to the other groups.

[0040] Figure 4 The results showed that the serum levels of IL-1β, IL-6, and TNF-α in the model group C57 mice were significantly higher than those in the sham-operated group (P < 0.05); the serum levels of IL-1β, IL-6, and TNF-α in the PLT group and dexamethasone group C57 mice were significantly lower than those in the model group.

[0041] Pancreatic tissue was harvested, fixed with 4% paraformaldehyde, and stained with hematoxylin and eosin (HE). The Schmidt method was used to score the degree of acinar necrosis, hemorrhage, inflammation, and edema in the pancreatic tissue from 0 to 4 points (total score 16 points, higher scores indicate more severe pancreatic damage). Specific criteria are shown in Table 1. Figure 5 .

[0042] Table 1. Schmidt's scoring criteria for pancreatic tissue.

[0043] Figure 5 The results showed that the pancreatic structure of C57 mice in the sham-operated group was normal. In the model group, C57 mice showed significant inflammatory cell infiltration, severe pancreatic necrosis and hemorrhage, and their Schmidt scores were significantly higher than those in the sham-operated group (P < 0.05). The aforementioned pathological damage in the pancreatic tissue of C57 mice in the dexamethasone group, fusion protein group, and fusion protein selenium chelate group was alleviated compared to the model group, and their Schmidt scores were significantly lower (P < 0.05).

[0044] Western blot was used to detect the expression of NF-κB, Nrf2, and HO-1 proteins in pancreatic tissue, with β-actin as an internal control. Appropriate amounts of pancreatic tissue were collected from C57 mice in each group, and total protein was extracted. The concentration was determined using the BCA method, and the tissue was denatured by boiling at 100 °C for 5 min. An appropriate amount of the denatured protein was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a membrane, blocked with blocking buffer for 2 h. After washing, primary antibodies against NF-κB, Nrf2, HO-1, and β-actin were added proportionally, and the membrane was incubated overnight at 4 °C. After washing, secondary antibodies were added, and the membrane was incubated at room temperature for 2 h. After ECL staining, the membrane was imaged using a gel imaging system. Using β-actin as an internal control, the gray values ​​of the bands of each target protein were analyzed using ImageJ software. The expression level of the target protein was represented by the ratio of the gray value of each target protein to the internal control protein (β-actin). Figure 6 .

[0045] Figure 6 The results showed that, compared with the sham-operated group, the expression of Nrf2 and HO-1 proteins in the colonic mucosa of C57 mice in the model group was significantly downregulated, while the expression of NF-κB protein was significantly upregulated, suggesting that pancreatic tissue damage in C57 mice with acute pancreatitis may be related to abnormal changes in the Nrf2 / HO-1 and NF-κB signaling pathways. After intervention with the fusion protein selenium chelate, the expression of Nrf2 and HO-1 proteins in the colonic mucosa of C57 mice with acute pancreatitis was significantly upregulated, while the expression of NF-κB protein was significantly downregulated.

[0046] The results of this invention show that after intervention with TAT-14-PTD-p65 fusion protein and its selenium chelate, the levels of lipase and amylase in C57 mice with acute pancreatitis were significantly reduced, the inflammatory response and oxidative stress were inhibited, and pancreatic tissue damage was significantly alleviated.

[0047] The TAT-14-PTD-p65 fusion protein and its selenium chelate can reduce inflammatory response and oxidative stress in C57 mice with acute pancreatitis by inhibiting the activation of the NF-κB inflammatory pathway in pancreatic tissue and upregulating the pancreatic Nrf2 / HO-1 antioxidant pathway. The selenium chelate is more effective.

[0048] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A fusion protein comprising selenomethionine, characterized in that, The fusion protein was prepared by in vivo biochelation of the TAT-14-PTD-p65 fusion protein with organic selenium. The chelation preparation method involved activating and culturing *E. coli* BL21(DE3) containing the gene for the TAT-14-PTD-p65 fusion protein with the target amino acid sequence shown in SEQ ID NO:

3. When the bacteria were in mid-logarithmic growth phase, the OD... 600 When the concentration reached 0.6-0.8, an appropriate amount of L-selenomethionine stock solution and IPTG were added to the culture medium to make the final concentration of L-selenomethionine in the culture medium reach 0.1-1 mmol / L and the final concentration of IPTG in the culture medium reach 0.5 mmol / L. After induction culture, the fusion protein selenium chelate was obtained by cell disruption, centrifugation, and Ni²⁺-NTA affinity chromatography purification, with a selenium chelation efficiency of 87.5%.

2. A method for preparing a targeted selenium-coupled anti-inflammatory fusion protein, characterized in that, Escherichia coli BL21(DE3) containing the TAT-14-PTD-p65 fusion protein gene with the target amino acid sequence shown in SEQ ID NO: 3 was activated and cultured. When the bacteria were in mid-logarithmic growth phase, the OD was... 600 When the concentration reached 0.6-0.8, an appropriate amount of L-selenomethionine stock solution and IPTG were added to the culture medium to make the final concentration of L-selenomethionine in the culture medium reach 0.1-1 mmol / L and the final concentration of IPTG in the culture medium reach 0.5 mmol / L. After induction culture, the fusion protein selenium chelate was obtained by cell disruption, centrifugation, and Ni²⁺-NTA affinity chromatography purification, with a selenium chelation efficiency of 87.5%.

3. The use of the fusion protein according to any one of claims 1-2 in the preparation of medicaments for the treatment and / or prevention of acute pancreatitis.

Images