A recombinant protein of pacific oyster galactose / rhamnose binding lectin CGL-1 and a preparation method and application thereof

Abstract

The application discloses a Pacific oyster ga lactose / rhamnose binding lectin CGL-1 recombinant protein and a preparation method and application thereof, and belongs to the technical field of biological medicines. In a diabetic rat model, the CGL-1 recombinant protein can significantly promote the healing of MRSA infectious wounds, and the healing rate reaches 92.44% to 94.51%. Histological analysis shows that the inflammatory cell infiltration of the wound of the CGL-1 treatment group is reduced, the collagen deposition is increased, and the new blood vessel formation is good, and a good healing effect is shown. In addition, the bacterial amount of the wound of the CGL-1 treatment group is significantly lower than that of the model group, and is reduced by 25.3% to 88.55% and 68.45% to 95.83% at the fifth day and the thirteenth day, respectively. These results show that the CGL-1 lectin protein has the potential to be used as a new type of non-antibiotic therapeutic agent, can be used for the treatment of diabetic infectious wounds, and provides a new idea for the application of marine biological resources in the field of wound healing.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a recombinant protein of Pacific oyster galactose / rhamnose-binding lectin CGL-1, its preparation method, and its application. Background Technology

[0002] Diabetes mellitus is a group of chronic metabolic diseases caused by defects in insulin secretion (such as type 1 β-cell autoimmune destruction) or impaired insulin action (such as type 2 insulin resistance). The core pathological manifestation is persistent hyperglycemia, and its etiology encompasses a multi-dimensional interaction of genetic susceptibility, environmental factors, and lifestyle. Long-term uncontrolled glycemia leads to systemic microvascular (retinopathy, nephropathy, neuropathy) and macrovascular (coronary artery disease, stroke, peripheral artery disease) complications, significantly increasing the risk of disability (amputation, blindness) and death, and has become one of the major challenges facing global public health. Among these complications, diabetic wounds are particularly common, with a slow and difficult healing process and an extremely high recurrence rate. These wounds often lead to ulcers that are difficult to heal, and may even result in amputation, and in severe cases, endanger the patient's life, causing extremely serious negative impacts on the patient's quality of life. Delayed wound healing in diabetic patients is the result of multiple factors, including oxidative stress caused by hyperglycemia, chronic inflammation, impaired angiogenesis, neuropathy, delayed reepithelialization, and metabolic disorders. These factors interact, hindering the wound healing process and increasing the risk of chronic wounds and infection. Because diabetic patients have impaired immune function, poor local blood circulation, and a hyperglycemic environment that provides favorable conditions for bacterial growth, the incidence of wound infection in diabetic patients is significantly higher than in the general population. Infection not only exacerbates the inflammatory response of the wound but can also lead to a series of serious consequences, further hindering wound healing. Therefore, the prevention and treatment of diabetic wound infection are crucial for improving patient prognosis and reducing the risk of amputation.

[0003] Diabetic wound infections pose serious risks, not only exacerbating local inflammation, leading to tissue necrosis, ulcer enlargement, and prolonged healing time, but also potentially triggering systemic inflammatory response syndrome, sepsis, and even significantly increasing the risk of amputation. In the early stages of diabetic wound infections, the main pathogens are staphylococci (such as Staphylococcus aureus) and streptococci (such as β-hemolytic streptococci). However, as the depth and severity of the infection increase, the spectrum of pathogens causing wound infections changes significantly: Gram-negative bacterial infections (such as Pseudomonas aeruginosa) gradually increase; mixed infections become increasingly complex, for example, mixed infections of Staphylococcus aureus and Pseudomonas aeruginosa are common in severe wounds; anaerobic infections begin to appear; and the risk of fungal infections also increases significantly. Furthermore, with the increasing complexity of infections, the frequency of drug-resistant strains (such as methicillin-resistant Staphylococcus aureus, MRSA) is constantly rising. These drug-resistant strains are resistant to commonly used antibiotics and can form biofilms on the wound surface to protect themselves from antibiotic attack, further increasing the difficulty and medical cost of treating diabetic wounds.

[0004] The pathological characteristics of diabetic infected wounds are mainly characterized by a significantly prolonged inflammatory phase. This process is closely related to the imbalance of inflammatory factors, impaired immune function, inhibited cell proliferation, impaired angiogenesis, reduced collagen synthesis, and delayed epithelialization. These pathological changes interact, disrupting the homeostasis of the wound microenvironment, leading to persistent wound infection. Drug-resistant bacteria easily form refractory biofilms, and the imbalance of inflammatory factors and cellular dysfunction result in excessive accumulation of reactive oxygen species, damaging cell structure and function, further aggravating wound damage, and causing delayed wound healing. Current clinical treatment strategies for diabetic infected wounds rely on single, independent methods, which may be both expensive and have limited effectiveness. For example, long-term use of antibiotics may lead to increased drug resistance, and since they are mainly administered systemically, it is difficult to achieve effective concentrations locally at the wound site. Debridement may damage surrounding healthy tissue, and the wound may still face adverse factors such as hyperglycemia and infection after debridement, leading to slow healing. Growth factor therapy also has certain limitations; its efficacy may be unstable due to individual differences and variations in the wound microenvironment, and it is also expensive. Therefore, the development of multifunctional non-antibiotic therapeutics is particularly urgent. These therapeutics can achieve efficient sterilization and accelerate the healing of diabetic wounds by modulating the infection microenvironment.

[0005] In recent years, with the increasing severity of antibiotic resistance, bioactive proteins, as a novel non-antibiotic therapeutic agent, have gradually attracted attention. Bioactive proteins possess diverse biological functions and play a crucial role in regulating the infection microenvironment, showing promise as an effective approach to addressing the challenges of treating diabetic infected wounds. Among numerous bioactive proteins, lectins have become a research hotspot due to their unique sugar recognition capabilities and immunomodulatory functions. Lectins can specifically recognize and bind to glycosyl groups, exerting antibacterial and immunomodulatory effects through interactions with glycosyl groups on the surface of pathogens, thus demonstrating great potential in the treatment of diabetic infected wounds. For example, Dos Santos Silva L et al. found that Cramoll, a lectin from Cratylia mollis, significantly improved diabetic wounds infected with multidrug-resistant Staphylococcus aureus. It promotes wound healing by reducing inflammatory cells, increasing revascularization and collagen production, decreasing bacterial load and inflammatory factor levels, and activating the ERK pathway, demonstrating its potential as a healing agent. Nunes MAS et al. discovered that Brazilian pepper (Schinus terebinthifolius) leaf lectin (SteLL) can effectively reduce the number of bacteria in wounds and decrease the infiltration of inflammatory cells through its antibacterial and immunomodulatory properties. At the same time, it promotes wound re-epithelialization and collagen synthesis, thus accelerating the wound healing process and showing potential clinical application value in the treatment of infected wounds.

[0006] Marine invertebrates, as an important part of Earth's biodiversity, contain abundant lectin resources. These lectins not only play a crucial role in the physiological processes of marine invertebrates, such as immune defense, anti-inflammation, and antibacterial activity, but also provide valuable resources for biomedical research and applications due to their unique structural and functional characteristics. For example, Liu Peng et al. reported a galactose-binding lectin (PFL-96) from the pearl oyster *Pinus hupehensis*, whose recombinant *Escherichia coli* protein showed significant antibacterial activity against *Bacillus subtilis*, *Staphylococcus aureus*, *Candida albicans*, and *Vibrio alginolyticus*, and also significantly inhibited the proliferation of HeLa tumor cells, HepG2 tumor cells, and C666-1 tumor cells. Olvera-Lucio F et al. reported a tandem repeat lectin (rMe'exLec1) from the horseshoe crab (*Limulus polyphemus*), which inhibited the growth of Gram-negative (*Vibrio parahaemolyticus*, *Escherichia coli*) and Gram-positive bacteria. Fujii Y et al. isolated a 15 kDa lectin (SeviL) from the striped mussel (*Mytilisepta virgata*) and found it to have antitumor activity. Although there are reports on the use of plant-derived lectins in the repair of infected wounds, research on marine invertebrate lectins in this field is relatively limited, and their potential application value remains to be further explored. Summary of the Invention

[0007] The purpose of this invention is to provide a recombinant galactose / rhamnose-binding lectin CGL-1 from Pacific oysters, its preparation method, and its applications, thereby addressing the problems existing in the prior art. This invention achieves efficient recombinant expression of this protein in *Escherichia coli* and explores its various biological activities in depth, including antibacterial, antioxidant, and ability to promote the healing of infected wounds in type 1 diabetes.

[0008] To achieve the above objectives, the present invention provides the following solution:

[0009] One of the technical solutions of the present invention is a CGL-1 lectin protein derived from Pacific oysters, the amino acid sequence of which is shown in SEQ ID NO.2.

[0010] The second technical solution of the present invention is a DNA molecule encoding the CGL-1 lectin protein, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0011] The third technical solution of the present invention is the application of the CGL-1 lectin protein in the preparation of drugs for the prevention and / or treatment of methicillin-resistant Staphylococcus aureus infection.

[0012] The fourth technical solution of the present invention is the application of the CGL-1 lectin protein in the preparation of a drug that promotes the healing of methicillin-resistant Staphylococcus aureus (MRSA) infected wounds in diabetic patients.

[0013] The fifth technical solution of the present invention is a drug for preventing and / or treating methicillin-resistant Staphylococcus aureus infection, comprising the CGL-1 lectin protein.

[0014] The sixth technical solution of the present invention is a drug that promotes the healing of methicillin-resistant Staphylococcus aureus (MRSA) infected wounds in diabetic patients, comprising the CGL-1 lectin protein.

[0015] Based on the above technical solution, the present invention has the following technical effects:

[0016] This invention marks the first successful preparation of CGL-1 lectin protein from Pacific oyster (Magnallanagigas) using an Escherichia coli recombinant expression system, and its antibacterial, antioxidant, and wound-repairing effects on infected wounds in diabetic rats were systematically evaluated. In vitro experiments showed that the CGL-1 recombinant protein exhibited significant antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA), with a minimum inhibitory concentration (MIC) of 64 μg / mL and a minimum bactericidal concentration (MBC) of 128 μg / mL. Scanning electron microscopy and transmission electron microscopy revealed that the CGL-1 recombinant protein could disrupt the cell structure of MRSA bacteria, induce reactive oxygen species (ROS) generation, inhibit biofilm formation, and promote the elimination of mature biofilms. Furthermore, the CGL-1 recombinant protein also demonstrated good antioxidant capacity, scavenging DPPH and ABTS free radicals at half-maximal effective concentrations (EC50). 50 The concentrations of CGL-1 lectin were 71.94 μg / mL and 7.77 μg / mL, respectively. In a diabetic rat model, recombinant CGL-1 protein significantly promoted the healing of MRSA-infected wounds, with a healing rate of 92.44%-94.51%, significantly higher than that of the model group (68.07%). Histological analysis showed that the wounds treated with CGL-1 had reduced inflammatory cell infiltration, increased collagen deposition, and good angiogenesis, demonstrating a good healing effect. Furthermore, the bacterial load in the wounds treated with CGL-1 was significantly lower than that in the model group, decreasing by 25.3%-88.55% and 68.45%-95.83% on days 5 and 13, respectively. These results indicate that CGL-1 lectin protein has the potential as a novel non-antibiotic therapeutic agent for the treatment of diabetic infected wounds, providing new insights into the application of marine biological resources in wound healing. Attached Figure Description

[0017] Figure 1This study describes the cloning, expression vector construction, and bioinformatics analysis of the CGL-1 lectin protein from Pacific oysters. The images include: (A) the original and codon-optimized sequences of the CGL-1 lectin gene; (B) the amino acid sequence of the CGL-1 lectin protein, with cysteine ​​residues forming disulfide bonds shown in red; (C) a schematic diagram of the pET-30a-CGL-1 recombinant plasmid; (D) the results of double enzyme digestion verification of the pET-30a-CGL-1 recombinant plasmid; (E) a predicted secondary structure diagram of the CGL-1 lectin protein; (F) a distribution diagram of conserved domains in the CGL-1 lectin protein; (G) physicochemical properties of the CGL-1 lectin protein; and (H) a predicted tertiary structure diagram of the CGL-1 lectin protein and a display of its four disulfide bonds.

[0018] Figure 2 Expression and purification of recombinant CGL-1 lectin protein from Pacific oyster. (A) Expression analysis of CGL-1 recombinant protein under different temperature induction conditions. M: Protein molecular weight standard; 1: Uninduced culture at 15℃ for 16 h; 2: Induced culture at 15℃ for 16 h; 3: Induced culture at 37℃ for 16 h; 4: Supernatant collected after whole-cell disruption following 16 h induction at 15℃; 5: Precipitate collected after whole-cell disruption following 16 h induction at 15℃; (B) Expression pattern analysis of CGL-1 recombinant protein under 37℃ induction condition. M: Protein molecular weight standard; 1: Precipitate collected after whole-cell disruption following 16 h induction at 37℃; 2: Supernatant collected after whole-cell disruption following 16 h induction at 37℃; (C) Inclusion body treatment and purification of CGL-1 recombinant protein. M: Protein molecular weight standard; 1-5: Elution fractions of 500mM imidazole; 6: Elution fractions of 50mM imidazole; 7: Flow-through buffer after incubation with Ni-IDA and centrifugation of the supernatant collected after inclusion body dissolution; 8: Supernatant collected after centrifugation of the inclusion body dissolution; (D) SDS-PAGE quality control results of purified CGL-1 recombinant protein. M: Protein molecular weight standard; 1: BSA (1.00μg); 2: CGL-1 recombinant protein (1.0μg).

[0019] Figure 3 Molecular weight, mass spectrometry, and circular dichroism analysis of recombinant CGL-1 lectin protein from Pacific oyster. (A) Mass spectrum of recombinant CGL-1 protein; (B) LC / MS analysis results of recombinant CGL-1 protein; (C) Circular dichroism spectrum of recombinant CGL-1 protein.

[0020] Figure 4This study analyzed the hemolytic activity and cytotoxicity of recombinant CGL-1 lectin protein from Pacific oysters. The results included: (A) hemolytic activity analysis of recombinant CGL-1 protein; (B) quantitative analysis of the hemolysis rate of recombinant CGL-1 protein; (C) cytotoxicity assessment of recombinant CGL-1 protein on HaCaT cells (24 hours); and (D) cytotoxicity assessment of recombinant CGL-1 protein on HaCaT cells (48 hours). In the figure, "*" indicates significant differences (P<0.05) between the 256, 128, and 64 μg / mL treatment groups and the negative control group (CGL-1 recombinant protein concentration of 0 μg / mL); "ns" indicates no significant differences (P>0.05) between the 32, 16, 8, 4, and 2 μg / mL treatment groups and the negative control group.

[0021] Figure 5 This study analyzed the in vitro antibacterial activity of CGL-1 recombinant lectin protein against MRSA (Metastatic spirulina) from Pacific oysters. The results included: (A) inhibition zone determination of CGL-1 recombinant protein against MRSA (PBS was the negative control); (B) quantitative analysis of the inhibition zone diameter of CGL-1 recombinant protein against MRSA; (C) evaluation of the MIC of CGL-1 recombinant protein against MRSA using a TTC colorimetric reaction; (D) quantitative analysis of OD485 after CGL-1 recombinant protein treatment of MRSA; (E) MBC determination of CGL-1 recombinant protein against MRSA; and (F) the effect of CGL-1 recombinant protein on the growth curve of MRSA. In the figures, "*" indicates a significant difference compared to the negative control group (CGL-1 recombinant protein concentration of 0 μg / mL) (P < 0.05); "ns" indicates no significant difference compared to the negative control group (P > 0.05).

[0022] Figure 6 This study investigated the effects of the CGL-1 lectin recombinant protein from Pacific oysters on the morphology and structure of MRSA bacteria. The images show: (A) Scanning electron microscopy (SEM) image of MRSA bacteria in the control group; (B) SEM image of MRSA bacteria treated with 64 μg / mL CGL-1; (C) SEM image of MRSA bacteria treated with 32 μg / mL CGL-1; (D) SEM image of MRSA bacteria treated with 16 μg / mL CGL-1; (E) Transmission electron microscopy (TEM) image of MRSA bacteria in the control group; and (F) TEM image of MRSA bacteria treated with 64 μg / mL CGL-1. The red arrows in the figures highlight the morphological and structural changes observed in MRSA bacteria after treatment with the CGL-1 recombinant protein, indicating that the CGL-1 recombinant protein has a significant cytotoxic effect on MRSA bacteria.

[0023] Figure 7 The effect of recombinant CGL-1 protein on the cell membrane integrity of MRSA was observed using DAPI / PI fluorescence staining. The scale bar in the figure is 100 μm.

[0024] Figure 8 The effect of CGL-1 lectin recombinant protein on MRSA biofilm formation in Pacific oysters was determined using the crystal violet method. The results show: (A) the inhibitory effect of CGL-1 recombinant protein on MRSA biofilm formation; (B) the quantitative analysis of the inhibitory effect of CGL-1 recombinant protein on MRSA biofilm formation; (C) the elimination effect of CGL-1 recombinant protein on mature MRSA biofilm; and (D) the quantitative analysis of the elimination effect of CGL-1 recombinant protein on mature MRSA biofilm. In Figures A and C, numbers 1-5 represent the blank group, negative control group, and the treatment groups with 64, 32, and 16 μg / mL CGL-1 recombinant protein, respectively. An asterisk "*" indicates a statistically significant difference (P < 0.05) between the CGL-1 recombinant protein treatment groups and the negative control group.

[0025] Figure 9 The effects of recombinant CGL-1 protein on intracellular contents leakage and reactive oxygen species (ROS) generation in MRSA cells were investigated. The results show: (A) concentration-dependent effect of CGL-1 protein inducing nucleic acid leakage in MRSA; (B) concentration-dependent effect of CGL-1 protein inducing protein leakage in MRSA; and (C) effect of CGL-1 protein inducing ROS generation in MRSA. Asterisks "*" indicate statistically significant differences (P < 0.05) between the CGL-1 protein treatment group and the negative control group.

[0026] Figure 10 The antioxidant activity of recombinant CGL-1 protein was evaluated. The results included: (A) scavenging capacity of recombinant CGL-1 protein against DPPH free radicals; (B) scavenging capacity of vitamin C against DPPH free radicals; (C) scavenging capacity of recombinant CGL-1 protein against DPPH free radicals; (D) scavenging capacity of vitamin C against ABTS free radicals; (E) reducing capacity of recombinant CGL-1 protein against ferric ions; and (F) reducing capacity of vitamin C against ferric ions. Asterisks “*” indicate statistically significant differences (P<0.05) between the CGL-1 protein treatment group and the vitamin C treatment group compared to the negative control group (CGL-1 protein concentration of 0 μg / mL); “ns” indicates no statistically significant difference (P>0.05) compared to the negative control group.

[0027] Figure 11This study investigated the promoting effect of recombinant CGL-1 protein on the healing of MRSA-infected wounds in diabetic rats. The figures show: (A) representative images of wound healing in each group; (B) quantitative analysis of wound healing rate in each group; (C) trends in rat body weight in each group; (D) changes in non-fasting blood glucose levels in each group; (E) detection of bacterial count in the wounds in each group; and (F) quantitative analysis of bacterial count in the wounds in each group. Letters (a, b, c) in Figures B and F indicate statistically significant differences. The same letter indicates no significant difference between groups (P>0.05), while different letters indicate significant differences between groups (P<0.05). The asterisks "*" in Figures C and D indicate statistically significant differences between the model group and the drug-treated group compared to the normal group (P<0.05).

[0028] Figure 12 This study investigated the histological effects of recombinant CGL-1 protein on the healing of MRSA-infected wounds in diabetic rats. The results included: (A) H&E staining of wound tissue from normal rats; (B) H&E staining of wound tissue from model rats; (C) H&E staining of wound tissue from KFX-treated rats; (D) H&E staining of wound tissue from rats treated with 16 μg / mL CGL-1; (E) H&E staining of wound tissue from rats treated with 32 μg / mL CGL-1; (F) H&E staining of wound tissue from rats treated with 64 μg / mL CGL-1; and (G) Statistical analysis of wound diameter across different treatment groups. In Figures A and B, red arrows represent neovascularization, blue arrows represent collagen fibers, black arrows represent adipose tissue, and yellow arrows represent inflammatory cells. Letters (a, b, c, d) in Figure G indicate statistically significant differences. The same letter indicates no significant difference between groups (P>0.05), while different letters indicate significant differences between groups (P<0.05).

[0029] Figure 13 This study investigated the effect of recombinant CGL-1 protein on collagen deposition in MRSA-infected wounds of diabetic rats. Figure G shows: (A) Masson staining of wound tissue from normal rats; (B) Masson staining of wound tissue from model rats; (C) Masson staining of wound tissue from KFX-treated rats; (D) Masson staining of wound tissue from rats treated with 16 μg / mL CGL-1; (E) Masson staining of wound tissue from rats treated with 32 μg / mL CGL-1; (F) Masson staining of wound tissue from rats treated with 64 μg / mL CGL-1; and (G) Statistical analysis of collagen volume fraction in wounds from different treatment groups. Letters (a, b, c) in Figure G indicate statistically significant differences. The same letter indicates no significant difference between groups (P>0.05), while different letters indicate significant differences between groups (P<0.05). Detailed Implementation

[0030] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0031] This invention provides a CGL-1 lectin protein derived from Pacific oysters, the amino acid sequence of which is shown in SEQ ID NO.2.

[0032] Embodiments of the present invention also provide a DNA molecule encoding the CGL-1 lectin protein, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0033] This invention also provides the use of the CGL-1 lectin protein in the preparation of medicaments for the prevention and / or treatment of methicillin-resistant Staphylococcus aureus infections.

[0034] This invention also provides the application of the CGL-1 lectin protein in the preparation of a drug that promotes the healing of methicillin-resistant Staphylococcus aureus (MRSA) infected wounds in diabetic patients.

[0035] This invention also provides a drug for preventing and / or treating methicillin-resistant Staphylococcus aureus infection, comprising the CGL-1 lectin protein.

[0036] In some specific implementation plans, pharmaceutically acceptable excipients are also included.

[0037] This invention also provides a drug for promoting the healing of methicillin-resistant Staphylococcus aureus (MRSA) infected wounds in diabetic patients, comprising the CGL-1 lectin protein.

[0038] In some specific implementation plans, pharmaceutically acceptable excipients are also included.

[0039] This invention marks the first successful preparation of CGL-1 lectin protein from Pacific oyster (Magallanagigas) using a recombinant expression system in *Escherichia coli*, and its antibacterial, antioxidant, and promoting effects on the repair of infected wounds in diabetic rats were comprehensively evaluated. In vitro experiments showed that the recombinant CGL-1 protein exhibited significant antibacterial activity against methicillin-resistant *Staphylococcus aureus* (MRSA), disrupting bacterial cell structure, inducing reactive oxygen species (ROS) generation, inhibiting biofilm formation, and promoting the elimination of mature biofilms. Furthermore, the recombinant CGL-1 protein also demonstrated good antioxidant capacity, which may help alleviate oxidative stress and inflammatory responses in diabetic wounds. In a diabetic rat model, the recombinant CGL-1 protein significantly promoted the healing of MRSA-infected wounds, reduced bacterial load, improved tissue structure, and increased collagen deposition. These findings suggest that the CGL-1 lectin protein has the potential as a novel non-antibiotic therapeutic agent for the treatment of diabetic infected wounds.

[0040] Data analysis was performed using IBM SPSS Statistics 19.0, and statistical graphing was performed using GraphPad Prism 8 software. All experiments were repeated three times, and quantitative results are expressed as mean ± standard deviation (X ± S). Independent samples t-tests were used to compare quantitative values ​​between two groups, and one-way ANOVA was used to compare quantitative values ​​among multiple groups. In all tests, P < 0.05 was considered statistically significant.

[0041] Example 1

[0042] Codon optimization, synthesis, construction of recombinant plasmid, and double enzyme digestion verification of the Pacific oyster lectin CGL-1 gene.

[0043] The CGL-1 lectin protein from Pacific oyster (Magnallanagigas) (NCBI reference sequence: XP_034338847.1) and its corresponding Gene ID (105329071) were retrieved from the NCBI database. Using NCBI's Open Reading Frame Finder, the open reading frame of the gene was determined, and the sequence encoding the mature protein was submitted to NCBI GenBank (accession number PV878053.1). Codon optimization was performed based on the codon usage preferences of *Escherichia coli*, and the entire genome was synthesized by Nanjing GenScript Biotech Co., Ltd. Using seamless cloning technology, the gene was inserted between Nde I and Hin dIII of the pETa-30 vector to construct the recombinant plasmid pET-30a-CGL-1. This plasmid was subsequently verified by double digestion with Xho I and Apa I, as well as sequencing. Bioinformatics analysis of the CGL-1 protein from Pacific oysters was performed as follows: the basic physicochemical properties of the CGL-1 lectin protein were predicted using the ProtParam online software (https: / / web.expasy.org / protparam / ); the conserved domains of the protein were analyzed using the NCBI Conserved Domains database (https: / / www.ncbi.nlm.nih.gov / Structure / cdd / wrpsb.cgi) and plotted using the functional domain mapping software DOG (v1.0); the secondary structure of the CGL-1 protein was predicted using the Jpred 4 online software (https: / / www.compbio.dundee.ac.uk / jpred / ); and the tertiary structure of the CGL-1 protein was predicted using the I-TASSER online software (https: / / zhanggroup.org / I-TASSER / ) and visualized using PyMOL software.

[0044] The gene sequence of the CGL-1 lectin protein (NCBI reference sequence: XP_034338847.1) from Pacific oyster (Magallanagigas) was analyzed using the NCBI Open Reading Frame Finder tool to determine its open reading frame (ORF). This ORF is 330 bp in length and encodes 109 amino acid residues. Figure 1 Based on the codon bias of E. coli, the ORF of the CGL-1 lectin protein was codon optimized. Figure 1 (A)

[0045] Optimized gene sequence such as SEQ ID Shown in NO.1: 5'-ATGGTTTGTCGTCTGCCGGGTAAAATTCAGACCGCTTGCGAAAACAAAGGCATCATCCTGCACTGCGGTATGCATCAGGTTCTGCACGTTTTCGACGCAATGTACGGTCGCGAAGATAAACACACCTGCAGTCGTAGCGGCAAACATAAAAGCACCAGCTGT ACCGCACGTAACGTTCTGAAAAAAGTCAAAAGCAAATGCAACGGCCGCCGTAGTTGTCGTCTGCGCGCACGTAATAGCGTTTTGGCGATCCGTGTCACGGTACCCTGAAATATCTGCGCGTTCGTTACGAGTGTCGTCGTCGTTATCACGGTTGGTGGCACGGTTAA-3'.

[0046] The amino acid sequence of the protein it encodes is shown in SEQ ID NO.2: MVCRLPGKIQTACENKGIILHCGMHQVLHVFDAMYGREDKHTCSRSGKHKSTSCTARNVLKKVKSKCNGRRSCRLRARNSVFGDPCHGTLKYLRVRYECRRRYHGWWHG.

[0047] The optimized gene sequence was synthesized in its entirety by Nanjing GenScript Biotech Co., Ltd., and then inserted into the Nde I and HindIII restriction sites of the pET-30a vector using seamless cloning technology, successfully constructing the recombinant plasmid pET-30a-CGL-1. Figure 1 (C). The recombinant plasmid pET-30a-CGL-1 was verified by double digestion with Xho I and Apa I. The digestion results showed that the recombinant plasmid produced the correct digestion fragments at the expected positions, indicating that the target gene had been successfully inserted into the vector (C). Figure 1 (D). Further sequencing confirmed that the recombinant plasmid sequence was completely identical to the designed sequence, with no mutations or insertions / deletions. The basic physicochemical properties of the CGL-1 lectin protein were predicted using the ProtParam online software. The results showed that the protein has a molecular weight of 12659.77 Da and a theoretical isoelectric point (pI) of 10.6. Its extinction coefficient is 15500 M. -1 cm -1The results indicate that the protein exhibits good absorption properties under ultraviolet light. The protein's instability index is 66.75, suggesting potential instability in vitro. Its gravy value is -0.756, indicating that the protein is generally hydrophilic. Figure 1 Analysis of the NCBI Conserved Domains database revealed that the CGL-1 lectin protein contains a typical lectin domain (galactose / rhamnose-binding lectin domain) located between amino acid residues 99-109. This domain shows high homology with known members of the lectin family, suggesting that the CGL-1 lectin protein may possess typical functions of the lectin family. A conserved domain diagram drawn using DOG software (v1.0) further visualizes the distribution of this domain within the protein sequence. Figure 1 (F). The secondary structure of the CGL-1 lectin protein was predicted using the online software Jpred4. The prediction results showed that the protein mainly consists of one α-helix and seven β-sheets. The secondary structure prediction diagram clearly shows the distribution of these secondary structure elements in the protein sequence. Figure 1 (E). The tertiary structure of the CGL-1 lectin protein was predicted using the I-TASSER online software. The prediction results showed that the protein presents a compact globular structure with one α-helix, six β-sheets, and random coils as secondary structures. Further visualization of the predicted tertiary structure was performed using PyMOL software, generating a high-quality three-dimensional structural model. This model demonstrates the tertiary structural features of the CGL-1 lectin protein, which has four pairs of disulfide bonds, namely Cys... 13 -Cys 43 Cys 22 -Cys 99 Cys 54 -Cys 86 Cys 67 -Cys 73 This provides an important structural foundation for subsequent functional research. Figure 1 (H).

[0048] Example 2

[0049] Expression and purification of recombinant CGL-1 protein from Pacific oyster

[0050] Single clones of *E. coli* containing the recombinant plasmid were inoculated into LB medium containing 50 μg / mL kanamycin sulfate and cultured at 37°C with shaking at 180 rpm for 3-4 hours until OD500 was reached. 600The protein concentration was reached to 0.6-0.8. IPTG was then added to a final concentration of 0.2 mM, and expression was induced for 16 hours at 15℃ and 37℃, respectively, with a control group at 15℃ without induction. After induction, bacterial cells were collected and sonicated in an ice-water bath (400W, 5 seconds sonication, 5 seconds interval, total duration 20 minutes). The cells were then centrifuged at 12000 rpm for 10 minutes at 4℃, and the supernatant and precipitate were collected for SDS-PAGE gel electrophoresis. For inclusion body processing, the cells were first washed with inclusion body washing buffer and then dissolved in inclusion body buffer. The Ni-IDA column was equilibrated, followed by elution with equilibration buffers of different concentrations of imidazole. Each eluted fraction was collected and analyzed by SDS-PAGE. Eluted fractions with higher purity were first dialyzed to refolding buffer for refolding, and then further dialyzed to 1×PBS (pH 7.4). The dialyzed protein solution was filtered through a 0.22 μm filter for sterilization and then processed using Toxin Eraser. TM Endotoxin Removal Kit removes endotoxins (the entire process is performed at low temperatures). Toxin Sensor is used. TM Endotoxin content was determined using the Gel Clot Endotoxin Assay Kit, and protein concentration was determined using the Bradford Protein Assay Kit. Finally, the proteins were aliquoted and stored at -20°C for later use.

[0051] The expression of recombinant CGL-1 protein in Pacific oysters under different temperature induction conditions was analyzed by 12% SDS-PAGE gel electrophoresis. The results showed that the highest recombinant protein expression was observed at 37℃ for 16 hours, while the expression levels were relatively low or almost nonexistent in the 15℃ induced and uninduced control groups. Furthermore, after sonication disruption of the bacterial cells induced at 15℃, the supernatant and precipitate were collected for gel electrophoresis, and the results showed that the expression level of CGL-1 recombinant protein was very low. Figure 2 (A). After bacterial cell disruption induced at 37℃ by sonication, the supernatant and precipitate were collected for gel electrophoresis. The results showed that the CGL-1 recombinant protein was mainly found in inclusion bodies. Figure 2 (B) Inclusion bodies were then collected from 1 L of bacterial culture. The CGL-1 recombinant protein in the inclusion bodies was washed, dissolved, and purified using a Ni-IDA affinity chromatography column. The purified CGL-1 recombinant protein was obtained by 12% SDS-PAGE analysis. Figure 2 (C) Select high-purity eluent fractions for dialysis refolding, and after filtration sterilization, use Toxin Eraser. TMEndotoxin removal was performed using an Endotoxin Removal Kit. Endotoxin content determination showed that the endotoxin level of the treated CGL-1 recombinant protein was less than 1 EU / μg. The concentration of the CGL-1 recombinant protein was determined using a Bradford protein assay kit, showing a concentration of 0.50 mg / mL. Ten tubes were prepared, each containing 1 mL. Further quality control of the CGL-1 recombinant protein was performed using 12% SDS-PAGE, showing a molecular weight of approximately 13.48 kDa, consistent with the expected size. In the SDS-PAGE electrophoresis pattern, the target protein band was clear and uniform, with no obvious impurity protein bands, indicating high protein purity. Figure 2 (D).

[0052] Example 3

[0053] Molecular weight determination, mass spectrometry, and circular dichroism analysis of CGL-1 recombinant protein

[0054] Molecular weight determination, mass spectrometry, and circular dichroism (CD) analysis of the recombinant protein were performed according to previous studies. The relative molecular mass of the CGL-1 recombinant protein was analyzed using an ultra-high resolution time-of-flight mass spectrometer (AB 5800MALDI-TOF / TOF, AB SCIEX, USA). The molecular weight of the CGL-1 recombinant protein was accurately determined by analyzing the mass-to-charge ratio (m / z) value in the obtained mass spectra. To further analyze the sequence information of the CGL-1 recombinant protein, it was digested with trypsin. After digestion, the generated peptides were analyzed using liquid chromatography-mass spectrometry (LC / MS). LC / MS analysis used nano-level liquid chromatography (EASY-nLC 1200, Thermo Fisher Scientific, USA) coupled with an electrostatic field orbital trap mass spectrometer (Q Exactive, Thermo Fisher Scientific, USA). The chromatographic column was a reversed-phase C18 column (Acclaim PepMap RSLC, 75 μm × 25 cm C18 - 2 μm). The mobile phase consisted of an aqueous solution containing 0.1% formic acid and an 80% acetonitrile solution. Mass spectrometry was performed in positive ion mode. Raw mass spectrometry data were processed and analyzed using PEAKSStudio 8.5 software (Bioinformatics Solutions Inc., Waterloo, Canada). The database used was the Magallanagigas protein database downloaded from Uniprot (Taxon ID: 29159). The secondary structure of the CGL-1 recombinant protein was analyzed using a circular dichroism spectroscopy (Chirascan Plus, Applied Photophysics, UK). The CGL-1 recombinant protein was dissolved in an appropriate buffer solution to prepare a sample solution of a specific concentration, which was then placed in a quartz cuvette for ultraviolet scanning. The raw data were processed using Pro-Data Viewer software, and the secondary structure of the sample was calculated using CDNN software.

[0055] Mass spectrometry analysis showed that the main mass-to-charge ratio (m / z) of the CGL-1 recombinant protein was concentrated at 13390.836 Da (13.39 kDa), which was highly consistent with the theoretical value (13.48 kDa), indicating that the expression and purification process of the CGL-1 recombinant protein did not damage its molecular structure. Figure 3 (A). LC / MS analysis after trypsin digestion showed that the purified CGL-1 recombinant protein had a high degree of matching with the theoretical peptides of the expected target protein (47 matching peptides, 68% coverage), indicating that its amino acid sequence was accurate and the purification effect was good, providing a reliable basis for subsequent functional studies. Figure 3 (B) Circular dichroism spectroscopy analysis showed that the CGL-1 recombinant protein had characteristic absorption peaks in the wavelength range of 180-260 nm. Calculations using CDNN software showed its secondary structure composition as follows: α-helix (3.3%-7.6%), β-sheet (45.4%-58.5%), β-turn (14.7%-19.3%), and random coil (25.2%-35.7%). The β-sheet structure had the highest proportion, indicating that the CGL-1 recombinant protein has a relatively abundant β-sheet structure, which may be closely related to its biological function. Figure 3 (C)

[0056] Example 4

[0057] Evaluation of hemolytic activity and cytotoxicity of recombinant CGL-1 protein

[0058] Different concentrations of CGL-1 recombinant protein solution were added to 96-well plates to achieve final concentrations of 256, 128, 64, 32, 16, 8, 4, and 2 μg / mL, with three replicates per group. PBS was used as a negative control, and 1.0% Triton X-100 as a positive control. An equal volume of 2% rabbit erythrocyte suspension was added to each well to ensure adequate contact between the erythrocytes and the recombinant protein. The 96-well plates were incubated at 37°C for 1 hour, followed by centrifugation at 1500 rpm for 10 minutes. The supernatant was carefully transferred to a new 96-well plate, and the absorbance was measured at 450 nm. The hemolysis rate was calculated using the formula: Hemolysis rate = (Experimental group OD) / (Obstacles of experimental group OD) / (Obstacles of experimental group OD). 450 - Negative control group OD 450 ) / (Positive control group OD 450 - Negative control group OD 450 )×100%.

[0059] The cytotoxicity of recombinant CGL-1 protein against HaCaT cells was detected using the CCK-8 assay. Logarithmic growth phase HaCaT cells were harvested and their concentration adjusted to 1 × 10⁶ cells / year using DMEM medium containing 1% penicillin / streptomycin and 10% fetal bovine serum (FBS). 4 Cells / mL. Add 200 μL of cell suspension to each well of a 96-well plate and incubate at 37°C and 5% CO2 for 24 hours to allow cell adhesion. After discarding the culture medium, add CGL-1 recombinant protein diluted with complete culture medium, setting final concentrations of 256, 128, 64, 32, 16, 8, 4, and 2 μg / mL. A blank control group containing only culture medium and a negative control group treated with PBS were also set up. After 24 and 48 hours of incubation, aspirate the culture medium, add 100 μL of serum-free DMEM medium to each well, followed by 10 μL of CGL-8 solution to each well, and continue incubation for 2 hours. Measure absorbance at 450 nm using a microplate reader (Synergy H1, BioTek, Winooski, VT, USA). Cell viability was calculated using the formula: [OD(drug group) - OD(blank)] / [OD(PBS control group) - OD(blank)] × 100%.

[0060] Hemolytic activity was assessed using 4% rabbit erythrocytes. Different concentrations of recombinant thymosin β4 protein were mixed with erythrocytes. PBS served as the negative control, and erythrocytes treated with 1.0% Triton X-100 served as the positive control. After incubation at 37°C for 1 hour, the mixture was centrifuged at 25°C and 1500 rpm for 10 minutes. The supernatant was collected, and the OD was measured. 450 The absorbance under the given conditions is calculated using the formula: [Experimental group OD] 450 -Negative control group] / [Positive control group OD 450The hemolysis rate of recombinant thymosin β4 protein was calculated by multiplying the negative control group by 100%. All experiments were repeated three times.

[0061] The hemolytic activity test results of the CGL-1 recombinant protein showed that it had certain hemolytic activity (42.23%) at a concentration of 256 μg / mL, while the hemolysis rate was less than 5% at different concentrations (128, 64, 32, 16, 8, 4, and 2 μg / mL). Figure 4 (A, B). This indicates that the CGL-1 recombinant protein may exhibit some hemolytic activity at higher concentrations (256 μg / mL), but shows good blood compatibility at lower concentrations. The toxicity assessment of CGL-1 recombinant protein on HaCaT cells showed that during incubation periods of 24 and 48 hours, cell viability was significantly inhibited and almost completely lost its growth ability at concentrations of 256, 128, and 64 μg / mL. However, at lower concentrations (32, 16, 8, 4, and 2 μg / mL), cell viability was not significantly affected, and there was no statistically significant difference compared to the negative control group. This indicates that the toxicity of CGL-1 recombinant protein to HaCaT cells is concentration-dependent, exhibiting strong cytotoxicity at high concentrations and good cell compatibility at low concentrations. Figure 4 (C, D)

[0062] Example 5

[0063] In vitro antibacterial activity of CGL-1 recombinant protein against MRSA

[0064] The antibacterial activity of recombinant CGL-1 protein was detected using the double-layer agar clear zone method. Single colonies of MRSA were picked and inoculated into LB liquid medium, and cultured at 37°C with shaking at 180 rpm for 16 hours to obtain a bacterial count of approximately 1.0 × 10⁻⁶. 6 LB agar medium containing CFU / mL. Add 100 μL of recombinant CGL-1 protein (0.50 mg / mL) to each well, and use PBS as a negative control. After pre-diffusion at 4°C for 3 hours, incubate at 37°C for 16 hours. Measure the diameter of the inhibition zone using calipers. Repeat the experiment three times.

[0065] The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of recombinant CGL-1 protein against MRSA were determined using the microbroth dilution method. 100 μL of 1.0 × 10⁻⁶ broth solution was added to each well of a 96-well plate. 6CFU / mL MRSA bacterial suspension was added, followed by 100 μL of CGL-1 recombinant protein to achieve final concentrations of 128, 64, 32, 16, 8, 4, 2, 1, and 0.5 μg / mL. Negative and blank controls were included, with three replicates per group. The 96-well plate was incubated at 37°C for 18 hours. Then, 1.5 μL of 1% 2,3,5-triphenyltetrazolium chloride (TTC) was added to each well, and the plate was incubated at 37°C for another 3 hours. The OD values ​​of each well were measured using a microplate reader. 485 The color of each well was observed. The minimum concentration at which no red color appeared was taken as the MIC. 20 μL of the clear well was taken and spread onto LB solid medium. The concentration at which no single colony grew or the number of colonies was ≤5 was taken as the MBC. The experiment was repeated 3 times.

[0066] The effect of recombinant CGL-1 protein on the growth curve of MRSA was determined using the microbroth dilution method. 100 μL of 1.0 × 10⁻⁶ protein was added to each well of a 96-well plate. 6 CFU / mL MRSA bacterial suspension was prepared, and 100 μL of CGL-1 recombinant protein was added to achieve final concentrations of 128, 64, and 32 μg / mL, respectively. A negative control group was also included, with three replicates per group. The 96-well plate was placed in a microplate reader and incubated at 37°C with shaking for 24 hours. OD was measured every 2 hours. 600 Plot the growth curve. The experiment was repeated 3 times.

[0067] CGL-1 recombinant protein (0.50 mg / mL) showed significant antibacterial activity against MRSA, with an inhibition zone diameter (22.60 ± 0.71 mm) significantly larger than that of the PBS negative control group (0 mm). Figure 5 (A). This result indicates that recombinant CGL-1 protein can effectively inhibit the growth of MRSA. The MIC value of recombinant CGL-1 protein against MRSA was 64 μg / mL and the MBC value was 128 μg / mL, determined by microbroth dilution and plate coating methods. Figure 5 (B, C, D). Growth curve analysis showed that at concentrations of 128, 64, and 32 μg / mL, recombinant CGL-1 protein significantly inhibited the growth of MRSA. Figure 5 (F) Among them, the 128 μg / mL treatment group almost completely inhibited bacterial proliferation within 24 hours, while the 64 μg / mL and 32 μg / mL treatment groups showed varying degrees of growth delay. The MRSA growth curve of the negative control group (PBS) conformed to a typical logarithmic growth trend. In summary, the CGL-1 recombinant protein has concentration-dependent antibacterial and bactericidal effects against MRSA, and its antibacterial activity is particularly significant at higher concentrations (≥32 μg / mL).

[0068] Example 6

[0069] Scanning electron microscopy and transmission electron microscopy observation

[0070] Prepare 1×10 8 CFU / mL MRSA bacterial suspension was mixed with CGL-1 recombinant protein to a final concentration of 1×MIC (64 μg / mL). PBS was used as a negative control. The suspension was incubated at 37°C with shaking at 100 rpm for 6 h. After centrifugation at 4°C and 4000 rpm for 5 min, the supernatant was discarded, and the cells were washed three times with sterile physiological saline. After centrifugation at 4000 rpm for 10 min, the cells were collected and fixed overnight in 2.5% glutaraldehyde at 4°C. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) samples were prepared for observation under both microscopes.

[0071] Scanning electron microscopy results showed that the MRSA bacteria (negative control) without CGL-1 treatment were morphologically intact and exhibited typical spherical or short chain aggregates. Figure 6 (A) The bacterial surface was smooth, and the cell wall membrane structure was continuous, without obvious damage or shrinkage, consistent with the morphological characteristics of normal MRSA. After treatment with CGL-1 recombinant protein (64, 32, and 16 μg / mL), the cells clumped together, and a large amount of cell contents leaked out. Most of the bacteria were in a lysed state, and some bacteria showed obvious cell membrane damage such as shrinkage and depression on their surface. Figure 6 (B, C, D). Similarly, transmission electron microscopy results showed that untreated MRSA bacteria (negative control) exhibited typical ultrastructural characteristics of Gram-positive bacteria, with a uniform and dense cell wall, a smooth and continuous outer surface, and the cell membrane tightly adhered to the cell wall without separation. The cytoplasmic electron density was uniform. Figure 6 (E). After treatment with CGL-1 recombinant protein, although the ultrastructure of MRSA did not undergo significant pathological changes, most bacterial cells showed complete lysis. Figure 6 (F). These results indicate that the CGL-1 recombinant protein can effectively disrupt the cell structure of MRSA bacteria, leading to leakage of cell contents and cell lysis, thereby exerting its antibacterial effect.

[0072] Example 7

[0073] DAPI / PI fluorescence staining observation

[0074] Prepare 1×10 8CFU / mL MRSA bacterial suspension was mixed with CGL-1 recombinant protein to a final concentration of 1×MIC (64 μg / mL). A PBS-treated group was set up as a negative control. After incubation at 37°C and 100 rpm for 6 hours, the bacterial cells were collected by centrifugation at 4°C and 4000 rpm for 5 minutes and washed three times with PBS. 200 μL DAPI (10 μg / mL) and 200 μL PI (10 μg / mL) were added, and the bacterial cells were gently resuspended. The mixture was incubated at 37°C in the dark for 30 minutes. After washing twice with PBS, the bacterial cells were resuspended in 400 μL PBS. 5 μL of the bacterial suspension was smeared and the staining was observed using a fluorescence microscope.

[0075] DAPI / PI fluorescence staining results showed that MRSA strains treated with recombinant CGL-1 protein (1×MIC, 64 μg / mL) exhibited significantly increased cell membrane permeability under a fluorescence microscope, with a large amount of PI dye entering the cells and displaying red fluorescence, indicating impaired cell membrane integrity and cell death. In contrast, the negative control group strains treated with PBS only showed blue fluorescence stained with DAPI, indicating intact cell membranes and viable cells. Figure 7 This result indicates that the CGL-1 recombinant protein can effectively disrupt the integrity of MRSA cell membranes, leading to cell death.

[0076] Example 8

[0077] Crystal violet staining was used to observe the effect of recombinant CGL-1 protein on MRSA biofilm formation.

[0078] Prepare 1.0 × 10⁻⁶ liters of glucose using LB medium (LB-G) containing a final glucose concentration of 0.5%. 8 MRSA bacterial suspension at CFU / mL. Add 100 μL of bacterial suspension to a 96-well plate, and add CGL-1 recombinant protein to achieve final concentrations of 64, 32, and 16 μg / mL, respectively. Set up a negative control group (LB-G medium containing bacteria with PBS) and a blank control group (LB-G medium only, without bacterial suspension and CGL-1 recombinant protein). Each group has three replicates. Incubate the 96-well plate at 37°C for 48 hours. After incubation, aspirate the bacterial suspension from each well, wash three times with PBS, fix with methanol for 20 minutes, dry at room temperature, and stain with 0.1% crystal violet at room temperature for 10 minutes. After staining, wash with sterile ultrapure water until clear, dry at 37°C, and photograph to observe biofilm adhesion. Subsequently, add 33% glacial acetic acid to each well, incubate at 37°C to dissolve the crystal violet, and measure the absorbance at 595 nm using a microplate reader. In the biofilm elimination experiment, MRSA was first pre-cultured to form a biofilm, and then treated with recombinant CGL-1 protein for 24 hours. A negative control group and a blank control group were set up to observe the elimination of the biofilm. The experiment was repeated 3 times.

[0079] Crystal violet staining results showed that recombinant CGL-1 protein significantly inhibited MRSA biofilm formation. At 64 μg / mL, biofilm formation was significantly reduced, and the staining color was significantly lighter than that of the negative control group. While biofilm formation increased slightly as the protein concentration decreased to 32 and 16 μg / mL, it remained significantly lower than that of the control group. Figure 8 (A) OD 595 Measurements of the OD values ​​further confirmed this inhibitory effect; the OD value at 64 μg / mL was significantly lower than that of the control group, and significant inhibition was also observed at 32 and 16 μg / mL. Figure 8 (B) indicates that the inhibitory effect is concentration-dependent. In the biofilm elimination experiment, after 24 hours of treatment with recombinant CGL-1 protein, the pre-formed biofilm was significantly reduced, and the crystal violet staining color became significantly lighter, confirming that recombinant CGL-1 protein has an effective elimination effect on existing biofilms. Figure 8 (C, D)

[0080] Example 9

[0081] The effect of CGL-1 recombinant protein on MRSA inclusion leakage and ROS generation

[0082] Pick a single colony of MRSA and inoculate it into LB liquid medium. Incubate overnight at 37°C with shaking at 180 rpm. Centrifuge at 4°C and 4000 rpm for 5 minutes to collect the bacterial cells. Wash the bacterial cells twice with PBS and adjust the bacterial concentration to 1×10⁻⁶ with PBS. 6 CFU / mL. CGL-1 recombinant protein was added to the bacterial culture to final concentrations of 128, 64, and 32 μg / mL, respectively. A negative control group (PBS only) and a blank group (no bacterial culture or CGL-1 recombinant protein) were also set up. All groups were mixed thoroughly and incubated at 37°C and 100 rpm for 6 hours. The supernatant was collected by centrifugation at 4°C and 4000 rpm for 5 minutes. The absorbance (OD) of the supernatant at 260 nm and 280 nm was measured using a UV-Vis spectrophotometer. 260 and OD 280 The experiment was repeated 3 times.

[0083] Adjust the bacterial concentration to 1×10 8CFU / mL. CGL-1 recombinant protein was added to the bacterial suspension to final concentrations of 128, 64, and 32 μg / mL, respectively. A negative control group (bacterial suspension with PBS) and a blank group (containing only blank medium) were set up. All groups were mixed thoroughly and incubated at 37°C and 100 rpm for 1 hour. After incubation, the bacterial cells were washed twice with PBS. ROS detection was performed using a ROS detection kit (S0033S, Beyotime, Shanghai, China). The obtained bacterial cells were resuspended in PBS, and the DCFH-DA probe was added to a final concentration of 10 μM. The cells were incubated at 37°C in the dark for 30 minutes. The bacterial cells were washed twice with PBS to remove unbound probe. Finally, the bacterial cells were resuspended again with PBS, and the resulting bacterial suspension was added to 96-well plates, with 3 replicates per group. Fluorescence intensity was measured using a microplate reader at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. The experiment was repeated 3 times.

[0084] UV spectrophotometer measurements showed that treatment with 128 μg / mL CGL-1 recombinant protein significantly increased the OD260 value of MRSA, indicating a significant increase in nucleic acid leakage. As the CGL-1 concentration decreased to 64 μg / mL and 32 μg / mL, the OD260 value decreased further. 260 The value gradually decreased, but remained higher than the control group, indicating that the degree of nucleic acid leakage had decreased but still existed. Figure 9 (A). This result indicates that the CGL-1 recombinant protein can effectively promote nucleic acid leakage of MRSA, and this effect is concentration-dependent. The effect of the CGL-1 recombinant protein on MRSA protein leakage is shown in Figure A. Figure 9 As shown in Figure B, the OD of MRSA was [data missing] under treatment with 128 μg / mL CGL-1 recombinant protein. 280 The significantly increased OD280 value indicates a significant increase in protein leakage. As the CGL-1 concentration decreased to 64 μg / mL and 32 μg / mL, the OD280 value gradually decreased, but remained higher than the control group, indicating that protein leakage was reduced but still present. This result further confirms the promoting effect of recombinant CGL-1 protein on MRSA protein leakage, and this effect is also concentration-dependent. The effect of recombinant CGL-1 protein on MRSA reactive oxygen species generation is shown in the figure below. Figure 9 As shown in Figure C, the fluorescence intensity significantly increased with increasing CGL-1 concentration, reaching peak values ​​at 64 μg / mL and 32 μg / mL. This indicates that recombinant CGL-1 protein can effectively induce reactive oxygen species (ROS) production in MRSA. Compared with the control group, the blank group showed the lowest fluorescence intensity, validating the specificity and accuracy of the experimental results. This result demonstrates that recombinant CGL-1 protein can significantly enhance ROS generation in MRSA, further supporting its potential antibacterial activity.

[0085] Example 10

[0086] Antioxidant activity detection

[0087] The in vitro antioxidant activity of CGL-1 recombinant protein was assessed using a DPPH radical scavenging activity assay kit, an ABTS radical scavenging ability assay kit (micro-method), and ferric reducing power. CGL-1 recombinant protein was diluted to final concentrations of 256, 128, 64, 32, 16, 8, 4, and 2 μg / mL and added to 96-well plates. A blank control group (solvent added only) and a negative control group (no CGL-1 recombinant protein added) were set up. Following the kit instructions, DPPH or ABTS radical scavenging solution was added to each well, mixed thoroughly, and the 96-well plate was incubated at room temperature in the dark for 30 minutes. The absorbance of each well was measured using a microplate reader at 517 nm or 405 nm. The DPPH and ABTS radical scavenging rates were calculated using the following formula: (1 - (A assay - A control) ÷ A blank) × 100%. In the iron reducing power assay, 0.5 mL of CGL-1 recombinant protein at different concentrations, vitamin C solution, 0.2 mol / L PBS buffer (pH 6.6, as a negative control), and deionized water (as a blank group) were first mixed thoroughly with 1.25 mL of 1% potassium cyanide solution. The mixtures were then incubated at 50°C for 20 minutes. After incubation, 1.25 mL of 10% trichloroacetic acid solution was quickly added and thoroughly mixed to terminate the reaction. The mixtures were then centrifuged at 3000 rpm for 10 minutes, and the supernatant was collected. 1.25 mL of the supernatant was added to 1.25 mL of deionized water and 0.25 mL of 1% ferric chloride solution, mixed thoroughly, and allowed to stand at room temperature for 10 minutes. Finally, the absorbance of each solution was measured at 700 nm to characterize the iron reducing power of the samples. The experiment was repeated three times.

[0088] The results showed that the DPPH radical scavenging rate significantly increased with increasing CGL-1 concentration. Figure 10 Similarly, in the positive control vitamin C, the DPPH free radical scavenging rate gradually increased with increasing concentration, reaching the maximum inhibition level at ≥16 μg / mL. Figure 10 (B) Based on calculations of the DPPH free radical scavenging experiment, the EC50 of CGL-1 and vitamin C was determined. 50 The concentrations were 71.94 μg / mL (0.53 μM) and 3.97 μg / mL (22.54 μM), respectively. With increasing CGL-1 concentration, the ABTS radical scavenging rate significantly increased, reaching a maximum inhibition level at 32 μg / mL, and then no longer increased with further concentration. Figure 10 In the positive control group, vitamin C reached its maximum inhibitory level at 4 μg / mL. Figure 10In the study of ABTS free radical scavenging experiments, the EC50 of CGL-1 and vitamin C was calculated. 50 The values ​​were 7.77 μg / mL (0.57 μM) and 1.82 μg / mL (10.33 μM), respectively. Iron reducing power assay results showed that the CGL-1 recombinant protein exhibited significant iron reducing power at concentrations of 64 μg / mL and above, which increased with increasing concentration. At the highest concentration of 256 μg / mL, its absorbance reached 1.16 ± 0.05, significantly higher than the negative control group (0.647 ± 0.01) (P < 0.05). The positive control vitamin C also exhibited significant iron reducing power at concentrations of 16 μg / mL and above, which increased with increasing concentration. At the highest concentration of 256 μg / mL, its absorbance reached 3.43 ± 0.14, significantly higher than the negative control group (0.57 ± 0.0036) (P < 0.05). Figure 10 The absorbance of CGL-1 at its highest concentration (E, F) was 18.99 μM, while the absorbance of vitamin C at this concentration was 3.34 μg, with a value of approximately 0.58 ± 0.01, significantly lower than that of CGL-1 (P < 0.05). In conclusion, the recombinant CGL-1 protein exhibits significant antioxidant activity in a concentration-dependent manner.

[0089] Example 11

[0090] Observation on the effect of CGL-1 on promoting the healing of MRSA infected wounds in diabetic rats

[0091] The experimental animals were male Sprague-Dawley (SD) rats, 4-5 weeks old, weighing 170-200 grams, purchased from Hunan Silek Jingda Experimental Animal Co., Ltd. The rats were housed in an environment with a temperature of 22-25℃, humidity of 50%-60%, and 12 hours of light / 12 hours of darkness, with free access to standard experimental animal feed and water. The experimental protocol was approved by the Animal Ethics Committee of Guangxi University of Traditional Chinese Medicine (Approval No.: DW20240919-191; Approval Date: September 19, 2024) and strictly followed the national guidelines for the management of experimental animals. The diabetic model was induced by a single intraperitoneal injection of streptozotocin (STZ, dose 65 mg / kg body weight). After injection, fasting blood glucose levels were measured continuously for 7 days using a blood glucose meter (Accu-Chek, Roche Diagnostics, USA). Rats with blood glucose levels exceeding 16.7 mmol / L were included in the experiment. After isoflurane inhalation anesthesia, the rats were fixed in a prone position on the operating table. Hair was removed from the backs of rats using an electric hair removal device. Depilatory cream was applied, and after 6 minutes, it was scraped off. Any residue was wiped away with warm water and then dried with gauze. The area was then disinfected with povidone-iodine, and a surgical drape was laid. Two circular, full-thickness skin defects, each 2 x 2 cm in diameter, were created 2 cm to each side of the spine. Two wounds were prepared for each rat. Immediately, 100 μL of a 1.0 x 10⁻⁶ solution was administered to each wound. 8 Methicillin-resistant Staphylococcus aureus (MRSA) at CFU / mL was evenly applied using a sterile, yellow pipette tip sterilized by flaming, without covering with a dressing to simulate a chronic wound environment. Rats were randomly divided into six groups (n=6 per group): normal wound (treated with PBS); diabetic wound (treated with PBS); diabetic wound (treated with Kangfuxin solution); diabetic wound (treated with 16 μg / mL CGL-1); diabetic wound (treated with 32 μg / mL CGL-1); and diabetic wound (treated with 64 μg / mL CGL-1). Twenty-four hours after infection, 100 μL of the corresponding drug was evenly applied to the wound once daily, with free access to food and water. Wounds were photographed on days 0, 3, 5, 7, 9, 11, and 13. The wound area was calculated using ImageJ image analysis software, and the wound healing rate was calculated using the following formula: Wound healing rate (%) = (Original wound area - Area at each time point) ÷ Original wound area × 100. Wound healing curves were plotted. Rats' body weight and blood glucose levels were measured daily, and their changes were plotted to assess diabetic status and the impact of treatment on overall health.

[0092] On days 5 and 13 after modeling and drug administration, two rats from each group were harvested, with a total of four wounds. Superficial tissue (including scabs) was excised from the wounds on a sterile laminar flow hood, weighed, and recorded. 1 mL of physiological saline was added, and the sample was homogenized using a 2 mL sterile glass homogenizer. The resulting homogenate was diluted tenfold. For the normal control group, 10... -3 10 -4 10 -5 Three gradients were used for plating, with 10 samples taken from the model group, positive group, and CGL-1 group. -5 10 -6 10 -7 Three gradients were used for plating. 100 μL of each gradient was spread onto mannitol-sodium chloride agar plates and incubated upside down at 37°C for 18 hours. The colony count was observed, and the gram-forming units (CFU) per gram of tissue were calculated. CFU / g = Colony count × Dilution factor × 10 ÷ Tissue weight (g).

[0093] ImageJ was used to monitor and quantify the effects of postoperative drug administration on days 0, 3, 5, 7, 9, 11, and 13. Figure 11 In the study (B), statistical results showed that 13 days after surgery, the wound healing rates in the normal group, KFX-positive group, and the CGL-1 recombinant protein treatment groups (16 μg / mL, 32 μg / mL, and 64 μg / mL) were 92.93% ± 3.35%, 92.91% ± 0.95%, 92.44% ± 2.8%, 95.15% ± 1.97%, and 94.51% ± 1.76%, respectively. In contrast, the wound healing rate in the model group was only 68.07% ± 6.67% (P < 0.05). Regarding weight change... Figure 11 In the normal group (C), the body weight of the rats gradually increased, reaching 363.67±11.06g at day 13, while the body weight of all diabetic rats remained essentially unchanged, ranging from 179.00±6.56g to 203.33±2.08g, significantly lower than that of the normal group (P<0.05). Changes in non-fasting blood glucose levels ( Figure 11 The results of the study (D) showed that the blood glucose levels in the normal group rats remained stable and normal, with a blood glucose concentration of 8.27±0.32 mM at day 13. In contrast, the blood glucose levels in the diabetic rats were significantly elevated, ranging from 24.50±0.53 mM to 26.97±1.72 mM at day 13, all exceeding 16.7 mM. The results of wound bacterial load detection showed (…). Figure 11 In the middle E, F), on day 5, the MRSA content in the wound of the model group rats was 1.0×10 7The CFU / g level was 3.32 ± 0.24. The MRSA content in the wounds of the normal group, KFX-positive group, and the CGL-1 recombinant protein treatment groups (16 μg / mL, 32 μg / mL, and 64 μg / mL) was 1.0 × 10⁻⁶. 7 The CFU / g levels in the model group rats were 0.18±0.01, 1.41±0.17, 2.48±0.40, 0.76±0.38, and 0.38±0.08, respectively, significantly lower than those in the model group (P<0.05). Similarly, at day 13, the MRSA content in the wound of the model group rats (1.0×10⁻⁶) was significantly lower than that in the model group rats. 7 The CFU / g level was 1.68 ± 0.76, while the MRSA content in the wound of the normal group, the KFX-positive group, and the CGL-1 recombinant protein treatment groups (16 μg / mL, 32 μg / mL, and 64 μg / mL) was 1.0 × 10⁻⁶. 7 The CFU / g concentrations were 0.07±0.01, 0.26±0.12, 0.53±0.23, 0.40±0.05, and 0.07±0.07, respectively, all significantly lower than the model group (P<0.05). Furthermore, the number of bacterial colonies in the wound treated with CGL-1 gradually decreased with increasing CGL-1 concentration, especially in the 64 μg / mL CGL-1 treatment group, where the number of bacterial colonies was significantly lower than the model group and approached the level of the normal group. These results indicate that CGL-1 can effectively reduce the bacterial load in MRSA-infected wounds of diabetic rats, thereby significantly promoting wound healing.

[0094] Example 12

[0095] Tissue staining observation

[0096] On day 13 after modeling and drug administration, two rats from each group were treated with four wounds. Wound tissue, including 0.5 cm of normal skin around the wound, was collected. The tissue was immediately fixed in 4% paraformaldehyde solution, dehydrated, cleared, and embedded in paraffin after 24 hours. The paraffin-embedded tissue blocks were cut into 5 μm thick sections using a microtome (Leica RM2245, Leica Microsystems, Germany). The sections were placed on glass slides and dried overnight at 37°C. The paraffin sections were dewaxed with xylene and rehydrated to distilled water by decreasing the ethanol concentration (100%, 95%, 90%, 80%, 70%). HE staining: sections were stained with hematoxylin (Sigma-Aldrich) for 5 minutes, differentiated with 1% acid ethanol for 30 seconds, blued with Scott's tap water substitute (Sigma-Aldrich) for 2 minutes, and then stained with eosin (Sigma-Aldrich) for 2 minutes. Sections were dehydrated using an increasing series of ethanol solutions (70%, 80%, 90%, 95%, 100%), cleared with xylene, and mounted with DPX mounting medium (Sigma-Aldrich). Masson staining: Sections were dewaxed with xylene and then rehydrated to distilled water using a decreasing series of ethanol solutions (100%, 95%, 90%, 80%, 70%). Sections were then stained with Weigert iron hematoxylin staining solution (Sigma-Aldrich) for 10 minutes, rinsed with running water for 5 minutes, differentiated with 1% acidic ethanol for 1 minute, rinsed with running water for 5 minutes, and then blued with Scott's tap water substitute (Sigma-Aldrich) for 5 minutes, rinsed with running water for 5 minutes. The sections were then immersed in solution B (Masson's Trichrome Staining Solution B, Sigma-Aldrich) for 10 minutes, rinsed with running water for 5 minutes, and then immersed in solution C (Masson's Trichrome Staining Solution C, Sigma-Aldrich) for 10 minutes, rinsed with running water for 5 minutes. Finally, the sections were dehydrated with an increasing series of ethanol solutions (70%, 80%, 90%, 95%, 100%), cleared with xylene, and mounted with DPX mounting medium (Sigma-Aldrich). Subsequently, the tissue sections were scanned under white light using a microscope slide scanner (Pannoramic MIDI II, 3DHISTECH Ltd., Budapest, Hungary) to obtain high-resolution panoramic images.

[0097] On day 13 after drug administration to establish the model, histological H&E staining was performed on the wound tissues of rats in each group. The results showed that the normal group ( Figure 12 In group A), the intact epidermis and dermis structure were observed, with good wound healing, minimal inflammatory cell infiltration, abundant collagen fibers, and a large amount of neovascularization and adipose tissue. In group A (…), the wound showed good healing, minimal inflammatory cell infiltration, abundant collagen fibers, and significant neovascularization and adipose tissue. Figure 12Group B showed a significant inflammatory response, with disordered epidermal and dermal structures, extensive infiltration of inflammatory cells, fewer collagen fibers, and poor wound healing. Group KFX ( Figure 12 (C) and different concentrations of CGL-1 recombinant protein treatment groups ( Figure 12 All groups (D-F) showed varying degrees of inflammation reduction and tissue structure improvement. Among them, the 16 μg / mL CGL-1 treatment group ( Figure 12 The treatment groups (D) and 32 μg / mL CGL-1 ( Figure 12 The wound healing in the E group was better than that in the model group, with reduced inflammatory cell infiltration and the beginning of tissue structure recovery. The 64 μg / mL CGL-1 treatment group ( Figure 12 The wound healing effect of the middle F) is the most significant, with less inflammatory cell infiltration, more collagen fibers, and a large amount of new blood vessels and adipose tissue, showing the best healing effect. Figure 12 The figure shows the quantitative results of wound diameter in each group. Compared with the normal group (5223.20±197.31 μm), the wound diameter in the model group (6835.78±332.21 μm) was significantly increased (P<0.05). The wound diameters in the KFX group and the CGL-1 treatment groups at various concentrations were 4359.53±2.44 μm, 5752.50±51.65 μm, 4291.10±198.32 μm, and 2726.88±202.41 μm, respectively, all significantly smaller than those in the model group (P<0.05). Among them, the 64 μg / mL CGL-1 treatment group had the smallest wound diameter, showing the best healing effect. These results indicate that recombinant CGL-1 protein can effectively promote the healing of MRSA-infected wounds in diabetic rats, and its effect is positively correlated with concentration.

[0098] Masson staining of wound tissue from normal rats showed intact tissue structure and abundant collagen fibers (blue), indicating good collagen deposition during normal wound healing. Figure 13 (A) In the model group rats, the collagen fibers (blue) in the wound tissue were significantly reduced, and the tissue structure was disordered, indicating that collagen deposition was reduced during the healing process of MRSA infected wounds in diabetic rats. Figure 13 (B) Compared with the model group, the KFX-treated rat wound tissue showed an increase in collagen fibers (blue) and improved tissue structure, indicating that KFX has a significant promoting effect on wound healing. Figure 13Masson staining of rat wound tissues treated with different concentrations of recombinant CGL-1 protein showed that with increasing CGL-1 concentration, collagen fiber (blue) deposition gradually increased, and tissue structure gradually improved. In particular, the 32 and 64 μg / mL CGL-1 treatment groups showed the richest collagen fiber (blue) deposition and the most intact tissue structure, demonstrating the best healing effect. Statistical analysis of the collagen volume fraction in the wounds of different treatment groups showed that the model group had the lowest collagen volume fraction (19.62±9.33%), significantly lower than the other groups. The collagen volume fractions in the normal group, KFX group, and 16 μg / mL CGL-1 treatment group were 44.16±7.85%, 54.36±12.51%, and 45.45±2.89%, respectively, but there were no significant differences among them. Among them, the collagen volume fractions of the 32 and 64 μg / mL CGL-1 treatment groups were the highest, at 84.40±1.09% and 78.20±3.21%, respectively, indicating that higher concentrations of recombinant CGL-1 protein can effectively promote collagen deposition in MRSA infected wounds of diabetic rats, thereby improving wound healing.

[0099] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. The use of CGL-1 lectin protein in the preparation of drugs for the prevention and / or treatment of methicillin-resistant Staphylococcus aureus infections, characterized in that, The amino acid sequence of the CGL-1 lectin protein is shown in SEQ ID NO.

2.

2. The application of CGL-1 lectin protein in the preparation of drugs that promote the healing of methicillin-resistant Staphylococcus aureus (MRSA) infected wounds in diabetic patients, characterized in that... The amino acid sequence of the CGL-1 lectin protein is shown in SEQ ID NO.2.

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