Pacific oyster galactose / rhamnose binding lectin CGL-1 recombinant protein as well as preparation method and application thereof

Abstract

The invention discloses pacific oyster galactose / rhamnose binding lectin CGL-1 recombinant protein as well as 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%-94.51%. Histological analysis shows that inflammatory cell infiltration of a wound surface of a CGL-1 treatment group is reduced, collagen deposition is increased, neovascularization is good, and a good healing effect is shown. In addition, the bacterial quantity of a wound surface of a CGL-1 treatment group is obviously lower than that of a model group, and the bacterial quantity is respectively reduced by 25.3%-88.55% and 68.45%-95.83% on the fifth day and the thirteenth day. The results show that the CGL-1 lectin protein has the potential of serving as a novel non-antibiotic therapeutic agent, can be used for treating diabetes infected wounds, and provides a new thought for application of marine biological resources in the field of wound healing.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a Pacific oyster ga lactose / rhamnose binding lectin CGL-1 recombinant protein and a preparation method and application thereof. BACKGROUND

[0002] Diabetes mellitus is a group of chronic metabolic diseases caused by insulin secretion defects (such as type 1 beta cell autoimmune destruction) or dysfunction (such as type 2 insulin resistance), the core pathological manifestations of which are persistent hyperglycemia, and the causes of which cover multi-dimensional interactions of genetic susceptibility, environmental factors and lifestyle, etc. Long-term blood glucose uncontrolled leads to systemic microvascular (retinopathy, nephropathy, neuropathy) and macrovascular (coronary heart disease, stroke, peripheral arterial disease) complications, significantly increases the risk of disability (amputation, blindness) and death, and has become one of the major challenges in the field of global public health. Among these complications, diabetic wounds are particularly common, and the healing process is slow and difficult, with a very high recurrence rate. Such wounds often lead to non-healing ulcers, and may even cause amputation, and in severe cases, even endanger the patient's life, causing a very serious negative impact on the patient's quality of life. Delayed wound healing in diabetic patients is the result of the combined action of multiple factors, including oxidative stress caused by high blood sugar, chronic inflammation, impaired angiogenesis, neuropathy, delayed reepithelialization, and metabolic disorders, etc. These factors interact with each other, causing the wound healing process to be blocked, increasing the risk of chronic wounds and infections. Due to the impaired immune function of diabetic patients, poor local blood circulation, and the high blood sugar environment providing a good breeding ground for bacteria, the incidence of diabetic wound infection is significantly higher than that of the general population. Infection not only aggravates the inflammatory response of the wound, but also can trigger a series of serious consequences, further hindering wound healing. Therefore, the prevention and treatment of diabetic wound infection is a key link to improve the prognosis of patients and reduce the risk of amputation.

[0003] Diabetic wound infection is a serious problem, not only exacerbating local inflammatory response, leading to tissue necrosis, ulcer expansion, and prolonged healing time, but also causing systemic inflammatory response syndrome, sepsis, and even significantly increasing the risk of amputation. In the early stage of diabetic wound infection, the main pathogenic bacteria are Staphylococcus (such as Staphylococcus aureus) and Streptococcus (such as beta-hemolytic streptococcus). However, as the depth and severity of infection increase, the pathogen spectrum of wound infection changes significantly: gram-negative bacteria (such as Pseudomonas aeruginosa) infection gradually increases; mixed infection becomes increasingly complex, such as the mixed infection of Staphylococcus aureus and Pseudomonas aeruginosa, which is common in severe wounds; anaerobic bacteria infection begins to appear; the risk of fungal infection also increases significantly. In addition, as the infection becomes more complex, the frequency of drug-resistant strains (such as methicillin-resistant Staphylococcus aureus, MRSA) is rising. These drug-resistant strains are resistant to commonly used antibiotics and can form biofilms on wounds, protecting themselves from antibiotic attack, further increasing the difficulty of diabetic wound treatment and medical costs.

[0004] The main pathological feature of diabetic infected wounds is a significantly prolonged inflammatory phase, which is closely related to the imbalance of inflammatory factors, impaired immune function, blocked cell proliferation, impaired angiogenesis, reduced collagen synthesis, and delayed epithelialization. These pathological changes interact with each other, disrupting the homeostasis of the wound microenvironment, leading to persistent wound infection, drug-resistant bacteria forming refractory biofilms, and the imbalance of inflammatory factors and cell dysfunction leading to excessive accumulation of reactive oxygen species, damaging cell structure and function, further exacerbating wound damage, and delaying wound healing. The current main clinical treatment strategy for diabetic infected wounds relies on single independent methods, which may be both expensive and limited in effect. For example, antibiotic therapy, which may lead to increased drug resistance with long-term use, and mainly through systemic administration, making it difficult to achieve an effective concentration locally in the wound. Debridement treatment may cause damage to surrounding healthy tissues, and the wound may still face adverse factors such as high blood sugar and infection after debridement, leading to slow healing. Growth factor therapy also has certain limitations, and the efficacy may be unstable due to individual differences and different wound microenvironments, and the price is relatively high. Therefore, it is particularly urgent to develop multifunctional non-antibiotic therapeutic agents that can regulate the infected microenvironment, achieve efficient sterilization, and accelerate the healing of diabetic wounds.

[0005] In recent years, with the increasing problem of antibiotic resistance, bioactive proteins as a new type of non-antibiotic therapeutic agent have gradually attracted attention. Bioactive proteins have multiple biological functions and can play an important role in regulating the microenvironment of infection, and are expected to become an effective way to solve the treatment problem of diabetic infected wounds. Among the many bioactive proteins, lectins have become one of the research hotspots due to their unique sugar recognition ability and immune regulation function. Lectins can specifically recognize and bind to glycosyl, and play an antibacterial and immune regulation role by interacting with the glycosyl on the surface of pathogens, thus showing great potential in the treatment of diabetic infected wounds. For example, Dos Santos Silva L et al. found that Cratylia mollis lectin (Cramoll) could significantly improve the diabetic wound infected with multi-drug resistant Staphylococcus aureus. It could promote wound healing by reducing inflammatory cells, increasing revascularization and collagen production, reducing bacterial load and inflammatory factor levels, and activating the ERK pathway, thus showing the potential as a healing agent. Nunes MAS et al. found that Schinus terebinthifolius leaf lectin (SteLL) could effectively reduce the number of bacteria in the wound, reduce the infiltration of inflammatory cells, and promote re-epithelialization and collagen synthesis in the wound, thus accelerating the wound healing process and having potential clinical application value in the treatment of infected wounds.

[0006] Marine invertebrates, as an important part of biodiversity on earth, contain abundant resources of lectins. These lectins not only play a key role in physiological processes such as immune defense, anti-inflammatory and antibacterial of marine invertebrates, but also provide valuable resources for biomedical research and application due to their unique structural and functional characteristics. For example, Liu Peng et al. reported a galactose-binding lectin (PFL-96) from Pinctada chemasiensis. The recombinant protein expressed in E. coli showed significant antibacterial activity against Bacillus subtilis, Staphylococcus aureus, Candida albicans and Vibrio alginolyticus, and also showed significant inhibitory effect on 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 Limulus polyphemus, which could inhibit the growth of gram-negative bacteria (Vibrio parahaemolyticus, Escherichia coli) and gram-positive bacteria. Fujii Y et al. isolated a 15kDa lectin (SeviL) from Mytilisepta virgata, which was found to have anti-tumor activity. Although the research on lectins from plants in the field of infectious wound repair has been reported, the research on lectins from marine invertebrates in this field is relatively less, and its potential application value still needs to be further explored. SUMMARY

[0007] The purpose of the present application is to provide a galactose / muramyl binding lectin CGL-1 from Crassostrea gigas and its preparation method and application, so as to solve the problems existing in the prior art. The present application realizes the efficient recombinant expression of the protein in Escherichia coli, and further explores its various biological activities, including antibacterial, antioxidant and the ability to promote the healing of infectious wounds in type I diabetes.

[0008] To achieve the above purpose, the present application provides the following solutions:

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

[0010] The second technical solution of the present application 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 application is the use of the CGL-1 lectin protein in the preparation of a drug for preventing and / or treating methicillin-resistant Staphylococcus aureus infection.

[0012] The CGL-1 lectin protein is used for preparing a medicine for promoting the healing of a methicillin-resistant Staphylococcus aureus infected wound of a diabetic patient.

[0013] The CGL-1 lectin protein is used for preparing a medicine for preventing and / or treating a methicillin-resistant Staphylococcus aureus infection.

[0014] The CGL-1 lectin protein is used for preparing a medicine for promoting the healing of a methicillin-resistant Staphylococcus aureus infected wound of a diabetic patient.

[0015] Based on the above technical solutions, the present application has the following technical effects:

[0016] The present application first successfully prepared the CGL-1 lectin protein from Magallana gigas by using an Escherichia coli recombinant expression system, and systematically evaluated the antibacterial, antioxidant activities and the repair effect on an infected wound of a diabetic rat. In vitro experiments showed that the CGL-1 recombinant protein had 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 observation showed that the CGL-1 recombinant protein could destroy the cell structure of MRSA bacteria, induce the generation of reactive oxygen species (ROS), inhibit the formation of biofilm and promote the elimination of mature biofilm. In addition, the CGL-1 recombinant protein also showed good antioxidant capacity, with a half maximal effective concentration (EC 50 ) of 71.94 μg / mL and 7.77 μg / mL for scavenging DPPH free radicals and ABTS free radicals, respectively. In a diabetic rat model, the CGL-1 recombinant protein could significantly promote the healing of an MRSA infected wound, with a healing rate of 92.44%-94.51%, which was significantly higher than that of the model group (68.07%). Histological analysis showed that the inflammatory cell infiltration of the wound in the CGL-1 treatment group was reduced, the collagen deposition was increased, and the new blood vessel formation was good, showing a good healing effect. In addition, the bacterial amount of the wound in the CGL-1 treatment group was significantly lower than that of the model group, which was reduced by 25.3%-88.55% and 68.45%-95.83% at the 5th day and the 13th day, respectively. These results showed that the CGL-1 lectin protein had the potential to be used as a new type of non-antibiotic therapeutic agent, and could be used for the treatment of diabetic infected wounds, providing a new idea for the application of marine biological resources in the field of wound healing. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1Figures for gene cloning, expression vector construction and bioinformatics analysis of CGL-1 lectin protein from Crassostrea gigas. (A) Original sequence and codon-optimized sequence of CGL-1 lectin gene; (B) Amino acid sequence of CGL-1 lectin protein, in which cysteine forming disulfide bond is indicated by red color; (C) Schematic diagram of pET-30a-CGL-1 recombinant plasmid; (D) Double enzyme digestion verification results of pET-30a-CGL-1 recombinant plasmid; (E) Secondary structure prediction map of CGL-1 lectin protein; (F) Distribution map of conserved domains of CGL-1 lectin protein; (G) Physicochemical property parameters of CGL-1 lectin protein; (H) Prediction map of tertiary structure of CGL-1 lectin protein and four pairs of disulfide bond display.

[0018] Figure 2 Figures for expression and purification of CGL-1 lectin recombinant protein from Crassostrea gigas. (A) Expression analysis of CGL-1 recombinant protein under different temperature induction conditions. M: protein molecular weight marker; 1: uninduced culture for 16 h at 15°C; 2: induced culture for 16 h at 15°C; 3: induced culture for 16 h at 37°C; 4: supernatant collected after whole bacteria disruption after induced for 16 h at 15°C; 5: precipitate collected after whole bacteria disruption after induced for 16 h at 15°C; (B) Expression form analysis of CGL-1 recombinant protein under 37°C induction condition. M: protein molecular weight marker; 1: precipitate collected after whole bacteria disruption after induced for 16 h at 37°C; 2: supernatant collected after whole bacteria disruption after induced for 16 h at 37°C; (C) Inclusion body treatment and purification of CGL-1 recombinant protein. M: protein molecular weight marker; 1-5: elution components of 500 mM imidazole; 6: elution component of 50 mM imidazole; 7: flow-through liquid after incubation of supernatant collected after centrifugation after inclusion body dissolution with Ni-IDA; 8: supernatant collected after centrifugation after inclusion body dissolution; (D) SDS-PAGE quality inspection results of CGL-1 recombinant protein after purification. M: protein molecular weight marker; 1: BSA (1.00 μg); 2: CGL-1 recombinant protein (1.0 μg).

[0019] Figure 3 Figures for molecular weight, mass spectrometry and circular dichroism spectrum analysis of CGL-1 lectin recombinant protein from Crassostrea gigas. (A) Mass spectrometry graph of CGL-1 recombinant protein; (B) LC / MS analysis results of CGL-1 recombinant protein; (C) Circular dichroism spectrum graph of CGL-1 recombinant protein.

[0020] Figure 4Hemolytic activity and cytotoxicity analysis of CGL-1 lectin recombinant protein from Crassostrea gigas. Among them, (A) hemolytic activity analysis of CGL-1 recombinant protein; (B) quantitative analysis of hemolysis rate of CGL-1 recombinant protein; (C) toxicity evaluation of CGL-1 recombinant protein on HaCaT cells (24 hours); (D) toxicity evaluation of CGL-1 recombinant protein on HaCaT cells (48 hours). In the figure, "*" indicates that the difference between the 256, 128, 64 μg / mL treatment groups and the negative control group (CGL-1 recombinant protein concentration is 0 μg / mL) is significant (P<0.05); "ns" indicates that the difference between the 32, 16, 8, 4, 2 μg / mL treatment groups and the negative control group is not significant (P>0.05).

[0021] Figure 5 Antibacterial activity analysis of CGL-1 lectin recombinant protein from Crassostrea gigas on MRSA in vitro. Among them, (A) inhibition zone determination of CGL-1 recombinant protein on MRSA (PBS as negative control); (B) quantitative analysis of inhibition zone diameter of CGL-1 recombinant protein on MRSA; (C) MIC evaluation of CGL-1 recombinant protein on MRSA by TTC color reaction; (D) OD485 quantitative analysis of MRSA after treated by CGL-1 recombinant protein; (E) MBC determination of CGL-1 recombinant protein on MRSA; (F) effect of CGL-1 recombinant protein on growth curve of MRSA. In the figure, "*" indicates that the difference between the CGL-1 recombinant protein treatment group and the negative control group (CGL-1 recombinant protein concentration is 0 μg / mL) is significant (P<0.05); "ns" indicates that the difference between the treatment group and the negative control group is not significant (P>0.05).

[0022] Figure 6 Effect of CGL-1 lectin recombinant protein from Crassostrea gigas on the morphology and structure of MRSA bacteria. Among them, (A) scanning electron microscope image of MRSA bacteria in the control group; (B) scanning electron microscope image of MRSA bacteria after treated by 64 μg / mL CGL-1; (C) scanning electron microscope image of MRSA bacteria after treated by 32 μg / mL CGL-1; (D) scanning electron microscope image of MRSA bacteria after treated by 16 μg / mL CGL-1. (E) transmission electron microscope image of MRSA bacteria in the control group; (F) transmission electron microscope image of MRSA bacteria after treated by 64 μg / mL CGL-1. The red arrows in the figure are used to highlight the morphological and structural changes of MRSA bacteria after treated by CGL-1 recombinant protein, which indicates that CGL-1 recombinant protein has a significant killing effect on MRSA bacteria.

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

[0024] Figure 8 The effect of CGL-1 recombinant protein on MRSA biofilm was detected by crystal violet method. Among them, (A) the inhibitory effect of CGL-1 recombinant protein on MRSA biofilm formation; (B) 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 MRSA mature biofilm; (D) quantitative analysis of the elimination effect of CGL-1 recombinant protein on MRSA mature biofilm. The numbers 1-5 in figures A and C represent the blank group, the negative control group and the 64, 32, 16 μg / mL CGL-1 recombinant protein treatment groups, respectively. The asterisk "*" mark indicates that the difference between the CGL-1 recombinant protein treatment group and the negative control group is statistically significant (P<0.05).

[0025] Figure 9 The effect of CGL-1 recombinant protein on MRSA intracellular content leakage and reactive oxygen species generation. Among them, (A) the concentration-dependent effect of CGL-1 recombinant protein on MRSA nucleic acid leakage; (B) the concentration-dependent effect of CGL-1 recombinant protein on MRSA protein leakage; (C) the effect of CGL-1 recombinant protein on MRSA reactive oxygen species generation. The asterisk "*" mark in the figure indicates that the difference between the CGL-1 recombinant protein treatment group and the negative control group is statistically significant (P<0.05).

[0026] Figure 10 The evaluation of antioxidant activity of CGL-1 recombinant protein. Among them, (A) the scavenging ability of CGL-1 recombinant protein on DPPH free radicals; (B) the scavenging ability of vitamin C on DPPH free radicals; (C) the scavenging ability of CGL-1 recombinant protein on DPPH free radicals; (D) the scavenging ability of vitamin C on ABTS free radicals; (E) the reducing ability of CGL-1 recombinant protein on iron ions; (F) the reducing ability of vitamin C on iron ions. The asterisk "*" mark in the figure indicates that the difference between the CGL-1 recombinant protein treatment group or the vitamin C treatment group and the negative control group (CGL-1 recombinant protein concentration is 0 μg / mL) is statistically significant (P<0.05); "ns" indicates that the difference is not statistically significant (P>0.05) compared with the negative control group.

[0027] Figure 11The promoting effect of CGL-1 recombinant protein on the healing of MRSA infected wounds in diabetic rats. Among them, (A) is a representative image of the healing of the wound in each group; (B) is the quantitative analysis of the healing rate of the wound in each group; (C) is the weight change trend of rats in each group; (D) is the change of blood glucose level of rats in each group under non-fasting condition; (E) is the detection of bacterial amount in the wound of each group; (F) is the quantitative analysis of the bacterial amount in the wound of each group. The letters (a, b, c) in figures B and F are used to represent the statistical significance. Among them, the same letter indicates that there is no significant difference between groups (P>0.05), and different letters indicate that there is a significant difference between groups (P<0.05). The asterisk "*" mark in figures C and D indicates that the difference between the model group and the drug treatment group is statistically significant compared with the normal group (P<0.05).

[0028] Figure 12 The histological effect of CGL-1 recombinant protein on the healing of MRSA infected wounds in diabetic rats. Among them, (A) is the H&E staining of the wound tissue of the normal group of rats; (B) is the H&E staining of the wound tissue of the model group of rats; (C) is the H&E staining of the wound tissue of the KFX treatment group of rats; (D) is the H&E staining of the wound tissue of the 16 μg / mL CGL-1 treatment group of rats; (E) is the H&E staining of the wound tissue of the 32 μg / mL CGL-1 treatment group of rats; (F) is the H&E staining of the wound tissue of the 64 μg / mL CGL-1 treatment group of rats; (G) is the wound diameter statistics of different treatment groups. The red arrow in figures A-F indicates the newly formed blood vessels, the blue arrow indicates the collagen fibers, the black arrow indicates the adipose tissue, and the yellow arrow indicates the inflammatory cells. The letters (a, b, c, d) in figure G are used to represent the statistical significance. Among them, the same letter indicates that there is no significant difference between groups (P>0.05), and different letters indicate that there is a significant difference between groups (P<0.05).

[0029] Figure 13 The effect of CGL-1 recombinant protein on the collagen deposition of MRSA infected wounds in diabetic rats. Among them, (A) is the Masson staining of the wound tissue of the normal group of rats; (B) is the Masson staining of the wound tissue of the model group of rats; (C) is the Masson staining of the wound tissue of the KFX treatment group of rats; (D) is the Masson staining of the wound tissue of the 16 μg / mL CGL-1 treatment group of rats; (E) is the Masson staining of the wound tissue of the 32 μg / mL CGL-1 treatment group of rats; (F) is the Masson staining of the wound tissue of the 64 μg / mL CGL-1 treatment group of rats; (G) is the collagen volume fraction statistics of the wound tissue of different treatment groups. The letters (a, b, c) in figure G are used to represent the statistical significance. Among them, the same letter indicates that there is no significant difference between groups (P>0.05), and different letters indicate that there is a significant difference between groups (P<0.05). DETAILED DESCRIPTION

[0030] The technical solutions described in the present application are conventional solutions in the art if not specifically stated, and the reagents or raw materials used are purchased from commercial channels or are already disclosed if not specifically stated.

[0031] The present application provides a CGL-1 lectin protein from Pacific oyster, and the amino acid sequence of the CGL-1 lectin protein is shown in SEQ ID NO. 2.

[0032] The present application also provides a DNA molecule encoding the CGL-1 lectin protein, and the nucleotide sequence of the DNA molecule is shown in SEQ ID NO. 1.

[0033] The present application also provides an application of the CGL-1 lectin protein in preparing a medicine for preventing and / or treating methicillin-resistant Staphylococcus aureus infection.

[0034] The present application also provides an application of the CGL-1 lectin protein in preparing a medicine for promoting the healing of a methicillin-resistant Staphylococcus aureus infected wound of a diabetic patient.

[0035] The present application also provides a medicine for preventing and / or treating methicillin-resistant Staphylococcus aureus infection, which comprises the CGL-1 lectin protein.

[0036] In some specific embodiments, a pharmaceutically acceptable excipient is further included.

[0037] The present application also provides a medicine for promoting the healing of a methicillin-resistant Staphylococcus aureus infected wound of a diabetic patient, which comprises the CGL-1 lectin protein.

[0038] In some specific embodiments, a pharmaceutically acceptable excipient is further included.

[0039] The present application successfully prepared Magallana gigas CGL-1 lectin protein by using E. coli recombinant expression system for the first time, and comprehensively evaluated its antibacterial, antioxidant activity and promoting effect on infected wound repair of diabetic rats. The results of in vitro experiment showed that CGL-1 recombinant protein had significant antibacterial activity on methicillin-resistant Staphylococcus aureus (MRSA), could destroy the bacterial cell structure, induce reactive oxygen species (ROS) generation, inhibit the formation of biofilm, and promote the elimination of mature biofilm. In addition, CGL-1 recombinant protein also showed good antioxidant capacity, which may help to reduce oxidative stress and inflammatory response of diabetic wounds. In the diabetic rat model, CGL-1 recombinant protein could significantly promote the healing of MRSA infected wound, reduce the amount of bacteria on the wound, improve the tissue structure, and increase the deposition of collagen. These findings suggest that CGL-1 lectin protein has the potential as a new non-antibiotic therapeutic agent, which can be used for the treatment of diabetic infected wounds.

[0040] The statistical software IBM SPSS Statistics 19.0 was used for data analysis, and the GraphPad Prism 8 software was used for statistical plotting. All experiments were repeated 3 times, and the quantitative results were expressed as mean ± standard deviation (X ± S). Independent sample t test was used for comparison of quantitative values between two groups, and one-way ANOVA was used for comparison of quantitative values between multiple groups. In all tests, P<0.05 was considered to be significantly different, which had statistical significance.

[0041] Example 1

[0042] Codon optimization, synthesis and construction of recombinant plasmid of Magallana gigas lectin CGL-1 gene and double enzyme digestion verification

[0043] The CGL-1 lectin protein of Magallana gigas (NCBI Reference Sequence: XP_034338847.1) and its corresponding Gene ID (105329071) were retrieved in the NCBI database. The open reading frame of the gene was determined using the Open Reading Frame Finder of NCBI, and the sequence encoding the mature protein was submitted to NCBI GenBank with the sequence number PV878053.1. According to the codon usage bias of Escherichia coli, the codon optimization was performed and the full gene synthesis was entrusted to Nanjing Kingsriver Biotechnology Co., Ltd. The gene was inserted into the pETa-30 vector between Nde I and Hin dIII by seamless cloning technology to construct the recombinant plasmid pET-30a-CGL-1, followed by Xho I and Apa I double enzyme digestion verification and sequencing verification. The bioinformatics analysis of the CGL-1 protein of Magallana gigas is 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 protein conserved domain was analyzed using the NCBI Conserved domains (https: / / www.ncbi.nlm.nih.gov / Structure / cdd / wrpsb.cgi) and mapped 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 / ); the tertiary structure of the CGL-1 protein was predicted using the I-TASSER online software (https: / / zhanggroup.org / I-TASSER / ) and visualized using the PyMOL software.

[0044] The open reading frame (ORF) of the CGL-1 lectin protein of Magallana gigas (NCBI Reference Sequence: XP_034338847.1) was determined by analyzing the gene sequence of the protein using the Open Reading Frame Finder tool of NCBI. The ORF is 330 bp in length, encoding 109 amino acid residues (aa) of the CGL-1 protein. Figure 1 The ORF of the CGL-1 lectin protein was codon-optimized based on the codon bias of Escherichia coli (E. coli) (Middle B). Figure 1

[0045] ​The optimized gene sequence is shown as SEQ ID NO. 1: 5'-ATGGTTTGTCGTCTGCCGGGTAAAATTCAGACCGCTTGCGAAAACAAAGGCATCATCCTGCACTGCGGTATGCATCAGGTTCTGCACGTTTTCGACGCAATGTACGGTCGCGAAGATAAACACACCTGCAGTCGTAGCGGCAAACATAAAAGCACCAGCTGTACCGCACGTAACGTTCTGAAAAAAGTCAAAAGCAAATGCAACGGCCGCCGTAGTTGTCGTCTGCGCGCACGTAATAGCGTTTTTGGCGATCCGTGTCACGGTACCCTGAAATATCTGCGCGTTCGTTACGAGTGTCGTCGTCGTTATCACGGTTGGTGGCACGGTTAA-3'.

[0046] The encoded protein amino acid sequence is shown as SEQ ID NO. 2: MVCRLPGKIQTACENKGIILHCGMHQVLHVFDAMYGREDKHTCSRSGKHKSTSCTARNVLKKVKSKCNGRRSCRLRARNSVFGDPCHGTLKYLRVRYECRRRYHGWWHG.

[0047] The optimized gene sequence was synthesized by Nanjing Kingsriver Biotechnology Co., Ltd., and inserted into the Nde I and Hin dIII enzyme cutting sites of the pET-30a vector by seamless cloning technology, and the recombinant plasmid pET-30a-CGL-1 Figure 1 was successfully constructed. The recombinant plasmid pET-30a-CGL-1 was verified by Xho I and Apa I double enzyme digestion. The enzyme digestion results showed that the correct enzyme digestion fragments were generated at the expected position, indicating that the target gene had been successfully inserted into the vector Figure 1 . Further verification by sequencing confirmed that the sequence of the recombinant plasmid was completely consistent with the designed sequence, without mutation or insertion / deletion phenomenon. The basic physicochemical properties of CGL-1 lectin protein were predicted using ProtParam online software. The results showed that the molecular weight of the protein was 12659.77 Da, and the theoretical isoelectric point (pI) was 10.6. Its extinction coefficient was 15500 M -1 cm -1, indicating that the protein has good absorption characteristics under ultraviolet light. The instability index of the protein is 66.75, indicating that it may have certain instability in vitro. The hydrophobicity (GRAVY) value of the protein is -0.756, indicating that the protein is hydrophilic as a whole Figure 1 The CGL-1 lectin protein contains a typical lectin domain (galactose / rhamnose binding lectin domain) between amino acid residues 99-109, as found by NCBI Conserved domains analysis. This domain has high homology with known members of the lectin family, indicating that CGL-1 lectin protein may have the typical function of the lectin family. The conserved domain map drawn using DOG software (v1.0) further visually displays the distribution of the domain in the protein sequence Figure 1 Figure 1 The secondary structure of CGL-1 lectin protein was predicted using Jpred4 online software. The prediction results show that the protein mainly contains 1 alpha-helix and 7 beta-folds. The secondary structure prediction map clearly shows the distribution of these secondary structure elements in the protein sequence Figure 1 The tertiary structure of CGL-1 lectin protein was predicted by I-TASSER online software. The prediction results show that the protein as a whole presents a compact globular structure with 1 alpha-helix, 6 beta-folds and random coils. Further visualization of the predicted tertiary structure was performed using PyMOL software, generating a high-quality three-dimensional structure model map. The model shows the tertiary structure characteristics of CGL-1 lectin protein, with four pairs of disulfide bonds, Cys 13 -Cys 43 , Cys 22 -Cys 99 , Cys 54 -Cys 86 , Cys 67 -Cys 73 , which provides an important structural basis for subsequent functional research

[0048] Example 2

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

[0050] The E. coli monoclonal containing the recombinant plasmid was inoculated into LB medium containing 50 μg / mL kanamycin and cultured at 37°C, 180 rpm for 3-4 hours, until the OD 600 ​0.6-0.8. Then IPTG was added to a final concentration of 0.2 mM, and the expression was induced at 15°C and 37°C for 16 hours, respectively, and the 15°C uninduced group was set as a control. After induction, the bacteria were collected and placed in an ice water bath for ultrasonic disruption (400 W, 5 seconds of ultrasonic, 5 seconds of interval, 20 minutes in total). Then the supernatant and the precipitate were collected by centrifugation at 12000 rpm for 10 minutes at 4°C, and were used for SDS-PAGE gel electrophoresis detection. For the treatment of inclusion bodies, the inclusion body washing solution was used for washing, and the inclusion body buffer was used for dissolving. Meanwhile, the Ni-IDA column was equilibrated, and then eluted with equilibration buffer containing different concentrations of imidazole. Each elution fraction was collected and subjected to SDS-PAGE analysis. The elution fraction with higher purity was selected, first dialyzed into refolding solution for refolding, and then further dialyzed into 1x PBS (pH 7.4). The protein solution after dialysis was filtered with a 0.22 μm filter to remove bacteria, and then subjected to ToxinEraser TM Endotoxin Removal Kit to remove endotoxin (the whole process was carried out at low temperature). The endotoxin content was determined by using ToxinSensor TM Gel Clot Endotoxin Assay Kit, and the protein concentration was determined by using Bradford protein concentration determination kit. Finally, the protein was aliquoted and stored at -20°C for standby.

[0051] The expression of CGL-1 recombinant protein under different temperature induction conditions was analyzed by 12% SDS-PAGE gel electrophoresis. The results showed that the expression amount of recombinant protein was the highest under the condition of induction at 37°C for 16 hours, while the expression amount of recombinant protein was relatively low or almost no expression in the 15°C induction and uninduced control groups. For the bacteria induced at 15°C, the supernatant and the precipitate were collected after ultrasonic disruption for gel electrophoresis detection, and the results showed that the expression amount of CGL-1 recombinant protein was very low Figure 2 A). While for the bacteria induced at 37°C, the supernatant and the precipitate were collected after ultrasonic disruption for gel electrophoresis detection, and the results showed that CGL-1 recombinant protein mainly existed in the inclusion body Figure 2 B). Then the inclusion bodies were collected from 1 L of bacterial culture, and the CGL-1 recombinant protein in the inclusion body was washed, dissolved, and purified by using Ni-IDA affinity chromatography column, and the CGL-1 recombinant protein with higher purity was obtained by 12% SDS-PAGE detection Figure 2 C). The elution fraction with higher purity was selected for dialysis refolding, and after filtering to remove bacteria, ToxinEraser TMEndotoxin Removal Kit. The endotoxin content determination results 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 the Bradford protein concentration determination kit, and the results showed that the protein concentration was 0.50 mg / mL, a total of 10 tubes were prepared, 1 mL per tube. Further, 12% SDS-PAGE was used to detect the quality of the CGL-1 recombinant protein, and the results showed that the molecular weight of the CGL-1 recombinant protein was about 13.48 kDa, which was consistent with the expected size. In the SDS-PAGE electrophoretogram, the target protein band was clear and single, and no obvious impurity protein band was observed, indicating that the protein had high purity Figure 2 D) in the middle.

[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 analysis of recombinant protein were performed according to previous studies. The relative molecular mass of CGL-1 recombinant protein was analyzed using an ultra-high resolution time-of-flight mass spectrometer (AB 5800 MALDI-TOF / TOF, AB SCIEX, USA). By analyzing the mass-to-charge ratio (m / z) values in the obtained mass spectrum, the molecular weight of CGL-1 recombinant protein was accurately determined. In order to further analyze the sequence information of CGL-1 recombinant protein, trypsin was used to perform enzyme digestion of the recombinant protein. After enzyme digestion, liquid chromatography-mass spectrometry (LC / MS) was used to analyze the generated peptides. LC / MS analysis used a nanoscale liquid chromatograph (EASY-nLC 1200, Thermo Fisher Scientific, USA) combined with an electrostatic field orbitrap mass spectrometer (Q Exactive, Thermo Fisher Scientific, USA), and the chromatographic column was a reversed-phase C18 column (Acclaim PepMap RSLC, 75 μm×25 cm C18-2 μm ), the mobile phase was 0.1% formic acid aqueous solution and 80% acetonitrile solution, the mass spectrometry detection mode was positive ion mode, the raw spectrum file collected by mass spectrometry was processed and analyzed by PEAKS Studio 8.5 (Bioinformatics Solutions Inc. Waterloo, Canada) software, and the database was Magallanagigas protein database (Taxon ID: 29159) downloaded from Uniprot. The secondary structure of CGL-1 recombinant protein was analyzed by circular dichroism spectrometer (Chirascan Plus, Applied Photophysics Ltd., UK). The CGL-1 recombinant protein was dissolved in a suitable buffer solution, prepared into a sample solution with a certain concentration, and loaded into a quartz cuvette for ultraviolet scanning. The raw data were processed by Pro-Data Viewer software, and the secondary structure of the test sample was fitted and calculated by CDNN software.

[0055] The mass spectrometry analysis results showed that the main mass-to-charge ratio (m / z) value of CGL-1 recombinant protein was concentrated in 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 CGL-1 recombinant protein did not cause damage to its molecular structure Figure 3 The LC / MS analysis results after trypsin digestion showed that the purified CGL-1 recombinant protein had high matching degree with the theoretical peptide segment of the expected target protein (the number of matching peptide segments was 47, and the coverage rate was 68%), indicating that its amino acid sequence was accurate and the purification effect was good, which provided a reliable basis for subsequent functional research Figure 3 The circular dichroism spectrum analysis showed that CGL-1 recombinant protein had characteristic absorption peaks in the wavelength range of 180-260 nm. The secondary structure composition calculated by CDNN software fitting was as follows: α-helix accounted for 3.3%-7.6%, β-sheet accounted for 45.4%-58.5%, β-turn accounted for 14.7%-19.3%, and random coil accounted for 25.2%-35.7%. Among them, the proportion of β-sheet structure was the highest, indicating that CGL-1 recombinant protein had relatively rich β-sheet structure, which might be closely related to its biological function Figure 3

[0056] Example 4

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

[0058] ​In 96-well plates, different concentrations of CGL-1 recombinant protein solution were added, with a final concentration of 256, 128, 64, 32, 16, 8, 4, 2 μg / mL, and 3 replicates were set for each group. At the same time, PBS was set as a negative control, and 1.0% Triton X-100 was set as a positive control. An equal volume of 2% rabbit red blood cell suspension was added to each well to allow the red blood cells to fully contact the recombinant protein. The 96-well plate was incubated at 37°C for 1 hour, and then centrifuged at 1500 rpm for 10 minutes. The supernatant was carefully removed to a new 96-well plate, and the absorbance was measured at 450 nm. The hemolysis rate calculation formula is: Hemolysis rate = (experimental group OD 450 - negative control group OD 450 ) / (positive control group OD 450 - negative control group OD 450 ) x 100%.

[0059] The toxicity of CGL-1 recombinant protein on HaCaT cells was detected by CCK-8 method. The HaCaT cells in the logarithmic growth phase were taken, and the cell concentration was adjusted to 1 x 10 4 / mL using DMEM medium containing 1% penicillin / streptomycin and 10% fetal bovine serum (FBS). 200 μL of cell suspension was added to each well of a 96-well plate, which was incubated at 37°C, 5% CO2 for 24 hours to allow the cells to adhere. After discarding the culture medium, CGL-1 recombinant protein diluted with complete culture medium was added, with a final concentration of 256, 128, 64, 32, 16, 8, 4, 2 μg / mL. At the same time, a blank group containing only medium and a negative control group treated with PBS were set. After incubation for 24 hours and 48 hours, the culture medium was aspirated, 100 μL of DMEM medium without serum was added to each well, followed by the addition of 10 μL of CCK-8 solution to each well, and the incubation was continued for 2 hours. The absorbance was measured at 450 nm using an enzyme marker (Synergy H1, BioTek, Winooski, VT, USA). The cell viability calculation formula is as follows: [OD (drug group) - OD (blank)] / [OD (PBS control group) - OD (blank)] x 100%.

[0060] 4% rabbit red blood cells were used for hemolytic activity detection. Different concentrations of recombinant thymosin beta 4 protein were mixed with red blood cells, PBS was used as a negative control group, and red blood cells treated with 1.0% Triton X-100 were used as a positive control group. After incubation at 37°C for 1 hour, centrifugation was performed at 25°C, 1500 rpm for 10 minutes, and the supernatant was collected to measure the absorbance at 450 nm. According to the formula: [experimental group OD 450 - negative control group] / [positive control group OD 450 - negative control group] / [positive control group OD 450The hemolysis rate of recombinant thymosin beta 4 protein was calculated by the following formula: (ODsample-ODnegative control) x 100%, and the experiment was repeated 3 times.

[0061] The results of the hemolytic activity test of CGL-1 recombinant protein showed that it had a 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, 2 μg / mL) Figure 4 A, B). This indicates that CGL-1 recombinant protein may exhibit a certain hemolytic activity at a higher concentration (256 μg / mL), but has good hemocompatibility at a lower concentration. The results of the toxicity evaluation of CGL-1 recombinant protein on HaCaT cells showed that the cell viability was significantly inhibited and almost completely lost the ability to grow when the concentration reached 256, 128, 64 μg / mL at incubation times of 24 hours and 48 hours. However, at lower concentrations (32, 16, 8, 4, 2 μg / mL), the cell viability was not significantly affected, with no statistical difference compared with the negative control group. This indicates that the toxicity of CGL-1 recombinant protein on HaCaT cells is concentration-dependent, with 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 on MRSA

[0064] The antibacterial activity of CGL-1 recombinant protein was detected by double-layer plate transparent circle method. MRSA single colony was inoculated in LB liquid medium and cultured at 37°C, 180 rpm for 16 hours to prepare LB agar medium containing about 1.0 x 10 6 CFU / mL of bacteria. 100 μL of CGL-1 recombinant protein (0.50 mg / mL) was added to the hole, and PBS was set as the negative control. After 3 hours of pre-diffusion at 4°C, it was cultured at 37°C for 16 hours. Vernier caliper was used to measure the diameter of the inhibition circle, and the experiment was repeated 3 times.

[0065] The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of CGL-1 recombinant protein on MRSA were determined by micro-broth dilution method. 100 μL of 1.0 x 10 6CFU / mL MRSA bacterial suspension, and then 100 μL of CGL-1 recombinant protein was added to make the final concentration of 128, 64, 32, 16, 8, 4, 2, 1 and 0.5 μg / mL, respectively, and negative and blank controls were set up, with 3 replicates in each group. The 96-well plate was placed in a 37°C incubator for 18 hours. Then 1.5 μL of 1% 2,3,5-chlorinated triphenyl tetrazolium chloride (TTC) was added to each well, and the incubation was continued at 37°C for 3 hours. The OD 485 of each well was determined using a microplate reader, and the color of each well was observed. The minimum concentration of the well without red color was taken as the MIC, and 20 μL of the clear well was spread on LB solid medium. The concentration corresponding to the well with no single colony or with ≤5 colonies was taken as the MBC. The experiment was repeated 3 times.

[0066] The effect of CGL-1 recombinant protein on the growth curve of MRSA was determined by micro-broth dilution method. 100 μL of 1.0 x 10 6 CFU / mL MRSA bacterial suspension was added to a 96-well plate, and then 100 μL of CGL-1 recombinant protein was added to make the final concentration of 128, 64 and 32 μg / mL, respectively, and a negative control group was set up, with 3 replicates in each group. The 96-well plate was placed in a microplate reader for incubation at 37°C with shaking for 24 hours, and the OD 600 was determined every 2 hours, and the growth curve was plotted. The experiment was repeated 3 times.

[0067] CGL-1 recombinant protein (0.50 mg / mL) showed significant antibacterial activity against MRSA, with a zone diameter (22.60 ± 0.71 mm) significantly larger than that of the PBS negative control group (0 mm) Figure 5 in Figure A). This result indicates that CGL-1 recombinant protein can effectively inhibit the growth of MRSA. Determination by micro-broth dilution method and plate spread method showed that the MIC value of CGL-1 recombinant protein against MRSA was 64 μg / mL, and the MBC value was 128 μg / mL Figure 5 in Figures B, C and D). Growth curve analysis results showed that CGL-1 recombinant protein significantly inhibited the growth of MRSA at concentrations of 128, 64 and 32 μg / mL Figure 5 in Figure 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 different degrees of growth delay effect. The growth curve of MRSA in the negative control group (PBS) showed a typical logarithmic growth trend. In summary, CGL-1 recombinant protein has concentration-dependent antibacterial and bactericidal effects on 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 biofilm elimination. The experiment was repeated three times.

[0079] Crystal violet staining results showed that CGL-1 recombinant protein significantly inhibited the formation of MRSA biofilm. At 64 μg / mL, the biofilm formation was significantly reduced, and the staining color was significantly lighter than the negative control group. With the decrease of protein concentration to 32 and 16 μg / mL, the biofilm formation increased, but was still significantly lower than the control group Figure 8 A). The measurement of OD 595 value further confirmed this inhibitory effect, and the OD value at 64 μg / mL was significantly lower than the control group, and at 32 and 16 μg / mL also showed significant inhibition Figure 8 B), indicating that the inhibitory effect was concentration-dependent. In the biofilm elimination experiment, after 24 hours of treatment with CGL-1 recombinant protein, the pre-formed biofilm was significantly reduced, and the crystal violet staining color was significantly lighter, confirming that CGL-1 recombinant protein had effective elimination effect on the formed biofilm Figure 8 C, D).

[0080] Example 9

[0081] Effect of CGL-1 recombinant protein on MRSA content leakage and ROS production

[0082] Pick MRSA single colony and inoculate into LB liquid medium, incubate at 37°C, 180 rpm overnight. Centrifuge at 4°C, 4000 rpm for 5 minutes, collect the bacterial cells. Wash the bacterial cells with PBS for 2 times, and adjust the bacterial liquid concentration to 1 × 10 6 CFU / mL with PBS. Add CGL-1 recombinant protein to the bacterial liquid, so that the final concentration is 128, 64, 32 μg / mL. At the same time, set up negative control group (only add PBS) and blank group (without bacterial liquid and CGL-1 recombinant protein). After mixing evenly, incubate at 37°C, 100 rpm for 6 hours. Centrifuge at 4°C, 4000 rpm for 5 minutes, collect the supernatant. Use ultraviolet-visible spectrophotometer to measure the absorbance (OD 260 and OD 280 ) of the supernatant at 260 nm and 280 nm. The experiment was repeated 3 times.

[0083] Adjust the bacterial liquid 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 Slike Jingda Experimental Animal Co., Ltd. The rats were raised in an environment with a temperature of 22-25°C, humidity of 50%-60%, 12 hours of light / 12 hours of darkness, and free access to standard experimental animal feed and water. The experimental protocol was approved by the Animal Ethics Committee of Guangxi University of Chinese Medicine (Approval No. DW20240919-191; Approval Date: September 19, 2024), and strict adherence to the National Experimental Animal Management Guidelines. The diabetic model was induced by a single intraperitoneal injection of streptozotocin (STZ, dose of 65 mg / kg body weight). After injection, the fasting blood glucose level was measured using a blood glucose meter (Accu-Chek, Roche Diagnostics, USA) for 7 consecutive days, and rats with blood glucose exceeding 16.7 mmol / L were included in the experiment. After anesthesia with isoflurane inhalation, the rats were fixed in a prone position on the operating table. The rat's back hair was removed using an electric pusher, and depilatory cream was applied. After 6 minutes, the depilatory cream was scraped off, and the remaining residue was wiped off with warm water and then dried with gauze. Then, iodophor was used for disinfection, and surgical towels were laid out. Two centimeters on both sides of the spine were made into full-thickness skin defect circular wounds with a diameter of 2 x 2 cm, and two wounds were prepared for each rat. Immediately after wound creation, 100 μL of methicillin-resistant Staphylococcus aureus (MRSA) with a concentration of 1.0 x 10 8 CFU / mL was uniformly applied with a sterilized yellow sterile gun head, and no dressing was applied to simulate a chronic wound environment. The rats were randomly divided into six groups (6 rats per group): normal group wounds (treated with PBS); diabetic infected group wounds (treated with PBS); diabetic infected group wounds (treated with Kangfuxin liquid); diabetic infected group wounds (treated with 16 μg / mL CGL-1); diabetic infected group wounds (treated with 32 μg / mL CGL-1); diabetic infected group wounds (treated with 64 μg / mL CGL-1). After 24 hours of infection, 100 μL of the corresponding drug was uniformly applied to the wound, and the rats were given medication once a day, with free access to water and food. The wounds were photographed on days 0, 3, 5, 7, 9, 11, and 13, and the wound area was calculated using Image J image analysis software. The wound healing rate was calculated according to the following formula: Wound healing rate (%) = (wound original area - area at each time point) ÷ wound original area x 100, and the wound healing curve was plotted. The body weight and blood glucose level of the rats were measured daily, and the change curve was plotted to evaluate the effect of diabetes and 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 7CFU / g) was 3.32 ± 0.24, and the MRSA content (1.0 x 10 7 CFU / g) were 0.18 ± 0.01, 1.41 ± 0.17, 2.48 ± 0.40, 0.76 ± 0.38, and 0.38 ± 0.08, respectively, which were significantly lower than that of the model group (P < 0.05). Similarly, at 13 days, the MRSA content (1.0 x 10 7 CFU / g) was 1.68 ± 0.76, and the MRSA content (1.0 x 10 7 CFU / g) were 0.07 ± 0.01, 0.26 ± 0.12, 0.53 ± 0.23, 0.40 ± 0.05, and 0.07 ± 0.07, respectively, which were significantly lower than that of the model group (P < 0.05). Moreover, the MRSA content of the CGL-1 treatment groups gradually decreased with the increase of the CGL-1 concentration, and the MRSA content of the 64 μg / mL CGL-1 treatment group was significantly lower than that of the model group and close to that of the normal group. These results show that CGL-1 can effectively reduce the MRSA content of the MRSA infected wounds of the diabetic rats and significantly promote the wound healing.

[0094] Example 12

[0095] Tissue staining observation

[0096] On day 13 after modeling and drug administration, two rats were taken from each group, and a total of four wound surfaces were collected. The wound tissues, including normal skin 0.5 cm around the wound surface, were immediately fixed in 4% paraformaldehyde solution. After 24 hours, the tissues were dehydrated, transparentized, and paraffin-embedded. 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 at 37°C overnight. The paraffin sections were deparaffinated with xylene and rehydrated to distilled water through a decreasing ethanol series (100%, 95%, 90%, 80%, 70%). HE staining: the sections were stained with hematoxylin (Sigma-Aldrich) for 5 minutes, differentiated with 1% acid alcohol 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. The sections were dehydrated through an increasing ethanol series (70%, 80%, 90%, 95%, 100%) and transparentized with xylene, and then mounted with DPX mounting medium (Sigma-Aldrich). Masson staining: the sections were deparaffinated with xylene and rehydrated to distilled water through a decreasing ethanol series (100%, 95%, 90%, 80%, 70%). The sections were immersed in Weigert's iron hematoxylin staining solution (Sigma-Aldrich) for 10 minutes and then rinsed with running water for 5 minutes; then differentiated with 1% acid alcohol for 1 minute and rinsed with running water for 5 minutes; and then blued with Scott's tap water substitute (Sigma-Aldrich) for 5 minutes and rinsed with running water for 5 minutes. Subsequently, the sections were immersed in solution B (Masson's trichrome staining solution B, Sigma-Ald) for 10 minutes and rinsed with running water for 5 minutes; and then immersed in solution C (Masson's trichrome staining solution C, Sigma-Aldrich) for 10 minutes and rinsed with running water for 5 minutes. Finally, the sections were dehydrated through an increasing ethanol series (70%, 80%, 90%, 95%, 100%) and transparentized with xylene, and then mounted with DPX mounting medium (Sigma-Aldrich). Subsequently, the tissue sections were subjected to white light scanning using a microscope slide scanner (Pannoramic MIDI II, 3DHISTECH Ltd., Budapest, Hungary) to obtain high-resolution panoramic images.

[0097] On day 13 after modeling and drug administration, the wound tissues of the rats in each group were observed by histological H&E staining. The results showed that the normal group (A) showed complete epidermal and dermal structures, good wound healing, less inflammatory cell infiltration, more collagen fibers, and a large amount of new blood vessels and adipose tissue. Figure 12 Figure 12 ​The middle B) showed obvious inflammatory reaction, epidermis and dermis structure disorder, and a large number of inflammatory cell infiltration, less collagen fibers, and poor wound healing. KFX group( Figure 12 The middle C) and different concentrations of CGL-1 recombinant protein treatment group( Figure 12 The middle D ~ F) showed different degrees of inflammation and tissue structure improvement. Among them, 16 μg / mL CGL-1 treatment group( Figure 12 The middle D) and 32 μg / mL CGL-1 treatment group( Figure 12 The middle E) of wound healing was better than the model group, inflammatory cell infiltration decreased, and tissue structure began to recover. 64 μg / mL CGL-1 treatment group( Figure 12 The middle F) of wound healing effect was the most significant, less inflammatory cell infiltration, more collagen fibers, and visible amount of new blood vessels and adipose tissue, showing the best healing effect. Figure 12 The middle G showed the quantification results of the wound diameter of each group. Compared with the normal group(5223.20±197.31 μm), the wound diameter of the model group(6835.78±332.21 μm) was significantly increased(P<0.05). The wound diameter of KFX group and each concentration of CGL-1 treatment group was 4359.53±2.44 μm, 5752.50±51.65 μm, 4291.10±198.32 μm, and 2726.88±202.41 μm, respectively, which were significantly smaller than that of the model group(P<0.05), and the wound diameter of the 64 μg / mL CGL-1 treatment group was the smallest, showing the best healing effect. These results showed that CGL-1 recombinant protein could effectively promote the healing of MRSA infected wounds in diabetic rats, and its effect was positively correlated with the concentration.

[0098] The Masson staining results of the wound tissue of the normal group of rats showed that the tissue structure was complete, and the collagen fibers(blue) were rich, indicating that the deposition of collagen was good in the normal wound healing process( Figure 13 The model group of rats showed that the collagen fibers(blue) were significantly reduced, and the tissue structure was disordered, indicating that the deposition of collagen was reduced in the healing process of MRSA infected wounds in diabetic rats( Figure 13 The KFX treatment group of rats showed that the collagen fibers(blue) were increased, and the tissue structure was improved, indicating that KFX had a significant promoting effect on wound healing( Figure 13Masson staining of the wound tissue of the rats in different CGL-1 recombinant protein treatment groups showed that with the increase of CGL-1 concentration, the deposition of collagen fibers (blue) gradually increased, and the tissue structure gradually improved. In particular, the 32 and 64 μg / mL CGL-1 treatment groups, the deposition of collagen fibers (blue) was the most abundant, the tissue structure was the most complete, and the best healing effect was shown. Statistical analysis of the collagen volume fraction of the wound tissue of different treatment groups showed that the collagen volume fraction of the model group was the lowest (19.62 ± 9.33%), which was significantly lower than that of other groups. Among them, the collagen volume fractions of the normal group, the KFX group and the 16 μg / mL CGL-1 treatment group were 44.16 ± 7.85%, 54.36 ± 12.51% and 45.45 ± 2.89%, respectively, but there was no significant difference between them. Among them, the collagen volume fractions of the 32 and 64 μg / mL CGL-1 treatment groups were the highest, which were 84.40 ± 1.09% and 78.20 ± 3.21%, respectively, indicating that higher concentration of CGL-1 recombinant protein could effectively promote the deposition of collagen protein in the MRSA infected wound of diabetic rats, thereby improving the wound healing.

[0099] Obviously, the above embodiments of the present application are only examples for clearly illustrating the present application, and are not intended to limit the embodiments of the present application. Based on the above description, other different forms of changes or variations can also be made by those skilled in the art. Here, it is not necessary and impossible to exhaust all the embodiments. Any modification, equivalent replacement and improvement made within the spirit and principle of the present application shall be included in the protection scope of the claims of the present application.

Claims

1. A CGL-1 lectin protein derived from Pacific oysters, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

2.

2. A DNA molecule encoding the CGL-1 lectin protein of claim 1, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.

1.

3. The use of the CGL-1 lectin protein of claim 1 in the preparation of medicaments for the prevention and / or treatment of methicillin-resistant Staphylococcus aureus infections.

4. The use of the CGL-1 lectin protein of claim 1 in the preparation of a medicament for promoting the healing of methicillin-resistant Staphylococcus aureus (MRSA) infected wounds in diabetic patients.

5. A drug for the prevention and / or treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections, characterized in that, Includes the CGL-1 lectin protein as described in claim 1.

6. The drug according to claim 5, characterized in that, It also includes pharmaceutically acceptable excipients.

7. A drug for promoting the healing of methicillin-resistant Staphylococcus aureus (MRSA) infected wounds in diabetic patients, characterized in that, Includes the CGL-1 lectin protein as described in claim 1.

8. The medicament according to claim 7, characterized in that, It also includes pharmaceutically acceptable excipients.

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