Method for synthesizing recombinant human defensin with antibacterial activity by using chlamydomonas and application of recombinant human defensin

Abstract

The invention discloses a method for synthesizing recombinant human defensin with antibacterial activity by using chlamydomonas and application of the recombinant human defensin, compared with a traditional expression system, the method has the advantages in the aspects of cost effectiveness, expandability and safety, and the recombinant human defensin produced by the method shows effective antibacterial activity on various pathogenic bacteria and can be used for preparing the recombinant human defensin with antibacterial activity. Under the action of different temperatures, pH values and proteases, the compound has good stability, low hemolytic activity and extremely low cytotoxicity to mammalian cells. The invention also relates to the use of the recombinant human defensin as an antibacterial agent in a variety of applications, including medicaments, disinfectants and agricultural products.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to a method for synthesizing recombinant human defensins with antibacterial activity using Chlamydomonas and its application. Background Technology

[0002] With the widespread use of antibiotics, bacterial resistance has become an increasingly serious problem, making the development of novel antibacterial substances an urgent priority. Human defensins are a class of small molecule peptides with broad-spectrum antibacterial activity, playing a crucial role in the body's innate immune defense and effectively resisting the invasion of pathogens such as bacteria, fungi, and viruses. However, the content of natural human defensins in the body is extremely low, and traditional extraction methods suffer from low yield, high cost, and complex processes, which greatly limit their industrial application.

[0003] Currently, using genetic engineering technology to produce recombinant proteins has become an effective way to solve this problem. Various expression systems have been used for the expression of human defensins, such as the *E. coli* expression system. While it has advantages such as simple operation and low cost, it also has drawbacks such as the inability to correctly fold and modify eukaryotic proteins, the tendency to form inclusion bodies, and the expression products often being inactive or cytotoxic. Although the yeast expression system can perform a certain degree of protein modification, its heterotrophic fermentation process consumes a large amount of resources and energy and easily causes environmental pollution.

[0004] Chlamydomonas, a single-celled eukaryotic green alga, has attracted considerable attention in recent years as a novel expression chassis cell. It combines the advantages of both bacteria and higher plants, possessing benefits such as a post-translational modification system, low production cost, short cycle time, and small batch-to-batch variation. Furthermore, it is free of toxic substances such as endotoxins and microcystins, has a clear genetic background, and boasts a stable nuclear genetic transformation system that is currently widely used and technologically mature. In addition, Chlamydomonas was included in the New Food Resources Catalogue in 2022, demonstrating its unique biosafety and application prospects. Therefore, utilizing Chlamydomonas to efficiently express human defensins with antibacterial activity will provide a new solution for the development and application of novel antibacterial drugs in clinical use. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, the present invention aims to provide a method for synthesizing recombinant human defensins with antibacterial activity using Chlamydomonas and its applications. Compared with traditional expression systems, this method has advantages in cost-effectiveness, scalability, and safety. The recombinant human defensins produced by this method exhibit effective antibacterial activity against a variety of pathogenic bacteria, good stability at different temperatures, low hemolytic activity, and minimal cytotoxicity to mammalian cells. The present invention also relates to the use of this recombinant human defensin as an antibacterial agent in various applications, including pharmaceuticals, disinfectants, and agricultural products.

[0006] To achieve the above objectives, this application adopts the following technical solution: In a first aspect, the present invention provides a recombinant human defensin gene, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0007] Secondly, the present invention provides a recombinant expression vector or transformant or transgenic Chlamydomonas containing the above-mentioned recombinant human defensin gene.

[0008] Thirdly, the present invention provides the application of the above-mentioned transgenic Chlamydomonas in the preparation of health foods, fish feed, or livestock and poultry feed additives.

[0009] Fourthly, the present invention provides a method for constructing a transgenic Chlamydomonas expressing the above-mentioned recombinant human defensin gene, comprising the following steps: Step A1, constructing the recombinant expression vector: The above-mentioned recombinant human defensin gene is ligated with an expression vector containing a Chlamydomonas endogenous strong promoter, terminator and protein expression tag sequence to construct the recombinant expression vector; Step A2, genetic transformation of Chlamydomonas: Using Chlamydomonas as the background algal strain, the recombinant expression vector constructed in step A1 is introduced into the background algal strain to obtain genetic transformants; Step A3, Screening and Identification: The genetic transformants obtained in Step A2 are screened and identified to obtain the transgenic Chlamydomonas that has been successfully integrated into the Chlamydomonas genome.

[0010] Fifthly, the present invention provides a method for preparing recombinant human defensins with antibacterial activity using Chlamydomonas, comprising the following steps: Step B1: The transgenic Chlamydomonas is obtained using the above construction method; Step B2 involves expanding the transgenic Chlamydomonas culture. After the culture is completed, the algal cells or culture medium are collected by centrifugation. Soluble proteins are released by cell disruption and then purified using affinity chromatography to obtain high-purity recombinant human defensin.

[0011] In a sixth aspect, the present invention provides the application of the recombinant human defensin prepared by the above method in the preparation of antibacterial agents.

[0012] In the above technical solutions, the antibacterial agent is a medical, daily chemical, industrial, or agricultural antibacterial agent.

[0013] Seventhly, the present invention provides a method for preparing Chlamydomonas recombinant human defensin algal powder, comprising the following steps: Step C1: The transgenic Chlamydomonas is obtained using the above construction method; Step C2: The transgenic Chlamydomonas is cultured on a large scale until the concentration reaches 2-4 × 10⁻⁴. 6 When the number of cells / mL is flame-inoculated into the fermenter, fermentation culture is carried out; Step C3, ferment to 5~9×10 8 After reaching a cell / mL concentration, fermentation was terminated, cells were collected by rapid low-temperature centrifugation, and then freeze-dried to obtain the Chlamydomonas recombinant human defensin algal powder.

[0014] Eighthly, the present invention provides a method for preparing recombinant human defensin protein from Chlamydomonas, comprising the following steps: Step D1: The Chlamydomonas recombinant human defensin algal powder is prepared using the above preparation method; Step D2: Perform affinity chromatography on the Chlamydomonas recombinant human defensin algal powder obtained in step D1 to obtain the Chlamydomonas recombinant human defensin protein.

[0015] Ninthly, the present invention provides the application of the Chlamydomonas recombinant human defensin algal powder prepared by the above preparation method or the Chlamydomonas recombinant human defensin protein prepared by the above preparation method in the fields of medicine, food, industry, agriculture or chemical industry.

[0016] The beneficial effects of this invention are as follows: 1. High-efficiency expression: Innovatively utilizing Chlamydomonas as chassis cells for recombinant protein expression and combining it with clinical applications, a strong promoter and optimized codon sequence are used to achieve high-efficiency and stable expression of recombinant human defensins in Chlamydomonas, significantly improving production efficiency and product purity.

[0017] 2. Cost advantage: Chlamydomonas grows rapidly and has low cultivation cost. It does not require expensive culture medium components and can be obtained with fermentation equipment commonly used in industrial production, which greatly reduces production costs. In addition, compared with traditional expression systems, it also has a post-translational modification mechanism, resulting in high product activity, which is conducive to large-scale industrial production.

[0018] 3. High biosafety: Chlamydomonas itself is edible and does not contain endotoxins. The recombinant human defensin it expresses has high biosafety and can be directly applied in food, medicine and other fields, reducing safety hazards in subsequent application and processing of the product.

[0019] 4. Excellent functional properties: Through multi-faceted performance tests of the expressed product, it has been confirmed that the recombinant human defensin has good antibacterial activity, stability and biosafety, and has broad application prospects in the field of antibacterial medicine. It is expected to become an effective component of new antibacterial drugs. Attached Figure Description

[0020] Figure 1 This is a technical roadmap for using Chlamydomonas to express recombinant human defensins with antibacterial activity.

[0021] Figure 2 It is a construction map of recombinant expression vectors.

[0022] Figure 3 This is an experimental diagram illustrating the identification of Chlamydomonas recombinant human defensin-expressing algal strains.

[0023] Figure 4 This is an experimental diagram illustrating the genetic stability analysis of Chlamydomonas recombinant human defensin-expressing algal strains.

[0024] Figure 5 This is an experimental diagram showing the purification and identification of recombinant human defensin from Chlamydomonas.

[0025] Figure 6 This is a diagram showing the antibacterial activity of recombinant 3×HNP2 against Gram-negative bacteria.

[0026] Figure 7 This is a graph showing the antibacterial activity of 3×HNP2.

[0027] Figure 8 This is a diagram of the biochemical stability experiment of recombinant human defensin 3×HNP2.

[0028] Figure 9 This is a diagram from an experiment demonstrating the hemolytic activity of 3×HNP2 on mouse erythrocytes.

[0029] Figure 10 This is a diagram of the cytotoxicity experiment of 3×HNP2 against NIH / 3T3 and HEK293T mammalian cell lines.

[0030] Figure 11 This is a membrane permeability analysis diagram based on propidium iodide (PI) staining.

[0031] Figure 12 This is a scanning electron microscope image showing the changes in cell membrane morphology of Pseudomonas aeruginosa and Escherichia coli before and after treatment with recombinant human defensin. Detailed Implementation

[0032] To better illustrate the objectives, technical solutions, and advantages of this invention, the invention will be further described below in conjunction with specific embodiments. This invention can be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the inventive concept to those skilled in the art. This invention will be defined only by the claims.

[0033] Figure 1 This is a technical roadmap for using Chlamydomonas to express recombinant human defensins with antibacterial activity. Figure 2This is a construction map of the recombinant expression vector. It shows the structure of the recombinant plasmid after the recombinant human defensin gene (HNP2) was ligated to a vector backbone containing the TUB2 promoter, rbcS2 intron, HA tag, paromomycin resistance gene (Paro), and terminator (rbcS2-3'UTR). The experimental procedure of this invention is as follows: 1. Construction of Chlamydomonas recombinant expression vector: Based on the recombinant human defensin gene sequence (SEQ ID NO.2), the target gene fragment was artificially synthesized through codon optimization. The target gene was then ligated into an expression vector containing a strong endogenous promoter (such as TUB2, rbcS2), a terminator, and a protein expression tag sequence of Chlamydomonas to construct the recombinant expression vector.

[0034] 2. Genetic transformation of Chlamydomonas: Using common algal strains such as Chlamydomonas sp. as recipient cells, the constructed recombinant expression vector was introduced into Chlamydomonas cells using electroporation or gene gun methods.

[0035] 3. Screening and identification of recombinant Chlamydomonas strains: Using the resistance gene carried by the vector (such as the paromomycin resistance gene), screening was performed on a culture medium containing the corresponding antibiotic to obtain algal strains that may contain the recombinant expression vector. Positive algal strains were further identified by Western blot, confirming that the recombinant human defensin gene had been successfully integrated into the Chlamydomonas genome.

[0036] 4. Purification of the target protein: Positive algal strains after screening and identification are collected and cultured in TAP medium for large-scale production. High-density fermentation production can be achieved by culturing in TAP + acetic acid or sodium acetate medium. After culture, algal cells or culture medium are collected by centrifugation (if secretory expression is desired). For intracellularly expressed recombinant human defensins, soluble proteins are released through cell disruption (e.g., low-temperature homogenization, ultrasonic disruption, etc.), and then the target protein is purified using affinity chromatography to obtain high-purity recombinant human defensins.

[0037] 5. Western blotting detection and genetic stability analysis: Western blotting was used to detect the accuracy and expression level of the purified protein. The target protein band was detected by chemiluminescence immunoassay using anti-HA as the primary antibody. Simultaneously, the expression level of recombinant Chlamydomonas strains from continuous subcultures was analyzed to observe the genetic and expression stability of the recombinant human defensin gene in Chlamydomonas.

[0038] 6. Antibacterial Efficacy Detection: The antibacterial efficacy of the purified recombinant human defensin was detected using the inhibition zone method. Common pathogenic bacteria such as *Pseudomonas aeruginosa* and *Escherichia coli* were used as indicator bacteria. A certain concentration of recombinant human defensin solution was added dropwise to solid culture plates containing the indicator bacteria. After incubation for a period of time, the size of the inhibition zone was observed to evaluate the antibacterial activity of the recombinant human defensin against different bacteria.

[0039] 7. Stability Study: Recombinant human defensin was treated for a certain period at different temperatures (e.g., 4℃, 25℃, 37℃, 65℃, 90℃) and different pH values ​​(pH range set to 2, 4, 6, 8, 10) to detect changes in its antibacterial activity and investigate its temperature and pH stability. Simultaneously, recombinant human defensin was enzymatically hydrolyzed using proteases (e.g., trypsin, proteinase K, papain, etc.). Changes in protein activity and structure before and after enzymatic hydrolysis were detected to analyze its resistance to protease hydrolysis.

[0040] 8. Safety Evaluation: The hemolytic activity of recombinant human defensin on erythrocytes was assessed using a hemolysis assay to determine its safety to blood cells during in vivo application. The cytotoxicity of recombinant human defensin to mammalian cells (such as HEK293T and NIH / 3T3 cell lines) was detected using the CCK-8 assay, and its impact on normal cell viability was analyzed to comprehensively evaluate its safety.

[0041] 9. Mechanism of action study: Scanning electron microscopy was used to observe the morphological changes of bacterial cell membranes after treatment with recombinant human defensin, and the destructive effect on the cell membrane was analyzed directly.

[0042] The technical solution of the present invention will be described below using specific embodiments. Unless otherwise specified, the experimental or testing methods in the following embodiments are conventional methods; reagents and materials, unless otherwise specified, are obtained from conventional commercial sources or prepared using conventional methods. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0043] Example 1: Screening and identification of Chlamydomonas recombinant human defensin algal strains 1. Based on the amino acid sequence of human α-defensin 2 (α-HNP2, accession number XP_011533042.1) in the NCBI database (SEQ ID NO.1), codon optimization was performed using the Chlamydomonas reinhardtii codon usage frequency database (https: / / www.novopro.cn / tools / codon-optimization.html), and a triple tandem repeat sequence (3×HNP2) was designed and synthesized by Shanghai Sangon Biotech Co., Ltd. (SEQ ID NO.2).

[0044] 2. The pMO508 plasmid was used as the backbone. Simultaneously, the target gene fragment was amplified using primer pairs 3×HNP2-S and 3×HNP2-AS. The nucleotide sequence of 3×HNP2-S is shown in SEQ ID NO.3, and the nucleotide sequence of 3×HNP2-AS is shown in SEQ ID NO.4. 20 bp homologous arms complementary to both ends of the linearized vector were introduced. The PCR program is shown in Table 1 below: Table 1 PCR Procedure Temperature (°C) time Cycle number 95 5min 1 95 30s 35 54 30s 72 30s 72 10min 1 16 ∞ PCR products were separated by 2% agarose gel electrophoresis and then purified using a DNA gel extraction kit. Homologous recombination was performed using the Gibson Assembly method.

[0045] 3. Positive clones were screened on LB solid medium containing ampicillin, and plasmids were extracted for PCR identification and sequencing verification to obtain the correct recombinant expression vector pMO508-Ptub-3×HNP2-HA-paro. The culture was shaken, plasmids were extracted, and linearized using the restriction endonuclease KpnI.

[0046] 4. The background algal strain *Chlamydomonas* sp. was cultured on TAP medium until the logarithmic growth phase reached approximately 1-2 × 10⁻⁶. 7 1 cell / mL, then diluted to 2-3×10 6 Cells / mL overnight, centrifuged at 2500 rpm for 5 minutes to collect algal cells in the logarithmic growth phase, and calculated to add 2 × 10⁶ cells / mL to each electroporation cuvette. 8 Cells and 250-500 ng of linearized plasmid were pre-cooled and electroporated using a BTX ECM 630 electroporator. The cells were then placed in the dark at 50 μmol photons / (m²). 2 •s) Overnight recovery.

[0047] 5. Spread the transformed algal cell suspension evenly on TAP solid medium plates containing paromomycin (10 mg / L) and culture for 10-14 days under the same culture conditions as above. After single clones grow, pick single clones and transfer them to TAP liquid medium for expansion culture. Collect the cells and boil the samples with SDS to obtain total protein from Chlamydomonas.

[0048] 6. Total protein was separated by 12% SDS-PAGE electrophoresis and transferred to a PVDF membrane by semi-dry transfer. Rabbit anti-HA monoclonal antibody was used as the primary antibody and HRP-labeled goat anti-rabbit IgG (1:5000 dilution) was used as the secondary antibody. Positive transformants that stably expressed 3×HNP2-HA fusion protein were screened by chemiluminescence detection.

[0049] Figure 3 This is an illustration of the identification experiment of Chlamydomonas recombinant human defensin-expressing algal strain. Western blot results show that Chlamydomonas sp. served as the control group (represented by ctrl). When Chlamydomonas sp. was transfected with the 3×HNP2 plasmid, the HA-tagged 3×HNP2 protein was specifically expressed in the "3×HNP2" sample, but not in the Chlamydomonas sp. control sample.

[0050] Example 2: Preparation of Chlamydomonas recombinant human defensin algal powder 1. As described in Example 1, the recombinant human defensin-producing Chlamydomonas strain was screened and obtained. The strain was inoculated into 300 mL of liquid seed culture medium and cultured until the concentration reached 4~7×10⁻⁶. 6 Flame inoculation was performed into the fermenter at a rate of 100 cells / mL for large-scale fermentation culture.

[0051] 2. Ferment to 3~7×10 8 After reaching a cell / mL concentration, fermentation was terminated, cells were collected by rapid low-temperature centrifugation, and then freeze-dried to obtain Chlamydomonas recombinant human defensin algal powder.

[0052] Example 3: Purification of the target protein 1. As described in Example 2.1, algal cells were first obtained by fermentation culture. After collecting the algal cell pellet by centrifugation as described in 2.2, the pellet was resuspended in an appropriate amount of TAP medium and the cells were lysed using a low-temperature high-pressure cell disruptor (1500 bar, 4°C).

[0053] 2. Centrifuge the cell lysate at 15000×g for 15 min (4℃) and collect the cell lysate supernatant containing the target protein. Purify the recombinant human defensin (3×HNP2) using Anti-HA Nanobody Magarose Beads. Mix the supernatant with magnetic agarose beads conjugated with HA tag-specific monoclonal antibodies at a volume ratio of 10:1 and incubate at 4℃ and 120 rpm for 1 h with shaking. Separate the magnetic beads using a magnetic rack and wash three times with PBS buffer (pH 7.4) containing 0.1% Tween-20 for 5 min each time to remove non-specific binding impurities.

[0054] 3. Add 50 μL of 200 mM glycine (pH 2.5) to resuspend the anti-HA beads and incubate for 10 minutes while maintaining good mixing. Separate the beads using a magnetic rack, transfer the supernatant to a new centrifuge tube, add 25 μL of 1M Tris-HCl (pH 10.4) to neutralize the glycine, and the purified target protein will be obtained.

[0055] Figure 5 This is an experimental diagram of the purification and identification of recombinant human defensin from Chlamydomonas. Western blot results show: M represents the protein marker; Lane 1 is the insoluble fraction after cell lysis, and no band was observed in this lane, indicating that most 3×HNP2 proteins did not form inclusion bodies or aggregate; Lane 2 is the supernatant after cell lysis, and a clear and relatively strong band was observed in this lane, with a molecular weight of approximately 22 kDa, consistent with the expected size of HA-tagged 3×HNP2 protein, proving that the target protein mainly exists in the soluble fraction after cell lysis, suitable for downstream purification; Lane 3 is the supernatant after magnetic bead incubation, and no band was observed in this lane, indicating that the target protein almost completely bound to the magnetic beads after incubation; Lane 4 is the eluent after magnetic bead incubation, and this lane shows a very bright and single band with a molecular weight of approximately 22 kDa, consistent with the expected size of HA-tagged 3×HNP2 protein, indicating that the magnetic bead purification yielded high-purity 3×HNP2 target protein.

[0056] Example 4: Detection of genetic stability and analysis of expression levels using Western blotting technique for proteins. 1. As described in Example 1, the Chlamydomonas recombinant human defensin algal strain was screened and obtained. The algal strain was cultured in TAP medium for 1 to 5 months, and samples were taken on the 1st of each month to collect cells.

[0057] 2. The samples were treated with SDS to obtain total protein from Chlamydomonas.

[0058] 3. Total protein was separated by 12% SDS-PAGE electrophoresis and transferred to a PVDF membrane by semi-dry transfer. Rabbit anti-HA monoclonal antibody and histone H3 antibody were used as primary antibodies, and HRP-labeled goat anti-rabbit IgG and goat anti-mouse IgG were used as secondary antibodies. The protein was then analyzed by chemiluminescence detection.

[0059] 4. The grayscale values ​​of the strips were analyzed using the ImageJ software.

[0060] Figure 4 This is a diagram from an experiment analyzing the genetic stability of a Chlamydomonas recombinant human defensin-expressing algal strain. Western blot results showed that, using H3 as an internal control protein to assess the total protein loading, the recombinant human defensin protein (with the HA tag) was stably expressed at different growth stages (1 to 5 months) without a significant upward or downward trend, and its molecular weight was approximately 22 kDa.

[0061] Example 5: Demonstrating the antibacterial activity of recombinant human defensin against Pseudomonas aeruginosa and Escherichia Coli using growth curves. 1. Incubate Pseudomonas aeruginosa ATCC 10145 and Escherichia coli ATCC 10305 at 37℃ and 200 rpm until OD500. 600 =1.0, diluted to 10 5 In CFU / mL culture medium.

[0062] 2. Conduct the experiment in a well plate, setting up an experimental group, a negative control group, and a positive control group.

[0063] 3. Add 100 μL of bacterial suspension to each well. For the experimental group, the protein purified in Example 3 was diluted to 64, 32, 16, 8 and 4 μg / mL, respectively, with 3 replicates for each group. For the positive control group, 20 μL of cefotaxime sodium (CEF) was added to each well, and for the negative control group (NC), 20 μL of bacterial suspension was added to each well.

[0064] 4. Incubate at 37℃ and 200 rpm for 12 h with shaking. OD values ​​are measured at the beginning, 2, 4, 8, 12, and 24 hours. 600 Absorbance value.

[0065] Figure 6This is a graph showing the antibacterial activity of recombinant 3×HNP2 against Gram-negative bacteria. Figures A and B show the growth curves of *Pseudomonas aeruginosa* and *Escherichia coli* at different concentrations of 3×HNP2 (4–64 μg / mL), respectively. A negative control (NC, bacterial buffer) and a positive control (CEF, 1 mg / mL cefotaxime) were included in the experiment. The absorbance (OD) at 600 nm was measured. 600 The growth of bacteria was monitored. Data showed that the growth of both bacteria was significantly inhibited with increasing 3×HNP2 concentration.

[0066] Example 6: Observation of the antibacterial effect against Pseudomonas aeruginosa and Escherichia coli using inhibition zones. 1. Spread the suspensions of Pseudomonas aeruginosa ATCC 10145 and Escherichia coli ATCC 10305 evenly on agar plates and let stand.

[0067] 2. Gently press a sterile Oxford cup (a round tube with an inner diameter of 6 nm, an outer diameter of 8 nm, and a height of 10 nm) into the plate to ensure that there are no gaps between it and the culture medium.

[0068] 3. On a Pseudomonas aeruginosa agar plate, inject less than 80 μL of solution into each Oxford cup. The experimental group is purified 8 μg / mL recombinant human defensin protein (as described in Example 3), the positive control group is 1 μg / mL CEF, the negative control is 20 μg / mL Chlamydomonas sp. total algal protein, and the blank control is 50 mM Tris-HCl solution.

[0069] 4. On E. coli agar plates, inject less than 80 μL of solution into Oxford cups. The experimental group is purified 16 μg / mL recombinant human defensin protein, the positive control group is 1 ng / mL CEF, the negative control is 20 μg / mL Chlamydomonas sp. total algal protein, and the blank control is 50 mM Tris-HCl solution.

[0070] 5. Incubate the plates at 37 ℃ for 24 hours, take photos and observe their inhibition zones.

[0071] Figure 7This is a graph showing the antibacterial activity of 3×HNP2. Figures A and B show agar plates inoculated with *Pseudomonas aeruginosa* (ATCC 10145) and *Escherichia coli* (ATCC 10305), respectively. The following substances were added to Oxford cups: a: cefotaxime sodium (positive control; *Pseudomonas aeruginosa* 1 µg / mL, *Escherichia coli* 1 ng / mL); b: purified 3×HNP2 protein (1× minimum inhibitory concentration, MIC); c: *Chlamydomonas* sp. cell lysate (negative control); d: buffer solution (50 mM Tris / HCl, negative control). Inhibition zones are indicated by the red dashed circles in the graph. The results show that 3×HNP2 exhibits significant antibacterial activity against both bacteria, while no inhibition zone was observed in the negative control group.

[0072] Example 7: Biochemical stability test of recombinant human defensin protein 1. Take the purified recombinant human defensin protein described in Example 3 and prepare protein solutions with a final concentration of 80 μg / mL using Tris-HCl buffer (pH 7.8) as the solvent. Incubate the solutions at 4, 25, 37, 65 and 90 °C for 1 h.

[0073] 2. Take the purified recombinant human defensin protein described in Example 3, and prepare solutions with pH 2, 4, 6, 8 and 10 using the Tris-HCl buffer system. Dissolve 3×HNP2 in each solution to prepare a protein solution with a final concentration of 80 μg / mL, and incubate at 37℃ for 4 h.

[0074] 3. The recombinant human defensin protein was dissolved in 0.2U trypsin, proteinase K and papain solution respectively to make the final concentration of 3×HNP2 protein 80 μg / mL, and incubated at 37℃ for 2 h.

[0075] 4. Take Pseudomonas aeruginosa and Escherichia coli in the logarithmic growth phase and mix them with the 3×HNP2 protein solutions after treatment in Examples 7.1, 7.2 and 7.3, respectively, so that the final concentration of 3×HNP2 in the Pseudomonas aeruginosa suspension is 8 μg / mL and the final concentration of 3×HNP2 in the Escherichia coli suspension is 16 μg / mL.

[0076] 5. The mixture system in Example 7.4 was incubated at a constant temperature of 37 °C for 10 hours, and its OD600 value was measured and recorded.

[0077] Figure 8This is a diagram showing the biochemical stability of recombinant human defensin 3×HNP2. The antibacterial activity of 3×HNP2 against *Pseudomonas aeruginosa* and *Escherichia coli* (expressed as OD500 of cultures) was measured after exposure to different conditions. 600 The values ​​represent bacterial growth status. (A) Temperature stability: 3×HNP2 maintained stable antibacterial activity after pretreatment at 4, 25, 37, 65, and 90°C. (B) pH stability: 3×HNP2 maintained stable antibacterial activity after pretreatment in buffers at pH -2, 4, 6, 8, and 10. (C) Protease stability: After pretreatment with trypsin, proteinase K, or papain, 3×HNP2 retained more than 80% of its inhibitory activity, although trypsin slightly reduced its function. Tris-HCl buffer was used as a negative control (NC) in all experiments. Data are presented as mean ± standard deviation (mean ± SD) of three biological replicates. Statistical analysis was performed using Tukey's post-hoc test (**p < 0.01, ***p < 0.001).

[0078] Example 8: Safety evaluation of recombinant human defensin protein on erythrocytes 1. Collect whole blood from mice in anticoagulant tubes, centrifuge at 4℃ and 1500×g for 5 min to separate red blood cells, and prepare red blood cell suspension (5%, v / v) after washing 3 times with PBS.

[0079] 2. Take the purified 3×HNP2 protein solution described in Example 3 and mix it with 90 μL of red blood cell suspension to make the final concentrations 20, 40, 80, 160 and 320 μg / mL, respectively. Use the Triton X-100 treatment group as a positive control and the PBS treatment group as a negative control. Incubate the mixed solution at 37℃ for 2 h and take pictures to record.

[0080] 3. After incubation, centrifuge to remove the precipitate, and measure the absorbance of the supernatant at 540 nm using a multi-functional microplate reader (Spectra Max i3x, Molecular Devices, USA). Hemolysis rate is calculated using the formula: Hemolysis rate (%) = (OD0.05)2 实验组 -OD PBS ) / (OD Triton X-100 - OD PBS ) × 100%.

[0081] Figure 9This is a graph showing the hemolytic activity of 3×HNP2 against mouse erythrocytes. The biosafety of 3×HNP2 was assessed using a hemolysis assay. Phosphate-buffered saline (PBS) was used as a negative control (N), and 1% Triton X-100 as a positive control (P). The bar graph shows that the hemolysis rate remained extremely low with increasing 3×HNP2 concentrations (0–320 μg / mL): only 0.1% at 160 μg / mL (10–20 times higher than the minimum inhibitory concentration (MIC), and still below 0.5% even at 320 μg / mL. The data indicate that 3×HNP2 has high selectivity for mammalian cell membranes, and its hemolytic toxicity is negligible. The data are from three independent experiments.

[0082] Example 9: Safety evaluation of recombinant human defensin protein in animal cells 1. The cytotoxicity of 3×HNP2 protein against NIH / 3T3 and HEK293T was evaluated using the CCK8 assay. Cells were cultured first, and 100 μL of suspension containing 5000 cells was added to each well of the plate.

[0083] 2. Add the 3×HNP2 protein described in Example 3 to each well, with final concentrations of 10, 20, 40, 80, and 160 μg / mL, respectively. Use PBS as a negative control, 1% Triton X-100 as a positive control, and culture medium only as a blank control. Incubate at 37°C and 5% CO2 for 24 h.

[0084] 3. Add 10 μL of CCK8 solution to each well, mix well, and incubate at 37℃ and 5% CO2 for 1 h. Shake the plate for 10 min, and measure the OD using a microplate reader. 450 Cell viability is calculated using the following formula: Cell viability (%) = (OD sample / OD control) × 100%.

[0085] Figure 10 This is a graph showing the cytotoxicity of 3×HNP2 on NIH / 3T3 and HEK293T mammalian cell lines. Cell viability was assessed 24 hours after exposure to 3×HNP2 protein using the CCK-8 assay. Data are presented as mean ± standard deviation (Mean ± SD) from three independent biological replicates. The results indicate that 3×HNP2 did not significantly affect the viability of either cell line at concentrations up to 160 µg / mL.

[0086] Example 10: Observation of the effect of recombinant human defensin protein on the cell membrane of Gram-negative bacteria using propidium iodide (PI). 1. Take Pseudomonas aeruginosa and Escherichia coli in the logarithmic growth phase, add the recombinant 3×HNP2 protein described in Example 3, and make their final concentrations reach 8 and 16 μg / mL, respectively. Incubate at 37°C for 2 h.

[0087] 2. Collect bacterial cells by centrifugation at 5000×g for 10 min at 4℃, wash twice with pre-cooled 0.1 M PBS (pH 7.4), add 1 mL of 2.5% glutaraldehyde (dissolved in 0.1 M PBS), and fix at 4℃ for 12 h to maintain cell morphology.

[0088] 3. Discard the fixative, wash three times with PBS for 5 min each time, resuspend the bacterial cells in 100 μL of PBS containing 5 μL of PI (1 μg / mL), and incubate at 37°C in the dark for 30 min.

[0089] 4. Centrifuge at 5000×g for 5 min to remove unbound dye. Take 12 μL of bacterial suspension and add it to a glass slide. After covering with a coverslip, observe immediately with an inverted fluorescence microscope.

[0090] Figure 11 This is a membrane permeability analysis diagram based on propidium iodide (PI) staining. To elucidate the antibacterial mechanism of 3×HNP2, its effect on bacterial membrane integrity was assessed by propidium iodide (PI) staining. *Pseudomonas aeruginosa* and *Escherichia coli* were treated with either 1× minimum inhibitory concentration (MIC) of 3×HNP2 or phosphate-buffered saline (PBS, control group). Fluorescence microscopy images showed that both bacteria exhibited significant red fluorescence in the 3×HNP2 treatment group, indicating substantial PI uptake and disruption of cell membrane integrity; no red fluorescence was observed in the PBS control group, confirming that the cell membrane remained intact.

[0091] Example 11: Observation of the effect of recombinant human defensin protein on the cell membrane of Gram-negative bacteria by scanning electron microscopy (SEM) 1. Take Pseudomonas aeruginosa and Escherichia coli in the logarithmic growth phase, add the recombinant 3×HNP2 protein described in Example 3, and make their final concentrations reach 8 and 16 μg / mL, respectively. Incubate at 37℃ for 1 h, and collect the bacterial cells by centrifugation at 4℃ and 5000×g for 10 minutes.

[0092] 2. Gently wash the bacterial cells twice with pre-cooled 0.1 M PBS (pH 7.4), discard the supernatant, add 1 mL of 4% paraformaldehyde (dissolved in 0.1 M PBS), and fix at 4°C for 12 h.

[0093] 3. Wash the fixed sample three times with 0.1 M PBS for 5 min each time, and dehydrate it step by step: dehydrate it with 30%, 50%, 70%, 80% and 90% ethanol in sequence (10 min each time), and then dehydrate it twice with 100% ethanol (10 min each time).

[0094] 4. Perform critical point drying: Place the sample in an ethanol-tert-butanol (1:1) mixture and replace it twice (10 min each time), then transfer it to tert-butanol for 15 min, freeze it at -80℃ and then vacuum dry it.

[0095] 5. After the dried sample is sputter-coated with gold, it is placed under an ultra-high resolution cold field emission scanning electron microscope (SU8100, HITACHI, Japan) for observation and image acquisition.

[0096] Figure 12 These are SEM images of the changes in cell membrane morphology of Pseudomonas aeruginosa and Escherichia coli before and after treatment with human defensin. The left image shows the untreated group with intact and smooth cell membranes, while the right image shows the treated group, which exhibits significant morphological damage, including cell membrane shrinkage, deformation, and rupture (indicated by arrows).

[0097] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A recombinant human-derived defensin gene, characterized in that, The nucleotide sequence of the recombinant human-derived defensin gene is shown as SEQ ID NO.

2.

2. A recombinant expression vector or transformant or transgenic Chlorella containing the recombinant human-derived defensin gene of claim 1.

3. Use of the transgenic Chlorella of claim 2 in the preparation of health food, bait for fish or feed additive for livestock and poultry.

4. A method for constructing a transgenic Chlamydomonas expressing the recombinant human defensin gene of claim 1, characterized by, The method comprises the following steps: Step A1, constructing a recombinant expression vector: connecting the recombinant human-derived defensin gene of claim 1 with an expression vector containing an endogenous strong promoter, a terminator and a protein expression tag sequence of Chlorella to construct the recombinant expression vector; Step A2, genetic transformation of Chlorella: introducing the recombinant expression vector constructed in step A1 into a background strain of Chlorella to obtain a genetic transformant; Step A3, screening and identification: screening and identifying the genetic transformant obtained in step A2 to obtain the transgenic Chlorella in which the recombinant human-derived defensin gene has been successfully integrated into the genome of Chlorella.

5. A method for preparing a recombinant human defensin having antibacterial activity using Chlamydomonas, characterized in that, The method comprises the following steps: Step B1, obtaining the transgenic Chlorella by the construction method of claim 4; Step B2, expanding the culture of the transgenic Chlorella, and after the culture is completed, centrifuging to collect the algal cells or culture solution, releasing soluble proteins by cell disruption, and then purifying by affinity chromatography to obtain high-purity recombinant human-derived defensin.

6. Use of the recombinant human-derived defensin prepared by the method of claim 5 in the preparation of an antibacterial agent.

7. Use according to claim 6, characterized in that, The antibacterial agent is a medical, daily chemical, industrial or agricultural antibacterial agent.

8. A method for preparing Chlamydomonas reinhardtii recombinant human defensin Chlamy powder, characterized by, The method comprises the following steps: Step C1, obtaining the transgenic Chlorella by the construction method of claim 4; Step C2, the transgenic Chlamydomonas is expanded to a concentration of 2-4 x 10 6 cells / mL and then flame inoculated into a fermentor for fermentation culture. Step C3, after fermentation to 5~9x10 8 After fermentation is terminated, the cells are collected by rapid low-temperature centrifugation and freeze-dried to obtain the Chlamydomonas reinhardtii recombinant human defensin algae powder.

9. A method for preparing a recombinant human defensin protein from Chlamydomonas, characterized by, The method comprises the following steps: Step D1, preparing the Chlorella recombinant human-derived defensin algal powder by the preparation method of claim 8; Step D2, affinity chromatography of the Chlorella recombinant human-derived defensin algal powder obtained in step D1 to obtain the Chlorella recombinant human-derived defensin protein.

10. Use of the Chlorella recombinant human-derived defensin algal powder prepared by the preparation method of claim 8 or the Chlorella recombinant human-derived defensin protein prepared by the preparation method of claim 9 in the fields of medicine, food, industry and agriculture.

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