Application method of chitosan nanoparticle gene delivery system in fish sex proportion regulation

Abstract

The invention belongs to the technical field of biological medicine, discloses an application method of a chitosan nano-particle gene delivery system in fish sex proportion regulation, and provides an oral chitosan nano-gene delivery system aiming at the problems of complicated operation, strong fish body stress response and high treatment cost in the existing gene delivery technology. The preparation method comprises the following steps: compounding a key gonad differentiation gene cyp19a1a of lateolabrax japonicus with chitosan, so as to construct the chitosan nanoparticles loaded with pcDNA3.1-CYP19A1A. Results show that the gene-loaded chitosan nanoparticles can effectively enter cells and realize the expression of exogenous genes, almost have no toxic effect, and can achieve an instantaneous transgenosis effect without modifying the genome of an organism. According to the system, feminization phenotypes are induced by regulating and controlling the estradiol content, and the female proportion of bred populations is remarkably increased. The method has high efficiency, safety and economical efficiency, and provides a reproducible solution for industrialization of an aquaculture sex control technology.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to an application method of a chitosan nanoparticle gene delivery system in regulating the sex ratio of fish. Background Art

[0002] Chitosan is a naturally occurring polycationic alkaline polysaccharide that forms stable polyelectrolyte complexes with genetic material and exhibits excellent biocompatibility, antibacterial properties, and adhesion. It is widely used in drug delivery systems. Chitosan nanoparticles are an important form of chitosan, offering advantages such as low toxicity, sustainability, biodegradability, biocompatibility, and low cost. They can improve the stability of genetic material in vivo and in vitro. Compared to traditional liposome transfection reagents, chitosan nanoparticles are virtually non-toxic to cells, offering significant advantages in gene delivery.

[0003] In recent years, chitosan nanoparticles have been used as delivery vehicles primarily in pharmaceuticals and medicine, biomaterials, agricultural applications, gene delivery, and food and nutrition applications. Currently, chitosan nanoparticles have demonstrated broad application potential in poultry, humans, and plants. These include their use as feed additives in poultry for the delivery of traditional Chinese medicines (CN116391798A) and vaccines (CN110251483A, CN108383913A). In plants, chitosan nanoparticles are delivered via leaf penetration or root absorption, enabling efficient gene editing to enhance stress resistance (CN119613179A). However, while the delivery mechanisms and application scenarios of chitosan nanoparticles in terrestrial organisms are relatively well established, research on their application in aquatic organisms such as fish remains exploratory. Existing reports are mostly limited to preliminary experiments in fish models, focusing on the delivery of RNA interference molecules or gene editing tools via intramuscular injection or immersion, but systematic conclusions on their delivery efficiency, tissue targeting, and long-term safety have yet to be drawn. The unique physiological environment of fish and the complexity of water exposure stimulate the stress response and protective humoral response of fish, increase time and labor costs, and further increase the challenges of the application of chitosan nanoparticles in fish. Summary of the Invention

[0004] In response to the problems existing in the prior art, the present invention provides an application method of a chitosan nanoparticle gene delivery system in regulating the sex ratio of fish.

[0005] The present invention is achieved by a chitosan nanoparticle gene delivery system for controlling fish sex ratio, comprising:

[0006] Step 1: Prepare chitosan nanoparticles by ionic crosslinking method: dissolve chitosan in acetic acid solution and stir magnetically at 37°C for 1 hour;

[0007] Step 2: adding the sodium tripolyphosphate solution dropwise to the chitosan solution in a certain ratio, stirring at a constant temperature, filtering the solution after the reaction is completed to remove unreacted insoluble impurities, and obtaining a chitosan nano solution;

[0008] Step 3: construct the vector, extract the plasmid, and prepare the chitosan nanoparticles of the vector gene by complex coacervation method. Add the chitosan solution to the diluted plasmid solution in a certain proportion, place it in a metal bath under certain conditions, mix it, and vortex it for 30 seconds to obtain the gene-loaded chitosan nanoparticles.

[0009] Furthermore, the mass ratio of the chitosan solution to the sodium tripolyphosphate solution is 6:1, the average particle size of the chitosan nanoparticles is about 130 nm, and the PDI is less than 0.3.

[0010] Further, the plasmid solution was diluted to 100 μg / mL with 6 mmoL / L Na2SO4;

[0011] Chitosan nanoparticles prepared under the conditions of pH = 5.5 and chitosan / plasmid = 1:1 had a moderate particle size. Gel electrophoresis and encapsulation efficiency determination confirmed that they could effectively encapsulate pDNA and had low toxicity.

[0012] Furthermore, a nanoparticle complex is formed through electrostatic adsorption.

[0013] Furthermore, the chitosan solution was mixed with an equal volume of the plasmid solution under the following reaction conditions: preheating at 55°C for 20 min, mixing, and vortexing for 30 s.

[0014] Furthermore, the mode of intake is oral administration, where it is absorbed through the gastrointestinal mucosa and then enters the epithelial cells for expression;

[0015] The cyp19a1a gene was loaded into the chitosan nanoparticle gene delivery system to regulate the sex ratio of fish;

[0016] After stopping the delivery of exogenous genes by chitosan nanoparticles for a period of time, the tissues in the body will no longer express the gene;

[0017] The fish sex ratio regulation refers to increasing the estradiol content in fish, affecting the gonadal differentiation of fish, and increasing the female ratio of fish;

[0018] The species is Lateolabrax maculatus.

[0019] Another object of the present invention is to provide a chitosan nanoparticle gene delivery system for fish sex ratio regulation application system comprising:

[0020] A stirring module was used to prepare chitosan nanoparticles using an ionic crosslinking method. Chitosan was dissolved in acetic acid solution and magnetically stirred at 37°C for 1 h.

[0021] The filtration module is used to add the sodium tripolyphosphate solution dropwise into the chitosan solution according to a certain ratio, stir at a constant temperature, filter the solution after the reaction is completed, remove unreacted insoluble impurities, and obtain the chitosan nano solution;

[0022] The extraction module is used to construct the vector, extract the plasmid, and prepare the chitosan nanoparticles containing the vector gene by complex coacervation. The chitosan solution is added to the diluted plasmid solution in a certain proportion, placed in a metal bath under certain conditions, and mixed. The mixture is vortexed for 30 seconds to obtain the gene-loaded chitosan nanoparticles.

[0023] In combination with the above technical solutions and the technical problems solved, please analyze the advantages and positive effects of the technical solutions to be protected by the present invention from the following aspects:

[0024] First, the present invention addresses a technical issue currently facing fish transgenic technology: the significant limitations of microinjection systems used in fish transgenic technology. Due to the unique ootheca structure of teleost oocytes and their tiny size (typically less than 1 mm in diameter), traditional microinjection techniques suffer from limitations such as insufficient positioning accuracy, high cell damage rates, low efficiency, and long processing times. Therefore, to improve the efficiency of fish transgenic technology, the present invention has developed a chitosan nanoparticle-based transient transgenic technology. By loading genes into a chitosan nanoparticle gene delivery system and orally absorbing them through the fish gastrointestinal mucosa, the genes enter epithelial cells and are expressed, achieving transient transgenic technology. This overcomes the limitations of traditional fish transgenic technology and enables a non-invasive transgenic method.

[0025] This invention addresses the safety concerns inherent in transgenic organisms: the biosafety of transgenic aquatic animals has become a significant factor restricting their industrial application. Transgenic species can lead to genetic homogeneity and biodiversity loss, as well as food safety issues such as allergic reactions or metabolic disruptions caused by exogenous gene products. The transient transgenic technology developed by this invention differs from traditional transgenic organisms in that the exogenous genes introduced through oral ingestion are not integrated into the host genome but exist in a free form. Gene expression is gradually diluted with cell division and gradually degraded and lost over time. This transient expression characteristic reduces the potential impact of exogenous genes on host genome stability, avoids biosafety issues such as gene escape and niche competition that can occur with traditional transgenic fish, and is more in line with environmental ethics.

[0026] Second, the expected benefits and commercial value of the technical solution of the present invention after transformation are as follows: the present invention uses chitosan nanoparticles loaded with pcDNA3.1-CYP190A1A as feed additives, and induces feminization of Lateolabrax japonicus in a non-invasive delivery manner, thereby accurately achieving sex control of Lateolabrax japonicus, breaking through the limitations of traditional intramuscular injection on fish farming specifications, increasing the scale of single processing, and reducing processing costs, providing a replicable technical paradigm for the industrialization of sex control technology, and significantly improving the economic benefits of farming.

[0027] This invention successfully solves the long-standing problem of lengthy breeding cycles in fish sex control technology. Traditional sex selection techniques rely on multi-generation backcrossing or hormone induction, and have inherent drawbacks such as long operating cycles, endocrine disruption, and uncontrollable ecological risks. By feeding chitosan nanoparticles loaded with CYP19A1A to increase the estradiol content of Lateolabrax japonicus, the present invention induces female-oriented gonadal differentiation in Lateolabrax japonicus within 40 days, breaking through the technical bottleneck of rapid breeding of sexually dimorphic species and providing an innovative paradigm for the aquatic breeding industry that combines high efficiency and biosafety.

[0028] Does the present invention's technical solution overcome technological bias? By constructing a transient transgenic system, the present invention innovatively achieves the spatiotemporal controllable expression of exogenous genes, effectively circumventing the biosafety concerns associated with the permanent integration of genetic material in traditional transgenic technologies. Validation experiments demonstrated that no exogenous gene expression was detectable in fish stomach tissue on day 6. This transient expression system does not trigger genomic insertional mutagenesis or epigenetic memory effects, completely eliminating the risk of transgenerational transmission of exogenous genes and providing an innovative solution to the ethical controversies surrounding transgenic technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a flow chart of a method for applying the chitosan nanoparticle gene delivery system provided by an embodiment of the present invention to regulate the sex ratio of fish.

[0030] Figure 2 This is a structural block diagram of the chitosan nanoparticle gene delivery system provided by an embodiment of the present invention for use in regulating the sex ratio of fish.

[0031] Figure 3 This is a rendering of an embodiment of the present invention.

[0032] Figure 4 The nucleotide sequence and the amino acid sequence encoded by the cyp19a1a gene of Lateolabrax japonicus provided in the embodiment of the present invention are shown in FIG. The stop codon is marked with an "*".

[0033] Figure 5This is a Western blot analysis of CYP19A1A protein expression in HEK-293T cells using the methods provided in the examples of the present invention. (+): Positive cells transfected with pcDNA3.1-CYP19A1A-His; (-): Control group without plasmid transfection.

[0034] Figure 6 1 is a graph showing the particle size, PDI, and Zeta potential of CS-CYP19A1A-NPs provided in an embodiment of the present invention.

[0035] Figure 7 This is a transmission electron microscope image of CS-CYP19A1A-NPs provided in an embodiment of the present invention.

[0036] Figure 8 This is a DNA binding and DNA protection experiment for CS-CYP19A1A-NPs and CS-GH1-NPs, as provided in the examples of the present invention. Lane numbers are: 1. Naked plasmid pcDNA3.1-CYP19A1A; 2. Naked plasmid pcDNA3.1-GH1; 3. CS-CYP19A1A-NPs; 4. CS-GH1-NPs; 5. DNase I-treated naked plasmid pcDNA3.1-CYP19A1A; 6. DNase I-treated naked plasmid pcDNA3.1-GH1; 7. DNase I-treated CS-CYP19A1A-NPs; 8. DNase I-treated CS-GH1-NPs.

[0037] Figure 9 This is a HEK-293T cell uptake experiment provided in an embodiment of the present invention. The uptake effect diagram of CS-CYP19A1A-mNG-NPs and Lipo8000 by HEK-293T cells at 24h and 48h.

[0038] Figure 10 Figures A and B show the cytotoxic effects of CS-CYP19A1A-NPs, naked plasmids, and liposomes Lipo8000 on HEK-293T cells after treatment with different plasmid concentrations (ng / well) for 24 and 48 hours (*P < 0.05, **P < 0.01, ***P < 0.001).

[0039] Figure 11 Western blot analysis of CYP19A1A protein expression in the gastrointestinal tract of Lateolabrax japonicus (Japonicus japonicus). A and B show the Lateolabrax japonicus intestine; C and D show the Lateolabrax japonicus stomach.

[0040] Figure 12This is the immunohistochemical localization of CYP19A1A in the intestine and stomach of Lateolabrax japonicus, as provided in the examples of the present invention. A, D: CYP19A1A-loaded chitosan nanoparticles (red arrows indicate positive signals); B, E: Negative controls without primary antibody. C, F: Blank controls.

[0041] Figure 13 This is a diagram showing the effect of CS-CYP19A1A-NPs provided in an embodiment of the present invention on the estradiol content in Lateolabrax japonicus.

[0042] Figure 14 This is a diagram showing the effect of CYP19A1A-loaded chitosan nanoparticles on the sex ratio of Lateolabrax japonicus provided by an embodiment of the present invention.

[0043] Figure 15 This is a diagram showing the effects of CYP19A1A-loaded chitosan nanoparticles provided in an embodiment of the present invention on serum physiological and biochemical indices of Lateolabrax japonicus.

[0044] Figure 16 3. HE staining of various organs of Lateolabrax japonicus in the experimental group of chitosan nanoparticles loaded with CYP19A1A and the blank control group provided in the examples of the present invention. DETAILED DESCRIPTION

[0045] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0046] like Figure 1 As shown, the application method of a chitosan nanoparticle gene delivery system in regulating the sex ratio of fish provided by an embodiment of the present invention includes the following steps:

[0047] S101, chitosan nanoparticles were prepared by ionic crosslinking method. Chitosan was dissolved in acetic acid solution and magnetically stirred at 37 °C for 1 h.

[0048] S102, adding the sodium tripolyphosphate solution dropwise to the chitosan solution in a certain ratio, stirring at a constant temperature, filtering the solution after the reaction is completed to remove unreacted insoluble impurities, and obtaining a chitosan nano solution;

[0049] S103, constructing the vector, extracting the plasmid, and preparing chitosan nanoparticles containing the vector gene by complex coacervation method. Chitosan solution is added to the diluted plasmid solution in a certain proportion, placed in a metal bath under certain conditions for mixing, and vortexed for 30 seconds to obtain gene-loaded chitosan nanoparticles.

[0050] The chitosan solution and sodium tripolyphosphate (TPP) solution provided in the embodiment of the present invention have a mass ratio of 6:1, the average particle size of the chitosan nanoparticles is about 130 nm, and the polydispersity index (PDI) is less than 0.3.

[0051] The plasmid solution provided in the embodiment of the present invention was diluted to 100 μg / mL with 6 mmol / L Na2SO4;

[0052] Chitosan nanoparticles prepared under the conditions of pH = 5.5 and chitosan / plasmid = 1:1 had a moderate particle size. Gel electrophoresis and encapsulation efficiency determination confirmed that they could effectively encapsulate pDNA and had low toxicity.

[0053] The nanoparticle complex provided by the embodiment of the present invention is formed by electrostatic adsorption, does not require the use of organic solvents, and does not damage the structure and function of the plasmid.

[0054] The chitosan solution provided in the embodiment of the present invention was mixed with an equal volume of the plasmid solution under the following reaction conditions: preheating at 55° C. for 20 minutes, mixing, and vortexing for 30 seconds.

[0055] The method of administration provided by the embodiment of the present invention is oral administration. After being absorbed by the gastrointestinal mucosa, the gene enters the epithelial cells and is expressed, thereby achieving transient transgenic fish.

[0056] The cyp19a1a gene was loaded into the chitosan nanoparticle gene delivery system to regulate the sex ratio of fish;

[0057] After a period of time when chitosan nanoparticles are stopped from delivering exogenous genes, the tissues in the body will no longer express the gene. This technology does not modify the genome of the organism and does not fall into the category of gene editing, so there is no ecological risk.

[0058] The fish sex ratio regulation refers to increasing the estradiol content in fish, affecting the gonadal differentiation of fish, and increasing the female ratio of fish;

[0059] The species is Lateolabrax maculatus.

[0060] like Figure 2 As shown, the chitosan nanoparticle gene delivery system provided by the embodiment of the present invention is applied in the fish sex ratio regulation system, including:

[0061] A stirring module was used to prepare chitosan nanoparticles using an ionic crosslinking method. Chitosan was dissolved in acetic acid solution and magnetically stirred at 37°C for 1 h.

[0062] The filtration module is used to add the sodium tripolyphosphate solution dropwise into the chitosan solution according to a certain ratio, stir at a constant temperature, filter the solution after the reaction is completed, remove unreacted insoluble impurities, and obtain the chitosan nano solution;

[0063] The extraction module is used to construct the vector, extract the plasmid, and prepare the chitosan nanoparticles containing the vector gene by complex coacervation. The chitosan solution is added to the diluted plasmid solution in a certain proportion, placed in a metal bath under certain conditions, and mixed. The mixture is vortexed for 30 seconds to obtain the gene-loaded chitosan nanoparticles.

[0064] The present invention loads gene plasmids into chitosan nanoparticles to construct a chitosan nanogene delivery system. The chitosan nanoparticles were characterized and tested for performance. The results showed that the chitosan nanoparticles were evenly distributed, the system was stable, the gene plasmid encapsulation efficiency reached more than 70%, and the plasmids could be effectively encapsulated. The results of agarose gel electrophoresis analysis showed that the chitosan nanoparticles successfully bound to DNA, had a protective effect on DNA, and could effectively resist degradation by nucleases. HEK-293T cells were transfected to determine the expression and cytotoxicity. The results showed that the gene-loaded chitosan nanoparticles could effectively enter the cells and achieve the expression of exogenous genes, and had almost no inhibitory effect on HEK-293T cells, indicating that they had almost no toxic effects. At the same time, a chitosan nano solution feeding experiment was carried out on Lateolabrax maculatus to verify its transient transgenic effect. The results showed that the target gene can be normally expressed in the gastrointestinal tract of the striped sea bass, and as the feeding end time continues to increase, the expression of the transfected exogenous gene is also continuously reduced. On the 5th day after the end of feeding, the expression of the target gene cannot be detected in the intestine, and on the 6th day after the end of feeding, the expression of the target gene cannot be detected in the stomach.

[0065] Based on the above research, the present invention proposes the application of chitosan nanogene delivery system in regulating the sex ratio of fish. The cyp19a1a gene of Lateolabrax japonicus was loaded into the chitosan nanogene delivery system to construct chitosan nanoparticles loaded with pcDNA3.1-CYP19A1A.

[0066] Among them, preferably, the fish sex ratio regulation refers to increasing the estradiol content of fish, affecting the gonadal differentiation of fish, and increasing the female ratio of fish.

[0067] Preferably, the restriction enzyme cleavage sites of pcDNA3.1-CYP19A1A-His are HindIII and BamH I.

[0068] Furthermore, the present invention also proposes a method for constructing the pcDNA3.1-CYP19A1A chitosan nanoparticles, comprising the following steps:

[0069] Step 1: Construction of the eukaryotic expression vector pcDNA3.1-CYP19A1A-His

[0070] pcDNA3.1(+) served as the eukaryotic expression vector backbone. PCDNA3.1-C-terminal-6×His was modified with Hind III and BamH I restriction sites. After agarose gel electrophoresis, the linearized vector was recovered by gel excision and purification. Using a correctly sequenced cyp19a1a-positive clone as a template, primers with homology arms were designed. The target DNA fragment was amplified by PCR. After gel excision and recovery, homologous recombination was performed. The product was transformed into competent Escherichia coli DH5α. Positive colonies were screened on plates containing ampicillin, and sequence accuracy was verified by sequencing.

[0071] Step 2: Plasmid extraction and eukaryotic vector expression verification

[0072] (1) The bacterial suspension was inoculated into 150 mL of LB liquid medium containing ampicillin, and cultured in a constant temperature shaker at 37°C for 12 to 16 hours to extract the eukaryotic expression plasmid.

[0073] (2) HEK-293T cells at passage 4 were plated and transfection experiments were performed after the cell coverage reached 60% to 70%. A Lip8000 experimental group and a blank control group were set up. Before transfection, a mixed solution of transfection reagent and plasmid was prepared. After completion, the culture medium was replaced and the mixed solution of transfection reagent and plasmid was added according to the requirement of 2.5 μg plasmid concentration per well. Cell proteins were collected 24h and 48h after transfection and immunoblotting experiments were performed.

[0074] Step 3: Chitosan nanoparticle preparation

[0075] Chitosan nanoparticles (CS-NPs) were prepared using an ionic crosslinking method. A 1% acetic acid solution was prepared, and the pH was adjusted to 2.8-4.0 with sodium hydroxide. 4 mg of sodium tripolyphosphate (TPP) powder was dissolved in 50 mL of pure water to a concentration of 0.8 mg / mL. 10 mg of chitosan was dissolved in 50 mL of 1% acetic acid solution and magnetically stirred at 37°C for 1 hour. The TPP solution was added dropwise to the CS solution at a 6:1 mass ratio of CS to TPP, with constant stirring at 37°C and 800 rpm. After the addition was complete, constant stirring was continued for 1 hour. The resulting solution was filtered through a 0.45 μm filter membrane to remove unreacted insoluble impurities, yielding a CS-NPs solution.

[0076] Step 4: Preparation of CYP19A1A-loaded chitosan nanoparticles

[0077] Gene-loaded chitosan nanoparticles were prepared using a complex coacervation method. A pcDNA3.1-CYP19A1A plasmid solution was diluted to 100 μg / mL with 6 mmol / L Na₂SO₄. Equal volumes of chitosan solution and 100 μg / mL plasmid were placed in 1.5 mL centrifuge tubes. The tubes were preheated in a metal bath at 55°C for 20 minutes, mixed, and vortexed for 30 seconds to obtain CYP19A1A-loaded chitosan nanoparticles.

[0078] Figure 4 The nucleotide sequence and the amino acid sequence encoded by the cyp19a1a gene of Lateolabrax japonicus provided in the embodiment of the present invention are shown in FIG. The stop codon is marked with an "*".

[0079] Figure 5 This is a Western blot analysis of CYP19A1A protein expression in HEK-293T cells using the methods provided in the examples of the present invention. (+): Positive cells transfected with pcDNA3.1-CYP19A1A-His; (-): Control group without plasmid transfection.

[0080] Figure 6 1 is a graph showing the particle size, PDI, and Zeta potential of CS-CYP19A1A-NPs provided in an embodiment of the present invention.

[0081] Figure 7 This is a transmission electron microscope image of CS-CYP19A1A-NPs provided in an embodiment of the present invention.

[0082] Figure 8 This is a DNA binding and DNA protection experiment of CS-CYP19A1A-NPs provided in an embodiment of the present invention. Lane numbers are: 1. Naked plasmid pcDNA3.1-CYP19A1A; 3. CS-CYP19A1A-NPs; 5. Naked plasmid pcDNA3.1-CYP19A1A treated with DNase I; 7. CS-CYP19A1A-NPs treated with DNase I.

[0083] Figure 9 This is a HEK-293T cell uptake experiment provided in an embodiment of the present invention. The uptake effect diagram of CS-CYP19A1A-mNG-NPs and Lipo8000 by HEK-293T cells at 24h and 48h.

[0084] Figure 10Figures A and B show the cytotoxic effects of CS-CYP19A1A-NPs, naked plasmids, and liposomes Lipo8000 on HEK-293T cells after treatment with different plasmid concentrations (ng / well) for 24 and 48 hours (*P < 0.05, **P < 0.01, ***P < 0.001).

[0085] Example 1

[0086] Construction and preparation of CYP19A1A chitosan nanoparticle gene delivery system

[0087] Construction of the eukaryotic expression vector pcDNA3.1-CYP19A1A-His: pcDNA3.1(+) served as the eukaryotic expression vector backbone and was modified to obtain pcDNA3.1-C-terminal-6×His, with restriction sites Hind III and BamH I. After agarose gel electrophoresis, the linearized vector was recovered by gel excision and purification. Using a correctly sequenced cyp19a1a-positive clone as a template, primers with homology arms were designed. The target DNA fragment was amplified by PCR. After gel excision and gel recovery, homologous recombination was performed. The product was transformed into competent Escherichia coli DH5α, and positive colonies were selected on plates containing ampicillin. Sequence accuracy was verified by sequencing.

[0088] Plasmid extraction and eukaryotic vector expression verification: The bacterial suspension was inoculated into 150 mL of LB liquid medium containing ampicillin and incubated on a 37°C shaker for 12–16 hours to extract the eukaryotic expression plasmid. HEK-293T cells at passage 4 were plated and transfection experiments were performed after the cell confluence reached 60%–70%. Lip8000 experimental groups and blank controls were set up. Before transfection, a mixed solution of transfection reagent and plasmid was prepared. After transfection, the medium was replaced, and the mixed solution of transfection reagent and plasmid was added to achieve a plasmid concentration of 2.5 μg per well. Cell proteins were collected 24 and 48 hours after transfection for immunoblotting.

[0089] Preparation of Chitosan Nanoparticles: Chitosan nanoparticles (CS-NPs) were prepared using an ionic crosslinking method. A 1% acetic acid solution was prepared, and the pH was adjusted to 2.8-4.0 with sodium hydroxide. 4 mg of sodium tripolyphosphate (TPP) powder was dissolved in 50 mL of pure water to a concentration of 0.8 mg / mL. 10 mg of chitosan was dissolved in 50 mL of 1% acetic acid solution and magnetically stirred at 37°C for 1 hour. The TPP solution was added dropwise to the CS solution at a 6:1 mass ratio of CS to TPP, with constant stirring at 800 rpm at 37°C. After the addition was complete, constant stirring was continued for 1 hour. The reacted solution was filtered through a 0.45 μm filter membrane to remove unreacted insoluble impurities, yielding the CS-NPs solution.

[0090] Preparation of CYP19A1A-loaded chitosan nanoparticles: Gene-loaded chitosan nanoparticles were prepared using a complex coacervation method. The pcDNA3.1-CYP19A1A plasmid solution was diluted to 100 μg / mL with 6 mmol / L Na₂SO₄. Equal volumes of chitosan solution and 100 μg / mL plasmid were placed in 1.5 mL centrifuge tubes. The tubes were preheated in a metal bath at 55°C for 20 minutes, mixed, and vortexed for 30 seconds to obtain CYP19A1A-loaded chitosan nanoparticles.

[0091] Example 2

[0092] Evaluation of the effect of chitosan nanoparticle gene delivery on sex differentiation of Lateolabrax japonicus

[0093] The pcDNA3.1-CYP19A1A-His chitosan nanoparticle solution prepared by the complex coacervation method was evenly sprayed on the surface of the feed to prepare the feed. The expression and localization of CYP19A1A-loaded chitosan nanoparticles in the gastrointestinal tract of the Lateolabrax were detected through feeding experiments on Lateolabrax, and the estrogen content and toxic effects in vivo were determined.

[0094] Depend on Figure 11 It can be seen that the expression of CYP19A1A can be detected in the gastrointestinal tract of the experimental group of sea bass, while no expression of CYP19A1A was found in the control group, indicating that chitosan nanoparticles loaded with CYP19A1A can be normally expressed in the digestive tract of sea bass.

[0095] Depend on Figure 12 It can be seen that positive signals can be detected in the gastrointestinal tract of the experimental group of sea bass fed with CYP19A1A-loaded chitosan nanoparticles, while no positive signals were observed in the blank control group and the negative control group, indicating that the CYP19A1A-chitosan nanocomplex can penetrate the gastrointestinal mucosal barrier, be absorbed by epithelial cells and express active proteins.

[0096] Depend on Figure 13 It can be seen that the estrogen content of the sea bass fed with CYP19A1A chitosan nanoparticles feed was significantly higher than that of the control group.

[0097] Depend on Figure 14 It can be seen that 40 days of CS-CYP19A1A-NPs treatment can induce female gonadal differentiation of Lateolabrax japonicus, and the female proportion of the experimental group is 18% higher than that of the control group.

[0098] Depend on Figure 15 It can be seen that there was no significant difference in the levels of alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, albumin, total cholesterol, and blood glucose between the CS-CYP19A1A-NPs group and the control group.

[0099] Depend on Figure 16 As can be seen, no ventricular hypertrophy, wall thinning, or abnormalities such as congestion, hemorrhage, or infarction were observed in either the experimental or control groups. HE staining revealed regular myocardial fiber arrangement, normal nuclei with an oval shape and central location, and no obvious areas of cellular degeneration or necrosis. The livers of both the experimental and control groups were normal in size, texture, and color. HE staining revealed hepatocytes arranged in cords, with round or oval nuclei located centrally and uniformly stained. The cytoplasm was abundant, lightly stained, and contained granular material. The spleens of both the experimental and control groups were not grossly enlarged, with normal color, texture, and margins. HE staining revealed numerous erythrocytes and macrophages in the red pulp, with darkly stained nuclei and lightly stained cytoplasm. The white pulp was composed of densely packed lymphocytes, with darkly stained nuclei and less abundant, lightly stained cytoplasm. The branchial arches of both the experimental and control groups were neatly arranged and bright red, with no foreign matter or abnormal lesions. HE staining revealed neatly arranged gill filaments, clear gill lamellae, and intact epithelial cells with no apparent shedding or damage. The stomach structure, color, and texture were normal in both the experimental and control groups. HE staining revealed neatly arranged gastric epithelial cells with basally located nuclei; the connective tissue in the submucosa was evenly distributed, without edema or fibrosis, and with no apparent inflammatory cell infiltration between the muscularis layers. The intestinal tract of both the experimental and control groups showed normal thickness and texture by naked eye. HE staining revealed neatly arranged intestinal epithelial cells with basally located nuclei and uniform staining. The villi structure was clear, with tightly arranged villus epithelial cells and a moderate number of goblet cells. The smooth muscle fibers of the intestinal wall were neatly arranged, with no apparent inflammatory cell infiltration between the muscularis layers.

[0100] The chitosan nanoparticles loaded with pcDNA3.1-CYP19A1A-His prepared by the complex coacervation method were characterized, examined by transmission electron microscopy, and the encapsulation efficiency was determined to evaluate the degree of DNA protection of the nanoparticle complexes and to detect cytotoxicity.

[0101] The results showed that after the pcDNA3.1-CYP19A1A-His eukaryotic vector was constructed, it was transfected into HEK-293T cells. CYP19A1A was expressed normally after 24h and 48h, and no protein was expressed in the control group, indicating that the eukaryotic vector was successfully constructed. Figure 5 The particle size, PDI and zeta potential results of the CS-CYP19A1A-NPs nanoparticle complex showed that the average particle size of CS-CYP19A1A-NPs was 200 nm, the PDI was 0.137, which was less than 0.3, and the zeta potential was +18.9 mV, which was positive. The particle size distribution was good, indicating that the CS-CYP19A1A-NPs were successfully prepared ( Figure 6Transmission electron microscopy results showed that CYP19A1A-chitosan nanoparticles were mainly oval in shape with a smooth surface structure. The particle size observed by electron microscopy was consistent with the results of ZetaSizer particle size analyzer, with an average diameter of approximately 200 nm ( Figure 7 ). The encapsulation efficiency of chitosan nanoparticles for pcDNA3.1-CYP19A1A plasmid was calculated to be over 70% by ultramicro spectrophotometer detection, indicating that the CYP19A1A-chitosan nanocomplex can effectively encapsulate the plasmid, which is beneficial for gene delivery. No obvious aggregation and sedimentation were observed with the naked eye within 5 days, and continuous monitoring of the ZetaSizer particle size analyzer for 5 days showed that its particle size and potential were stable. The results of the gel retardation experiment showed that the plasmid pcDNA3.1-CYP19A1A in the CS-CYP19A1A-NPs group was retained in the sample well, and no DNA bands appeared on the corresponding lanes. In contrast, the naked plasmid in the control group showed a clear DNA band on the lane, indicating that the chitosan nanoparticles effectively bound to the DNA ( Figure 8 The results of the nuclease-resistant degradation experiment showed that the DNA in the CS-CYP19A1A-NPs group remained in the sample wells, while no DNA bands appeared in the lanes and sample wells of the naked plasmid group. The DNA was completely degraded by DNase I, indicating that chitosan nanoparticles have a good protective effect on DNA and can effectively resist nuclease degradation ( Figure 8 The results of the HEK-293T cell transfection experiment showed that no obvious green fluorescence was observed in the CS-CYP19A1A-mNG-NPs experimental group 24 hours after transfection. As the cell culture time increased, green fluorescence could be observed in the experimental group cells 48 hours after transfection. This indicates that CS-CYP19A1A-mNG-NPs can achieve effective delivery and expression of exogenous genes, and gene-loaded chitosan nanoparticles can enter cells and express ( Figure 9 The results of the toxicity of the nanoparticle complex to cells showed that after 24 hours of co-incubation, the chitosan nanoparticles loaded with CYP19A1A had almost no inhibitory effect on HEK-293T cells compared with Lipo8000, indicating that it had almost no toxic effect. After 48 hours of co-incubation, Lipo8000 and chitosan nanoparticles loaded with CYP19A1A had a certain inhibitory effect on HEK-293T cells as the plasmid concentration increased, but the inhibitory effect of Lipo8000 transfection reagent on cell proliferation was more significant ( Figure 10 ).

[0102] The above description is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with this technical field within the technical scope disclosed by the present invention and within the spirit and principles of the present invention should be covered by the scope of protection of the present invention.

Claims

1. A method for applying a chitosan nanoparticle gene delivery system to regulate fish sex ratio, characterized in that: The method for applying the chitosan nanoparticle gene delivery system to fish sex ratio regulation comprises the following steps: Step 1: Prepare chitosan nanoparticles by ionic crosslinking method: dissolve chitosan in acetic acid solution and stir magnetically at 37°C for 1 hour; Step 2: adding the sodium tripolyphosphate solution dropwise to the chitosan solution in a certain ratio, stirring at a constant temperature, filtering the solution after the reaction is completed to remove unreacted insoluble impurities, and obtaining a chitosan nano solution; Step 3: construct the vector, extract the plasmid, and prepare the chitosan nanoparticles of the vector gene by complex coacervation method. Add the chitosan solution to the diluted plasmid solution in a certain proportion, place it in a metal bath under certain conditions, mix it, and vortex it for 30 seconds to obtain the gene-loaded chitosan nanoparticles.

2. The method for applying the chitosan nanoparticle gene delivery system in regulating fish sex ratio according to claim 1, characterized in that: The mass ratio of the chitosan solution to the sodium tripolyphosphate solution is 6:1, the average particle size of the chitosan nanoparticles is about 130 nm, and the PDI is less than 0.

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3. The method for applying the chitosan nanoparticle gene delivery system in regulating fish sex ratio according to claim 1, characterized in that: The plasmid solution was diluted to 100 μg / mL with 6 mmoL / L Na2SO4; Chitosan nanoparticles prepared under the conditions of pH = 5.5 and chitosan / plasmid = 1:1 had a moderate particle size. Gel electrophoresis and encapsulation efficiency determination confirmed that they could effectively encapsulate pDNA and had low toxicity.

4. The method for applying the chitosan nanoparticle gene delivery system in regulating fish sex ratio according to claim 1, characterized in that: Nanoparticle complexes are formed through electrostatic adsorption.

5. The method for applying the chitosan nanoparticle gene delivery system in regulating fish sex ratio according to claim 1, characterized in that: The chitosan solution was mixed with an equal volume of the plasmid solution under the following reaction conditions: preheating at 55°C for 20 min, mixing, and vortexing for 30 s.

6. The method for applying the chitosan nanoparticle gene delivery system in regulating fish sex ratio according to claim 1, characterized in that: The route of intake is oral administration. After being absorbed through the gastrointestinal mucosa, it enters the epithelial cells and is expressed; The cyp19a1a gene was loaded into the chitosan nanoparticle gene delivery system to regulate the sex ratio of fish; After stopping the delivery of exogenous genes by chitosan nanoparticles for a period of time, the tissues in the body will no longer express the gene; The fish sex ratio regulation refers to increasing the estradiol content in fish, affecting the gonadal differentiation of fish, and increasing the female ratio of fish; The species is Lateolabrax maculatus.

7. A chitosan nanoparticle gene delivery system for controlling the sex ratio of fish, which implements the chitosan nanoparticle gene delivery system for controlling the sex ratio of fish according to any one of claims 1 to 6, characterized in that: The chitosan nanoparticle gene delivery system for fish sex ratio regulation application system includes: A stirring module was used to prepare chitosan nanoparticles using an ionic crosslinking method. Chitosan was dissolved in acetic acid solution and magnetically stirred at 37°C for 1 h. The filtration module is used to add the sodium tripolyphosphate solution dropwise into the chitosan solution according to a certain ratio, stir at a constant temperature, filter the solution after the reaction is completed, remove unreacted insoluble impurities, and obtain the chitosan nano solution; The extraction module is used to construct the vector, extract the plasmid, and prepare the chitosan nanoparticles containing the vector gene by complex coacervation. The chitosan solution is added to the diluted plasmid solution in a certain proportion, placed in a metal bath under certain conditions, and mixed. The mixture is vortexed for 30 seconds to obtain the gene-loaded chitosan nanoparticles.

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