Preparation method of pcDNA3.1-GH-loaded chitosan nanoparticle gene delivery system and application of pcDNA3.1-GH-loaded chitosan nanoparticle gene delivery system in fishes

Abstract

The invention belongs to the technical field of biological medicines, discloses a preparation method of a pcDNA3.1-GH-loaded chitosan nanoparticle gene delivery system and an application of the pcDNA3.1-GH-loaded chitosan nanoparticle gene delivery system in fishes, and aims to solve the problems that the existing gene delivery technology is complicated in operation, the public are not trusted to transgenic foods and the growth rate of the fishes is low. The invention provides an oral chitosan nano-gene delivery system, which is characterized in that pcDNA3.1-GH-loaded chitosan nanoparticles are constructed by compounding a lateolabrax japonicus growth key gene gh with chitosan. Results show that the gene-loaded chitosan nanoparticles can effectively enter cells and realize the expression of exogenous genes, almost have no toxic effect, can achieve an instantaneous transgenosis effect without modifying the genome of an organism, and can significantly improve the growth performance of cultured fishes at the same time. 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 a preparation method of a pcDNA3.1-GH-loaded chitosan nanoparticle gene delivery system and its application in 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 view of the problems existing in the prior art, the present invention provides a method for preparing a chitosan nanoparticle gene delivery system loaded with pcDNA3.1-GH and its application in fish.

[0005] The present invention is achieved by: a method for preparing a pcDNA3.1-GH-loaded chitosan nanoparticle gene delivery system 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 a eukaryotic expression vector pcDNA3.1-GH-His, extract the plasmid, and prepare GH-loaded chitosan nanoparticles by complex coacervation. Add the chitosan solution to the diluted GH plasmid solution in a certain proportion, place it in a metal bath and mix under certain conditions to obtain pcDNA3.1-GH-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 200 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 the drug is absorbed through the gastrointestinal mucosa and enters the epithelial cells for expression, thus achieving transient transgenic fish.

[0015] The gh gene was loaded into the chitosan nanoparticle gene delivery system;

[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 growth performance improvement refers to the increase in fish body weight, body length, weight gain rate and specific growth rate;

[0018] The species is Lateolabrax maculatus.

[0019] Another object of the present invention is to provide a preparation system of a chitosan nanoparticle gene delivery system loaded with pcDNA3.1-GH, 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 eukaryotic expression vector pcDNA3.1-GH-His, extract the plasmid, and prepare GH-loaded chitosan nanoparticles using a complex coacervation method. Chitosan solution is added to the diluted GH plasmid solution in a certain proportion, and the mixture is placed in a metal bath under certain conditions to obtain pcDNA3.1-GH-loaded chitosan nanoparticles.

[0023] In combination with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solutions to be protected by the present invention are as follows:

[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 present invention precisely activates the growth regulation pathway of Lateolabrax japonicus by feeding chitosan nanoparticles loaded with pcDNA3.1-GH in a non-invasive delivery manner, breaking through the limitations of traditional hormone injection or artificial breeding in regulating fish growth traits, and realizing large-scale, low-cost and efficient growth breeding.

[0027] This invention successfully solves the long-standing problem of lengthy breeding cycles in fish growth. Traditional growth breeding techniques rely on multi-generation backcrossing or hormone induction, and have inherent drawbacks such as long operating cycles, endocrine disruption, and uncontrollable ecological risks. This invention activates the growth regulation pathways of Lateolabrax japonicus by feeding them a GH-loaded chitosan nanoparticle feed, significantly promoting their growth performance in just 40 days. This overcomes the technical bottleneck of rapid breeding of growth-specific species and provides an innovative paradigm for the aquatic breeding industry that combines high efficiency with 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 the preparation method of the chitosan nanoparticle gene delivery system loaded with pcDNA3.1-GH provided in an embodiment of the present invention.

[0030] Figure 2 This is a structural block diagram of the preparation system of the pcDNA3.1-GH-loaded chitosan nanoparticle gene delivery system provided by an embodiment of the present invention.

[0031] Figure 3 It is a rendering of the technical solution of the present invention.

[0032] Figure 4 The nucleotide sequence and the encoded amino acid sequence of Lateolabrax japonicus gh1 provided in the embodiment of the present invention are shown in FIG. The predicted signal peptide is marked in gray, and the stop codon is marked with "*".

[0033] Figure 5 This is a Western blot analysis of GH1 protein expression in HEK-293T cells using the methods provided in the present invention. (+): Positive cells transfected with pcDNA3.1-GH1-His; (-): Control group without plasmid transfection.

[0034] Figure 63 is a graph showing the particle size, PDI, and Zeta potential of CS-GH1-NPs provided in an embodiment of the present invention.

[0035] Figure 7 This is a transmission electron microscope image of CS-GH1-NPs provided in an embodiment of the present invention.

[0036] Figure 8 This is a DNA binding and DNA protection experiment of CS-GH1-NPs. Lane numbers are: 2. Naked plasmid pcDNA3.1-GH1; 4. CS-GH1-NPs; 6. Naked plasmid pcDNA3.1-GH1 treated with DNase I; 8. CS-GH1-NPs treated with DNase I.

[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-GH1-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-GH1-NPs, naked plasmids, and liposome Lipo8000 on HEK-293T cells at different plasmid concentrations (ng / well) for 24 and 48 hours, respectively (*P < 0.05, **P < 0.01, ***P < 0.001).

[0039] Figure 11 Western blot analysis of GH1 protein expression in the gastrointestinal tract of Lateolabrax japonicus using the methods provided in this embodiment. Figures A and B represent the intestine, and C and D represent the stomach.

[0040] Figure 12 This is the immunohistochemical localization of GH1 in the intestine and stomach of Lateolabrax japonicus, as provided in the examples of the present invention. A, D: GH1-loaded chitosan nanoparticles group, red arrows indicate positive signals; B, E: negative controls without primary antibody. C, F: blank control groups.

[0041] Figure 13 This is a diagram showing the effects of GH1-loaded chitosan nanoparticles provided in an embodiment of the present invention on serum physiological and biochemical indices of Lateolabrax japonicus.

[0042] Figure 14 3. HE staining of various organs of Lateolabrax japonicus in the experimental group of GH1-loaded chitosan nanoparticles and the blank control group provided in the examples of the present invention. DETAILED DESCRIPTION

[0043] 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.

[0044] like Figure 1 As shown, the preparation method of a chitosan nanoparticle gene delivery system provided by an embodiment of the present invention includes the following steps:

[0045] 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.

[0046] 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;

[0047] S103, constructing a eukaryotic expression vector pcDNA3.1-GH-His, extracting the plasmid, and preparing GH-loaded chitosan nanoparticles using a complex coacervation method. Chitosan solution is added to the diluted GH plasmid solution in a certain proportion, and the mixture is placed in a metal bath under certain conditions to obtain pcDNA3.1-GH-loaded chitosan nanoparticles.

[0048] The mass ratio of the chitosan solution to the sodium tripolyphosphate (TPP) solution provided in the embodiment of the present invention is 6:1, the average particle size of the chitosan nanoparticles is about 200 nm, and the polydispersity index (PDI) is less than 0.3.

[0049] The plasmid solution provided in the embodiment of the present invention was diluted to 100 μg / mL with 6 mmol / L Na2SO4;

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] The gh gene was loaded into the chitosan nanoparticle gene delivery system;

[0055] 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.

[0056] The fish growth performance improvement refers to the increase in fish body weight, body length, weight gain rate and specific growth rate;

[0057] The species is Lateolabrax maculatus.

[0058] like Figure 2 As shown, the preparation system of a chitosan nanoparticle gene delivery system carrying pcDNA3.1-GH provided in an embodiment of the present invention includes:

[0059] 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.

[0060] 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;

[0061] The extraction module is used to construct the eukaryotic expression vector pcDNA3.1-GH-His, extract the plasmid, and prepare GH-loaded chitosan nanoparticles using a complex coacervation method. Chitosan solution is added to the diluted GH plasmid solution in a certain proportion, and the mixture is placed in a metal bath under certain conditions to obtain pcDNA3.1-GH-loaded chitosan nanoparticles.

[0062] The present invention proposes a method for constructing the GH chitosan nanoparticles, comprising the following steps:

[0063] Step 1: Construction of eukaryotic expression vector pcDNA3.1-GH-His

[0064] 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 gh1-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.

[0065] Step 2: Plasmid extraction and eukaryotic vector expression verification

[0066] (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.

[0067] (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.

[0068] Step 3: Chitosan nanoparticle preparation

[0069] 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.

[0070] Step 4: Preparation of GH-loaded chitosan nanoparticles

[0071] Gene-loaded chitosan nanoparticles were prepared using a complex coacervation method. A pcDNA3.1-GH 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 GH-loaded chitosan nanoparticles.

[0072] The present invention loads pcDNA3.1-GH into chitosan nanoparticles to construct a GH 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 plasmid 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 the degradation of nucleases. HEK-293T cells were transfected to measure 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.

[0073] On the basis of the above research, the present invention proposes the application of GH chitosan nanogene delivery system in fish growth regulation.

[0074] Among them, preferably, the fish sex ratio regulation refers to the fish growth performance improvement, which refers to increasing the fish body weight, body length, weight gain rate and specific growth rate.

[0075] Wherein, preferably, the restriction enzyme cleavage sites of the pcDNA3.1-GH-His are Hind III and BamH I.

[0076] Figure 4 The nucleotide sequence and the encoded amino acid sequence of Lateolabrax japonicus gh1 provided in the embodiment of the present invention are shown in FIG. The predicted signal peptide is marked in gray, and the stop codon is marked with "*".

[0077] Figure 5 This is a Western blot analysis of GH1 protein expression in HEK-293T cells using the methods provided in the present invention. (+): Positive cells transfected with pcDNA3.1-GH1-His; (-): Control group without plasmid transfection.

[0078] Figure 6 3 is a graph showing the particle size, PDI, and Zeta potential of CS-GH1-NPs provided in an embodiment of the present invention.

[0079] Figure 7This is a transmission electron microscope image of CS-GH1-NPs provided in an embodiment of the present invention.

[0080] Figure 8 This is a HEK-293T cell uptake experiment provided in an embodiment of the present invention. The uptake effect diagram of CS-GH1-mNG-NPs and Lipo8000 by HEK-293T cells at 24h and 48h.

[0081] Figure 9 Figures A and B show the cytotoxic effects of CS-GH1-NPs, naked plasmids, and liposome Lipo8000 on HEK-293T cells at different plasmid concentrations (ng / well) for 24 and 48 hours, respectively (*P < 0.05, **P < 0.01, ***P < 0.001).

[0082] Example 1

[0083] Preparation of GH chitosan nanoparticle gene delivery system

[0084] Construction of the eukaryotic expression vector pcDNA3.1-GH-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 GH1-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 screened on plates containing ampicillin. Sequence accuracy was verified by sequencing.

[0085] 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.

[0086] 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.

[0087] Preparation of GH-loaded chitosan nanoparticles: Gene-loaded chitosan nanoparticles were prepared using a complex coacervation method. The pcDNA3.1-GH 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 at 55°C in a metal bath for 20 minutes, mixed, and vortexed for 30 seconds to obtain GH-loaded chitosan nanoparticles.

[0088] Example 2

[0089] Growth regulation of Lateolabrax japonicus by GH chitosan nanogene delivery.

[0090] The pcDNA3.1-GH1-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 GH1-loaded chitosan nanoparticles in the gastrointestinal tract of the Lateolabrax were detected through feeding experiments on Lateolabrax, and the growth indicators and toxic effects in vivo were measured.

[0091] Depend on Figure 11 It can be seen that the expression of GH1 can be detected in the gastrointestinal tract of the experimental group of sea bass, while no expression of GH1 was found in the control group, indicating that GH1-loaded chitosan nanoparticles can be normally expressed in the digestive tract of sea bass.

[0092] Depend on Figure 12 It can be seen that positive signals can be observed in the gastrointestinal tract of the sea bass in the GH1-loaded chitosan nanoparticles experimental group, while no positive signals were observed in the blank control group and the negative control group, indicating that the GH1-chitosan nanocomplex can pass through the gastrointestinal mucosal barrier, be absorbed by epithelial cells and achieve the expression of functional proteins.

[0093] As shown in Table 1, the weight, body length, weight gain rate, and specific growth rate of the experimental group were significantly higher than those of the control group. Compared with the blank control group, the weight gain rate increased by 17.2% and the specific growth rate increased by 10.9%.

[0094] Table 1 Effects of CS-GH1-NPs on the growth performance of Lateolabrax japonicus

[0095]

[0096] Note: The data in the table are mean ± standard deviation (n = 45). Different letters in the upper right corner of the same row of data indicate significant differences (P < 0.05).

[0097] Depend on Figure 13 It can be seen that there is no significant difference between the experimental group and the control group in terms of alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, albumin, total cholesterol and blood glucose levels.

[0098] Depend on Figure 14 Neither the experimental nor the control groups observed ventricular hypertrophy, cardiac wall thinning, nor abnormalities such as congestion, hemorrhage, or infarction. HE staining revealed neatly arranged myocardial fibers, oval nuclei located centrally, with no obvious 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 darker nuclei and lighter cytoplasm. The white pulp was composed of densely packed lymphocytes, with darker nuclei and less abundant, lighter 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 obvious desquamation or damage. The stomach structure, color, and texture of the experimental and control groups were normal. HE staining showed that the gastric epithelial cells were neatly arranged with basally located nuclei, the connective tissue of the submucosal layer was evenly distributed, no edema or fibrosis was observed, and there was no obvious inflammatory cell infiltration between the muscularis layers. The intestinal tract of the experimental and control groups showed normal thickness and texture by naked eye. HE staining showed that the intestinal epithelial cells were neatly arranged with basally located nuclei and evenly stained, the villi structure was clear, the villus epithelial cells were tightly arranged, the number of goblet cells was moderate, the smooth muscle fibers of the intestinal wall were neatly arranged, and there was no obvious inflammatory cell infiltration between the muscularis layers.

[0099] Relevant evidence of the technical effects achieved by the embodiments of the present invention.

[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-GH1-His eukaryotic vector was constructed, it was transfected into HEK-293T cells. GH1 was expressed normally after 24h and 48h, and no protein was expressed in the control group, indicating that the eukaryotic expression vector was successfully constructed. Figure 5 The particle size, PDI and zeta potential results of the CS-GH-NPs nanoparticle complex showed that the particle size of CS-GH1-NPs was 200 nm, the PDI was 0.145, which was less than 0.3, and the zeta potential was +19 mV, which was positive. The particle size distribution range was good, indicating that the preparation of CS-GH1-NPs was successful ( Figure 6 The results of transmission electron microscopy showed that most of the GH1-chitosan nanocomposites were round or oval, with a few irregular in shape. The observed nanoparticle size was basically consistent with the results of ZetaSizer particle size potential instrument detection ( Figure 7 ). The encapsulation rate of chitosan nanoparticles for plasmid pcDNA3.1-GH1 was calculated by ultra-micro spectrophotometry and it was found that the encapsulation rate of chitosan nanoparticles reached 70%. At the same time, no aggregation and sedimentation of the nanoparticle suspension was observed within 5 days. The ZetaSizer particle size potential instrument was used for continuous detection for 5 days and found that the particle size, PDI and zeta potential of the nanoparticles were all within the normal range, indicating that the GH1-chitosan nanoparticle complex has good stability. The results of the gel retardation experiment showed that the pcDNA3.1-GH1 encapsulated by chitosan was retained in the sample well, and no DNA bands appeared on the corresponding lane, while the naked plasmid pcDNA3.1-GH1 in the control group ran out of the sample well, indicating that the chitosan nanoparticles effectively combined with the DNA and prevented the migration of the DNA ( Figure 8 The results of the anti-nuclease degradation experiment showed that the DNA in the CS-GH1-NPs group remained in the sample wells, showing bright bands, while the sample wells and lanes of the naked plasmid group had no bands, indicating that the DNA had been degraded by DNaseI enzyme. This shows 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 fluorescence signal was detected in the CS-GH1-mNG-NPs experimental group 24 hours after transfection. As the culture time prolonged, green fluorescence was observed in the experimental group cells 48 hours after transfection, indicating that CS-GH1-mNG-NPs can achieve the delivery and expression of exogenous genes, and the gene-loaded chitosan nanoparticles can enter the cells and express ( Figure 9The cytotoxicity of the nanoparticle complex prepared by the complex coacervation method was detected by the CCK8 kit. The results showed that after 24 hours of co-incubation, the chitosan nanoparticles loaded with GH1 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 GH1-loaded chitosan nanoparticles had a certain inhibitory effect on HEK-293T cells as the plasmid concentration increased, but Lipo8000 transfection reagent had a significant inhibitory effect on cell proliferation ( 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 preparing a chitosan nanoparticle gene delivery system loaded with pcDNA3.1-GH, characterized in that: The following steps are involved: 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 a eukaryotic expression vector pcDNA3.1-GH-His, extract the plasmid, and prepare GH-loaded chitosan nanoparticles by complex coacervation. Add the chitosan solution to the diluted GH plasmid solution in a certain proportion, place it in a metal bath and mix under certain conditions to obtain pcDNA3.1-GH-loaded chitosan nanoparticles.

2. The method for preparing the chitosan nanoparticle gene delivery system loaded with pcDNA3.1-GH 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 200 nm, and the PDI is less than 0.

3.

3. The method for preparing the pcDNA3.1-GH-loaded chitosan nanoparticle gene delivery system 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 preparing the pcDNA3.1-GH-loaded chitosan nanoparticle gene delivery system according to claim 1, wherein: Nanoparticle complexes are formed through electrostatic adsorption.

5. The method for preparing the pcDNA3.1-GH-loaded chitosan nanoparticle gene delivery system 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 preparing the pcDNA3.1-GH-loaded chitosan nanoparticle gene delivery system according to claim 1, characterized in that: The drug is taken orally. After being absorbed by the gastrointestinal mucosa, it enters the epithelial cells and expresses, thus achieving transient transgenic fish. The gh gene was loaded into the chitosan nanoparticle gene delivery system; 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 growth performance improvement refers to the increase in fish body weight, body length, weight gain rate and specific growth rate; The species is Lateolabrax maculatus.

7. A system for preparing a pcDNA3.1-GH-loaded chitosan nanoparticle gene delivery system for implementing the method for preparing a pcDNA3.1-GH-loaded chitosan nanoparticle gene delivery system according to any one of claims 1 to 6, characterized in that: The preparation system of the pcDNA3.1-GH-carrying chitosan nanoparticle gene delivery system comprises: 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 eukaryotic expression vector pcDNA3.1-GH-His, extract the plasmid, and prepare GH-loaded chitosan nanoparticles using a complex coacervation method. Chitosan solution is added to the diluted GH plasmid solution in a certain proportion, and the mixture is placed in a metal bath under certain conditions to obtain pcDNA3.1-GH-loaded chitosan nanoparticles.

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