DDX5 protein-targeted anti-fowl adenovirus veterinary drug composition and application thereof

Abstract

The invention discloses an anti-fowl adenovirus veterinary drug composition targeting DDX5 protein and application thereof, and relates to the technical field of antiviral veterinary drugs for fowls, biodegradable polylactic acid-glycolic acid copolymer (PLGA) nanoparticles are used as a core delivery carrier, and siRNA targeting DDX5 protein and a small molecule inhibitor are loaded, so that the anti-fowl adenovirus veterinary drug composition targeting DDX5 protein is prepared, and the anti-fowl adenovirus veterinary drug composition targeting DDX5 protein is prepared. According to the present invention, the dual antiviral effects of gene silencing and protein activity inhibition are achieved, in order to improve the targeting property, the poultry respiratory tract epithelial cell specific ligand is modified on the surface of the PLGA nanoparticle, such that the composition can accurately recognize and gather on the infected site, and the intracellular delivery efficiency is significantly improved; the stability and the long-acting effect of the composition are ensured.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of bioinformatics, in particular to a veterinary drug composition for targeting DDX5 protein and application thereof. BACKGROUND

[0002] Avian adenovirus is one of the important pathogens that endanger the poultry industry, which can cause various diseases such as inclusion body hepatitis, pericardial effusion syndrome, and respiratory tract infection in poultry. Different age groups of poultry are susceptible, especially 1-3 week old chicks. At present, the prevention and control measures for avian adenovirus infection are limited. Traditional vaccines have the problems of narrow immune protection spectrum, delayed immune response, and poor protection effect on variant strains. Conventional antiviral drugs are mostly broad-spectrum preparations with poor targeting, and large doses are needed to achieve certain prevention and control effect, which not only increases the breeding cost, but also easily leads to adverse reactions such as intestinal flora disorder and liver and kidney function damage in poultry. At the same time, it may induce drug resistance of the virus, further increasing the difficulty of prevention and control.

[0003] DDX5 protein is an RNA helicase that plays an irreplaceable key role in the replication of avian adenovirus. It can regulate the transcription, translation of viral genes and assembly of virions by uncoiling the secondary structure of viral genomic RNA, directly promoting the proliferation and spread of viruses in host cells. Therefore, targeting and inhibiting the activity of DDX5 protein has become a core target for developing specific antiviral drugs for avian adenovirus. Existing inhibitors targeting DDX5 protein include siRNA and small molecule compounds, but these drugs have obvious technical defects when used alone: first, the intracellular delivery efficiency is low, and it is difficult to penetrate the host cell membrane and play a role in the cytoplasm; second, it is easily cleared by the host immune system, with a short half-life in the body; third, it has poor stability in cells and is easily degraded by nucleases or proteases, and cannot maintain an effective concentration, ultimately resulting in poor antiviral effect.

[0004] PLGA (poly-lactic-co-glycolic acid) is a synthetic polymer material with good biocompatibility and biodegradability, but conventional PLGA nanoparticles lack targeting ability and are difficult to accurately recognize and bind to avian respiratory epithelial cells at the infection site, easily non-specifically distributed in whole body tissues, causing drug waste. At the same time, the drug release rate of conventional PLGA nanoparticles is difficult to control, and most of them are released by passive diffusion, which cannot achieve rapid and accurate release of drugs in cells, affecting the intracellular effective concentration of drugs. SUMMARY

[0005] The present application aims at solving the problems in the prior art, and provides an anti-avian adenovirus veterinary composition targeting DDX5 protein and an application thereof.

[0006] The present application aims at solving the problems in the prior art, and provides an anti-avian adenovirus veterinary composition targeting DDX5 protein and an application thereof.

[0007] The particle size of the PLGA nanoparticle is controlled to be 100-200 nm.

[0008] Further, the molar ratio of lactic acid LA to glycolic acid GA in the PLGA is 50:50-80:20; the weight average molecular weight of the PLGA is 10000-50000 Da, preferably 20000-30000 Da, the PLGA nanoparticle with the ratio and the molecular weight has good biocompatibility, degradability and drug loading capacity, and can ensure that the particle size of the nanoparticle is stably controlled within the range of 100-200 nm, which is beneficial to endocytosis in cells.

[0009] Further, the siRNA sequence targeting DDX5 protein is designed according to the conserved region of the avian DDX5 gene, the sequence of the sense strand is 5'-GGAUUAUGCUCGACUACAATT-3', and the sequence of the antisense strand is 5'-UUGUAGUCGAGCAUAAUCCTT-3'.

[0010] The loading amount of the siRNA is 5%-15% of the mass of the PLGA nanoparticle, preferably 8%-12%, the siRNA can specifically silence the expression of the DDX5 gene in the host cell, block the replication process of the avian adenovirus, and has reasonable loading amount, which can ensure good silencing effect and drug stability.

[0011] Further, the small molecule inhibitor targeting DDX5 protein is one or more of curcumin derivatives, quercetin derivatives or purine derivatives.

[0012] Further, the loading amount of the small molecule inhibitor is 10-25% of the mass of the PLGA nanoparticles, preferably 15-20%, which can ensure that the drug reaches an effective inhibitory concentration after being released in cells, and forms a synergistic effect with siRNA to improve the overall antiviral effect.

[0013] Further, the avian respiratory epithelial cell-specific ligand is one or more of sialic acid receptor ligand, mannose receptor ligand or avian transferrin receptor ligand.

[0014] Further, the modification amount of the avian respiratory epithelial cell-specific ligand is 10-30% of the number of molecules on the surface of the PLGA nanoparticles.

[0015] Further, in the veterinary composition, the mass ratio of the PLGA nanoparticles, siRNA, small molecule inhibitor and specific ligand is 100:(5-15):(10-25):(2-8), preferably the mass ratio is 100:(8-12):(15-20):(4-6), which is determined by multiple orthogonal experiments. Under the preferred ratio, the endocytosis efficiency of the composition is ≥92%, the DDX5 protein inhibition rate is ≥90%, and the drug use dose can be reduced by more than 60%, significantly reducing the impact of the drug on the intestinal flora of poultry.

[0016] Further, the preparation method of the composition is as follows:

[0017] The PLGA is dissolved in an organic solvent to prepare an oil phase with a concentration of 10-50 mg / mL; the surfactant is dissolved in deionized water to prepare an aqueous phase with a concentration of 0.5-2% (w / v), and the surfactant concentration can be adjusted to improve the dispersibility of the nanoparticles and avoid aggregation; the oil phase is slowly added to the aqueous phase at a volume ratio of 1:10-1:20, and ultrasonic emulsification is performed at a power of 100-300 W and a frequency of 20-40 kHz for 10-30 min; then the mixture is stirred at room temperature at a magnetic stirring speed of 100-200 r / min for 2-4 h, and the organic solvent is slowly removed to allow the PLGA to solidify in the water droplets to form nanoparticles; finally, centrifugation is performed at 8000-12000 r / min for 10-20 min, the precipitate is collected, washed with deionized water for 3-5 times to remove unreacted surfactant and organic solvent, and freeze-dried for 24 h to obtain blank PLGA nanoparticles.

[0018] The siRNA and small molecule inhibitor targeting DDX5 protein are dissolved in the PBS buffer solution according to the mass ratio, a drug mixed solution is prepared, the blank PLGA nanoparticles are added according to the mass-volume ratio of 1:10-1:20 (g / mL) of the blank PLGA nanoparticles and the drug mixed solution, and the drug is loaded on the surface and internal pores of the PLGA nanoparticles through electrostatic adsorption under the condition of magnetic stirring at 100-200 r / min at room temperature for 1-2 h; then, the drug loaded PLGA nanoparticles are obtained by centrifugation at 10,000 r / min for 15 min, and the drug loading rate is detected to be greater than or equal to 85%;

[0019] The ligand specific to the avian respiratory epithelial cells is dissolved in the PBS buffer solution to prepare a ligand solution with a concentration of 0.5-2 mg / mL; the ligand solution is added according to the mass-volume ratio of 1:5-1:10 of the drug loaded PLGA nanoparticles and the ligand solution, and the ligand is modified on the surface of the PLGA nanoparticles through covalent binding under the condition of magnetic stirring at 100-200 r / min at 37°C for 2-4 h; then, the ligand is removed by centrifugation at 10,000 r / min for 15 min, and the precipitate is washed with the PBS buffer solution for 2-3 times to remove the unbound free ligand, and the freeze-drying is performed for 24 h to obtain the anti-avian adenovirus veterinary composition targeting DDX5 protein.

[0020] In another aspect, the application relates to the anti-avian adenovirus veterinary composition targeting DDX5 protein, which can be prepared into an injection, an aerosol, a nasal drop or an oral preparation, preferably an aerosol or a nasal drop, and directly acts on the infection site of the avian respiratory tract.

[0021] Compared with the prior art, the anti-avian adenovirus veterinary composition targeting DDX5 protein and the application thereof have the following beneficial effects:

[0022] The core architecture of the PLGA nano-carrier + double targeted drugs + specific ligand is designed, and the efficient and low-toxicity prevention and treatment of the avian adenovirus infection are realized, the composition has strong targeting property, can accurately recognize and gather at the infection site, significantly improves the intracellular delivery efficiency, and reduces the damage to the normal tissues; the double drug combination has a synergistic effect, the virus replication is blocked through gene silencing and protein activity inhibition, and the antiviral effect is significantly better than that of the traditional drugs; the drug dosage is low, the safety is high, the poultry intestinal flora and liver and kidney functions have no obvious adverse effects, the preparation process is controllable, the large-scale production can be realized, the cost is reduced, the market blank of the specific anti-avian adenovirus veterinary drug can be effectively filled, and the healthy development of the poultry breeding industry is promoted.

[0023] Other advantages, objectives and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination or study, or may be learned from the practice of the invention. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0025] Figure 1 A flowchart illustrating the preparation method of an anti-avian adenovirus veterinary drug composition targeting the DDX5 protein;

[0026] Figure 2 This is a flowchart illustrating the detection of antiviral activity of an anti-avian adenovirus veterinary drug composition targeting the DDX5 protein. Detailed Implementation

[0027] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features, and effects of the present invention, in conjunction with the accompanying drawings and preferred embodiments, is provided below.

[0028] Example 1

[0029] PLGA, siRNA targeting DDX5 protein, small molecule inhibitor Cur-1, sialic acid receptor ligand, polyvinyl alcohol, dichloromethane, and PBS buffer were precisely selected. The PLGA had a lactate to glycolic acid molar ratio of 75:25 and a weight-average molecular weight of 25000 Da. The sense strand of the siRNA targeting DDX5 protein was 5'-GGAUUAUGCUCGACUACAATT-3', and the antisense strand was 5'-UUGUAGUCGAGCAUAAUCCTT-3'.

[0030] Take 0.2g of PLGA and place it in a sterile centrifuge tube. Add 5mL of dichloromethane and sonicate in an ultrasonic cleaner for 5min until the PLGA is completely dissolved, obtaining a homogeneous oil phase with a concentration of 40mg / mL. Store at room temperature in the dark for later use. Take 0.1g of PVA and place it in a beaker. Add 100mL of deionized water and place in a 60℃ constant temperature water bath. Stir and dissolve for 30min until the PVA is completely dissolved. After cooling to room temperature, obtain a homogeneous aqueous phase with a concentration of 1% (w / v). Filter through a 0.22μm filter membrane for sterilization and store in a sterile container for later use. Slowly add the oil phase dropwise to the aqueous phase at a volume ratio of 1:20, controlling the dropwise addition rate at 1mL / min. Simultaneously, sonicate in an ultrasonic homogenizer for 20min, maintaining an ice bath cooling period to prevent excessively high temperatures from causing rapid solvent evaporation, forming a homogeneous and stable oil-in-water emulsion. Transfer the emulsion to a three-necked flask and place it on a magnetic stirrer. The mixture was stirred at 150 rpm at room temperature for 3 hours to slowly evaporate dichloromethane. The emulsion was continuously observed during this period until it changed from milky white to translucent, indicating that PLGA had solidified into nanoparticles in the aqueous droplets. The solidified nanoparticle suspension was transferred to a centrifuge tube and centrifuged at 10,000 rpm at room temperature for 15 minutes. The supernatant was discarded, and the precipitate was collected. The precipitate was resuspended in deionized water and washed by centrifugation four times, each time at 10,000 rpm for 15 minutes, to thoroughly remove unreacted PVA and residual dichloromethane. The washed precipitate was placed in a freeze dryer and freeze-dried at -50°C and ≤10 Pa for 24 hours to obtain white, loose, blank PLGA nanoparticles. Dynamic light scattering analysis showed that the nanoparticles had a particle size of 152 nm, a dispersion coefficient of 0.18, and a uniform particle size distribution with no obvious agglomeration.

[0031] Take 20 mg of lyophilized siRNA targeting DDX5 protein, add 20 mL of PBS buffer, and gently shake to dissolve for 5 min to obtain a solution with an siRNA concentration of 1 mg / mL; separately, take 30 mg of the small molecule inhibitor Cur-1, add it to the siRNA solution, and magnetically stir at 150 rpm for 5 min until Cur-1 is completely dissolved to obtain a homogeneous drug mixture solution; take 100 mg of blank PLGA nanoparticles, add them to the above drug mixture solution, with a mass-to-volume ratio of blank PLGA nanoparticles to drug mixture solution of 1:20; place on a magnetic stirrer, and stir at 150 rpm at room temperature. The mixture was stirred and incubated for 1.5 h to allow the drug to be loaded onto the surface and internal pores of the PLGA nanoparticles via electrostatic adsorption. The incubated suspension was then transferred to a centrifuge tube and centrifuged at 10,000 r / min at room temperature for 15 min. The supernatant (containing unloaded free drug) was discarded, and the precipitate was collected to obtain the drug-loaded PLGA nanoparticles. The drug loading rate was detected by high performance liquid chromatography (HPLC). The detection wavelength for siRNA was 260 nm, and the detection wavelength for Cur-1 was 420 nm. The results showed that the siRNA loading was 10.2%, the Cur-1 loading was 18.5%, and the total drug loading rate was 89.3%.

[0032] Take 8 mg of sialic acid receptor ligand (N-acetylneuraminic acid), add 10 mL of PBS buffer, and stir magnetically at 150 rpm for 5 min until completely dissolved to obtain a ligand solution with a concentration of 0.8 mg / mL. Add drug-loaded PLGA nanoparticles to the above ligand solution, with a mass-to-volume ratio of drug-loaded PLGA nanoparticles to ligand solution of 1:10. Incubate in a 37°C water bath at 150 rpm for 3 h to allow the ligand to covalently modify the surface of the PLGA nanoparticles. The incubated suspension was transferred to a centrifuge tube and centrifuged at 10,000 r / min at room temperature for 15 min. The supernatant was discarded, and the precipitate was collected. The precipitate was resuspended in PBS buffer (pH 7.4) and centrifuged and washed three times, each time at 10,000 r / min for 15 min, to remove free ligands. The washed precipitate was placed in a freeze dryer and freeze-dried at -50℃ and a vacuum degree ≤10 Pa for 24 h to obtain a white, loose anti-avian adenovirus veterinary drug composition targeting DDX5 protein.

[0033] Multiple performance tests were performed on the prepared veterinary drug composition:

[0034] Particle size and dispersibility: Dynamic light scattering instrument test showed that the particle size of the composition was 165nm, the dispersibility coefficient was 0.22, and there was no obvious agglomeration, which met the particle size control requirements of 100-200nm.

[0035] Targeting: The endocytosis efficiency of the composition in avian respiratory epithelial cells was detected by flow cytometry. The results showed that the endocytosis efficiency reached 92.3%, which was significantly higher than that of unmodified ligand-loaded PLGA nanoparticles (endocytosis efficiency 65.1%), indicating a significant targeted recognition effect.

[0036] Drug release performance: The drug release rate was detected by dialysis. The dialysis medium was PBS buffer solution containing 10 mmol / L glutathione (simulating the intracellular environment of avian cells). The results showed that the drug release rate was 86.7% within 2 hours, which can achieve rapid intracellular release of the drug.

[0037] Cytotoxicity: The cytotoxicity of the composition to DF-1 cells was detected by the MTT assay. The results showed that the half-maximal toxic concentration (CC50) of the composition to DF-1 cells was 62.5 μg / mL, with no obvious cytotoxicity and good safety.

[0038] Example 2

[0039] Avian respiratory epithelial cells were seeded into 96-well cell culture plates, with 1×10⁶ cells per well. 4 Add 100 μL of LMEM medium to each cell and incubate at 37°C in a 5% CO2 incubator for 24 hours until the cell confluence reaches 80%.

[0040] Avian adenovirus FAdV-4 was inoculated into DF-1 cells at an MOI of 1. 100 μL of virus solution was added to each well. After adsorption at 37°C for 1 h, the virus solution was discarded. The cells were then washed twice with PBS buffer (pH 7.4), and the washing solution was discarded after each wash.

[0041] The composition prepared in Example 1 was diluted with DMEM medium to different concentrations (0.1 μg / mL, 0.5 μg / mL, 1 μg / mL, 5 μg / mL, 10 μg / mL), and 100 μL was added to each infected DF-1 cell. Three control groups were set up: control group 1 was the infection group without drug, with 100 μL of DMEM medium added; control group 2 was the drug-loaded PLGA nanoparticle group without modified ligand, with the same concentration as the experimental group; and control group 3 was the group using free siRNA + small molecule inhibitor, with the same concentration of siRNA and small molecule inhibitor as the experimental group. Each group was set up with 3 replicates and cultured in a 37°C, 5% CO2 incubator for 24 h.

[0042] Cell viability assay: The MTT assay was used. 20 μL MTT solution was added to each well and cultured for 4 h. The culture medium was then discarded, and 150 μL LDMSO was added to each well. The mixture was shaken for 10 min, and the absorbance (OD value) of each well was measured at 490 nm using a microplate reader. Cell viability was calculated as (OD value of experimental group / OD value of normal cell control group) × 100%.

[0043] Virus inhibition rate detection: Real-time quantitative PCR was used to collect cells from each well, extract total RNA from the cells, and reverse transcribe it into cDNA. Using cDNA as a template, specific primers (upstream primer: 5'-GCGGCTTCTACGACTACCT-3', downstream primer: 5'-GCGGCTTCTACGACTACCT-3') were designed for the avian adenovirus Hexon gene and qPCR amplification was performed. The virus inhibition rate was calculated as (1 - viral gene copy number in the experimental group / viral gene copy number in the control group) × 100%.

[0044] MTT assay results showed that when the concentration of the composition of the present invention was 1 μg / mL, the cell viability reached 89.2%, while the cell viability of control group 2 and control group 3 at the same concentration were 76.5% and 68.3%, respectively. qPCR assay results showed that when the concentration of the composition of the present invention was 1 μg / mL, the intracellular avian adenovirus genome copy number was reduced by 92.6% compared with control group 1, which was significantly higher than that of control group 2 (reduction of 68.4%) and control group 3 (reduction of 52.1%). When the concentration of the composition was 10 μg / mL, the cell viability remained at 85.3%, with no obvious cytotoxicity, indicating that the composition of the present invention has significant antiviral activity and good cell compatibility in vitro.

[0045] Example 3

[0046] One hundred and twenty healthy one-day-old SPF chicks, weighing 35-40g, were randomly divided into four groups of 30 chicks each: experimental group, control group 1, control group 2, and blank control group (saline group). Control group 1 was the unmodified ligand-loaded PLGA nanoparticle group, control group 2 was the traditional antiviral drug group (ribavirin), and the blank control group was the saline group. The experimental animals were housed in an SPF animal house at a temperature of 28-30℃ and a humidity of 60%-70%, with free access to feed and water, and were acclimatized for three days.

[0047] The experimental group of chicks was administered the drug via nasal drops at a dose of 0.5 mg / kg body weight, once daily for 3 consecutive days, with each nasal drop volume being 0.1 mL / chick. The control group 1 received the same dosage, administration method, and administration period as the experimental group. The control group 2 received the traditional antiviral drug ribavirin orally at a dose of 2 mg / kg body weight, once daily for 3 consecutive days, with each oral dose being 0.1 mL / chick. The blank control group received an equal volume of physiological saline via nasal drops once daily for 3 consecutive days.

[0048] 24 hours after the administration of the drug, all experimental chicks were inoculated with avian adenovirus FAdV-4 via intranasal drip, with a challenge dose of 10. 6 TCID 50 / each, each nasal drop volume is 0.1mL / each.

[0049] After challenge, chicks were observed for 7 consecutive days, and the incidence and mortality of chicks were recorded daily to calculate the morbidity and mortality rates. On the 7th day after challenge, 10 chicks were randomly selected from each group, and blood was collected from the subwing vein. Serum was separated, and the avian adenovirus load in the serum was detected by qPCR. At the same time, the contents of the cecum of the chicks were collected, and the intestinal flora diversity was detected by high-throughput sequencing.

[0050] Test results:

[0051] Morbidity and mortality: The morbidity rate of chicks in the experimental group was 13.3%, and the mortality rate was 3.3%; the morbidity rate of control group 1 was 36.7%, and the mortality rate was 16.7%; the morbidity rate of control group 2 was 43.3%, and the mortality rate was 20.0%; the morbidity rate of the blank control group was 90.0%, and the mortality rate was 76.7%, indicating that the composition of the present invention can significantly reduce the morbidity and mortality rate of chicks infected with avian adenovirus.

[0052] Serum viral load: The avian adenovirus load in the serum of chicks in the experimental group was (1.2×10³±0.3×10³) copies / mL, which was significantly lower than that in control group 1 (8.5×10³±1.2×10³ copies / mL) and control group 2 (15.6×10³±2.1×10³ copies / mL), indicating that the composition of the present invention can effectively clear avian adenovirus from chicks.

[0053] Gut microbiota diversity: The number of lactic acid bacteria in the gut of chicks in the experimental group was (8.2×10⁻⁶). 9 ±0.5×10 9 CFU / g, Bifidobacterium count was (6.5×10) 8 ±0.4×10 8 CFU / g, compared with the blank control group (lactic acid bacteria: 8.5×10⁻⁶ CFU / g). 9 ±0.6×10 9 CFU / g; Bifidobacteria: 6.8 × 10⁻⁶ 8 ±0.5×10 8 There was no significant difference in CFU / g (P>0.05); the number of lactic acid bacteria in the intestines of control group 1 and control group 2 were 4.1×10⁻⁶ and 4.1×10⁻⁶, respectively. 9 ±0.3×10 9 ) CFU / g and (3.5×10 9 ±0.4×10 9 The CFU / g and the number of Bifidobacteria were 3.2 × 10⁻⁶ CFU / g and 3.2 × 10⁻⁶ CFU / g, respectively. 8 ±0.3×10 8 ) CFU / g and (2.8×10 8 ±0.2×10 8The levels of CFU / g in the experimental group were significantly lower than those in the blank control group (P<0.05); meanwhile, the number of Escherichia coli in the intestines of the experimental group was 1.2×10⁻⁶. 6 ±0.2×10 6 The CFU / g of the control group was not significantly different from that of the blank control group, while the number of Escherichia coli in control group 1 and control group 2 was significantly increased (P<0.05), indicating that the composition of the present invention has no significant adverse effects on the intestinal flora of poultry and its safety is significantly better than that of the control group drugs.

[0054] Test group Morbidity (%) Mortality (%) Serum viral load reduction rate (%) Changes in the number of intestinal beneficial bacteria Test group 13.3 3.3 ≥92.6 No significant difference Control group 1 36.7 16.7 68.4 Significantly reduced Control group 2 43.3 20.0 52.1 Significantly reduced Blank control group 90.0 76.7 0 Normal

[0055] In summary, this invention successfully constructed an anti-avian adenovirus veterinary drug composition targeting the DDX5 protein by precisely designing a PLGA nanocarrier + dual-targeting drug + specific ligand architecture and optimizing the specific parameters and preparation process of each component. Example 1 precisely prepared the composition according to the steps of raw material preparation, blank carrier preparation, drug loading, surface modification, and performance testing, verifying that the composition can be stably prepared according to the optimized process, and the particle size, dispersibility, drug loading rate, and intracellular delivery efficiency all meet the design requirements. Example 2 in vitro experiments confirmed that the antiviral activity of this composition is significantly better than that of the unmodified ligand carrier group and the traditional free drug group, and it has low cytotoxicity. Example 3 in vivo experiments further proved that the dosage of this composition is only 25% of that of traditional antiviral drugs, which can significantly reduce the morbidity and mortality of avian adenovirus infection, while having no significant adverse effects on the intestinal flora of poultry. The safety and efficacy both meet the requirements for clinical application.

[0056] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. An avian adeno virus veterinary pharmaceutical composition targeting DDX5 protein, characterized in that, The composition takes biodegradable polylactic acid-glycolic acid copolymer PLGA nanoparticles as a delivery carrier, and the carrier is loaded with siRNA and small molecule inhibitors targeting DDX5 protein, and the surface of the carrier is modified with a ligand specific to avian respiratory epithelial cells; The particle size of the PLGA nanoparticles is controlled to be 100-200 nm.

2. The veterinary pharmaceutical composition for targeting DDX5 protein according to claim 1, characterized in that, The molar ratio of lactic acid LA to glycolic acid GA in the PLGA is 50:50-80:20; the weight average molecular weight of the PLGA is 10,000-50,000 Da, preferably 20,000-30,000 Da.

3. The veterinary pharmaceutical composition for targeting DDX5 protein according to claim 1, characterized in that, The siRNA sequence targeting DDX5 protein is designed according to the conserved region of avian DDX5 gene, and the sequence of the sense strand is 5'-GGAUUAUGCUCGACUACAATT-3', and the sequence of the antisense strand is 5'-UUGUAGUCGAGCAUAAUCCTT-3'; The loading amount of siRNA is 5%-15% of the mass of PLGA nanoparticles, and the loading amount of siRNA is 5%-15% of the mass of PLGA nanoparticles, preferably 8%-12%.

4. The veterinary pharmaceutical composition for targeting DDX5 protein according to claim 1, wherein, The small molecule inhibitor targeting DDX5 protein is one or more of curcumin derivatives, quercetin derivatives or purine derivatives.

5. The veterinary pharmaceutical composition for targeting DDX5 protein according to claim 1, wherein, The loading amount of the small molecule inhibitor is 10%-25% of the mass of the PLGA nanoparticles, preferably 15%-20%.

6. The veterinary pharmaceutical composition for targeting DDX5 protein according to claim 1, wherein, The ligand specific to avian respiratory epithelial cells is one or more of sialic acid receptor ligand, mannose receptor ligand or avian transferrin receptor ligand.

7. The veterinary pharmaceutical composition for targeting DDX5 protein according to claim 1, wherein, The modification amount of the ligand specific to avian respiratory epithelial cells is 10%-30% of the number of molecules on the surface of the PLGA nanoparticles.

8. The veterinary pharmaceutical composition for targeting DDX5 protein according to claim 1, wherein, In the veterinary composition, the mass ratio of PLGA nanoparticles, siRNA, small molecule inhibitors and specific ligand is 100:(5-15):(10-25):(2-8).

9. The veterinary pharmaceutical composition for targeting DDX5 protein according to claim 1, wherein The preparation method of the composition is: PLGA is dissolved in an organic solvent to obtain an oil phase; a surfactant is dissolved in deionized water to obtain an aqueous phase; the oil phase is slowly added to the aqueous phase, ultrasonically emulsified for 10-30 min, then magnetically stirred for 2-4 h to remove the organic solvent, centrifuged and washed 3-5 times, and freeze-dried to obtain blank PLGA nanoparticles; The siRNA and small molecule inhibitors targeting DDX5 protein are dissolved in a buffer solution, and the blank PLGA nanoparticles are added, incubated at room temperature for 1-2 h, centrifuged to collect the precipitate, and the drug-loaded PLGA nanoparticles are obtained; The ligand specific to avian respiratory epithelial cells is dissolved in a buffer solution, and the drug-loaded PLGA nanoparticles are added, incubated at 37°C for 2-4 h, centrifuged and washed 2-3 times, and freeze-dried to obtain the anti-avian adenovirus veterinary composition targeting DDX5 protein.

10. The use of an anti-avipoxvirus veterinary composition targeting a DDX5 protein, characterized in that, The composition can be prepared into an injection, an aerosol, a nasal drop or an oral preparation, preferably an aerosol or a nasal drop, which directly acts on the site of avian respiratory infection.

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