CN112675147A - Natural traditional Chinese medicine polysaccharide-loaded zinc finger antiviral protein sustained and controlled release preparation and preparation method thereof - Google Patents
Natural traditional Chinese medicine polysaccharide-loaded zinc finger antiviral protein sustained and controlled release preparation and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a natural traditional Chinese medicine polysaccharide zinc finger antiviral protein-loaded sustained and controlled release preparation and a preparation method thereof, belonging to the technical field of sustained and controlled release preparation. The zinc finger antiviral protein with specific antiviral activity is loaded into the nano self-assembly body of the natural traditional Chinese medicine polysaccharide, the broad-spectrum antibacterial and antiviral activity of the traditional Chinese medicine polysaccharide plays a role in synergism, the immune environment in an animal body is improved, the specific inhibition of the zinc finger antiviral protein on the avian leukosis virus is realized through a sustained and controlled release technology, and finally the effects of remarkably promoting the healthy growth of the animal and improving the production performance of the animal can be achieved.
Description
Technical Field
The invention relates to the technical field of preparation of sustained and controlled release preparations, in particular to a natural traditional Chinese medicine polysaccharide zinc finger antiviral protein-loaded sustained and controlled release preparation and a preparation method thereof.
Background
Zinc finger antiviral protein (ZAP) is a natural host endogenous immune factor, mainly distributed in animals, plants and microorganisms, and the ZAP can inhibit virus replication by mediating degradation of virus mRNA and inhibiting translation. Wherein the CCCH type zinc finger antiviral protein (CCCH-ZAP) exhibits a specific inhibitory activity against a virus. Research has found that CCCH type zinc finger antiviral protein as natural defense factor in chicken can inhibit the replication of leukemia J subgroup virus (ALV-J) effectively.
However, the current zinc finger antiviral protein is mainly used in an injection mode, so that the stress effect of livestock and poultry is easily generated.
The traditional Chinese medicine polysaccharide is a complex natural high molecular compound existing in natural plants, microorganisms and seaweed organisms. Research shows that the Chinese medicinal polysaccharide such as Atractylodis rhizoma polysaccharide and radix Platycodi polysaccharide is an immunopotentiator, can enhance or regulate immunity, and has bioactivity of enhancing immunity, resisting virus, tumor, oxidation and aging. However, the traditional Chinese medicine polysaccharide is a broad-spectrum antibacterial antiviral agent, and cannot realize specific antiviral activity.
Disclosure of Invention
Aiming at the prior art, the invention aims to provide a natural traditional Chinese medicine polysaccharide zinc finger antiviral protein-loaded sustained-release preparation and a preparation method thereof. The zinc finger antiviral protein with specific antiviral activity is loaded into the nano self-assembly body of the natural traditional Chinese medicine polysaccharide, the broad-spectrum antibacterial and antiviral activity of the traditional Chinese medicine polysaccharide plays a role in synergism, the immune environment in an animal body is improved, the specific inhibition of the zinc finger antiviral protein on the avian leukosis virus is realized through a sustained and controlled release technology, and finally the effects of remarkably promoting the healthy growth of the animal and improving the production performance of the animal can be achieved.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a preparation method of a natural traditional Chinese medicine polysaccharide-loaded zinc finger antiviral protein sustained-release preparation, which comprises the following steps:
(1) dissolving Chinese medicinal polysaccharide in deionized water to obtain 5-30mg/ml Chinese medicinal polysaccharide water solution;
(2) dripping a solvent into the aqueous solution of the traditional Chinese medicine polysaccharide obtained in the step (1) to ensure that the volume content of the solvent is 30-70%, and carrying out ultrasonic treatment to ensure that traditional Chinese medicine polysaccharide molecules are self-assembled to form a nano self-assembly body; adding zinc finger antiviral protein, and performing oscillation treatment in ice water bath to obtain a natural traditional Chinese medicine polysaccharide zinc finger antiviral protein-loaded sustained-release preparation;
or adding zinc finger antiviral protein into the aqueous solution of the traditional Chinese medicine polysaccharide obtained in the step (1), then dropwise adding a solvent to make the volume percentage of the solvent be 30-70%, and carrying out oscillation treatment in an ice-water bath to co-assemble traditional Chinese medicine polysaccharide molecules and the zinc finger antiviral protein to form a nano-assembly, thus preparing the natural traditional Chinese medicine polysaccharide zinc finger antiviral protein-loaded sustained-release preparation.
Preferably, the traditional Chinese medicine polysaccharide is atractylodes polysaccharide or platycodon polysaccharide.
Preferably, in the step (2), the solvent is absolute ethyl alcohol or Dimethylformamide (DMF).
Preferably, the zinc finger antiviral protein is a CCCH type zinc finger antiviral protein.
Preferably, the adding amount ratio of the zinc finger antiviral protein to the traditional Chinese medicine polysaccharide aqueous solution is 50 μ L: 50 μ L.
Preferably, in step (2), the power of the ultrasonic treatment is 150W.
In a second aspect of the invention, the sustained-release preparation of natural traditional Chinese medicine polysaccharide-loaded zinc finger antiviral protein prepared by the method is provided.
In a third aspect of the invention, the use of the sustained-release preparation in the preparation of a product for treating avian leukemia is provided.
The invention has the beneficial effects that:
the invention utilizes the special functional group structures of the traditional Chinese medicine polysaccharide and the zinc finger antiviral protein to realize the effective assembly of the traditional Chinese medicine polysaccharide and the zinc finger antiviral protein in a mixed solvent system through non-covalent bond acting force; the slow controlled release of the protein is realized by loading the zinc finger antiviral protein by the traditional Chinese medicine polysaccharide; makes full use of the synergistic effect of the broad-spectrum antibacterial immunocompetence of the traditional Chinese medicine polysaccharide on the specific antiviral performance of the zinc finger antiviral protein, realizes the selective treatment and the comprehensive immunity of animal diseases, and has wide application prospect in the field of animal disease prevention and treatment.
Drawings
FIG. 1: in the embodiment 1 of the invention, when the volume ratio of ethanol to water is 1: assembly morphology at 1; FIG. 1a is a low power scanning electron micrograph of the self-assembled structure, from which it can be seen that the obtained self-assembled structure of the Atractylodis rhizoma polysaccharide is substantially flat pea-like structure with uniform size distribution; FIG. 1b is a high magnification photograph of the self-assembled structures, from which it can be seen that each pea structure is about 1.5 μm long, about 1 μm wide, and about 500nm thick. The special self-assembly structure not only plays a good role in loading zinc finger antiviral proteins, but also is easier to carry and convey drugs in animal bodies.
FIG. 2: in embodiment 3 of the present invention, when the volume ratio of DMF to water is 1: assembly morphology at 1; FIG. 2a is a low power scanning electron micrograph of the self-assembled structure, from which it can be seen that the obtained self-assembled structure of the Atractylodis rhizoma polysaccharide is substantially a relatively uniform particle aggregate; fig. 2b is a high magnification photograph of the self-assembled structure, from which it can be seen that each particle aggregate surface has an uneven micro-nano structure. The special self-assembly structure is more beneficial to the loading of zinc finger antiviral protein.
FIG. 3: in order to test the sustained and controlled release performance of traditional Chinese medicine polysaccharide-loaded zinc finger antiviral protein, the sustained and controlled release performance of the traditional Chinese medicine polysaccharide-loaded zinc finger antiviral protein in an aqueous solution is researched by taking the atractylodes macrocephala polysaccharide-loaded protein as an example. Firstly, adding the solid of the white atractylodes rhizome polysaccharide load protein into a deionized water solution, then placing the solution into a constant-temperature water bath shaking table at 37 ℃ for sustained and controlled release, taking out 10 mu L of the solution every 10 minutes, and measuring the protein concentration by using a BCA method to obtain a protein sustained and controlled release curve. Wherein, the curves a and b respectively correspond to the sustained and controlled release curves of the samples obtained in the embodiments 1 and 3, namely, the sustained and controlled release experimental curves of the samples obtained by firstly adding ethanol and DMF to obtain the atractylodes macrocephala polysaccharide assembly and then loading zinc finger antiviral protein; curves c and d correspond to the sustained and controlled release curves of the samples obtained in examples 2 and 4, respectively, that is, the sustained and controlled release experimental curves of the samples obtained by adding ethanol and DMF to the solution of the atractylodes macrocephala polysaccharide and the zinc finger antiviral protein.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
As introduced in the background art, zinc finger antiviral proteins can show specific inhibitory activity against viruses, but the current zinc finger antiviral proteins are mainly used in an injection mode, so that the stress action of livestock and poultry is easily caused; the traditional Chinese medicine polysaccharide has broad-spectrum antibacterial and antiviral activity, but cannot realize specific inhibition on viruses.
Based on the above, in order to solve the problems existing when the zinc finger antiviral protein and the traditional Chinese medicine polysaccharide are used independently, the invention adopts a mixed solvent co-assembly technology, utilizes the precipitation self-assembly of the traditional Chinese medicine polysaccharide in the mixed solvent and the non-covalent bond acting force between the traditional Chinese medicine polysaccharide and the zinc finger antiviral protein to load the zinc finger antiviral protein into a natural traditional Chinese medicine polysaccharide nano-assembly body with various biological activities, and regulates and controls the size and the shape of the traditional Chinese medicine polysaccharide self-assembly body through the regulation of conditions such as concentration, a solvent system and the like so as to improve the load capacity of zinc finger antiviral protein molecules in the traditional Chinese medicine polysaccharide. The sustained and controlled release of the zinc finger antiviral protein loaded with the traditional Chinese medicine polysaccharide in vitro or in vivo of animals is realized by regulating and controlling different solvents, temperatures and the like. The invention combines the traditional Chinese medicine polysaccharide and the zinc finger antiviral protein through the co-assembly to obtain the sustained-release technology, fully utilizes the broad-spectrum antibacterial immunocompetence of the traditional Chinese medicine polysaccharide and the specific antiviral property of the zinc finger antiviral protein, and has wide application prospect in the field of animal disease prevention and treatment.
In one embodiment of the invention, the preparation of the natural traditional Chinese medicine polysaccharide zinc finger antiviral protein-loaded sustained-release preparation comprises the following steps:
(1) dissolving 0.25-1.50mg of Chinese medicinal polysaccharide (Atractylodis rhizoma polysaccharide, radix Platycodi polysaccharide, etc.) in 50 μ L deionized water at concentration range of 5-30 mg/mL. And adding solvent such as anhydrous ethanol or DMF dropwise into the solution to make the volume content of the solvent in the solution be 30-70%. With the dropwise slow addition of the solvent, the solubility of the traditional Chinese medicine polysaccharide in the solution is gradually reduced, and solids are separated out. After ultrasonic treatment, the traditional Chinese medicine polysaccharide molecules are self-assembled to form a nano assembly with a specific nano structure. Adding zinc finger antiviral protein 50 μ L before or after adding the above solvent, shaking in ice water bath, and loading protein with polysaccharide for more than 6 hr.
(2) Zn in zinc finger antiviral protein molecule while traditional Chinese medicine polysaccharide self-assembles to form nano-assembly2+-NH in cysteine (Cys) and histidine (His)2And the-COOH, -SH and imidazole rings are co-assembled with-OH in the traditional Chinese medicine polysaccharide through non-covalent acting forces such as hydrogen bonds, pi-pi accumulation and the like, so that the loading of the traditional Chinese medicine polysaccharide on the zinc finger antiviral protein is realized.
(3) The size and the appearance of the loading system are adjusted to improve the loading capacity. The size and the shape of the loading system are adjusted through different solvent systems, concentrations, loading modes and the like, and the loading capacity of the zinc finger antiviral protein is improved.
In the step (2), the zinc finger antiviral protein is CCCH type zinc finger antiviral protein (according to Zn)2+Binding conserved amino acid residues of cysteine (Cys) and histidine (His) in different combinationsIs of 9 major classes: C2H2, C8, C6, C3HC4, C2HC, C2HC5, C4, C4HC3, and CCCH).
In the step (3), the content of the solvent is 30-70%; the loading mode is co-assembly, coating, surface adsorption and the like.
The sustained and controlled release preparation prepared by the invention can adjust the solubility of polysaccharide by regulating a solution system, temperature and the like, and realize the sustained and controlled release of in vitro zinc finger antiviral protein. The purpose of sustained and controlled release of protein is achieved by utilizing the unique body fluid, pH value, temperature and other environments in the animal body. If the zinc finger antiviral protein loaded by the atractylis ovata polysaccharide is dispersed into the aqueous solution, the atractylis ovata polysaccharide in the assembly structure is gradually dissolved in the aqueous solution, and the sustained and controlled release of the zinc finger antiviral protein is realized.
In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments.
The test materials used in the examples of the present invention are all conventional in the art and commercially available. The experimental procedures, for which no detailed conditions are indicated, were carried out according to the usual experimental procedures or according to the instructions recommended by the supplier. Wherein, the atractylodes macrocephala polysaccharide [ the extraction, purification and activity research of the atractylodes macrocephala polysaccharide, Anhui agri. Sci.2012, 40(24): 12011-.
The protein loading in the examples of the invention was determined according to the BCA method. Firstly, Bovine Serum Albumin (BSA) is used as a model protein to draw a standard curve, and then the concentration of the protein in the solution before and after loading is obtained according to the absorbance values of zinc finger antiviral proteins in the solution before and after loading, so that the loading capacity can be obtained.
Example 1:
dissolving 500 mu g of atractylenovata polysaccharide in 50 mu L of deionized water, and carrying out ultrasonic treatment at 150W power for 30 minutes to obtain a solution with the concentration of 10 mg/mL. Then, 50. mu.L of absolute ethyl alcohol is added dropwise into the solution, so that the content of the absolute ethyl alcohol in the solution is 50%. With the dropwise slow addition of anhydrous ethanol, the solubility of the traditional Chinese medicine polysaccharide in the solution is gradually reduced, the traditional Chinese medicine polysaccharide molecules gradually perform self-assembly under the ultrasonic treatment condition (ultrasonic power of 150W), and a nano-assembly with a specific nano-structure is formed after the self-assembly is performed for 6h (as shown in figure 1). And continuously adding 50 mu L of zinc finger antiviral protein into the ethanol-water mixed solution, and performing oscillation treatment in ice-water bath for 6h to obtain the slow/controlled release preparation of the bighead atractylodes rhizome polysaccharide-loaded zinc finger antiviral protein, so that the loading of the zinc finger antiviral protein is realized. The maximum protein loading can reach 19.14 mu g/mg.
Example 2:
dissolving 500 mu g of atractylenovata polysaccharide in 50 mu L of deionized water, and carrying out ultrasonic treatment at 150W power for 30 minutes to obtain a solution with the concentration of 10 mg/mL. And continuously adding 50 mu L of zinc finger antiviral protein into the solution and uniformly mixing. Then, absolute ethyl alcohol was added dropwise to the mixed solution so that the volume content of ethyl alcohol in the mixed solution was 50%. Gradually reducing the solubility of the traditional Chinese medicine polysaccharide in the solution along with the dropwise slow addition of the absolute ethyl alcohol, and carrying out oscillation treatment in an ice-water bath to gradually carry out co-assembly on the traditional Chinese medicine polysaccharide molecules and the zinc finger antiviral protein for 6 hours to form a nano-assembly body with a specific nano-structure. Realizes the loading of zinc finger antiviral protein. The maximum protein loading was 10.28. mu.g/mg.
Example 3:
dissolving-500 mu g of atractylenovata polysaccharide in 50 mu L of deionized water, and carrying out ultrasonic treatment at 150W power for 30 minutes to obtain a solution with the concentration of 10 mg/mL. Then, DMF was added dropwise to the above solution so that the volume content of DMF in the solution was 50%. With the dropwise and slow addition of DMF, the solubility of the traditional Chinese medicine polysaccharide in the solution is gradually reduced, the traditional Chinese medicine polysaccharide molecules are gradually self-assembled under the ultrasonic treatment condition (the ultrasonic power is 150W), and the self-assembly lasts for 6h to form a nano-assembly with a specific nano-structure. And continuously adding 50 mu L of zinc finger antiviral protein into the mixed solution, and carrying out oscillation treatment in an ice-water bath for 6h to realize the loading of the zinc finger antiviral protein. The maximum protein loading was 24.54. mu.g/mg.
Example 4:
dissolving 500 mu g of atractylenovata polysaccharide in 50 mu L of deionized water, and carrying out ultrasonic treatment at 150W power for 30 minutes to obtain a solution with the concentration of 10 mg/mL. Adding 50 mu L of zinc finger antiviral protein into the solution and mixing evenly. Then, DMF was added dropwise to the above solution so that the volume content of DMF in the solution was 50%. Gradually reducing the solubility of the traditional Chinese medicine polysaccharide in the solution along with the dropwise and slow addition of DMF, and carrying out oscillation treatment in an ice-water bath to gradually carry out co-assembly on the traditional Chinese medicine polysaccharide molecules and the zinc finger antiviral protein for 6h to form a nano assembly body with a specific nano structure. Realizes the loading of zinc finger antiviral protein. The maximum protein loading was 14.84. mu.g/mg.
Example 5:
dissolving 500 mu g of platycodon grandiflorum polysaccharide in 50 mu L of deionized water, and carrying out ultrasonic treatment at 150W power for 30 minutes to obtain a solution with the concentration of 10 mg/mL. Then, absolute ethyl alcohol is added into the solution drop by drop, so that the volume content of the absolute ethyl alcohol in the solution is 50%. With the dropwise slow addition of the absolute ethyl alcohol, the solubility of the traditional Chinese medicine polysaccharide in the solution is gradually reduced, the traditional Chinese medicine polysaccharide molecules are gradually self-assembled under the ultrasonic treatment condition (the ultrasonic power is 150W), and a nano-assembly with a specific nano-structure is formed after the self-assembly is carried out for 6 hours. And continuously adding 50 mu L of zinc finger antiviral protein into the ethanol-water mixed solution, and carrying out oscillation treatment in an ice water bath for 6h to realize the loading of the zinc finger antiviral protein. The maximum protein loading was 19.26. mu.g/mg.
Example 6:
dissolving 500 mu g of platycodon grandiflorum polysaccharide in 50 mu L of deionized water, and carrying out ultrasonic treatment at 150W power for 30 minutes to obtain a solution with the concentration of 10 mg/mL. And continuously adding 50 mu L of zinc finger antiviral protein into the solution and uniformly mixing. Then, absolute ethyl alcohol was added dropwise to the above mixed solution so that the volume content of the absolute ethyl alcohol in the mixed solution was 50%. Gradually reducing the solubility of the traditional Chinese medicine polysaccharide in the solution along with the dropwise slow addition of the ethanol, and carrying out oscillation treatment in an ice-water bath to gradually carry out co-assembly on the traditional Chinese medicine polysaccharide molecules and the zinc finger antiviral protein for 6h to form a nano-assembly with a specific nano-structure. Realizes the loading of zinc finger antiviral protein. The maximum protein load was 11.58. mu.g/mg.
Example 7:
dissolving 500 mu g of platycodon grandiflorum polysaccharide in 50 mu L of deionized water, and carrying out ultrasonic treatment at 150W power for 30 minutes to obtain a solution with the concentration of 10 mg/mL. Then, DMF was added dropwise to the above solution so that the volume content of DMF in the solution was 50%. With the dropwise and slow addition of DMF, the solubility of the traditional Chinese medicine polysaccharide in the solution is gradually reduced, the traditional Chinese medicine polysaccharide molecules are gradually self-assembled under the ultrasonic treatment condition (the ultrasonic power is 150W), and a nano-assembly with a specific nano-structure is formed after the self-assembly is carried out for 6 h. And continuously adding 50 mu L of zinc finger antiviral protein into the mixed solution, and carrying out oscillation treatment in an ice-water bath for 6h to realize the loading of the zinc finger antiviral protein. The maximum protein loading was 22.23. mu.g/mg.
Example 8:
dissolving 500 mu g of platycodon grandiflorum polysaccharide in 50 mu L of deionized water, and carrying out ultrasonic treatment at 150W power for 30 minutes to obtain a solution with the concentration of 10 mg/mL. Adding 50 mu L of zinc finger antiviral protein into the solution and mixing evenly. Then, DMF was added dropwise to the above solution so that the volume content of DMF in the solution was 50%. Gradually reducing the solubility of the traditional Chinese medicine polysaccharide in the solution along with the dropwise and slow addition of DMF, and carrying out oscillation treatment in an ice-water bath to gradually carry out co-assembly on the traditional Chinese medicine polysaccharide molecules and the zinc finger antiviral protein for 6h to form a nano assembly body with a specific nano structure. Realizes the loading of zinc finger antiviral protein. The maximum protein loading was 13.79. mu.g/mg.
Test example:
in order to test the sustained and controlled release performance of the traditional Chinese medicine polysaccharide-loaded zinc finger antiviral protein prepared in the embodiment of the invention, the sustained and controlled release performance of the traditional Chinese medicine polysaccharide-loaded zinc finger antiviral protein in an aqueous solution is researched by taking the atractylodes macrocephala polysaccharide-loaded protein as an example.
The method for testing the sustained and controlled release performance comprises the following steps: firstly, adding the solid of the white atractylodes rhizome polysaccharide load protein into a deionized water solution, then placing the solution into a constant-temperature water bath shaking table at 37 ℃ for sustained and controlled release, taking out 10 mu L of the solution every 10 minutes, measuring the protein concentration by using a BSA method, and drawing a protein sustained and controlled release curve (figure 3).
It can be seen from the controlled release curve (fig. 3) that the curves a and b release protein more easily than the curves c and d, which indicates that the zinc finger antiviral protein is adsorbed after the traditional Chinese medicine polysaccharide assembly is formed by adding the solvent (ethanol and DMF), mainly by surface adsorption, so that desorption is easier; and the traditional Chinese medicine polysaccharide and the zinc finger antiviral protein are mixed firstly, and then the solvent (ethanol and DMF) is added, so that a co-assembly of the traditional Chinese medicine polysaccharide and the zinc finger antiviral protein is easier to form and is relatively more stable, and the desorption of c and d is more difficult than that of a and b.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims (8)
1. A preparation method of a natural traditional Chinese medicine polysaccharide-loaded zinc finger antiviral protein sustained-release preparation is characterized by comprising the following steps:
(1) dissolving traditional Chinese medicine polysaccharide in deionized water to obtain 5-30mg/mL traditional Chinese medicine polysaccharide water solution;
(2) dripping a solvent into the aqueous solution of the traditional Chinese medicine polysaccharide obtained in the step (1) to ensure that the volume content of the solvent is 30-70%, and carrying out ultrasonic treatment to ensure that traditional Chinese medicine polysaccharide molecules are self-assembled to form a nano self-assembly body; adding zinc finger antiviral protein, and performing oscillation treatment in ice water bath to obtain a natural traditional Chinese medicine polysaccharide zinc finger antiviral protein-loaded sustained-release preparation;
or adding zinc finger antiviral protein into the aqueous solution of the traditional Chinese medicine polysaccharide obtained in the step (1), then dropwise adding a solvent to make the volume percentage of the solvent be 30-70%, and carrying out oscillation treatment in an ice-water bath to co-assemble traditional Chinese medicine polysaccharide molecules and the zinc finger antiviral protein to form a nano-assembly, thus preparing the natural traditional Chinese medicine polysaccharide zinc finger antiviral protein-loaded sustained-release preparation.
2. The method for preparing the polysaccharide of claim 1, wherein the polysaccharide of traditional Chinese medicine is polysaccharide of white atractylodes rhizome or polysaccharide of platycodon grandiflorum.
3. The method according to claim 1, wherein in the step (2), the solvent is absolute ethanol or DMF.
4. The method according to claim 1, wherein the zinc finger antiviral protein is a CCCH-type zinc finger antiviral protein.
5. The method according to claim 1 or 4, wherein the ratio of the zinc finger antiviral protein to the aqueous solution of the traditional Chinese medicine polysaccharide is 50 μ L: 50 μ L.
6. The production method according to claim 1, wherein in the step (2), the power of the ultrasonic treatment is 150W.
7. The sustained-release preparation of natural traditional Chinese medicine polysaccharide-loaded zinc finger antiviral protein prepared by the method of any one of claims 1 to 6.
8. Use of the sustained or controlled release formulation of claim 7 in the manufacture of a product for the treatment of avian leukemia.
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| CN114699532A (en) * | 2022-04-02 | 2022-07-05 | 山东农业大学 | Sodium alginate/traditional Chinese medicine polysaccharide-loaded zinc finger antiviral protein co-assembly medicine and preparation method thereof |
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| CN114699532A (en) * | 2022-04-02 | 2022-07-05 | 山东农业大学 | Sodium alginate/traditional Chinese medicine polysaccharide-loaded zinc finger antiviral protein co-assembly medicine and preparation method thereof |
| CN114699532B (en) * | 2022-04-02 | 2024-02-02 | 山东农业大学 | Sodium alginate/traditional Chinese medicine polysaccharide loaded zinc finger antiviral protein co-assembled medicine and preparation method thereof |
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