CN104706580A - Veterinary interferon inducer and preparation method thereof - Google Patents

Abstract

The invention relates to a veterinary interferon inducer. The veterinary interferon inducer is prepared from the following components: a sodium chloride-phosphate buffer solution with the pH value of 7.2, a 10-80g/L calcium chloride aqueous solution, a 0.1-5g/L chitosan hydrochloride solution, polyinosinic acid and polycytidysic acid. The invention further provides a preparation method of the veterinary interferon inducer. The veterinary interferon inducer provided by the invention is few in side reaction, low in irritation and high in safety, and the preparation method can avoid precipitates generated by calcium chloride and other components.

Description

A kind of veterinary interferon inducer and preparation method thereof

technical field

The invention relates to an interferon inducer, in particular to a veterinary interferon inducer base and a preparation method thereof.

Background technique

Interferon (Interferon, IFN) is a kind of cytokine, which has multiple functions such as inhibiting cell division, regulating immunity, anti-virus, and anti-tumor. As a broad-spectrum antiviral agent, interferon does not directly kill or inhibit viruses, mainly through the action of cell surface receptors to make cells produce antiviral proteins, thereby inhibiting virus replication; at the same time, it can also enhance natural killer cells (NK cells) ), the vitality of macrophages and T lymphocytes, thereby playing an immune regulatory role and enhancing the antiviral ability. They have broad-spectrum anti-virus, affect cell growth, differentiation, regulation of immune function and other biological activities on the same kind of cells.

Polyinosinic acid, also known as polyinosinic acid-cytidylic acid, is a synthetic double-stranded RNA polymer and a strong inducer of interferon. In addition to having broad-spectrum antiviral effects, it also has antibacterial, antiprotozoal , anti-tumor, anti-radiation and immune regulation and other biological effects. In addition, because of its unique chemical structure, polymyocytes can be degraded in the body without causing residues, endangering human health, and are safe and environmentally friendly. Polymyocytes have been proven effective in treating diseases in experiments on experimental animals, and in phase I and phase II clinical trials of human medicine. At present, polymyocytes are widely used in the prevention and treatment of herpes virus, influenza virus, hepatitis virus and other viral diseases and certain tumor diseases in human clinical practice. It is mainly used in veterinary clinical research for the treatment of viral diseases.

The current polymuscular injection products contain kanamycin as a cationic stabilizer, and the use of kanamycin is likely to cause problems such as antibiotic residues. Although chitosan can be used as a cationic stabilizer to replace kanamycin, chitosan is a kind of amino polysaccharide. Due to the strong intermolecular force, it can only be dissolved in some special acidic solutions, and chitosan acid The solution is prone to degradation during storage, and the viscosity and molecular weight will change greatly, which will affect the quality stability, which greatly limits the wide application of chitosan. It has been reported to use chitosan to coat polymyocytes to form nano-microcapsules. The basis is to use a laser pointer to test the Tyndall phenomenon of the final preparation. Since chitosan dissolves in an acidic solution, it forms a viscous colloid. The Tyndall phenomenon will appear in itself, which is not necessarily related to the formation of nano-capsules containing polysarcoma. Therefore, this method is not enough to prove that polysarcoma is encapsulated by chitosan. In addition, because polysarcoma is easily degraded in acidic solution , polyinosinocyte-chitosan acid solution is not suitable for long-term storage.

Calcium ions also play an important role in stabilizing the secondary structure of polymyocytes. In the prior art, calcium chloride powder is directly added to the reaction solution during the synthesis process. Since calcium chloride itself is relatively insoluble, directly adding powder will lead to high concentrations Calcium ions are easy to form precipitation with hydrogen phosphate, free polyinosinic acid and polycytidylic acid in the reaction system, which affects product quality and synthesis concentration.

In addition, the existing polymyocyte preparation technology also uses substances such as sodium pyrosulfite and excipients, such as the invention patent application document with the publication number CN101810638A, which discloses an injection of endogenous interferon inducers and nano-microcapsules The preparation method of chemical solution, which specifically discloses: add sodium pyrophosphite, sodium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate to the water for injection, then heat at 20-60 degrees Celsius, then add polycytidylic acid to stir, dissolve , keep warm for 10min, then add polyinosinic acid, stir and dissolve, keep warm for 15-30min. Substances such as sodium pyrosulfite and excipients are likely to irritate the body to varying degrees, especially sodium pyrosulfite, which is highly toxic and should not be swallowed. It will cause serious damage to the eyes of humans and animals if it comes into contact with it, and it is not conducive to the production and operation of workshop personnel. The polymuscular cell product prepared by the above-mentioned prior art has the risk of side effects and environmental pollution when it is injected into animals, dipped in medicine, or mixed with bait. If it is used for meat economic animals, it will also involve food safety issues and it is difficult to promote it.

Contents of the invention

The purpose of the present invention is to provide a veterinary interferon inducer with less side effects, low irritation and high safety, and meanwhile, the present invention also provides a preparation method of the veterinary interferon inducer.

In order to solve the above problems, the technical scheme adopted in the present invention is as follows:

A veterinary interferon inducer, which is made of the following components: sodium chloride-phosphate buffer solution with a pH value of 7.2, calcium chloride aqueous solution with a concentration of 10-80g/L, and a concentration of 0.1-5g/L L chitosan hydrochloride aqueous solution, polyinosinic acid, polycytidylic acid, the volume ratio of the polyinosinic acid quality and sodium chloride-phosphate buffer solution is 0.5-10g/L, the polycytosinic acid The volume ratio of the glycoside quality to the sodium chloride-phosphate buffer solution is 0.5-10g/L, the volume ratio of the calcium chloride aqueous solution to the sodium chloride-phosphate buffer solution is 2-16:1000, and the shell The volume ratio of polysaccharide hydrochloride aqueous solution and sodium chloride-phosphate buffer saline solution is 984-998:1000; The volume ratio of liquid is 1:1.

The sodium chloride-phosphate buffer solution with a pH value of 7.2 described in the present invention is formed by mixing sodium dihydrogen phosphate dihydrate, disodium hydrogen phosphate, sodium chloride and water; every 1L of sodium chloride-phosphate buffer solution Contains 0.262g sodium dihydrogen phosphate dihydrate, 1.546g disodium hydrogen phosphate, 8.768g sodium chloride.

Veterinary interferon inducer among the present invention, its preparation method comprises the steps:

a. Get polyinosinic acid, then dissolve it with the described sodium chloride-phosphate buffer solution (half of the total volume of the sodium chloride-phosphate buffer solution in the formula) of 1/2 volume, and then dissolve it at 40-70 Heating at ℃ for 5-10 minutes;

B. get polycytidylic acid, then dissolve with the described sodium chloride-phosphate buffer saline (half of the total volume of sodium chloride-phosphate buffer saline in the formula) of 1/2 volume, then in 40- Heating at 70°C for 5-10 minutes;

c. Mix the liquid obtained in step a and step b, then stir at a speed of 10-40 rpm for 3-10 minutes; then react at a temperature of 40-70°C for 20-60 minutes, and keep for 10-10 minutes during the reaction Stir at a speed of 40 rpm to obtain a reaction solution;

d. The reaction solution is cooled to room temperature, and then every 5-20s is added dropwise to the calcium chloride aqueous solution, and the volume ratio of the total volume of the calcium chloride aqueous solution to the sodium chloride-phosphate buffer solution is 2 -16:1000, keep stirring at a speed of 10-40 rpm during the dropping process;

e. slowly add the chitosan hydrochloride aqueous solution of formula quantity (the volume ratio of the above-defined chitosan hydrochloride aqueous solution and sodium chloride-phosphate buffer saline buffer solution to 984% in the reaction solution processed through d step) -998:1000; the volume ratio of the total volume of the calcium chloride aqueous solution and the chitosan hydrochloride aqueous solution to the sodium chloride-phosphate buffer solution is 1:1), and the addition process maintains 10-40 rpm speed stirring;

f. Filter the reaction solution treated in step e with a filter membrane with a pore size of 0.2-0.45 μm, and collect the filtrate to obtain the product.

Among the present invention, preferred scheme is that the calcium chloride aqueous solution in the described d step is made by following steps: according to the concentration (i.e. 10-80g/L) of described calcium chloride aqueous solution, get calcium chloride and be dissolved in water, Then filter with a 0.2-0.45 μm filter membrane, collect the filtrate, and obtain the obtained product.

In the present invention, the preferred scheme is that the chitosan hydrochloride aqueous solution in the e step is prepared by the following steps: according to the concentration (ie 0.1-5g/L) of the chitosan hydrochloride aqueous solution, the shell The polysaccharide hydrochloride is dissolved in water, then filtered through a 0.2-0.45 μm filter membrane, and the filtrate is collected.

Compared with prior art, the present invention has following advantage:

1, use soluble chitosan hydrochloride to replace kanamycin and chitosan in the present invention, has following advantage: 1) chitosan hydrochloride is all nontoxic to animal body and environment, and cost is low; 2) Chitosan hydrochloride has strong cationicity and can be used as a stabilizer for polykinases to effectively resist the degradation of RNases in the body or the environment, without antibiotic residues, and to promote the absorption of pharmaceutical ingredients by somatic cells; 3) chitosan Hydrochloride can be quickly dissolved in neutral water, and the pH value of its aqueous solution is close to neutral. It can be compatible with sodium chloride-phosphate buffer solution with pH = 7.2, which is very beneficial for comparison of irritation requirements in veterinary injections, etc. Harsh field applications also avoid the degradation of polysarcoma by acidic solutions; 4) Chitosan hydrochloride aqueous solution is stable and easy to store, which avoids the need to prepare and use traditional chitosan to restrict mass production disadvantages of development;

2. There is no need to add sodium metabisulfite, kanamycin, excipients and other substances. While ensuring the efficacy of the medicine, it can reduce the side effects on the body, avoid antibiotics and toxic residues, have no food safety problems, and do not pollute the environment. It is especially suitable for Used in meat economic animal breeding industry;

3) The preparation method of the veterinary interferon inducer of the present invention optimizes the addition process of auxiliary materials such as calcium chloride, avoids precipitation with other components when it dissolves, and ensures the synthesis effect. The concentration is between 0.1-20g/L The polyinosin solution can be successfully prepared.

The present invention will be described in detail below in combination with specific embodiments.

Detailed ways

Example 1

A veterinary interferon inducer, which is made of the following components: a volume of 500mL of sodium chloride-phosphate buffer solution with a pH value of 7.2, a volume of 1mL of calcium chloride aqueous solution with a concentration of 44.4g/L , a volume of 499mL chitosan hydrochloride aqueous solution with a concentration of 0.65g/L, 0.5g of polyinosinic acid, and 0.5g of polycytidylic acid.

The preparation method of the veterinary interferon inducer comprises the following steps:

a. Take 0.5g of polyinosinic acid, then dissolve it with 250mL of sodium chloride-phosphate buffer, and then heat it at 45°C for 5 minutes;

b. Take 0.5g of polycytidylic acid, then dissolve it with 250mL of sodium chloride-phosphate buffer, and then heat at 45°C for 5 minutes;

c. Mix the liquids obtained in steps a and b, and then stir at a speed of 30 rpm for 3 minutes; then react at a temperature of 45°C for 30 minutes, and keep stirring at a speed of 30 rpm during the reaction to obtain the reaction liquid;

d. When the reaction solution is cooled to room temperature, then add calcium chloride aqueous solution dropwise every 5s, the total volume of the calcium chloride aqueous solution added dropwise is 1mL, and the dropping process keeps stirring at a speed of 30 rpm;

E. slowly add the chitosan hydrochloride aqueous solution of 499mL in the reaction solution that is processed through d step, add process and keep the speed stirring of 30 rpm;

f. filter the reaction solution treated in step e with a filter membrane with a pore size of 0.22 μm, and collect the filtrate to obtain final product;

The calcium chloride aqueous solution in the described d step is made by following steps: according to the concentration (i.e. 44.4g/L) of described calcium chloride aqueous solution, get calcium chloride and be dissolved in water, then filter with the filter membrane of 0.22 μm, Collect the filtrate, that is, too.

Among the present invention, preferred scheme is that the chitosan hydrochloride aqueous solution in the e step is made by the following steps: according to the concentration (i.e. 0.65g/L) of the chitosan hydrochloride aqueous solution, take chitosan Dissolve sugar hydrochloride in water, then filter with a 0.22 μm filter membrane, and collect the filtrate to obtain the product.

The veterinary interferon inducer prepared in this example is compared with the quality of a commercially available certain brand of human polykinocyte injection, and the comparison is based on the National Drug Standard of the State Drug Administration [WS1-XG-050-2000 ], the specific test results are shown in Table 1:

Table 1: Comparison of the quality test results between the interferon inducer prepared by this technology and similar samples (1mg/ml)

As can be seen from the test results in Table 1, the veterinary interferon inducer prepared by the present invention meets the national drug standard [WS1-XG-050-2000] of the State Drug Administration, and is clean and sterile, can be used directly, and the effective content High, stable product quality.

The veterinary interferon inducer prepared in this example was tested for its resistance to RNase degradation stability. The veterinary interferon inducer prepared in this example and a commercially available certain brand of polymuscular cell injection group for humans were tested. Then according to the formula and method of the present embodiment but do not add the veterinary interferon inducer that chitosan hydrochloride makes as a control group, all samples are diluted to an effective concentration of 0.08mg/ml, add 20ul of 10mg/ml RNase, in a 37°C water bath, and measure the 248nm absorbance value every 15 minutes (see Table 2 for the test results).

It can be seen from the test results that the ability of the veterinary interferon inducer prepared by the invention to resist RNase degradation is better than that of the control group, and also better than that of the commercially available polymuscular cell injection for human use. It is suggested that it has good tolerance to the degradation of RNase in the environment and in animals, and is suitable for drinking water, bait mixing and medicinal bath in breeding.

Table 2: 248nm absorbance value of anti-RNase degradation test

Alpha interferon induction test

30 21-day-old SPF chickens were randomly divided into three groups, 10 in each group, respectively injected with the veterinary interferon inducer of the present embodiment, a commercially available certain brand of human polymyocyte injection, and another group injected with chlorine NaCl-phosphate buffer was used as the blank control group. Each chicken was injected at 0.01 mg/kg body weight, and injected again at intervals of 3 days. Blood was collected at the 12th and 36th hours after the last injection, and the content of α-interferon in the blood was determined by ELISA method. The specific results are detailed in Table 3:

Table 3: α-interferon induction test results

Note: peers ab means significant difference compared with the control group, ac means extremely significant difference compared with the control group, same letters have no significant difference.

It can be seen from the test results that the 0.01mg/kg body weight injection of the veterinary interferon inducer group prepared in this embodiment and the commercially available certain brand of human polykinocyte injection group have higher ability to induce alpha interferon than The blank control group and the difference are extremely significant, while the veterinary interferon inducer group prepared in this example is higher than that of a commercially available brand of human polykinocyte injection group, but the difference is not significant.

safety test

Thirty 14-day-old SPF chickens were randomly divided into three groups, 10 in each group, respectively injected with the veterinary interferon inducer of this implementation, a commercially available certain brand of human polymyocyte injection, and the other group was injected with "chlorine Sodium NaCl-phosphate buffer saline” was used as the blank control group. Each chicken was injected at 0.1 mg/kg body weight for 3 consecutive days. After the injection, it was observed whether the animals had abnormal reactions. On the 7th day after the last injection, all the animals were killed, and the injection sites and internal organs were checked.

The results showed that the animals in each group had no abnormal reaction after the injection. After the experiment was over, all the animals were killed, and the injection sites and internal organs were normal, which indicated that the preparation was safe for chickens.

Example 2

A veterinary interferon inducer, which is made of the following components: a sodium chloride-phosphate buffer solution with a pH value of 7.2, a calcium chloride aqueous solution with a concentration of 10g/L, and a shell with a concentration of 0.1g/L Polysaccharide hydrochloride aqueous solution, polyinosinic acid, polycytidylic acid, the volume ratio of described polyinosinic acid quality and sodium chloride-phosphate buffer solution is 0.5g/L, described polycytidylic acid quality and The volume ratio of sodium chloride-phosphate buffer solution is 0.5g/L, and the volume ratio of described calcium chloride aqueous solution and sodium chloride-phosphate buffer solution is 16:1000, and described chitosan hydrochloride aqueous solution and The volume ratio of the sodium chloride-phosphate buffer solution is 984:1000; the volume ratio of the total volume of the calcium chloride aqueous solution and the chitosan hydrochloride aqueous solution to the sodium chloride-phosphate buffer solution is 1:1.

Veterinary interferon inducer among the present invention, its preparation method comprises the steps:

a. Take polyinosinic acid, then dissolve it with 1/2 volume of the sodium chloride-phosphate buffer, and then heat it at 40°C for 10 minutes;

b. Take polycytidylic acid, then dissolve it with 1/2 volume of the sodium chloride-phosphate buffer, and then heat at 70°C for 10 minutes;

c. Mix the liquids obtained in steps a and b, and then stir at a speed of 10 rpm for 10 minutes; then react at a temperature of 40°C for 60 minutes, and keep stirring at a speed of 10 rpm during the reaction to obtain the reaction liquid;

d. The reaction solution is cooled to room temperature, and then the calcium chloride aqueous solution is added dropwise at intervals of 10s, and the volume ratio of the total volume of the calcium chloride aqueous solution added dropwise to the sodium chloride-phosphate buffer solution is 16:1000 , the dropping process keeps stirring at a speed of 10 rpm;

E. slowly add the chitosan hydrochloride aqueous solution of formula quantity in the reaction liquid that is processed through d step, add process and keep the speed stirring of 10 revs/min;

f. filter the reaction solution treated in step e with a filter membrane with a pore size of 0.2 μm, and collect the filtrate to obtain final product;

The calcium chloride aqueous solution in the described d step is obtained by the following steps: according to the concentration (i.e. 10g/L) of the calcium chloride aqueous solution, calcium chloride is dissolved in water, then filtered with a filter membrane of 0.2 μm, and collected Filtrate, that is.

In the present invention, the preferred scheme is that the chitosan hydrochloride aqueous solution in the e step is prepared by the following steps: according to the concentration (ie 0.1g/L) of the chitosan hydrochloride aqueous solution, the chitosan Dissolve sugar hydrochloride in water, then filter through a 0.2 μm filter membrane, and collect the filtrate to obtain the product.

The veterinary interferon inducer prepared in this example is compared with the quality of a commercially available certain brand of human polykinocyte injection, and the comparison is based on the National Drug Standard of the State Drug Administration [WS1-XG-050-2000 ], the specific test results are shown in Table 4:

Table 4: Comparison of the quality test results between the interferon inducer prepared by this technology and similar samples (1mg/ml)

As can be seen from the test results in Table 4, the veterinary interferon inducer prepared by the embodiment of the present invention meets the national drug standard [WS1-XG-050-2000] of the State Drug Administration, and is clean and sterile, can be used directly, and is effective High content, stable product quality.

The veterinary interferon inducer prepared in this example was tested for its resistance to RNase degradation stability. The veterinary interferon inducer prepared in this example and a commercially available certain brand of polymuscular cell injection group for humans were tested. Then according to the formula and method of the present embodiment but do not add the veterinary interferon inducer that chitosan hydrochloride makes as a control group, all samples are diluted to an effective concentration of 0.08mg/ml, add 20ul of 10mg/ml RNase, in a 37°C water bath, and measure the 248nm absorbance value every 15 minutes (see Table 5 for the test results).

It can be seen from the test results that the ability of the veterinary interferon inducer prepared by the invention to resist RNase degradation is better than that of the control group, and also better than that of the commercially available polymuscular cell injection for human use. It is suggested that it has good tolerance to the degradation of RNase in the environment and in animals, and is suitable for drinking water, bait mixing and medicinal bath in breeding.

Table 5: 248nm absorbance value of anti-RNase degradation test

Alpha interferon induction test

30 21-day-old SPF chickens were randomly divided into three groups, 10 in each group, respectively injected with the veterinary interferon inducer prepared in this embodiment, a commercially available certain brand of human polymyocyte injection, and another group Sodium chloride-phosphate buffer was injected as a blank control group. Each chicken was injected at 0.01 mg/kg body weight, and injected again at intervals of 3 days. Blood was collected at the 12th and 36th hours after the last injection, and the content of α-interferon in the blood was determined by ELISA method. The specific results are detailed in Table 6:

Table 6: α-interferon induction test results

Note: peers ab means significant difference compared with the control group, ac means extremely significant difference compared with the control group, same letters have no significant difference.

It can be seen from the test results that the 0.01 mg/kg body weight injection of the veterinary interferon inducer group prepared in this example and the commercially available human polykinocyte injection group have higher ability to induce the production of α-interferon than the blank control group And the difference is extremely significant, while the veterinary interferon inducer group prepared in this example is higher than the commercially available human polykinocyte injection group, but the difference is not significant.

safety test

30 14-day-old SPF chickens were randomly divided into three groups, 10 in each group, respectively injected with the veterinary interferon inducer prepared in this embodiment, a commercially available certain brand of human polymyocyte injection, and another group Inject "sodium chloride-phosphate buffer saline" as a blank control group. Each chicken was injected at 0.1 mg/kg body weight for 3 consecutive days. After the injection, it was observed whether the animals had abnormal reactions. On the 7th day after the last injection, all the animals were killed, and the injection sites and internal organs were checked.

The results showed that the animals in each group had no abnormal reaction after the injection. After the experiment was over, all the animals were killed, and the injection sites and internal organs were normal, which indicated that the preparation was safe for chickens.

Example 3

A veterinary interferon inducer, which is made of the following components: a sodium chloride-phosphate buffer solution with a pH value of 7.2, a calcium chloride aqueous solution with a concentration of 80g/L, and chitosan with a concentration of 5g/L Sugar hydrochloride aqueous solution, polyinosinic acid, polycytidylic acid, the volume ratio of described polyinosinic acid quality and sodium chloride-phosphate buffer solution is 10g/L, described polycytidylic acid quality and chloride The volume ratio of sodium-phosphate buffer saline is 10g/L, and the volume ratio of described calcium chloride aqueous solution and sodium chloride-phosphate buffer saline is 2:1000, and described chitosan hydrochloride aqueous solution and sodium chloride -The volume ratio of the phosphate buffer solution is 998:1000; the volume ratio of the total volume of the calcium chloride aqueous solution and the chitosan hydrochloride aqueous solution to the sodium chloride-phosphate buffer solution is 1:1.

Veterinary interferon inducer among the present invention, its preparation method comprises the steps:

a. Take polyinosinic acid, then dissolve it with 1/2 volume of the sodium chloride-phosphate buffer, and then heat it at a temperature of 70° C. for 6 minutes;

b. Take polycytidylic acid, then dissolve it with 1/2 volume of the sodium chloride-phosphate buffer, and then heat at 70°C for 6 minutes;

c. Mix the liquids obtained in steps a and b, and then stir at a speed of 40 rpm for 3 minutes; then react at a temperature of 70°C for 20 minutes, and keep stirring at a speed of 40 rpm during the reaction to obtain the reaction liquid;

d. The reaction solution is cooled to room temperature, and then the calcium chloride aqueous solution is added dropwise at intervals of 10s, and the volume ratio of the total volume of the calcium chloride aqueous solution added dropwise to the sodium chloride-phosphate buffer solution is 2:1000 , the dropping process keeps stirring at a speed of 40 rpm;

E. slowly add the chitosan hydrochloride aqueous solution (being that the volume ratio of chitosan hydrochloride aqueous solution and sodium chloride-phosphate buffer saline buffer solution is 998:1000) of formula quantity in the reaction solution processed through d step, Keep stirring at a speed of 40 rpm during the addition process;

f. filter the reaction solution treated in step e with a filter membrane with a pore size of 0.45 μm, and collect the filtrate to obtain final product;

The calcium chloride aqueous solution in the described d step is made by following steps: according to the concentration (i.e. 80g/L) of described calcium chloride aqueous solution, get calcium chloride and be dissolved in water, filter with the filter membrane of 0.45 μ m then, collect Filtrate, that is.

Among the present invention, preferred scheme is that the chitosan hydrochloride aqueous solution in the e step is made by the following steps: according to the concentration (i.e. 5g/L) of the chitosan hydrochloride aqueous solution, take chitosan Dissolve hydrochloride in water, then filter with a 0.45 μm membrane filter, and collect the filtrate, that is, too.

The veterinary interferon inducer prepared in this example is compared with the quality of a commercially available certain brand of human polykinocyte injection, and the comparison is based on the National Drug Standard of the State Drug Administration [WS1-XG-050-2000 ], the specific test results are shown in Table 7:

Table 7: Comparison of the quality test results between the interferon inducer prepared by this technology and similar samples (1mg/ml)

As can be seen from the test results in Table 7, the veterinary interferon inducer prepared in this embodiment meets the National Drug Standard [WS1-XG-050-2000] of the State Drug Administration, and is clean and sterile, can be used directly, and the effective content High, stable product quality.

The veterinary interferon inducer prepared in this example was tested for its resistance to RNase degradation stability. The veterinary interferon inducer prepared in this example and a commercially available certain brand of polymuscular cell injection group for humans were tested. Then according to the formula and method of the present embodiment but do not add the veterinary interferon inducer that chitosan hydrochloride makes as a control group, all samples are diluted to an effective concentration of 0.08mg/ml, add 20ul of 10mg/ml RNase, in a 37°C water bath, and measure the absorbance value at 248nm every 15 minutes (see Table 8 for the test results).

It can be seen from the test results that the ability of the veterinary interferon inducer prepared by the invention to resist RNase degradation is better than that of the control group, and also better than that of the commercially available polymuscular cell injection for human use. It is suggested that it has good tolerance to the degradation of RNase in the environment and in animals, and is suitable for drinking water, bait mixing and medicinal bath in breeding.

Table 8: 248nm absorbance value of anti-RNase degradation test

Alpha interferon induction test

30 21-day-old SPF chickens were randomly divided into three groups, 10 in each group, respectively injected with the veterinary interferon inducer prepared in this embodiment, a commercially available certain brand of human polymyocyte injection, and another group Sodium chloride-phosphate buffer was injected as a blank control group. Each chicken was injected at 0.01 mg/kg body weight, and injected again at intervals of 3 days. Blood was collected at the 12th and 36th hours after the last injection, and the content of α-interferon in the blood was determined by ELISA method. The specific experimental results are shown in Table 9:

Table 9: α-interferon induction test results

Note: peers ab means significant difference compared with the control group, ac means extremely significant difference compared with the control group, same letters have no significant difference.

It can be seen from the test results that the 0.01mg/kg body weight injection of the veterinary interferon inducer group prepared in this embodiment and the commercially available certain brand of human polykinocyte injection group have higher ability to induce alpha interferon than The blank control group and the difference are extremely significant, while the veterinary interferon inducer group prepared in this example is higher than that of a commercially available brand of human polykinocyte injection group, but the difference is not significant.

safety test

30 14-day-old SPF chickens were randomly divided into three groups, 10 in each group, respectively injected with the veterinary interferon inducer prepared in this embodiment, a commercially available certain brand of human polymyocyte injection, and another group Inject "sodium chloride-phosphate buffer saline" as a blank control group. Each chicken was injected at 0.1 mg/kg body weight for 3 consecutive days. After the injection, it was observed whether the animals had abnormal reactions. On the 7th day after the last injection, all the animals were killed, and the injection sites and internal organs were checked.

The results showed that the animals in each group had no abnormal reaction after the injection. After the experiment was over, all the animals were killed, and the injection sites and internal organs were normal, which indicated that the preparation was safe for chickens.

The above-mentioned embodiment is only a preferred embodiment of the present invention, and cannot be used to limit the protection scope of the present invention. Any insubstantial changes and substitutions made by those skilled in the art on the basis of the present invention belong to the scope of the present invention. Scope of protection claimed.

Claims (4)

1. an interferon inducers for animals, it is characterized in that being made up of following component: pH value is the sodium chloride-phosphate buffer of 7.2, concentration is the calcium chloride water of 10-80g/L, concentration is the chitosan hydrochlorate aqueous solution of 0.1-5g/L, polyinosinic acid, poly, the volume ratio of described polyinosinic acid quality and sodium chloride-phosphate buffer is 0.5-10g/L, the volume ratio of described poly quality and sodium chloride-phosphate buffer is 0.5-10g/L, the volume ratio of described calcium chloride water and sodium chloride-phosphate buffer is 2-16:1000, the volume ratio of described chitosan hydrochlorate aqueous solution and sodium chloride-phosphate buffer is 984-998:1000, the cumulative volume of described calcium chloride water and chitosan hydrochlorate aqueous solution and the volume ratio of sodium chloride-phosphate buffer are 1:1.

2. a preparation method for interferon inducers for animals according to claim 1, is characterized in that comprising the steps:

A. get polyinosinic acid, then dissolve with the described sodium chloride-phosphate buffer of 1/2 volume, then at the temperature of 40-70 DEG C, heat 5-10 minute;

B. get poly, then dissolve with the described sodium chloride-phosphate buffer of 1/2 volume, then at the temperature of 40-70 DEG C, heat 5-10 minute;

C. by the liquid mixing that a step and b step obtain, then 3-10 minute is stirred with the speed of 10-40 rev/min; Then at the temperature of 40-70 DEG C, react 20-60 minute, keep the speed of 10-40 rev/min to stir in course of reaction, obtain reactant liquor;

D. question response liquid is cooled to room temperature, then dropwise calcium chloride water is dripped at interval of 5-20s, the cumulative volume of the calcium chloride water of described dropping and the volume ratio of sodium chloride-phosphate buffer are 2-16:1000, and the process that drips keeps the speed of 10-40 rev/min to stir;

E. in the reactant liquor through Step d process, slowly add the chitosan hydrochlorate aqueous solution of formula ratio, adition process keeps the speed of 10-40 rev/min to stir;

F. by the reactant liquor membrane filtration of aperture 0.2-0.45 μm through step e process, collect filtrate, to obtain final product.

3. the preparation method of interferon inducers for animals according to claim 2, it is characterized in that the calcium chloride water in described Step d is obtained by following steps: according to the concentration of described calcium chloride water, get calcium chloride water-soluble, then the membrane filtration of 0.2-0.45 μm is used, collect filtrate, to obtain final product.

4. the preparation method of interferon inducers for animals according to claim 2, it is characterized in that the chitosan hydrochlorate aqueous solution in described step e is obtained by following steps: according to the concentration of described chitosan hydrochlorate aqueous solution, get chitosan hydrochlorate water-soluble, then the membrane filtration of 0.2-0.45 μm is used, collect filtrate, to obtain final product.