阅读说明：本技术 一种兽用免疫疫苗的稀释剂及其制备方法 (Diluent of veterinary immunity vaccine and its preparing method ) 是由 车传燕 车晓青 胡忠泽 王旋 许金根 蔡治华 于 2021-11-19 设计创作，主要内容包括：本发明提供一种兽用免疫疫苗的稀释剂及其制备方法,其由以下组分按质量百分比组成：匹多莫德1-5％、油相1-10％、表面活性剂5-30％、助表面活性剂2.5-18％、余量为蒸馏水。本发明制备的稀释剂是W/O/W型双连续相匹多莫德纳米乳,其主要成分匹多莫德以单分子状态分布于该纳米乳中,这种分布状态使得该制剂注入畜禽机体后,有效成分匹多莫德可被组织吸收、分布,进而迅速提高免疫系统中免疫因子或免疫细胞的活性。本发明制备的稀释剂为匹多莫德纳米乳,其主要成分匹多莫德以单分子状态分布于该纳米乳中。作为一种新型药物载体,匹多莫德纳米乳免疫佐剂具有缓释、协同作用。(The invention provides a diluent of a veterinary immune vaccine and a preparation method thereof, wherein the diluent comprises the following components in percentage by mass: 1-5% of pidotimod, 1-10% of oil phase, 5-30% of surfactant, 2.5-18% of cosurfactant and the balance of distilled water. The diluent prepared by the invention is W/O/W type bicontinuous phase pidotimod nanoemulsion, the main component pidotimod is distributed in the nanoemulsion in a monomolecular state, and the distribution state ensures that the effective component pidotimod can be absorbed and distributed by tissues after the preparation is injected into livestock organisms, so that the activity of immune factors or immune cells in an immune system is rapidly improved. The diluent prepared by the invention is the pidotimod nanoemulsion, and the major component pidotimod of the pidotimod is distributed in the nanoemulsion in a monomolecular state. As a novel drug carrier, the pidotimod nanoemulsion immunologic adjuvant has slow-release and synergistic effects.)

1. A diluent of an immunization vaccine for livestock, which is characterized in that: the composition comprises the following components in percentage by mass:

pidotimod 1-5%

1 to 10 percent of oil phase

5 to 30 percent of surfactant

2.5 to 18 percent of cosurfactant

The balance of distilled water.

2. A diluent for a veterinary vaccine according to claim 1, wherein: the oil phase comprises one or a mixture of corn oil, isopropyl myristate, oleic acid, cinnamaldehyde, oleic acid polyethylene glycol glyceride, glyceryl triacetate and liquid paraffin.

3. A diluent for a veterinary vaccine according to claim 1, wherein: the surfactant comprises one or a mixture of Tween-80, span-80, OP-10, polyoxyethylene hydrogenated castor oil RH-40 and castor oil polyoxyethylene ether EL-40.

4. A diluent for a veterinary vaccine according to claim 1, wherein: the cosurfactant comprises one of absolute ethyl alcohol, n-butyl alcohol, 1, 2-propylene glycol, isopropanol and glycerol.

5. A process for the preparation of a diluent for veterinary vaccines according to any one of claims 1 to 4 wherein: the preparation steps are as follows:

(1) adding pidotimod into distilled water, dissolving by ultrasonic waves to form a water phase, and placing the water phase in a water bath at 70 ℃ for later use;

(2) mixing the oil phase, the surfactant and the cosurfactant, putting the mixture into a container, and stirring the mixture in a water bath at 70 ℃ for at least 30min to obtain uniform mixed liquid for later use;

(3) and (3) slowly adding the water phase into the mixed solution in the 70 ℃ water bath in a sprayer while stirring for at least 30min, stopping stirring, and cooling to room temperature to prepare the W/O/W type bicontinuous phase nano-emulsion.

Technical Field

The invention relates to the field of preparation of diluents special for livestock vaccines, in particular to a diluent for veterinary vaccines and a preparation method thereof.

Background

Under the current complex feeding environment, when the body of a livestock and poultry group is in a sub-health state due to the influence of feeding management, poor environment or malnutrition, the immune response can be inhibited, and immune failure is often caused by the occurrence of some immunosuppressive diseases. Therefore, the special diluent which can improve the immunity of the organism, protect the vaccine and improve the antibody titer of the vaccine is very important, and the pidotimod is taken as an excellent immunopotentiator and a protective agent.

Pidotimod is an artificially synthesized immune promoter, which has a structure similar to a dipeptide and has a chemical name of (R) -3- (S) - (5-oxo-2-pyrrolidinyl) carbonyl ] -thiazolidine-4-carboxylic acid. Pidotimod does not have a direct antimicrobial effect per se, and the drug effect is mainly realized by regulating the activity of immune factors or immune cells in the immune system; the traditional Chinese medicine composition has the advantages that the traditional Chinese medicine composition can remarkably treat bacterial (pneumococcus, escherichia coli, pseudomonas aeruginosa, proteus and the like) and viral (influenza virus, herpes simplex virus, myocarditis virus and the like) infection mainly by promoting the immune function of an organism; it can be used together with antibacterial agent for treating respiratory tract infection, otorhinolaryngology infection, and urinary system infection with repeated attack of cellular immunity inhibition. The vaccine diluent is important for protecting and improving the immune effect of the vaccine, and the pidotimod immune accelerator is added into the immune diluent to further improve the immune effect.

Because the pidotimod is rapidly distributed and excreted in the body, the slow release becomes an important factor for restricting the pidotimod to be used as an immunopotentiator. The nanoemulsion is used as a novel drug carrier, and the pidotimod-containing immune diluent is prepared by utilizing the carrier, so that the metabolism of pidotimod is slowed down, and a new direction is provided for the development of immune vaccine diluents.

Pidotimod is mainly applied to the field of human medicines at present, and is rarely reported in the field of veterinary medicines. The application of pidotimod as an immunopotentiator in livestock and poultry is introduced in Chinese patent application No. CN103182067A, pidotimod raw material is added into the feed of livestock and poultry, and no related dosage forms are reported, and no continuous use with vaccines and protective action on the vaccines are reported. At present, no report on the preparation of the pidotimod nanoemulsion and the application of the pidotimod nanoemulsion as an immunopotentiator in the field of special diluents for veterinary vaccines exists.

Disclosure of Invention

The invention aims to provide a diluent of an immune vaccine for livestock and a preparation method thereof.

The technical scheme of the invention is as follows:

a diluent of an immune vaccine for livestock comprises the following components in percentage by mass:

pidotimod 1-5%

1 to 10 percent of oil phase

5 to 30 percent of surfactant

2.5 to 18 percent of cosurfactant

The balance of distilled water.

In a further embodiment, the oil phase comprises one or a mixture of corn oil, isopropyl myristate, oleic acid, cinnamaldehyde, oleic acid macrogol glyceride, triacetin or liquid paraffin.

In a further scheme, the surfactant comprises one or a mixture of Tween-80, span-80, OP-10, polyoxyethylene hydrogenated castor oil RH-40 and castor oil polyoxyethylene ether EL-40.

In a further scheme, the cosurfactant comprises one of ethanol, n-butanol, 1, 2-propylene glycol and glycerol.

The invention also aims to provide a preparation method of the diluent of the veterinary immunity vaccine, which comprises the following steps:

(1) adding pidotimod into distilled water, dissolving by ultrasonic waves to form a water phase, and placing the water phase in a water bath at 70 ℃ for later use;

(2) mixing the oil phase, the surfactant and the cosurfactant, putting the mixture into a container, and stirring the mixture in a water bath at 70 ℃ for at least 30min to obtain uniform mixed liquid for later use;

(3) and (3) slowly adding the water phase into the mixed solution in the 70 ℃ water bath in a sprayer while stirring for at least 30min, stopping stirring, and cooling to room temperature to prepare the W/O/W type bicontinuous phase nano-emulsion.

The diluent prepared by the invention is W/O/W type bicontinuous phase pidotimod nanoemulsion, the proportion of an oil phase, a surfactant and a cosurfactant is reasonable, the major component pidotimod is distributed in the nanoemulsion in a monomolecular state, and the distribution state ensures that after the preparation is injected into a livestock organism, the active component pidotimod can be rapidly absorbed and distributed by tissues, so that the activity of immune factors or immune cells in an immune system is rapidly improved, the change of the immune activity of the animal body can greatly improve the immune antibody titer and the vaccine protection efficacy of the vaccine, the success rate of the vaccine immunity is ensured, and the W/O/W type bicontinuous phase pidotimod nanoemulsion is the best substitute of the existing conventional diluent.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

Preparing 100kg of 1% W/O/W type bicontinuous pidotimod nanoemulsion:

(1) adding 1kg of pidotimod into 46kg of distilled water, dissolving by ultrasonic waves to form a water phase, and placing the water phase in a water bath at 70 ℃ for later use;

(2) weighing 5kg of sterilized IPM, 20kg of RH-40, 10kg of EL-40 and 18kg of absolute ethyl alcohol, and stirring for 30min at 70 ℃ to obtain uniform mixed liquor for later use;

(3) and (3) slowly adding the water phase into the mixed solution in the 70 ℃ water bath in a sprayer while stirring for at least 30min, stopping stirring, and cooling to room temperature to prepare the W/O/W type bicontinuous phase nano-emulsion.

The clinical usage amount is 500ml of 1 percent pidotimod nanoemulsion diluent used per 1000 feather parts.

Example 2

Preparing 100kg of 1.5% W/O/W type pidotimod nanoemulsion:

preparing 100kg of 1% W/O/W type bicontinuous pidotimod nanoemulsion:

(1) adding 1.5kg of pidotimod into 53kg of distilled water, ultrasonically dissolving to form a water phase, and placing in a water bath at 70 ℃ for later use;

(2) weighing 6kg of cinnamaldehyde after sterilization, 25kg of EL-40 and 15kg of isopropanol, and stirring for 30min at 70 ℃ to obtain a uniform mixed solution for later use;

(3) and (3) slowly adding the water phase into the mixed solution in the 70 ℃ water bath in a sprayer while stirring for at least 30min, stopping stirring, and cooling to room temperature to prepare the W/O/W type bicontinuous phase nano-emulsion.

The clinical usage amount is 334ml of 1.5 percent pidotimod nanoemulsion diluent used per 1000 feather parts.

Example 3

Preparing 100kg of 2% W/O/W type pidotimod nanoemulsion:

(1) adding 2kg of pidotimod into 60kg of distilled water, ultrasonically dissolving to form a water phase, and placing the water phase in a water bath at 70 ℃ for later use;

(2) weighing sterilized 7kg of liquid paraffin, 21kg of tween-80, 7kg of span-80 and 3kg of propylene glycol, and stirring for 30min at 70 ℃ to obtain a uniform mixed solution for later use;

(3) and (3) slowly adding the water phase into the mixed solution in the 70 ℃ water bath in a sprayer while stirring for at least 30min, stopping stirring, and cooling to room temperature to prepare the W/O/W type bicontinuous phase nano-emulsion.

The clinical usage amount is 250ml of 2 percent pidotimod nanoemulsion diluent used per 1000 feather parts.

Example 4

Preparing 100kg of 2.5% W/O/W type pidotimod nanoemulsion:

(1) adding 2.5kg of pidotimod into 65kg of distilled water, ultrasonically dissolving to form a water phase, and placing in a water bath at 70 ℃ for later use;

(2) weighing 10kg of sterilized oleic acid, 12kg of OP-10 and 10.5kg of propylene glycol, and stirring for 30min at 70 ℃ to obtain a uniform mixed solution for later use;

(3) and (3) slowly adding the water phase into the mixed solution in the 70 ℃ water bath in a sprayer while stirring for at least 30min, stopping stirring, and cooling to room temperature to prepare the W/O/W type bicontinuous phase nano-emulsion.

The clinical usage amount is 200ml of 2.5 percent pidotimod nanoemulsion diluent used per 1000 feather parts.

Example 5

Preparing 100kg of 3% W/O/W type pidotimod nanoemulsion:

(1) adding 3kg of pidotimod into 68kg of distilled water, ultrasonically dissolving to form a water phase, and placing in a water bath at 70 ℃ for later use;

(2) weighing 1kg of sterilized corn oil, 14kg of tween-80 and 14kg of anhydrous ethanol, and stirring at 70 deg.C for 30min to obtain uniform mixed solution;

(3) and (3) slowly adding the water phase into the mixed solution in the 70 ℃ water bath in a sprayer while stirring for at least 30min, stopping stirring, and cooling to room temperature to prepare the W/O/W type bicontinuous phase nano-emulsion.

The clinical usage amount is 167ml of 3 percent pidotimod nanoemulsion diluent used per 1000 feather parts.

Example 6

Preparing 100kg of 3.5% W/O/W type pidotimod nanoemulsion:

(1) adding 3.5kg of pidotimod into 75kg of distilled water, ultrasonically dissolving to form a water phase, and placing in a water bath at 70 ℃ for later use;

(2) weighing 2kg of sterilized polyethylene glycol oleate, 7.5kg of EL-40, 6kg of span-80 and 6kg of glycerol, and stirring for 30min at 70 ℃ to obtain a uniform mixed solution for later use;

(3) and (3) slowly adding the water phase into the mixed solution in the 70 ℃ water bath in a sprayer while stirring for at least 30min, stopping stirring, and cooling to room temperature to prepare the W/O/W type bicontinuous phase nano-emulsion.

The clinical usage amount is 143ml of pidotimod nanoemulsion diluent with the concentration of 3.5 percent per 1000 feather.

Example 7

Preparing 100kg of 4% W/O/W type pidotimod nanoemulsion:

(1) adding 4kg of pidotimod into 78kg of distilled water, ultrasonically dissolving to form a water phase, and placing in a water bath at 70 ℃ for later use;

(2) weighing 1kg of sterilized IPM, 1kg of sterilized corn oil, 8kg of sterilized gRH-40, 4kg of sterilized span-80 and 4kg of sterilized glycerol, and stirring for 30min at 70 ℃ to obtain a uniform mixed solution for later use;

(3) and (3) slowly adding the water phase into the mixed solution in the 70 ℃ water bath in a sprayer while stirring for at least 30min, stopping stirring, and cooling to room temperature to prepare the W/O/W type bicontinuous phase nano-emulsion.

The clinical usage amount is 125ml of 4 percent pidotimod nanoemulsion diluent used per 1000 feather parts.

Example 8

Preparing 100kg of 4.5% W/O/W type pidotimod nanoemulsion:

(1) adding 4.5kg of pidotimod into 81kg of distilled water, ultrasonically dissolving to form a water phase, and placing in a water bath at 70 ℃ for later use;

(2) weighing sterilized 0.5kg of liquid paraffin, 8kg of tween-80 and 6kg of absolute ethyl alcohol, and stirring at 70 ℃ for 30min to obtain a uniform mixed solution for later use;

(3) and (3) slowly adding the water phase into the mixed solution in the 70 ℃ water bath in a sprayer while stirring for at least 30min, stopping stirring, and cooling to room temperature to prepare the W/O/W type bicontinuous phase nano-emulsion.

The clinical usage amount is about 111ml of 4.5 percent pidotimod nanoemulsion diluent used per 1000 feather.

Example 9

Preparing 100kg of 5% W/O/W type pidotimod nanoemulsion:

(1) adding 5kg of pidotimod into 85.5kg of distilled water, ultrasonically dissolving to form a water phase, and placing in a water bath at 70 ℃ for later use;

(2) weighing sterilized 2kg glyceryl triacetate, 2.5kg tween-80, 2.5kg span-80 and 2.5kg n-butanol, and stirring at 70 deg.C for 30min to obtain uniform mixed solution;

(3) and (3) slowly adding the water phase into the mixed solution in the 70 ℃ water bath in a sprayer while stirring for at least 30min, stopping stirring, and cooling to room temperature to prepare the W/O/W type bicontinuous phase nano-emulsion.

The clinical usage amount is 100ml of 5 percent pidotimod nanoemulsion diluent used per 1000 feather parts.

The W/O/W type pidotimod nanoemulsion prepared by the embodiment can effectively improve the immune antibody titer of the veterinary vaccine and enhance the protective efficacy of the vaccine.

The experimental design and results are as follows:

(1) test method

36 ICR female mice were randomly divided into 6 groups of 6 mice each. Each mouse was immunized nasally with 20ul of vaccine dilution containing 1 wing of Newcastle disease vaccine. The concentrations of the pidotimod nanoemulsion of the 5 groups of diluents are 0.125, 0.25, 0.5, 1.0 and 2.0mg/ml respectively. Another set of conventional vaccine dilutions was used as a control (0 mg/ml). D0 primary immunization and D14 secondary immunization.

(2) Sampling and antibody detection

Sampling and serum preparation: blood collection was performed on mice 7, 14, 21d after the secondary immunization, and the whole blood was allowed to stand at 37 ℃ for 2 hours. After the mixture is placed at 4 ℃ for 3h, the mixture is centrifuged at 2500r/min for 8 min. Sucking the upper layer serum, and freezing at-20 deg.C.

Indirect ELISA determination of total IgG levels in serum: the newcastle disease hemagglutination test standard antigen (according to HA experiment 2 unit antigen dilution, CBS dilution) is coated on a 96-well enzyme label plate, each well is 100ul, and the plate is placed at 4 ℃ overnight. PBST was washed 5 times for 3min each. Add blocking solution (5% skim milk-PBS diluted) 300. mu.l to each well, work at 37 ℃ for 1h, wash as above. The freshly prepared test serum (diluted 1:400, 5% skim milk-PBS) was added to each well at 100ul, incubated at 37 ℃ for 1h, and washed. Adding the enzyme-labeled antibody (diluted by 1:2000, 5% skim milk-PBS), reacting at 37 deg.C for 1h and washing, wherein each well is 100 ul. Adding ready-prepared TMB, 100ul per well, and developing at 37 deg.C for 10-15 min. Adding 2M sulfuric acid 50ul into each hole, terminating the reaction, and detecting OD value at 450nm (the antigen coating concentration, serum dilution and enzyme-labeled antibody dilution required by ELASA are obtained by performing matrix test on a pre-test)

(3) Statistical analysis

SPSS statistical software is adopted to perform one-way analysis of variance on the 3-week data. Multiple comparisons were tested using one-way anova with differences in lower case letters indicating significant differences (p <0.05) and differences in upper case letters indicating significant differences (p < 0.01).

(4) Test results

The effects of the O/W pidotimod nanoemulsion prepared by the three methods on the newcastle disease virus antibody level in the mouse serum are shown in tables 1,2 and 3 respectively.

Table 1 example 1 changes in newcastle disease virus antibody levels in mouse serum

Table 2 example 2 changes in newcastle disease virus antibody levels in mouse serum

Table 3 example 3 changes in newcastle disease virus antibody levels in mouse serum

According to the statistical results of the mouse serum antibody levels of the three embodiments, the level of the specific antibody in the mouse serum after vaccine immunization can be remarkably improved in a certain concentration range after the Newcastle disease vaccine immunization is carried out on the mouse by using the pimod nanoemulsion as the vaccine diluent. By taking the example 1 as an example, compared with the control group, the levels of the newcastle disease antibodies in the serum of the mice can be remarkably improved when the pidotimod nanoemulsion of 0.125mg/mL, 0.25mg/mL, 0.5mg/mL and 1mg/mL is added, and the level is remarkably reduced when the pidotimod nanoemulsion of 2mg/mL is added.

The effect of the pidotimod nanoemulsion prepared in examples 1-3 on the newcastle disease virus antibody level in the mouse serum is consistent, that is, the newcastle disease antibody level in the mouse serum shows a trend of increasing and then decreasing with the increase of the added concentration of the pidotimod nanoemulsion. The test result indicates that the pimoda nanoemulsion can effectively improve the immune antibody titer and the vaccine protection efficacy of the vaccine, and can achieve the optimal immune promotion effect under the appropriate concentration.

The original data information of the influence of the W/O/W pidotimod nanoemulsion prepared by the three methods on the level of the mouse serum Newcastle disease virus antibody is shown in the following tables 4-6:

table 4 table for analysis of antibody level in example 1

Table 5 table for analysis of antibody level in example 2

Table 6 table for analysis of antibody level in example 3

The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other. The device disclosed by the embodiment corresponds to the method disclosed by the embodiment, so that the description is simple, and the relevant points can be referred to the method part for description.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.