Poultry attenuated drinking water immune vaccine nano adjuvant as well as preparation method and application thereof

Abstract

The invention discloses a nano adjuvant for a poultry attenuated drinking water immune vaccine as well as a preparation method and application of the nano adjuvant, and relates to the technical field of preparation of immune adjuvants. The adjuvant is a catecholamine-modified functional silicon dioxide microsphere, and then the attenuated drinking water immune vaccine for poultry is prepared by electrostatic adsorption of an attenuated antigen. The preparation conditions are mild, the process is simple, the method is stable and reliable, the production cost is low, and the prepared poultry attenuated drinking water immune vaccine can promote production of secretory immune globulin A, effectively stimulate immune response of intestinal mucosa and realize efficient immune protection. The problems that an existing poultry attenuated vaccine is poor in drinking water immunization effect and tedious in immunization procedure are solved.

Description

Technical Field

[0001] The present invention relates to the technical field of immunoadjuvant preparation, and particularly relates to a nanoadjuvant for attenuated live poultry drinking water immunization vaccine, a preparation method thereof, and an application thereof. Background Art

[0002] Vaccination, as the most effective measure against poultry infectious diseases, plays an important role in modern intensive farming; among them, the drinking water immunization method can not only greatly reduce the time cost and labor input of immunization, but also effectively avoid the mechanical damage and physiological stress caused by traditional immunization methods to poultry. Drinking water immunization vaccines are convenient to use, have high compliance, save costs and can induce systemic or local mucosal immune responses, which is more in line with actual production. However, drinking water immunization vaccines have not been widely applied in actual production, limited by many factors: 1. Antigen degradation in the gastrointestinal environment (digestive enzymes, gastric acid); 2. Low permeability of the mucus barrier; 3. Nonspecific binding of antigens at the intestinal mucosal site, etc.

[0003] In recent years, innovative nano-delivery technologies have brought opportunities to improve the effectiveness and applicability of oral vaccines. Nano-delivery systems, with clear components, stable structures, and special physical and chemical properties, have greatly promoted the uptake of antigens at the mucosal site, transported vaccine components to key immune cells or lymphoid tissues, stimulated the maturation of immune cells, and ultimately achieved efficient immune protection. However, traditional adjuvants are difficult to meet the requirements of the oral immunization route. Therefore, it is particularly important to develop new adjuvants to adapt to the vaccine drinking water immunization method. Summary of the Invention

[0004] In order to solve the above technical problems, the object of the present invention is to provide a nanoadjuvant for attenuated live poultry drinking water immunization vaccine, a preparation method thereof, and an application thereof, so as to solve the problems of poor drinking water immunization effect and cumbersome immunization procedures existing in existing attenuated live poultry vaccines.

[0005] The technical solution for solving the above technical problems of the present invention is as follows: Provide a nanoadjuvant for attenuated live poultry drinking water immunization vaccine, which is a catecholamine-modified functionalized silica microsphere.

[0006] On the basis of the above technical solution, the present invention can also be improved as follows:

[0007] Further, the catecholamine is dopamine, norepinephrine or epinephrine.

[0008] Further, the functional group is amino group, mercapto group or phenyl group.

[0009] Further, the particle size of the nanoadjuvant for attenuated live poultry drinking water immunization vaccine is 361±88nm, and the potential is 13.3±2.3mV.

[0010] The present invention also provides a method for preparing the nano - adjuvant for attenuated live poultry drinking water immunization vaccine, comprising the following steps:

[0011] Disperse the functionalized silica microspheres in the aqueous solution of catecholamine, add the NaOH solution, stir, wash the obtained microspheres and freeze - dry them to obtain the catecholamine - modified functionalized silica microspheres, which are the nano - adjuvant for attenuated live poultry drinking water immunization vaccine.

[0012] The beneficial effect of the present invention is that: under the alkaline condition of NaOH, catecholamine oxidatively self - polymerizes to form a polycatecholamine coating, which covalently binds to the surface of the functionalized silica microspheres through Michael addition or Schiff base reaction, and finally the catecholamine - modified functionalized silica microspheres are obtained.

[0013] Furthermore, the concentration of the aqueous solution of catecholamine is 4 - 6 wt%.

[0014] Furthermore, the concentration of the aqueous solution of catecholamine is 5 wt%.

[0015] Furthermore, the mass ratio of the functionalized silica microspheres to catecholamine is 0.6:0.5 - 0.6.

[0016] Furthermore, add the NaOH solution to adjust the pH value to 10 - 14.

[0017] Furthermore, the concentration of the NaOH solution is 5 wt%.

[0018] Furthermore, stir at room temperature for 6 h.

[0019] Furthermore, the functionalized silica microspheres are prepared by the following method:

[0020] (1) Mix the chloride salt, deionized water, ethanol and tetraethyl orthosilicate, dropwise add ammonia water, and stir to obtain a white emulsion A;

[0021] (2) Drop the ethanol solution of tetraethyl orthosilicate into the white emulsion A obtained in step (1), add ammonia water to obtain an emulsion B;

[0022] (3) Dissolve the functionalized raw materials in ethanol to obtain a mixed solution, and drop the mixed solution into the emulsion B obtained in step (2), and stir to obtain an emulsion C;

[0023] (4) Centrifuge, wash and dry the emulsion C obtained in step (3) to obtain the functionalized silica microspheres.

[0024] Furthermore, in step (1), the mass - volume ratio of the chloride salt, deionized water, ethanol, tetraethyl orthosilicate and ammonia water is 0.02 - 0.03 g:9 - 10 g:75 - 76 g:1.6 - 1.7 g:3 - 5 mL.

[0025] Further, in step (1), the mass-volume ratio of the chloride salt, deionized water, ethanol, tetraethyl orthosilicate, and ammonia water is 0.0248 g: 9.45 g: 75.39 g: 1.645 g: 4 mL.

[0026] Further, in step (1), the chloride salt is KCl or NaCl.

[0027] Further, in step (1), stirring is carried out at room temperature and 200 - 250 rpm for 18 - 22 min.

[0028] Further, in step (1), stirring is carried out at room temperature and 240 rpm for 20 min.

[0029] Further, in step (2), in the ethanol solution of tetraethyl orthosilicate, the mass ratio of tetraethyl orthosilicate to ethanol is 2 - 3: 13 - 14.

[0030] Further, in step (2), in the ethanol solution of tetraethyl orthosilicate, the mass ratio of tetraethyl orthosilicate to ethanol is 2.06: 13.12.

[0031] Further, in step (2), the mass-volume ratio of tetraethyl orthosilicate, tetraethyl orthosilicate in step (1), and ammonia water is 2 - 3 g: 1.6 - 1.7 g: 0.8 - 1.2 mL.

[0032] Further, in step (2), the mass-volume ratio of tetraethyl orthosilicate, tetraethyl orthosilicate in step (1), and ammonia water is 2.06 g: 1.645 g: 1 mL.

[0033] Further, in step (3), in the mixed solution, the mass ratio of the functionalized raw material to ethanol is 2 - 3: 13 - 14.

[0034] Further, in step (3), in the mixed solution, the mass ratio of the functionalized raw material to ethanol is 2.52: 13.12.

[0035] Further, in step (3), the mass ratio of the mixed solution to tetraethyl orthosilicate in step (2) is 15 - 17: 2 - 3.

[0036] Further, in step (3), the mass ratio of the mixed solution to tetraethyl orthosilicate in step (2) is 15.64: 2.06.

[0037] Further, in step (3), the functionalized raw material is a mixture of tetraethyl orthosilicate and 3-aminopropyltriethoxysilane, mercaptopropyltriethoxysilane, or a mixture of 3-aminopropyltriethoxysilane and phenyltriethoxysilane.

[0038] Furthermore, in the mixture of tetraethyl orthosilicate and 3-aminopropyltriethoxysilane, the mass ratio of tetraethyl orthosilicate to 3-aminopropyltriethoxysilane is 2.06:0.46.

[0039] Furthermore, in step (3), stir for 0.8 - 1.2 h.

[0040] Furthermore, in step (3), stir for 1 h.

[0041] Furthermore, in step (4), wash twice with absolute ethanol and deionized water respectively, then disperse in Tris-HCl with a pH value of 6, add HCl solution to adjust the pH value to 4 - 5, stir, and then remove the excessive HCl to complete the washing process.

[0042] The beneficial effects of adopting the above further technical solutions are as follows: The functions of HCl are as follows: 1. Adjust the pH value to activate surface functional groups, such as amino groups. After emulsion C is washed, it is dispersed in Tris-HCl buffer solution (pH = 6), and the pH is adjusted to 4 - 5 with HCl solution. This acidic environment can protonate the amino group (-NH2) to -NH3+, enhancing the positive charge on the surface of the microspheres, thereby promoting the subsequent adsorption of catecholamines. Catecholamines are easily adsorbed onto the positively charged aminated surface through electrostatic interaction or hydrogen bonding under weakly acidic to neutral conditions (pH 4 - 8); 2. Improve the surface reaction activity: Provide favorable conditions for the oxidative self-polymerization of catecholamines in the next step; 3. Remove residual alkaline substances. Ammonia water (alkaline catalyst) is used in steps (1) - (3) to promote the hydrolysis and condensation of TEOS to form SiO2 microspheres. HCl can neutralize the residual ammonia water to avoid the interference of the alkaline environment on subsequent functionalization modifications (such as dopamine polymerization requires controllable pH conditions). After removing the excessive HCl, it is ensured that only the protonated amino group (-NH3+) remains on the surface of the microspheres, without introducing free HCl molecules. This reaction enhances the electrostatic interaction between the microspheres and catecholamines (containing phenolic hydroxyl groups, showing negative charges).

[0043] Furthermore, the concentration of the HCl solution is 4 - 5 wt%.

[0044] The beneficial effects of adopting the above further technical solutions are as follows: The concentration of HCl needs to be strictly controlled to avoid the dissolution or structural damage of SiO2 microspheres caused by excessive acidification. The choice of adjusting the pH value to 4 - 5 balances the amino protonation requirement and the material stability (too low pH may corrode the SiO2 framework). HCl plays a key role in charge regulation and cleaning in the surface functionalization modification of microspheres, laying the foundation for the efficient loading of catecholamines.

[0045] Furthermore, stir for 25 - 35 min.

[0046] Furthermore, stir for 30 min.

[0047] Further, wash away the excess HCl with a Tris-HCl solution having a pH of 6.

[0048] Further, in step (4), lyophilization is adopted.

[0049] The present invention also provides the application of the above-mentioned nanoadjuvant for attenuated live poultry drinking water immunization vaccine in the preparation of attenuated live poultry drinking water immunization vaccine.

[0050] Further, the attenuated live poultry drinking water immunization vaccine is prepared by the following method:

[0051] Dilute the antigen step by step with PBS buffer to the immunization titer to obtain a mixed solution, add catecholamine-modified functionalized silica microspheres, stir and mix, centrifuge, and recover the precipitate to obtain the attenuated live poultry drinking water immunization vaccine.

[0052] Further, the antigen is infectious bronchitis virus, Newcastle disease virus, infectious bursal disease virus or infectious laryngotracheitis virus.

[0053] Further, the mass-volume ratio of the catecholamine-modified functionalized silica microspheres to the PBS buffer is 0.2 g: 55 - 65 mL.

[0054] Further, the mass-volume ratio of the catecholamine-modified functionalized silica microspheres to the mixed solution is 0.1 - 0.2 g: 20 - 45 mL.

[0055] Further, the dose of the antigen in the mixed solution is 10 4 -10 5 EID 50 / 0.2 mL.

[0056] Further, the dose of the antigen in the mixed solution is 10 4.5 EID 50 / 0.2 mL.

[0057] The present invention has the following beneficial effects:

[0058] 1. Silica microspheres (SM) have shown broad application prospects in drug delivery systems and the biomedical field due to their high specific surface area, excellent stability, high adsorption capacity, easy surface functionalization, and good biocompatibility. In particular, the abundant silanol groups on their surface facilitate various functionalizations, enabling them to meet different biomedical application requirements. Catecholamines, such as dopamine (DA), as an important biogenic amine neurotransmitter, not only have strong hydrophilicity but also exhibit unique adhesion properties. The introduction of catecholamines can significantly enhance the adhesion ability of the delivery system to mucosal tissues, thereby prolonging the retention time of antigens in the body. Based on this, for example, in the present invention, SMP-DA microparticles are used as a delivery carrier, utilizing the sustained and controlled release and adhesion ability of DA, combined with the immobilization and protection of SMP on attenuated antigens, to play the role of attenuated vaccine drinking water immunization.

[0059] 2. The virus loading rate of the attenuated poultry drinking water immunization vaccine prepared in the present invention is 99%. The relatively high virus loading rate can reduce the dosage of the virus, reducing its toxicity and stimulatory effects on chicken body cells. Therefore, the present invention has characteristics such as a relatively high loading rate and pH-controlled release, and has a good immune protection effect against IB, which can provide ideas for the industrialization of chicken oral vaccines. At the same time, the present invention uses catecholamines as adhesives, which can successfully adhere the virus loaded on SMP nanoparticles in the intestine, and can controllably release the loaded virus in a weakly alkaline environment, enhancing intestinal mucosal immunity.

[0060] 3. The preparation conditions of the present invention are mild, the process is simple, the method is stable and reliable, the production cost is low, and the prepared attenuated poultry drinking water immunization vaccine can promote the production of secretory immunoglobulin A, effectively stimulate intestinal mucosal immune responses, and achieve efficient immune protection. Brief Description of the Drawings

[0061] Figure 1 SEM images of SMP@DA and D90-SMP@DA in Example 1;

[0062] Figure 2 Effect of different concentrations of SMP@DA on the viability of DF-1 cells in Example 1;

[0063] Figure 3 Body weights of chickens after oral gavage with SMP@DA at 0d, 10d, and 17d in Example 1;

[0064] Figure 4 Virus production amount of D90 in chicken embryos at different time points of the vaccine in Example 1;

[0065] Figure 5 Acid-base release of the vaccine in simulated gastrointestinal fluids in Example 1;

[0066] Figure 6 The tracheal sIgA content after vaccination with the vaccine in Example 1;

[0067] Figure 7 The sIgA content in the intestinal lavage fluid after immunization in Example 1. Detailed implementation manners

[0068] The principles and features of the present invention will be described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention. For those conditions not specified in the examples, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. For those reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained by purchasing in the market.

[0069] Infectious bronchitis virus, Newcastle disease virus, infectious bursal disease virus and infectious laryngotracheitis virus are all purchased from Guangdong Wens Dahuayuan Biotechnology Co., Ltd.

[0070] Example 1:

[0071] A nano - adjuvant for attenuated live poultry drinking water immunization vaccine, and its preparation method includes the following steps:

[0072] (1) Mix KCl, deionized water, ethanol and tetraethyl orthosilicate (TEOS) and add them to a three - necked round - bottom flask. Slowly dropwise add ammonia water, and stir at room temperature and 240 rpm for 20 min to obtain a white emulsion A; among them, the mass - to - volume ratio of KCl, deionized water, ethanol, tetraethyl orthosilicate and ammonia water is 0.0248 g: 9.45 g: 75.39 g: 1.645 g: 4 mL;

[0073] (2) Drop an ethanol solution of tetraethyl orthosilicate (the mass ratio of tetraethyl orthosilicate to ethanol is 2.06: 13.12) into the white emulsion A obtained in step (1), and add ammonia water to obtain emulsion B; among them, the mass - to - volume ratio of tetraethyl orthosilicate, tetraethyl orthosilicate in step (1) and ammonia water is 2.06 g: 1.645 g: 1 mL;

[0074] (3) Dissolve tetraethyl orthosilicate and 3 - aminopropyltriethoxysilane (APTE) in ethanol (the mass ratio of tetraethyl orthosilicate, 3 - aminopropyltriethoxysilane, and ethanol is 2.06: 0.46: 13.12) to obtain a mixed solution. Drop the mixed solution into emulsion B obtained in step (2) and stir for 1 h to obtain emulsion C; among them, the mass ratio of tetraethyl orthosilicate to tetraethyl orthosilicate in step (2) is 2.06: 2.06;

[0075] (4) Centrifuge the emulsion C prepared in step (3) to obtain microspheres, wash them twice with anhydrous ethanol and deionized water respectively to remove residues, then disperse them in 20 mL of Tris-HCl with a pH value of 6, add HCl solution (concentration: 4 wt%) to adjust the pH value to 4, stir for 30 min, and then wash away the excess HCl with Tris-HCl solution with a pH value of 6 to complete the washing process. Freeze-dry for 24 h to obtain amino-functionalized silica microspheres (SMP).

[0076] (5) Disperse the amino-functionalized silica microspheres in an aqueous dopamine solution (concentration of the aqueous dopamine solution: 5 wt%), with the mass ratio of the amino-functionalized silica microspheres to dopamine being 0.6:0.6. Add NaOH solution (5 wt%) to adjust the pH value to 12, stir at room temperature for 6 h. Wash the obtained microspheres twice with pure water and then freeze-dry to obtain grey dopamine-modified amino-functionalized silica microspheres (SMP@DA), which are the nanoadjuvants for poultry live attenuated drinking water vaccines.

[0077] A poultry live attenuated drinking water vaccine, and its preparation method comprises the following steps:

[0078] S1: Propagation and culture of the virus

[0079] Dilute the infectious bronchitis virus D90 (Guangdong Wens Dahuayuan Bio-Science Co., Ltd.) 10-fold with sterile PBS, inoculate the allantoic cavity of 9-day-old SPF chicken embryos at a dose of 0.2 mL / embryo, and culture them in an incubator at 37°C. Examine the embryos 24 h after inoculation, discard the dead embryos; after 48 h, place the chicken embryos in a refrigerator at 4°C. After the blood vessels contract, collect the allantoic fluid, aliquot it, and store it at -80°C. The collected allantoic fluid is the propagated D90, and use the Reed-Muench method to perform EID 50 Calculate the virus titer, 10 6.5 EID 50 / 0.2 mL;

[0080] S2: Vaccine preparation

[0081] Gradually dilute the antigen with PBS buffer to the immunological titer (10 4.5 EID 50 / 0.2 mL) to obtain a mixed solution. Add the dopamine-modified amino-functionalized silica microspheres, and stir and mix them uniformly in a magnetic stirrer to enable SMP@DA to fully adsorb D90. Centrifuge and recover the precipitate to obtain the poultry live attenuated drinking water vaccine (D90-SMP@DA). Among them, the mass-volume ratio of the dopamine-modified amino-functionalized silica microspheres to the mixed solution is 0.5 g:100 mL.

[0082] Example 2:

[0083] A nanoadjuvant for attenuated live poultry drinking water immunization vaccine, and its preparation method comprises the following steps:

[0084] (1) Mix NaCl, deionized water, ethanol and tetraethyl orthosilicate (TEOS) and add them into a three-necked round-bottom flask. Slowly add ammonia water dropwise, and stir at room temperature and 200 rpm for 22 min to obtain a white emulsion A; wherein, the mass-volume ratio of NaCl, deionized water, ethanol, tetraethyl orthosilicate and ammonia water is 0.02 g: 9 g: 75 g: 1.6 g: 3 mL;

[0085] (2) Drop an ethanol solution of tetraethyl orthosilicate (the mass ratio of tetraethyl orthosilicate to ethanol is 2:13) into the white emulsion A prepared in step (1), and add ammonia water to obtain an emulsion B; wherein, the mass-volume ratio of tetraethyl orthosilicate, tetraethyl orthosilicate and ammonia water in step (1) is 2 g: 1.6 g: 0.8 mL;

[0086] (3) Dissolve mercaptopropyltriethoxysilane (STOES) in ethanol (the mass ratio of mercaptopropyltriethoxysilane to ethanol is 2:13) to obtain a mixed solution. Drop the mixed solution into the emulsion B prepared in step (2) and stir for 0.8 h to obtain an emulsion C; wherein, the mass ratio of tetraethyl orthosilicate to tetraethyl orthosilicate in step (2) is 1:1;

[0087] (4) Centrifuge the emulsion C prepared in step (3) to separate the microspheres, wash with anhydrous ethanol and deionized water twice each to remove the residues, then disperse them in 20 mL of Tris-HCl with a pH value of 6, add an HCl solution (concentration: 5 wt%) to adjust the pH value to 4, stir for 30 min, and then wash away the excess HCl with a Tris-HCl solution with a pH value of 6 to complete the washing process. Freeze-dry for 22 h to obtain mercapto-functionalized silica microspheres;

[0088] (5) Disperse the mercapto-functionalized silica microspheres in an aqueous solution of norepinephrine (the concentration of the aqueous solution of norepinephrine is 4 wt%). The mass ratio of the mercapto-functionalized silica microspheres to norepinephrine is 0.6:0.6. Add a NaOH solution (5 wt%) to adjust the pH value to 14, stir at room temperature for 6 h. Wash the obtained microspheres twice with pure water and then freeze-dry to obtain gray norepinephrine-modified mercapto-functionalized silica microspheres, namely the nanoadjuvant for attenuated live poultry drinking water immunization vaccine.

[0089] An attenuated live poultry drinking water immunization vaccine, and its preparation method comprises the following steps:

[0090] S1: Propagation and culture of virus

[0091] The Newcastle disease virus (Guangdong Wenshi Dahuanong Biotechnology Co., Ltd.) was diluted 10-fold with sterile PBS and inoculated into the allantoic cavity of 9-day-old SPF chicken embryos at a dose of 0.2 mL / embryo. The embryos were then placed in an incubator at 37°C for cultivation. The embryos were candled 24 hours after inoculation, and the dead embryos were discarded. After 48 hours, the chicken embryos were placed in a refrigerator at 4°C. After the blood vessels contracted, the allantoic fluid was collected, aliquoted, and stored at -80°C. The collected allantoic fluid was the propagated virus, and the EID was calculated using the Reed-Muench method. 50 Calculate 10 6.5 EID 50 / 0.2 mL;

[0092] S2: Vaccine preparation

[0093] The antigen was stepwise diluted with PBS buffer to the immunopotency (10 4.5 EID 50 / 0.2 mL) to prepare a mixed solution. Noradrenaline-modified mercapto-functionalized silica microspheres were added, and the mixture was stirred evenly in a magnetic stirrer to allow the noradrenaline-modified mercapto-functionalized silica microspheres to fully adsorb the Newcastle disease virus. After centrifugation, the precipitate was recovered to prepare a live attenuated drinking water vaccine for poultry. Among them, the mass-volume ratio of the noradrenaline-modified mercapto-functionalized silica microspheres to the mixed solution was 0.5 g:100 mL.

[0094] Example 3:

[0095] A nanoadjuvant for a live attenuated drinking water vaccine for poultry, and its preparation method includes the following steps:

[0096] (1) KCl, deionized water, ethanol, and tetraethyl orthosilicate (TEOS) were mixed and added to a three-necked round-bottom flask, and ammonia water was slowly added dropwise. The mixture was stirred at room temperature and 250 rpm for 18 minutes to obtain a white emulsion A; among them, the mass-volume ratio of KCl, deionized water, ethanol, tetraethyl orthosilicate, and ammonia water was 0.03 g:10 g:76 g:1.7 g:5 mL;

[0097] (2) An ethanol solution of tetraethyl orthosilicate (the mass ratio of tetraethyl orthosilicate to ethanol was 3:14) was dropped into the white emulsion A prepared in step (1), and ammonia water was added to obtain emulsion B; among them, the mass-volume ratio of tetraethyl orthosilicate, tetraethyl orthosilicate in step (1), and ammonia water was 3 g:1.7 g:1.2 mL;

[0098] (3) Dissolve 3-aminopropyltriethoxysilane and phenyltriethoxysilane (PTOES) in ethanol (the mass ratio of 3-aminopropyltriethoxysilane, phenyltriethoxysilane, and ethanol is 2.06:0.46:13.12) to obtain a mixed solution. Drop the mixed solution into the emulsion B prepared in step (2), and stir for 1.2 h to obtain emulsion C; wherein, the mass ratio of tetraethyl orthosilicate to tetraethyl orthosilicate in step (2) is 1:1;

[0099] (4) Centrifuge the emulsion C prepared in step (3) to separate the microspheres, wash with anhydrous ethanol and deionized water twice each to remove the residues, then disperse them in 20 mL of Tris-HCl with a pH value of 6, add HCl solution (concentration 4 wt%) to adjust the pH value to 5, stir for 35 min, and then wash away the excess HCl with Tris-HCl solution with a pH value of 6 to complete the washing process. Freeze-dry for 26 h to obtain phenylated silica microspheres;

[0100] (5) Disperse the phenylated silica microspheres in an aqueous dopamine solution (the concentration of the aqueous dopamine solution is 6 wt%), the mass ratio of the phenylated silica microspheres to dopamine is 0.6:0.6, add NaOH solution (5 wt%) to adjust the pH value to 10, stir at room temperature for 6 h, wash the obtained microspheres twice with pure water, and freeze-dry to obtain gray dopamine-modified phenylated silica microspheres, which are the nanoadjuvants for poultry live attenuated drinking water vaccines.

[0101] A poultry live attenuated drinking water vaccine, and its preparation method includes the following steps:

[0102] S1: Propagation and cultivation of the virus

[0103] Dilute the infectious bursal disease virus (Guangdong Wens Dahuannong Biotechnology Co., Ltd.) 10-fold with sterile PBS, inoculate the allantoic cavity of 9-day-old SPF chicken embryos, inoculate 0.2 mL per embryo, and place them in an incubator at 37 °C for cultivation. Examine the embryos once 24 h after inoculation, and discard the dead embryos; after 48 h, place the chicken embryos in a refrigerator at 4 °C. After the blood vessels contract, collect the allantoic fluid, aliquot it, and store it at -80 °C. The collected allantoic fluid is the propagated virus, and use the Reed-Muench method to perform EID 50 Calculation, 10 6.5 EID 50 / 0.2 mL;

[0104] S2: Vaccine preparation

[0105] Stepwise dilute the antigen with PBS buffer to the immunopotency (10 4.5 EID 50(0.2 mL), a mixed solution was prepared, and dopamine-modified phenylated silica microspheres were added. The mixture was stirred evenly in a magnetic stirrer to allow the dopamine-modified phenylated silica microspheres to fully adsorb infectious bursal disease virus. After centrifugation, the precipitate was recovered to obtain a live attenuated poultry drinking water immunization vaccine. Among them, the mass-volume ratio of the dopamine-modified phenylated silica microspheres to the mixed solution was 0.5 g: 100 mL.

[0106] Example 4:

[0107] A nanoadjuvant for live attenuated poultry drinking water immunization vaccine, and its preparation method includes the following steps:

[0108] Same as Example 1.

[0109] A live attenuated poultry drinking water immunization vaccine, and its preparation method includes the following steps:

[0110] Replace the infectious bronchitis virus in Example 1 with infectious laryngotracheitis virus.

[0111] S1: Propagation and culture of the virus

[0112] Inoculate the infectious laryngotracheitis virus on the chorioallantoic membrane (CAM) of 9-day-old SPF chicken embryos. Incubate at 37 °C for 4 days, and pox-like lesions can be seen. Collect the CAM tissue, homogenize it, centrifuge to take the supernatant, aliquot it, and store it at -80 °C. The supernatant collected is the propagated virus. Calculate the EID50 using the Reed-Muench method, 10 6.5 EID 50 / 0.2 mL;

[0113] The rest is the same as Example 1.

[0114] Test example

[0115] I. Morphology detection

[0116] The morphologies of the SMP@DA and D90-SMP@DA products prepared in Example 1 were detected by scanning electron microscopy, and the results are shown in Figure 1 ( Figure 1 In it, A is the morphology of SMP@DA, and B is the morphology of D90-SMP@DA).

[0117] From Figure 1 it can be seen that the D90 virus was successfully adsorbed on SMP@DA.

[0118] II. Particle size and zeta potential detection

[0119] The SMP, SMP@DA, and D90-SMP@DA prepared in Example 1 were detected for particle size and zeta potential using a Malvern laser particle size analyzer, and the results are shown in Table 1.

[0120] As can be seen from Table 1, SMP@DA is in the form of nanoparticles with average particle sizes of 301±15 nm and 361±88 nm. After the modification and adsorption of D90 on the microspheres, the average particle size of the microspheres increased, which is consistent with the results of scanning electron microscopy. From the change of the Zeta potential, it can be seen that the potential of SMP@DA is 13.3±2.3 mV, but it still maintains a certain positive charge to ensure the effective adsorption of D90. At the same time, the adjacent hydroxyl groups brought by it effectively increase the adhesion of the microspheres and the adsorption ability of D90 through hydrogen bond effect and electronegativity.

[0121] Table 1 Average particle size and Zeta potential

[0122]

[0123] III. Cytotoxicity test

[0124] To verify the biosafety of SMP@DA for the preparation of live attenuated poultry drinking water vaccines, we detected the effect of different concentrations of SMP@DA (solvent: PBS) on the viability of DF-1 cells and evaluated the biocompatibility of SMP@DA, with PBS as the blank control. The specific method was the CCK-8 method, and the results are shown in Figure 2 .

[0125] As Figure 2 can be seen, cell viability is closely related to the concentration of SMP@DA. As the concentration increases, the cell survival rate gradually decreases. When the concentration of SMP@DA reaches 1 mg / mL, the cell survival rate can still reach 83%.

[0126] IV. Effects on chickens and chicken embryos

[0127] 1. After inoculating the allantoic cavity of chicken embryos with the SMP@DA suspension in Example 1 (0.1 g SMP@DA and 20 mL PBS), observations were made daily. All survived at 20 days of age, and all chicks hatched at 21 - 22 days of age, indicating that SMP@DA has no obvious adverse effects on chicken embryos.

[0128] 2. After orally gavage-feeding 5-day-old SPF chickens with the SMP@DA suspension in Example 1 (0.1 g SMP@DA and 20 mL PBS), observations were made within 17 days. The body weight of chickens in each group at 0 d, 10 d, and 17 d after oral gavage-feeding is shown in Figure 3 (using PBS as the blank control, setting up the D90 eye-drop and nasal-drop group and the D90 oral group).

[0129] The results showed that all chicks were healthy, active, and had no obvious abnormalities. When weighed at 5 d, 15 d, and 22 d of age, there was no significant difference in body weight between the SMP@DA group and the blank control group;

[0130] In summary, SMP@DA has no obvious adverse effects on chickens and chicken embryos and is a safe candidate material.

[0131] V. Influence on virus activity

[0132] 1. The adsorption rate of the SMP@DA prepared in Example 1 to the infectious bronchitis virus D90 was detected. The method was as follows: According to the results of fluorescence quantitative PCR, before adsorption, the measured virus copy number of the D90 strain was 2212107.36 copies / μL, and after adsorption, the virus copy number in the supernatant of D90-SMP@DA was 2091.78 copies / μL. According to the formula: Adsorption rate = (Original virus copy number before non-adsorption - Supernatant copy number) / Original virus copy number before non-adsorption, the adsorption rate of the poultry live attenuated drinking water immune vaccine D90-SMP@DA to D90 was calculated to be 0.999.

[0133] 2. To prove that SMP@DA does not affect virus activity, the poultry live attenuated drinking water immune vaccine prepared in Example 1 was inoculated into 9-11-day-old SPF chicken embryos, with PBS and D90 as control groups, and the virus copy number in the allantoic fluid of the chicken embryos was detected. The results are shown in Figure 4 .

[0134] It can be seen from Figure 4 that after the mixture of SMP@DA and the D90 strain was adsorbed and then inoculated into chicken embryos, D90 could still reproduce in the chicken embryos, indicating that the silica microsphere material does not affect virus activity.

[0135] VI. Acid-base release results

[0136] The acid and alkali resistance of the poultry live attenuated drinking water immune vaccine D90-SMP@DA prepared in Example 1 was detected. The method was as follows: The acid-base environment of the chicken gastrointestinal tract was artificially simulated using simulated gastrointestinal fluid. The results are shown in Figure 5 .

[0137] It can be seen from Figure 5 that in the simulated gastric juice (pH = 2) environment, the virus copy number of D90-SMP@DA was below 1000 copies / μL within 6 h; in the simulated intestinal juice (pH = 6.8) environment, the virus copy number of the D90-SMP@DA vaccine reached above 5000 copies / μL after 30 min, and the virus copy number increased slowly in the subsequent time period. At 6 h, the virus copy number of D90-SMP@DA reached 1.62×106 copies / μL, indicating that D90-SMP@DA was stable in the simulated gastric juice environment and the antigen was slowly released over time in the simulated intestinal juice environment.

[0138] VII. Immune protection experiment of poultry live attenuated drinking water immune vaccine

[0139] The immune protection performance of the poultry live attenuated drinking water immune vaccine D90-SMP@DA in Example 1 was detected. The specific method was as follows:

[0140] A total of 160 5-day-old SPF chickens were randomly divided into 5 groups (3 immunization groups: the D90 eye-drop and nasal-drop group, the D90 oral group, and the D90-SMP@DA oral group; 1 challenge control group; 1 blank control group). At 5 days old, the first immunization was carried out. The D90 eye-drop and nasal-drop group was immunized by eye-drop and nasal-drop, and the D90 oral group and the D90-SMP@DA oral group were immunized orally; the blank control group and the challenge control group were given saline by eye-drop and nasal-drop for control; the immunization dose for each chicken was 10 4.0 EID 50 / 0.2 mL. The second immunization was carried out at 15 days old.

[0141] 1. At 10 and 17 days after the first immunization (i.e., when the chickens were 15 days old and 22 days old. The 15-day-old chickens had received the first immunization, and the 22-day-old chickens had received the second immunization), the sIgA secretion in the tracheal and intestinal lavage fluids after immunization with different vaccines is shown in Figures 6 - 7 .

[0142] It can be seen from Figures 6 - 7 that compared with the control group, the D90-SMP@DA oral group significantly increased the sIgA secretion in chickens (P<0.001). The secretory sIgA in the trachea and intestine induced by the D90-SMP@DA oral group was extremely significantly higher than that of the D90 eye-drop and nasal-drop group (P<0.01) and significantly higher than that of the pure D90 oral group (P<0.05).

[0143] 2. 14 days after the second immunization (i.e., when the chickens were 29 days old), the D90 eye-drop and nasal-drop group, the D90 oral group, the D90-SMP@DA oral group, and the challenge control group were challenged (challenged with the ZSD strain by nasal drop, and the challenge dose for each chicken was 10 5.0 EID 50 / 0.2 mL), and the blank control group was not treated. During the feeding period, the chickens had free access to food and water. At 10 days after the challenge, autopsy was carried out to observe and evaluate the immune protection effect of the chickens. The relative protection rate of the vaccine was calculated according to the following formula: RPS = [1 - (morbidity and mortality rate of the immunization group / morbidity and mortality rate of the control group)] × 100%. Protection rate = 1 - number of sick and dead chickens in the immunization group / total number of chickens in the immunization group.

[0144] Within 10 days after the challenge, there were no obvious adverse symptoms in the blank control group and the D90-SMP@DA oral group. Chickens in the challenge control group began to die on the 5th day, and until the 10th day, 7 chickens died. During this period, the chicks showed symptoms such as listlessness, slow movement, loss of appetite, and dyspnea. After the challenge, chickens in the D90 eye-drop and nasal-drop group and the D90 oral group died within 5 - 7 days. During this period, some chickens showed obvious open-mouth breathing and listlessness, and the rest of the chickens had no abnormal symptoms. All chickens were dissected. The kidneys of the sick but not dead chickens were swollen and pale, and the kidneys of all dead chickens were mottled kidneys. The challenge protection results are shown in Table 2.

[0145] Table 2 Incidence and protection rate of chickens in each group after ZSD challenge

[0146]

[0147] As can be seen from Table 2, the oral administration of D90-SMP@DA has the best effect, and the protection rate reaches 100%.

[0148] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A nano - adjuvant for attenuated live poultry drinking water immunization vaccine, characterized in that, The nano - adjuvant for attenuated live poultry drinking water immunization vaccine is catecholamine - modified functionalized silica microspheres.

2. The nanoadjuvant for attenuated live poultry drinking water immunization vaccine according to claim 1, characterized in that, The catecholamine is dopamine, norepinephrine or epinephrine.

3. The nano - adjuvant for attenuated live poultry drinking water immunization vaccine according to claim 1, characterized in that, The functional group is amino group, mercapto group or phenyl group.

4. The nanoadjuvant for attenuated live poultry drinking water immunization vaccine according to claim 1, characterized in that, The particle size of the nano - adjuvant for attenuated live poultry drinking water immunization vaccine is 361±88 nm, and the zeta potential is 13.3±2.3 mV.

5. The preparation method of the nano - adjuvant for the live attenuated poultry drinking water immunization vaccine according to any one of claims 1 - 4, characterized in that, It includes the following steps: Disperse the functionalized silica microspheres in the catecholamine aqueous solution, add NaOH solution, stir, wash and freeze - dry the obtained microspheres to prepare the catecholamine - modified functionalized silica microspheres, which are the nano - adjuvant for attenuated live poultry drinking water immunization vaccine.

6. The preparation method of the nano - adjuvant for attenuated live poultry drinking water immunization vaccine according to claim 5, wherein, The functionalized silica microspheres are prepared by the following method: (1) Mix the chloride salt, deionized water, ethanol and tetraethyl orthosilicate, dropwise add ammonia water, and stir to obtain a white emulsion A; (2) Drop the ethanol solution of tetraethyl orthosilicate into the white emulsion A obtained in step (1), add ammonia water to obtain an emulsion B; (3) Dissolve the functionalized raw material in ethanol to obtain a mixed solution, drop the mixed solution into the emulsion B obtained in step (2), and stir to obtain an emulsion C; (4) Centrifuge, wash and dry the emulsion C obtained in step (3) to prepare the functionalized silica microspheres.

7. Use of the nano - adjuvant for attenuated live poultry drinking water immunization vaccine according to any one of claims 1 - 4 in the preparation of attenuated live poultry drinking water immunization vaccine.

8. Use of the nano - adjuvant for attenuated live poultry drinking water immunization vaccine according to claim 7 in the preparation of attenuated live poultry drinking water immunization vaccine, characterized in that, The attenuated live poultry drinking water immunization vaccine is prepared by the following method: Dilute the antigen step - by - step with PBS buffer solution to the immunization titer to obtain a mixed solution, add the catecholamine - modified functionalized silica microspheres, stir and mix, centrifuge, and recover the precipitate to prepare the attenuated live poultry drinking water immunization vaccine.

9. Use of the nanoadjuvant for attenuated live poultry drinking water immunization vaccine according to claim 8 in the preparation of an attenuated live poultry drinking water immunization vaccine, characterized in that, The antigen is infectious bronchitis virus, Newcastle disease virus, infectious bursal disease virus or infectious laryngotracheitis virus.

10. Use of the poultry live attenuated drinking water immunization vaccine nano - adjuvant according to claim 8 in the preparation of a poultry live attenuated drinking water immunization vaccine, characterized in that, The mass - volume ratio of the catecholamine - modified functionalized silica microspheres to the mixed solution is 0.1 - 0.2 g: 20 - 45 mL.

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