Preparation method of oral inactivated rahnella aquatic sustained-release vaccine for fish

Abstract

The invention discloses a method for preparing an oral inactivated rahnella aquatic sustained-release vaccine for fish. According to the preparation method, on the basis of the principle that metal ions and high-molecular compounds are crosslinked, the three-dimensional network hydrogel is prepared by adopting a high-speed shearing process, and the inactivated rahnella aquatica is covered with the three-dimensional network hydrogel. The coating rate of Rahnella aquatica is measured by virtue of a spectrophotometric method and is subjected to decapsulation through decapsulation liquid, and the coating rate of thalli can reach 82.0%. A simulated gastric juice environment and a simulated intestinal juice environment are utilized, the thallus sustained release performance of the inactivated aquatic rahnella sustained release gel is measured in vitro through simulation, the thallus sustained release rate in 3h in the simulated gastric juice environment is about 2.85%, and the thallus sustained release rate in 5h in the simulated intestinal juice environment is about 84.98%. Through a zebra fish challenge immune test, the protection rate can reach 91.5%.

Description

Technical Field

[0001] The invention belongs to the fields of molecular biology and immunology, and specifically relates to a method for preparing an oral inactivated aquatic Rahnella sustained-release vaccine for fish. Background Art

[0002] Rahnella aquatilis belongs to the family Enterobacteriaceae, genus Rahnella, is a Gram-negative straight bacillus with flagella, size (0.5-0.7) μm × (2-3) μm, and is a facultative anaerobic organic nutrient. As a conditional pathogen, Rahnella aquatilis is pathogenic to aquatic animals and humans, and can cause diseases in crucian carp, damselfish, Chinese forest frogs, etc. During the infection process, it causes major clinical symptoms such as bleeding in the pectoral fins and caudal fins of fish and ascites. Studies have shown that Rahnella aquatilis is more common in contaminated muscles, intestines, and visceral tissues and organs.

[0003] There are currently three main methods for research on fish vaccines: injection, immersion, and oral administration. The injection method is only suitable for large fish, and has high requirements for the injection volume of the vaccine. This method limits the size range of the fish. The immersion method is difficult to achieve effective immune effects for fish absorption, and there are strict regulations on the immersion environment. The oral method can be used for large-scale fish vaccination and is suitable for a wider range of fish sizes. The current problem with oral vaccines is that the vaccine will be degraded by the digestive enzymes present in the gastric and intestinal juices in the body, thereby failing to induce an immune response and achieving the ideal immune effect.

[0004] The current oral vaccine immunization patent technology (patent publication number CN116617380A) adopts the method of enriching and encapsulating vaccine proteins with Artemia, which can protect the vaccine from the destruction of gastric acid and various digestive enzymes, and at the same time, the antigen substance can be released slowly and persistently, but this technology has certain technical requirements for the hatching and breeding of Artemia. In addition, in patent CN111748020A, Bacillus subtilis is used as a carrier for the delivery of targeting ligands and specific antigens in the digestive tract of fish, which has high technical requirements and there are still limitations on its application; patent CN112618709A adopts poly 2-vinyl pyridine or poly 4-vinyl pyridine, immune adjuvant mixed coating antigen, which requires specific containers and microfluidic control technology.

[0005] In view of the problems of high technical requirements and poor immune effect of oral vaccines for fish, the present invention utilizes the principle of forming a three-dimensional network structure by physical crosslinking of polymer compounds and metal ions, prepares gel-encapsulated inactivated aquatic Laenella by high-speed shearing method, determines the encapsulation rate of bacteria by simple method, and invents a method for in vitro simulation of the sustained release rate of bacteria. The inactivated bacteria sustained-release gel obtained by encapsulation is used to immunize fish orally, which can not only protect the inactivated vaccine from being destroyed by digestive enzymes in gastric juice, but also achieve the effect of sustained release of the vaccine.

[0006] The inactivated bacteria are wrapped with polymer materials to reduce the degradation of the vaccine by digestive enzymes in gastric and intestinal juices. When the vaccine is delivered to the target location, it is slowly released to exert its immune effect. Summary of the invention

[0007] The present invention provides a method for preparing an oral sustained-release vaccine for fish. The oral sustained-release vaccine prepared by the method can be used in large quantities for the preparation of vaccines for fish, and utilizes the sustained-release principle to deliver the vaccine to the immune target position, thereby improving the immune effect of the vaccine.

[0008] The following technical solutions are included to achieve the above purpose

[0009] 1. The present invention provides a method for preparing an oral sustained-release vaccine of aquatic Laenella, which is characterized by a process for encapsulating inactivated bacteria;

[0010] 2. The present invention provides a method for determining the encapsulation efficiency of gel-coated inactivated bacteria;

[0011] 3. The present invention provides a method for simulating the sustained release of gel-coated inactivated bacteria in vitro.

[0012] The present invention has the following advantages:

[0013] 1. The preparation method of the inactivated aquatic Laenella oral sustained-release vaccine for fish is simple and easy to operate, with high encapsulation efficiency and relatively accurate determination;

[0014] 2. The inactivated aquatic Laenella oral sustained-release vaccine for fish can be released at the target site of the organism through sustained release, so that the vaccine can play the maximum role and reduce the number of immunizations and the amount of vaccine used;

[0015] 3. The in vitro simulated sustained-release operation method and sustained-release rate determination method are highly accurate and reliable;

[0016] 4. This technology uses an oral method rather than a direct injection method, which can effectively reduce the stress response of fish and make the whole process proceed smoothly. DETAILED DESCRIPTION

[0017] The present invention is further described below in conjunction with the examples. The examples are intended to illustrate the present invention, but are not intended to limit the present invention in any form.

[0018] The conventional experimental methods and experimental materials involved in the embodiments of the present invention are as follows:

[0019] 1. Inactivation of aquatic Laenella: Prepare 200-1000mL of LB liquid culture medium, place the culture medium in an autoclave, and sterilize at 120℃ for 2-4h. After the culture medium cools to room temperature, take 4-20mL of activated aquatic Laenella, inoculate it into 200-1000mL of LB liquid culture medium, and shake and culture it at 30℃ 50-150rpm for 24h. After the culture is completed, add 0.4% formaldehyde solution and inactivate it for 24-48h. Place the completely inactivated bacterial solution in a centrifuge, centrifuge it at 10000-15000rpm for 5-10min, wash it with physiological saline and resuspend it to obtain the inactivated aquatic Laenella solution, and store it at 4℃ for later use.

[0020] 2. Experimental reagents: Metal ion liquids with different concentrations were prepared by the laboratory; sodium alginate (SA) and sodium carboxymethyl cellulose (CMC);

[0021] Sodium citrate; normal saline.

[0022] Embodiment 1:

[0023] The syringe method determines whether ions and polymer compounds can form gels through physical cross-linking

[0024] (1) Preparation of CMC system gel balls

[0025] Prepare 2% (w / v) CMC solution and add it dropwise to CuCl 2 , CaCl 2 、FeCl 3 、FeSO 4 、ZnCl 2 、AlCl 3 ion solution, solidified for 30 minutes, and the gel state was observed.

[0026] (2) Preparation of SA system gel spheres

[0027] Prepare 2% (w / v) SA solution and add it dropwise to CuCl 2 , CaCl 2 、FeCl 3 、FeSO 4 、ZnCl 2 、AlCl 3 ion solution, solidified for 30 minutes, and the gel state was observed.

[0028] Embodiment 2:

[0029] Preparation of sustained-release gel by high-speed shearing method

[0030] (1) Preparation of inactivated bacterial solution of CMC system and SA system

[0031] Prepare 2% (w / v) CMC and SA solutions respectively, add inactivated aquatic Laenella suspension respectively, and mix well.

[0032] (2) Preparation of sustained-release gel suspensions of CMC and SA inactivated bacterial liquid

[0033] The prepared CMC system and SA system inactivated bacterial solutions were added into ion solutions of different concentrations respectively, and sheared for 10 minutes using a high-speed shearing machine and allowed to stand for 30 minutes.

[0034] Embodiment 3:

[0035] Determination of gel encapsulation efficiency by decapsulating the gel sustained-release body with sodium citrate solution

[0036] (1) preparing sodium citrate deblocking solution;

[0037] (2) The prepared CMC system and SA system inactivated bacteria liquid sustained-release gel suspensions were added to 25 mL of sodium citrate solution and placed on a shaker for 30 minutes and then unsealed;

[0038] (3) The unblocking solution was placed in a centrifuge and centrifuged at 4000 rpm to collect the inactivated bacterial precipitate;

[0039] (4) The inactivated bacterial precipitate obtained by centrifugation was resuspended in physiological saline and the bacterial solution concentration was determined by spectrophotometry;

[0040] (5) The encapsulation efficiency was calculated based on the concentration of the inactivated bacterial solution before and after encapsulation.

[0041] Embodiment 4:

[0042] In vitro simulated sustained release in gastric and intestinal fluids

[0043] (1) Preparation of simulated gastric fluid: Weigh 10 g of pepsin and dissolve it in water. Transfer it to a 1 L volumetric flask, mix well, dilute it with water to 1000 mL, and add hydrochloric acid to adjust the pH to between 1 and 2.

[0044] (2) Preparation of simulated intestinal fluid: Weigh 6.8 g of potassium dihydrogen phosphate, add 500 mL of water to dissolve, adjust the pH value to between 6 and 7 with 0.1 mol / L sodium hydroxide, take another 10 g of trypsin, dissolve it in water, transfer it to a volumetric flask together with the potassium dihydrogen phosphate, and adjust the volume to 1000 mL.

[0045] (3) In vitro sustained release experiment in simulated gastric fluid: Prepare multiple groups of equal amounts of blank gel suspension and bacteria-containing gel suspension, add 5 mL of simulated gastric fluid to each group after filtration, and place on an oscillator at 60 rpm for 3 h. Take one tube every 1 h, centrifuge at 2000 rpm for 5 min, collect the supernatant, and use the blank gel sample as a control. The absorbance of the bacteria-containing filtrate is measured by a spectrophotometer to determine the release amount of aquatic Laenella.

[0046] (4) In vitro sustained-release experiment in simulated intestinal fluid: Prepare multiple equal amounts of blank gel suspension and bacteria-containing gel suspension, add 10 mL of simulated gastric fluid to each suspension after filtration, and oscillate at 60 rpm for 3 h. After centrifugation, replace the simulated gastric fluid with simulated intestinal fluid, and then sustain release for 3 h. Measure the released bacterial content every 1 h. The steps are the same as those for the simulated gastric fluid group.

[0047] Experimental Results

[0048] 1: Metal ion encapsulation rate

[0049] This technology uses a variety of metal ions to perform physical cross-linking to obtain ions that can produce normal cross-linking, and then conducts experiments on the ions at different concentrations. 2 Take the following table as an example.

[0050] <![CDATA[Ca 2+ Concentration (mol / L)]]> Encapsulation efficiency 0.1 73.37％ 0.2 75.90％ 0.3 82.04％

[0051] It can be seen from the table that when Ca 2+ When the concentration increases, the encapsulation rate also increases, and the encapsulation effect is better.

[0052] 2: In vitro simulated sustained release

[0053] According to step (3) of Example 4, the sustained-release gel in gastric juice has a sustained-release rate of 0.44% at 1 hour and a sustained-release rate of 2.85% at 3 hours. According to step (4) of Example 4, the sustained-release gel in intestinal juice has a sustained-release rate of 59.32% at 1 hour, a sustained-release rate of 72.48% at 3 hours and a sustained-release rate of 84.98% at 5 hours. The experimental results show that the technology can effectively protect the vaccine and enable the vaccine to achieve the best sustained-release effect.

[0054] 3: Challenge immunity test

[0055] Three groups of 60 zebrafish were taken as samples for experiment. The fish in the first group were continuously fed with fish food mixed with inactivated aquatic Laenella sustained-release gel for 2 weeks. During the same period, the fish in the second and third groups were fed with fish food without inactivated aquatic Laenella sustained-release gel. On the 15th day, the fish in the first and third groups were artificially injected with live aquatic Laenella liquid for virus attack, and the fish in the second group were injected with physiological saline. The mortality of fish in each group was observed within 48 hours after virus attack. The research results are shown in the table below. The vaccine protection rate reached 91.5%.

[0056]

Claims

1. A method for preparing an oral inactivated aquatic Rahnella sustained-release vaccine for fish, characterized in that: Preparation method and process of encapsulated inactivated bacteria; determination method of encapsulation rate; determination method of in vitro sustained release rate.

2. The preparation of the oral inactivated aquatic Raenella sustained-release vaccine for fish according to claim 1, characterized in that: The gel encapsulating the inactivated bacteria is formed by mixing sodium alginate solution or sodium carboxymethyl cellulose solution or a mixture of inactivated aquatic Laenella with metal ion solutions such as iron ions, copper ions, calcium ions, aluminum ions, zinc ions, etc. under the shearing action of a high-speed homogenizer to form a gel.

3. The method for determining the encapsulation efficiency of the oral inactivated aquatic Raenella sustained-release vaccine for fish according to claim 1 is characterized in that: In the determination of the encapsulation rate, the gel containing the inactivated aquatic bacteria of Laenella described in claim 2 is unsealed, the bacteria are separated by centrifugation, resuspended, and the absorbance of the bacterial solution is measured at 600nm to obtain the concentration of the encapsulated bacteria.

4. The method for determining the in vitro sustained-release rate of the oral inactivated aquatic Rahnella sustained-release vaccine for fish according to claim 1 is characterized in that: The in vitro sustained-release gel is a blank sustained-release gel and a bacteria-containing sustained-release gel prepared by the method of claim 2. The gel is filtered with a filter membrane and then sustained-released in simulated gastric fluid and simulated intestinal fluid. After a certain period of sustained release, the sustained-release liquid is filtered and centrifuged to obtain released bacteria, and then the bacteria are resuspended, and the absorbance of the bacterial solution is measured to obtain the bacterial solution concentration.

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