A recombinant yeast beta-glucan particle and its use in preparing a trichinosis vaccine

Abstract

The application provides a recombinant yeast beta-glucan particle and application thereof in preparation of a trichinosis vaccine, and belongs to the technical field of parasitic disease prevention and control. The application solves the problem of how to improve the effective prevention of trichinosis. The application comprises a recombinant yeast beta-glucan particle, wherein the yeast beta-glucan particle is composed of a trichinella DNase-II protein, a yeast beta-glucan particle and a yeast tRNA. The yeast beta-glucan particle is mainly used for preventing trichinosis.

Description

Technical Field

[0001] This invention belongs to the field of parasitic disease prevention and control technology, and in particular relates to a recombinant yeast β-glucan particle and its application in the preparation of trichinosis vaccine. Background Technology

[0002] Trichinosis is a global foodborne zoonotic disease caused by the nematode Trichinella spiralis. It has a broad host spectrum, infecting over 150 mammal species, including humans. Sporadic cases and localized outbreaks still exist in some parts of my country. The life cycle of Trichinella spiralis is complex and elusive. After invading the host through the digestive tract, the larvae undergo several stages, including intestinal parasitism, migration via the bloodstream, and cyst formation within striated muscle. The cysted larvae can survive in the host for several years, making them difficult to eradicate completely with conventional treatments. At the immune level, Trichinella spiralis can actively regulate the host's immune microenvironment through secretions and excretions, inducing Th2 immune shift, inhibiting antigen-presenting cell function, and inducing regulatory immune cell differentiation, achieving highly efficient immune evasion and making it difficult for the host to establish long-term effective anti-infection protection. The gut is the host's first line of defense against Trichinella spiralis infection. In the early stages of infection, various immune cells work closely together to resist the parasite's invasion, but the host's own immunity alone is insufficient to resist infection. Currently, trichinosis control measures are limited, and vaccines represent the most promising approach for prevention and control.

[0003] Existing trichinella protein vaccines generally have many shortcomings: they contain a mixture of crude antigen components, which can easily trigger non-specific immune responses, have poor safety, and have weak antigen stability, requiring high storage and transportation conditions. Summary of the Invention

[0004] This invention provides a recombinant yeast β-glucan granule and its application in the preparation of a trichinosis vaccine. The purpose of this invention is to address the problem of how to improve the effective prevention of trichinosis.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a recombinant yeast β-glucan particle, which is composed of Trichinella spiralis DNase-II protein, yeast β-glucan particles and yeast tRNA; the gene encoding Trichinella spiralis DNase-II protein has the accession number GenBank: AY963701.1.

[0006] Further specifying, the Trichinella DNase-II protein is precipitated inside the yeast β-glucan particles via electrostatic interaction, and then yeast tRNA is added.

[0007] Further specifying, the ratio of Trichinella DNase-II protein, yeast β-glucan particles and yeast tRNA content is 1 mg: 10 mg: 25 mg / ml.

[0008] This invention provides a method for preparing the above-mentioned recombinant yeast β-glucan particles, the preparation method being as follows: Step 1: Obtain yeast β-glucan granules by acid treatment of active yeast powder; Step 2: Mix Trichinella DNase-II protein with yeast β-glucan particles obtained in Step 1. Trichinella DNase-II protein precipitates inside the yeast β-glucan particles through electrostatic adsorption. Yeast tRNA is then added to react and obtain the final product.

[0009] Further specifying the method for obtaining yeast β-glucan particles in step 1: Weigh 100 g of active yeast powder, disperse it evenly in 1 L of sodium hydroxide solution, and stir continuously for 1 h under constant temperature water bath conditions of 85 ℃; after the reaction is completed, centrifuge at 1000 g for 5 min, discard the supernatant and collect the yeast precipitate, add 500 mL of hydrochloric acid solution with pH 6.0 to the precipitate, stir in a water bath at 60 ℃ for 1 h, centrifuge at 1500 g for 5 min to obtain the primary precipitate, wash 4 times with isopropanol and 2 times with acetone, and obtain 8 g of grayish-white yeast β-glucan particles after spray drying.

[0010] Further specifying, in step 2, the ratio of Trichinella DNase-II protein, yeast β-glucan particles and yeast tRNA is 1 mg: 10 mg: 25 mg / ml.

[0011] The present invention provides a vaccine for preventing trichinosis infection, the vaccine comprising the above-mentioned recombinant yeast β-glucan particles.

[0012] Furthermore, the dosage form of the vaccine can be any one of tablets, capsules, granules, powders, or liquid preparations.

[0013] This invention provides the application of the above-mentioned recombinant yeast β-glucan particles in the preparation of a vaccine to prevent trichinosis.

[0014] Compared with the prior art, the beneficial effects of the present invention are: 1. The yeast β-glucan particles in the present invention can serve as a protein carrier, protect the protein from gastrointestinal degradation, and improve the bioavailability of the protein.

[0015] 2. Yeast β-glucan particles loaded with Trichinella DNase-II protein are used to prevent Trichinella infection.

[0016] 3. Yeast β-glucan particle vaccines loaded with Trichinella DNase-II protein have a worm-reducing effect, and the worm-reducing effect is better than that of Trichinella single antigen.

[0017] 4. The yeast β-Glucan granule vaccine of the present invention is a monovalent oral vaccine in the form of dry powder, which does not contain adjuvants and has a simple preparation method.

[0018] 5. The vaccine immunization program is a single immunization, and the immunization program is simple and easy to operate. Attached Figure Description

[0019] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings: Figure 1 Transmission electron microscopy (TEM) results; A shows the morphology and structure of the original yeast cells under TEM; B shows the morphology and structure of β-glucan particles; C shows the morphology and structure of DNase-II-BGP loaded particles. Figure 2 The images show the fluorescence microscopy results of the RB-DNase-II-CFW-BGP particle vaccine; A is CFW-BGP; B is RB-DNase-II; C is MERGE. Figure 3 The adult load in each group; Figure 4 The reduction rate of adult insects in each group. Detailed Implementation

[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. It should be noted that, unless otherwise specified, the embodiments and features in the embodiments of the present invention can be combined with each other, and the described embodiments are only some embodiments of the present invention, not all embodiments.

[0021] Source of insects: Trichinella spiralis species from Henan, China (International Standard Species Number: ISS534), passaged and preserved in our laboratory; Rat source: 4-6 week old female SD rats from Changchun Yisi Experimental Animal Technology Co., Ltd. Mouse source: 6-8 week old female Balb / c mice from Changchun Yisi Experimental Animal Technology Co., Ltd. Yeast source: Purchased from Angel Yeast Co., Ltd., active bread yeast powder; Yeast tRNA: Purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd., Yeast tRNA Type VI.

[0022] Example 1. Method for preparing a vaccine loaded with yeast-derived β-glucan containing Trichinella spiralis DNase-II 1. Preparation of yeast β-glucan granules using the acid-base method: 100 g of active yeast powder was weighed and uniformly dispersed in 1 L of sodium hydroxide solution. The mixture was stirred continuously in an 85 ℃ water bath for 1 h. After the reaction, the mixture was centrifuged at 1000 g for 5 min, and the supernatant was discarded to collect the yeast precipitate. 500 mL of pH 6.0 hydrochloric acid solution was added to the precipitate, and the mixture was stirred in a 60 ℃ water bath for 1 h. After centrifugation at 1500 g for 5 min, the primary precipitate was obtained. The precipitate was washed four times with isopropanol and twice with acetone, and then spray-dried to obtain 8 g of grayish-white yeast β-glucan particles.

[0023] 2. Expression and purification of Trichinella spiralis DNase-II protein: (1) Construction of pET28a-T3223-7: The DNase-II gene sequence was ligated into the pET28a vector to obtain the recombinant vector; the encoding gene of DNase-II protein (GenBank: AY963701.1).

[0024] (2) From the recombinant vector (pET28a-T3223-7) containing the Trichinella DNase-II gene stored at -80 ℃, an appropriate amount of bacterial culture was streaked onto LB solid medium containing the corresponding ampicillin antibiotic and cultured overnight at 37 ℃ to activate the strain. Single colonies were picked and inoculated into LB liquid medium and cultured at 37 ℃ with shaking until the logarithmic growth phase, as the seed culture. The seed culture was inoculated into fresh LB medium at a ratio of 1:100 and cultured until the OD 600 value was 0.8. IPTG was added to a final concentration of 1 mM and expression was induced at 28 ℃ for 6 hours. After induction, the bacterial culture was collected and centrifuged to obtain the bacterial cell pellet. The pellet was resuspended in lysis buffer, and the cells were lysed by sonication. The supernatant was collected after centrifugation. DNase-II protein in the supernatant was purified using a Ni-NTA affinity chromatography column. Impurities were removed with washing buffer (containing 20 mM imidazole), followed by elution with elution buffer (containing 500 mM imidazole). Elution peaks were collected, and protein purity was assessed by SDS-PAGE electrophoresis. Protein concentration was determined using the BCA method.

[0025] 3. β-glucan particles loaded with Trichinella DNase-II protein: 10 mg of β-glucan particles were thoroughly mixed with 1 mg of DNase-II protein solution, shaken at 4 ℃ for 3 h, and then frozen at -80 ℃ overnight. Trichinella spiralis muscle precipitated inside the yeast β-glucan particles via electrostatic adsorption. 25 mg / ml tRNA solution was added to the lyophilized product, and the mixture was incubated in a 50 ℃ metal bath for 30 min. Then, 400 μL of 10 mg / ml tRNA solution was added, and the reaction continued for 1 h. The precipitate was then lyophilized again, yielding 5 mg of grayish-white powder DNase-II-BGP. The antigen protein concentration in the supernatant was determined using the BCA method, and the antigen encapsulation efficiency was calculated. The calculation process was: Encapsulation efficiency = [(DNase-II content 100 μg, DNase-II content in supernatant 48.7 μg) / DNase-II content 100 μg × 100%]. The encapsulation efficiency of β-glucan particles for Trichinella spiralis DNase-II protein was 51.3%, and the loading rate was 5%.

[0026] 4. Detection of Trichinella DNase-II protein loading on yeast β-glucan particles using transmission electron microscopy: The results are as follows Figure 1 Under transmission electron microscopy, Figure A shows the original yeast cells, and Figure B shows the yeast β-glucan particles obtained after acid-base treatment. Compared with Figure A, Figure B shows a decrease in density in the central region and a porous structure on the cell wall surface, indicating that the acid-base treatment effectively removed some of the yeast cell contents, forming a carrier structure with a porous surface and loose internal structure, creating conditions for loading Trichinella spiralis DNase-II protein. In contrast to Figure B, Figure C shows DNase-II-BGP particles, with a smoother porous surface and a significantly increased internal density, confirming that the yeast β-glucan particles have been successfully loaded with Trichinella spiralis DNase-II protein.

[0027] 5. Observe the loading of Trichinella DNase-II protein onto yeast β-glucan particles using a fluorescence microscope: To verify the antigen loading effect, yeast β-glucan particles and Trichinella spiralis DNase-II protein were fluorescently labeled, respectively. β-glucan was labeled with blue fluorescence using calcium fluorescein white (CFW), and DNase-II protein was labeled with red fluorescence using rhodamine B (RB). Both were purified after labeling. The labeled DNase-II protein was loaded onto the labeled β-glucan to prepare the RB-DNase-II-CFW-BGP particle vaccine. A small amount of the particle vaccine was dispersed, and the fluorescence signals of CFW and RB were observed under a fluorescence microscope at excitation wavelengths of 355 nm and 553 nm, respectively. The antigen loading was evaluated using image fusion technology.

[0028] The results are as follows Figure 2The images showed blue fluorescence at an excitation wavelength of 355 nm and red fluorescence at an excitation wavelength of 553 nm. Fusion of images from the same field of view revealed dual fluorescence signals, confirming the successful loading of Trichinella spiralis DNase-II protein onto yeast β-glucan particles.

[0029] Example 2. Animal experiments to verify the immunogenicity of yeast-derived β-glucan loaded with Trichinella DNase-II. This experiment was divided into four groups: PBS group, BGP group, DNase-II group, and DNase-II-BGP group, with 6 female Balb / c mice in each group.

[0030] PBS group: PBS; BGP group: β-glucan particles; DNase-II group: DNase-II protein; DNase-Ⅱ-BGP group: Particle-coated protein obtained in Example 1.

[0031] 1. Collect Trichinella spiralis muscle larvae: Pre-preparation of digestion solution: Add 5 mL of concentrated hydrochloric acid and 5 g of pepsin to 500 mL of distilled water at 37 ℃, mix well, and let stand at a constant temperature of 37 ℃ for later use. Take SD rats infected with Trichinella spiralis, anesthetize and sacrifice them, remove fur, head and internal organs, and collect skeletal muscle, which is then pulverized into a paste. Add the paste to the above digestion solution and digest at a constant temperature of 37 ℃ for 2 h with stirring until the tissue is completely digested. Filter the digestion solution through a stainless steel sieve to remove undigested residue. Let the filtrate settle for 1 h, discarding the supernatant. Continue to let the remaining filtrate settle for 30 min, repeating the settling and supernatant discarding process until the filtrate volume is concentrated to 25 mL. Wash the precipitate repeatedly with sterile physiological saline 5-6 times until the washing liquid is clear, and collect the lower sediment to obtain purified Trichinella spiralis muscle larvae.

[0032] 2. Prepare Trichinella spiralis DNase-II protein solution, DNase-II-BGP particle solution, and BGP particle solution: Under aseptic conditions, a sterile PBS solution was prepared, and the Trichinella spiralis DNase-II protein solution, BGP particles, and DNase-II-BGP particle powder were diluted with sterile PBS to prepare concentrations of 50 μg / ml Trichinella spiralis muscle larvae DNase-II protein solution, 2.5 mg / ml BGP solution, and 50 μg / ml DNase-II-BGP solution, respectively.

[0033] 3. Immunization procedures for animal experiments and collection of adult Trichinella spiralis: Mice in the DNase-II group were administered the above-prepared Trichinella myolarvae DNase-II protein solution by gavage, with an immunization dose of 0.5 mg / kg per mouse; mice in the DNase-II-BGP group were administered a yeast β-glucan granule solution loaded with Trichinella myolarvae DNase-II protein by gavage, with an immunization dose of 10 mg / kg per mouse; mice in the yeast β-glucan granule group were administered the above-prepared β-glucan granule solution by gavage, with an immunization dose of 10 mg / kg per mouse; mice in the PBS group were administered an equal volume of sterile PBS solution by gavage.

[0034] Seven days after immunization, all mice were orally infected with 300 Trichinella muscle larvae.

[0035] Seven days after infection, mice were anesthetized and euthanized. The small intestine was completely separated, longitudinally dissected, and rinsed thoroughly with sterile saline. A small intestine incubation solution was prepared: penicillin and streptomycin sulfate were added to saline to a final concentration of 200 U / mL. The treated small intestine was suspended in a beaker containing the incubation solution and incubated at 37 °C for 3 h. The small intestine was removed, and the intestinal wall was gently rinsed with saline containing both antibiotics. All incubation solution was collected and allowed to stand at room temperature for 1 h. The supernatant was slowly aspirated, and the lower suspension containing the worms was transferred to a glass petri dish, thus completing the collection of adult Trichinella spiralis. The number of adult worms in the petri dish was counted under a microscope. Figure 3 And calculate whether there is a statistically significant difference in the insect reduction rate between the groups based on the number of adult insects, such as... Figure 4 .

[0036] result: Figure 3 , 4 As shown, the worm reduction rates in the BGP group, DNase-II group, and DNase-II-BGP group were 25.97%, 45.18%, and 65.79%, respectively. This demonstrates that oral immunization of mice with yeast β-Glucan granules loaded with Trichinella spiralis DNase-II protein can reduce the adult worm load and has a good adult worm reduction rate, showing significant differences compared with the Trichinella spiralis muscle larvae crude antigen group and the yeast β-Glucan granule group. Statistical analysis and visualization were performed using GraphPadPrism 9. Independent samples t-tests were used to compare means and determine statistically significant differences between different conditions. P-values ​​are expressed as *P<0.05, ***P<0.001, and ****P<0.0001.

[0037] The specific embodiments of the present invention disclosed above are merely illustrative of the invention. These embodiments do not exhaustively describe all details, nor do they limit the invention to the specific embodiments described. Many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention.

Claims

1. A recombinant yeast β-glucan granule, characterized in that, The yeast β-glucan particles are composed of Trichinella spiralis DNase-II protein, yeast β-glucan particles, and yeast tRNA; the gene encoding the Trichinella spiralis DNase-II protein has the accession number GenBank: AY963701.

1.

2. The recombinant yeast β-glucan granules according to claim 1, characterized in that, The Trichinella DNase-II protein is deposited inside the yeast β-glucan particles via electrostatic interaction, and then yeast tRNA is added.

3. The recombinant yeast β-glucan granules according to claim 1, characterized in that, The ratio of Trichinella spiralis DNase-II protein, yeast β-glucan particles, and yeast tRNA was 1 mg: 10 mg: 25 mg / ml.

4. The method for preparing recombinant yeast β-glucan granules according to any one of claims 1-3, characterized in that, The preparation method is as follows: Step 1: Obtain yeast β-glucan granules by acid treatment of active yeast powder; Step 2: Mix Trichinella DNase-II protein with yeast β-glucan particles obtained in Step 1. Trichinella DNase-II protein precipitates inside the yeast β-glucan particles through electrostatic adsorption. Yeast tRNA is then added to react and obtain the final product.

5. The preparation method according to claim 4, characterized in that, The method for obtaining yeast β-glucan particles in step 1 is as follows: 100 g of active yeast powder is weighed and evenly dispersed in 1 L of sodium hydroxide solution. The mixture is stirred continuously in a constant temperature water bath at 85 ℃ for 1 h. After the reaction is completed, the mixture is centrifuged at 1000 g for 5 min, the supernatant is discarded and the yeast precipitate is collected. 500 mL of hydrochloric acid solution with pH 6.0 is added to the precipitate, and the mixture is stirred in a water bath at 60 ℃ for 1 h. After centrifugation at 1500 g for 5 min, the primary precipitate is obtained. The precipitate is washed 4 times with isopropanol and 2 times with acetone. After spray drying, 8 g of grayish-white yeast β-glucan particles are obtained.

6. The preparation method according to claim 4, characterized in that, In step 2, the ratio of Trichinella DNase-II protein, yeast β-glucan particles, and yeast tRNA was 1 mg: 10 mg: 25 mg / ml.

7. A vaccine for preventing trichinosis infection, characterized in that, The vaccine comprises recombinant yeast β-glucan particles as described in any one of claims 1-3.

8. The vaccine according to claim 7, characterized in that, The vaccine can be in any of the following dosage forms: tablets, capsules, granules, powders, or liquid preparations.

9. The vaccine according to claim 7, characterized in that, The immunization dose is 10 mg / kg.

10. The use of the recombinant yeast β-glucan particles according to any one of claims 1-3 in the preparation of a vaccine for the prevention of trichinosis.

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