Echinococcus granulosus PLGA-EgG1Y162 nanometer subunit vaccine and preparation method thereof

Abstract

The invention discloses an echinococcus granulosus PLGA-EgG1Y162 nano subunit vaccine and a preparation method thereof, and relates to the field of biomedical engineering and parasitic disease prevention and treatment. According to the method, echinococcus granulosus EgG1Y162 recombinant protein (the amino acid sequence is as shown in SEQ ID NO.1) is taken as an antigen core, and is stably coated in a polylactic acid-glycolic acid copolymer (PLGA) nano carrier through an innovative embedding process, so that the nano subunit vaccine in a brand new form is constructed. Compared with pure EgG1Y162 recombinant protein, the nanoparticles prepared by the preparation method disclosed by the invention not only have innovated components, but also obviously improve the slow release performance of drugs, and can realize precise enrichment and long-acting release of antigens. The invention fills up the blank of research on the echinococcus granulosus PLGA nano subunit vaccine, provides a novel technical scheme for prevention and treatment of echinococcosis, and has important application value and market prospect in the field of biological medicine.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a fine-grained Echinococcus granulosus PLGA-EgG1Y162 nano-subunit vaccine and its preparation method. Background Technology

[0002] Cystic echinococcosis (CE) is a zoonotic parasitic disease caused by infection with *Echinococcus granulosus* (Eg). It is endemic in many countries in Asia, South America, the Middle East, and Australia, and has a global distribution. Currently, treatment and prevention mainly rely on chemotherapy, in addition to surgery. However, issues such as drug resistance and side effects make immunoprophylaxis one of the most promising and ideal control measures for managing the disease's spread.

[0003] Previous studies have shown that the EgG1Y162 protein has low antigen presentation and immunogenicity, resulting in limited long-term protective effects. Its application is also limited by its difficulties in in vitro storage and transportation, short in vivo half-life, and rapid clearance.

[0004] Poly(D,L-lactic-co-glycolic acid) (PLGA) has garnered significant attention in the field of nanomedicine carriers due to its favorable safety, biocompatibility, excellent drug delivery capabilities, and sustained-release effects. The U.S. Food and Drug Administration (FDA) has approved PLGA for clinical use as a biomedical material and pharmaceutical adjuvant. Early studies have shown that encapsulating soluble parasitic antigens in PLGA nanocarriers can protect mice from experimental parasitic diseases. Encapsulating EgG1Y162 within PLGA nanoparticles holds promise for enhancing the immunogenicity of antigens and improving their targeting and sustained-release properties within the body. Summary of the Invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide a fine-grained Echinococcus granulosus PLGA-EgG1Y162 nano-subunit vaccine and its preparation method, which addresses the shortcomings of the prior art.

[0006] To solve the above-mentioned technical problems, the present invention discloses the following technical solution:

[0007] In one aspect, this invention discloses a recombinant nanosubunit vaccine against Echinococcus granulosus. The vaccine is composed of nanoparticles formed by encapsulating recombinant protein EgG1Y162 in PLGA nanoparticles. The recombinant protein EgG1Y162 is a protective antigen expressed in both the protocercariae and adult larvae of Echinococcus granulosus, and its amino acid sequence is shown in SEQ ID NO.1.

[0008] The recombinant protein EgG1Y162 is preferably obtained by transforming the recombinant expression plasmid pET30a(+)-EgG1Y162 into BL21 *Escherichia coli* for induced expression, resulting in bacteria containing the pET30a(+)-EgG1Y162 recombinant expression plasmid. The expressed recombinant protein EgG1Y162 is then purified by Ni column affinity chromatography. More specifically, bacteria containing the pET30a(+)-EgG1Y162 recombinant expression plasmid are inoculated into LB liquid medium for culture to obtain a bacterial suspension. When the OD600 value of the bacterial suspension reaches 0.4-0.6, IPTG is added to the bacterial suspension to induce recombinant protein expression. The induction is continued for 14-18 hours, and after induction, the bacterial cells are collected by centrifugation. The obtained bacterial cells are washed, resuspended in PBS, centrifuged, and sonicated to lyse the bacteria. The supernatant is collected, and the recombinant protein is purified by Ni column affinity chromatography.

[0009] The preferred particle size of the fine-particle Echinococcus recombinant EgG1Y162 protein nano-subunit vaccine is 360-420nm, such as 380nm, 390nm, 395nm, 400nm, 405nm, and 410nm.

[0010] The nano-subunit vaccine is prepared by the following method: a PLGA dichloromethane solution is vortexed with 2-5 wt% polyvinyl alcohol, and subjected to a first sonication under ice bath conditions to obtain a mixed solution; recombinant protein EgG1Y162 is vortexed with the mixed solution, and subjected to a second sonication under ice bath conditions to form a milky white liquid; the milky white liquid is vortexed with 2-5 wt% polyvinyl alcohol, and subjected to a third sonication under ice bath conditions to obtain a complex emulsion; the organic solvent in the complex emulsion is evaporated, and the liquid is collected; the obtained liquid is centrifuged, the precipitate is collected, the precipitate is resuspended in deionized water, placed at -70 to -90°C for standing, and then freeze-dried to obtain the fine-grained Echinococcus nano-subunit vaccine.

[0011] Secondly, this invention discloses a method for preparing the recombinant EgG1Y162 protein nanounit vaccine of Echinococcus granulosus described in the first aspect above.

[0012] The method includes the following steps:

[0013] (1) Transform E. coli BL21 competent cells with the pET30a(+)-EgG1Y162 recombinant expression plasmid to obtain bacteria containing the pET30a(+)-EgG1Y162 recombinant expression plasmid;

[0014] (2) Expression and purification of Echinococcus granulosus recombinant protein EgG1Y162;

[0015] (3) Preparation of nano-subunit vaccine PLGA-EgG1Y162.

[0016] In step (2), the expression purification involves inoculating bacteria containing the pET30a(+)-EgG1Y162 recombinant expression plasmid into LB liquid medium for culture to obtain a bacterial culture; when the OD600 value of the bacterial culture reaches 0.4-0.6, IPTG is added to the bacterial culture to induce the expression of the recombinant protein, and the culture is continued for 14-18 hours until the expression is induced, centrifuged, and the bacterial cells are collected; after washing the obtained bacterial cells, PBS is added to resuspend the precipitate, centrifuged and sonicated to lyse the bacteria, the supernatant is collected, and the recombinant protein is purified by Ni column affinity chromatography.

[0017] The bacteria containing the pET30a(+)-EgG1Y162 recombinant expression plasmid are inoculated into LB liquid medium at a volume ratio of 1:80-120, preferably at a volume ratio of 1:100; the culture is carried out at 35-40℃ and 160-200 rpm, such as 37℃ and 180 rpm; the final concentration of IPTG is 0.2-1 mmol / ml.

[0018] In step (3), the dichloromethane solution of PLGA is vortexed with 2-5 wt% polyvinyl alcohol and subjected to a first sonication under ice bath conditions to obtain a mixed solution; the recombinant protein EgG1Y162 is vortexed with the mixed solution and subjected to a second sonication under ice bath conditions to form a milky white liquid; the milky white liquid is vortexed with 2-5 wt% polyvinyl alcohol and subjected to a third sonication under ice bath conditions to obtain a complex emulsion; the organic solvent in the complex emulsion is evaporated and the liquid is collected; the obtained liquid is centrifuged, the precipitate is collected, the precipitate is resuspended with deionized water, placed at -70 to -90℃ and then freeze-dried to obtain the fine-grained Echinococcus nanounit vaccine.

[0019] Wherein, the concentration of PLGA in the dichloromethane solution of PLGA is 40-60 mg / mL, preferably 45-55 mg / mL, such as 50 mg / mL; the total volume ratio of the 2-5% polyvinyl alcohol to dichloromethane is 2-6:1, preferably 3-5:1, and more preferably 4:1; the 2-5% polyvinyl alcohol added before the first ultrasound is 40%-60% of the total volume of 2-5% polyvinyl alcohol, preferably 45%-55%, and more preferably 50%; the mass-volume ratio of the recombinant protein EgG1Y162 to dichloromethane is 3-5 mg:1 mL; the 2-5 wt% polyvinyl alcohol is preferably 2-4 wt% polyvinyl alcohol, and more preferably 3 wt% polyvinyl alcohol.

[0020] The power of the first, second, and third ultrasounds is independently selected from 35-45W, preferably 40W; the total time of the first, second, and third ultrasounds is independently selected from 3-8min, preferably 5-6min; the time of each ultrasound is independently selected from 3-7s, preferably 4-6s, preferably 5s; and the time of each interval is independently selected from 3-7s, preferably 4-6s, preferably 5s.

[0021] The centrifugation speed is 30,000-50,000 r / min, preferably 35,000-45,000 r / min, and more preferably 4,000 r / min; the centrifugation time is 25-35 min, preferably 28-32 min, and more preferably 30 s; the settling time is 1.5-3 h; and the freeze-drying time is 20-24 h.

[0022] In some specific embodiments, a 40-50 mg / mL PLGA solution in dichloromethane is mixed with 2-5 wt% PVA and vortexed for 1-2 min; under ice bath conditions, a first ultrasonic disruption is performed for 3-6 min to obtain a mixed solution; while vortexing, the recombinant protein EgG1Y162 solution prepared in step (2) is added dropwise and mixed with the mixed solution by vortexing for 1-2 min; under ice bath conditions, a second ultrasonic disruption is performed for 3-6 min to form a milky white primary emulsion; while vortexing, 2-5 wt% PVA is added to the primary emulsion, and under ice bath conditions, a third ultrasonic disruption is performed for 3-6 min to form a secondary emulsion; the secondary emulsion obtained after ultrasonication is stirred and evaporated until the organic solvent is dissolved. After the agent has evaporated completely, centrifuge at 30,000-40,000 r / min for 30-40 min using a refrigerated ultracentrifuge. After centrifugation, collect the supernatant and precipitate separately. Resuspend the precipitate obtained after ultracentrifugation in deionized water to obtain a PLGA-encapsulated recombinant protein suspension. Place it in a vial and incubate at -80℃ for 1.5-3 h. Then transfer it to a vacuum freeze dryer and freeze-dry for 20-24 h to obtain the fine-particle echinococcosis recombinant EgG1Y162 protein nano-subunit vaccine. The volume of 2-5 wt% PVA added each time is 2-2.5 times the volume of the PLGA dichloromethane solution. The ultrasonic disruption power is 40-50 W each time, and the ultrasonication time is 3-5 s with a 5 s interval.

[0023] Thirdly, the present invention discloses the application of the recombinant EgG1Y162 protein nanosubunit vaccine of Echinococcus granulosus described in the first aspect above in the preparation of drugs for preventing Echinococcus granulosus infection.

[0024] The drugs mentioned include vaccines.

[0025] Beneficial effects:

[0026] (1) There are currently no studies or reports on Echinococcus granulosus PLGA nanosubunit vaccines. This invention fills the gap in the research on Echinococcus granulosus PLGA nanosubunit vaccines.

[0027] (2) This invention uses PLGA nanomaterials to encapsulate the recombinant protein EgG1Y162, forming a novel vaccine formulation that differs from the standalone EgG1Y162 recombinant protein, as their components are different. Furthermore, after the EgG1Y162 recombinant protein is coated with PLGA nanomaterials, its immunoprotective effect is significantly enhanced, such as a significant reduction in lesions, a significant decrease in liver-to-body weight ratio, a significant and substantial reduction in vesicle count, and a decrease in CD4 count. + T, CD8 + Significantly increased T lymphocytes, etc.

[0028] (3) This invention improves the encapsulation process of nanomaterials, specifically by optimizing the concentration of polyvinyl alcohol (PVA) to 3%. Experimental results show that this process improvement can significantly reduce the particle size of nano-vaccines while maintaining a high encapsulation rate, effectively solving the technical problem of low antigen loading efficiency. Attached Figure Description

[0029] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0030] Figure 1 Electrophoresis diagram of double enzyme identification of recombinant plasmid pET30a(+)-EgG1Y162.

[0031] Figure 2 Expression conditions for recombinant protein EgG1Y162 in *Escherichia coli* BL21 optimized and purified products analyzed by SDS-PAGE: A. Optimized and soluble analysis results; Lane M: Protein Marker; Lane 1: Sample induced by 0.2 mM IPTG at 15℃; Lane 2: Sample induced by 1.0 mM IPTG at 15℃; Lane 3: Sample induced by 0.2 mM IPTG at 37℃; Lane 4: Sample induced by 1.0 mM IPTG at 37℃; Lane 5: Uninduced sample; Lane 6: Precipitate after induced by 1.0 mM IPTG at 37℃; Lane 7: Supernatant after induced by 1.0 mM IPTG at 37℃; Lane 8: Precipitate after induced by 0.2 mM IPTG at 37℃; Lane 9: Supernatant after induced by 0.2 mM IPTG at 37℃; Lane 10: Precipitate after induced by 1.0 mM IPTG at 15℃; Lane 11: Precipitate after induced by 1.0 mM IPTG at 15℃. IPTG induction supernatant, lane 12: 0.2mM IPTG induction precipitate at 15℃, lane 13: 0.2mM IPTG induction supernatant at 15℃; B. Affinity purification SDS-PAGE results, lane M: Protein Marker, lane 1: precipitate after disruption, lane 2: supernatant after disruption, lane 3: eluent, lane 4: washing sample, lane 5: elution sample; C. Protein dialysis SDS-PAGE results, lane M: Protein Marker, lane S: dialysis sample.

[0032] Figure 3 This is a particle size distribution diagram of the PLGA-EgG1Y162 nanoparticle vaccine.

[0033] Figure 4 The scanning electron microscope results for the PLGA-EgG1Y162 nanoparticle vaccine are shown in the figure. The scale bar is 5 μm.

[0034] Figure 5The transmission electron microscopy results for the PLGA-EgG1Y162 nanoparticle vaccine are shown in the figure. The scale bar is 100 nm.

[0035] Figure 6 This is a diagram illustrating the in vitro sustained-release characteristics of the PLGA-EgG1Y162 nanoparticle vaccine.

[0036] Figure 7 The toxicity results for the PLGA-EgG1Y162 nanoparticle vaccine.

[0037] Figure 8 The distribution results of the PLGA-EgG1Y162 nanovaccine in mice.

[0038] Figure 9 The changes in serum total IgG antibody levels in mice after immunization with PLGA-EgG1Y162 nanovaccine.

[0039] Figure 10 The results of liver lesions (10A, 10C) and histopathological (10B, 10D) in mice infected with Echinococcus granulosus after immunization with PLGA-EgG1Y162 nano-vaccine are shown.

[0040] Figure 11 The results show the changes in liver weight ratio after infection in mice.

[0041] Figure 12 The result is the number of vesicles (mean ± SD).

[0042] Figure 13 The effects of infection on biochemical indicators AST (13A, 13C) and ALT (13B, 13D) in mice.

[0043] Figure 14 CD4+ splenic lymphocytes after mouse immunization with PLGA-EgG1Y162 nanoparticle vaccine + T(14A, 14B, 14E, 14F), CD8 + Differentiation of T(14C, 14D, 14G, 14F) lymphocytes. Detailed Implementation

[0044] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the descriptions in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as detailed in the claims.

[0045] The basic materials used in the following embodiments are as follows:

[0046] 1. Experimental animals: BABL / c mice weighing approximately 20g were purchased from the Experimental Animal Center of Xinjiang Medical University. The breeding environment was strictly disinfected, and the animals had free access to food and water.

[0047] 2. Plasmids and bacterial strains: The recombinant expression plasmid pET30a(+)-EgG1Y162 and the bacteria containing the recombinant expression plasmid pET30a(+)-EgG1Y162 were synthesized by Anhui Global Gene Technology Co., Ltd.

[0048] 3. Main reagents: Restriction endonucleases were purchased from TaKaRa (Japan); HisTrap™ FF protein affinity chromatography column was a product of GE (USA); BCA protein quantification kit was a product of Thermo Scientific (USA); and Cy5.5 labeled fluorescence was purchased from BD (USA).

[0049] 4. Major Instruments and Equipment: The general-purpose PCR instrument is a product of TaKaRa Corporation (Japan); the benchtop constant temperature shaker is a product of Suzhou Peiying Experimental Equipment Co., Ltd.; the benchtop room temperature centrifuge (5418) and benchtop refrigerated centrifuge (5417R) are both products of Eppendorf Corporation (Germany); the horizontal nucleic acid electrophoresis system is a product of Tanon Corporation (Shanghai); the floor-standing high-speed centrifuge is a product of Backman Coulter Corporation (USA); the protein electrophoresis system is purchased from Bio-Rad Corporation (USA); the gel imaging analysis system (ImageQuant300) is a product of GE (USA); the vertical pressure steam sterilizer (MLS-3870) is a product of Sanyo Corporation (Japan); the 37℃ water-jacketed constant temperature incubator is a product of Shanghai Senxin Experimental Instrument Co., Ltd.; the ultrasonic homogenizer is from Ningbo Xinzhi Corporation; the ultracentrifuge is a product of Beckman Coulter Corporation (USA); the freeze dryer is a product of Labconco Corporation (USA); and the IVIS Spectrum in vivo imaging system is a product of PerkinElmer Corporation.

[0050] Example 1. Purification and expression of recombinant EgG1Y162 protein from Echinococcus granulosus larvae

[0051] The fragment size obtained after double enzyme digestion was consistent with the theoretical value, indicating that the EgG1Y16 gene had been correctly inserted between the BamHI / HindIII restriction sites of the pET30a(+) vector. Figure 1 The clear bands and lack of tails in the electrophoresis image indicate high plasmid DNA purity and complete enzyme digestion, making it suitable for subsequent transformation and expression experiments. Figure 2 ).

[0052] EgG1Y162 positive clones with correct sequencing were inoculated with the recombinant expression plasmid pET30a(+)-EgG1Y162 at a volume ratio of 1:100 in 1L of liquid LB medium containing Kana (100 μg / mL). The culture was incubated at 37℃ with shaking at 180 rpm until the logarithmic growth phase (OD600 approximately 0.6), at which point IPTG (working concentration: 1 mM) was added. The culture was continued for 16 h. After induction of expression, the cells were centrifuged at 8000 rpm for 10 min, and the supernatant was discarded. The cells were washed twice with PBS and resuspended in binding buffer, undergoing freeze-thaw cycles 2-3 times to facilitate cell disruption. The lysate was centrifuged at 4℃ and 8000 rpm for 15 min, and the supernatant was collected. The precipitate was dissolved overnight at 4℃ in 30–40 mL of inclusion body binding buffer. SDS-PAGE electrophoresis analysis of the supernatant and precipitate samples was performed to determine the expression distribution of the recombinant protein. The supernatant was purified by Ni column affinity chromatography of the EgG1Y162 recombinant protein according to the instructions of the GE protein purification kit. The washing and elution samples were dialyzed into buffer (50mM Tris, 300mM NaCl, 10% Glycerol, pH 8.0), filtered, sterilized, aliquoted, and stored at -20°C.

[0053] Example 2. Preparation and formulation screening of PLGA-EgG1Y162 nanovaccine

[0054] 2.1 Preparation of PLGA-EgG1Y162 nanovaccine

[0055] Weigh 50 mg of PLGA into a 10 mL EP tube, add 1 mL of dichloromethane (operate in a fume hood), and dissolve the PLGA in the dichloromethane. Add 2 mL of 3 wt% PVA dropwise to the dissolved PLGA while vortexing, mixing for 1 min. Under ice bath conditions, sonicate for 5 min (40 W, 5 s sonication, 5 s interval). While vortexing, add 4 mL of 1 mg / mL protein solution dropwise, mixing for 1 min. Under ice bath conditions, sonicate for 5 min (40 W, 5 s sonication, 5 s interval), forming a milky white primary emulsion. Add 2 mL of PVA to the primary emulsion while vortexing. 3% PVA was ultrasonically disrupted for 6 min under ice bath conditions (ultrasonic power 40W, 5s sonication, 5s interval) to form a complex emulsion. The complex emulsion obtained after sonication was placed in a fume hood and stirred while evaporating until the organic solvent was completely evaporated. It was then centrifuged at 40000 rpm for 30 min in a refrigerated ultracentrifuge. After centrifugation, the precipitate obtained after ultracentrifugation was resuspended in 2 mL of deionized water to obtain a PLGA-encapsulated recombinant protein suspension. This suspension was placed in a 5 mL vial and incubated at -80℃ for 2 h. Then, it was transferred to a vacuum freeze dryer and freeze-dried for 24 h. The sample was removed and the PLGA nano-subunit vaccine was stored at 4℃ for later use.

[0056] 2.2 Investigation of PVA concentration in the preparation process of PLGA-EgG1Y162 nano-vaccine

[0057] Following the formulation process, the PLGA concentration was fixed at 50 mg / mL, the colostrum ultrasonic power was 40 W, and the ultrasonic time was 5 min. The EgG1Y162 concentration in 4 mL of protein was 1 mg / mL. The re-emulsion ultrasonic power was 40 W, and the ultrasonic time was 6 min. Keeping other parameters constant, the effects of PVA concentrations of 2%, 3%, 4%, and 5% (volume constant, only PVA mass changed) on nanoparticles were investigated. Particle size, potential, PDI, and encapsulation efficiency (EE) were used as evaluation indicators. The results are shown in Table 1. With increasing PVA concentration, the particle size increased, and the encapsulation efficiency decreased. Therefore, 3% PVA was selected for subsequent experiments.

[0058] Table 1. Investigation of PVA concentration

[0059]

[0060] Example 3. Characteristics of the PLGA-EgG1Y162 nanovaccine containing Echinococcus granulosus larvae

[0061] 3.1 Physical characteristics of the Echinococcus granulosus PLGA-EgG1Y162 nano-vaccine

[0062] Analysis using a nanolaser particle size analyzer and dynamic light scattering method revealed that the particle size of the PLGA-EgG1Y162 nanovaccine was 394.4 ± 7.6 nm. Figure 3 A small amount of the PLGA-EgG1Y162 nanoparticle vaccine was taken and sent to the Public Technology Center of the Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences, for observation using scanning electron microscopy and transmission electron microscopy. The results are attached. Figure 4 , 5 As shown, the PLGA-EgG1Y162 nanovaccine is a uniform spherical shape with a relatively smooth surface. To determine the encapsulation efficiency of the PLGA-EgG1Y162 nanovaccine, the supernatant was recovered after ultracentrifugation, and the total volume of the supernatant was recorded. The concentration of free protein in the supernatant was determined using the BCA method, and the encapsulation efficiency of the PLGA-EgG1Y162 nanovaccine was calculated according to formula (1). After three independent replicate experiments, the encapsulation efficiency of the PLGA-EgG1Y162 nanovaccine was 79.05±0.3%.

[0063] Encapsulation ratio = (W 总 -W 游离 ) / W 总 ×100%(1)

[0064] 3.2 In vitro sustained-release properties of the Echinococcus granulosus PLGA-EgG1Y162 nanovaccine

[0065] 20 μg of PLGA-EgG1Y162 nano-vaccine lyophilized powder was weighed and placed in a sealed centrifuge tube. 5 mL of PBS solution was used as the sustained-release medium. The constant-temperature water bath shaker was set to 37℃ and 180 rpm. For sampling, the centrifuge tube was centrifuged at 15000 rpm for 5 min, and the volume of the supernatant was recorded. 20 μL of the supernatant was collected as the sample for protein concentration determination. The sample was stored at -20℃ for later use. 20 μL of PBS was added to the centrifuge tube to resuspend the precipitate, and the tube was placed in its original environment. Samples were collected at 0, 6 h, 12 h, 1 d, 3 d, 5 d, 7 d, 9 d, and 11 days. The protein concentration in the samples was determined by the BCA method, and the total protein content in the supernatant was calculated. The results are attached. Figure 6 As shown, the PLGA-EgG1Y162 nanoparticle vaccine gradually releases its coated recombinant protein during the first three days, and the release gradually stops after the seventh day. This suggests that the PLGA-EgG1Y162 nanoparticle vaccine has good sustained-release properties.

[0066] 3.3 Toxicity of the PLGA-EgG1Y162 nano-vaccine containing Echinococcus granulosus

[0067] To systematically evaluate the toxicity of the PLGA-EgG1Y162 nanoparticle vaccine, the following experimental procedures were followed: Twenty healthy BABL / c mice were randomly divided into four experimental groups, with five mice in each group. Specific grouping and treatment methods are as follows:

[0068] (1) Control group: As a blank control, each mouse was injected subcutaneously with an equal volume of PBS buffer on days 0, 14, 28 and 42.

[0069] (2) EgG1Y162 group: At the same time points as above, each mouse was subcutaneously injected with 25 μg of recombinant EgG1Y162 protein;

[0070] (3) FCA-EgG1Y162 group: At the same time point as above, each mouse was subcutaneously injected with FCA-EgG1Y162 nano-vaccine containing 25 μg EgG1Y162 (after thoroughly mixing EgG1Y162 solution and Freund's adjuvant at a volume ratio of 1:1 and then sonicating it to emulsify it evenly).

[0071] (3) PLGA-EgG1Y162 group: At the same time point as above, each mouse was subcutaneously injected with PLGA-EgG1Y162 nano-vaccine containing 25μgEgG1Y162.

[0072] Throughout the experiment, the health status, behavior, and mental state of the mice were closely observed and recorded in detail. Three days after immunization on day 45, the mice were sacrificed, and tissues from the heart, liver, spleen, lungs, and kidneys were collected. These tissues were fixed in 4% paraformaldehyde, stained with hematoxylin and eosin (HE), and prepared as tissue sections. The histological morphological changes in each organ were observed to systematically evaluate whether the PLGA-EgG1Y162 nanovaccine induced pathological damage. Histopathological analysis showed that, compared with the Control group, no significant changes in tissue structure, abnormal cell morphology, or inflammatory cell infiltration were observed in the HE-stained sections of the heart, liver, spleen, lungs, and kidneys of mice in the EgG1Y162 group, FCA-EgG1Y162 group, and PLGA-EgG1Y162 group. Figure 7 Throughout the toxicity experiment, continuous dynamic behavioral monitoring revealed that mice in all experimental groups maintained normal spontaneous activity, eating and drinking, and social behavior, without exhibiting toxicity-related symptoms such as lethargy, anorexia, or abnormal gait.

[0073] Based on the above histological observations and behavioral assessments, it was confirmed that the PLGA-EgG1Y162 nanovaccine did not have a significant adverse effect on the function of vital organs in mice during repeated immunization, nor did it induce systemic toxicity. This indicates that the PLGA-EgG1Y162 nanovaccine has good biosafety and provides reliable safety data support for subsequent clinical translation and application.

[0074] Example 4. Distribution of PLGA-EgG1Y162 nanoparticles in mice.

[0075] The distribution of PLGA-EgG1Y162 nanovaccine in mice was dynamically monitored using an IVIS spectral visible light imager. PLGA-EgG1Y162 nanovaccine, FCA-EgG1Y162 adjuvanted vaccine, and free EgG1Y162 protein (control) labeled with Cy5.5 fluorescent dye were subcutaneously injected into SPF-grade BABL / c mice at a dose of 100 μL, with each mouse containing 15 μg of Cy5.5 fluorescent dye. At 0 h, 12 h, 24 h, 3 d, 5 d, 7 d, 9 d, and 11 d post-injection, mice were placed in an imaging chamber with an excitation wavelength of 675 nm and an emission wavelength of 710 nm. Fluorescence signals were acquired at the injection site and draining lymph nodes, and quantitative analysis of fluorescence intensity was performed using LivingImage software. The results showed that in the initial injection period (0-12h), the PLGA-EgG1Y162 nanovaccine exhibited high-intensity fluorescence signals at the injection sites (8A, 8B), indicating good local retention characteristics. At 24h post-injection, the fluorescence intensity in the draining lymph nodes (8C, 8D) significantly increased and remained at a high level until day 5, confirming the effective targeting and accumulation of the vaccine in the lymph nodes. In contrast, free EgG1Y162 protein diffused rapidly at the injection site, and the fluorescence intensity in the lymph nodes was significantly lower than that of the nanovaccine group. Compared with the FCA-EgG1Y162 adjuvant group, the PLGA-EgG1Y162 group maintained a higher trend in lymph node fluorescence intensity even after 9 days, indicating that the nanovaccine has sustained-release properties, continuously activating local immune responses and regulating systemic immune distribution. This further validates the unique in vivo distribution advantages of the PLGA-EgG1Y162 nanovaccine.

[0076] Example 5. Humoral immunity induced in mice by Echinococcus granulosus PLGA-EgG1Y162 nanovaccine.

[0077] 5.1 Experimental Design

[0078] Twenty healthy SPF-grade BABL / c mice were randomly divided into four experimental groups (n=5 per group). The experimental period was set at 49 days. The specific grouping and intervention protocols are as follows:

[0079] (1) Model group: On day 0, day 14, day 28 and day 42, each mouse was injected with an equal volume of PBS buffer via subcutaneous route;

[0080] (2) EgG1Y162 group: At the same time points as above, each mouse was subcutaneously injected with 25 μg of recombinant EgG1Y162 protein;

[0081] (3) FCA-EgG1Y162 group: At the same time point as above, each mouse was subcutaneously injected with FCA-EgG1Y162 nano-vaccine containing 25μgEgG1Y162.

[0082] (3) PLGA-EgG1Y162 group: At the same time point as above, each mouse was subcutaneously injected with PLGA-EgG1Y162 nano-vaccine containing 25μgEgG1Y162.

[0083] The model group and the three experimental groups (EgG1Y162, FCA-EgG1Y162, PLGA-EgG1Y162) underwent four immunizations on days 0, 14, 28, and 42. Seven days after the last immunization (day 49), portal vein infection modeling was performed simultaneously.

[0084] 5.2 Detection of antibody titers in serum

[0085] Mice were sacrificed after serum collection on days 14, 28, and 42. The dynamic changes in antibody titers were assessed using ELISA. In short, EgG1Y162 was diluted with coating buffer, and 100 μL of a solution containing 2 μg was added to each well of a 96-well ELISA plate. The EgG1Y162 recombinant protein was coated with buffer and incubated overnight at 4°C. After washing three times with PBS containing 5% skim milk, each well was incubated with 20 μL of mouse serum for 2 h at room temperature. After washing three times with PBS, each well was incubated with HRP-coupled anti-mouse total IgG antibody (1:156.25, 1:312.5, 1:625, 1:1250, 1:2500, 1:5000, 1:10000, 1:20000, 1:40000, 1:80000, and 1:160000 dilutions) for 2 h. After washing three times with PBS again, 100 μL of TMB chromogenic solution was added to each well. After incubation at room temperature for 15 min, 100 μL of freshly prepared 2 mol / L H2SO4 was added to each well to stop the incubation. The OD450 values ​​of each well were then read using a microplate reader. (See attached image) Figure 9As shown, on days 14, 28, and 42, mice in the PLGA-EgG1Y162 group exhibited high levels of total IgG, significantly higher than those in the EgG1Y162 and FCA-EgG1Y162 groups (P<0.01). The FCA-EgG1Y162 group induced high levels of total IgG antibodies on days 14 and 28, but these levels peaked on day 28 and then significantly decreased. In contrast, the PLGA-EgG1Y162 group maintained high antibody expression on day 42, demonstrating a long-lasting immune response. These data indicate that the PLGA-EgG1Y162 nanovaccine prepared in this invention can effectively stimulate the body's immune system, continuously inducing the production of specific total IgG antibodies, thereby providing immune protection against Echinococcus granulosus infection.

[0086] Example 6. Echinococcus granulosus PLGA-EgG1Y162 nanovaccine and its immunoprotective effect against Echinococcus granulosus infection.

[0087] 6.1 Establishment of the fine-grained echinococcosis model

[0088] Twenty-four healthy SPF-grade BABL / c mice were randomly divided into four experimental groups (n=6 per group). The experimental period was set at 49 days. The specific grouping and intervention protocols are as follows:

[0089] (1) Model group: On day 0, day 14, day 28 and day 42, each mouse was injected with an equal volume of PBS buffer via subcutaneous route;

[0090] (2) EgG1Y162 group: At the same time points as above, each mouse was subcutaneously injected with 25 μg of recombinant EgG1Y162 protein;

[0091] (3) FCA-EgG1Y162 group: At the same time point as above, each mouse was subcutaneously injected with FCA-EgG1Y162 nano-vaccine containing 25μgEgG1Y162.

[0092] (3) PLGA-EgG1Y162 group: At the same time point as above, each mouse was subcutaneously injected with PLGA-EgG1Y162 nano-vaccine containing 25μgEgG1Y162.

[0093] The model group and three experimental groups (EgG1Y162, FCA-EgG1Y162, PLGA-EgG1Y162) underwent four immunizations on days 0, 14, 28, and 42. Seven days after the last immunization (day 49), 0.2 mL of physiological saline suspension containing 3000 protocercariae was injected into mice via the portal vein to establish an infection model.

[0094] 6.2 Sample collection and histological observation of mice after being challenged by protocercariae

[0095] Mice were dissected at weeks 16 and 24 after being infected with protocercariae. Blood was collected by enucleation of the eyeballs, and cystic lesions on the livers of the mice were photographed, recorded, and weighed. Figure 10 The number of vesicles was recorded. Liver tissues were collected from mice in each group, fixed with 4% paraformaldehyde, and stained with hematoxylin and eosin (HE) to prepare tissue sections. The histological morphological changes of the liver tissues in each group were observed. The results at week 16 showed that the liver-to-body weight ratio in the model group was (6.37±0.44%), which was significantly higher than that in the PLGA-EgG1Y162 group (4.56±0.016%), and the difference was statistically significant (P<0.0001). Figure 11 A) Liver vesicle count: The average number of liver vesicles in the model group mice was (17.88±5.8), while that in the PLGA-EgG1Y162 group was only (3±2.08), and the difference was statistically significant (P<0.01). Figure 12 A) Histological morphology: Obvious cystic lesions were observed in the liver of mice in the model group, while the severity of liver lesions was significantly reduced in the PLGA-EgG1Y162 group. Results at week 24 showed that the liver-to-body weight ratio in the model group was (21.9±4.9%), significantly higher than that in the PLGA-EgG1Y162 group (5.6±0.5%), with a highly statistically significant difference (P<0.0001). Figure 11 B); Liver vesicle count: The average number of liver vesicles in the model group mice was (66.5±17.6), while that in the PLGA-EgG1Y162 group was only (2.2±0.6), and the difference was statistically significant (P<0.0001). Figure 12 B) Tissue Morphology: With prolonged infection time, the cystic lesions in the liver of the model group further aggravated, while the PLGA-EgG1Y162 group could still effectively inhibit the development of cystic lesions in the liver and maintain a relatively normal morphology of the liver tissue. Experimental data indicate that the PLGA-EgG1Y162 nanovaccine can provide long-term and stable protection against liver damage caused by protocephala infection by inhibiting the formation of cystic lesions, reducing inflammatory response, and maintaining the integrity of liver tissue. It has potential application value as a preventive and therapeutic agent for liver diseases related to echinococcosis infection.

[0096] 6.3 Biochemical Detection

[0097] The biosafety of PLGA-EgG1Y162 in vivo was systematically evaluated by comparing serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels in mice at weeks 16 and 24. Data showed that compared to the model group, the PLGA-EgG1Y162 group exhibited a highly significant decrease in AST and ALT levels (P<0.05). This significant reduction in these indicators fully demonstrates that PLGA-EgG1Y162 can effectively reduce hepatocyte damage, maintain hepatocyte membrane integrity, and provide good protection for liver function. Figure 13 This also indirectly confirms that the vaccine has good biosafety in animals, which is expected to provide a reliable basis for subsequent clinical applications.

[0098] 6.4 Differentiation detection of mouse splenic lymphocytes

[0099] To assess the differentiation of T lymphocyte subsets in mouse spleen lymphocytes, splenic lymphocytes were isolated from mice at weeks 16 and 24 after protocercariae infection. The specific procedures were as follows: The isolated lymphocytes were stained with FITC-conjugated anti-mouse CD3, PE-conjugated anti-mouse CD4, and PerCP-Cy5.5-conjugated anti-mouse CD8 antibodies. After staining, the cells were washed three times with PBS buffer, and then CD4+ was analyzed by flow cytometry. + T, CD8 + Quantitative analysis was performed on the differentiation ratio of T lymphocytes.

[0100] Experimental results are as follows Figure 14 As shown: At week 16 post-infection, compared with the model group, CD4+ in splenic lymphocytes of mice in the PLGA-EgG1Y162 nanovaccine group was significantly higher than that in the model group. + The proportion of T cells significantly increased to (68.96±1.02)%, an increase of approximately 7.1% compared to the model group (64.36±2.48)% (P<0.001); CD8 + The proportion of T cells was (28.91±1.27)%, which was approximately 21.9% higher than that in the model group (23.71±1.07)% (P<0.0001). At week 24 post-infection, the regulatory effect of the PLGA-EgG1Y162 nanovaccine on splenic lymphocyte subsets remained stable and significantly different from the model group, even with prolonged infection cycles. Compared to the model group, the CD4+ level in splenic lymphocytes of mice in the PLGA-EgG1Y162 nanovaccine group was significantly higher. + The proportion of T cells significantly increased to (74±1.61)%, an increase of approximately 7.9% compared to the model group (68.6±1.24)% (P<0.01); CD8 +The proportion of T cells was (27.13±3.82)%, which was approximately 37.5% higher than that in the model group (19.73±3.17)% (P<0.05). Splenic lymphocytes activated by vaccine immunization further differentiated into CD4+ cells. + T and CD8 + T lymphocytes, CD8 + T lymphocytes further differentiate into CTL cells, secrete specific antibodies, target and kill Echinococcus granulosus larvae, and enhance the body's anti-parasitic immune response. These results confirm that the PLGA-EgG1Y162 nanovaccine specifically induces CD4+... + T cell dominant differentiation, while enhancing CD8 + The effector function of T cells was utilized to construct a multi-layered immune network against Echinococcus granulosus, providing cellular mechanism support for the clinical application of the vaccine.

[0101] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A nano-subunit vaccine against Echinococcus granulosus, characterized in that, The aforementioned Echinococcus nanounit vaccine is composed of PLGA nanoparticles coated with recombinant protein EgG1Y162, the amino acid gene sequence of which is shown in SEQ ID NO.

1.

2. The nano-subunit vaccine according to claim 1, characterized in that, The recombinant protein EgG1Y162 was obtained by transforming the recombinant expression plasmid pET30a(+)-EgG1Y162 into BL21 Escherichia coli for induced expression, resulting in bacteria containing the pET30a(+)-EgG1Y162 recombinant expression plasmid, followed by expression and purification to obtain the recombinant protein EgG1Y162.

3. The nano-subunit vaccine according to claim 2, characterized in that, The expression and purification process involved inoculating bacteria containing the pET30a(+)-EgG1Y162 recombinant expression plasmid into LB liquid medium for culture to obtain a bacterial suspension. When the OD600 value of the bacterial suspension reached 0.4-0.6, IPTG was added to the bacterial suspension to induce the expression of the recombinant protein. After 14-18 hours of induction, the bacterial cells were collected by centrifugation. The obtained bacterial cells were washed, the precipitate was resuspended in PBS, and the bacterial cells were lysed by centrifugation and sonication. The supernatant was collected, and the recombinant protein was purified by Ni column affinity chromatography.

4. The nano-subunit vaccine according to claim 1, characterized in that, The aforementioned nano-subunit vaccine is a regular sphere with a diameter of 360-420 nm.

5. The nano-subunit vaccine according to claim 1, characterized in that, The nano-subunit vaccine was prepared by the following method: a PLGA dichloromethane solution was vortexed with 2-5 wt% polyvinyl alcohol, and subjected to a first sonication under ice bath conditions to obtain a mixed solution; recombinant protein EgG1Y162 was vortexed with the mixed solution, and subjected to a second sonication under ice bath conditions to form a milky white liquid; the milky white liquid was mixed with 2-5 wt% polyvinyl alcohol, vortexed, and subjected to a third sonication under ice bath conditions to obtain a complex emulsion; the organic solvent in the complex emulsion was evaporated, and the liquid was collected; the obtained liquid was centrifuged, the precipitate was collected, the precipitate was resuspended with deionized water, placed at -70 to -90°C for standing, and then freeze-dried to obtain the fine-grained Echinococcus nano-subunit vaccine.

6. The method for preparing the nano-subunit vaccine according to any one of claims 1-4, characterized in that, A PLGA solution in dichloromethane was vortexed with 2-5 wt% polyvinyl alcohol and subjected to a first sonication under ice bath conditions to obtain a mixed solution. Recombinant protein EgG1Y162 was vortexed with the mixed solution and subjected to a second sonication under ice bath conditions to form a milky white liquid. The milky white liquid was then vortexed with 2-5 wt% polyvinyl alcohol and subjected to a third sonication under ice bath conditions to obtain a complex emulsion. The organic solvent in the complex emulsion was evaporated, and the liquid was collected. The resulting liquid was centrifuged, the precipitate was collected, resuspended in deionized water, allowed to stand at -70 to -90°C, and then freeze-dried to obtain a fine-grained Echinococcus nanounit vaccine.

7. The nano-subunit vaccine according to claim 5 or the preparation method according to claim 6, characterized in that, The concentration of PLGA in the dichloromethane solution is 40-60 mg / mL, preferably 45-55 mg / mL, such as 50 mg / mL; the total volume ratio of the 2-5% polyvinyl alcohol to dichloromethane is 2-6:1, preferably 3-5:1, and more preferably 4:1; the 2-5% polyvinyl alcohol added before the first ultrasound is 40%-60% of the total volume of the 2-5% polyvinyl alcohol, preferably 45%-55%, and more preferably 50%; the mass-volume ratio of the recombinant protein EgG1Y162 to dichloromethane is 3-5 mg:1 mL; the 2-5 wt% polyvinyl alcohol is preferably 2-4 wt% polyvinyl alcohol.

8. The nano-subunit vaccine according to claim 5 or the preparation method according to claim 6, characterized in that, The power of the first, second, and third ultrasounds is independently selected from 35-45W, preferably 40W; the total time of the first, second, and third ultrasounds is independently selected from 3-8min, preferably 5-6min; the time of each ultrasound is independently selected from 3-7s, preferably 4-6s, preferably 5s; and the time of each interval is independently selected from 3-7s, preferably 4-6s, preferably 5s.

9. The nano-subunit vaccine according to claim 5 or the preparation method according to claim 6, characterized in that, The centrifugation speed is 30,000-50,000 r / min, preferably 35,000-45,000 r / min, and more preferably 4,000 r / min; the centrifugation time is 25-35 min, preferably 28-32 min, and more preferably 30 s; the settling time is 1.5-3 h; and the freeze-drying time is 20-24 h.

10. The use of the nanosubunit vaccine according to any one of claims 1-4 or any one of claims 7-9 in the preparation of a medicament for preventing infection with Echinococcus granulosus; preferably, the medicament comprises a vaccine.

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