Canine toxocara canis rTcMUCs-plga nano vaccine, preparation method and application thereof

Abstract

The application discloses a canine toxocara cati rTcMUCs-PLGA nano vaccine, a preparation method and application thereof, and is characterized in that the nano vaccine takes PLGA nanospheres as a carrier and loads TcMUC-2 and / or TcMUC-3 recombinant proteins. According to the animal protection test result, compared with the PBS and PLGA control groups, the number of canine toxocara cati L3 stage larvae in the liver and lung of the mice in the immunization groups inoculated with rTcMUC-2-PLGA and rTcMUC-3-PLGA is obviously reduced, the worm reduction rates of the rTcMUC-2 and rTcMUC-3 immunization groups using saponin as an adjuvant are 55.31% and 53.61% respectively, after the rTcMUC-2 and rTcMUC-3 are coated by using PLGA as a nano carrier, the immunization frequency is reduced from three times to twice, and the worm reduction rates are 65.13% and 55.9% respectively, and the result proves that the PLGA nano vaccine can be used as an adjuvant of the recombinant protein to reduce the immunization cycle and induce the host to generate better immunoprotection to inhibit the invasion and migration of the canine toxocara cati larvae.

Description

Technical Field

[0001] This invention relates to the field of molecular immunology, specifically to a Toxocara canis rTcMUCs-PLGA nanovaccine, its preparation method, and its application. Background Technology

[0002] Canine toxocariasis is a global zoonotic parasitic disease caused by infection with *Toxocara canis* (T. canis). Although chemical drugs can effectively prevent and treat the disease, long-term drug treatment easily leads to drug-resistant strains and cannot prevent repeated infections in animals. This makes vaccine development crucial for the control of canine toxocariasis. Unfortunately, there is currently no commercially available vaccine for *Toxocara canis*.

[0003] The literature (Veterinary. Diagnostic value and immunogenicity evaluation of parasite-specific high-expression antigens TcHB / TcSP of Toxocara canis [D]. 2023.) reported that using the parasite-specific high-expression candidate antigen genes hemoglobin (Tchb) and serine protease (Tcsp) of Toxocara canis as research objects, the biological characteristics of the two proteins in Toxocara canis were preliminarily explored through cloning, expression and molecular identification. Furthermore, the serological diagnostic value of recombinant rTcHB and rTcSP proteins was evaluated using a "Toxocara canis-mouse / dog" infection model.

[0004] The excretion and secretion antigens (TES) of *Toxocara canis* are widely involved in the immune interaction between the parasite and the host, making them ideal vaccine candidates. However, these antigens exhibit low immunogenicity and rapid metabolism during immunization testing, making the enhancement of their immunogenicity a current challenge in *Toxocara canis* TES vaccine antigen research. Notably, the hatching of L3-stage eggs and the migration of larvae into L3-stage intestinal and lung tissues are key pathogenic processes, and TES plays a crucial role in these two stages. Furthermore, these antigens possess significant immunogenicity, thus making them ideal vaccine candidates. As important members of the TES protein family, mucins play vital roles in protection, immune defense, environmental adaptation, reproduction, and development during worm infection through unique glycosylation modifications. These functions enable nematodes to evade the host's immune response and survive and reproduce in complex and variable environments, making them potential targets for developing new antiparasitic agents. Although the five mucins TcMUC1-5 of *Toxocara canis* were discovered early on, and some researchers, through studies of several of these proteins, have found that *Toxocara canis* mucins TcMUC2-4 can stimulate Th1 and Th2 responses in the body, and that the interaction proteins between TcMUC1-4 and mouse macrophages are mainly involved in biological processes such as DNA replication, metabolism, and cell adhesion, a comprehensive and systematic comparative study on the molecular characteristics and immune effects of these five proteins is still lacking. Summary of the Invention

[0005] Based on the above reasons, this invention proposes a *Toxocara canis* rTcMUCs-PLGA nanovaccine and its preparation method. Specifically, to achieve the objectives of this invention, the following technical solution is proposed:

[0006] This invention relates to a Toxocara canis rTcMUCs-PLGA nanovaccine, characterized by using PLGA nanospheres as a carrier to load TcMUC-2 and / or TcMUC-3 recombinant proteins, wherein the TcMUC-2 recombinant protein contains the following amino acid sequence:

[0007] MNVRVVILLTVLISVVKPQPGAQTTTTAATTTTAAATTTTAAATTTTAAATTTTAAATTTTAAPMTTTAGATTTAAGATTTAAGATTTAGGPTTTAAGAITTAAGATTTAAVMTTTPACIDTANDCQLFMPLCFVQPYSRAIQGRCRRTCNICSCQDSANDCANFVSVCLNPTYQPVLRSRCALTCGFC;

[0008] The TcMUC-3 recombinant protein contains the following amino acid sequence:

[0009] MNACACALLLLFIGVVRHQSIFAAATMMTTSAPCVDSASDCQQHTSLCFMQPYSRSMQSRCQRTCNICNCRDDANDCARLVTFCGNPMYQPVLRTRCTLTCGFCSNDTLATTVAGTTTTPAPTTTTAAATTTTAA ATTTAAPTTTTAAPTTTTATPTTTTAAPTTTTAAPTTTTAAPTTTTAAPTTTTGAIVTTTAACSDAAMDCQRYAGMCFTQPYSRAIQGRCRRTCNICNCHDSANNCGSLISYCDDPTLQPVLRSRCPLSCGFCS.

[0010] In a preferred embodiment of the present invention, the nanovaccine is loaded with 2-3 wt% recombinant protein.

[0011] In another preferred embodiment of the present invention, the average size of the nanovaccine is 100-300 nm.

[0012] Another aspect of the present invention relates to a method for preparing the above-mentioned nano-vaccine, which includes the following steps:

[0013] (1) Recombinant proteins rTcMUC-2 and / or rTcMUC-3 were dissolved in PBS solution to form an inner aqueous phase; PLGA was dissolved in dichloromethane to form an organic phase;

[0014] (2) The aqueous phase is slowly dripped into the organic phase while vortexing to mix, and the mixture is ultrasonically dissolved in ice water to form a water-in-oil (w / o) emulsion.

[0015] (3) Transfer the w / o emulsion to an aqueous solution containing 1-3 wt% PVA and sonicate again to obtain a w / o / w emulsion;

[0016] (4) Add the prepared w / o / w emulsion to an aqueous solution containing 0.2-0.4 wt% PVA and evaporate the organic solvent by magnetic stirring at room temperature;

[0017] (5) Centrifuge the nano vaccine solution, then wash the precipitate three times with ultrapure water, and finally resuspend it with ultrapure water.

[0018] (6) Freeze the nano-vaccine in a freeze dryer.

[0019] Another aspect of the present invention relates to the application of the above-mentioned nano-vaccine in the preparation of a vaccine against Toxocara canis.

[0020] In a preferred embodiment of the invention, the vaccine is used to reduce the number of Toxocara canis in the liver and lungs.

[0021] Combined with the results of animal protection experiments, it was found that compared with the PBS and PLGA control groups, the number of L3 stage Toxocara canis larvae in the liver and lungs of mice immunized with rTcMUC-2-PLGA and rTcMUC-3-PLGA was significantly reduced. Compared with the 55.31% and 53.61% reduction rates of rTcMUC-2 and rTcMUC-3 immunization groups using saponins as adjuvants, the number of immunizations was reduced from three to two after rTcMUC-2 and rTcMUC-3 were coated with PLGA as a nanocarrier, and the reduction rates reached 65.13% and 55.9%, respectively. These results confirm that PLGA nanovaccines can act as adjuvants for recombinant proteins to reduce the immunization cycle and induce the host to produce better immune protection to inhibit the invasion and migration of Toxocara canis larvae.

[0022] The beneficial effects of this invention are as follows:

[0023] This invention discloses a Toxocara canis rTcMUCs-PLGA nanovaccine, its preparation method and application, characterized in that the nanovaccine uses PLGA nanospheres as a carrier to load recombinant TcMUC-2 and / or TcMUC-3 proteins. Combined with the results of animal protection experiments, it was found that compared with the PBS and PLGA control groups, the number of L3 stage Toxocara canis larvae in the liver and lungs of mice immunized with rTcMUC-2-PLGA and rTcMUC-3-PLGA was significantly reduced. Compared with the 55.31% and 53.61% reduction rates of rTcMUC-2 and rTcMUC-3 immunization groups using saponins as adjuvants, the number of immunizations was reduced from three to two after rTcMUC-2 and rTcMUC-3 were coated with PLGA as a nanocarrier, and the reduction rates reached 65.13% and 55.9%, respectively. These results confirm that PLGA nanovaccines can act as adjuvants for recombinant proteins to reduce the immunization cycle and induce the host to produce better immune protection to inhibit the invasion and migration of Toxocara canis larvae. Attached Figure Description

[0024] Figure 1 PCR amplification of the Tcmuc1-5 gene and pET-32a, where: (A) represents PCR amplification of the Tcmuc1-5 gene. M: DL2000 DNA molecular weight standard; 1: blank control; 2: Tcmuc-1; 3: Tcmuc-2; 4: Tcmuc-3; 5: Tcmuc-4; 6: Tcmuc-5. (B) represents reverse PCR amplification of the pET-32a vector. M: DL8000 DNA molecular weight standard; 1: pET-32a linearized plasmid;

[0025] Figure 2 The expression and purification analysis of rTcMUC1-5 was performed, including: (A) PAGE detection of rTcMUC1-5 expression and purification. M: 10-200 kDa protein molecular weight standard; 1: pET-32a empty vector; 2: IPTG-induced pET-32a-Tcmuc-1 expression bacteria; 3: purified recombinant rTcMUC-1 protein; 4: IPTG-induced pET-32a-Tcmuc-2 expression bacteria; 5: purified recombinant rTcMUC-2 protein; 6: IPTG-induced pET-32a-Tcmuc-3 expression bacteria; 7: purified recombinant rTcMUC-3 protein; 8: IPTG-induced pET-32a-Tcmuc-4 expression bacteria; 9: purified recombinant rTcMUC-4 protein; 10: IPTG-induced pET-32a-Tcmuc-5 expression bacteria; 11: purified recombinant rTcMUC-5 protein. (B) Detection of His-tagged proteins rTcMUC (1-5). 1: Purified recombinant rTcMUC-1 protein; 2: Purified recombinant rTcMUC-2 protein; 3: Purified recombinant rTcMUC-3 protein; 4: Purified recombinant rTcMUC-4 protein; 5: Purified recombinant rTcMUC-5 protein;

[0026] Figure 3 Immunogenicity of rTcMUC1-5 is shown in the following figures: (A) Imprint bands of recombinant protein rTcMUC1-5 with positive serum from mice infected with Toxocara canis; (B) Imprint bands of recombinant protein rTcMUC(1-5) with negative serum from mice. M: 10-200 kDa protein molecular weight standard; 1-5: from left to right, rTcMUC-1, rTcMUC-2, rTcMUC-3, rTcMUC-4, and rTcMUC-5.

[0027] Figure 4 The relative expression levels of the Tcmuc(1-5) genes at different developmental stages of *Toxocara canis* are given, where: (A) Tcmuc-1; (B) Tcmuc-2; (C) Tcmuc-3; (D) Tcmuc-4; (E) Tcmuc-5. ns indicates that compared to the stage with the lowest expression level for each gene, P > 0.05, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001.

[0028] Figure 5Immunofluorescence localization of TcMUC-1 in adult Toxocara canis, where TD represents white light, green fluorescence represents FITC-labeled protein, and red fluorescence represents autofluorescence of Toxocara canis tissue. Cu: epidermis; Mu: muscle; Tw: testicular wall; S: spermatocyte; Iw: intestinal wall; Uw: uterine wall; E: eggs in the uterus. Scale bar = 25 μm;

[0029] Figure 6 Immunofluorescence localization of TcMUC-2 in adult Toxocara canis, where: TD represents white light, green fluorescence represents FITC-labeled protein, and red fluorescence represents autofluorescence of Toxocara canis tissue. Scale bar = 25 μm;

[0030] Figure 7 Immunofluorescence localization of TcMUC-3 in adult Toxocara canis, where: TD represents white light, green fluorescence represents FITC-labeled protein, and red fluorescence represents autofluorescence of Toxocara canis tissue. Scale bar = 25 μm;

[0031] Figure 8 Immunofluorescence localization of TcMUC-4 in adult Toxocara canis, where: TD represents white light, green fluorescence represents FITC-labeled protein, and red fluorescence represents autofluorescence of Toxocara canis tissue. Scale bar = 25 μm;

[0032] Figure 9 Immunofluorescence localization of TcMUC-5 in adult Toxocara canis, where: TD represents white light, green fluorescence represents FITC-labeled protein, and red fluorescence represents autofluorescence of Toxocara canis tissue. Scale bar = 25 μm;

[0033] Figure 10 The reduction in liver and lung larvae in mice immunized with rTcMUC1-5 and infected with Toxocara canis is shown in the figure. The results are: (A) rTcMUC-1; (B) rTcMUC-2; (C) rTcMUC-3; (D) rTcMUC-4; (E) rTcMUC-1. ns indicates P > 0.05 compared to the PBS group, * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

[0034] Figure 11 The effects of different PLGA concentrations on cell viability were investigated, including: (A) the effect of different PLGA concentrations on the viability of 293T cells; and (B) the effect of different PLGA concentrations on the viability of ANA-1 cells. ns indicates P > 0.05 compared to 0 μg / mL.

[0035] Figure 12The binding of rTcMUC-2 and rTcMUC-3 to PLGA is shown below, where: M: 10-200 kDa protein molecular weight standard; 1: PLGA nanovaccine; 2: purified recombinant protein rTcMUC-2; 3: rTcMUC-2-PLGA nanovaccine; 4: purified recombinant protein rTcMUC-3; 5: rTcMUC-3-PLGA nanovaccine.

[0036] Figure 13 Scanning electron microscope (SEM) images of rTcMUC-2-PLGA and rTcMUC-3-PLGA, where: (A): rTcMUC-2-PLGA nanovaccine; (B): rTcMUC-3-PLGA nanovaccine. Scale bar = 500 nm;

[0037] Figure 14 The size distribution diagrams of rTcMUC-2-PLGA and rTcMUC-3-PLGA are shown, where: (A): rTcMUC-2-PLGA nanovaccine; (B): rTcMUC-3-PLGA nanovaccine;

[0038] Figure 15 Size distribution diagrams of rTcMUC-2-PLGA and rTcMUC-3-PLGA;

[0039] Figure 16 The in vitro release rates of rTcMUC-2-PLGA and rTcMUC-3-PLGA;

[0040] Figure 17 The changes in blood counts in mice immunized with rTcMUC-2-PLGA and rTcMUC-3-PLGA 28 days later were shown, including: (A): white blood cell count (WBC); (B): red blood cell count (RBC); (C): platelet count (PLT). ns indicates P > 0.05 compared to the PBS group;

[0041] Figure 18 Comparison of histopathological findings in mice immunized with rTcMUC-2-PLGA and rTcMUC-3-PLGA 28 days later, where: scale bar = 50 μm;

[0042] Figure 19 The liver and lung lesions of mice immunized with rTcMUC-2-PLGA and rTcMUC-3-PLGA after infection with Toxocara canis;

[0043] Figure 20 Pathological findings of liver and lung tissues in mice immunized with rTcMUC-2-PLGA and rTcMUC-3-PLGA after infection with Toxocara canis;

[0044] Figure 21The changes in serum-specific IgG and IgG1 / IgG2a antibodies in mice immunized with rTcMUC-2-PLGA and rTcMUC-3-PLGA from *Toxocara canis* were shown. Specifically: (A) changes in rTcMUC-2-specific IgG; (B) changes in rTcMUC-2-specific IgG1; (C) changes in rTcMUC-2-specific IgG2a; (D) IgG1 / IgG2a ratio. ns indicates P > 0.05 compared to the PBS group, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001.

[0045] Figure 22 The changes in serum-specific IgG and IgG1 / IgG2a antibodies in mice immunized with rTcMUC-2-PLGA and rTcMUC-3-PLGA from *Toxocara canis* were shown. Specifically: (A) Changes in rTcMUC-2-specific IgG; (B) Changes in rTcMUC-2-specific IgG1; (C) Changes in rTcMUC-2-specific IgG2a; (D) IgG1 / IgG2a ratio. * indicates P < 0.05 compared to the PBS group, *** indicates P < 0.001, **** indicates P < 0.0001;

[0046] Figure 23 Splenic CD28 days after immunizing mice with rTcMUC-2-PLGA and rTcMUC-3-PLGA 4+ Cell differentiation trends, including: (A) T-bet, GATA3, and RORγt in CD 4+ (B) Flow cytometry plot of positive percentage in cell population; (B) T-bet, GATA3 and RORγt in CD 4+ Statistical chart of the percentage of positive cells in the cell population. ** indicates P < 0.01 compared to the PBS group, *** indicates P < 0.001, **** indicates P < 0.0001;

[0047] Figure 24 Mesenteric lymph node CD 28 days after mice were immunized with rTcMUC-2-PLGA and rTcMUC-3-PLGA 4+ Cell differentiation trends, including: (A) T-bet, GATA3, and RORγt in CD 4+ (B) Flow cytometry plot of positive percentage in cell population; (B) T-bet, GATA3 and RORγt in CD 4+ Statistical graph of the percentage of positive cells in the cell population. ** indicates P < 0.01 compared to the PBS group, **** indicates P < 0.0001;

[0048] Figure 25The levels of cytokines IFN-γ, IL-4, IL-9, IL-10, IL-12, IL-13, and IL-17 in the serum of mice immunized with rTcMUC-2-PLGA and rTcMUC-3-PLGA from *Toxocara canis* were measured. (A) IFN-γ; (B) IL-12; (C) IL-4; (D) IL-9; (E) IL-10; (F) IL-13; (G) IL-17. ns indicates P > 0.05 compared to the PBS group, * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

[0049] Figure 26 The reduction in liver and lung larvae in mice immunized with rTcMUC-2-PLGA and rTcMUC-3-PLGA is shown in the figures: (A) rTcMUC-2-PLGA; (B) rTcMUC-3-PLGA; (C) rTcMUC-2-PLGA + rTcMUC-3-PLGA. ns indicates P > 0.05 compared to the PBS group, * indicates P < 0.05, *** indicates P < 0.001, and **** indicates P < 0.0001. Detailed Implementation

[0050] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0051] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0052] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.

[0053] Example 1:

[0054] 1. Experimental Materials

[0055] 1.1 Parasites, laboratory animals, and serum

[0056] (1) Parasites: Toxocara canis eggs, L3 stage eggs, IL3, LL3, L4 and L5 stage larvae and adults (♂, ♀) were preserved by the Animal Parasitic Diseases Research Center of Sichuan Agricultural University.

[0057] (2) Experimental animals: Six-week-old male C57BL / 6 mice were purchased from Beijing Spaford Experimental Animal Technology Co., Ltd. All animal use protocols of this invention have been reviewed and approved by the Animal Ethics and Welfare Committee of Sichuan Agricultural University (SYXK2019-187).

[0058] (3) Serum: Positive and negative serum from mice infected with Toxocara canis were provided by the Animal Parasitic Diseases Research Center of Sichuan Agricultural University.

[0059] 1.2 Strains and Plasmid Vectors

[0060] (1) Competent cells: Escherichia coli DH5α and Escherichia coli Rosetta were purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0061] (2) Plasmid vector: pET-32a vector was provided by the Animal Parasitic Diseases Center of Sichuan Agricultural University (commercially purchased).

[0062] 1.3 Cells

[0063] Human embryonic kidney cells (293T) and mouse macrophages (ANA-1) were preserved by the Animal Parasitic Diseases Research Center of Sichuan Agricultural University.

[0064] 1.4 Main Reagents

[0065] RNA Extraction Kit (Accurate Biology AG21102), Reverse Transcription Kit (CWBIO CW2020M), Gel Extraction Kit (TIANGEN DP219), Seamless Cloning Kit (Takara 638947), Plasmid Mini-Extraction Kit (TIANGEN DP103), High-Protein Enzyme Retention Kit (Takara R045A), TaqPCR MasterMix (Vazyme P222), DNA Marker (TRANS BM101), Protein Marker (Servicebio G2058), Nucleic Acid Dye (Life-iLab AN34L022), High-Sensitivity Chemiluminescence Detection Kit (CWBIO CW0049S); HRP-conjugated Goat anti-Mouse IgG (H+L) (ABclonalAS003); FITC-conjugated Goat anti-Mouse IgG (H+L) (ABclonalAS001); Hoechst 33258 (Beyotime) C1011); Antifluorescence quenching mounting solution (Beyotime P0126), Mouse antiHis-Tag mAb (Abclonal AE003); IPTG (Solarbio I8070); Protein quantification kit (BCA method) (Abbkine KTD3001), 12.5% ​​SDS-PAGA kit (Epizyme PE213), AMP (Sangon Biotech A610028), BSA (Biosharp BS114), PBS phosphate buffer (Biosharp BL601A), CFA (Beyotime P2036), IFA (Beyotime P2031), Coomassie Brilliant Blue R-250 (Solarbio C8430), 5×SDS-PAGA protein loading buffer (Servicebio G2075), saponin (MACKLIN S817818), polylactic acid-glycolic acid copolymer (Sigma P2066), polyvinyl alcohol (aladdin) P434367), dichloromethane (Knowles 75-09-2), DMEM (biosharpBL304A), fetal bovine serum (Excell FSP500), penicillin-streptomycin mixture (100×) (Servicebio G4003-100ML), 0.25% trypsin digestion solution (Servicebio G4011-100ML), CCK-8 kit (oriscience CB101), HRP-conjugated Goat anti-Mouse IgG1 (Abclonal AS066), HRP-conjugated Goat anti-Mouse IgG2a (Abclonal AS065), soluble single-component TMB substrate chromogenic solution (TIANGENPA107), erythrocyte lysis buffer (Beyotime) (C3702), FITC-CD3+, PE-Cy5.5-CD4+, PE-T-bet, PacificBlue-GATA3, APC-Cy7-RORγt, Mouse IFN-γ ELISA Kit (RX203097M), Mouse IL-4 ELISA Kit (RX203051M), Mouse IL-9 ELISA Kit (RX203045M), Mouse IL-10 ELISA Kit (RX203075M), Mouse IL-12 ELISA Kit (RX2D201666), Mouse IL-13 ELISA Kit (RX203070M), Mouse IL-17 ELISA Kit (RX203067M).

[0066] 1.5 Commonly Used Solutions and Culture Media

[0067] The commonly used solutions and culture media of this invention are shown in Table 1:

[0068] Table 1 Commonly Used Solutions and Culture Media

[0069]

[0070] 2. Experimental Methods

[0071] 2.1 Total RNA extraction from Toxocara canis

[0072] Total RNA was extracted from *Toxocara canis* L0-L5 and adult worms (male and female) according to the instructions of the total RNA extraction kit. The concentration was determined using a Nanodrop 2000 and then stored at -80°C.

[0073] 2.2 cDNA Synthesis of Toxocara canis

[0074] Using total RNA extracted from Toxocara canis as a template, cDNA was synthesized using a reverse transcription kit. The resulting product was stored at -80°C.

[0075] 2.3 Cloning of the Tcmuc1-5 gene

[0076] 2.3.1 Primer Design and Synthesis

[0077] The gene sequences of five mucins Tcmuc1-5 from *Toxocara canis* were downloaded from NCBI. After deleting the signal peptide and transmembrane region, Tcmuc1-5 with a 19bp pET-32a homologous arm was designed using SnapGene 6.0.2 software. PCR amplification primers and reverse amplification primers for pET-32a were synthesized by Sangon Biotech Co., Ltd., as shown in Table 2.

[0078] Table 2 PCR primer sequences

[0079]

[0080] Where: the underlined part indicates a sequence that overlaps with pET-32a.

[0081] 2.3.2 Amplification of the target fragment

[0082] Using Toxocara canis cDNA and plasmid pET-32a as templates, PCR amplification and reverse amplification of the five target genes Tcmuc1-5 were performed.

[0083] 2.3.3 Purification and recovery of amplification products

[0084] The amplified product was mixed with 10× DNA Loading buffer by vortexing and electrophoresis on a 1% agarose gel at 120V and 90mA for 30 min. After cutting the correct-sized band using a gel cutter, the target gene was purified and recovered according to the instructions of the agarose gel DNA recovery kit. After determining the concentration using Nanodrop 2000, the sample was stored at -20℃.

[0085] 2.3.4 Sequencing

[0086] 10 μL of purified and recovered target gene was sent to SnapGene for sequencing. The sequencing results were compared with the genomic data using SnapGene 6.0.2. Plasmid construction was performed after confirming correct sequencing.

[0087] Construction of 2.4pET-32a-Tcmuc(1-5) vector

[0088] 2.4.1 Seamless Cloning

[0089] Using a seamless cloning kit, following the seamless cloning system shown in Table 3, the purified target gene Tcmuc1-5 was mixed with the linearized plasmid pET-32a at a molar ratio of 3:1. After vortexing, the mixture was incubated at 50°C for 15 minutes in a PCR instrument, and then removed and placed on ice.

[0090] Table 3 Seamless Cloning System

[0091]

[0092] 2.4.2 Transformation into DH5α competent cells

[0093] Add 50 μL of DH5α competent cells to a sterile EP tube, then transfer 10 μL of seamless cloning product into the EP tube. Gently pipette to mix, then incubate on ice for 30 min, followed by heat shock at 42°C for 90 s, and then on ice for another 3 min. Add 900 μL of LB liquid medium to the EP tube and incubate at 37°C with shaking at 160 rpm for 2 h. Spread the cultured bacterial solution evenly onto LB solid medium containing Amp and incubate upside down at 37°C until single colonies appear.

[0094] 2.4.3 Identification

[0095] Select 10-20 single colonies from plates and inoculate them into 1 mL of LB medium containing Amp, then incubate for 3-6 hours. Use the bacterial culture as a PCR template for colony PCR identification. Subsequently, 500 μL of the selected positive bacterial culture is sent to Qingke Biotechnology for sequencing. SnapGene is used to compare the sequencing results with the genomic data. Once the sequencing is confirmed to be correct, the culture is transferred into expression bacteria.

[0096] 2.4.4 Plasmid Extraction

[0097] The bacterial cultures with correctly sequenced target genes were expanded and cultured separately. Plasmids were extracted according to the instructions of the plasmid extraction kit, and the concentration was determined by Nanodrop 2000 before storage at -20℃.

[0098] 2.5Tcmuc1-5 prokaryotic expression and protein purification

[0099] 2.5.1 Transformation

[0100] Following the method described in Section 2.4.2, 1 μL of each of the five pET-32a-Tcmuc1-5 plasmids and the empty pET-32a plasmid were transformed into competent Escherichia coli Rosetta cells.

[0101] 2.5.2 Induced Expression

[0102] After overnight culture of the expression bacteria and empty vector bacteria, they were inoculated at a 1:100 ratio into fresh Amp LB medium and cultured until OD600 = 0.6-0.8. Then, 1 mmol / L IPTG inducer was added, and the mixture was induced for 6 h at 160 rpm in an air shaker at 37°C. One mL of the induced bacterial culture was used to prepare a sample, and the expression was detected by 12.5% ​​SDS-PAGE.

[0103] 2.6 Optimization of Recombinant Protein Expression Conditions

[0104] 2.6.1 IPTG Concentration Optimization

[0105] Different amounts of IPTG were added to LB culture medium containing 5 mL of positive bacterial suspension to achieve final concentrations of 0 mmol / L, 0.2 mmol / L, 0.4 mmol / L, 0.6 mmol / L, 0.8 mmol / L, and 1 mmol / L, respectively. After incubation at 37°C in a shaker for 6 h, 1 mL of bacterial suspension was taken for SDS-PAGE electrophoresis detection.

[0106] 2.6.2 Optimization of Induction Time

[0107] An appropriate concentration of IPTG was added to each LB culture medium containing 5 mL of positive bacterial solution. After induction at 37°C for 0 h, 3 h, 6 h, and 9 h, 1 mL of bacterial solution was taken from each culture, centrifuged, stained, and then detected by SDS-PAGE electrophoresis.

[0108] 2.6.3 Optimization of Induction Temperature

[0109] Add appropriate concentrations of IPTG to each LB culture medium containing 5 mL of positive bacterial solution. Induce expression at 37℃, 25℃, and 16℃ for appropriate times (the induction time at 25℃ and 16℃ should be extended to 20 h). After induction, take 1 mL of bacterial solution from each culture medium for SDS-PAGE electrophoresis detection.

[0110] 2.7 Solubility analysis and purification of recombinant proteins

[0111] 2.7.1 Solubility Analysis

[0112] Based on optimized induction conditions, 200 mL of expression bacteria were cultured and induced. The bacterial suspension was centrifuged at 7,000 rpm for 10 min, the supernatant was discarded, and 10 mL of Tris-HCl (50 mM, pH 8.0) was added, followed by repeated shaking to resuspend the bacterial cells. 40 μL of the supernatant was collected for sample preparation. The bacterial cells were disrupted using an ultrasonic cell disruptor. After the suspension changed from viscous to clear, it was centrifuged at 7,000 rpm for 10 min at 4°C, and the supernatant and precipitate were collected. 10 mL of inclusion body washing buffer containing 2 M, 4 M, and 8 M urea were added sequentially to the precipitate, followed by shaking and centrifugation at 7,000 rpm for 10 min at 4°C. 40 μL of each collected supernatant was collected for SDS-PAGE electrophoresis analysis.

[0113] 2.7.2 Purification

[0114] According to the results in 2.7 of this chapter, add appropriate washing buffer, centrifuge at 7,000 r / min, 4℃ for 20 min, take the supernatant and filter it through a 0.45 μm filter membrane, perform Ni+ affinity chromatography column purification, prepare samples for SDS-PAGE electrophoresis analysis, then perform ultrafiltration according to protein concentration, and store at -80℃.

[0115] 2.8 Preparation of mouse anti-rTcMUC1-5 hyperimmune serum

[0116] The purified rTcMUC1-5 recombinant protein was mixed with Freund's complete adjuvant (CFA) and Freund's incomplete adjuvant (IFA) at a 1:1 ratio to prepare emulsion vaccines. Six 5-week-old male C57BL / 6 mice were immunized four times subcutaneously, one week apart. The injection dose was 50 μg of recombinant protein per mouse per injection. Two weeks after the final immunization, blood was collected, serum was separated, and stored at -80℃, as shown in Table 4.

[0117] Table 4 Immunization Schedule

[0118]

[0119] 2.9 Immunoblotting

[0120] 2.9.1 Detection of His Tag on Recombinant Proteins

[0121] After the purified rTcMUC1-5 was separated by 12.5% ​​SDS-PAGE electrophoresis, the gel was cut to an appropriate size, and a 0.45 μm cellulose acetate membrane (PVDF membrane) of the same size was cut off and soaked in methanol for 5 min. The sponge, filter paper, PVDF membrane, and gel were placed in transfer buffer and layered in the following order: sponge → filter paper → gel → PVDF membrane → filter paper → sponge, from bottom (black side), avoiding air bubbles. The assembled transfer tank was covered with ice, and the transfer current was set to 200 mA for 1 h. The transferred membrane was then incubated with antibodies: the PVDF membrane was washed three times with TBST (5 min each time); blocked with 5% skim milk powder at room temperature for 2 h; washed three times with TBST (5 min each time); incubated overnight at 4°C with mouse anti-His tag antibody diluted 1:5000; washed three times with TBST (5 min each time); added HRP-goat anti-mouse IgG diluted 1:3000, and incubated at room temperature for 2 h; washed four times with TBST (5 min each time). Finally, ECL was used for color development and images were acquired.

[0122] 2.9.2 Immunogenicity of recombinant proteins

[0123] The purified rTcMUC1-5 was separated by 12.5% ​​SDS-PAGE electrophoresis and transferred to a membrane, followed by incubation according to procedure 2.10.1. The primary antibody was replaced with 1:100 diluted positive and negative sera from mice infected with *Toxocara canis*. ECL staining was performed and images were acquired.

[0124] 2.10 Immunohistochemistry

[0125] Tissue sections (5 μm thick) of adult Toxocara canis (♂, ♀) were baked in a 60℃ incubator for 1 hour, and then dewaxed and hydrated in the following order: xylene I (15 min); xylene II (15 min); 100% ethanol I (4 min); 100% ethanol II (4 min); 95% ethanol (4 min); 85% ethanol (4 min); 75% ethanol (4 min); distilled water (8 min). The tissue sections were then incubated with antibodies: after blocking with 5% BSA for 1 hour, mouse anti-rTcMUC1-5 hyperimmune serum was diluted 1:100 with PBS and incubated overnight at 4°C in a humidified chamber; washed three times with PBS for 4 minutes each time; FITC-labeled goat anti-mouse IgG (1:100) diluted with PBS and Hoechst were added, and the sections were incubated at 37°C in the dark for 1 hour; washed three times with PBS for 4 minutes each time; after adding anti-fluorescence quencher, a coverslip was placed on top, and the sections were mounted with nail polish. Finally, the sections were observed and photographed under a fluorescence microscope.

[0126] 2.11 Expression levels of Tcmuc1-5 at different stages of Toxocara canis

[0127] 2.11.1 Primer Design and Synthesis

[0128] qPCR primers for the Tcmuc1-5 and 18S RNA internal reference genes were designed using Primer Premier 5 software and synthesized by Shanghai Sangon Biotech Co., Ltd., as shown in Table 5.

[0129] Table 5 qPCR primer sequences

[0130]

[0131] 2.11.2 qPCR reaction system and procedure

[0132] Using Toxocara canis eggs, L3 stage eggs, IL3, LL3, L4 and L5 stage larvae and adult (♂, ♀) cDNA as templates, the transcription levels of Tcmuc1-5 at different stages were detected.

[0133] 2.11.3 Data Processing

[0134] SPSS Statistics 23.0 software was used for statistical analysis, and GraphPad Prism 8.0 software was used to plot the results. The relative expression level of the target gene was calculated using the formula fold change = 2 - ΔΔCt, as follows:

[0135] △△Ct=(Target gene C value - Internal reference Ct value) Experimental group - (Target gene Ct value - Internal reference Ct value) Control group

[0136] 2.12rTcMUC1-5 Animal Protective Test

[0137] 2.12.1 Immunization with vaccines

[0138] Six-week-old male C57BL / 6 mice were randomly divided into seven groups: a PBS control group, a saponin control group, and an rTcMUC1-5+ saponin group, with five mice in each group receiving subcutaneous injections. The saponin was prepared at a concentration of 2 mg / mL, with a 1:1 mixture of recombinant protein and saponin, 50 μg / mouse, and an immunization dose of 0.1 mL. After the initial immunization, booster immunizations were administered at 2-week intervals, for a total of three immunizations.

[0139] 2.12.2 Insect attack and sampling

[0140] Two weeks after their final immunization, mice were challenged with the parasite via gavage. Each mouse received 1000 L3-stage infective eggs (dose controlled at 0.1 ml). Before challenge, the eggs were placed at 28°C to restore their viability. Five days after challenge, the mice were euthanized by cervical dislocation, and necropsy was performed, photographed, and liver and lung tissues were collected.

[0141] 2.12.3 Reduction rate of L3 stage larvae in mice

[0142] The collected mouse liver and lung tissues were minced and the number of worms was counted under a microscope. The worm reduction rate was then calculated. The calculation method is as follows:

[0143]

[0144] 2.13 Preparation of rTcMUC-2-PLGA and rTcMUC-3-PLGA nanovaccines

[0145] rTcMUC-2-PLGA and rTcMUC-3-PLGA nanovaccines were prepared using a two-emulsion (w / o / w) solvent evaporation method.

[0146] (1) 2 mg of recombinant proteins rTcMUC-2 and rTcMUC-3 were dissolved in 2 mL of PBS solution to form an internal aqueous phase. No recombinant protein was added to the blank PLGA. Subsequently, 50 mg of PLGA was dissolved in 1 mL of dichloromethane to form an organic phase.

[0147] (2) Slowly drop the aqueous phase into the organic phase while vortexing to mix. Sonicate in ice water (60w, 5s, 5s) for 5min to form a water-in-oil (w / o) emulsion with a total volume of 3mL.

[0148] (3) Transfer the w / o emulsion to 2 ml of 2% PVA aqueous solution and sonicate again (60w, 5s, 5s) for 5 min to obtain the final w / o / w emulsion with a total volume of 5 ml.

[0149] (4) Add the prepared w / o / w emulsion to 30 ml of 0.3% PVA aqueous solution and stir magnetically at room temperature for 8 h to evaporate the organic solvent.

[0150] (5) The PLGA, rTcMUC-2-PLGA and rTcMUC-3-PLGA nanovaccine solutions were centrifuged at 15,000 r / min for 30 min at 4 °C. The supernatant was collected, and the amount of protein in the supernatant was determined using a BCA protein assay kit. The precipitate was then washed three times with ultrapure water by centrifugation and finally resuspended in 5 mL of ultrapure water.

[0151] (6) After freezing the nano-vaccine in a freeze dryer for 24 hours, store it at -80°C.

[0152] 2.14 PLGA Cytotoxicity Assay

[0153] The cytotoxicity of PLGA nanovaccines was assessed using 293T and ANA-1 assays. When cells reached 80-90% confluence, they were digested with trypsin, diluted with DMEM, and added to 96-well plates at 2 × 10⁵ cells per well (100 μL per well). After culturing at 37°C in a 5% CO₂ incubator for 24 h, the 96-well plates were removed, the culture medium in the wells was carefully discarded, and the plates were washed with PBS. Different final concentrations of PLGA nanovaccines (1 μg / mL, 10 μg / mL, and 100 μg / mL) were diluted with 100 μL of DMEM. A blank control group was added with 100 μL of DMEM. Incubation was performed at 37°C for 0 h, 12 h, 24 h, 36 h, and 48 h, with three parallel wells for each concentration and time point. At the corresponding time point, remove the 96-well plate, add 10 μL of CCK8, and continue to incubate at 37℃ for 1.5 h. Then, use a microplate reader to detect the absorbance of each well at a wavelength of 450 nm, record the results, and analyze the data.

[0154] 2.15 Determination of morphology, particle size and zeta potential

[0155] The prepared nanovaccine was dropped onto a silicon wafer, dried by evaporation at room temperature, sputtered with gold, and observed under a scanning electron microscope. The prepared nanovaccine was dispersed in deionized water, and the particle size and zeta potential were measured using a laser particle size analyzer.

[0156] 2.16 Load Capacity and Encapsulation Efficiency Measurement

[0157] The protein loading efficiency (LE) and encapsulation efficiency (EE) of the nanovaccine were determined using the following equations.

[0158]

[0159] Finally, SDS-PAGE gel electrophoresis was used to identify whether the proteins were encapsulated within the nanovaccine.

[0160] 2.17 Release rate determination

[0161] Nanoparticles containing 100 mg of protein were dispersed in 1 mL of 0.1 M sterile PBS and incubated at 37 °C and 120 rpm. The suspension was centrifuged at 15,000 rpm for 30 min daily, and 50 μL of the supernatant was immediately replaced with the same volume of fresh PBS. The concentrations of free rTcMUC-2 and rTcMUC-3 in the supernatant were determined using a BCA protein assay kit. All analyses were performed in triplicate.

[0162] 2.18 Immunization with vaccines

[0163] Six-week-old male C57BL / 6 mice were randomly divided into five groups: a PBS control group, a PLGA control group, and experimental groups consisting of rTcMUC-2-PLGA, rTcMUC-3-PLGA, and rTcMUC-2-PLGA+rTcMUC-3-PLGA. Twelve mice in each group received subcutaneous injections. The PLGA dose was 5 mg / mouse, the rTcMUC-2-PLGA and rTcMUC-3-PLGA doses were 50 μg / mouse, and the rTcMUC-2-PLGA+rTcMUC-3-PLGA dose was 25 μg / mouse for each protein. The immunization dose was 0.1 mL. Two booster immunizations were administered 2 weeks after the initial immunization, for a total of two immunizations.

[0164] 2.19 Insect attack and sampling

[0165] Two weeks after the second immunization, heart, liver, spleen, lung, and kidney tissues were collected for pathological examination. Subsequently, each group of mice was challenged with the parasite via gavage, with 1000 L3-stage infective eggs per mouse (dose controlled at 0.1 mL). Before challenge, the eggs were placed at 28°C to restore their viability. Blood samples were collected from mice after each immunization, challenge, and 5 days after challenge for serum separation, and stored at -80°C. Mice were euthanized 5 days after challenge by cervical dislocation, and necropsy was performed, photographed, and liver and lung tissues were collected for pathological examination and calculation of larval reduction rate.

[0166] 2.20 Evaluation Indicators

[0167] 2.20.1 Changes in routine blood tests

[0168] Two weeks after the second immunization, blood was collected from the tail tip of mice in anticoagulant tubes containing EDTA, and the mice's blood routine was tested.

[0169] 2.20.2 Comparison of lesion conditions

[0170] The liver and lung tissues and their sections of infected mice were photographed and recorded. The pathological changes of liver and lung tissues in the PBS control group, PLGA control group, rTcMUC-2-PLGA, rTcMUC-3-PLGA and rTcMUC-2-PLGA+rTcMUC-3-PLGA experimental groups were observed and compared.

[0171] 2.20.3 Antibody Level Analysis

[0172] Using the checkerboard method, rTcMUC-2 and rTcMUC-3 were serially diluted (40 μg / mL, 20 μg / mL, 10 μg / mL, 5 μg / mL, 2.5 μg / mL, 1.25 μg / mL, 0.625 μg / mL, and 0.3125 μg / mL) and added to ELISA plates, 100 μL / well, and incubated overnight at 4°C. The next day, the coating solution was discarded, and the plates were washed four times (5 min each time) with 200 μL / well of PBST. 5% skim milk powder was added, 300 μL / well, and the plates were blocked at 37°C for 2 h. Wash 4 wells with 200 μL PBST (5 min each time) by shaking. Serially dilute mouse serum positive for *Toxocara canis* and mouse negative serum with PBS (1:25, 1:50, 1:100, 1:200, 1:400, and 1:800), add 100 μL / well to each well of the ELISA plate, and incubate at 37°C for 1 h. Wash 4 wells with 200 μL PBST (5 min each time) by shaking. Add 100 μL / well of HRP-goat anti-mouse IgG diluted 1:3000, and incubate at 37°C for 1 h. Wash 4 wells with 200 μL PBST (5 min each time) by shaking. Add 100 μL of TMB substrate chromogenic solution, and incubate at room temperature in the dark for 20 min. Add 100 μL of stop solution to each well to terminate the reaction. The OD450 value is measured using an ELISA reader. The optimal antigen coating concentration and serum dilution ratio are determined based on the calculated P / N value. The P / N value calculation formula is: P / N value = Positive serum OD450 value / Negative serum OD450 value.

[0173] Select the optimal protein coating concentration, and serially dilute the immunized (0w, 2w, and 4w) mouse serum (1:3200, 1:6400, 1:12800, 1:25600, 1:51200, and 1:102400). Detect antibody titers (P / N > 2.1) according to the steps described above.

[0174] To select the optimal protein coating concentration, the serum from immunized mice (0d, 14d, and 28d) was diluted to the optimal antibody dilution concentration, and the antibody levels were detected using HRP-goat anti-mouse IgG, HRP-goat anti-mouse IgG1, and HRP-goat anti-mouse IgG2a diluted 1:3000, respectively.

[0175] 2.20.4 Flow Cytometry Analysis

[0176] Spleens or mesenteric lymph nodes were collected from mice in each group, with three replicates per group. Cells were ground in PBS, filtered through a 200-mesh filter, and centrifuged at 450×g for 5 min. The supernatant was discarded, and erythrocyte lysis buffer was added. The cells were lysed for 10 min, then centrifuged at 450×g for 5 min. Cells were resuspended in PBS and counted, yielding 1×10⁶ cells. The cells were centrifuged at 450×g for 5 min. The supernatant was discarded, and 30 μL of a 1:200 dilution of CD3+ and CD4+ antibodies was added to the cell pellet. The antibody was diluted 1:200 with PBS and incubated at 4°C in the dark for 25 min. The cells were centrifuged at 450×g for 5 min. The supernatant was discarded, and 100 μL of fixative was added to each tube. The tubes were incubated at room temperature in the dark for 1 h. 300 μL of washing buffer was added to each fixative tube, and the cells were centrifuged at 450×g for 5 min, followed by centrifugation at 8,500×g for 2 min. Add 30 μL of GATA3, T-bet, and RORγt antibodies diluted 1:200 to the cell pellet, incubate at room temperature in the dark for 1 h, centrifuge at 450 × g for 5 min, followed by centrifugation at 8,500 × g for 2 min. Discard the supernatant, add 200 μL of PBS, and filter through a 200-mesh filter into a flow cytometry tube. Detect antibody-stained cells by flow cytometry and analyze using NovoExpress.

[0177] 2.20.5 Cytokine Level Analysis

[0178] The levels of serum cytokines IFN-γ, IL-4, IL-9, IL-10, IL-12, IL-13, and IL-17 in immunized mice (0d, 14d, and 28d) were detected using the corresponding mouse cytokine EILSA kit, and the concentrations of the cytokines were calculated using a standard curve.

[0179] 2.20.6 Reduction rate of L3 stage larvae in mice

[0180] The calculation method is the same as 2.11.

[0181] 2.20.7 Data Analysis

[0182] Statistical analysis was performed using GraphPad Prism 8 software. T-tests were used to analyze the differences between the two groups of data. Significance was indicated as follows: P < 0.05 was statistically significant; ns indicated P > 0.05; * indicated P < 0.05; ** indicated P < 0.01; *** indicated P < 0.001; **** indicated P < 0.0001.

[0183] 3 Results and Analysis

[0184] 3.1 Amplification and Cloning of Tcmuc1-5 and pET-32a

[0185] After removing the signal peptide and transmembrane region of the Tcmuc1-5 gene, the gene was amplified using cDNA from *Toxocara canis* as a template, with primers containing a 19bp pET-32a homologous arm. The vector pET-32a was then linearized using reverse amplification primers. Sequencing revealed the target fragment sizes to be 515bp, 554bp, 779bp, 533bp, 920bp, and 5848bp, respectively. Figure 1 After seamless cloning and recombination, the sequencing results were consistent with expectations.

[0186] 3.2 Expression and purification of recombinant rTcMUC1-5 protein

[0187] Five plasmids of pET-32a-Tcmuc1-5 were constructed and transformed into Rosetta protein. Expression was induced for 20 h at 16 °C with 1 mM IPTG. The predicted sizes of the recombinant protein rTcMUC1-5 with thioredoxin (TrxA) and His tag were 29.4 kDa, 29.8 kDa, 39.1 kDa, 29.9 kDa, and 43.6 kDa, respectively. The expressed protein was expressed via Ni... + Column affinity chromatography purification and SDS-PAGE electrophoresis showed that, although the results were approximately 5 kDa larger than predicted, the size trends of the five proteins were consistent. Mouse anti-His tag detection revealed distinct protein bands at the same locations. Figure 2 Combined with the sequencing results of the five plasmid vectors above, it can be confirmed that the recombinant protein rTcMUC1-5 was successfully expressed and purified, which can meet the needs of subsequent experiments.

[0188] 3.3 Immunogenicity of recombinant rTcMUC1-5 protein

[0189] The purified recombinant rTcMUC1-5 protein was subjected to immunoblotting analysis with positive and negative sera from mice infected with Toxocara canis, respectively. The results showed that rTcMUC1-5 exhibited a specific immune response with positive Toxocara canis serum, but did not react with negative sera, indicating that it possesses good immunogenicity. Figure 3 ).

[0190] 3.4 Transcription levels at different stages of Tcmuc1-5

[0191] Using 18S RNA as an internal control, qPCR analysis of the expression levels of five genes (Tcmuc1-5) in eight stages of *Toxocara canis* showed that all five genes were expressed at all stages, with the highest expression levels in L3 stage eggs (EL3) and adult males. Tcmuc-2 and Tcmuc-5 also showed high expression levels in eggs and adult females. Except for Tcmuc-1, the other four genes showed very low expression levels in the intestinal L3 stage (IL3), but also relatively high expression levels in the L4 stage. Notably, Tcmuc-3 showed high expression levels in the L3 stage (LL3) of lung migration, followed by Tcmuc-2. Figure 4 ).

[0192] 3.5TcMUC1-5 Immunofluorescence Tissue Localization

[0193] Immunofluorescence histochemical localization analysis was performed on the distribution of TcMUC1-5 in the small intestine of canine adult females and in vitro. The results showed that in male adults, TcMUC1-5 was mainly distributed in the intestine and muscle, with some differences in the distribution of different proteins. All five proteins were almost entirely absent from the body wall and spermatocytes.

[0194] In female adult worms, TcMUC1-5 is mainly distributed in the intestine, muscle, and uterine wall, but its distribution in the intestinal wall of female adult worms in the canine small intestine is relatively low. Notably, TcMUC2-5 is abundantly distributed on eggs and intestinal villi within the uterus of female adult worms in the canine small intestine, but not significantly in female adult worms outside the body. Meanwhile, TcMUC-2, TcMUC-3, and TcMUC-5 are also found in small amounts in the ovary. Figures 5 to 9 ).

[0195] 3.6rTcMUC1-5 Animal Protective Test

[0196] To evaluate the immunoprotective effect of rTcMUC1-5 against Toxocara canis infection in mice, mice were subcutaneously injected with PBS, saponin, and rTcMUC1-5 + saponin, respectively. Five days after challenge, liver and lung tissues were collected to count the Toxocara canis larvae. Figure 10 (Among them, rTcMUC1-2 and rTcMUC3-5 were from different batches of mice, so blank control group and adjuvant control group were set up in the two experiments respectively. The results show that compared with the control group, the number of larvae of rTcMUC-2, rTcMUC-3 and rTcMUC-5 was significantly reduced, with reduction rates of 55.31%, 53.61% and 50.52% respectively; the immunoprotective effect of rTcMUC-1 was low, only 18.48%, while the reduction rate of rTcMUC-4 was only 1.03%, with almost no immunoprotective effect.)

[0197] 3.7 Safety of PLGA Nanoparticle Vaccines

[0198] Stimulation of human embryonic kidney cells (293T) and mouse macrophages (ANA-1) with different concentrations of PLGA nanovaccine showed that even after stimulation with the highest concentration of 100 μg / mL for 48 hours, the activity of 293T and ANA-1 cells remained unaffected. Figure 11 ).

[0199] 3.8 Coating of rTcMUC-2-PLGA and rTcMUC-3-PLGA nanovaccines

[0200] After PLGA coating of recombinant proteins rTcMUC-2 and rTcMUC-3, the nanovaccine was precipitated by high-speed centrifugation, and the content of free rTcMUC-2 and rTcMUC-3 in the supernatant was determined by BCA method. The loading capacity of rTcMUC-2-PLGA was calculated to be 2.43%, with an encapsulation efficiency of 81.54%; the loading capacity of rTcMUC-3-PLGA was 2.58%, with an encapsulation efficiency of 87.73% (Table 6).

[0201] Table 6 Loading capacity and encapsulation efficiency of rTcMUC-2-PLGA and rTcMUC-3-PLGA nanovaccines

[0202]

[0203] SDS-PAGA assays showed that both rTcMUC-2 and rTcMUC-3 were successfully encapsulated in PLGA, with encapsulation efficiency similar to that determined by the BCA method. Figure 12 ).

[0204] 3.9 Characteristics of rTcMUC-2-PLGA and rTcMUC-3-PLGA nanovaccines

[0205] Scanning electron microscopy results show that the prepared rTcMUC-2-PLGA and rTcMUC-3-PLGA nanovaccines are uniform in size, smooth in appearance, and spherical. Figure 13 ).

[0206] The rTcMUC-2-PLGA has a particle size of 107nm-283nm and a polydispersity index (PDI) of 0.037; the rTcMUC-3-PLGA has a particle size of 116nm-283nm and a PDI of 0.074. Figure 14 ).

[0207] Both rTcMUC-2-PLGA and rTcMUC-3-PLGA carry negative charges, with values ​​of -8.31 mV and -14.24 mV, respectively. Figure 15 ).

[0208] 3.10 Release rates of rTcMUC-2-PLGA and rTcMUC-3-PLGA nanovaccines

[0209] The prepared nano-vaccines were subjected to cumulative release assays to determine their in vitro protein release kinetics. After continuous release at pH 7.0 and 37°C for 14 days, approximately 81% and 75% of the antigens were released from rTcMUC-2-PLGA and rTcMUC-3-PLGA, respectively. Figure 16 ).

[0210] 3.11 Safety Assessment of PLGA Nanoparticle Vaccine

[0211] Blood routine tests were performed on mice after immunization, and the white blood cell count (WBC), red blood cell count (RBC), and platelet count (PLT) were compared. No significant changes were found in these three counts across the five groups. However, compared to the PBS and PLGA control groups, the WBC and PLT counts in the rTcMUC-2-PLGA, rTcMUC-3-PLGA, and rTcMUC-2-PLGA+rTcMUC-3-PLGA experimental groups all showed a certain increase. Figure 17 Subsequently, histopathological changes in the heart, liver, spleen, lungs, and kidneys of mice in each group were observed by HE staining. It was found that, compared with the PBS control group, no significant lesions were observed. Figure 18 ).

[0212] 3.12 Comparison of lesion conditions

[0213] 3.12.1 Liver and lung diseases

[0214] Comparison of organ lesions in the liver and lungs of mice infected with *Toxocara canis* in PBS, PLGA, rTcMUC-2-PLGA, rTcMUC-3-PLGA, and rTcMUC-2-PLGA+rTcMUC-3-PLGA groups revealed that, compared with the PBS and PLGA control groups, mice immunized with rTcMUC-2-PLGA and rTcMUC-3-PLGA showed significantly fewer hemorrhages and milder lesions in the lungs. Compared with the two groups immunized alone, the rTcMUC-2-PLGA+rTcMUC-3-PLGA mixed immunization group, although showing some reduction in hemorrhages, still exhibited more severe lesions. Compared to the lungs with clearly defined lesions, the liver showed no visible lesions. Figure 19 ).

[0215] 3.12.2 Liver and lung tissue lesions

[0216] Tissue sections of the liver and lungs of mice infected with *Toxocara canis* in PBS, PLGA, rTcMUC-2-PLGA, rTcMUC-3-PLGA, and rTcMUC-2-PLGA+rTcMUC-3-PLGA groups were stained with hematoxylin and eosin (HE). The PBS and PLGA groups showed significant liver hemorrhage, thickened bronchial and vascular walls, and the lumens were filled with large amounts of exudate, erythrocytes, and inflammatory cells; in severely affected areas, no obvious tissue structure was visible. The rTcMUC-2-PLGA and rTcMUC-3-PLGA groups showed a small amount of hemorrhage and serous exudate, but the severity of the lesions was significantly reduced compared to the control group. The rTcMUC-2-PLGA+rTcMUC-3-PLGA mixed immunization group showed no significant difference in lesions compared to the control group.

[0217] The liver showed extensive inflammatory cell infiltration, with some hemorrhage between hepatic cords and blood vessels, and a significant increase in intercellular contents in some tissues, with the hepatic cords almost completely disappearing. In contrast, mice immunized with rTcMUC-2-PLGA and rTcMUC-3-PLGA showed a significant reduction in erythrocytes and inflammatory cells, while the hepatic cord structure remained clearly defined. However, the lesions in the mixed immunization group were not significantly different from those in the control group. Figure 20 ).

[0218] Where: Scale bar = 50μm.

[0219] 3.13 Antibody Level Analysis

[0220] The antibody titers of rTcMUC-2 and rTcMUC-3 specific antibodies IgG were detected in mice in five groups (PBS, PLGA, rTcMUC-2-PLGA, rTcMUC-3-PLGA and rTcMUC-2-PLGA+rTcMUC-3-PLGA) 2 weeks after primary and secondary immunization.

[0221] Two weeks after the first immunization, the antibody titers of rTcMUC-2-PLGA-specific IgG in mice from all three experimental groups reached 1:12800, with the rTcMUC-2-PLGA group reaching 1:25600, while the mixed immunization groups had relatively lower titers, all around 1:12800. Two weeks after the second booster immunization, the highest antibody titers of rTcMUC-2-PLGA-specific IgG in all three groups increased to 1:102400 (Table 7).

[0222] Table 7. Serum-specific IgG antibody titers of rTcMUC-2 mice immunized with rTcMUC-2-PLGA and rTcMUC-3-PLGA.

[0223]

[0224] Each group has 3 replicates.

[0225] Subsequently, analysis of serum from mice infected with *Toxocara canis* revealed that rTcMUC-2-PLGA-specific antibodies IgG, IgG1, and IgG2a increased rapidly 2 weeks after the first immunization, then leveled off around the peak 2 weeks after the second immunization. Conversely, rTcMUC-2-PLGA-specific antibody IgG2a increased relatively slowly 2 weeks after the first immunization, only beginning to rise rapidly 2 weeks after the second immunization. The levels of the three specific antibodies in the rTcMUC-2-PLGA group were higher than those in the rTcMUC-3-PLGA and rTcMUC-2-PLGA+rTcMUC-3-PLGA groups. The IgG1 / IgG2a ratio showed a significant difference at 2 weeks, but decreased by 4 weeks, indicating a potential equilibrium. While the levels of all three antibodies decreased somewhat after infection with *Toxocara canis*, the ratios did not change significantly. Figure 21 ).

[0226] Two weeks after the first immunization, the rTcMUC-3-PLGA-specific antibody IgG in the experimental group mice ranged from 1:3200 to 1:12800, which was lower than the results of the rTcMUC-2-PLGA-specific antibody IgG. The rTcMUC-3-PLGA group had the highest antibody titer, reaching 1:12800. Two weeks after the second booster immunization, the highest antibody titer of rTcMUC-3-PLGA-specific antibody IgG in all three groups increased to 1:102400 (Table 8).

[0227] Table 8. Serum-specific IgG antibody titers of rTcMUC-3 mice immunized with rTcMUC-2-PLGA and rTcMUC-3-PLGA.

[0228]

[0229] Each group has 3 replicates.

[0230] The trends of specific antibodies IgG, IgG1, and IgG2a in the three experimental groups of rTcMUC-3-PLGA were not significantly different from those in rTcMUC-2-PLGA. However, compared to the rTcMUC-2-PLGA and rTcMUC-2-PLGA+rTcMUC-3-PLGA groups, the rTcMUC-3-PLGA group had higher levels of heterologous antibodies. Figure 22 As shown.

[0231] 3.14CD 4+ Cell differentiation trend

[0232] Flow cytometry analysis was performed on the spleen (SP) and mesenteric lymph nodes (ML) of five groups of mice 28 days after immunization.4+ It can differentiate into Th1, Th2, and Th17 cells, and T-bet, GATA3, and RORγt are specific transcription factors secreted by these three cell types. The three transcription factors were detected in CD4+ cells after cell rupture. 4+ The percentage of positive results in cells revealed that T-bet and RORγt transcription factors were significantly increased in the spleen of mice immunized with rTcMUC-2-PLGA, with a more than three-fold increase in positivity rate, while GATA3 transcription factor was significantly decreased. The cytokine trends in mice immunized with rTcMUC-3-PLGA were consistent with those in the rTcMUC-2-PLGA immunized group, but the growth rate of RORγt was lower than that in rTcMUC-2-PLGA. Notably, the percentage of positive results for the three transcription factors in the rTcMUC-2-PLGA + rTcMUC-3-PLGA mixed immunization group was consistent with that in the control group, showing no significant change. Figure 23 ).

[0233] In the mesenteric lymph nodes, the changes in T-bet, GATA3, and RORγt transcription factors in the rTcMUC-2-PLGA immunized mice were not significant, but the general trend was consistent with that in the spleen. In contrast, the T-bet and RORγt transcription factors in the rTcMUC-3-PLGA immunized mice were significantly increased in the mesenteric lymph nodes, while the GATA3 transcription factor was significantly decreased. In the mixed immunization group, the three transcription factors remained largely unchanged. Figure 24 This indicates that immunization with rTcMUC-2-PLGA and rTcMUC-3-PLGA alone induces Th1 and Th17 type immunity, but mixed immunization has no effect.

[0234] 3.15 Cytokine Level Analysis

[0235] The levels of cytokines in serum samples from five groups of mice before and after the insect challenge were detected. Before the challenge, compared with the PBS control group, the levels of cytokines IFN-γ, IL-12, IL-13, and IL-17 in mice immunized with rTcMUC-2-PLGA were significantly increased (P < 0.01), and the level of IL-9 was also increased (P < 0.05), while the levels of IL-4 and IL-10 were significantly decreased (P < 0.01). The trends of serum cytokine changes in mice in the PLGA group, the rTcMUC-3-PLGA group, and the rTcMUC-2-PLGA+rTcMUC-3-PLGA mixed immunization group were consistent with those in the rTcMUC-2-PLGA group, but the significance of rTcMUC-3-PLGA was slightly lower than that in the rTcMUC-2-PLGA group. In the rTcMUC-2-PLGA+rTcMUC-3-PLGA mixed immunization group, only IL-4, IL-9, and IL-10 showed significant changes (P < 0.05), while no significant changes were observed in the PLGA group. After challenge with the parasite, the cytokine levels in the rTcMUC-2-PLGA and rTcMUC-3-PLGA groups decreased slightly compared to before challenge, but the levels of IL-12, IL-13, and IL-17 in the rTcMUC-2-PLGA + rTcMUC-3-PLGA mixed immunization group showed significant changes (P < 0.05). Figure 25 ).

[0236] 3.16 Count of L3 stage larvae in mice

[0237] To further evaluate the immunization effects of rTcMUC-2-PLGA and rTcMUC-3-PLGA, liver and lung tissues from five groups were collected 5 days after infection to count L3 stage larvae. The study found that compared with the PBS control group, the number of liver and lung larvae in the rTcMUC-2-PLGA and rTcMUC-3-PLGA immunization groups alone was significantly reduced (P < 0.001), with reduction rates of 65.13% and 55.9%, respectively. In section 2.1.7, the reduction rates of rTcMUC-2 and rTcMUC-3 were 55.31% and 53.61%, respectively. It can be seen that using PLGA as a nanocarrier to coat the recombinant protein improved the reduction effect, with rTcMUC-2-PLGA even improving it by 10%. However, unfortunately, the reduction effect of rTcMUC-2-PLGA + rTcMUC-3-PLGA was only 15.38% (…). Figure 26 ).

[0238] 4 Discussion

[0239] This invention compared and analyzed the sequences of five proteins, finding that their mucin domains were all rich in Ser and Thr, but 3D prediction showed that this domain exhibited an irregular coiled shape. Furthermore, the predicted regions of the B antigen linear epitopes were concentrated in the mucin domain, rather than the shKT domain, suggesting that the shKT domain is the main domain involved in the interaction between TcMUCs and mouse macrophage proteins. Each TcMUC contains two or four shKT domains. Studies have shown that proteins containing shKT domains from parasitic nematodes can block the voltage-gated potassium 1.3 channel on human effector memory T cells. This channel has immunomodulatory activity and can regulate membrane potential and calcium. 2+ Signal transmission.

[0240] qPCR results showed that five genes were highly expressed in L3 stage eggs and adult males of *Toxocara canis*, with Tcmuc-2 and Tcmuc-5 also highly expressed in L0 stage eggs and adult females. Tcmuc-1 was highly expressed in the intestinal L3 stage, while Tcmuc-2 and Tcmuc-3 were highly expressed in the L3 stage of lung migration. It is speculated that these proteins are related to biological functions such as egg hatching, larval invasion, and migration. Subsequently, immunofluorescence histochemical localization analysis was performed on the distribution of *Toxocara canis* TcMUC1-5 in female and male adult worms in the canine small intestine and in vitro. The results showed that in male adults, TcMUC1-5 was mainly distributed in the intestine and muscle of *Toxocara canis*, with some differences in the distribution of different proteins. Meanwhile, TcMUC-2 was also abundant on the testicular wall, and was more pronounced in male adult worms in the canine small intestine. In female adult worms, TcMUC1-5 are mainly distributed in the intestine, muscle, and uterine wall, but TcMUC-5 has a lower distribution in the intestinal wall of female adult worms in the canine small intestine. Notably, TcMUC-2-5 is abundantly distributed on eggs and intestinal villi in the uterus of female adult worms in the canine small intestine, but not significantly in female adult worms outside the body. Meanwhile, TcMUC-2, TcMUC-3, and TcMUC-5 are also found in small amounts in the ovary. Based on the differences in the distribution of these five proteins inside and outside the canine small intestine, it is speculated that they play important functions in motility, lipid transport, and adaptation to the host intestinal environment. Furthermore, TcMUC-2, TcMUC-3, and TcMUC-5 also play important roles in reproduction and embryonic development.

[0241] Recombinant protein subunit vaccines are widely used in parasite vaccine research due to their simple composition and ease of mass production; however, they suffer from drawbacks such as low immunogenicity and rapid in vivo metabolism. Saponins are a class of natural plant compounds that can simultaneously stimulate Th1 and Th2 immune responses, enhancing vaccine immunogenicity. This invention successfully prepared the recombinant protein rTcMUC1-5 through cloning and prokaryotic expression. Western blotting confirmed its good immunogenicity, suggesting its potential as a candidate vaccine for canine toxocara canis infection. To screen for recombinant proteins with immunoprotective effects, a *Toxocara canis*-mouse infection model was constructed, and saponins were selected as an adjuvant for immunoprotective experiments. The protective effect against *Toxocara canis* infection in mice was preliminarily evaluated by the reduction rate of *Toxocara canis* in mouse liver and lung tissues. The results showed that mice immunized with rTcMUC-2, rTcMUC-3, and rTcMUC-5 exhibited a significant reduction in larval numbers, with reduction rates of 55.31%, 53.61%, and 50.52%, respectively. Specifically, rTcMUC-2 significantly reduced larval numbers in both the liver and lungs, while rTcMUC-3 and rTcMUC-5 also significantly reduced larval numbers in the lungs. This is likely because rTcMUC-2, rTcMUC-3, and rTcMUC-5 induce host immune protection, delaying larval migration in the liver and lung tissues. Therefore, based on the immunoprotective effects of the five proteins and combined with the preceding molecular and biological characterization analyses, rTcMUC-2 and rTcMUC-3 were selected as candidate vaccines for canine toxocaraxia.

[0242] Adjuvants offer advantages such as stimulating the innate immune system, regulating immune response patterns, reducing antigen dosage, and improving vaccine stability; however, their safety and long-term effects remain concerns. Currently, CFA+IFA and saponins are commonly used adjuvants in canine Toxocara canis vaccine research. CFA+IFA has drawbacks, including difficulty in preparation and a tendency to trigger strong local / systemic reactions and immunopathological reactions. While saponins offer relatively better safety and immunogenicity, they can still cause adverse reactions. In contrast, PLGA, as an FDA-approved safe material, possesses excellent characteristics such as biocompatibility, biodegradability, plasticity, and sustained-release properties. Its nanoparticle vaccine preparation method is simple and reproducible, demonstrating great potential in recombinant protein carrier applications.

[0243] This invention successfully prepared PLGA nanovaccines using a double emulsion-solvent evaporation method. Safety was verified using 293T cells (commonly used to verify the cytotoxicity of nanovaccines) and ANA-1 cells (used in animal protective immunization experiments). The results again confirmed that even at a high concentration (100 μg / mL) of PLGA for 48 hours, the nanovaccines had no significant effect on the activity of either 293T or ANA-1 cells. Subsequently, rTcMUC-2-PLGA and rTcMUC-3-PLGA nanovaccines were successfully prepared using this method, with encapsulation efficiencies of 81.54% and 87.73%, respectively. Scanning electron microscopy and particle size analysis showed that the nanovaccines were uniform in size, with similar particle sizes of 107 nm-283 nm and 116 nm-283 nm, respectively. Their PDI values ​​were 0.037 and 0.074, respectively, both less than 0.3, indicating uniform dispersion in solution. It is noteworthy that the particle size of the nanovaccines is closely related to their in vivo delivery pathway. Larger nanoparticles typically interact with antigen-presenting cells (APCs) surrounding tissues, while smaller nanoparticles can provide better antigen presentation through venous and lymphatic circulation. Studies have shown that particles of 200-500 nm have higher enrichment efficiency in the lungs, while particles of 50-200 nm are more easily captured by the liver, indicating that the nanovaccine prepared in this invention can effectively target the liver and lungs. The absolute value of zeta reflects the stability of nanomaterials, and usually (absolute value <20 mV) may lead to particle aggregation. Both measured values ​​were negatively charged, -8.31 mV and -14.24 mV, respectively, consistent with the results of electron microscopy, indicating some aggregation, but the measured particle size is within a suitable range and does not affect the targeting to the liver and lungs. After culturing at 37°C for 14 days, the cumulative release of rTcMUC-2 and rTcMUC-3 reached 81% and 75%, respectively. This sustained-release characteristic is beneficial for the targeted delivery of antigens through the circulatory system and concentrated release at the site of action, thereby enhancing the immune effect.

[0244] After immunizing mice with the nanovaccine, there were no significant changes in blood routine tests, and no significant changes in histopathological sections compared to the PBS group, further verifying the safety of the PLGA nanovaccine. The challenge test results showed that lung lesions were significantly reduced in mice immunized with rTcMUC-2-PLGA and rTcMUC-3-PLGA. Although liver lesions were not obvious, hemorrhage and a reduction in inflammatory cells were observed in histopathological sections. Subsequent analysis of the antibody titers of rTcMUC-2 and rTcMUC-3 specific IgG, as well as the antibody levels of IgG, IgG1, and IgG2a in the protein immunization group and the control group, revealed that in all three experimental groups, the specific IgG titers of the two proteins reached at least 1:3200 within 2 weeks after the first immunization, and almost all reached as high as 1:102400 within 2 weeks after the second immunization, indicating a certain degree of cross-protection between the two proteins. Comparing the antibody levels of IgG, IgG1, and IgG2a in the experimental and control groups, it was observed that the serum antibody levels in the experimental group mice were significantly higher than those in the PBS and PLGA control groups, and remained at a high level from the second immunization to day five after challenge. In mice, elevated IgG1 levels are generally associated with Th2-type immune responses, while elevated IgG2a levels are generally associated with Th1-type immune responses. The IgG1 / IgG2a ratio is often used to assess Th1 / Th2 balance; a high ratio indicates a dominant Th2 response, while a low ratio indicates that a dominant Th1 response is beginning to emerge. Two weeks after the first immunization, serum IgG1 levels in the experimental group mice first increased significantly and then leveled off, while the increase in IgG2a was relatively gradual, only beginning to rise significantly two weeks after the second immunization. The IgG1 / IgG2a ratio first increased and then decreased, indicating a gradual shift from a Th1-type immune response to a Th2-type response. The trends of high IgG1 levels and gradually increasing IgG2a are consistent with the trends of TES antigen-specific antibodies in Toxocara canis infection.

[0245] CD 4+ T cells are key regulators of the immune response, with their three subtypes—Th1, Th2, and Th17—playing unique roles in maintaining the balance of the immune system. Transcription factors T-bet, GATA3, and RORγt are considered specific markers of Th1, Th2, and Th17 cells, respectively. Flow cytometry was used to detect the relative levels of these three transcription factors in the spleen and mesenteric lymph nodes of five groups of mice after immunization, compared to CD4+. 4+The percentage of cells showed that the transcription factors T-bet and RORγt were significantly increased in the rTcMUC-2-PLGA and rTcMUC-3-PLGA groups, while GATA3 was significantly decreased, indicating that both cytokines induced Th1 / Th17 immune responses. To further verify this conclusion, the levels of serum cytokines IFN-γ, IL-4, IL-9, IL-10, IL-12, IL-13, and IL-17 in mice were detected using an ELISA kit. The results showed that the levels of these cytokines were significantly increased in the rTcMUC-2-PLGA and rTcMUC-3-PLGA groups, while the levels of IL-4 and IL-10 were significantly decreased. IFN-γ and IL-12 are two key factors in Th1 immune responses. Studies have shown that IL-12 can induce IFN-γ production and promote T cells, participating in cellular immunity and antigen presentation. The increase in both IL-4 and IL-13 indicates that rTcMUC-2-PLGA and rTcMUC-3-PLGA can induce humoral immunity and participate in antigen presentation on target cells. IL-4 and IL-13 are mainly produced by Th2 cells and participate in humoral immunity and tissue repair. The cytokine IL-4 forms an IL-4 / GATA3 positive feedback loop with the transcription factor GATA3, and the two complement each other. In the test results, IL-4 and GATA3 also showed a consistent decrease. However, IL-13, also a Th2 cytokine, showed an increase, and this factor is considered to play a dominant role in antiparasitic immunity. This trend may indicate the end of the early stage of Th2 cell differentiation and the entry of the immune response into a later stage, focusing on tissue repair and barrier function. IL-9, previously thought to be associated with Th2 cells, has recently been reassessed as a Th9 cell-associated factor, playing an important role in combating extracellular parasites. An increase in IL-9 can synergize with Th2 cytokines to achieve better anti-worm effects. IL-10 is an immunosuppressive factor, mainly produced by Th2 and Treg cells, and antagonizes cytokines such as IFN-γ and Th17. Its decrease indicates a preference for a pro-immune state. IL-17 is a key factor in the Th17 immune response, and the presence of its receptor IL-17RA is associated with reducing the parasite load during the acute phase of lung infections. Increased IL-17 levels can better inhibit larval migration within the body. In summary, these results indicate that immunization with rTcMUC-2-PLGA and rTcMUC-3-PLGA alone primarily induces a Th1 / Th17 immune response, accompanied by some Th2 immune response, with relatively low changes after challenge, and can sustainably maintain the body's immune homeostasis.The changes in cytokine levels in the rTcMUC-2-PLGA+rTcMUC-3-PLGA mixed immunization group were lower than those in the two individual immunization groups, but a certain increase was observed five days after challenge. This may be because the concentration of individual antigens in the rTcMUC-2 and rTcMUC-3 mixed immunization is insufficient, and multiple antigens compete for the immune system's attention, resulting in ineffective stimulation of cytokine production. Meanwhile, B cells can be activated through a T cell-independent pathway, leading to high antibody levels but minimal changes in cytokines.

[0246] Combined with animal protection experiments, it was found that compared with the PBS and PLGA control groups, the number of L3 stage Toxocara canis larvae in the liver and lungs of mice immunized with rTcMUC-2-PLGA and rTcMUC-3-PLGA was significantly reduced. Compared with the 55.31% and 53.61% reduction rates of the rTcMUC-2 and rTcMUC-3 immunization groups using saponins as adjuvants, the PLGA nanocarrier-coated rTcMUC-2 and rTcMUC-3 reduced the number of immunizations from three to two, with reduction rates of 65.13% and 55.9%, respectively. This result confirms that PLGA nanovaccines can act as adjuvants to recombinant proteins, reducing the immunization cycle and inducing better host immune protection to inhibit the invasion and migration of Toxocara canis larvae. Unfortunately, the protective effect of the rTcMUC-2-PLGA + rTcMUC-3-PLGA mixed immunization group was not significant, similar to the results for transcription factors and cytokines. In summary, both PLGA-coated rTcMUC-2 and rTcMUC-3 can induce better immune protection, but rTcMUC-2-PLGA has a better immune protection effect on mice.

[0247] Therefore, those skilled in the art will recognize that although embodiments of the present invention have been shown and described in detail herein, many other variations or modifications conforming to the principles of the present invention can be directly determined or derived from the disclosure of the present invention without departing from the spirit and scope of the invention. Therefore, the scope of the present invention should be understood and recognized as covering all such other variations or modifications.

Claims

1. A canine toxocara canis rTcMUCs-PLGA nanovaccine, characterized by, PLGA nanospheres were used as carriers to load either TcMUC-2 or TcMUC-3 recombinant proteins. The TcMUC-2 recombinant protein contained the following amino acid sequence: MNVRVVILLTVLISVVKPQPGAQTTTTAATTTTAAATTTTAAATTTTAAATTTTAAATTTTAAPMTTTAGATTTAAGATTTAAGATTTAGGPTTTAAGAITTAAGATTTAAVMTTTPACIDTANDCQLFMPLCFVQPYSRAIQGRCRRTCNICSCQDSANDCANFVSVCLNPTYQPVLRSRCALTCGFC; The TcMUC-3 recombinant protein contains the following amino acid sequence: MNACACALLLLFIGVVRHQSIFAAATMMTTSAPCVDSASDCQQHTSLCFMQPYSRSMQSRCQRTCNICNCRDDANDCARLVTFCGNPMYQPVLRTRCTLTCGFCSNDTLATTVAGTTTTPAPTTTTAAATTTTAA ATTTAAPTTTTAAPTTTTATPTTTTAAPTTTTAAPTTTTAAPTTTTAAPTTTTGAIVTTTAACSDAAMDCQRYAGMCFTQPYSRAIQGRCRRTCNICNCHDSANNCGSLISYCDDPTLQPVLRSRCPLSCGFCS; The nanovaccine is loaded with 2-3 wt% recombinant protein, and the average size of the nanovaccine is 100-300 nm.

2. The nanovaccine according to claim 1, wherein the average size of the nanovaccine is 150-250 nm.

3. A method for preparing the nanovaccine according to any one of claims 1-2, comprising the following steps: (1) Recombinant proteins rTcMUC-2 and / or rTcMUC-3 were dissolved in PBS solution to form an inner aqueous phase; PLGA was dissolved in dichloromethane to form an organic phase; (2) The aqueous phase is slowly dripped into the organic phase while vortexing to mix, and then ultrasonically mixed in ice water to form a water-in-oil (w / o) emulsion; (3) Transfer the w / o emulsion to an aqueous solution containing 1-3 wt% PVA and sonicate again to obtain a w / o / w emulsion; (4) Add the prepared w / o / w emulsion to an aqueous solution containing 0.2-0.4wt% PVA and evaporate the organic solvent by magnetic stirring at room temperature; (5) The nano vaccine solution was centrifuged, then the precipitate was washed three times with ultrapure water by centrifugation, and finally resuspended with ultrapure water. (6) Freeze the nano-vaccine in a freeze dryer.

4. The use of the nano-vaccine according to any one of claims 1-3 in the preparation of a canine Toxocara canis vaccine.

5. The application according to claim 4, wherein the vaccine is used to reduce the number of Toxocara canis in the liver and lungs.

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