Application of lycium barbarum polysaccharide in preparing vaccine adjuvant

Abstract

The application provides application of Chinese wolfberry polysaccharide or L-arabinose in preparation of a vaccine adjuvant, and belongs to the technical field of immunoregulation. A Chinese wolfberry polysaccharide is separated from Chinese wolfberry, and research finds that the Chinese wolfberry polysaccharide is a Trichinella spiralis CPF1 gene recombinant plant lactobacillus vaccine immune promoter; the Chinese wolfberry polysaccharide and single component L-arabinose thereof as an adjuvant combined with a rTs-CPF1 vaccine can significantly improve the secretion of CD4 + IL-4 + and CD4 + IFN-gamma + specific antibody levels in intestinal lavage fluid and specific IgG antibody levels in serum; the Chinese wolfberry polysaccharide and single component L-arabinose thereof as anadjuvant combined with a rTs-CPF1 vaccine improve the worm reduction rate in the adult stage and muscle larva stage and reduce the pathological damage degree of the intestine, lingual muscle and masseter muscle.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and in particular relates to the application of Lycium barbarum polysaccharide and L-arabinose in the preparation of vaccine adjuvants. Background Technology

[0002] The statements in this section are merely background information related to the present invention and do not necessarily constitute prior art.

[0003] Trichinosis is a zoonotic parasitic disease that poses a significant threat to human health. People are primarily infected through consuming raw or undercooked meat containing Trichinella spiralis (Ts) cysts. Trichinosis is an important animal infectious disease with a substantial impact on agriculture, animal husbandry, and aquaculture.

[0004] Current treatments for trichinosis include albendazole and mebendazole, but traditional chemotherapy typically targets only the adult stage of the trichinella worm, failing to effectively prevent secondary infections. Furthermore, long-term use can lead to drug resistance, drug residues, and low bioavailability. Vaccination has proven to be an effective and safe method for preventing trichinosis. The trichinella worm development process includes the adult stage, the new larval migration stage, and the myolarval stage. However, most current vaccine research targets only one stage of the trichinella worm, failing to provide complete immune protection. In the development of trichinella vaccines, in addition to developing and screening different antigens, selecting the optimal adjuvant to enhance the vaccine's specificity and protective efficacy is also an effective way to improve vaccine efficacy.

[0005] Studies have shown that polysaccharides from traditional Chinese medicine (TCM) can play a promoting role in vaccines as adjuvants. Furthermore, TCM polysaccharides are low-toxicity, biodegradable compounds. Several natural polysaccharides from TCM herbs, including Poria cocos polysaccharide, ginseng polysaccharide, lentinan, and Astragalus membranaceus polysaccharide, have already been used as adjuvants in vaccines. However, research on the use of Lycium barbarum polysaccharide as a vaccine adjuvant is limited.

[0006] Lycium barbarum polysaccharide (LBP) is a water-soluble complex containing sugar chains and proteins. The main sugar components are arabinose, galactose, glucose, rhamnose, mannose, and xylose, accounting for approximately 70% of the total sugars. Studies have demonstrated the important role of L-arabinose (L-Ara) in the field of functional sugars, but research on its use as a vaccine adjuvant is limited. Summary of the Invention

[0007] In this invention, cathepsin F-like protease 1 (CPF1), which can be expressed in the excretory-secretory products (ESP) of Trichinella spiralis at different developmental stages, was selected as the antigen gene to construct a recombinant Lactobacillus plantarum vaccine based on the Trichinella spiralis CPF1 gene. This invention also discovered that Lycium barbarum polysaccharides can act as an immunostimulant, enhancing the immune efficacy of the recombinant Lactobacillus plantarum vaccine based on the Trichinella spiralis CPF1 gene. Furthermore, it was discovered for the first time that L-Ara, the monosaccharide component with the highest content in Lycium barbarum polysaccharides, can enhance the immune efficacy of the recombinant Lactobacillus plantarum vaccine based on the Trichinella spiralis CPF1 gene.

[0008] To achieve the above objectives, one or more embodiments of the present invention provide the following technical solutions:

[0009] A first aspect of the present invention provides the use of Lycium barbarum polysaccharide in the preparation of vaccine adjuvants.

[0010] Preferably, the vaccine is a recombinant Lactobacillus plantarum vaccine containing the Trichinella spiralis CPF1 gene.

[0011] Preferably, the application includes protection against Trichinella infection by combining Lycium barbarum polysaccharide with a recombinant Lactobacillus plantarum (rTs-CPF1) vaccine containing the Trichinella spiralis CPF1 gene.

[0012] Preferably, the application includes inducing a Th1 / Th2 mixed immune response in the body; improving pathological damage to the intestine, tongue muscles, and masseter muscles; and reducing the parasite burden.

[0013] Preferably, the wolfberry polysaccharide is obtained by alcohol precipitation.

[0014] Preferably, the preparation method of the wolfberry polysaccharide includes the following steps:

[0015] 1) Dry the goji berries and grind them into powder;

[0016] 2) Mix the goji berries from step 1) with water to make a homogenate;

[0017] 3) After homogenizing the slurry in step 2), centrifuge it in a water bath and collect the supernatant;

[0018] 4) Take the supernatant and mix it with anhydrous ethanol. After standing, centrifuge to collect the precipitate. Dissolve the precipitate with water to obtain crude wolfberry polysaccharide solution.

[0019] Preferably, in step 2), the mass ratio of goji berries to water is 3-8:100.

[0020] Preferably, in step 3), the water bath conditions are: extraction in a 100℃ water bath for 1.5-2.5 hours.

[0021] Preferably, in step 4), the ratio of the supernatant to anhydrous ethanol is 1:4; and the standing is carried out overnight at 4°C.

[0022] A second aspect of the invention provides the use of L-Ara in the preparation of a vaccine adjuvant; said vaccine is a recombinant Lactobacillus plantarum vaccine containing the Trichinella spiralis CPF1 gene.

[0023] The above one or more technical solutions have the following beneficial effects:

[0024] Lycium barbarum polysaccharides or L-Ara play an auxiliary role in improving the body's immune response as adjuvants. As immune enhancers, they can not only enhance immunogenicity and improve the body's innate and acquired immunity, but also save on antigen dosage or reduce the number of vaccinations, thereby reducing vaccine costs.

[0025] The combination of Lycium barbarum polysaccharide and LBP vaccine induced a Th1 / Th2 mixed immune response in the body, enhancing the levels of specific sIgA, total IgG, IgG1, and IgG2a antibodies. The combination of LBP and Trichinella spiralis CPF1 gene recombinant Lactobacillus plantarum vaccine induced cellular, humoral, and mucosal immune responses in mice, enhancing the protective efficacy of the vaccine against Trichinella spiralis infection. After Trichinella spiralis infection, the LBP combination vaccine improved the pathological damage to the intestines, tongue muscles, and masseter muscles, enhancing the immunoprotective effect of the Trichinella spiralis vaccine. The adult worm reduction rate reached 47.31%, and the larval worm reduction rate reached 68.88%, which was superior to the LBP-immunized group or the vaccine-immunized group alone.

[0026] L-Ara combined with the vaccine promoted the production of specific IgG in the serum and specific sIgA in the intestinal lavage fluid of immunized mice. It also increased the expression of IL-4 and IFN-γ in the spleen and mesenteric lymph nodes (MLN), inducing humoral immunity, cellular immunity, and mucosal immunity. L-Ara, as an immune enhancer, has a certain anti-Trichinella spiralis effect. Combined with the Trichinella spiralis CPF1 gene recombinant Lactobacillus plantarum vaccine, it improved the immunoprotective effect of the recombinant Lactobacillus plantarum vaccine. The L-Ara combined with CPF1 gene recombinant Lactobacillus plantarum vaccine group showed a significant reduction in the worm load of adult mice and muscle larvae, with worm reduction rates of 32.19% and 36.58%, respectively. Attached Figure Description

[0027] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0028] Figure 1 This is the PCR amplification result of the TsCPF1 gene in Example 2 of the present invention.

[0029] Figure 2 This is a schematic diagram of the construction pattern of the pValac-TsCPF1 recombinant plasmid in Example 2 of the present invention.

[0030] Figure 3 This is the result of double enzyme digestion identification of the pValac-TsCPF1 recombinant plasmid in Example 2 of the present invention.

[0031] Figure 4 This is a schematic diagram of the immunization procedure and experimental design in Embodiment 3 of the present invention.

[0032] Figure 5 This is an example of the changes in mouse body weight after immunization in Example 3 of the present invention.

[0033] Figure 6 This describes the changes in mouse body weight after parasite attack in Example 3 of the present invention.

[0034] Figure 7 Example 3 of the present invention: Intestinal adult worm load

[0035] Figure 8 This invention relates to the three muscle larvae load of embodiment three.

[0036] Figure 9 This invention presents the mRNA transcription levels of IL-4 and IFN-γ in the spleen at different time points following immunization in Example 3. (A) Transcription level of IL-4 in the spleen on day 37 post-immunization; (B) Transcription level of IFN-γ in the spleen on day 37 post-immunization; (C) Transcription level of IL-4 on days 44 and 79 post-immunization; (D) Transcription level of IFN-γ on days 44 and 79 post-immunization.

[0037] Figure 10 In Embodiment 3 of the present invention, CD4 in the spleen after three immunizations + IL-4 + T cells and CD4 + IFN-γ + Number of T cells

[0038] Figure 11 CD4 in MLN after three-stage exemption in Embodiment 3 of the present invention + IL-4 + T cells and CD4 + IFN-γ + Number of T cells

[0039] Figure 12 CD4 in the spleen during the adult stage of the present invention, as described in Embodiment 3 of the present invention. + IL-4 + T cells and CD4 + IFN-γ + Number of T cells

[0040] Figure 13 CD4 in adult MLN of Embodiment 3 of the present invention + IL-4 + T cells and CD4 + IFN-γ + Number of T cells

[0041] Figure 14 This invention relates to CD4 in the spleen during the larval stage of the third muscle larvae. + IL-4 + T cells and CD4 + IFN-γ + Number of T cells

[0042] Figure 15 CD4 in MLN during the larval stage of the third muscle of this invention. + IL-4 + T cells and CD4 + IFN-γ + Number of T cells

[0043] Figure 16 The levels of IL-4 and IFN-γ in mouse serum in Example 3 of this invention.

[0044] Figure 17 The level of specific sIgA in the intestinal lavage fluid of mice in Example 3 of this invention.

[0045] Figure 18 The specific IgG, IgG1, and IgG2a levels in mouse serum in Example 3 of this invention.

[0046] Figure 19 This is an example of the histopathological changes in the intestinal tract of adult mice in Example 3 of the present invention.

[0047] Figure 20 This is a description of the pathological and histological changes of the tongue muscle in mice during the larval stage in Example 3 of the present invention.

[0048] Figure 21 This describes the pathological and histological changes of the masseter muscle in mice during the larval stage in Example 3 of the present invention.

[0049] Figure 22 This is an example of the colonization of *Lactobacillus plantarum* in the gut after three immunizations according to Embodiment 3 of the present invention.

[0050] Figure 23 This is a schematic diagram of the immunization procedure and experimental design in Embodiment 4 of the present invention.

[0051] Figure 24 This is an example of the changes in mouse body weight after immunization in Example 4 of the present invention.

[0052] Figure 25 This is a description of the changes in mouse body weight after parasite attack in Example 4 of the present invention.

[0053] Figure 26 Example 4 of the present invention: Intestinal adult worm load

[0054] Figure 27 The four-muscle larvae load of this invention embodiment

[0055] Figure 28 This invention presents the mRNA transcription levels of IL-4 and IFN-γ in the spleen at different time points following infection with Trichinella spiralis in Example 4 of this invention. (A) Transcription level of IL-4 in the spleen on day 37 post-immunization; (B) Transcription level of IFN-γ in the spleen on day 37 post-immunization; (C) Transcription level of IL-4 on days 44 and 79 post-immunization; (D) Transcription level of IFN-γ on days 44 and 79 post-immunization.

[0056] Figure 29 In Embodiment 4 of the present invention, CD4 in the spleen after immunization + IL-4 + T cells and CD4 + IFN-γ + Number of T cells

[0057] Figure 30 In Embodiment 4 of the present invention, CD4 in MLN after three-stage removal + IL-4 + T cells and CD4 + IFN-γ + Number of T cells

[0058] Figure 31 This invention relates to CD4+ in the spleen during the adult stage of worms, as described in Example 4. + IL-4 + T cells and CD4 + IFN-γ + Number of T cells

[0059] Figure 32 CD4 in adult MLN of Embodiment 4 of the present invention + IL-4 + T cells and CD4 + IFN-γ + Number of T cells

[0060] Figure 33 This invention relates to CD4 in the spleen during the larval stage of the fourth muscle worm. + IL-4 + T cells and CD4 + IFN-γ + Number of T cells

[0061] Figure 34 CD4 in MLN during the larval stage of the four-muscle larvae in this embodiment of the invention. + IL-4+ T cells and CD4 + IFN-γ + Number of T cells

[0062] Figure 35 The levels of IL-4 and IFN-γ in mouse serum in Example 4 of this invention.

[0063] Figure 36 The level of specific sIgA in the intestinal lavage fluid of mice in Example 4 of this invention.

[0064] Figure 37 The specific IgG, IgG1, and IgG2a levels in mouse serum in Example 4 of this invention.

[0065] Figure 38 This is an example of the histopathological changes in the intestinal tract of adult mice in Example 4 of the present invention.

[0066] Figure 39 This is a histopathological change of the tongue muscle in the mouse muscle larva stage in Example 4 of the present invention.

[0067] Figure 40 This is a description of the pathological and histological changes of the masseter muscle in mice during the larval stage in Example 4 of the present invention.

[0068] Figure 41 This is an example of the colonization of *Lactobacillus plantarum* in the gut after immunization in Embodiment 4 of the present invention. Detailed Implementation

[0069] 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.

[0070] It should be noted that the terminology used herein is for the purpose of describing particular implementations only and is not intended to limit the exemplary implementations of the present invention.

[0071] Where there is no conflict, the embodiments and features in the embodiments of the present invention can be combined with each other.

[0072] Main reagents and antibodies used in this invention

[0073] Table 1-1 Reagents and Antibodies

[0074]

[0075] Main instruments, equipment and consumables used in this invention

[0076] Table 1-2 Instruments and Equipment

[0077]

[0078]

[0079] Main reagents and preparation methods used in this invention

[0080] (1) MRS liquid culture medium: 10g beef powder, 10g peptone, 10g glucose, 5g yeast powder, 5g sodium acetate, 2g triamine citrate, 2g dipotassium hydrogen phosphate, 0.2g anhydrous magnesium sulfate, 0.05g manganese sulfate monohydrate, 1mL Tween-80, add ddH2O and mix thoroughly, bring the volume to 1L, autoclave at 115℃ for 20 minutes, and store at room temperature.

[0081] (2) Erythromycin (20mg / mL): 1g of erythromycin is thoroughly mixed with ddH2O, and the volume is adjusted to 50mL. After dissolving, it is sterilized by a 0.22μm filter, dispensed into 500μl tubes, and stored at -20℃.

[0082] (3) SppIP-induced peptide (20 mg / mL): The synthesized SppIP-induced peptide was prepared into a liquid of 20 mg / mL, then sterilized with a 0.22 μm filter, dispensed into 50 μl tubes, and stored at -20 °C. It should not be repeatedly frozen and thawed before use.

[0083] (4) 0.05M Coating Buffer (CBS, pH 9.6): Na2CO3 1.59g, NaHCO3 2.93g, mix thoroughly with ddH2O, bring to a final volume of 1L, and store at room temperature.

[0084] (5) Washing solution PBST (pH 7.0-7.2): Add 1 mL Tween-20 to 2 L of 0.01 M PBS, store at room temperature, and prepare fresh before use.

[0085] (6) Termination solution (2M H2SO4): Take 178.3mL of ddH2O, add 21.7mL of concentrated sulfuric acid (analytical grade), mix thoroughly, and store in a brown bottle at room temperature away from light.

[0086] This invention uses FlowJo V10 software to analyze flow cytometry results.

[0087] This invention uses GraphPad Prism software for graphing and statistical analysis. Data processing employs one-way ANOVA with at least 5 independent trials and statistically significant differences (extremely significant: ***, P<0.001; significant: **, P<0.01; *, P<0.05).

[0088] Example 1: Preparation of Lycium barbarum polysaccharides

[0089] 1.1 Extraction of Lycium barbarum polysaccharides by water extraction and alcohol precipitation

[0090] (1) Drying: Weigh 100g of wolfberry sample and dry it in an oven at 80℃ for 4h;

[0091] (2) Homogenization: The dried goji berries were ground into powder using a high-speed blender and mixed thoroughly at a ratio of 0.05g goji berries to 1mL ddH2O.

[0092] (3) Water bath: The homogenized liquid is extracted in a 100℃ water bath for 2 hours (the lid must be tightly closed to prevent moisture loss);

[0093] (4) Centrifugation: After cooling the liquid in the water bath to room temperature, centrifuge at 10,000g for 10 minutes and collect the supernatant;

[0094] (5) Alcohol precipitation: Mix the supernatant with anhydrous ethanol at a ratio of 1:4, and then let it stand overnight at 4°C.

[0095] (6) Dissolution: Centrifuge 10,000g of the overnight liquid for 10min, collect the precipitate, and dissolve the precipitate with ddH2O to obtain crude wolfberry polysaccharide solution.

[0096] 1.2 Determination of Lycium barbarum polysaccharides by phenol-sulfuric acid method

[0097] (1) 5% phenol solution: Distill an appropriate amount of analytical grade phenol at high temperature, collect the fraction extracted at 180℃, weigh 5.0g, dissolve in ddH2O, make up to 100mL, store in a brown bottle, and store in the dark and refrigerated.

[0098] (2) Glucose standard solution (200mg / L): Accurately weigh 20.0mg of glucose at constant weight at 110℃, dissolve it with ddH2O, and finally dilute it to 100mL. Accurately pipette 2.0, 4.0, 6.0, 8.0 and 10.0mL of liquid into 100mL volumetric flasks, dilute to the mark, mix well and obtain glucose dilutions of different concentrations.

[0099] (3) Standard curve: Accurately weigh 2 mL of each of the above-mentioned dilution solutions and place them in stoppered test tubes. Add 1 mL of 5% phenol solution (prepared fresh for use) to each tube, mix well, and then quickly add 5.0 mL of concentrated sulfuric acid. Then place the tubes in a 40℃ water bath for 30 min, and then place them in a cold water bath for 5 min. Using the blank solution prepared by 2.0 mL of ddH2O as a reference, measure the absorbance of each solution at a wavelength of 490 nm. Plot the standard curve with the concentration of glucose dilution solution (μg / mL) as the x-axis and the absorbance as the y-axis.

[0100] (4) Sample determination: Take 2.0 mL of the obtained crude polysaccharide solution (which can be diluted to a certain ratio) and place it in a test tube. Add 1.0 mL of 5% phenol solution and quickly add 5.0 mL of concentrated sulfuric acid. Perform three replicates for each sample. After mixing evenly, place it in a 40℃ water bath for 30 min, then place it in a cold water bath for 5 min. Zero the absorbance at 490 nm using a blank and then substitute it into the standard curve to calculate the total content of Lycium barbarum polysaccharides.

[0101] Example 2: Preparation of a recombinant Lactobacillus plantarum vaccine based on the Trichinella spiralis CPF1 gene

[0102] The recombinant Lactobacillus plantarum vaccine using the Trichinella spiralis CPF1 gene used in this invention was previously constructed in our laboratory. For details, please refer to the article: Xue Y, Zhang B, Huang HB, et al. Immunoprotective effects of invasive Lactobacillus plantarum delivered nucleic acid vaccine coexpressing Trichinella spiralis CPF1 and murine interleukin-4. Vet Parasitol. 2021; 298:109556. The specific steps are briefly described below:

[0103] 1 Experimental Methods

[0104] 1.1 Amplification of the Trichinella spiralis CPF1 gene

[0105] The GeneBank number for CPF1 is XM-003378197.1, and its nucleotide sequence is shown in SEQ ID NO.1, while its amino acid sequence is shown in SEQ ID NO.2. Mice infected with Trichinella spiralis for 35 days were sacrificed, and Trichinella spiralis muscle larvae were collected. Total RNA was extracted from the muscle larvae using the Trizol method and then reverse transcribed into cDNA. Specific primers F: 5'-GG were designed. GGTACC ATGGTCAGCGTGAAGTGCAC-3' (SEQ ID NO.3), R: 5'-GC TCTAGA GATGACGACGACGGAGGAAGCCA-3' (SEQ ID NO.4) was used to amplify the Trichinella spiralis CPF1 gene by PCR; GGTACC for KpnI restriction site TCTAGA is XbaI restriction site; the target gene was obtained, gel-cleaved and ligated into the PMD-18T cloning vector to construct the PMD-18T-TsCPF1 plasmid and transformed into DH5α competent cells. After double enzyme digestion identification and sequencing confirmation, it was cultured and amplified in large quantities, and the plasmid was extracted for later use.

[0106] 1.2 Construction of recombinant plasmid pValac-TsCPF1

[0107] The PMD-18T-TsCPF1 plasmid and the pValac-GFP plasmid were digested with restriction endonucleases KpnI and XbaI, respectively. The TsCPF1 and pValac gene fragments were recovered by gel extraction using a DNA gel extraction kit, and the two gene fragments were ligated using T4 ligase to construct the pValac-TsCPF1 plasmid. The plasmid was then transformed into E. coli TG1, and after double enzyme digestion and sequencing confirmation, it was cultured and amplified. The plasmid was then extracted and stored for later use.

[0108] 1.3 Construction of Recombinant Lactobacillus plantarum with Trichinella spiralis CPF1 gene

[0109] The plasmid pValac-TsCPF1 was electroporated into *Lactobacillus plantarum* NC8 to construct a recombinant *Lactobacillus plantarum* strain containing the *Trichinella spiralis* CPF1 gene. The recombinant bacteria were cultured in MRS medium containing 10 μg / mL erythromycin and chloramphenicol for positive screening. After 6 hours of culture, the plasmid was extracted for PCR verification and sequencing identification. Successfully transformed and correctly identified *Trichinella spiralis* CPF1 gene recombinant *Lactobacillus plantarum* strains were cultured at 30°C for 3 hours in MRS medium containing 10 μg / mL erythromycin and chloramphenicol. SppIP inducible peptide was added, and the culture continued for 24 hours to induce the expression of the target gene CPF1. The recombinant *Lactobacillus plantarum* strains were collected, counted, and preserved for subsequent experiments.

[0110] 2 Experimental Results

[0111] 2.1 Successful amplification of the Trichinella spiralis CPF1 gene

[0112] The results are as follows Figure 1 As shown, the full-length TsCPF1 gene obtained by PCR amplification is 1095 bp.

[0113] 2.2 Successful construction of pValac-TsCPF1 recombinant plasmid

[0114] The pValac-TsCPF1 recombinant plasmid map is shown below. Figure 2 As shown; the results of double enzyme digestion identification of the recombinant plasmid after ligation are as follows. Figure 3 As shown, clear bands were visible at 3758bp and 1095bp, and the recombinant plasmid sequencing alignment was 100% consistent with the gene sequence. These results indicate that the pValac-TsCPF1 recombinant plasmid was successfully constructed in this experiment. The recombinant plasmid was extracted and electroporated into Lactobacillus plantarum NC8, and after the plasmid was correctly sequenced, it was induced to express and used for subsequent immunization experiments.

[0115] Example 3: Immunization of mice with LBP combined with rTs-CPF1 vaccine

[0116] 3.1 Grouping and Immunization Protocol of Experimental Animals

[0117] Six-week-old female (specific pathogen-free, SPF grade) BALB / c mice were purchased from Beijing Huafukang Biotechnology Co., Ltd. All purchased mice were housed at the SPF-grade Laboratory Animal Center of Jilin Agricultural University, using autoclaved water, bedding, and a maintenance diet. The mice had free access to food and water and were allowed to acclimatize to the animal barrier environment for one week before the experiments were conducted. The animal experiments complied with the relevant provisions of the "Regulations on the Protection of Laboratory Animals in China" and the charter of the "Jilin Agricultural University Laboratory Animal Welfare and Ethics Committee" (2019-11-12 001).

[0118] 125 BALB / c mice were randomly divided into 5 groups: negative control (PBS), positive control Trichinella spiralis infection (Ts), immunization with Lycium barbarum polysaccharide alone (LBP), immunization with Trichinella spiralis CPF1 gene recombinant Lactobacillus plantarum vaccine alone (rTs-CPF1), and immunization with Trichinella spiralis CPF1 gene recombinant Lactobacillus plantarum vaccine combined with Lycium barbarum polysaccharide (rTs-CPF1+LBP), with 25 mice in each group. Each mouse in the LBP group was orally administered Lycium barbarum polysaccharide for 37 consecutive days; each mouse in the rTs-CPF1 group was orally administered 1.0 × 10⁻⁶ LBP. 9 CFU live bacteria were administered orally for three consecutive days after each immunization, with a 10-day interval between each immunization. Mice in the rTs-CPF1+LBP group were immunized three times with recombinant Lactobacillus plantarum vaccine and simultaneously orally administered Lycium barbarum polysaccharide for 37 consecutive days. Mice in the PBS and Ts groups were orally administered PBS solution for 37 consecutive days. Ten days after the third immunization, mice in each group (except the PBS control group) were orally infected with 350 Trichinella spiralis muscle larvae (ML). Adult worms and muscle larvae were collected and counted on days 7 and 42 post-infection. Pathological histological methods were used to examine the intestinal lesions of mice in each group on day 7 post-infection and the number of cysts in the tongue and masseter muscles of mice in each group on day 42 post-infection. The immunization groups and dosages are shown in Table 1, and the immunization schedule is as follows. Figure 4 As shown.

[0119] Table 1 Grouping of experimental animals

[0120]

[0121] 3.2 Effects of LBP combined with rTs-CPF1 vaccine on mouse body weight

[0122] The results are as follows Figure 5As shown, throughout the immunization cycle, the weight trends of mice in the LBP, rTs-CPF1, and rTs-CPF1+LBP groups were consistent with those in the PBS group, exhibiting a steady increase. Subsequently, to detect weight changes in mice after Trichinella infection, the weight of mice was recorded from 0 to 42 days post-infection, and the results are as follows: Figure 6 As shown, compared with the PBS group from day 0 to day 7, the body weight of mice in the LBP, rTs-CPF1, and rTs-CPF1+LBP groups showed a decreasing trend; compared with the PBS group from day 28 to day 35, the body weight of mice in the rTs-CPF1 and rTs-CPF1+LBP groups showed a decreasing trend, and the body weight trends of mice in each group at other times were consistent with those of the PBS group, showing a steady increase.

[0123] 3.3 Adult Trichinella spiralis load during intestinal stage

[0124] To evaluate the protective effect of LBP combined with rTs-CPF1 vaccine against Trichinella spiralis infection, we isolated and counted adult trichinella worms in the intestinal stage of mice in each group. The specific procedures are as follows:

[0125] (1) On the 7th day after infection with Trichinella spiralis, mice in each group (n=5) were sacrificed, and their small intestines were collected. The intestines were then dissected longitudinally and washed with water to remove the intestinal contents. (2) The cleaned small intestines were suspended in a 0.9% NaCl solution containing double the amount of antibiotics and incubated at 37°C for 2 hours. (3) The intestines were removed, and after standing for 30 minutes, the supernatant was aspirated with a syringe. The intestines were repeatedly washed with a 0.9% NaCl solution containing double the amount of antibiotics until they settled. The worms were then collected and counted under a microscope. All experimental materials contaminated with Trichinella spiralis required high-temperature and high-pressure treatment.

[0126] Adult worm reduction rate = (Number of Trichinella infection group members - Number of immune group members) / Number of Trichinella infection group members × 100%

[0127] The results are shown in Table 2 and Figure 7 As shown, LBP, rTs-CPF1, and rTs-CPF1+LBP groups all reduced the adult trichinella infection load. The rTs-CPF1+LBP group had a higher adult reduction rate (47.31%), which was higher than that of the rTs-CPF1 group. This indicates that LBP and rTs-CPF1 vaccines can synergistically inhibit the growth and reproduction of adult trichinella, suggesting that LBP can serve as an adjuvant to the rTs-CPF1 vaccine to enhance its immunogenicity.

[0128] Table 2. Reduction rate of adult worms in the intestinal stage

[0129]

[0130] 3.4 Trichinella spiralis muscle larvae load

[0131] Mice in each group (n=5) were sacrificed on day 42 post-Trichinella infection. Muscle larvae were collected from the muscles of the mice, and the larval reduction rate was calculated to evaluate the immunomodulatory effect of LBP on the rTs-CPF1 vaccine. The specific procedures are as follows:

[0132] (1) Infected mice were euthanized by dislocation of the neck, and the muscle tissue was minced using a meat grinder. Then, each mouse meat sample was placed in 300 mL of artificial gastric juice (containing 1% concentrated hydrochloric acid and 1% pepsin) preheated at 37°C and digested for 3 hours using a magnetic stirrer.

[0133] (2) After complete digestion, filter the liquid through a 100-mesh sieve, let it stand for 30 minutes, then remove the supernatant with a syringe and add water. Wash repeatedly until the solution is clear, collect the muscle larvae in the precipitate and count them under a microscope. All experimental items contaminated with Trichinella spiralis need to be treated with high temperature and high pressure.

[0134] (3) The reduction rate of muscle larvae is calculated based on the number of muscle larvae per gram of muscle (LPG) = (LPG of muscle larvae in the Trichinella infection group - LPG of muscle larvae in the immune group) / LPG of muscle larvae in the Trichinella infection group × 100%.

[0135] The results are shown in Table 3 and Figure 8 As shown, LBP, rTs-CPF1, and rTs-CPF1+LBP groups all reduced the number of muscle larvae after Trichinella spiralis infection. The rTs-CPF1 and rTs-CPF1+LBP groups had higher muscle larvae reduction rates, reaching 35.35% and 68.88%, respectively, with the rTs-CPF1+LBP group showing significantly higher rates than the rTs-CPF1 group. This indicates that LBP can synergistically inhibit the growth and reproduction of Trichinella spiralis muscle larvae with the rTs-CPF1 vaccine, suggesting that LBP can serve as an adjuvant to the rTs-CPF1 vaccine to enhance its immunogenicity.

[0136] Table 3. Insect reduction rate during the larval stage.

[0137]

[0138] Effects of 3.5LBP combined with rTs-CPF1 vaccine on IL-4 and IFN-γ transcriptional levels in mouse spleen

[0139] (1) Total RNA was extracted from the spleens of mice (n=5) in each group using the Trizol method and then reverse transcribed.

[0140] (2) The reverse transcription system is as follows. After adding the corresponding reagents, it is placed in a water bath at 37°C for 3 hours.

[0141]

[0142] (3) The RT-PCR experiment was performed using an Applied Biosystems 7500 Real-Time PCR System to detect the mRNA transcription levels of IL-4 and IFN-γ. The specific conditions were as follows: pre-denaturation: 95℃, 2 min; denaturation: 95℃, 15 s; extension: 60℃, 15-30 s; repeat denaturation and extension for 40 cycles.

[0143] The primer sequences are as follows:

[0144]

[0145]

[0146] The test results after three immunizations are as follows Figure 9 As shown, compared with the PBS control group, the LBP, rTs-CPF1, and rTs-CPF1+LBP groups all increased the mRNA transcription levels of IL-4 and IFN-γ in the spleen; compared with the rTs-CPF1 group, the levels of IL-4 and IFN-γ in the rTs-CPF1+LBP group were significantly increased.

[0147] The results of IL-4 and IFN-γ mRNA transcription levels during the adult stage (day 44 of the cycle) and the myolarval stage (day 79 of the cycle) after Trichinella infection are as follows: Figure 9 As shown, compared with the Ts control group, the rTs-CPF1+LBP group significantly increased the mRNA transcription levels of IL-4 and IFN-γ in the spleen on days 44 and 79; compared with the rTs-CPF1 group, the rTs-CPF1+LBP group significantly increased the mRNA transcription levels of IL-4 and IFN-γ in the spleen on day 44 and significantly increased the mRNA transcription levels of IL-4 and IFN-γ on day 79, indicating that LBP can promote the immune effect of the rTs-CPF1 vaccine.

[0148] 3.6 Effects of LBP combined with rTs-CPF1 vaccine on the expression levels of IL-4 and IFN-γ in the spleen and mesenteric lymph nodes of mice

[0149] Mice in each group (n=5) were euthanized by cervical dislocation, and spleens and MLNs were removed. Spleen cells and MLN cells were obtained by grinding, centrifugation, washing, and lysis. Cells were then counted, incubated with surface antibodies, fixed, and stained with intracellular cytokine antibodies. The cell suspension was then analyzed by flow cytometry.

[0150] The expression levels of IL-4 and IFN-γ cytokines in the spleen and MLN of mice in each group were detected. After three immunizations, the results were as follows: Figure 10 , Figure 11As shown, compared with the PBS control group, the levels of IL-4 in the spleen and MLN were significantly increased in the rTs-CPF1+LBP group, and the level of IFN-γ in the MLN was significantly increased; compared with the rTs-CPF1 group, the level of IL-4 in the MLN was significantly increased in the rTs-CPF1+LBP group.

[0151] In the adult stage following Trichinella infection, compared with the Ts control group, the rTs-CPF1+LBP group showed significantly elevated levels of IL-4 and IFN-γ in the spleen. The difference in IL-4 levels was statistically significant, and the difference in IFN-γ levels was extremely significant. The results in the MLN were consistent with the trend in the spleen. Compared with the rTs-CPF1 group, the rTs-CPF1+LBP group showed an increasing trend in IL-4 levels in the spleen, but the difference was not statistically significant. IFN-γ levels were significantly elevated. The results in the MLN were consistent with the trend in the spleen. (See attached figures). Figure 12 , Figure 13 .

[0152] During the muscle larval stage following Trichinella infection, compared with the Ts control group, the rTs-CPF1+LBP group showed significantly elevated levels of IL-4 and IFN-γ in the spleen, and significantly elevated levels of both IL-4 and IFN-γ in the MLN. Compared with the rTs-CPF1 group, there was no significant difference in IL-4 and IFN-γ levels in the spleen of the rTs-CPF1+LBP group. In the MLN, IL-4 levels showed an increasing trend but the difference was not significant, while IFN-γ levels were significantly elevated. (See attached figures). Figure 14 , Figure 15 .

[0153] 3.7 Effects of LBP combined with rTs-CPF1 vaccine on serum IL-4 and IFN-γ expression levels in mice

[0154] On days 0, 13, 25, 37, 44, and 79 post-immunization, blood was collected from the eyeballs of mice in each group (n=5), and the mice were euthanized by cervical dislocation. Blood samples were incubated at 37°C for 1 hour, then at 4°C for 4 hours, followed by centrifugation at 1500 rpm for 10 minutes. Serum was collected, and the levels of IFN-γ and IL-4 in the serum of each group of mice at different time points were detected using an ELISA kit. Specific procedures are detailed in the instruction manual. Results are as follows: Figure 16After the first immunization (day 13 post-immunization), compared with the Ts control group, the levels of IL-4 and IFN-γ in the rTs-CPF1+LBP group were significantly increased; compared with the rTs-CPF1 group, the level of IL-4 in the rTs-CPF1+LBP group was slightly increased but the difference was not significant, while the level of IFN-γ was significantly increased. After the second immunization (day 25 post-immunization), compared with the Ts control group, the levels of IL-4 and IFN-γ in the rTs-CPF1+LBP group were significantly increased; compared with the rTs-CPF1 group, the levels of IL-4 and IFN-γ in the rTs-CPF1+LBP group were slightly increased but the difference was not significant. After the third immunization (day 37 post-immunization), compared with the Ts control group, the levels of IL-4 and IFN-γ in the rTs-CPF1+LBP group were significantly increased; compared with the rTs-CPF1 group, the level of IL-4 in the rTs-CPF1+LBP group was significantly increased, while the level of IFN-γ was slightly increased but the difference was not significant.

[0155] During the adult stage (day 44 post-immunization), compared with the Ts control group, the levels of IL-4 and IFN-γ in the rTs-CPF1+LBP group were significantly increased; compared with the rTs-CPF1 group, the level of IL-4 in the rTs-CPF1+LBP group was significantly increased, and the level of IFN-γ was significantly increased. During the muscle larval stage (day 79 post-immunization), compared with the Ts control group, the levels of IL-4 and IFN-γ in the rTs-CPF1+LBP group were significantly increased; compared with the rTs-CPF1 group, the level of IL-4 in the rTs-CPF1+LBP group was significantly increased, while the level of IFN-γ did not differ significantly.

[0156] In this invention, mice were immunized with LBP combined with a recombinant Lactobacillus plantarum vaccine containing the Trichinella spiralis CPF1 gene. The levels of IL-4 and IFN-γ in the spleen and MLN increased, indicating that a Th1 / Th2 mixed response was induced. LBP can be used as an adjuvant for Trichinella spiralis vaccine to enhance the immune response.

[0157] 3.8 Effect of LBP combined with rTs-CPF1 vaccine on the level of specific sIgA antibody in mouse intestinal lavage fluid

[0158] On days 0, 37, 44, and 79 post-immunization, intestinal lavage fluid was collected from each group of mice (n=5). Specifically, 500 μL of PBS solution containing 1% PMSF was added to every 10 cm of small intestine. One end of the small intestine was held with forceps, and the intestine was gently flushed with a syringe to avoid puncturing it. The intestinal lavage fluid was then collected for specific sIgA detection. In this invention, ELISA was used to detect sIgA, and the results are as follows: Figure 17After three immunizations, there were no significant differences in antibody levels among the groups. However, on days 44 and 79, compared to the Ts control group, the level of specific sIgA antibodies in the rTs-CPF1+LBP group was significantly higher, with a highly significant difference on day 44 and a significant difference on day 79. Compared to the rTs-CPF1 group, the level of specific antibody sIgA in the rTs-CPF1+LBP group was slightly higher, but the difference was not significant. This indicates that the recombinant Lactobacillus plantarum vaccine containing the Trichinella spiralis CPF1 gene can effectively induce mucosal immunity in mice.

[0159] 3.9 Effects of LBP combined with rTs-CPF1 vaccine on the levels of specific IgG, IgG1, and IgG2a antibodies in mouse serum

[0160] The serum samples obtained in step 3.7 were subjected to specific IgG, IgG1 and IgG2a antibody detection. The serum samples to be tested were diluted at a ratio of 1:50.

[0161] The results are as follows Figure 18 As shown, compared with the Ts control group, the level of specific IgG antibodies in the rTs-CPF1+LBP group was significantly increased after the first immunization (day 13 post-immunization) and the second immunization (day 25 post-immunization). Compared with the rTs-CPF1 group, the level of specific IgG antibodies in the rTs-CPF1+LBP group was increased, but the difference was not significant. After the third immunization (day 37 post-immunization), the adult stage (day 44 post-immunization), and the muscle larva stage (day 79 post-immunization), the level of specific IgG antibodies in the rTs-CPF1+LBP group was significantly increased compared with the Ts control group. Compared with the rTs-CPF1 group, the level of specific IgG antibodies in the rTs-CPF1+LBP group was significantly increased. The increasing trend of IgG1 and IgG2a antibody levels was basically consistent with that of IgG antibodies.

[0162] This indicates that immunization of mice with LBP combined with the recombinant Lactobacillus plantarum vaccine containing the Trichinella spiralis CPF1 gene can effectively induce a humoral immune response.

[0163] 3.10 Effects of LBP combined with rTs-CPF1 vaccine on pathological damage to the intestine, tongue muscle, and masseter muscle in mice.

[0164] Duodenal tissue samples were collected from mice in each group (n=5) on day 44 post-immunization (adult stage), and tongue and masseter muscle tissue samples were collected from mice in each group (n=5) on day 79 (muscle larva stage). Paraffin sections were prepared, H&E stained, and observed under a microscope.

[0165] The pathological damage to the intestines of mice in different groups during the adult stage of Trichinella spiralis infection is as follows: Figure 19As shown, in the Ts control group, the duodenum had a large number of inflammatory cells in the intestinal mucosa, some epithelial cells showed edema, the intestinal villi were significantly shortened, and local defects appeared, showing vacuolar changes; the duodenal pathological damage in the LBP group was similar to that in the Ts group mice; the duodenal pathological damage in the rTs-CPF1 group was alleviated to a certain extent, and the defects and atrophy of intestinal villi were also alleviated, and the number of inflammatory cells in the mucosa was reduced; the duodenal pathological damage in the rTs-CPF1+LBP group was significantly improved, and the histological appearance of the duodenum in the PBS group mice was close to that observed by the naked eye.

[0166] On day 79 post-immunization, pathological damage to the tongue and masseter muscles of mice in each group was observed 42 days after Trichinella infection. Figure 20 and Figure 21 As shown, in the Ts control group mice, muscle fibers in the tongue and masseter muscles showed degeneration, sarcoplasm dissolution, and myofibril cysts of varying sizes and numbers within the muscle fibers, surrounded by significant inflammatory cell aggregation. Compared to the Ts control group, the number of myofibril cysts in the tongue and masseter muscles of the rTs-CPF1 and rTs-CPF1+LBP groups was significantly reduced, with the rTs-CPF1+LBP group exhibiting milder inflammatory cell infiltration in the tongue and masseter muscles. This indicates that the LBP and rTs-CPF1 vaccines can synergistically inhibit the growth and reproduction of adult Trichinella spiralis and myofibril larvae, improving intestinal and muscle damage.

[0167] 3.11 Immunofluorescence detection of rTs-CPF1 recombinant Lactobacillus plantarum colonization in the intestine

[0168] After three immunizations, colon samples were collected from each group of mice (n=5) for immunofluorescence staining. Paraffin sections were prepared as in step 3.10, dewaxed, and then subjected to antigen retrieval, washing, and permeabilization. Afterward, the sections were blocked, incubated with antibodies, and stained with DAPI. After washing, the sections were mounted with an anti-fluorescence attenuator and observed under a fluorescence microscope in the dark.

[0169] The results are as follows Figure 22 As shown, the cell nucleus was stained blue by DAPI, and the recombinant Lactobacillus plantarum appeared green. No green recombinant Lactobacillus plantarum was found in the PBS, Ts and LBP groups. Recombinant Lactobacillus plantarum could be clearly found in the rTs-CPF1 and rTs-CPF1+LBP groups, but the difference between the two groups was not significant.

[0170] The above results indicate that LBP combined with the Trichinella spiralis CPF1 gene recombinant Lactobacillus plantarum vaccine induces cellular, humoral, and mucosal immune responses in mice, enhancing the vaccine's protective efficacy against Trichinella spiralis infection.

[0171] Example 4: Immunization of mice with L-Ara combined with rTs-CPF1 vaccine

[0172] 4.1 Grouping and Immunization Protocol of Experimental Animals

[0173] Six-week-old female BALB / c mice were used as experimental animals. 125 mice were randomly divided into five groups: negative control (PBS), positive control (Trichinella spiralis infection (Ts), L-Ara immunization alone, Trichinella spiralis CPF1 gene recombinant Lactobacillus plantarum vaccine immunization alone (rTs-CPF1), and Trichinella spiralis CPF1 gene recombinant Lactobacillus plantarum vaccine combined with L-Ara immunization (rTs-CPF1+L-Ara), with 25 mice in each group. After purchase, the mice were housed normally in an SPF-grade barrier environment for 7 days before the experiment. Each mouse in the L-Ara group was orally administered L-Ara for 37 consecutive days; each mouse in the rTs-CPF1 group was orally administered 1.0 × 10⁻⁶ L-Ara. 9 CFU live bacteria were administered orally for three consecutive days after each immunization, with a 10-day interval between each immunization. Mice in the rTs-CPF1+L-Ara group received three immunizations with recombinant Lactobacillus plantarum vaccine and were orally administered L-Ara for 37 consecutive days. Mice in the PBS and Ts groups received PBS solution orally for 37 consecutive days. Ten days after the third immunization, mice in each group (except the PBS control group) were orally infected with 350 Trichinella spiralis muscle larvae (ML). Adult worms and muscle larvae were collected and counted on days 7 and 42 post-infection. Histopathological methods were used to detect intestinal pathological changes in each group on day 7 post-infection and the number of cysts in the tongue and masseter muscles of each group on day 42 post-infection. The immunization groups and dosages are shown in Table 4, and the immunization schedule is as follows. Figure 23 As shown.

[0174] Table 4 Grouping of experimental animals

[0175]

[0176] 4.2 Effect of L-Ara combined with rTs-CPF1 vaccine on mouse body weight

[0177] The results are as follows Figure 24 and Figure 25As shown in the figure, throughout the immunization cycle, the mice in each group showed a steady growth trend. The weight trends of the L-Ara, rTs-CPF1, and rTs-CPF1+L-Ara groups were consistent with those of the PBS group, showing a steady increase. Subsequently, to detect the weight changes of the mice in each group after Trichinella infection, the weight of the mice was recorded from 0 to 42 days post-infection. The results showed that from 0 to 7 days, compared with the PBS group, the weight of the L-Ara, rTs-CPF1, and rTs-CPF1+L-Ara groups showed a decreasing trend; from 21 to 28 days, compared with the PBS group, the weight of the rTs-CPF1 and rTs-CPF1+L-Ara groups showed a decreasing trend; at other times, the weight trends of the mice in each group were consistent with those of the PBS group, showing a steady increase.

[0178] 4.3 Adult Trichinella spiralis load during the intestinal stage

[0179] The method is the same as step 3.3.

[0180] The results are shown in Table 5 and Figure 26 As shown, the L-Ara, rTs-CPF1, and rTs-CPF1+L-Ara groups all reduced the body load of adult Trichinella spiralis after infection, and the rTs-CPF1+L-Ara group had a higher adult reduction rate (32.19%).

[0181] Table 5. Intestinal worm reduction rate

[0182]

[0183] 4.4 Trichinella spiralis muscle larvae load

[0184] The method is the same as step 3.4.

[0185] The results are shown in Table 6 and Figure 27 As shown, the insect reduction rate of the rTs-CPF1 group was 39.32%, but the insect reduction rate of the rTs-CPF1+L-Ara group was not significantly different from that of the rTs-CPF1 group, and was 36.58%.

[0186] Table 6. Reduction rate of muscle larvae

[0187]

[0188] Effects of 4.5L-Ara combined with rTs-CPF1 vaccine on IL-4 and IFN-γ transcriptional levels in mouse spleen

[0189] The method is as shown in step 3.5.

[0190] After three immunizations were completed, the test results were as follows: Figure 28As shown, compared with the PBS control group, both the rTs-CPF1 and rTs-CPF1+L-Ara groups significantly increased the mRNA transcription levels of IL-4 and IFN-γ in the spleen; compared with the rTs-CPF1 group, there was no significant difference in the transcription levels of IL-4 and IFN-γ in the rTs-CPF1+L-Ara group.

[0191] The results of IL-4 and IFN-γ mRNA transcription levels during the adult stage (day 44 of the cycle) and the myolarval stage (day 79 of the cycle) after Trichinella infection are as follows: Figure 28 As shown, compared with the Ts control group, the rTs-CPF1+L-Ara group significantly increased the mRNA transcription levels of IL-4 and IFN-γ in the spleen on day 44. On day 79, the mRNA transcription level of IL-4 in the spleen increased slightly but the difference was not significant, while the mRNA transcription level of IFN-γ was significantly increased. Compared with the rTs-CPF1 group, the rTs-CPF1+L-Ara group significantly increased the mRNA transcription level of IL-4 in the spleen on day 44, while the mRNA transcription level of IFN-γ showed a significant decreasing trend. On day 79, the mRNA transcription level of IL-4 in the spleen showed a significant decreasing trend, while the mRNA transcription level of IFN-γ was significantly increased.

[0192] Effects of 4.6L-Ara combined with rTs-CPF1 vaccine on the expression levels of IL-4 and IFN-γ in the spleen and mesenteric lymph nodes of mice.

[0193] The method is as shown in step 3.6.

[0194] After three immunizations, the levels of IL-4 and IFN-γ cytokines in the spleen and MLN of mice in each group (n=5) were detected by flow cytometry. Results are as follows: Figure 29 and Figure 30 As shown, compared with the PBS control group, the levels of IL-4 and IFN-γ in the MLN were significantly increased in the rTs-CPF1+L-Ara group; there was no significant difference in the levels of IL-4 and IFN-γ in the MLN between the rTs-CPF1+L-Ara group and the rTs-CPF1 group.

[0195] In the adult stage following Trichinella infection, compared with the Ts control group, the rTs-CPF1+L-Ara group showed significantly increased IL-4 levels and extremely significantly increased IFN-γ levels in the spleen, and extremely significantly increased IL-4 and IFN-γ levels in the MLN. Compared with the rTs-CPF1 group, the rTs-CPF1+L-Ara group showed a decreasing trend in IL-4 levels in the spleen, and a slight increase in IFN-γ levels, but the difference was not significant. In the MLN, IL-4 levels showed a significant decreasing trend, while IFN-γ levels showed no significant difference. The results are as follows. Figure 31 and Figure 32 As shown.

[0196] During the muscle larval stage following Trichinella infection, compared with the Ts control group, the levels of IL-4 and IFN-γ in the spleen and MLN were significantly increased in the rTs-CPF1+L-Ara group; however, there were no significant differences in the levels of IL-4 and IFN-γ in the spleen and MLN between the rTs-CPF1 group and the rTs-CPF1+L-Ara group. Figure 33 and 34 As shown.

[0197] 4.7 Effects of L-Ara combined with rTs-CPF1 vaccine on serum IL-4 and IFN-γ expression levels in mice

[0198] The method is as shown in step 3.7.

[0199] This invention detected the expression levels of IL-4 and IFN-γ in the serum of mice in each group (n=5) at different time points. The detection results are as follows: Figure 35 As shown, after the first immunization (day 13 post-immunization), compared with the Ts control group, the level of IL-4 in the rTs-CPF1+L-Ara group was increased, but the difference was not significant, while the level of IFN-γ was significantly increased; compared with the rTs-CPF1 group, the level of IL-4 in the rTs-CPF1+L-Ara group was increased, but the difference was not significant, while the level of IFN-γ was significantly increased.

[0200] After the second immunization (day 25 post-immunization), compared with the Ts control group, the level of IL-4 in the rTs-CPF1+L-Ara group was significantly increased, while the level of IFN-γ was not significantly different; compared with the rTs-CPF1 group, the levels of IL-4 and IFN-γ in the rTs-CPF1+L-Ara group were not significantly different.

[0201] After the third immunization (day 37 post-immunization), compared with the Ts control group, the level of IL-4 in the rTs-CPF1+L-Ara group was not significantly different, while the level of IFN-γ was significantly increased; compared with the rTs-CPF1 group, there was no significant difference in the levels of IL-4 and IFN-γ in the rTs-CPF1+L-Ara group.

[0202] During the adult stage (day 44 post-immunization), the levels of IL-4 and IFN-γ in the rTs-CPF1+L-Ara group were not significantly different compared to the Ts control group and the rTs-CPF1 group. During the muscle larval stage (day 79 post-immunization), compared to the Ts control group, the IL-4 level in the rTs-CPF1+L-Ara group was not significantly different, but the IFN-γ level was significantly increased; compared to the rTs-CPF1 group, the levels of IL-4 and IFN-γ in the rTs-CPF1+L-Ara group were not significantly different.

[0203] 4.8 Effect of L-Ara combined with rTs-CPF1 vaccine on the level of specific sIgA antibody in mouse intestinal lavage fluid

[0204] The method is as shown in step 3.8.

[0205] In this invention, the levels of specific sIgA antibodies in the intestinal lavage fluid of mice in each group (n=5) were detected at different time points (0, 37, 44, and 79 days post-immunization). The results are as follows: Figure 36 As shown, after the third immunization, the rTs-CPF1+L-Ara group showed a significant increase compared to the Ts control group; there was no significant difference between the rTs-CPF1 group and the rTs-CPF1+L-Ara group; and there were no significant differences in the adult and myolar stages after infection with Trichinella spiralis.

[0206] 4.9 Effects of L-Ara combined with rTs-CPF1 vaccine on the levels of specific IgG, IgG1 and IgG2a antibodies in mouse serum

[0207] The method is as shown in step 3.9.

[0208] The results are as follows Figure 37 As shown, after the first immunization (day 13 post-immunization), compared with the Ts control group, the specific IgG antibody level in the rTs-CPF1+L-Ara group was significantly increased; compared with the rTs-CPF1 group, the specific IgG antibody level in the rTs-CPF1+L-Ara group was not significantly different. After the second immunization (day 25 post-immunization), compared with the Ts control group, the specific IgG antibody level in the rTs-CPF1+L-Ara group was significantly increased; compared with the rTs-CPF1 group, the specific IgG antibody level in the rTs-CPF1+L-Ara group was not significantly different. After the third immunization (day 37 post-immunization), compared with the Ts control group, the specific IgG antibody level in the rTs-CPF1+L-Ara group was significantly increased; compared with the rTs-CPF1 group, the specific IgG antibody level in the rTs-CPF1+L-Ara group was not significantly different. During the adult stage (day 44 post-immunization), compared with the Ts control group, the rTs-CPF1+L-Ara group showed a significantly higher level of specific IgG antibodies; however, there was no significant difference in specific IgG antibody levels between the rTs-CPF1 group and the rTs-CPF1+L-Ara group. During the muscle larval stage (day 79 post-immunization), compared with the Ts control group, the rTs-CPF1+L-Ara group showed a highly significantly higher level of specific IgG antibodies; however, there was no significant difference in specific IgG antibody levels between the rTs-CPF1 group and the rTs-CPF1+L-Ara group. The increasing trends of IgG1 and IgG2a antibody levels were basically consistent with those of IgG antibodies.

[0209] 4.10 Effects of L-Ara combined with rTs-CPF1 vaccine on pathological damage to the intestine, tongue muscle, and masseter muscle in mice.

[0210] The method is as shown in step 3.10.

[0211] The pathological damage in the intestines of mice in different groups infected with Trichinella spiralis during the adult stage was as follows: Figure 38 As shown, the Ts control group showed an increase in inflammatory cells in the duodenal mucosa, significant shortening and partial absence of intestinal villi, exhibiting vacuolar changes; the degree of duodenal pathological damage in the L-Ara group was similar to that in the Ts group mice; the duodenal pathological damage in the rTs-CPF1 group and the rTs-CPF1+L-Ara group was slightly improved, with reduced villi defects and atrophy, and a decrease in the number of inflammatory cells in the mucosa. The histological features of the duodenum in the rTs-CPF1 group and the rTs-CPF1+L-Ara group mice were similar upon visual inspection.

[0212] The pathological damage to the tongue and masseter muscles of mice in each group was observed 42 days after Trichinella infection. The results are as follows: Figure 39 and Figure 40 As shown, in the Ts control group mice, inflammatory cells accumulated in the tongue and masseter muscles, sarcoplasmic resorption occurred, muscle fibers degenerated, and muscle larvae of varying sizes and numbers were present. Compared with the Ts infection group, the number of muscle larvae cysts in the tongue and masseter muscles of the rTs-CPF1 and rTs-CPF1+L-Ara groups was significantly reduced, and the degree of cellular inflammation was milder. Histopathological results showed that the rTs-CPF1+L-Ara group showed the most significant improvement in the pathological damage after Trichinella spiralis infection, indicating that L-Ara can enhance the immune effect of the rTs-CPF1 vaccine.

[0213] 4.11 Immunofluorescence detection of the colonization of rTs-CPF1 recombinant Lactobacillus plantarum in the intestine

[0214] The method is as shown in step 3.11.

[0215] After the third immunization was completed, the results were as follows: Figure 41 As shown, the cell nucleus was stained blue by DAPI, and the recombinant Lactobacillus plantarum appeared green. No green recombinant Lactobacillus plantarum was found in the PBS, Ts, and L-Ara groups. Recombinant Lactobacillus plantarum could be clearly found in the rTs-CPF1 and rTs-CPF1+L-Ara groups, but the difference between the two groups was not significant.

[0216] Following infection with Trichinella spiralis, the main immune responses produced by the host include antibody-mediated humoral immunity, mucosal immunity, cellular immunity, cytokine action, and intestinal mucosal immune responses. The results of this invention indicate that mice in the L-Ara combined with the CPF1 gene recombinant Lactobacillus plantarum vaccine group showed increased expression of IL-4 and IFN-γ in the spleen and MLN, inducing a mixed Th1 / Th2 immune response after Trichinella spiralis infection. The L-Ara combined with the CPF1 gene recombinant Lactobacillus plantarum vaccine significantly increased the levels of specific IgG antibodies in mouse serum and specific sIgA antibodies in intestinal lavage fluid, exerting an anti-Trichinella spiralis effect by inducing humoral and mucosal immunity.

[0217] After infection with Trichinella spiralis, the worm load in adult mice in the L-Ara combined with CPF1 recombinant Lactobacillus plantarum vaccine group was significantly reduced, with a worm reduction rate of 32.19%, which was better than the L-Ara group or the vaccine group alone. This indicates that the L-Ara combined with CPF1 recombinant Lactobacillus plantarum vaccine can stimulate the body to produce a stronger immune response and exert an immune protective effect.

[0218] While the specific embodiments of the present invention have been described above in conjunction with the accompanying drawings, this is not intended to limit the scope of protection of the present invention. Those skilled in the art should understand that various modifications or variations that can be made by those skilled in the art without creative effort based on the technical solutions of the present invention are still within the scope of protection of the present invention.

Claims

1. Application of L-arabinose in the preparation of vaccine adjuvants; the vaccine is a recombinant Lactobacillus plantarum r of the Trichinella spiralis CPF1 gene. Ts -CPF1 vaccine.

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