Recombinant lactococcus lactis oral vaccine, its preparation method and application

Abstract

The application discloses a recombinant lactococcus lactis oral vaccine, a preparation method and application thereof, characterized in that the recombinant lactococcus lactis expresses TcCTL-2 and / or TcCTL-5. The oral vaccine can not only effectively induce a mouse to produce a high-efficiency intestinal mucosal immune response, but also can stimulate the body to produce cellular immunity. The recombinant lactococcus lactis expressing TcCTL-2 and TcCTL-5 has the best protection effect on the mouse, and can be used as a preferred oral vaccine strain for preventing canine toxocariasis.

Description

Technical Field

[0001] This invention relates to the field of molecular immunology, specifically to a recombinant lactococcal oral vaccine, 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] When *Toxocara canis* invades and colonizes the intestines, it not only damages the host's intestinal epithelial barrier but also induces inflammation, ulcers, or intestinal obstruction, seriously endangering the life and health of humans and animals. Studies have confirmed that *Toxocara canis* larvae release large amounts of ES antigens. Type C lectin, one of the most abundant ES products secreted during the intestinal invasion larval stage, participates in nutrient acquisition, growth and reproduction, and the parasite-host immune interaction, and mediates immune evasion, thus possessing important biological functions. However, current research on the function of *Toxocara canis* type C lectin remains relatively scarce. Summary of the Invention

[0005] Based on the above reasons, this invention proposes a recombinant lactococcus oral vaccine and its preparation method. Specifically, to achieve the objectives of this invention, the following technical solution is proposed:

[0006] This invention relates, in one aspect, to a recombinant Lactococcus lactis oral vaccine, characterized in that the recombinant Lactococcus lactis expresses TcCTL-2 and / or TcCTL-5, wherein,

[0007] The amino acid sequence of TcCTL-2 is as follows:

[0008] MFAIFIICLCFHFSTIDACARDADCNLFQVCVNNVCVANNQGCNPPCVAPMVCVAPNCVAVPAPQPQPATTTTAAPAATTTAAPAATTTTARRTCPPSWSLFNNNCYIASVAGRFLF NQASDWCTQTGSRVVWFDQSNAANFNSELAFVNNLAISGGSSRYWIGVNRQFGQWVWTNGSPVILSNWRPSQPDGCCGSNVTCVFVNYANFLGQWDDASCGGLFTNPQGFVCKRPL;

[0009] The amino acid sequence of TcCTL-5 is as follows:

[0010] MIAFCLLLTLAISVANANRCDPGWRYSPFTRKCYRFYDHETMWPSAEFSCLFKGGHLISIHSYTDNRFAIELARGAETVWLGNAQFGSSKEYIWSDHTAYNYGSWPDRKRPEKIKTKPCTKLNTTSGEWFQSCCKDPAPYICQKELSDSNAMYRNSEELLGHTSEDRRLESNEDFRRRFFL.

[0011] In a preferred embodiment of the present invention, the recombinant lactococcus simultaneously expresses TcCTL-2 and TcCTL-5.

[0012] Another aspect of the present invention relates to a method for preparing recombinant lactococcus, comprising the following steps:

[0013] Recombinant plasmids loaded with TcCTL-2 and / or TcCTL-5 nucleotide sequences were transfected into Lactococcus lactis.

[0014] In a preferred embodiment of the present invention, the preparation method includes the following steps:

[0015] Pre-cool the electroporation cuvette on ice and irradiate it with UV light in a clean bench. Remove the NZ3900 competent cells from the freezer and thaw them on ice. Add the recombinant plasmid to the thawed competent cells, gently pipette to mix, and then place in an ice bath. Transfer the mixed bacterial solution to the bottom of the electroporation cuvette, remove air bubbles, and wipe the outer wall of the cuvette dry. Set the electroporation parameters to 2200V, 200Ω, 25μF, and 2mm diameter cuvette. After setting the parameters, proceed with the electroporation. Electroporation; after the electroporator completes the electroporation, immediately add pre-cooled GM17-MC medium to the electroporation cup, gently aspirate 3-5 times to thoroughly mix the bacterial suspension with the medium, and transfer the mixture to a pre-cooled sterile centrifuge tube, place it on ice, and then transfer it to a 30°C incubator for static incubation for 2-3 hours; remove the bacterial suspension, centrifuge, discard the supernatant, retain approximately the bacterial suspension, resuspend it, and then evenly spread the bacterial suspension on the surface of Elliker solid medium, incubate at 30°C upside down for 24 hours, and screen for positive strains.

[0016] In a preferred embodiment of the present invention, the recombinant plasmid is prepared by the following steps: homologous recombination of the target genes expressing TcCTL-2 and TcCTL-5 with the pNZ8149 plasmid.

[0017] Another aspect of the present invention relates to the use of the above-mentioned recombinant lactococcus in the preparation of Toxocara canis vaccine.

[0018] In a preferred embodiment of the invention, the vaccine is used to reduce the number of Toxocara canis in the liver and lungs.

[0019] The oral vaccine of this invention can not only effectively induce mice to produce a high-efficiency intestinal mucosal immune response, but also stimulate the body to produce cellular immunity. The recombinant lactococcus expressing TcCTL-2 and TcCTL-5 has the best protective effect on mice and can be used as a preferred oral vaccine strain for the prevention of canine toxocariasis.

[0020] The beneficial effects of this invention are as follows:

[0021] This invention discloses a recombinant Lactococcus lactis oral vaccine, its preparation method, and its application. The key feature is that the recombinant Lactococcus lactis expresses TcCTL-2 and / or TcCTL-5. This oral vaccine not only effectively induces a high-efficiency intestinal mucosal immune response in mice but also stimulates cellular immunity. The recombinant Lactococcus lactis expressing both TcCTL-2 and TcCTL-5 exhibits the best protective effect in mice and can be considered a preferred oral vaccine strain for the prevention of canine toxocara disease. Attached Figure Description

[0022] Figure 1PCR amplification of the Tcctl-2 and Tcctl-5 genes, where A represents the PCR amplification result of Tcctl-2; B represents the PCR amplification result of Tcctl-5; M represents the DNA molecular weight standard; and 1 represents the amplified product of the target gene.

[0023] Figure 2 PCR amplification of pET32a(+) vector, where M is the DNA molecular weight standard; 1 is the pET32a(+) vector amplification product;

[0024] Figure 3 Bioinformatics analysis for Tcctl-2, including: A, signal peptide prediction; B, transmembrane region prediction; C, amino acid multiple sequence alignment. The genera were *Toxocara canis* (Genbank accession number: KHN74601.1); *Toxocara catica* (Genbank accession number: MH183028.1); *Draconis medina* (Genbank accession number: VDN60627.1); and *Anisakis simplex* (Genbank accession number: UYRR01036511.1). The depth of the blue background indicates the degree of sequence conservation; the darker the color, the more conserved the sequence.

[0025] Figure 4 Bioinformatics analysis for Tcctl-5, including: A, signal peptide prediction; B, transmembrane region prediction; C, amino acid multiple sequence alignment. The genera included: *Toxocara canis* (Genbank accession number: KHN74601.1); *Nematodeus worm* (Genbank accession number: VDD85501.1); *Filarial worm* (Genbank accession number: VDK89318.1); *Nematodeus fasciatus* (Genbank accession number: VDN60627.1); and *Brucella malayi* (Genbank accession number: KAK6107812.1). The depth of the blue background indicates the degree of sequence conservation; the darker the color, the more conserved the sequence.

[0026] Figure 5 For the expression and purification of rTcCTL-2, M represents the protein molecular weight standard; 1 represents the expression results of pET-32a empty vector; lanes 2-3 represent the expression results of rTcCTL-2.

[0027] Figure 6 For rTcCTL-5 expression and purification, where M represents the protein molecular weight standard; lanes 1-2 show the pET-32a empty vector expression results; lanes 3-4 show the rTcCTL-5 expression results;

[0028] Figure 7The images show the immunoblotting results of recombinant proteins, including: A, rTcCTL-2 immunoblotting results; B, rTcCTL-5 immunoblotting results; M, protein molecular weight standard; and 1, immunoblotting analysis of recombinant proteins with positive serum from *Toxocara canis* infection.

[0029] Figure 8 Immunofluorescence localization of TcCTL-2 in *Toxocara canis*, where Cu represents the epidermis; Iw represents the intestinal wall; Iv represents the intestinal villi; Mu represents the muscle; Uw represents the uterine wall; and Tw represents the testicular wall. The scale bar "-" indicates 100 μm.

[0030] Figure 9 Immunofluorescence localization of TcCTL-5 in *Toxocara canis*, where Cu represents the epidermis; Iw represents the intestinal wall; Iv represents the intestinal villi; Mu represents the muscle; Uw represents the uterine wall; and Tw represents the testicular wall. The scale bar "-" indicates 100 μm.

[0031] Figure 10 The relative expression levels of Tcctl-2 and Tcctl-5 genes at different developmental stages were analyzed. A represents the relative expression level of Tcctl-2, and B represents the relative expression level of Tcctl-5. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and ns indicates no significant difference. Each gene's expression level is relative to the stage with the lowest expression level.

[0032] Figure 11 PCR amplification of the Tcctl-2 and Tcctl-5 genes, where A represents the PCR amplification result of Tcctl-2; B represents the PCR amplification result of Tcctl-5; M represents the DNA molecular weight standard; and 1 represents the amplified product of the target gene.

[0033] Figure 12 For the validation and linearization amplification of pNZ8149 plasmid, where A, pNZ8149 plasmid validation results; B, pNZ8149 plasmid linearization amplification product; M, DNA molecular weight standard; 1, pNZ8149 plasmid amplification product.

[0034] Figure 13 The results of electroporation of recombinant Lactococcus lactis are shown in Figure 1. A shows the electroporation results of recombinant Lactococcus lactis pNZ8149 / TcCTL-2; B shows the electroporation results of recombinant Lactococcus lactis pNZ8149 / TcCTL-5.

[0035] Figure 14This document describes the PCR identification of recombinant *Lactococcus lactis*, where A represents the PCR identification results of recombinant *Lactococcus lactis* pNZ8149 / TcCTL-2; B represents the PCR identification results of recombinant *Lactococcus lactis* pNZ8149 / TcCTL-5; M represents the DNA molecular weight standard; and 1-2 represent the PCR amplification products of positive bacteria.

[0036] Figure 15 SDS-PAGE analysis of recombinant Lactococcus lactis expression products: A, analysis of recombinant pNZ8149 / TcCTL-2 expression products; B, analysis of recombinant pNZ8149 / TcCTL-5 expression products; M, protein molecular weight standard; 1, results of induction expression in bacterial supernatant; 2, results of induction expression in empty vector pNZ8149; 3, results of induction expression after 8 h of bacterial cell precipitation; 4, results of induction expression after 12 h of bacterial cell precipitation.

[0037] Figure 16 The protein expression of pNZ8149 / TcCTL-2 induced by different concentrations of Nisin was analyzed. Where M represents the molecular weight of the protein; 1 represents the empty vector pNZ8149; 2 represents induction with 1 ng / mL Nisin; 3 represents induction with 3 ng / mL Nisin; 4 represents induction with 5 ng / mL Nisin; and 5 represents induction with 10 ng / mL Nisin.

[0038] Figure 17 Protein expression analysis of pNZ8149 / TcCTL-5 induced by different concentrations of Nisin, where M represents the molecular weight of the protein; 1 represents induction with 1 ng / mL Nisin; 2 represents induction with 3 ng / mL Nisin; 3 represents induction with 5 ng / mL Nisin; 4 represents induction with 10 ng / mL Nisin; and 5 represents empty vector pNZ8149.

[0039] Figure 18 Analysis of intestinal colonization of recombinant lactococcus;

[0040] Figure 19 PCR detection of intestinal colonization of recombinant Lactococcus lactis pNZ8149 / TcCTL-2 was performed. A represents the colonization results at 7 days; B represents the colonization results at 14 days. M represents DNA molecular weight standards; 1-2 represent amplification products from duodenal lavage fluid; 3-4 represent amplification products from jejunal lavage fluid; 5-6 represent amplification products from ileal lavage fluid; 7-8 represent amplification products from fecal suspension.

[0041] Figure 20PCR detection of intestinal colonization of recombinant Lactococcus lactis pNZ8149 / TcCTL-5 was performed. A represents the colonization results at 7 days; B represents the colonization results at 14 days. M represents DNA molecular weight standards; 1-2 represent amplification products from duodenal lavage fluid; 3-4 represent amplification products from jejunal lavage fluid; 5-6 represent amplification products from ileal lavage fluid; 7-8 represent amplification products from fecal suspension.

[0042] Figure 21 The change in weight gain rate of mice in the control group and the immunized group is shown in the figure. * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001, and ns indicates no significant difference.

[0043] Figure 22 The denominator represents the organ index of mice, where * indicates P<0.05 and ns indicates no significant difference.

[0044] Figure 23 The number of L3 larvae recovered in mice is given by *, where * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001, and ns indicates no significant difference.

[0045] Figure 24 The images show pathological changes in mouse liver and lung tissue. The scale bar "—" represents 50 μm. Blue arrows indicate areas of inflammatory cell infiltration, and yellow arrows indicate areas of severe hemorrhage.

[0046] Figure 25 The image shows the pathological changes in the duodenum after worm attack. The scale bar "—" represents 50 μm; blue arrows indicate goblet cells; and yellow arrows indicate Paneth cells.

[0047] Figure 26 The image shows pathological changes in the jejunum after worm attack. The scale bar "—" indicates 50 μm; blue arrows represent goblet cells; yellow arrows represent Paneth cells.

[0048] Figure 27 For the analysis of interspecies differences, the horizontal axis represents time; the vertical axis represents relative abundance. Others represents the relative abundance of all organisms other than the 20 microbial categories shown in the figure;

[0049] Figure 28 The results are for complete blood cell counts. * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001, and ns indicates no significant difference.

[0050] Figure 29The results represent changes in antibody levels in mice, including: A, IgG antibody level detection; B, IgG1 antibody level detection; C, IgG2a antibody level detection; and D, sIgA antibody level detection. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and ns indicates no significant difference.

[0051] Figure 30 The changes in transcription factor levels in the mesenteric lymph nodes of immunized mice were represented by the percentages of AB and GTAT-3, CD and T-bet, and EF and RORγt. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and ns indicates no significant difference.

[0052] Figure 31 The changes in transcription factor levels in mouse spleen were represented by percentages of AB and GTAT-3, CD and T-bet, and EF and RORγt. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and ns indicates no significant difference.

[0053] Figure 32 The values ​​represent changes in Th1 and Th17 cytokine levels. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, **** indicates P < 0.0001, and ns indicates no significant difference.

[0054] Figure 33 The values ​​represent changes in Th2 cytokine levels. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, **** indicates P < 0.0001, and ns indicates no significant difference. Detailed Implementation

[0055] 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.

[0056] 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.

[0057] 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.

[0058] Example 1:

[0059] I. Materials and Reagents:

[0060] 1.1 Insect strains, bacterial strains and vectors

[0061] Escherichia coli BL21(DE3) and Escherichia coli Rosetta(DE3) were purchased from Beijing Tiangen Biotech Co., Ltd.; DH5α competent Escherichia coli cells were purchased from Shanghai Sangon Biotech Co., Ltd.; pET32a(+) vector was stored in our laboratory (commercially purchased); Lactococcus lactis strain NZ3900 and pNZ8149 plasmid were purchased from Hangzhou Baosai Biotechnology Co., Ltd.

[0062] 1.2 Experimental animals and serum

[0063] Six-week-old SPF-grade male C57BL / 6 mice were purchased from Beijing Spaford; positive serum for Toxocara canis infection was prepared using standard procedures and stored at -20°C.

[0064] 1.3 Culture medium

[0065] (1) GM17 broth medium: Weigh 16.92g of M17 medium dry powder, add ddH2O to make up to 390mL, autoclave at 115℃ for 20min, cool and add 10mL of 20% glucose storage solution, and store at 4℃.

[0066] (2) GM17 agar medium: Weigh 8.46g of M17 medium dry powder and 3g of agar powder, add ddH2O to make up to 195mL, autoclave at 115℃ for 20min, cool naturally to 60℃, add 5mL of 20% glucose storage solution and mix well, pour into sterile petri dishes, cool naturally to solidify and store at 4℃.

[0067] (4) GSGM17 medium: Weigh 8.46g M17 broth medium, 34.23g sucrose, and 3g glycine, add ddH2O to make up to 195mL, autoclave at 115℃ for 20min, cool to about 60℃, add 5mL 20% glucose storage solution, and store at 4℃.

[0068] (5) GM17-MC medium: Weigh 4.23g of M17 broth medium, 0.02g of CaCl2 and 0.19g of MgCl2, add ddH2O to make up to 92.5mL, autoclave at 115℃ for 20min, cool and add 7.5mL of 20% glucose storage solution, store at 4℃.

[0069] (6) Elliker medium: Weigh 4g tryptone, 1g yeast extract, 0.8g sodium chloride, 0.3g sodium acetate (anhydrous), 0.1g / L ascorbic acid, and 3g agar. Add ddH2O to bring the volume to 193mL. Autoclave at 121℃ for 20 minutes. After cooling to 60℃, add 5mL of 20% lactose stock solution and 2mL of 0.4% bromocresol purple to bring the final concentration to 0.004%. Pour into sterile Petri dishes, cool and solidify, and store at 4℃.

[0070] 1.4 Main Reagents

[0071] (1) 0.4% bromocresol purple: Weigh 0.4g of bromocresol purple and dissolve it in 20mL of 0.02mol / L NaOH solution. Add ddH2O to make up to 100mL. Filter through a 0.22μm microporous membrane to remove bacteria and store at 4℃.

[0072] (2) 0.02M NaOH solution: Dissolve 0.08g NaOH in 50mL ddH2O, make up to 100mL, and store at room temperature.

[0073] (3) 0.1 mg / mL Nisin stock solution: Weigh 0.01 g nisin, add 100 mL 0.05% acetic acid solution to dissolve, filter to sterilize, and store in aliquots at -20℃.

[0074] (4) 20% glucose storage solution: Weigh 20g of glucose, add 100mL of ddH2O, heat and stir until fully dissolved, filter to remove bacteria, and store at 4℃.

[0075] (5) Solution I: Weigh 34.23g of sucrose and 20mL of glycerol, add ddH2O to make up to 200mL, autoclave at 115℃ for 20min, and store at 4℃.

[0076] (6) Solution II: Weigh 17.12g sucrose, 10mL glycerol, 1.86g EDTA, add ddH2O to make up to 100mL, autoclave at 115℃ for 20min, and store at 4℃.

[0077] II. Test Methods

[0078] 2.1 Total RNA extraction from Toxocara canis

[0079] RNA was extracted from Toxocara canis bodies / eggs at different stages. The specific steps were as follows: Toxocara canis bodies / eggs at different stages stored in an ultra-low temperature freezer were used. 1 mL of Trizol was added to every 50-80 mg of tissue, and the mixture was homogenized and allowed to stand at room temperature for 15 min to allow for complete lysis. The mixture was then centrifuged at 12,000 rpm / min for 15 min at 4°C. The supernatant was transferred to a 1.5 mL EP tube, and 0.2 mL of chloroform was added to every 1 mL of supernatant. The mixture was shaken vigorously and allowed to stand at room temperature for 3 min. The mixture was then centrifuged at 12,000 rpm / min for 15 min at 4°C. The colorless upper layer was collected, and 0.5 mL of isopropanol was added to every 1 mL of supernatant. The mixture was repeatedly inverted and mixed, and allowed to stand at room temperature for 10 min. Finally, the mixture was centrifuged at 12,000 rpm / min for 15 min at 4°C to precipitate the RNA. The supernatant was discarded, and 1 mL of pre-cooled 75% ethanol was added to the tube to wash the precipitate twice, followed by vortexing to mix. Centrifuge at 7,500 rpm for 5 min at 4℃, discard the supernatant, allow the RNA to dry slightly, add 20 μL of RNase-Free to dissolve, and store at -80℃.

[0080] 2.2 Synthesis of first-strand cDNA

[0081] Using extracted total RNA from Toxocara canis as a template, cDNA was generated by reverse transcription and stored at -20℃.

[0082] 2.3 Primer Design and Synthesis

[0083] Based on GenBank data, primers for the full-length sequences of the Tcctl-2 (Genebank accession number: KR052876.1) and Tcctl-5 (Genebank accession number: JPKZ01002894.1) genes were designed using Primer 5.0 software. The sequence information is shown in Table 1. The primers were sent to Shanghai Sangon Biotech Co., Ltd. for synthesis.

[0084] Table 1 Primers for target gene amplification

[0085]

[0086] 2.4 Target gene amplification and cloning

[0087] (1) Amplification and recovery of target genes: Using cDNA from stage 3 larvae as templates, the Tcctl-2 and Tcctl-5 genes were amplified. After the PCR products were analyzed by 1% agarose gel electrophoresis, the gel block containing the target band was cut into a 1.5 mL EP tube and weighed. The gel was recovered according to the instructions of the TIANGEN gel recovery kit.

[0088] Primers for the target gene fragment containing the homologous arm of the pET-32a(+) E. coli expression vector (underlined to indicate the pET-32a homologous arm) were designed for PCR amplification and gel recovery. Primer information is as follows:

[0089] Tcctl-2 F1: 5'- cattcttctggtctggtg ATGTTCGCCATATTCATAATTTG-3'

[0090] Tcctl-2 R1: 5'- gatctgggctgtccatgt TTATAGAGGTCTCTTGCATACG-3'

[0091] Tcctl-5 F1: 5'- gatctgggctgtccatgt TTACAAGAAAAATCGCCTCCT-3'

[0092] Tcctl-5 R1: 5'- cattcttctggtctggtg ATGATCGCCTTTTGTCTGC-3'

[0093] (2) Ligation of the target gene fragment with the vector: using The seamless cloning kit allows for homologous recombination of the target gene product recovered from the gel with a vector. The reaction conditions are 50°C for 15 min followed by an ice bath for 5 min.

[0094] (3) Transformation: 10 μL of the ligation product was transferred to DH5α Escherichia coli competent cells that had been thawed on ice using a pipette. After gentle mixing, the cells were incubated on ice for 30 min, then heat-shocked in a water bath at 42°C for 60 s. The cells were then immediately placed on ice and allowed to stand for 5 min. 800 μL of liquid medium was then added and the cells were shaken to mix. The centrifuge tubes were placed in a 37°C constant temperature water bath and cultured at 140 rpm / min for 1 h. After centrifugation at 3,000 rpm / min for 1 min, the supernatant was discarded, and 100 μL of culture medium was retained. The medium was then mixed by pipetting and spread evenly on AMP-resistant LB plates using a sterile spreader and cultured overnight at 37°C.

[0095] (4) Sequencing analysis: A single colony was picked and inoculated into LB liquid medium containing AMP. It was incubated at 37°C for about 8 hours. 2 μL of bacterial solution was taken for PCR identification. Positive samples were sent to Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results were compared with the target gene sequences in the GenBank database. The pET32a / TcCTL-2 and pET32a / TcCTL-5 strains with correct sequence alignment were preserved in 20% glycerol.

[0096] 2.5 Bioinformatics Analysis

[0097] Transmembrane regions and signal peptides of the target gene were predicted using TMHMM 2.0 and SignalP 6.0; protein secondary structure prediction and amino acid multiple sequence alignment were performed using JalView software.

[0098] 2.6 Expression and purification of rTcCTL-2 and rTcCTL-5

[0099] (1) Extraction of recombinant plasmid: The preserved pET32a / TcCTL-2 and pET32a / TcCTL-5 bacterial cultures were expanded and cultured overnight in a 37℃ water bath at 140 rpm / min. The next day, the pET32a(+) expression vector plasmid was extracted according to the instructions of the plasmid miniplasmid kit (Tianprep miniplasmid kit).

[0100] (2) Transformation: The extracted recombinant plasmid was transformed into BL21 Escherichia coli infected cells. The specific operation steps were the same as in 2.1.2.4, and the positive monoclonal bacteria were identified by PCR.

[0101] (3) Induction of expression: Selected expression bacteria that were identified as positive by PCR were expanded and cultured. When the OD600 value of the bacterial culture reached about 0.6, IPTG was added to the culture medium to make the final concentration 1 mmol / L to start the induction. After continuous induction culture for 7 h, 1 mL of bacterial culture was taken for sample preparation, and the expression of rTcCTL-2 and rTcCTL-5 was detected by SDS-PAGE.

[0102] (4) Solubility analysis: The bacterial culture expressing the target protein was placed in a 37℃ incubator and cultured with shaking at 140 rpm / min. The OD600 value of the bacterial culture was monitored in real time. When it reached about 0.6, the target protein was induced to express according to the determined optimal induction time and temperature. After induction, 2 mL of bacterial culture was taken and centrifuged at 7,000 rpm / min for 10 min at 4℃. The bacterial pellet was collected, and 10 mL of 0.05 mM soluble protein solution was added to the pellet. The bacterial cell lysis buffer in Tris-HCl was repeatedly pipetted to ensure thorough mixing of the cells and lysis buffer. The cell suspension was then sonicated until the liquid became clear. The suspension was then centrifuged again at 7,000 rpm for 15 min, and the supernatant was collected. The remaining bacterial cell pellet was resuspended sequentially in 2M, 4M, and 8M urea solutions, and centrifuged at 7,000 rpm for 10 min after each resuspending, and the supernatant was collected. 20 μL of the supernatant was mixed with 5 μL of 5×SDS-PAGE protein loading buffer, and the mixture was boiled for 10 min. The solubility of rTcCTL-2 and rTcCTL-5 was analyzed by SDS-PAGE.

[0103] (5) Protein purification: The recombinant protein was purified based on the results of the solubility analysis. The specific operation steps were strictly carried out in accordance with the Ni+ affinity chromatography instructions. The purified protein was detected by SDS-PAGE, then concentrated by ultrafiltration, and after the protein purity was determined, it was aliquoted and stored in an ultra-low temperature freezer at -80℃.

[0104] 2.7 Immunogenicity of rTcCTL-2 and rTcCTL-5

[0105] (1) Preparation of hyperimmune serum: Ten 6-week-old male C57BL / 6 mice were randomly divided into two groups (n=5) to prepare hyperimmune serum for recombinant proteins rTcCTL-2 and rTcCTL-5, respectively. Freund's adjuvant and purified protein were mixed in the same proportion to adjust the protein concentration to 500 μg / mL. Mice were inoculated subcutaneously at a dose of 50 μg / mouse, immunized every 7 days for a total of 3 times. Seven days after the last immunization, whole blood was collected from mice by ocular venous plexus sampling. After being placed at room temperature for 2 hours, the serum was centrifuged at 3500 rpm / min for 5 min to separate the serum, aliquoted, and stored at -20℃.

[0106] (2) Immunoblot analysis: The purified recombinant protein was subjected to SDS-PAGE gel electrophoresis. After electrophoresis, a piece of gel from the target lane was cut and placed in transfer buffer for equilibration, repeated 3 times for 5 min each time; at the same time, the cut NC membrane and 3 layers of filter paper were soaked in transfer buffer for 15 min; the gel piece, NC membrane and filter paper were removed and transferred, and the 3 layers of filter paper, NC membrane, gel and 3 layers of filter paper were assembled from bottom to top to form a "sandwich" structure, which was then placed in the transfer tank at 1 mA / cm 2 Set the current magnitude; after the transfer is complete, peel off the NC membrane and wash it three times with TBST, 5 min each time; then add blocking solution containing 5% skim milk powder to the system and perform blocking treatment at room temperature for 2 hours to block non-specific binding sites; after blocking, discard the blocking solution and wash the NC membrane three times with TBST solution, 5 min each time; add positive serum of *Toxocara canis* infection diluted with PBS as primary antibody and incubate overnight at 4°C; the next day, discard the primary antibody solution and wash the NC membrane three times with TBST, 5 min each time to remove unbound primary antibody; then add HRP-labeled goat anti-mouse IgG diluted with PBS as secondary antibody, incubate at room temperature for 2 hours, discard the secondary antibody, wash the membrane with TBST solution, place the NC membrane in a dark box, evenly drop ECL chromogenic solution onto the surface of the NC membrane, incubate in the dark for 2-3 minutes, and then immediately perform development.

[0107] 2.8 Immunofluorescence Tissue Localization

[0108] Remove the preserved paraffin section of *Toxocara canis* and perform immunofluorescence tissue localization according to the following steps:

[0109] 1) Drying the slices: Place the paraffin slices in an oven at 60℃ for 1 hour;

[0110] 2) Dewaxing: Dewax the paraffin sections with xylene I for 15 minutes, and then dewax them with xylene II for 15 minutes;

[0111] 3) Rehydration: Immerse the paraffin sections in 100% alcohol I, 100% alcohol II, 95% alcohol, 85% alcohol and 75% alcohol in sequence for 5 minutes each time, and wash with PBS for 5 minutes after the end.

[0112] 4) Sealing: Use filter paper to absorb any residual liquid outside the specimen, add 5% skim milk powder, and seal at 37°C for 1 hour;

[0113] 5) Incubation with primary antibody: After blocking, wash 3 times with PBS for 5 minutes each time, wipe dry with filter paper, add 1:100 hyperimmune serum, and incubate overnight at 4°C;

[0114] 6) Incubation with secondary antibody: Wash 3 times with PBS for 5 min each time, wipe dry with filter paper, add 1:200 FITC-labeled goat anti-mouse IgG, and incubate at room temperature in the dark for 1 h.

[0115] 7) Nucleus exposure: Wash 3 times with PBS, 5 min each time, wipe dry with filter paper, add DAPI reagent, and incubate in the dark for 3-5 min;

[0116] 8) Mounting: Wash 3 times with PBS for 5 minutes each time, add anti-fluorescence quencher, apply a little nail polish around the coverslip and mount, let it dry and observe and photograph under a fluorescence microscope.

[0117] 2.9 Transcriptional Levels of Tcctl-2 and Tcctl-5 at Different Stages of the Parasite

[0118] Based on the Tcctl-2 and Tcctl-5 gene sequences, qPCR primers were designed (primer information is shown in Table 2). The designed primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. Using 18S RNA as an internal reference gene, the expression levels of Tcctl-2 and Tcctl-5 genes at different developmental stages of the insect were measured. The results were expressed using the 2-ΔΔct method, and the data were visualized using GraphPad 9.0 software.

[0119] Table 2 qPCR primer sequences

[0120]

[0121] 2.10 Primer Design and Synthesis

[0122] Linearized primers for the pNZ8149 vector were designed using Snapgene 6.0 software and a 6×His tag was added. Primers for the target gene with pNZ8149 homologous arms were also designed to amplify Tcctl-2 and Tcctl-5. Primer sequence information is shown in Table 3.

[0123] Table 3. Primers for gene amplification

[0124]

[0125] Note: Lowercase letters indicate vector homologous sequences; underscore letters indicate 6×His tags.

[0126] 2.11 Construction of recombinant Lactococcus lactis expression vector

[0127] (1) Amplification and gel extraction of target gene: Using plasmids pET-32a / TcCTL-2 and pET-32a / TcCTL-5 as templates, the target gene fragment was amplified by PCR using homologous arm primers. After agarose gel electrophoresis, the gel block containing the target gene was cut off, and TcCTL-2 and TcCTL-5 were extracted using a gel extraction kit.

[0128] (2) Extraction of pNZ8149 plasmid: Take the pNZ8149 / NZ3900 lactococcus lactis stored at 4℃, pick a single colony and inoculate it into 2mL of M17 medium. Incubate overnight at 30℃. The next day, take 1mL of the overnight culture and transfer it to 9mL of M17 broth medium. After culturing for 8h, extract the pNZ8149 plasmid according to the kit instructions. After determining the concentration, store at -20℃ for later use. The extraction procedure is as described in Chapter 2, 2.1.2.6. Note that after adding Buffer P1, 20mg / ml of lysozyme should be added. Incubate at 37℃ for 30min, gently shaking the liquid in the tube every 5min to ensure complete lysis of the cell wall.

[0129] (3) Linearization and gel recovery of pNZ8149 plasmid: Using the extracted pNZ8149 plasmid as a template, the linearized vector was amplified by reverse PCR using specific primers pNZ8149 F / R, and gel blocks of the same size as the target band were cut for gel recovery.

[0130] (4) Homologous recombination of target gene and vector: According to the instructions of ClonExpress Ultra One Step Cloning Kit V2, the molar ratio of vector to insert fragment is 1:2. React on an ice box, and then perform homologous recombination of the target gene recovered from the gel with the linearized vector.

[0131] After adding the above reagents to the reaction tube, gently mix them by pipetting. Then, react in a 50°C water bath for 5 minutes. After the reaction is complete, place the tube on ice to cool.

[0132] (5) Preparation of Lactococcus lactis NZ3900 competent cells:

[0133] Using a sterile inoculation loop, pick a single colony from the plate and inoculate it into 1 mL of GM17 medium. Incubate overnight at 30°C. Transfer the overnight culture to 9 mL of GM17 liquid medium and incubate at 30°C for approximately 10 hours. Inoculate the culture into 100 mL of GSGM17 medium and incubate at 30°C until the OD value reaches approximately 0.3. Remove the medium. Centrifuge at 4°C, 5000g for 15 min, discard the supernatant, and collect the bacterial precipitate. Add 25 mL of pre-chilled Solution I solution, and repeatedly pipette the precipitate to mix. Centrifuge at 4°C, 5000g for 15 min, and discard the supernatant. Add 20 mL of pre-chilled Solution II solution to the precipitate and gently resuspend until the precipitate is completely dissolved. Incubate the resuspended solution on ice for 15 min. Centrifuge at 4°C, 5000g for 15 min, discard the supernatant, and retain the precipitate. Add another 25 mL of pre-chilled Solution II solution to the precipitate. Mix Solution I solution by vortexing to resuspend the bacterial cells. Centrifuge at 5000g for 15 minutes at 4°C. Carefully discard the supernatant, keeping the bacterial precipitate at the bottom of the tube. Add 1 mL of pre-cooled Solution I solution and mix by repeatedly pipetting until the bacterial precipitate is completely dissolved. Then aliquot and store in an ultra-low temperature freezer at -80°C.

[0134] (6) Electroconversion:

[0135] Pre-cool the electroporation cuvette on ice and irradiate it with UV light in a clean bench for about 20 minutes. Remove the NZ3900 competent cells from the -80℃ ultra-low temperature freezer and thaw them on ice. Add 10 μL of ligation product to the thawed competent cells, gently pipette to mix, and then incubate on ice for 10 minutes. Transfer the mixed bacterial solution to the bottom of the electroporation cuvette, remove air bubbles, and wipe the outer wall of the cuvette dry. Set the electroporation parameters to 2200V, 200Ω, 25μF, and 2mm diameter of the electroporation cuvette. After setting the parameters, perform electroporation. Wait for the electroporator to complete the electroporation process. Immediately after the shock, add 900 μL of pre-chilled GM17-MC medium to the shock cup, gently aspirate 3-5 times to thoroughly mix the bacterial suspension with the medium, and transfer the mixture to a pre-chilled sterile centrifuge tube. Incubate on ice for 10 min, then transfer to a 30°C incubator and incubate for 2-3 h. Remove the bacterial suspension, centrifuge at 12,000 rpm for 3 min, discard the supernatant, retain approximately 100 μL of the bacterial suspension, resuspend it, and spread it evenly on the surface of Elliker solid medium. Incubate at 30°C upside down for 24 h, observe the morphology of single colonies, and screen for positive strains.

[0136] 2.12 Identification of recombinant Lactococcus lactis expression vector

[0137] (1) Extraction of recombinant plasmid: Pick a single yellow colony from Elliker medium and inoculate it into 2 mL of GM17 medium. Incubate overnight at 30°C. The next day, transfer 2 mL of bacterial culture to 5 mL of GM17 medium and incubate overnight at 30°C. Extract the recombinant plasmid according to the method in 3.1.2.2.

[0138] (2) PCR identification and sequencing of recombinant plasmid: PCR identification was performed using the extracted recombinant plasmid as a template. The primers for identification / sequencing of plasmid pNZ8149 are shown in Table 4.

[0139] Table 4. Primer sequences for pNZ8149

[0140]

[0141] The plasmids that were positive by PCR were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The plasmids with correct sequence alignment were named pNZ8149 / TcCTL-2 and pNZ8149 / TcCTL-5.

[0142] 2.13 Induction and Optimization of Recombinant Lactococcus lactis

[0143] (1) Induction and verification of recombinant lactococcus: The successfully constructed recombinant lactococcus pNZ8149 / TcCTL-2, pNZ8149 / TcCTL-5 and empty vector pNZ8149 were inoculated into 10 mL of water. In GM17 liquid medium, the culture was statically incubated at 30℃ for about 10 hours. The bacterial culture was then transferred to GM17 medium at a ratio of 1:10. When the OD600 value was about 0.4, the inducing agent Nisin was added to the bacterial culture at a working concentration of 10 ng / mL. The culture was statically incubated at 30℃ for 8 hours and 12 hours. After induction, the bacterial cells were lysed using an ultrasonic homogenizer. The lysate was centrifuged at 12,000 rpm / min for 10 minutes at 4℃. The supernatant and bacterial precipitate were carefully separated and collected. 5×SDS-PAGE protein loading buffer and the sample were added at a ratio of 1:4 and mixed thoroughly. The mixed sample was denatured in a boiling water bath for 10 minutes. The sample was then removed for SDS-PAGE electrophoresis analysis.

[0144] (2) Optimization of induction concentration of recombinant lactococcus: When the OD600 value of the bacterial solution is about 0.4, different concentrations of Nisin were added to the positive expression bacteria pNZ8149 / TcCTL-2, pNZ8149 / TcCTL-5 and empty vector bacteria pNZ8149, respectively, so that the final Nisin concentrations were 3ng / mL, 5ng / mL, 10ng / mL and 20ng / mL, respectively. After inducing expression for 8h, 1mL of bacterial solution was taken for each sample and tested to determine the optimal induction concentration.

[0145] 2.14 Intestinal colonization capacity test

[0146] Recombinant lactococci pNZ8149 / TcCTL-2 and pNZ8149 / TcCTL-5 were induced and cultured for 8 h, centrifuged at 12,000 rpm / min, 4 °C for 5 min, the supernatant was discarded, and the bacterial pellet was resuspended in 1 mL PBS. After vortexing and mixing, the pellet was serially diluted 10-fold. 200 μL of each 10-fold dilution was then extracted. 5 -10 8 The diluted solutions were spread evenly on GM17 plates and incubated overnight at 30°C, with three replicates for each concentration gradient. The colonies were counted using the plate count method the following day.

[0147] Twelve male C57BL / 6 mice were used, with six mice in each of the pNZ8149 / TcCTL-2 and pNZ8149 / TcCTL-5 groups. The concentration of the induced recombinant lactic acid bacteria was adjusted to 1×10⁻⁶. 9Mice were administered 0.3 mL of bacterial culture orally at CFU / mL, with normal water and food intake during the experiment. On days 7 and 14, three mice from each group were sacrificed. The mouse intestines were flushed with sterile PBS, and the flushing fluid was collected, vortexed, and allowed to stand for 30 min. After brief centrifugation, the supernatant was spread onto Elliker agar plates. After 24 h of incubation, colony morphology was observed, and single yellow colonies were selected for culture and PCR identification.

[0148] 2.15 Animal Immunization

[0149] Ninety 6-week-old SPF-grade male C57BL / 6 mice were randomly divided into five groups of 18 mice each: a PBS control group, a pNZ8149 / NZ3900 empty vector group, a pNZ8149 / TcCTL-2 immunization group, a pNZ8149 / TcCTL-5 immunization group, and a pNZ8149 / TcCTL-2 + pNZ8149 / TcCTL-5 mixed immunization group (hereinafter referred to as pNZ8149 / TcCTL-2+5). Recombinant Lactococcus lactis, the empty vector (prepared fresh for each immunization), and PBS were administered orally to the mice via gavage at a dose of 300 μL per mouse. Immunization was performed in three doses, with each immunization occurring 14 days apart. The immunization group information is shown in Table 5.

[0150] Table 5. Grouping information of immunized animals

[0151]

[0152] 2.16 Mouse challenge with parasites

[0153] Seven days after the last immunization, all mice were artificially infected with *Toxocara canis* via gavage, with each mouse infected with 1000 L3-stage eggs. Before challenge, the L3-stage eggs were incubated at 28°C for 1 hour to restore egg viability and ensure infection efficacy. Five days after challenge, all mice were euthanized by cervical dislocation, and liver and lung tissues were collected. The number of larvae in the tissues was recorded, and the larval load reduction rate was calculated.

[0154] 2.17 Weight Changes and Organ Indices

[0155] Starting with the first immunization, mouse weight changes were recorded weekly. On the 5th day after the parasite attack, the heart, liver, spleen, and lungs of the mice were dissected and weighed. The initial weight of the mice on day 0 was used as the initial weight. The weight gain rate was calculated using formula (1), and the organ coefficient was calculated using formula (2).

[0156]

[0157]

[0158] 3.9 Comparison of lesion conditions

[0159] To assess the pathological changes in the liver and lung tissues of mice in each group, liver and lung tissues of mice after parasite attack were collected, and tissue samples were fixed with 4% paraformaldehyde fixative and sent to Wuhan Boerfu Biotechnology Co., Ltd. for pathological section HE staining.

[0160] 2.18 Changes in gut microbiota

[0161] Small intestinal contents were collected from mice in the experimental and control groups at days 0, 14, 28, 35, and 40, with three replicates per group. Mice were euthanized by cervical dislocation, and the abdominal hair was moistened with 75% ethanol. Small intestinal contents were collected using sterile surgical scissors and cotton swabs and placed in cryovials, which were then rapidly placed in liquid nitrogen and subsequently transferred to -80°C for storage. The intestinal contents were stored on dry ice and sent to Chengdu Luoning Biotechnology Co., Ltd. for sequencing of the 16S rRNA gene V3-V4 region. Sequencing was performed using an Illumina NextSeq 2000 sequencing system.

[0162] 2.19 Complete Blood Count Analysis

[0163] On the 5th day after the parasite challenge, 0.1 mL of blood was collected from the tail vein of each group of mice in anticoagulated blood collection tubes. Blood cell counts were performed using a complete blood count analyzer, and the number of red blood cells, white blood cells (neutrophils, lymphocytes and monocytes) and platelets were statistically analyzed.

[0164] 2.20 Detection of antibody levels in the serum of immunized mice

[0165] (1) Sample collection and processing:

[0166] Serum: Tail vein blood was collected from mice before and 5 days after infection. After standing at room temperature for 1 hour, the serum was separated by centrifugation at 3500 rpm for 5 minutes, aseptically aliquoted, and stored at -20°C for use in IgG / IgG1 / IgG antibody testing. 2a Detection of antibodies and cytokines.

[0167] Intestinal contents: Before and 5 days after worm challenge, small intestines of approximately 5 cm in length were harvested from 3 mice in each group. Intestinal contents were collected in centrifuge tubes, and 500 μL of PBS solution containing 0.05 mol / L EDTA was added. The mixture was vortexed and incubated at 4°C for 12 h. The tubes were then centrifuged at 10,000 rpm / min for 5 min at 4°C. The supernatant was collected and stored at -20°C for later use in sIgA detection.

[0168] (2) IgG / IgG1 / IgG 2aDetection: Dilute the purified antigen to the working concentration using carbonate buffer. Add 100 μL of antigen solution to each well of a 96-well polystyrene microplate and coat at 4°C for 12-16 h. Discard the liquid in the wells and wash with PBST for 5 min each time, repeating 3 times. Then, add 100 μL of blocking buffer containing 5% skim milk powder to each well and incubate at 37°C for 2 h to block non-specific sites. After blocking, wash three times with PBST. Dilute the serum sample to be tested 1:20 with diluent and add 100 μL to each well, incubating at 37°C for 1 h. Discard the primary antibody solution, wash the plate, and add 100 μL of HRP-labeled secondary antibody (goat anti-mouse IgG / IgG1 / IgG) diluted 1:200 to each well. 2a Incubate at 37°C in the dark for 1 hour; discard the secondary antibody solution.

[0169] After washing three times with PBST, 100 μL of freshly prepared TMB substrate solution was added to each well, and the reaction was developed at room temperature in the dark for 20 min. Finally, 20% sulfuric acid stop solution was added to terminate the reaction, and the absorbance of each well was immediately measured at 450 nm using an ELISA reader.

[0170] (3) sIgA antibody detection: The intestinal contents dilution stored at -20℃ was taken out and the sIgA level was detected and analyzed according to the instructions of the mouse sIgA kit.

[0171] 2.21 CD4+ cell differentiation trend

[0172] (1) Cell isolation and counting:

[0173] 1. Sample collection: Mice were euthanized by cervical dislocation after blood collection 5 days after the parasite attack. The samples were disinfected with 75% alcohol. The spleen and mesenteric lymph nodes of the mice were separated using sterile surgical scissors and forceps and collected in 6-well plates. 1 mL of PBS was added to each well.

[0174] 2. Grind the tissue: Press and grind the tissue with the plunger of a 5ml syringe, then add 1mL of PBS to the centrifuge tube and mix by pipetting (spleen: add 1mL of PBS to the centrifuge tube; lymph nodes: no need to add PBS due to the small number of cells);

[0175] 3. Cell filtration: Filter the ground tissue suspension through a 200-mesh filter into a centrifuge tube, centrifuge at room temperature, 450g, for 5 minutes;

[0176] 4. Lysis of red blood cells: Discard the supernatant, add 1 mL of red blood cell lysis buffer to the spleen cell pellet, add 100 μL of red blood cell lysis buffer to the mesenteric lymphocyte pellet, mix well by pipetting and let stand for 10 min to lyse;

[0177] 5. Centrifugation: After lysis, add 3 mL of PBS to the spleen cells and 500 μL of PBS to the mesenteric lymphocytes. Centrifuge at 450 g for 5 min at room temperature.

[0178] 6. Resuspension: Discard the supernatant, add 1 mL of PBS and 500 μL of PBS to the spleen cells and mesenteric lymph node cells respectively, and resuspend the cells;

[0179] 7. Dilution: Take 10 μL of spleen cell resuspended solution and add 990 μL of PBS to dilute 100 times; take 100 μL of lymphocyte resuspended solution and add 900 μL of PBS to dilute 10 times.

[0180] 8. Counting: Add 10 μL trypan blue staining solution, 20 mL PBS, and 10 μL cell resuspension to a 1.5 mL EP tube, mix well, and take 10 μL of the mixture for counting.

[0181] (2) Cell staining:

[0182] 1. Antibody preparation: Dilute CD3+ and CD4+ antibodies with PBS at a ratio of 1:200. This step should be performed on ice.

[0183] 2. Antibody incubation: Based on cell count results, add 4 × 10⁻⁶ cells / mL. 6 One cell was placed in a 1.5 mL EP tube, and 200 μL of PBS was added. The tube was centrifuged at 450 g for 5 min at room temperature. The supernatant was discarded, and 30 μL of antibody was added to the cell pellet. The tube was then incubated at 4 °C in the dark for 25 min.

[0184] 3. Centrifugation: After incubation, add 200 μL of PBS, incubate at room temperature, 450 g, for 5 min;

[0185] 4. Fixation: Discard the supernatant, add 100 μL of fixative to each tube, and incubate at room temperature in the dark for 1 h. The fixative is prepared by mixing Perm Diluent and Permeablization Concentrate in a 3:1 ratio.

[0186] 5. Washing: Mix 10×Permeablization and ddH2O in a 1:9 ratio to prepare Washing Buffer. Add 300 μL of Washing Buffer to each tube of fixative, centrifuge at 450 g for 5 min at room temperature, then change the speed to 8500 g and continue centrifuging for 2 min.

[0187] 6. Antibody preparation: Dilute GATA3, T-bet, and RORγt antibodies with PBS at a ratio of 1:200. This step should be performed on ice.

[0188] 7. Antibody incubation: After centrifugation, carefully remove the supernatant, add 30 μL of antibody, and incubate at room temperature in the dark for 1 hour;

[0189] 8. Centrifugation: After incubation, add 300 μL of PBS, vortex to mix, centrifuge at 450g for 5 min, and then centrifuge at 8500g for 2 min.

[0190] 9. Filtration: Discard the supernatant, add 200 μL of PBS, filter through a 200-mesh filter into a flow cytometry tube, and then use a flow cytometer to count the cells.

[0191] 2.22 Cytokine Level Analysis

[0192] The levels of these cytokines in mouse serum were measured before and after insect challenge using an ELISA kit. The levels of Th1 cytokines IFN-γ and IL-12, Th2 cytokines IL-4, IL-9, IL-10 and IL-13, and Th17 cytokine IL-17 were measured according to the kit instructions. The concentrations of each cytokine were calculated by establishing a standard curve.

[0193] III. Test Results

[0194] 3.1 Amplification and Cloning of the Target Gene

[0195] Using cDNA reverse transcribed from total RNA of *Toxocara canis* as a template, specific primers were designed to amplify Tcctl-2 and Tcctl-5 by PCR. 1% agarose gel analysis showed the target bands were the expected size. Sequencing comparison revealed Tcctl-2 to be 702 bp. Figure 1 A), whose nucleotide sequence is SEQ ID No. 1, and Tcctl-5 is 546 bp ( Figure 1 B), whose nucleotide sequence is SEQ ID No. 2, is consistent with the reference sequence.

[0196] The pET32a(+) plasmid was reverse-amplified by PCR to obtain a linearized vector. The target band size was consistent with expectations, at 5900 bp. Figure 2 The purified target gene fragment and the linearized vector were homologously recombinated to successfully construct a cloning expression vector.

[0197] 3.2 Bioinformatics Analysis

[0198] Bioinformatics analysis was performed on the sequenced target gene. Signal peptide prediction revealed that the Tcctl-2 signal peptide is 18 amino acids in length. Figure 3 A), the Tcctl-5 signal peptide is 17 amino acids in length. Figure 4 A); Transmembrane region structure prediction analysis shows that Tcctl-2 ( Figure 3B) and Tcctl-5 ( Figure 4 B) No transmembrane regions were detected in either protein. Furthermore, protein secondary structure prediction showed that the proteins were predominantly composed of α-helices and β-sheets; TcCTL-2 contained 6 α-helices and 13 β-sheets, while TcCTL-5 contained 6 α-helices and 5 β-sheets. Multiple sequence alignment (MSA) showed that TcCTL-2 had the highest similarity to Toxocaracati C-type lectin, at 82.77%. Figure 3 C); Tcctl-5 showed the highest similarity to the CTLD protein of Enterobius Vermicularis, at 58.7%. Figure 4 C).

[0199] 3.3 Expression and purification of recombinant proteins rTcCTL-2 and rTcCTL-5

[0200] Recombinant plasmids pET32a / TcCTL-2 and pET32a / TcCTL-5 were transformed into BL21 expression bacteria for induced expression. SDS-PAGE electrophoresis was used to detect the expression of the recombinant protein, showing that the molecular weight of the recombinant protein rTcCTL-2 was approximately 30 kDa. Figure 5 The rTcCTL-5 is approximately 25 kDa in size. Figure 6 All of these values ​​are consistent with the expected protein molecular weight.

[0201] 3.4 Immunogenicity of recombinant proteins rTcCTL-2 and rTcCTL-5

[0202] The purified rTcCTL-2 and rTcCTL-5 proteins were analyzed by Western blot with positive serum from mice infected with Toxocara canis, as shown in the results. Figure 7 The results showed that both rTcCTL-2 and rTcCTL-5 proteins specifically reacted with positive serum from *Toxocara canis* infection, exhibiting clear blot bands, indicating that the two recombinant proteins had good immunogenicity.

[0203] 3.5 Immunofluorescence localization of TcCTL-2 and TcCTL-5

[0204] TcCTL-2 and TcCTL-5 are two important natural proteins of *Toxocara canis*, exhibiting specific distribution patterns within the parasite. Localization analysis of TcCTL-2 and TcCTL-5 revealed that TcCTL-2 (… Figure 8 ) and TcCTL-5 ( Figure 9Both TcCTL-2 and TcCTL-5 were detected in the epidermis, muscle tissue, and intestines of both female and male adult worms. In particular, TcCTL-2 and TcCTL-5 were located in the uterine wall of female adult worms and the testis wall of male adult worms, suggesting that they may play an important role in the reproductive process of the worms.

[0205] 3.6 Transcriptional levels of Tcctl-2 and Tcctl-5 in the parasite

[0206] To further investigate the transcriptional changes of Tcctl-2 and Tcctl-5 in *Toxocara canis*, we used RT-qPCR to detect the relative expression levels of Tcctl-2 and Tcctl-5 genes at different developmental stages. The results are as follows: Figure 10 As shown, Tcctl-2 was significantly highly expressed in L3 eggs and L3 larvae (gut and migration stages), with the highest expression level in L3 eggs; Tcctl-5 was highly expressed in L4 and L5 larvae as well as in both male and female adults.

[0207] 3.7 Amplification of the target gene and vector

[0208] Specific primers with the pNZ8149 homologous arm were designed, and the target gene fragment was amplified by PCR. 1% agarose gel electrophoresis showed that the target band matched the expected size, with Tcctl-2 measuring 678 bp. Figure 11 A), Tcctl-5 is 525bp ( Figure 11 B).

[0209] Electrophoresis of the extracted pNZ8149 plasmid nucleic acids revealed that, due to the different topological structures of the plasmid, the migration speeds varied, from fastest to slowest: supercoiled, linear, and open-circular, resulting in three bands. Figure 12 A). PCR amplification was performed on the validated plasmid using vector linearization primers. Nucleic acid electrophoresis confirmed that the target band matched the expected size, with pNZ8149 measuring 2550 bp. Figure 12 B).

[0210] 3.8 Electroporation and Identification of Recombinant Lactococcus lactis

[0211] The purified Tcctl-2 and Tcctl-5 gene fragments were homologously recombinated with a linearized vector to construct recombinant plasmids pNZ8149 / TcCTL-2 and pNZ8149 / TcCTL-5, respectively. The recombinant plasmids were then electroporated into Lactococcus lactis NZ3900 competent cells, and the results are as follows: Figure 13 As shown, both recombinant bacteria can grow yellow single colonies on Elliker medium, which indicates they are positive bacteria.

[0212] A single yellow-positive bacterium was picked from Elliker medium and cultured. PCR identification results of the positive bacteria are shown below. Figure 14 The target bands were consistent with the expected size, with Tcctl-2 and Tcctl-5 being 852bp and 699bp, respectively, and the sequencing results were consistent with the recombinant vector fragments.

[0213] 3.9 Induction and Optimization of Recombinant Lactococcus lactis

[0214] Empty vector bacteria pNZ8149, recombinant lactic acid bacteria pNZ8149 / TcCTL-2, and pNZ8149 / TcCTL-5 were induced with 10 ng / mL Nisin at 30℃ for 8 h and 12 h, respectively. After induction, the supernatant and precipitate were collected and subjected to SDS-PAGE electrophoresis. The target protein was not expressed in the supernatant of pNZ8149 / TcCTL-5, while the protein expressed in the bacterial precipitate had a molecular weight of approximately 20 kDa, consistent with the expected protein size. Furthermore, there was no significant difference in protein expression levels between 8 h and 12 h of induction. (See attached figures). Figure 15 A. Similarly, the target protein was not expressed in the supernatant of pNZ8149 / TcCTL-2, but a band of the target protein with a molecular weight of approximately 25 kDa appeared in the bacterial cell pellet. Furthermore, different induction times did not significantly affect the protein expression level. (See results below.) Figure 15 B.

[0215] To optimize the expression conditions of recombinant Lactococcus lactis proteins, this study induced expression using Nisin at different concentration gradients (1 ng / mL, 3 ng / mL, 5 ng / mL, and 10 ng / mL). Western blot analysis results are shown below. Figure 16 (pNZ8149 / TcCTL-2) and Figure 17 As shown in (pNZ8149 / TcCTL-5), all tested concentrations successfully induced the expression of the target protein in both recombinant Lactococcus lactis strains. Under the condition of strictly controlling the induction time and loading amount, the expression level of the target protein gradually increased with the increase of the inducing agent concentration. Therefore, 10 ng / mL was determined to be the optimal induction concentration.

[0216] 3.10 Intestinal colonization detection

[0217] During the 14-day continuous monitoring period, all mice administered recombinant Lactococcus lactis via gavage maintained normal water and food intake, exhibited stable physiological conditions, and did not die. Spreading mouse intestinal washes onto Elliker selection medium revealed that both recombinant strains could maintain effective colonization in the mouse intestine for at least 14 days. Recombinant Lactococcus lactis pNZ8149 / TcCTL-5 primarily colonized the duodenum and jejunum, but was not detected in the ileum or feces; however, pNZ8149 / TcCTL-2 successfully colonized all segments of the small intestine and was also distributed in feces. (See attached results). Figure 18Molecular identification of positive single colonies was performed by PCR, pNZ8149 / TcCTL-2 ( Figure 19 ) and pNZ8149 / TcCTL-2 ( Figure 20 The identification results were consistent with the plate screening results, confirming that recombinant lactococcus could colonize the mouse intestine for at least 14 days, providing data support for subsequent oral vaccine immunization programs based on this strain.

[0218] 3.11 Changes in mouse body weight and organ indices

[0219] To assess the effects of recombinant Lactococcus lactis on the growth and development of mice, this study recorded the body weight of mice weekly and calculated the weight gain rate after the first immunization. The results are as follows: Figure 21 As shown, compared with the PBS group (average weight gain of 28.94% over 40 days), the recombinant lactococcus immunization group showed a significant effect in promoting weight gain. Among them, the pNZ8149 / TcCTL-2 group had the most significant effect, with a weight gain of 45.87% over 40 days (P<0.001); the pNZ8149 / TcCTL-5 group was the second most effective, with a weight gain of 39.55% (P<0.051). However, the weight gain of mice in the pNZ8149 / TcCTL-2+5 mixed group (34.67%) was lower than that of the single-strain immunization group and the same as that of the empty vector control group (NZ3900 / pNZ8149, weight gain of 34.48%).

[0220] Further analysis of the indices of various organs in mice revealed ( Figure 22 Compared with the PBS control group, mice administered recombinant lactococcus showed an increasing trend in the heart, spleen, liver, and lung indices. Statistical analysis revealed that only the liver organ coefficients of mice in the empty vector group and the mixed immunization group were statistically significant (P < 0.05), while there were no significant differences in organ coefficients between the other immunization groups and the control group (P > 0.05).

[0221] 3.12 Larval Count in Mice

[0222] The number of three-stage migrating larvae in mouse liver and lung tissues was examined and counted using an optical microscope. The statistical analysis results are shown below. Figure 23 The average number of larvae recovered from mice in the PBS control group was 28, while in the NZ3900 / pNZ8149 empty vector group it was 18, representing a 35.7% reduction in larval count. In the pNZ8149 / TcCTL-2 and pNZ8149 / TcCTL-5 immunization groups, 11 and 15 larvae were recovered from mouse tissues, respectively, representing reductions in larval load of 60.7% and 46.4%. Interestingly, the combined immunization with two recombinant lactococci significantly improved the protective effect, with only 9 L3 migrating larvae found in the pNZ8149 / TcCTL-2+5 immunization group, representing a 67.9% reduction in larval load.

[0223] 3.13 Comparison of lesion conditions

[0224] To systematically evaluate the immunogenicity of recombinant Lactococcus lactis in mice, this study analyzed the pathological damage to the liver, lung, and intestinal tissues of infected mice using histopathological analysis. The liver and lung lesions are as follows: Figure 24 As shown, mice in the PBS group exhibited severe lung hemorrhage, with numerous red blood cells filling the bronchioles and alveolar cavities, thickened alveolar walls, and inflammatory cell infiltration. Hepatocytes were damaged and disorganized, with congested and dilated sinusoids containing numerous red blood cells, accompanied by inflammatory cell aggregation and macrophage proliferation. Mice in the NZ3900 / pNZ8149 and pNZ8149 / TcCTL-5 groups showed improvement in liver and lung lesions. While numerous red blood cells and inflammatory cells remained in the alveolar cavities, and hepatocytes showed vacuolation, there was no severe hemorrhage or extensive inflammatory cell infiltration. Mice in the pNZ8149 / TcCTL-2 and pNZ8149 / TcCTL-2+5 immunization groups showed significant improvement. Lung tissue structure was nearly normal, with a small number of diffusely distributed red blood cells and reduced inflammatory cell infiltration. Hepatocytes were tightly packed, morphologically normal, and radially distributed around the central vein without obvious abnormalities.

[0225] Intestinal tissue lesions showed that in the PBS group, the intestinal villi of the duodenum of mice were broken or even lost, resulting in exposure of the mucosal layer; while in the duodenum of mice orally administered lactic acid bacteria, the damage to the intestinal villi was milder, with some arranged in a scattered manner, but the overall morphology was relatively intact. A small amount of inflammatory cell infiltration was observed in the submucosa and muscularis propria, with an increased number of goblet cells and Paneth cells. The results are shown in […]. Figure 25 Compared to the duodenum, the severity of lesions in the jejunum was significantly improved, from... Figure 26 It was found that the intestinal villi of the jejunum in all groups of mice were mostly neatly arranged and relatively intact. In the pNZ8149 / TcCTL-2 group and the pNZ8149 / TcCTL-2+5 group, the intestinal epithelial cells were tightly arranged with few villi detachment, and the intestinal barrier was intact. In addition, the number of goblet cells and Paneth cells in the basal layer of mice that were orally administered lactic acid bacteria increased significantly.

[0226] 3.14 Analysis of differences among gut microbiota species

[0227] Gut microbiota species difference analysis results sequencing as follows Figure 27As shown, the dominant species in the PBS group were unclassified_g_Muribaculaceaes and unclassified_g_Escherichia-Shigella. Muribaculaceae is a family of bacteria in the Bacteroidetes family, which helps break down macromolecules and support the growth of other bacteria; Shigella and Escherichia are Gram-negative bacilli that parasitize the intestine and are common commensal and opportunistic pathogens. The two are closely related genetically, with nucleotide similarity of 80-90%. After oral administration of recombinant lactic acid bacteria, the dominant intestinal flora of mice became Lactococcus lactis, while the proportion and abundance of unclassified_g_Muribaculaceaes and unclassified_g_Escherichia-Shigella showed a decreasing trend.

[0228] 3.15 Complete blood cell count

[0229] Anticoagulated blood was collected from mice and analyzed using a fully automated hematology analyzer. Statistical analysis revealed that, compared with the PBS group, mice orally administered with Lactococcus lactis showed increased numbers of white blood cells, red blood cells, and platelets. The results are as follows: Figure 28 As shown; specifically, the pNZ8149 / TcCTL-2 group showed the most significant increase in erythrocyte count (P < 0.01), while the white blood cell and platelet counts also showed an increasing trend, but the differences were not statistically significant (P > 0.05). Furthermore, statistical analysis of neutrophil, monocyte, and lymphocyte counts ( Figure 28 B) The number of monocytes and lymphocytes in the three groups of mice treated with recombinant lactic acid bacteria increased significantly (P < 0.01). Although the number of neutrophils also increased, only the mice with pNZ8149 / TcCTL-2+5 showed a significant difference from the mice in the PBS group (P < 0.05).

[0230] 3.16 Antibody Level Analysis

[0231] To further evaluate the ability of recombinant Lactococcus lactis to stimulate an immune response, this study used ELISA to detect the dynamic changes in specific antibodies in mouse serum before and after challenge. The results showed that after challenge, the level of specific antibody IgG in the serum of mice immunized with recombinant Lactococcus lactis was significantly upregulated (P < 0.001); conversely, the antibody levels in mice in the PBS group and the empty vector NZ3900 / pNZ8149 group remained low, with no significant changes before and after challenge. Figure 29 A) Further testing for IgG1 and IgG 2a Antibody subtype discovery ( Figure 29 B / C), IgG1 and IgG in immunized mice 2aIt was also significantly upregulated, and IgG 2a The antibody content was greater than that of IgG1, indicating that recombinant lactococci pNZ8149 / TcCTL-2 and pNZ8149 / TcCTL-5 could induce effective specific immune responses in mice, and the immune stimulation effect was stronger when the two recombinant strains were used together.

[0232] Similarly, we also examined the dynamic changes in gut sIgA antibodies. Figure 29 D) To evaluate the intestinal mucosal immune effect. The results showed that before challenge, the intestinal sIgA level of all mice orally immunized with Lactococcus lactis (empty vector and recombinant strain) was significantly higher than that of the PBS group. After challenge, there was no significant change in sIgA in the PBS group, while the antibodies of mice orally immunized with the recombinant strain increased significantly. Among them, the pNZ8149 / TcCTL-2+5 mixed immunization group maintained the highest antibody level, followed by pNZ8149 / TcCTL-2 and pNZ8149 / TcCTL-5. This result confirms that recombinant Lactococcus lactis can not only effectively activate the adaptive immune response, but also significantly enhance the intestinal mucosal immune barrier function by enhancing sIgA secretion. Moreover, the mixed immunization strategy may further enhance the immune effect through inter-strain synergy.

[0233] 3.17 CD4+ T cell differentiation trend

[0234] Analysis of CD4+ T cell subsets in mesenteric lymph nodes and spleen cells using flow cytometry showed that recombinant lactococcus significantly affected the differentiation trend of CD4+ T cells. Figure 30 and Figure 31 As shown, the expression of Th17 cell transcription factor RORγt in the mesenteric lymph nodes and spleen of mice immunized with pNZ8149 / TcCTL-2, pNZ8149 / TcCTL-5, and pNZ8149 / TcCTL-2+5 was significantly upregulated (P < 0.05); at the same time, the proportion of T-bet, a key transcription factor of Th1 cells, was also significantly increased in mice immunized with pNZ8149 / TcCTL-2 and pNZ8149 / TcCTL-2+5 (P < 0.05), suggesting an enhanced Th17 and Th1 response; conversely, the proportion of GATA-3, a marker transcription factor of Th2 cells, remained at a low level in each group, and there was no significant difference. This result confirms that recombinant lactococcus can selectively promote the differentiation of CD4+ T cells in the mesenteric lymph nodes and spleen of mice into Th17 and Th1 subsets, ultimately forming a characteristic distribution pattern in the mesenteric lymph nodes and spleen dominated by Th17 cells, followed by Th1 cells, and with Th2 cells accounting for the least. This specific immunomodulatory effect may be closely related to the mucosal immune protection mechanism induced by the recombinant strain.

[0235] 3.18 Changes in cytokine levels

[0236] To comprehensively evaluate the immunomodulatory effects of recombinant lactococcus and further verify the results of flow cytometry, this study used ELISA to quantitatively analyze the dynamic changes of characteristic cytokines of Th1 (IFN-γ, IL-12), Th2 (IL-4, IL-9, IL-10 and IL-13), and Th17 (IL-17) in the serum of mice before and after challenge with the worm. Figure 32 and Figure 33 Data showed that after mice were challenged with parasites, the recombinant bacterial immunized mice showed a more significant increase in Th1 cytokines (IFN-γ and IL-12) and Th17 (IL-17) cytokines. At the same time, Th2 cytokines IL-4, IL-9 and IL-13 also showed an upward trend. The immunomodulatory cytokine IL-10 remained relatively stable before and after the challenge with parasites, but its level was still significantly higher than that of the PBS control group.

[0237] IV. Discussion

[0238] Previous comparative analysis of transcriptional data of C-type lectins from 17 species of *Toxocara canis* revealed that Tcctl-2 and Tcctl-5 are highly expressed genes during the parasitic stage of the parasite. This invention successfully amplified these genes via PCR. Molecular identification showed that Tcctl-2 is 702 bp in length, encoding a protein of approximately 28 kDa; Tcctl-5 is 546 bp in length, encoding a protein of approximately 23 kDa. Both genes contain signal peptides but lack transmembrane domains, and their secondary structures are primarily α-helices and β-sheets. TcCTL-2 and TcCTL-5 are mainly located in the epidermis, muscle, and intestinal tissues of both male and female parasites, as well as reproductive organs including the ovaries and testes. This result is similar to the tissue localization of TcCTL-4 reported by Wang Bingnan, suggesting that they may be involved in biological functions such as nutrient acquisition, growth and development, reproduction, and invasion of the parasite. Further investigation into the transcriptional levels at different developmental stages revealed that Tcctl-2 was significantly highly expressed in L3 eggs, L3 intestinal larvae, and L3 migrating larvae, but almost not expressed in L5 larvae and male adults. Tcctl-5 was significantly highly expressed in L3 eggs, L4 and L5 larvae, and both male and female adults, but almost not expressed in the L3 intestinal larvae and migrating larvae. Due to the complexity and difficulty in obtaining large quantities of natural TES antigens, commercialization needs cannot be met. Therefore, this study expressed recombinant proteins rTcCTL-2 and rTcCTL-5 using an E. coli prokaryotic expression system. Immunoblot analysis showed that both proteins were specifically recognized by positive serum from *Toxocara canis* infection, indicating good immunogenicity. Animal protection experiments showed that rTcCTL-2 and rTcCTL-5 had a good protective effect against Toxocara canis infection in mice, reducing the migrating larval load in liver and lung tissues by 51.7% and 41.4%, respectively. Compared with the larval load reduction rates of other candidate antigens—rTcSP (25.5%), rTcMUC-3 (20.4%), rTcCTL-1 (16.7%), rTcCTL-4 (18.7%), and rTc-SOD (37.6%)—rTcCTL-2 and rTcCTL-5 showed significantly improved protective efficiency, demonstrating their potential as candidate vaccine antigens. This conclusion provides data reference for subsequent research on recombinant Lactococcus lactis C-type lectin of Toxocara canis.

[0239] In constructing recombinant plasmids, current research often employs double enzyme digestion. However, in this experiment, we found that using enzyme digestion and ligation resulted in low vector recovery efficiency and ligation success rate. Homologous recombination technology, on the other hand, can improve the success rate of ligation between the vector and the target fragment. By adjusting the molar ratio of vector to insert fragment to 1:2, recombinant plasmids pNZ8149 / TcCTL-2 and pNZ8149 / TcCTL-5 were successfully constructed. When electroporating Lactococcus lactis NZ3900 competent cells, we followed the method of Liu Yaxin et al. with some adjustments, and determined the optimal transformation conditions: maintaining a voltage of 2200V, a resistance of 200Ω, and a capacitance of 25μF. After electroporation, the cells were incubated on ice for approximately 10 minutes, followed by incubation at 30℃ for approximately 3 hours. Centrifugation was then performed to collect the cell pellet, which was plated. Under these conditions, the positive transformation rate was the highest. Because the *Lactococcus lactis* NZ3900 lacks the lacF gene, it cannot produce acid through lactose fermentation. However, after transforming the *Lactococcus lactis* with the plasmid pNZ8149 carrying the lacF gene, its acid-producing ability was restored. Therefore, bromocresol purple in Elliker medium under acidic conditions showed a color reaction (changing from purple to yellow), which can serve as an indicator of successful integration of the recombinant plasmid. Recombinant *Lactococcus lactis* were induced to express protein using 10 ng / mL of Nisin. SDS-PAGE and Western blot analysis confirmed that the protein size expressed by recombinant *Lactococcus lactis* pNZ8149 / TcCTL-2 was 25 kDa, and that of recombinant *Lactococcus lactis* pNZ8149 / TcCTL-5 was 20 kDa, consistent with the expected protein size. Furthermore, the induction time had no significant effect on the recombinant protein expression level. Subsequently, the recombinant lactic acid bacteria were induced and validated using different concentrations of inducing agents. It was found that Nisin concentrations between 1 ng / mL and 10 ng / mL could induce the expression of the target protein, with the thickest band observed at a final concentration of 10 ng / mL, representing the optimal induction concentration. To develop a reasonable immunization program, this study evaluated the dosage and colonization time of the recombinant lactic acid bacteria using bacterial counting and intestinal colonization assays, using a concentration of 1×10⁻⁶ Nisin. 9 Recombinant lactic acid bacteria at CFU / mL were orally administered to mice. Intestinal contents of the mice were collected at 7 and 14 days for selective culture and PCR identification. It was found that yellow monoclonal antibodies grew on the selective culture medium at both 7 and 14 days, and the target bands of the same size were found after PCR identification with specific primers. This indicates that the two recombinant lactic acid bacteria can stably colonize the small intestine for at least 14 days. This result is crucial for the timing of immunization programs.

[0240] During intestinal invasion by *Toxocara canis*, intestinal hemorrhage and inflammation are common. *Lactococcus lactis* colonization in the small intestine may reduce intestinal mucosal damage by inducing mucus secretion, strengthening tight junctions, or secreting antimicrobial peptides. Mouse protection experiments showed that recombinant *Lactococcus lactis* pNZ8149 / TcCTL-2 and pNZ8149 / TcCTL-5 achieved 46.4% and 60.7% larval clearance rates in mouse liver and lung tissue, respectively. Interestingly, the combined use of the two strains resulted in better immunization efficacy, with a larval clearance rate of 67.9%. Histopathological analysis showed that the liver and lung tissues of mice in the PBS group were severely damaged, with alveolar erythrocytes filling the alveolar cavities, inflammatory cells clustering together, and hepatic sinusoidal congestion and dilation. The lesions in mice in the NZ3900 / pNZ8149 group were alleviated, and the lesions in the recombinant lactococcus immunization group were significantly improved, with a decrease in the number of alveolar erythrocytes and inflammatory cells, and normal hepatocyte morphology, arranged radially around the central vein. In addition, the duodenum in the PBS group was severely damaged, with broken and detached villi and exposed mucosa, while mice orally administered lactococcus showed only partial breakage of intestinal villi, and the overall structure was relatively intact. Compared with the duodenum, the jejunum of mice in all groups was not significantly damaged. The villi of the jejunum in the immunization group were mostly neatly arranged with little detachment, especially with a significant increase in goblet cells and a significant increase in the number of Paneth cells in the basal layer. The increase in these key cell populations is particularly important for maintaining the intestinal mechanical and immune barriers. Analysis of gut microbiota species differences showed that the dominant species in the gut microbiota of immunized mice changed, with an increase in the abundance of *Lactococcus lactis*, which may reduce damage caused by *Toxococcus canis* larvae invasion by stabilizing the intestinal barrier and microbiota homeostasis. Blood routine statistical analysis showed that the number of red blood cells in the PBS group was much lower than that in the immunized group, possibly due to anemia caused by *Toxococcus canis* infection. Simultaneously, the number of white blood cells in immunized mice also increased, suggesting that recombinant lactic acid bacteria may reduce the damage to the body caused by parasites by mediating the host inflammatory response. Studies have shown that neutrophils may synergistically recruit other immune cells to promote the worm clearance and killing effect. Xuan et al.'s research confirmed that monocytes play a protective role in antimalarial infection through phagocytosis, mediating cytokine production, and antigen presentation. In this study, an increase in monocytes, lymphocytes, and neutrophils was observed in immunized mice, but whether they play a similar role in anti-*Toxococcus canis* infection requires further investigation. The levels of specific IgG, IgG1, and IgG2a antibodies in mice were detected by ELISA. The results showed that the level of specific antibody IgG in the serum of mice orally administered with recombinant lactococcus showed a significant upward trend, indicating that the recombinant lactococcus constructed in this study can induce an effective specific immune response in mice.The study suggests that IgG2a and IgG1 subtypes serve as markers for Th1 and Th2 cells, respectively. The recombinant subunit vaccine constructed by Chen Yijun and Salazar induced a predominantly Th2-type immune response in mice. Further analysis of the IgG subtypes in this study revealed that mice orally administered the recombinant Lactococcus lactis vaccine had slightly higher IgG2a antibody levels than IgG1, indicating that Th1 immunity is primarily generated in mice, with preferential secretion of IgG2a. Notably, a significant increase in sIgA antibody concentration was observed in the mouse intestines, particularly after challenge with worms. Since sIgA is a marker of mucosal immunity, this demonstrates that recombinant Lactococcus lactis can efficiently stimulate a mucosal immune response in the intestines. Flow cytometry analysis revealed a significant polarization in the transcription factor expression profiles of CD4+ T cell subsets in the mesenteric lymph nodes and spleen: RORγt+ cells had the highest proportion, followed by T-bet+ cells, while GATA-3+ cells had the lowest proportion, suggesting that CD4+ T cells preferentially differentiate into Th17 and Th1 subsets. Studies have shown that Th17 cells can not only secrete IL-17A to upregulate the expression of tight junction proteins and mucins in intestinal epithelial cells, but also promote plasma cells to secrete sIgA to enhance intestinal mucosal barrier function. In addition, Th17 cells maintain intestinal mucosal homeostasis by working in conjunction with intestinal epithelial cells, antigen-presenting cells, Treg cells, and intestinal symbiotic flora. Cytokine assays further confirmed that, compared with the PBS group, mice orally administered with recombinant lactic acid bacteria exhibited significant Th1 / Th17 immune polarization characteristics after larval infection, characterized by marked upregulation of IFN-γ, IL-12, and IL-17. These inflammatory cytokines primarily originated from Th1 and Th17 cells, followed by neutrophils and eosinophils. During the acute inflammatory phase, neutrophils were initially recruited to the infection site, followed by persistent infiltration of eosinophils that continued into the chronic infection phase. Studies have shown that both types of granulocytes can produce various pro-inflammatory cytokines, including IFN-γ, by expressing the IL-17A receptor. These cytokines may inhibit Toxocara canis infection and damage by enhancing the inflammatory response and intestinal mucosal immune response. In summary, the oral vaccines pNZ8149 / TcCTL-2 and pNZ8149 / TcCTL-5 can not only effectively induce a high-efficiency intestinal mucosal immune response in mice, but also stimulate the body to produce cellular immunity. pNZ8149 / TcCTL-2 / TcCTL-5 has the best protective effect on mice and can be used as the preferred oral vaccine strain for the prevention of canine toxocariasis.

[0241] 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. Thus, the scope of the present invention should be understood and recognized as covering all such other variations or modifications.

Claims

1. A recombinant lactococcal oral vaccine, characterized in that... Recombinant Lactococcus lactis expresses TcCTL-2 or TcCTL-5, among which, The amino acid sequence of TcCTL-2 is as follows: MFAIFIICLCFHFSTIDACARDADCNLFQVCVNNVCVANNQGCNPPCVAPMVCVAPNCVAVPAPQPQPATTTTAAPAATTTAAPAATTTTARRTCPPSWSLFNNNCYIASVAGRFLF NQASDWCTQTGSRVVWFDQSNAANFNSELAFVNNLAISGGSSRYWIGVNRQFGQWVWTNGSPVILSNWRPSQPDGCCGSNVTCVFVNYANFLGQWDDASCGGLFTNPQGFVCKRPL; The amino acid sequence of TcCTL-5 is as follows: MIAFCLLLTLAISVANANRCDPGWRYSPFTRKCYRFYDHETMWPSAEFSCLFKGGHLISIHSYTDNRFAIELARGAETVWLGNAQFGSSKEYIWSDHTAYNYGSWPDRKRPEKIKTKPCTKLNTTSGEWFQSCCKDPAPYICQKELSDSNAMYRNSEELLGHTSEDRRLESNEDFRRRFFL.

2. The recombinant lactococcus oral vaccine according to claim 1, wherein the recombinant lactococcus simultaneously expresses TcCTL-2 and TcCTL-5.

3. The method for preparing the recombinant lactococcal oral vaccine according to claim 1 or 2, comprising the following steps: Recombinant plasmids loaded with TcCTL-2 and / or TcCTL-5 nucleotide sequences were transfected into Lactococcus lactis.

4. The preparation method according to claim 3, wherein the preparation method comprises the following steps: Pre-cool the electroporation cuvette on ice and irradiate it with UV light in a clean bench. Remove the NZ3900 competent cells from the freezer and thaw them on ice. Add the recombinant plasmid to the thawed competent cells, gently pipette to mix, and then place in an ice bath. Transfer the mixed bacterial solution to the bottom of the electroporation cuvette, remove air bubbles, and wipe the outer wall of the cuvette dry. Set the electroporation parameters to 2200 V, 200 Ω, 25 μF, and 2 mm diameter of the electroporation cuvette. After setting the parameters, perform electroporation. Immediately after the electroporator completes the electroporation, add pre-cooled GM17-MC medium to the cuvette, gently pipette 3-5 times to thoroughly mix the bacterial solution with the medium, and transfer the mixture to pre-cooled sterile centrifuge tubes. Incubate on ice, then transfer to a 30°C incubator and incubate for 2-3 days. h; Take out the bacterial suspension, centrifuge, discard the supernatant, retain about 10% of the bacterial suspension, resuspend it, and spread the bacterial suspension evenly on the surface of Elliker solid medium. Incubate at 30°C upside down for 24 h to screen for positive strains.

5. According to the preparation method of claim 4, the recombinant plasmid is prepared by the following steps: homologous recombination of the target genes expressing TcCTL-2 and TcCTL-5 with the pNZ8149 plasmid.

6. The use of the recombinant lactococcal oral vaccine according to claim 1 or 2 in the preparation of a canine toxocara vaccine.

7. The application according to claim 6, wherein the vaccine is used to reduce the number of Toxocara canis in the liver and lungs.

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