Recombinant lactococcus lactis oral vaccine as well as preparation method and application thereof

Abstract

The invention discloses a recombinant lactococcus lactis oral vaccine as well as a preparation method and application thereof. The recombinant lactococcus lactis oral vaccine is characterized in that the recombinant lactococcus lactis expresses TcCTL-2 and / or TcCTL-5. The oral vaccine not only can effectively induce mice to generate efficient intestinal mucosa immune response, but also can stimulate the organism to generate cellular immunity, and the recombinant lactococcus lactis expressing the TcCTL-2 and the TcCTL-5 has an optimal protection effect on the mice, and can be used as a preferable oral vaccine strain for preventing dog bow head ascariasis.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of molecular immunology, in particular to a recombinant Lactococcus lactis oral vaccine, a preparation method and application thereof. BACKGROUND

[0002] Dog toxocariasis is a global zoonosis caused by Toxocara canis (T. canis) infection. Although chemical drugs can effectively prevent and treat the disease, long-term drug deworming is prone to produce drug-resistant worm strains, and cannot prevent repeated infection of animals, which makes vaccine development the key to prevent and control dog toxocariasis. Unfortunately, there is still a lack of commercialized T. canis vaccine on the market.

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

[0004] T. canis not only causes damage to the host intestinal epithelial barrier during intestinal invasion and colonization, but also induces inflammation, ulceration or intestinal obstruction, which seriously endangers the life and health safety of humans and animals. Studies have shown that T. canis larvae can release a large amount of ES antigen, and C-type lectin is one of the most secreted ES products during the intestinal invasion of larvae, which is involved in nutrient acquisition, growth and reproduction, and worm-host immune interaction, and mediates immune evasion, and has important biological functions. However, the current research on the function of C-type lectin of T. canis is still relatively scarce. SUMMARY

[0005] Based on the above reasons, the present application provides a recombinant Lactococcus lactis oral vaccine and a preparation method thereof. Specifically, in order to achieve the purpose of the present application, the present application proposes the following technical solutions:

[0006] In one aspect, the present application relates 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:

[0008] MFAIFIICLCFHFSTIDACARDADCNLFQVCVNNVCVANNQGCNPPCVAPMVCVAPNCVAVPAPQPQPATTTTAAPAATTTAAPAATTTTARRTCPPSWSLFNNNCYIASVAGRFLFNQASDWCTQTGSRVVWFDQSNAANFNSELAFVNNLAISGGSSRYWIGVNRQFGQWVWTNGSPVILSNWRPSQPDGCCGSNVTCVFVNYANFLGQWDDASCGGLFTNPQGFVCKRPL;

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

[0010] MIAFCLLLTLAISVANANRCDPGWRYSPFTRKCYRFYDHETMWPSAEFSCLFKGGHLISIHSYTDNRFAIELARGAETVWLGNAQFGSSKEYIWSDHTAYNYGSWPDRKRPEKIKTKPCTKLNTTSGEWFQSCCKDPAPYICQKELSDSNAMYRNSEELLGHTSEDRRLESNEDFRRRFFL.

[0011] In a preferred embodiment of the application, the recombinant Lactococcus lactis expresses both TcCTL-2 and TcCTL-5.

[0012] Another aspect of the application relates to a method for the preparation of a recombinant Lactococcus lactis, comprising the steps of:

[0013] transfecting a recombinant plasmid loaded with the TcCTL-2 and / or TcCTL-5 nucleotide sequence into Lactococcus lactis.

[0014] In a preferred embodiment of the application, the method for the preparation of a recombinant Lactococcus lactis comprises the steps of:

[0015] Pre-cool the electric shock cup on ice and irradiate it with ultraviolet light in a clean bench; take the NZ3900 competent cells out of the refrigerator and melt them on ice; take the recombinant plasmid and add it to the thawed competent cells, mix gently and then ice bath; transfer the mixed bacterial solution to the bottom of the electric shock cup, remove the bubbles, and wipe off the moisture on the outer wall of the electric shock cup; set the electric conversion parameters to voltage 2200V, resistance 200Ω, capacitance 25μF, and the diameter of the electric shock cup is 2mm; after the parameters are set, perform electric conversion; after the electric conversion instrument completes the electric conversion, immediately add pre-cooled GM17-MC medium to the electric shock cup, gently blow and suck 3-5 times to mix the bacterial solution and the medium, and then transfer the mixed solution to a pre-cooled sterile centrifuge tube, ice, and then transfer to a 30℃ constant temperature incubator, and incubate for 2-3h; take out the bacterial solution, centrifuge, discard the supernatant, and reserve about 1 / 10 of the bacterial solution, resuspend the bacterial solution, and evenly spread it on the surface of Elliker solid medium, and incubate at 30℃ for 24h, and screen positive strains.

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

[0017] Another aspect of the present application also relates to the use of the above-mentioned recombinant Lactococcus lactis in the preparation of a Toxocara canis vaccine.

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

[0019] The oral vaccine of the present application not only can effectively induce mice to produce efficient intestinal mucosal immune response, but also can stimulate the body to produce cellular immunity, and the recombinant Lactococcus lactis expressing TcCTL-2 and TcCTL-5 has the best protective effect on mice, which can be used as a preferred oral vaccine strain for preventing Toxocara canis disease.

[0020] The present application has the following beneficial effects:

[0021] The present 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 of the present application not only can effectively induce mice to produce efficient intestinal mucosal immune response, but also can stimulate the body to produce cellular immunity, and the recombinant Lactococcus lactis expressing TcCTL-2 and TcCTL-5 has the best protective effect on mice, which can be used as a preferred oral vaccine strain for preventing Toxocara canis disease. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1PCR amplification of Tcctl-2 and Tcctl-5 genes, in which A, PCR amplification result of Tcctl-2; B, PCR amplification result of Tcctl-5. M, DNA molecular weight marker; 1, target gene amplification product;

[0023] Figure 2 PCR amplification of pET32a(+) vector, in which M, DNA molecular weight marker; 1, pET32a(+) vector amplification product;

[0024] Figure 3 Bioinformatics analysis of Tcctl-2, in which A, signal peptide prediction; B, transmembrane region prediction; C, amino acid multiple sequence alignment. Dog roundworm (Genbank accession number: KHN74601.1); cat roundworm (Genbank accession number: MH183028.1); Madina roundworm (Genbank accession number: VDN60627.1); simple heterodera (Genbank accession number: UYRR01036511.1). The lightness of the blue background indicates the degree of sequence conservation, the darker the color, the more conservative the sequence;

[0025] Figure 4 Bioinformatics analysis of Tcctl-5, in which A, signal peptide prediction; B, transmembrane region prediction; C, amino acid multiple sequence alignment. Dog roundworm (Genbank accession number: KHN74601.1); roundworm (Genbank accession number: VDD85501.1); cotton rat filaroid (Genbank accession number: VDK89318.1); conjunctival sucking roundworm (Genbank accession number: VDN60627.1); Malay bruchia (Genbank accession number: KAK6107812.1). The lightness of the blue background indicates the degree of sequence conservation, the darker the color, the more conservative the sequence;

[0026] Figure 5 Expression and purification of rTcCTL-2, in which M, protein molecular weight marker; 1, pET-32a empty expression result; lanes 2-3, rTcCTL-2 expression result;

[0027] Figure 6 Expression and purification of rTcCTL-5, in which M, protein molecular weight marker; lanes 1-2, pET-32a empty expression result; lanes 3-4, rTcCTL-5 expression result;

[0028] Figure 7Fig. 6 is a result of immunoblotting for recombinant proteins, wherein A, a result of immunoblotting for rTcCTL-2; B, a result of immunoblotting for rTcCTL-5. M, a protein molecular weight standard; 1, a result of blotting analysis of recombinant proteins with positive serum of A. caninum infection;

[0029] Figure 8 Fig. 7 is an immunofluorescence localization of TcCTL-2 in A. caninum, wherein Cu, cuticle; Iw, intestinal wall; Iv, intestinal villus; Mu, muscle; Uw, uterine wall; Tw, testis wall; scale bar "-" indicates 100 μm;

[0030] Figure 9 Fig. 8 is an immunofluorescence localization of TcCTL-5 in A. caninum, wherein Cu, cuticle; Iw, intestinal wall; Iv, intestinal villus; Mu, muscle; Uw, uterine wall; Tw, testis wall; scale bar "-" indicates 100 μm;

[0031] Figure 10 Fig. 9 is a relative expression analysis of Tcctl-2 and Tcctl-5 genes at different developmental stages, wherein A, a relative expression analysis of Tcctl-2; B, a relative expression analysis of Tcctl-5. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, ns indicates no significant difference, and each gene is relative to the stage with the lowest expression;

[0032] Figure 11 Fig. 10 is a PCR amplification of Tcctl-2 and Tcctl-5 genes, wherein A, a result of PCR amplification of Tcctl-2; B, a result of PCR amplification of Tcctl-5. M, a DNA molecular weight standard; 1, an amplification product of a target gene;

[0033] Figure 12 Fig. 11 is a verification and linear amplification of pNZ8149 plasmid, wherein A, a result of verification of pNZ8149 plasmid; B, a linear amplification product of pNZ8149 plasmid; M, a DNA molecular weight standard; 1, a pNZ8149 plasmid amplification product;

[0034] Figure 13 Fig. 12 is a result of electrotransformation of recombinant Lactococcus lactis, wherein A, a result of electrotransformation of recombinant Lactococcus lactis pNZ8149 / TcCTL-2; B, a result of electrotransformation of recombinant Lactococcus lactis pNZ8149 / TcCTL-5;

[0035] Figure 14PCR identification of recombinant Lactococcus lactis, wherein, A, PCR identification result of recombinant Lactococcus lactis pNZ8149 / TcCTL-2; B, PCR identification result of recombinant Lactococcus lactis pNZ8149 / TcCTL-5. M, DNA molecular weight marker; 1-2, PCR amplification product of positive bacteria;

[0036] Figure 15 SDS-PAGE analysis of expression products of recombinant Lactococcus lactis, wherein, A, analysis of expression products of recombinant bacteria pNZ8149 / TcCTL-2; B, analysis of expression products of recombinant bacteria pNZ8149 / TcCTL-5. M, protein molecular weight marker; 1, supernatant induction expression result of bacterial liquid; 2, induction expression result of empty vector pNZ8149; 3, induction expression result of bacterial body precipitate after 8h; 4, induction expression result of bacterial body precipitate after 12h;

[0037] Figure 16 Protein expression analysis of pNZ8149 / TcCTL-2 induced by different concentrations of Nisin, wherein, M, protein molecular weight marker; 1, empty bacteria pNZ8149; 2, 1ng / mL Nisin induction; 3, 3ng / mL Nisin induction; 4, 5ng / mL Nisin induction; 5, 10ng / mL Nisin induction;

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

[0039] Figure 18 Intestinal colonization analysis of recombinant Lactococcus lactis;

[0040] Figure 19 PCR detection of intestinal colonization of recombinant Lactococcus lactis pNZ8149 / TcCTL-2, wherein, A, 7-day colonization detection result; B, 14-day colonization detection result. M, DNA molecular weight marker; 1-2, amplification product of duodenum washing liquid; 3-4, amplification product of jejunum washing liquid; 5-6, amplification product of ileum washing liquid; 7-8, amplification product of fecal suspension;

[0041] Figure 20Detection of recombinant Lactococcus lactis pNZ8149 / TcCTL-5 intestinal colonization by PCR, in which A, 7-day colonization detection results; B, 14-day colonization detection results. M, DNA molecular weight marker; 1-2, duodenum flush liquid amplification product; 3-4, jejunum flush liquid amplification product; 5-6, ileum flush liquid amplification product; 7-8, fecal suspension amplification product;

[0042] Figure 21 Changes in the weight gain rate of mice in the control and immunized groups, in which * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and ns indicates no significant difference;

[0043] Figure 22 Organ index of mice, in which * indicates P < 0.05, and ns indicates no significant difference;

[0044] Figure 23 L3 larvae recovered from mice in vivo, in which * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and ns indicates no significant difference;

[0045] Figure 24 Changes in the liver and lung histopathology of mice, in which the scale “—” is 50 μm; the blue arrow indicates inflammatory cell infiltration; and the yellow arrow indicates severe hemorrhage;

[0046] Figure 25 Changes in the duodenum histopathology after challenge, in which the scale “—” is 50 μm; the blue arrow indicates goblet cells; and the yellow arrow indicates Paneth cells;

[0047] Figure 26 Changes in the jejunum histopathology after challenge, in which the scale “—” is 50 μm; the blue arrow indicates goblet cells; and the yellow arrow indicates Paneth cells;

[0048] Figure 27 Inter-species difference analysis, in which the horizontal axis is time; and the vertical axis is relative abundance. Others indicates the relative abundance of all other organisms other than the 20 microbial categories in the figure;

[0049] Figure 28 Complete blood count results, in which * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and ns indicates no significant difference;

[0050] Figure 29Fig. 6 shows the changes in the levels of antibodies in mice, wherein A, IgG antibody levels were detected; B, IgGl antibody levels were detected; C, IgG2a antibody levels were detected; and D, sIgA antibody levels were detected. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and ns indicates no significant difference.

[0051] Figure 30 Fig. 7 shows the changes in the levels of transcription factors in the mesenteric lymph nodes of immunized mice, wherein A-B, GTAT-3 percentages were detected; C-D, T-bet percentages were detected; and E-F, RORyt percentages were detected. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and ns indicates no significant difference.

[0052] Figure 31 Fig. 8 shows the changes in the levels of transcription factors in the spleen of mice, wherein A-B, GTAT-3 percentages were detected; C-D, T-bet percentages were detected; and E-F, RORyt percentages were detected. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and ns indicates no significant difference.

[0053] Figure 32 Fig. 9 shows the changes in the levels of Thl and Thl7 type cytokines, wherein * 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 Fig. 10 shows the changes in the levels of Th2 type cytokines, wherein * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, **** indicates P < 0.0001, and ns indicates no significant difference. DETAILED DESCRIPTION

[0055] In order to make the objects, technical solutions, and advantages of the present application clearer, the present application will be further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and should not be used to limit the present application.

[0056] It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the present application. 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 the present application belongs.

[0057] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments in accordance with the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and / or "comprising," when used in this specification, specify the presence of stated features, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof.

[0058] Example 1

[0059] I. Materials and reagents

[0060] 1.1 Strains, strains and vectors

[0061] Escherichia coli BL21 (DE3), Escherichia coli Rosetta (DE3) were purchased from Beijing Tiangen Biochemical Technology Co., Ltd.; DH5a competent cells of Escherichia coli were purchased from Shanghai Sunway Biotech Co., Ltd.; pET32a(+) vector was preserved in the laboratory (commercially purchased); Lactococcus lactis NZ3900 strain and pNZ8149 plasmid were purchased from Hangzhou Baosai Biotechnology Co., Ltd.

[0062] 1.2 Experimental animals and serum

[0063] 6-week-old SPF male C57BL / 6 mice were purchased from Beijing Sbieluo; positive serum of A. caninum infection was prepared by conventional process and stored at -20℃.

[0064] 1.3 Culture medium

[0065] (1) GM17 broth medium: weigh 16.92 g of M17 medium dry powder, add ddH2O to 390 mL, sterilize at 115℃ for 20 min, after cooling, add 10 mL of 20% glucose stock solution, and store at 4℃.

[0066] (2) GM17 agar medium: weigh 8.46 g of M17 medium dry powder, 3 g of agar powder, add ddH2O to 195 mL, sterilize at 115℃ for 20 min, naturally cool to 60℃, mix evenly with 5 mL of 20% glucose stock solution, pour into sterile culture dishes, naturally cool and solidify, and store at 4℃.

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

[0068] (5) GM17-MC medium: weigh M17 broth medium 4.23 g, CaCl20.02 g, MgCl20.19 g, add ddH2O to constant volume of 92.5 mL, high-pressure steam sterilization at 115°C for 20 min, after cooling, add 7.5 mL of 20% glucose stock solution, store at 4°C.

[0069] (6) Elliker medium: weigh 4 g of tryptone, 1 g of yeast extract, 0.8 g of sodium chloride, 0.3 g of sodium acetate (anhydrous), 0.1 g / L ascorbic acid, 3 g of agar, add ddH2O to constant volume of 193 mL, high-pressure sterilization at 121°C for 20 min, after cooling to 60°C, add 5 mL of 20% lactose stock solution, add 2 mL of 0.4% bromocresol purple to a final concentration of 0.004%, pour into sterilized flat dishes, cool and solidify, and store at 4°C.

[0070] 1.4 Main reagents

[0071] (1) 0.4% bromocresol purple: weigh 0.4 g of bromocresol purple and dissolve in 20 mL of 0.02 mol / L NaOH solution, add ddH2O to constant volume of 100 mL, filter sterilization with 0.22 μm microporous filter, store at 4°C.

[0072] (2) 0.02 M NaOH solution: 0.08 g NaOH is dissolved in 50 mL ddH2O, constant volume to 100 mL, store at room temperature.

[0073] (3) 0.1 mg / mL Nisin stock solution: weigh 0.01 g of nisin, dissolve in 100 mL of 0.05% acetic acid solution, filter sterilization, store at -20°C.

[0074] (4) 20% glucose stock solution: weigh 20 g of glucose, add 100 mL of ddH2O, heat and stir until completely dissolved, filter sterilization, store at 4°C.

[0075] (5) Solution I: weigh 34.23 g of sucrose, 20 mL of glycerol, add ddH2O to constant volume of 200 mL, high-pressure sterilization at 115°C for 20 min, store at 4°C.

[0076] (6) Solution II: weigh 17.12 g of sucrose, 10 mL of glycerol, EDTA 1.86 g, add ddH2O to constant volume of 100 mL, high-pressure steam sterilization at 115°C for 20 min, store at 4°C.

[0077] II. Test method

[0078] 2.1 Extraction of total RNA from canine toxocara

[0079] The RNA of each stage of canine Toxocara worm body / ova was extracted, and the specific operation steps were as follows: 50-80 mg of tissue was taken from the canine Toxocara worm of different stages stored in the ultra-low temperature refrigerator, 1 mL of Trizol was added, homogenized, and placed at room temperature for 15 min to fully lyse the tissue sample; centrifuged at 12,000 rpm / min at 4 DEG C for 15 min, and the supernatant was taken into a 1.5 mL EP tube; 0.2 mL of chloroform was added to each 1 mL of supernatant, and it was shaken vigorously and placed at room temperature for 3 min; centrifuged at 12,000 rpm / min at 4 DEG C for 15 min, and the upper colorless water sample was taken; 0.5 mL of isopropyl alcohol was added to each 1 mL of supernatant, and it was repeatedly inverted and mixed, and then placed at room temperature for 10 min; centrifuged at 12,000 rpm / min at 4 DEG C for 15 min to precipitate the RNA, and the supernatant was discarded; 1 mL of pre-cooled 75% ethanol was added to the tube to wash the precipitate twice, and it was vortexed and mixed. Centrifuged at 7,500 rpm / min at 4 DEG C for 5 min, the supernatant was discarded, and the RNA was slightly dried, 20 μL of RNase Free was added, and it was stored at -80 DEG C.

[0080] 2.2 Synthesis of first strand cDNA

[0081] The total RNA of the extracted canine Toxocara was used as a template to reverse transcribe cDNA, which was stored at -20 DEG C.

[0082] 2.3 Primer design and synthesis

[0083] According to the GenBank data, Tcctl-2 (Genebank accession number: KR052876.1) and Tcctl-5 (Genebank accession number: JPKZ01002894.1) gene full sequence primers were designed by using Primer 5.0 software, and the sequence information is shown in Table 1, and the primers were synthesized by Shanghai Shengong Biological Engineering Co., Ltd.

[0084] Table 1 Amplification primers of target genes

[0085]

[0086] 2.4 Amplification and cloning of target genes

[0087] (1) Amplification and recovery of target genes: 3rd instar larva cDNA was used as a template to amplify Tcctl-2 and Tcctl-5 genes. After 1% agarose gel electrophoresis analysis of the PCR product, the gel block containing the target band was cut and weighed in a 1.5 mL EP tube, and the gel recovery was performed 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 genes were predicted by TMHMM 2.0 and SignalP 6.0; protein secondary structure prediction and amino acid multiple sequence alignment were completed using JalView software.

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

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

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

[0101] (3) Induction of expression: The expression bacteria identified as positive by PCR were selected for expansion and culture. When the OD600 value of the bacterial liquid reached about 0.6, the inducer IPTG was added to the culture medium to make its final concentration 1 mmol / L, and the induction was started. After 7 h of continuous induction culture, 1 mL of bacterial liquid was taken for sample preparation, and SDS-PAGE was used to detect whether rTcCTL-2 and rTcCTL-5 were expressed.

[0102] (4) Solubility analysis: The bacterial liquid expressing the target protein was placed in a 37°C constant temperature incubator and cultured at 140 rpm / min. The OD600 value of the bacterial liquid was monitored in real time. When it reached about 0.6, the target protein was induced for expression according to the best induction time and temperature determined. After induction, 2 mL of bacterial liquid was taken and centrifuged at 7,000 rpm / min for 10 min at 4°C. The bacterial pellet was collected, 10 mL of bacterial lysis solution containing 0.05 mM Tris-HCl was added to the pellet, and the bacterial lysis solution was mixed well with the bacterial lysis solution by repeated blowing and sucking. Then the cell suspension was treated by ultrasonic crushing until the liquid became clear. The remaining bacterial pellet was resuspended with 2M, 4M and 8M urea, respectively, and centrifuged at 7,000 rpm / min for 10 min after each resuspension. The supernatant was collected. 20 μL of supernatant and 5 μL of 5×SDS-PAGE protein loading buffer were mixed and boiled for 10 min. The solubility of rTcCTL-2 and rTcCTL-5 was analyzed by SDS-PAGE.

[0103] (5) Protein purification: Based on the results of solubility analysis, the recombinant protein was purified according to the Ni+affinity chromatography instructions; the purified protein was detected by SDS-PAGE, then concentrated by ultrafiltration, and after determining the protein purity, it was aliquoted and stored in a -80°C ultra-low temperature refrigerator.

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

[0105] (1) Preparation of hyperimmune serum: 10 six-week-old male C57BL / 6 mice were selected and randomly divided into two groups (n=5) for the preparation of hyperimmune serum of recombinant proteins rTcCTL-2 and rTcCTL-5. The Freund's adjuvant was mixed with the purified protein at the same ratio, and the protein concentration was adjusted to 500 μg / mL. The mice were inoculated subcutaneously, with an immunization dose of 50 μg per mouse, and immunized every 7 days for a total of 3 times. Seven days after the last immunization, the whole blood of the mice was collected by the retro-orbital plexus method, and after being placed at room temperature for 2 h, it was centrifuged at 3500 rpm / min for 5 min to separate the serum, which was then aliquoted and stored at -20°C.

[0106] (2) Western blot analysis: SDS-PAGE gel electrophoresis was performed on the purified recombinant protein. After electrophoresis, the gel block of the target lane was cut and placed in the transfer buffer for equilibration treatment, repeated 3 times, each for 5 min. At the same time, the cut NC membrane and 3 layers of filter paper were soaked in the transfer buffer for 15 min. The gel block, 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 in order from bottom to top to form a "sandwich" structure, which was placed in the transfer slot, and the current size was set to 1 mA / cm 2 After transfer, the NC membrane was peeled off and washed with TBST 3 times for 5 min each time. Then blocking solution containing 5% skimmed milk was added to the system, and blocking treatment was carried out at room temperature for 2 h to block non-specific binding sites. After blocking, the blocking solution was discarded and the NC membrane was washed with TBST solution 3 times for 5 min each time. The positive serum of canine toxocara infection diluted with PBS was added as the primary antibody, and incubated overnight in a 4°C refrigerator. The next day, the primary antibody solution was discarded and the NC membrane was washed with TBST 3 times for 5 min each time to remove the unbound primary antibody. Then, the HRP-labeled goat anti-mouse IgG diluted with PBS was added as the secondary antibody, and after incubation at room temperature for 2 h, the secondary antibody was discarded, the membrane was washed with TBST solution, and the NC membrane was placed in a dark box. The ECL developing solution was evenly dropped onto the surface of the NC membrane, and after incubation in the dark for 2-3 min, the film was developed immediately.

[0107] 2.8 Immunofluorescence tissue localization

[0108] The preserved paraffin sections of canine Toxocara were taken out and the immunofluorescence tissue localization was completed according to the following steps:

[0109] 1) Baking the slice: the paraffin section was placed in an oven at 60°C for 1 h;

[0110] 2) De-waxing: the paraffin section was de-waxed with xylene I for 15 min and then de-waxed with xylene II for 15 min;

[0111] 3) Rehydration: the paraffin section was sequentially immersed in 100% alcohol I, 100% alcohol II, 95% alcohol, 85% alcohol and 75% alcohol for 5 min each time, and then washed with PBS for 5 min;

[0112] 4) Blocking: the residual liquid outside the sample was absorbed with filter paper, 5% skimmed milk powder was added dropwise, and the blocking was performed at 37°C for 1 h;

[0113] 5) Incubation of the first antibody: after the blocking was completed, the sample was washed with PBS for 3 times, 5 min each time, and then dried with filter paper, 1:100 high-titer serum was added dropwise, and the incubation was performed at 4°C overnight;

[0114] 6) Incubation of the second antibody: the sample was washed with PBS for 3 times, 5 min each time, and then dried with filter paper, 1:200 FITC-labeled goat anti-mouse IgG was added dropwise, and the incubation was performed at room temperature in the dark for 1 h;

[0115] 7) Nucleus staining: the sample was washed with PBS for 3 times, 5 min each time, and then dried with filter paper, DAPI reagent was added dropwise, and the incubation was performed in the dark for 3-5 min;

[0116] 8) Mounting: the sample was washed with PBS for 3 times, 5 min each time, anti-fluorescence quenching agent was added dropwise, and a small amount of nail polish was applied around the cover glass for mounting, and then the sample was observed and photographed under a fluorescence microscope after drying.

[0117] 2.9 Analysis of the transcription level of Tcctl-2 and Tcctl-5 in different stages of the worm

[0118] According to the gene sequences of Tcctl-2 and Tcctl-5, qPCR primers were designed, the primer information is shown in Table 2, and the designed primers were synthesized by Shengong Bioengineering (Shanghai) Co., Ltd. 18S RNA was used as an internal reference gene to determine the expression of Tcctl-2 and Tcctl-5 genes in different developmental stages of the worm, and the results were calculated by 2-△△ct method, and the data visualization was processed by using GraphPad 9.0 software.

[0119] Table 2 qPCR primer sequences

[0120]

[0121] 2.10 Primer design and synthesis

[0122] The linearization primers of pNZ8149 vector were designed using software Snapgene 6.0 and a 6xHis tag was added, while the primers for amplifying Tcctl-2 and Tcctl-5 with pNZ8149 homologous arms were designed. The primer sequence information is shown in Table 3.

[0123] Table 3 Gene amplification primers

[0124]

[0125] Note: Lowercase letters represent vector homologous sequences; underlined letters represent 6xHis tag.

[0126] 2.11 Construction of recombinant Lactococcus expression vector

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

[0128] (2) Extraction of pNZ8149 plasmid: The pNZ8149 / NZ3900 Lactococcus lactis stored at 4°C was taken out, a single colony was picked and inoculated into 2 mL M17 medium, and incubated at 30°C overnight. The next day, 1 mL of the overnight culture was transferred to 9 mL of M17 broth and incubated for 8 h. The pNZ8149 plasmid was extracted according to the kit instructions, and the concentration was determined and stored at -20°C for future use. The extraction steps are described in Chapter 2.1.2.6. It should be noted that after adding Buffer P1, 20 mg / ml lysozyme should be added, and the tube should be gently shaken every 5 min or so at 37°C water bath for 30 min to ensure complete lysis of the cell wall.

[0129] (3) Linearization of pNZ8149 plasmid and gel recovery: Using the extracted pNZ8149 plasmid as a template, specific primers pNZ8149 F / R were used to amplify the linearized vector by PCR, and the gel block with the same size as the target band was cut and recovered.

[0130] (4) Homologous recombination of target gene and vector: According to the ClonExpress Ultra One Step Cloning Kit V2 kit instructions, the molar ratio of vector to insert was 1:2, and the reaction was carried out on ice. The homologous recombination of the gel-recovered target gene and the linearized vector was carried out.

[0131] After the above reagent is added to the reaction tube, a pipette gun is used to gently blow and suck to mix, then a 50°C water bath is used for reaction for 5 min, and after the reaction is completed, the reaction tube is placed on ice to cool.

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

[0133] A single colony is picked from the plate using a sterile inoculation loop and inoculated into 1 mL of GM17 medium, which is then placed in a constant temperature condition at 30°C and incubated overnight. The overnight cultured bacterial solution is removed and transferred to 9 mL of GM17 liquid medium, which is then placed in a constant temperature condition at 30°C and incubated for about 10 hours. The bacterial solution is inoculated into 100 mL of GSGM17 medium, which is then incubated at 30°C until the OD value is about 0.3. The medium is removed and centrifuged at 4°C and 5000g for 15 min. The supernatant is discarded and the bacterial sediment is collected. 25 mL of pre-cooled Solution I solution is added, and the bacterial sediment is repeatedly blown and beaten with a pipette gun. After mixing, the mixture is centrifuged at 4°C and 5000g for 15 min, and the supernatant is discarded. 20 mL of pre-cooled Solution II solution is added to the bacterial sediment, and the sediment is gently resuspended until it is completely dissolved. The resuspended solution is placed on ice for 15 min. The mixture is centrifuged at 4°C and 5000g for 15 min, and the supernatant is discarded. 25 mL of pre-cooled Solution I solution is added to the sediment, and the mixture is vortexed to resuspend the bacteria. The mixture is centrifuged at 4°C and 5000g for 15 min, and the supernatant is carefully discarded. 1 mL of pre-cooled Solution I solution is added, and the mixture is repeatedly blown and mixed until the bacterial sediment is completely dissolved. The mixture is then aliquoted and stored in a -80°C ultra-low temperature refrigerator.

[0134] (6) Electroporation:

[0135] Pre-cool the electroporation cuvette on ice and irradiate it with UV light for about 20 min in a clean bench; take the NZ3900 competent cells from -80℃ ultra-low temperature freezer and melt them on ice; pipette 10 μL ligation product into the thawed competent cells, mix gently, then ice-bath for 10 min; transfer the mixed bacterial solution to the bottom of the electroporation cuvette, remove the air bubbles, and wipe off the moisture on the outer wall of the electroporation cuvette; set the electroporation parameters as follows: voltage 2200 V, resistance 200 Ω, capacitance 25 μF, and cuvette diameter 2 mm; after the parameters are set, perform the electroporation; after the electroporation instrument finishes the electroporation, immediately add 900 μL pre-cooled GM17-MC medium into the electroporation cuvette, gently blow and suck 3-5 times to mix the bacterial solution and the medium, and transfer the mixed solution to a pre-cooled sterile centrifuge tube, stand on ice for 10 min, then transfer to a 30℃ constant temperature incubator, and stand for 2-3 h; take out the bacterial solution, centrifuge at 12,000 rpm / min for 3 min, discard the supernatant, reserve about 100 μL of the bacterial solution, resuspend it, and evenly spread it on the surface of Elliker solid medium, and culture at 30℃ for 24 h, observe the single colony morphology, and screen the positive strain.

[0136] 2.12 Identification of recombinant Lactococcus lactis expression vector

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

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

[0139] Table 4 pNZ8149 primer sequences

[0140]

[0141] Send the plasmid with positive PCR identification to Beijing Qikang Biotechnology Co., Ltd. for sequencing. The plasmids with correct sequences are named as pNZ8149 / TcCTL-2 and pNZ8149 / TcCTL-5.

[0142] 2.13 Induced expression and optimization of recombinant Lactococcus lactis

[0143] (1) Recombinant Lactococcus lactis induced expression and verification: Take the successfully constructed recombinant Lactococcus lactis pNZ8149 / TcCTL-2, pNZ8149 / TcCTL-5 and empty bacteria pNZ8149 respectively inoculated into 10 mL GM17 liquid medium, 30℃ static culture for 10h or so; with 1:10 ratio of bacteria liquid to GM17 medium, when OD600 value is about 0.4, add inducer Nisin to the bacteria liquid, the working concentration is 10 ng / mL, 30℃ constant temperature conditions, static induction 8h and 12h; after induction, use ultrasonic disruptor to lyse the bacteria, the lysate is centrifuged at 12,000 rpm / min at 4℃ for 10 min, carefully separate and collect the supernatant and bacterial pellet, add 5x SDS-PAGE protein loading buffer and sample at a ratio of 1:4 and mix evenly, mix the sample in boiling water bath for 10 min, take out the sample for SDS-PAGE electrophoresis analysis.

[0144] (2) Recombinant Lactococcus lactis induced concentration optimization: when the OD600 value of the bacteria liquid is about 0.4, add different concentrations of Nisin to the positive expression bacteria pNZ8149 / TcCTL-2, pNZ8149 / TcCTL-5 and empty bacteria pNZ8149 respectively, so that the final concentration of Nisin is 3 ng / mL, 5 ng / mL, 10 ng / mL and 20 ng / mL, and then induce expression for 8h. Take 1 mL of bacteria liquid for sample preparation and detection to determine the optimal induction concentration.

[0145] 2.14 Detection of intestinal colonization ability

[0146] The recombinant Lactococcus lactis pNZ8149 / TcCTL-2 and pNZ8149 / TcCTL-5 were induced to culture for 8h, centrifuged at 12,000 rpm / min at 4℃ for 5 min, the supernatant was discarded, 1 mL PBS was added to resuspend the bacterial pellet, vortexed uniformly, and then diluted by 10 times gradient. Take 200 μL of dilution liquid with dilution degree of 10 5 -10 8 The dilution liquid was evenly coated on the GM17 plate and cultured at 30℃ overnight, and three repeats were made for each concentration gradient. The next day, the colonies were counted by plate counting method.

[0147] Take 12 C57BL / 6 male mice, pNZ8149 / TcCTL-2 and pNZ8149 / TcCTL-5, 6 in each group. The concentration of the induced recombinant Lactococcus lactis was adjusted to 1×10 9CFU / mL, each mouse was orally gavaged with 0.3 mL of bacteria solution, and normal drinking water and food were provided during the experiment. On the 7th day and the 14th day, 3 mice from each group were sacrificed, the intestinal tract of the mice was washed with sterile PBS, the washing solution was collected, vortexed and then left for 30 min. Supernatant was collected by centrifugation and spread on Elliker solid plates. After 24 h of culture, the colony morphology was observed, and yellow single colonies were picked for bacterial solution PCR identification.

[0148] 2.15 Animal immunization

[0149] Ninety 6-week-old SPF male C57BL / 6 mice were randomly divided into five groups, 18 mice in each group, namely PBS control group, pNZ8149 / NZ3900 empty vector group, pNZ8149 / TcCTL-2 immunization group, pNZ8149 / TcCTL-5 immunization group and pNZ8149 / TcCTL-2+pNZ8149 / TcCTL-5 mixed immunization group (hereinafter referred to as pNZ8149 / TcCTL-2+5). The prepared recombinant Lactococcus lactis, empty vector (freshly prepared for each immunization) and PBS were used to immunize mice by oral gavage, with a dose of 300 μL per mouse. Immunization was completed in three times, with an interval of 14 days between each immunization, and the immunization grouping information is shown in Table 5:

[0150] Table 5 Immunization animal grouping information

[0151]

[0152] 2.16 Mouse challenge

[0153] On the 7th day after the last immunization, all mice were artificially infected with Toxocara canis by gavage, with 1000 L3 stage worm eggs per mouse; before challenge, the L3 stage worm eggs were incubated in a 28°C constant temperature incubator for 1 h to restore the activity of the worm eggs and ensure the infection effect. On the 5th day after challenge, all mice were sacrificed by decapitation, and liver and lung tissues were collected, the number of larvae in the tissues was recorded and the reduction rate of larval load was calculated.

[0154] 2.17 Body weight change and organ index

[0155] The body weight change of the mice was recorded regularly every week after the first immunization; on the 5th day after challenge, the heart, liver, spleen and lung of the mice were dissected and weighed, and the body weight of the mice on the 0th day was taken as the initial weight. The weight gain rate was calculated by formula (1), and the organ coefficient was calculated by formula (2):

[0156]

[0157]

[0158] 3.9 Comparison of pathological changes

[0159] To evaluate the liver, lung tissue pathological changes of each group of mice, the liver, lung tissue of mice after infection was collected, the tissue samples were fixed with 4% paraformaldehyde fixing solution, and sent to Wuhan Bolif Biological Technology Co., Ltd. for pathological section HE staining.

[0160] 2.18 Changes in intestinal flora

[0161] The intestinal contents of the test group and the control group mice were taken at 0 days, 14 days, 28 days, 35 days and 40 days, respectively, with 3 samples repeated in each group. After the mice were executed by cervical dislocation, the abdominal hair of the mice was soaked with 75% ethanol, the intestinal contents were collected in a cryopreservation tube using sterile surgical scissors and cotton swabs, and quickly placed into liquid nitrogen, and then transferred to -80°C for storage. The intestinal contents were stored with dry ice and sent to Chengdu Luoning Biological Technology Co., Ltd. for 16S rRNA gene V3-V4 region sequencing. After sequencing, the Illumina NextSeq 2000 sequencing system was used for analysis.

[0162] 2.19 Blood routine analysis

[0163] On the 5th day after infection, 0.1 mL of tail vein blood of mice in each group was collected in an anticoagulant blood collection tube, and blood cell count was performed using a blood routine analyzer, and the number of red blood cells, white blood cells (neutrophils, lymphocytes and mononuclear cells) and platelets was counted.

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

[0165] (1) Sample collection and processing:

[0166] Serum: Collect the tail vein blood of mice before infection and on the 5th day after infection, and let stand at room temperature for 1 h, then centrifuge at 3500 rpm / min for 5 min to separate the serum, and store at -20°C after sterile packaging for IgG / IgG1 / IgG 2a antibody and cytokine detection.

[0167] Intestinal contents: Before infection and on the 5th day after infection, 3 mice were selected from each group to cut off the small intestine with a length of about 5 cm, collect the intestinal contents in a centrifuge tube, and add 500 μL of PBS solution containing 0.05 mol / L EDTA, shake well, and stand at 4°C for 12 h. 4°C, 10,000 rpm / min, 5 min, collect the supernatant, and store at -20°C for 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 mixture was incubated 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 a microplate 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 the end of lysis, 3 mL PBS was added to the spleen cells, and 500 μL PBS was added to the mesenteric lymphocytes, and centrifuged at 450 g for 5 min at room temperature;

[0178] 6. Resuspension: The supernatant was discarded, and 1 mL PBS and 500 μL PBS were added to the spleen cells and mesenteric lymph node cells, respectively, to resuspend the cells;

[0179] 7. Dilution: 10 μL of the spleen cell resuspension and 990 μL of PBS were taken, and diluted 100 times; 100 μL of the lymphocyte resuspension and 900 μL of PBS were taken, and diluted 10 times;

[0180] 8. Counting: 10 μL of trypan blue staining solution, 20 mL of PBS, and 10 μL of cell resuspension were added to a 1.5 mL EP tube, and mixed, and then 10 μL of the mixture was taken for counting.

[0181] (2) Cell staining:

[0182] 1. Preparation of antibodies: CD3+, CD4+ antibodies were diluted with PBS at a ratio of 1:200, and this step was performed on ice;

[0183] 2. Incubation of antibodies: According to the cell counting results, 4 x 10 6 cells were added to a 1.5 mL EP tube, and 200 μL of PBS was added, and centrifuged at 450 g for 5 min at room temperature, and the supernatant was discarded, and 30 μL of antibodies was added to the cell pellet, and incubated at 4°C for 25 min in the dark;

[0184] 3. Centrifugation: After the end of incubation, 200 μL of PBS was added, and centrifuged at 450 g for 5 min at room temperature;

[0185] 4. Fixation: The supernatant was discarded, and 100 μL of fixing solution was added to each tube, and incubated at room temperature in the dark for 1 h, and Perm Diluent and Permeablization Concentrate were mixed at a ratio of 3:1 to prepare the fixing solution;

[0186] 5. Washing: 10 x Permeablization and ddH2O were mixed at a ratio of 1:9 to prepare Washing Buffer, and 300 μL of Washing Buffer was added to each tube of fixing solution, and centrifuged at 450 g for 5 min at room temperature, and then the speed was changed to 8500 g, and centrifuged for another 2 min;

[0187] 6. Preparation of antibodies: GATA3, T-bet, and RORγt antibodies were diluted with PBS at a ratio of 1:200, and this step was performed on ice;

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

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

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

[0191] 2.22 Analysis of cytokine levels

[0192] The Th1-type cytokines IFN-γ and IL-12, the Th2-type cytokines IL-4, IL-9, IL-10, and IL-13, and the Th17-type cytokine IL-17 were detected using ELISA kits before and after infection, according to the kit instructions. The concentrations of the above cytokines in the mouse serum were determined by establishing a standard curve, and the concentrations of the cytokines were calculated.

[0193] III. 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. The target bands were consistent with the expected size, and sequencing alignment showed that Tcctl-2 was 702 bp (A) with the nucleotide sequence of SEQ ID No. 1, and Tcctl-5 was 546 bp (B) with the nucleotide sequence of SEQ ID No. 2, which was consistent with the reference sequence. Figure 1 Figure 1 A), and the signal peptide of Tcctl-5 was 17 amino acids (A) in length; transmembrane region structure prediction analysis showed that Tcctl-2 had a transmembrane region of 21 amino acids (B) and Tcctl-5 had a transmembrane region of 22 amino acids (C).

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

[0197] 3.2 Bioinformatics analysis

[0198] Bioinformatics analysis was performed on the sequences of the target genes obtained by sequencing. The signal peptide was predicted, and the results showed that the signal peptide of Tcctl-2 was 18 amino acids (A) in length, and the signal peptide of Tcctl-5 was 17 amino acids (A) in length; transmembrane region structure prediction analysis showed that Tcctl-2 had a transmembrane region of 21 amino acids (B) and Tcctl-5 had a transmembrane region of 22 amino acids (C). Figure 3 Figure 4 Figure 3 ​​​​B) and Tcctl-5 Figure 4 B) and Tcctl-5 Figure 3 C) and Tcctl-5 Figure 4 C) and Tcctl-5

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

[0200] The recombinant plasmids pET32a / TcCTL-2 and pET32a / TcCTL-5 were transformed into BL21 expression bacteria for induction expression. The expression of recombinant proteins was detected by SDS-PAGE electrophoresis, and the results showed that the molecular weight of recombinant protein rTcCTL-2 was about 30 kDa Figure 5 ), and the size of rTcCTL-5 was about 25 kDa Figure 6 ), which were 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 subjected to immunoblotting analysis with positive serum from mice infected with Toxocara cati, and the results are shown in Figure 7 It was found that both rTcCTL-2 and rTcCTL-5 proteins could specifically react with positive serum from mice infected with Toxocara cati, showing clear blotting 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 cati, and have specific distribution patterns in the worm body. By analyzing the localization of TcCTL-2 and TcCTL-5, it was found that TcCTL-2 Figure 8 ) and TcCTL-5 Figure 9) were detected in the epidermis, muscle tissue and intestinal tract of both female and male adult worms; in particular, TcCTL-2 and TcCTL-5 were both located in the uterine wall of female adult worms and the testis wall of male adult worms, which suggested that they might play an important role in the reproductive process of the worms.

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

[0206] To further explore the transcriptional level changes of Tcctl-2 and Tcctl-5 in Toxocara canis, we detected the relative expression of Tcctl-2 and Tcctl-5 genes in each developmental stage by RT-qPCR technology, and the results are shown in Figure 10 As shown in the table, Tcctl-2 was significantly highly expressed in L3 worm eggs and 3rd instar larvae (intestinal tract and migration) stages, and the highest expression was observed in 3rd instar worm eggs; Tcctl-5 was highly expressed in 4th and 5th instar larvae and female and male adult worms.

[0207] 3.7 Amplification of target genes and vectors

[0208] Specific primers with pNZ8149 homologous arms were designed to amplify the target gene fragments by PCR. The target bands were consistent with the expected size on 1% agarose gel, with Tcctl-2 being 678 bp Figure 11 (A) and Tcctl-5 being 525 bp Figure 11 (B).

[0209] The extracted pNZ8149 plasmid nucleic acid was verified by electrophoresis. Due to the different migration speeds of different topological structures of plasmids, the order from fast to slow was supercoiled, linear and open loop, showing three bands Figure 12 (A). The verified plasmid was amplified by PCR using vector linearization primers, and the target band was consistent with the expected size, with pNZ8149 being 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 subjected to homologous recombination with the linearized vector to construct recombinant plasmids pNZ8149 / TcCTL-2 and pNZ8149 / TcCTL-5, which were then electroporated into Lactococcus lactis NZ3900 competent cells. As shown in Figure 13 , both recombinant bacteria could grow into yellow single colonies on Elliker medium, which were positive bacteria.

[0212] Single yellow positive bacteria on Elliker medium were picked and cultured, and the PCR identification results of the positive bacteria are shown inFigure 14 The target band matched the expected size, and the sequencing results were consistent with the recombinant vector fragment.

[0213] 3.9 Induced expression and optimization of recombinant Lactococcus lactis

[0214] The empty bacteria pNZ8149, recombinant Lactococcus lactis pNZ8149 / TcCTL-2 and pNZ8149 / TcCTL-5 were induced at 30°C for 8h and 12h, respectively, using 10 ng / mL of Nisin. After induction, the supernatant and precipitate were sampled and subjected to SDS-PAGE electrophoresis. No target protein was expressed in the supernatant of pNZ8149 / TcCTL-5, but a protein with a molecular weight of about 20 kDa was expressed in the bacterial precipitate, which was consistent with the expected protein size. There was no significant difference in protein expression between 8h and 12h induction, and the results are shown in Figure 15 A. Similarly, no target protein was expressed in the supernatant of pNZ8149 / TcCTL-2, but a target protein band with a molecular weight of about 25 kDa appeared in the bacterial precipitate, and there was no significant difference in protein expression between different induction times, and the results are shown in Figure 15 B.

[0215] To optimize the protein expression conditions of recombinant Lactococcus lactis, different concentration gradients (1 ng / mL, 3 ng / mL, 5 ng / mL, 10 ng / mL) of Nisin were used for induction and expression, and the Western blot analysis results are shown in Figure 16 (pNZ8149 / TcCTL-2) and Figure 17 (pNZ8149 / TcCTL-5). All test concentrations successfully induced the expression of target proteins in the two recombinant Lactococcus lactis strains, and under the condition of strict control of induction time and consistent sample loading, the expression level of the target protein gradually increased with the increase of the concentration of the inducer, so 10 ng / mL was determined as the optimal induction concentration.

[0216] 3.10 Detection of intestinal colonization

[0217] During the 14-day continuous monitoring period, all mice fed with recombinant Lactococcus lactis drank water and ate normally, and their physiological state was stable, with no deaths. By coating the Elliker screening medium with the intestinal lavage of the mice, it was found that both recombinant strains could maintain effective colonization in the intestinal tract of mice for at least 14 days. Recombinant Lactococcus lactis pNZ8149 / TcCTL-5 mainly colonized in the duodenum and jejunum, but was not detected in the ileum and feces. However, pNZ8149 / TcCTL-2 not only successfully colonized in each segment of the small intestine, but also distributed in the feces, and the results are shown in Figure 18Positive single colonies were identified by PCR, pNZ8149 / TcCTL-2( Figure 19 ) and pNZ8149 / TcCTL-2( Figure 20 ) were consistent with the results of plate screening, confirming that the recombinant Lactococcus lactis could colonize the mouse intestine for at least 14 days, providing data support for the subsequent oral vaccine immunization program based on this strain.

[0218] 3.11 Changes in body weight and organ index of mice

[0219] To evaluate the effect of recombinant Lactococcus lactis on the growth and development of mice, the body weight of mice was recorded every week after the first immunization, and the weight gain rate was calculated, as shown in Figure 21 Compared with the PBS group of mice (average weight gain rate of 28.94% for 40 days), the recombinant Lactococcus lactis immunization group showed a significant effect on weight gain, with the pNZ8149 / TcCTL-2 group being the most effective, with a 40-day weight gain rate of 45.87% (P < 0.001); the pNZ8149 / TcCTL-5 group was second, with a weight gain rate of 39.55% (P < 0.051); however, the weight gain rate of the pNZ8149 / TcCTL-2+5 mixed group (34.67%) was lower than that of the single-strain immunization group, and was equal to that of the empty carrier control group (NZ3900 / pNZ8149, weight gain rate of 34.48%).

[0220] Further analysis of the organ index of mice found that Figure 22 Compared with the PBS control group, mice fed with recombinant Lactococcus lactis showed an upward trend in heart, spleen, liver and lung index. Statistical analysis found that only the liver organ coefficient of the empty group and the mixed immunization group mice was statistically significant (P < 0.05), and there was no significant difference between the organ coefficients of the other immunization groups and the control group (P > 0.05).

[0221] 3.12 Larvae count in mice

[0222] The number of stage 3 migrating larvae in the liver and lung tissue of mice was examined under an optical microscope and counted, and the statistical analysis results are shown in Figure 23 The average number of larvae recovered from the PBS control group mice was 28, and the NZ3900 / pNZ8149 empty carrier group was 18, with a reduction of 35.7% in the number of larvae; the pNZ8149 / TcCTL-2 immunization group and the pNZ8149 / TcCTL-5 immunization group recovered 11 and 15 larvae, respectively, with a reduction of 60.7% and 46.4% in the number of larvae; interestingly, the use of two recombinant Lactococcus lactis strains for mixed immunization significantly improved the protection effect, with only 9 L3 migrating larvae in the tissue of the pNZ8149 / TcCTL-2+5 immunization group mice, and the reduction rate of the number of larvae was 67.9%.

[0223] 3.13 Lesion comparison

[0224] To systematically evaluate the immune effect of recombinant Lactococcus lactis on mice, the histopathological analysis of liver, lung and intestinal tissues of infected mice was performed. As shown in Figure 24 , the lung of the PBS group mice showed severe hemorrhage, and a large number of red blood cells were visible in the bronchioles and alveolar cavities, the alveolar wall thickened, and inflammatory cells infiltrated. The liver cells were destroyed, arranged in disorder, and the liver sinus expanded with a large number of red blood cells, accompanied by inflammatory cell aggregation and macrophage proliferation. The liver and lung lesions of the NZ3900 / pNZ8149 group and the pNZ8149 / TcCTL-5 group were improved, and a large number of red blood cells and inflammatory cells were still visible in the alveolar cavities, and the liver cells appeared vacuoles, but there was no severe hemorrhage and a large number of inflammatory cell infiltration. The pNZ8149 / TcCTL-2 and pNZ8149 / TcCTL-2+5 immunized mice showed significant improvement in lesions, and the lung tissue structure was close to normal, with a small amount of red blood cell diffusion and reduced inflammatory cell infiltration. The liver cells were arranged closely, with normal morphology, and were distributed radially around the central vein, with no obvious abnormalities.

[0225] The intestinal tissue lesion showed that the intestinal villi of the duodenum of the PBS group mice were broken and even shed, leading to the exposure of the mucosa layer; while the intestinal villi of the mice orally administered with lactic acid bacteria showed slight damage, and the overall morphology was relatively complete, with a small amount of inflammatory cell infiltration in the submucosal layer and muscle layer, and an increase in the number of goblet cells and Paneth cells, as shown in Figure 25 . Compared with the duodenum, the jejunum lesions were significantly improved, as shown in Figure 26 , the intestinal villi of the jejunum of the mice in each group were mostly arranged in order, and the structure was relatively complete. The intestinal epithelial cells of the pNZ8149 / TcCTL-2 group and the pNZ8149 / TcCTL-2+5 group were arranged closely, and the intestinal villi were rarely shed, and the intestinal barrier was intact. In addition, the number of goblet cells in the mice orally administered with lactic acid bacteria increased significantly, and the number of Paneth cells in the basal layer increased significantly.

[0226] 3.14 Intestinal flora species difference analysis

[0227] The results of intestinal flora species difference analysis are shown in 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 effect of recombinant Lactococcus lactis and further verify the flow cytometry results, the dynamic changes of Th1 (IFN-γ, IL-12), Th2 (IL-4, IL-9, IL-10 and IL-13) and Th17 (IL-17) type characteristic cytokines in the serum of mice in each group before and after challenge were quantitatively analyzed by ELISA method. Figure 32 and Figure 33 The data showed that after challenge, the Th1 type cytokines (IFN-γ and IL-12) and Th17 (IL-17) cytokines of the recombinant bacteria immunized mice presented more significant response and increased, while the Th2 type cytokines IL-4, IL-9 and IL-13 also showed an upward trend, and the immunomodulatory cytokine IL-10 remained relatively stable before and after challenge, but the level was still significantly higher than that of the PBS control group.

[0237] IV. DISCUSSION

[0238] The previous comparative analysis of 17 canine Toxocara cati C-type lectin transcriptional data found that Tcctl-2 and Tcctl-5 are highly expressed genes in the parasitic stage of the worm body, and the present application successfully amplifies them by PCR. The molecular identification results show that Tcctl-2 is 702 bp in length and encodes a protein of about 28 kDa in size; Tcctl-5 is 546 bp in length and encodes a protein of about 23 kDa in size. Both genes contain a signal peptide but no transmembrane domain, and the secondary structure is mainly alpha helix and beta fold. TcCTL-2 and TcCTL-5 are mainly located in the epidermis, muscle and intestinal tissues of male and female worms, and the reproductive organs including ovary and testis. 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 worm body. Further exploration of their transcription levels in different worm development stages found that Tcctl-2 was significantly highly expressed in L3 worm eggs, L3 intestinal larvae and L3 migrating larvae, and almost not expressed in L5 larvae and male adult worms; Tcctl-5 was significantly highly expressed in L3 worm eggs, L4, L5 larvae and male and female adult worms, and almost not expressed in L3 intestinal and migrating larvae. Since the natural TES antigen components are complex and difficult to obtain in large quantities, it cannot meet the commercial demand. Therefore, in this study, recombinant proteins rTcCTL-2 and rTcCTL-5 were expressed by E. coli prokaryotic expression system, and Western blot analysis showed that both of them could be specifically recognized by canine Toxocara cati infection positive serum, indicating that they had good immunogenicity. Animal protection test data showed that rTcCTL-2 and rTcCTL-5 had good protective effect on mice against Toxocara cati infection, and the migrating larvae load in their liver and lung tissues was reduced by 51.7% and 41.4%, respectively, compared with other candidate antigens rTcSP(25.5%), rTcMUC-3(20.4%), rTcCTL-1(16.7%), rTcCTL-4(18.7%) and rTc-SOD(37.6%) The reduction rate of larval load of rTcCTL-2 and rTcCTL-5 significantly improved the protection efficiency, which has the potential to be used as a candidate vaccine antigen. This conclusion provides data reference for the subsequent research on C-type lectin recombinant Lactococcus lactis of Toxocara cati.

[0239] In the construction of recombinant plasmid, the current study mostly uses the method of double enzyme digestion, but in this experiment, it is found that the use of enzyme digestion enzyme connection method leads to low recovery efficiency and low success rate of vector connection, while the use of homologous recombination technology can improve the success rate of vector and target fragment connection, and by adjusting the molar ratio of vector and insert fragment to 1:2, the recombinant plasmids pNZ8149 / TcCTL-2 and pNZ8149 / TcCTL-5 are successfully constructed. In the electroporation of Lactococcus lactis NZ3900 competent cells, the best transformation conditions are found by referring to the method of Liu Yaxin et al. and making certain adjustments: keeping the voltage at 2200V, the resistance at 200Ω, and the capacitance at 25μF, and after electroporation, the cells are placed on ice for about 10min and then cultured at 30℃ for about 3h, then centrifuged to obtain the bacterial pellet and plated, at which time the positive transformation rate is the highest. Since the Lactococcus lactis NZ3900 lacF gene is deleted, it cannot produce acid by lactose fermentation, but after the plasmid pNZ8149 carrying the lacF gene is transformed into the lactobacillus, its acid-producing ability is restored, so the bromocresol purple in the Elliker medium undergoes color reaction under acidic conditions (from purple to yellow), which can be used as an indicator for successful integration of recombinant plasmid. The recombinant lactobacillus is induced for expression using 10ng / mL of inducer Nisin, and the SDS-PAGE and Western blot verification show that the recombinant Lactococcus lactis pNZ8149 / TcCTL-2 expresses a protein with a size of 25kDa, and the recombinant Lactococcus lactis pNZ8149 / TcCTL-5 expresses a protein with a size of 20kDa, which is consistent with the expected protein size, and the induction time has no obvious effect on the expression amount of recombinant protein. Subsequently, the recombinant lactobacillus is induced for expression and verification using different concentrations of inducer, and it is found that the Nisin concentration between 1ng / mL and 10ng / mL can induce the expression of target protein, and when the final concentration of Nisin is 10ng / mL, the target band is the thickest, which is the best induction concentration. In order to develop a reasonable immunization program, the use dose and colonization time of recombinant lactobacillus are evaluated by bacterial count and intestinal colonization detection, and the recombinant lactobacillus with a concentration of 1×10 9 CFU / mL is orally administered to mice, and the intestinal contents of mice are taken for selective culture and PCR identification at 7 days and 14 days, respectively, and it is found that yellow single colonies can grow on the selective medium at 7 days and 14 days, and the target bands with the same size appear after specific primer PCR identification, indicating that the two recombinant lactobacilli can stably colonize in the small intestine, and the colonization time is not less than 14 days, which is crucial for the selection of time for developing immunization program.

[0240] During the intestinal invasion of A. caninum, intestinal hemorrhage and inflammation are usually caused. Lactococcus lactis colonized in the small intestine may reduce the damage of intestinal mucosa by inducing mucus secretion, enhancing tight junction or secreting antibacterial peptides. The results of mouse protection test showed that the clearance rates of recombinant L. lactis pNZ8149 / TcCTL-2 and pNZ8149 / TcCTL-5 in the liver and lung tissues of mice were 46.4% and 60.7%, respectively. Interestingly, the clearance rate was 67.9% when the two strains were mixed, which was better than that of single strain. Histopathological analysis showed that the pathological damage of liver and lung tissues in the PBS group was serious, with red blood cells filling the alveolar cavity, inflammatory cells gathering into groups, and liver sinusoidal congestion and expansion. The pathological changes of the NZ3900 / pNZ8149 group were alleviated, and the pathological changes of the recombinant L. lactis immunization group were significantly improved, with the number of red blood cells and inflammatory cells in the alveolar cavity reduced, and the liver cells normal and arranged radially around the central vein. In addition, the duodenal structure of the PBS group was severely damaged, with intestinal villi breaking and falling off, and the mucosal layer exposed. However, the mice fed with Lactococcus lactis only showed partial intestinal villi breaking, and the overall structure was relatively complete. Compared with the duodenum, the damage of the jejunum of mice in each group was not obvious, and the intestinal villi of the jejunum of the immunized group were mostly arranged in order, with few falling off, especially the goblet cells and Paneth cells significantly increased. The increase of these key cell populations was particularly important for maintaining the mechanical and immune barriers of the intestinal tract. The analysis of intestinal flora species showed that the dominant species of intestinal flora in mice changed after immunization, and the abundance of Lactococcus lactis increased, which may reduce the damage caused by A. caninum invasion by stabilizing the intestinal barrier and flora homeostasis. The statistical analysis of blood routine showed that the number of red blood cells in the PBS group was much less than that in the immunized group, which may be due to anemia caused by A. caninum infection. At the same time, the number of white blood cells in the immunized mice also increased, which suggested that the recombinant Lactococcus lactis may mediate the inflammatory response to reduce the damage of the worm to the body. Studies have shown that neutrophils may cooperate with other immune cells to promote the killing effect of helminth clearance, and Xuan et al. found that monocytes played a protective role in anti-malaria infection by phagocytosis, mediation of cytokine production and antigen presentation. In this study, the increase of mononuclear, lymphocytes and neutrophils in immunized mice was observed, but whether they played a similar role in anti-A. caninum infection remained to be further studied. The detection of specific IgG, IgG1 and IgG2a antibody levels in mice by ELISA showed that the level of specific antibody IgG in the serum of mice fed with recombinant L. lactis increased significantly, indicating that the recombinant L. lactis constructed in this study could induce effective specific immune response in mice.The study suggests that IgG2a and IgG1 subtypes are markers of Th1 and Th2 cells, respectively, and the recombinant subunit vaccine constructed by Chen Y J and Salazar induces a Th2-type immune response in mice. Further analysis of IgG subtypes in this study found that the level of IgG2a antibody in mice administered the oral recombinant Lactococcus lactis vaccine was slightly higher than that of IgG1, suggesting that Th1 immune cells in mice primarily produce IgG2a. Notably, a significant increase in sIgA antibody concentration was observed in the mouse intestine, particularly after infection, and sIgA is a marker of mucosal immunity, indicating that recombinant Lactococcus lactis can efficiently stimulate the intestinal mucosal immune response. Flow cytometry results showed that the expression profile of CD4+ T cell subsets in the mesenteric lymph nodes and spleen exhibited significant polarization characteristics: RORγt+ cells accounted for the highest proportion, followed by T-bet+ cells, and GATA-3+ cells accounted for the smallest proportion, indicating that CD4+ T cells preferentially differentiate into Th17 and Th1 subsets. Studies have shown that Th17 cells not only secrete IL-17A to upregulate the expression of tight junction proteins and mucin in intestinal epithelial cells, but also promote the secretion of sIgA by plasma cells to enhance the intestinal mucosal barrier function. In addition, Th17 cells maintain intestinal mucosal homeostasis by coordinating with intestinal epithelial cells, antigen-presenting cells, Treg cells, and intestinal flora. Cytokine detection further confirmed that, compared with the PBS group, mice administered the oral recombinant Lactococcus lactis exhibited significant Th1 / Th17-type immune polarization characteristics after infection with larvae, as evidenced by the significant upregulation of IFN-γ, IL-12, and IL-17 expression. These inflammatory cytokines are mainly secreted by Th1 cells and Th17 cells, followed by neutrophils and eosinophils. During the acute phase of inflammation, neutrophils are first recruited to the infection site, followed by sustained infiltration of eosinophils and persistence during the chronic infection stage. Studies have shown that both types of granulocytes can produce various pro-inflammatory cytokines, including IFN-γ, by expressing IL-17A receptors, which may inhibit the infection and damage of A. caninum by enhancing the inflammatory response and intestinal mucosal immune response. In summary, pNZ8149 / TcCTL-2 and pNZ8149 / TcCTL-5 oral vaccines can effectively induce high-efficiency intestinal mucosal immune responses in mice and stimulate cellular immunity. pNZ8149 / TcCTL-2 / TcCTL-5 has the best protective effect on mice and can be used as an optimal oral vaccine strain for preventing canine A. caninum disease.

[0241] To the extent not already already described herein, it will be apparent from this detailed description of embodiments of the application, which can be made and used without departing from the spirit and scope of the application, many other embodiments of the application will be readily ascertainable to those skilled in the art and the inventor contemplates the use of only the preferred embodiments of the application in the following claims.

Claims

1. A recombinant lactococcal oral vaccine, characterized in that... Recombinant Lactococcus lactis expresses TcCTL-2 and / or TcCTL-5, wherein, The amino acid sequence of TcCTL-2 is as follows: MFAIFIICLCFHFSTIDACARDADCNLFQVCVNNVCVANNQGCNPPCVAPMVCVAPNCVAVPAPQPQPATTTTAAPAATTTAAPAATTTTARRTCPPSWSLFNNNCYIASVAGRFLF NQASDWCTQTGSRVVWFDQSNAANFNSELAFVNNLAISGGSSRYWIGVNRQFGQWVWTNGSPVILSNWRPSQPDGCCGSNVTCVFVNYANFLGQWDDASCGGLFTNPQGFVCKRPL; The amino acid sequence of TcCTL-5 is as follows: MIAFCLLLTLAISVANANRCDPGWRYSPFTRKCYRFYDHETMWPSAEFSCLFKGGHLIS IHSYTDNRFAIELARGAETVWLGNAQFGSSKEYIWSDHTAYNYGSWPDRKRPEKIKTKPCT KLNTTSGEWFQSCCKDPAPYICQKELSDSNAMYRNSEELLGHTSEDRRLESNEDFRRRFFL.

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 lactococcus 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 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.

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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