Application of Lawsonia intracellularis outer membrane protein in preparation of vaccines and diagnostic kits

Abstract

The invention belongs to the field of biology, and discloses application of a Lawsonia intracellularis outer membrane protein MltA protein in preparation of vaccines and diagnostic kits, the protein is used as an antigen to prepare the Lawsonia intracellularis vaccine, and the risk that pigs are infected with porcine ileitis can be effectively reduced; meanwhile, the titer of the MltA protein specific IgG antibody can reach 1: 1638400, and the MltA protein specific IgG antibody can be used as a core antigen of a subunit vaccine and a key component of a high diagnostic reagent at the same time and is a potential anti-LI drug target.

Description

Technical Field

[0001] The present application relates to the biological field, and in particular to the application of an intracellular Lawsonia intracellularis outer membrane protein in the preparation of vaccines and diagnostic kits. Background Art

[0002] Porcine ileitis, also known as porcine proliferative enteropathy (PPE), is an infectious disease of the swine intestine caused by Lawsonia intracellularis (LI), characterized by intestinal proliferation, inflammation, and hemorrhage. It primarily affects growing and finishing pigs, causing significant economic losses to the livestock industry. L. intracellularis is an obligate intracellular parasite, and currently only a handful of laboratories are able to sustain its growth through cell co-culture (such as the porcine intestinal epithelial cell line IPEC-J2 or the mouse fibroblast line McCoy). This technical bottleneck has directly hindered basic research on its infection mechanisms, virulence factors, and host interactions.

[0003] Bacterial lytic transglycosylases (LTs) play a key role in regulating the size of nascent peptidoglycan, peptidoglycan recycling, cell division, cell wall repair, integration of multiprotein structures such as flagella or secretion systems into the cell wall, and release of signaling molecules associated with virulence and antibiotic resistance during cell wall biosynthesis, and can serve as potential targets for antimicrobial drugs.

[0004] Numerous studies have elucidated the mechanisms by which pathogenic LTs interact with the host. LTs participate in the synthesis and remodeling of bacterial biofilms, and inhibiting their activity can reduce bacterial resistance to antibiotics and immune attack. Broad-spectrum antibacterial agents targeting LTs have been a key focus of antibacterial drug development in recent years. LTs also contribute to pathogenic pathogenicity. LTs from Haemophilus influenzae, Neisseria meningitidis, Shigella flexneri, and Pseudomonas syringae are upregulated during host infection. Furthermore, LtgC (Lytic transglycine osylase C) from Neisseria gonorrhoeae has been shown to be highly immunogenic, inducing specific antibodies and a Th1-polarized immune response.

[0005] Bioinformatics analysis of the LTs of Lawsonia intracellularis revealed the presence of a functional gene encoding membrane-bound lytic murein transglycosylase A (MltA), designated LI0723, in the L. intracellularis genome. However, the role of this protein in LI cell wall metabolism, its virulence mechanism, and its association with bacterial environmental adaptability remain largely unexplored.

[0006] The problem that this project needs to solve: How to propose an antigen against intracellular Lawsonia spp. Summary of the Invention

[0007] The purpose of this application is to propose an application of an intracellular Lawsonia outer membrane protein in the preparation of vaccines and diagnostic kits, wherein the intracellular Lawsonia outer membrane protein is MltA protein; by using this protein as an antigen, antibodies can be effectively produced against intracellular Lawsonia, thereby reducing the risk of pigs being infected with porcine ileitis.

[0008] To achieve the above objectives, the present application discloses the use of an outer membrane protein of Lawsonia intracellularis in preparing vaccines and diagnostic kits, wherein the outer membrane protein of Lawsonia intracellularis is MltA protein.

[0009] Preferably, the amino acid sequence of the MItA protein includes the amino acid sequence fragment shown in SEQ ID NO:1.

[0010] Preferably, the gene sequence of the MItA protein includes the gene sequence fragment shown in SEQ ID NO: 2.

[0011] Preferably, the amino acid sequence of the MItA protein is shown in SEQ ID NO: 1 or SEQ ID NO: 3.

[0012] Preferably, the gene sequence of the MItA protein is shown as SEQ ID NO: 2 or SEQ ID NO: 4.

[0013] In addition, the present application also discloses a recombinant protein, the amino acid sequence of the recombinant protein is shown in SEQ ID NO: 1 or SEQ ID NO: 3.

[0014] In addition, the present application also discloses a vaccine and a kit, in which the above-mentioned recombinant protein serves as an antigen.

[0015] The beneficial effects of this application are:

[0016] The present application provides a use of an outer membrane protein of intracellular Lawsoniae in preparing vaccines and diagnostic kits, wherein the outer membrane protein of intracellular Lawsoniae is MltA protein, the amino acid sequence of which includes the amino acid sequence fragment shown in SEQ ID NO: 1; the gene sequence of the MltA protein includes the gene sequence fragment shown in SEQ ID NO: 2; a vaccine for intracellular Lawsoniae can be prepared by using the protein as an antigen, which can effectively reduce the risk of pigs being infected with porcine ileitis; at the same time, the titer of MltA protein-specific IgG antibodies can reach 1:1638400, and the protein can be used as a core antigen for subunit vaccines and a key component for high-dose diagnostic reagents, and is a potential anti-LI drug target. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the signal peptide prediction result of MltA protein;

[0018] Figure 2 This is the prediction result of the transmembrane region of MltA protein;

[0019] Figure 3 This is the result of antigen epitope prediction of MltA protein;

[0020] Figure 4 This is the tertiary structure prediction result of MltA protein;

[0021] Figure 5 is the gene sequence amplification result of MltA protein, where M is DL2000 DNA Marker; 1 is the gene sequence fragment of MltA protein;

[0022] Figure 6 The following are the results of enzyme digestion identification of the pMAL-C6T-MltA recombinant vector, where M is the DL15000 DNA Marker; 1 is the result of enzyme digestion of the pMAL-C6T-MltA recombinant vector with the BamHI restriction enzyme;

[0023] Figure 7 The induced expression results of rMltA recombinant protein, where M is the protein molecular weight standard; 1 is the uninduced bacterial solution; 2 is the induced bacterial solution; 3 is the bacterial super-disruption precipitate; 4 is the bacterial super-disruption supernatant;

[0024] Figure 8 The figure shows the purification results of rMltA recombinant protein, where M is the protein molecular weight standard; 1 is the flow-through; 2 is the wash solution; 3 is the first eluent; 4 is the second eluent;

[0025] Figure 9 is the purification result of MltA protein, where M is the protein molecular weight standard; 1 is MltA protein;

[0026] Figure 10 is the Western blot identification result of rMltA recombinant protein, where M is the protein molecular weight standard; 1 is rMltA recombinant protein;

[0027] Figure 11 is the Western blot identification result of MltA protein, where M is the protein molecular weight standard; 1 is MltA protein;

[0028] Figure 12 The titer of MltA protein-specific IgG antibody was determined;

[0029] Figure 13The effect of different immunization times on the level of MltA protein-specific IgG antibodies, where P represents positive serum and N represents negative serum; ns, no significant difference (p>0.05);

[0030] Figure 14 is the serum total IgG antibody level, where * indicates significant difference (P<0.05) and **** indicates extremely significant difference (P<0.0001);

[0031] Figure 15 is the total serum IgG1 antibody level, where **** indicates extremely significant difference (P<0.0001);

[0032] Figure 16 is the level of total serum IgG2a antibody, where ** indicates extremely significant difference (P<0.01), **** indicates extremely significant difference (P<0.0001);

[0033] Figure 17 This is the map of the pMAL-C6T-MltA recombinant expression vector. DETAILED DESCRIPTION

[0034] In the description of this application, it should be noted that if specific conditions are not specified in the examples, the experiments were carried out according to conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used, if the manufacturer is not specified, are all conventional products that can be purchased commercially.

[0035] Main reagents and their sources

[0036] High-fidelity PCR enzyme Prime GXL DNA Polymerase and 5× In-Fusion Snap Assembly Master Mix were purchased from TakaRa Co., Ltd.;

[0037] The Tiangen bacterial genomic DNA extraction kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.;

[0038] Ileitis live attenuated vaccine was purchased from Shanghai Boehringer Ingelheim Pharmaceutical Co., Ltd.;

[0039] PMAL-C6T expression vector was purchased from New England Biolabs Co., Ltd.;

[0040] BamHI restriction endonuclease was purchased from Thermo Fisher Scientific;

[0041] Omega Gel Extraction Kit and Omega Plasmid Miniprep Kit II were purchased from Omega Bio-Tek;

[0042] Dextrin Beads were purchased from Changzhou Tiandiren Biotechnology Co., Ltd.;

[0043] Coomassie brilliant blue staining solution was purchased from Jiangsu Addison Biotechnology Co., Ltd.;

[0044] TEV protease, SDS-PAGE protein loading buffer, and One-Step PAGE Gel Fast Preparation Kit (10%) were purchased from Nanjing Novozymes Biotechnology Co., Ltd.;

[0045] E. coli DH5α, BL21 competent cells, HRP-labeled goat anti-rabbit IgG antibody, and HRP-labeled goat anti-mouse IgG antibody were purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0046] 0.45 μm filter membrane was purchased from Milipore Co., Ltd.;

[0047] Aluminum hydroxide adjuvant was purchased from Suzhou Botelon Immunotechnology Co., Ltd.;

[0048] Mouse IgG ELISA Kit, Mouse IgG1 ELISA Kit, and Mouse IgG2a ELISA Kit were purchased from Hangzhou Lianke Biotechnology Co., Ltd.

[0049] The His-MBP protein was obtained by transforming E. coli BL21 competent cells with the pMAL-C6T expression vector, induced and purified, and stored in the Swine Disease Laboratory of the Animal Health Institute of the Guangdong Academy of Agricultural Sciences;

[0050] Rabbit anti-LI high immune serum was 2mL The Ileitis live attenuated vaccine was prepared by collecting blood from New Zealand white rabbits after three subcutaneous immunizations and stored in the swine disease department of the Animal Health Institute of the Guangdong Academy of Agricultural Sciences.

[0051] New Zealand white rabbits and BALB / c mice were purchased from Guangdong Medical Experimental Center.

[0052] Reagent formula:

[0053] PBS: 2.08 g Na2HPO4, 0.2 g KH2PO4, 8 g NaCl, 1 g KCl, add ultrapure water to 1 L, pH 7.4;

[0054] Wash solution: 2mM Tris-HCl, 20mM NaCl, 1mM EDTA, add ultrapure water to 500mL, pH 7.4;

[0055] Eluent: 20 mM Tris-HCl, 1 mM EDTA, 10 mM maltose, add ultrapure water to 500 mL, adjust the pH to 7.4;

[0056] 1M IPTG: Dissolve 2 g IPTG in 8 mL deionized water, dilute to 10 mL with deionized water, filter through a 0.22 μm filter, and aliquot into 1 mL portions. Store at -20°C.

[0057] PBST: 2.08 g Na2HPO4, 0.2 g KH2PO4, 8 g NaCl, 1 g KCl, 0.1% Twee-20, add ultrapure water to 1 L of solute, pH 7.4;

[0058] 5% skim milk: 1 g skim milk dissolved in 20 mL PBST;

[0059] Coating solution: 0.05 M carbonate buffer, pH 9.6;

[0060] Blocking solution: 1 g BSA dissolved in 50 mL PBST;

[0061] Serum diluent: 0.5 g BSA dissolved in 50 mL PBST.

[0062] Example 1

[0063] 1.1 Bioinformatics analysis of MltA protein

[0064] The amino acid sequence of MltA protein (GenBank: CAJ54777.1) was downloaded from NCBI, and the bioinformatics information of MltA was analyzed using SignalP-5.0 (https: / / services.healthtech.dtu.dk / services / SignalP-5.0 / ), TMHMM (http: / / www.cbs.dtu.dk / services / TMHMM / ), Swiss-Model (https: / / swissmodel.expasy.org / ) online websites and Protean software. The characteristics are as follows: the full length of MltA protein is 403 amino acids, and the lipoprotein signal peptide (Sec / SPII) is present with a probability of 99.81%. The cleavage site is as follows: Figure 1 As shown, it is located between amino acids 22 and 23 (IVG-CS); Figure 2 As shown in Figure 2, the protein is a non-transmembrane protein; and Figure 3 As shown in Figure 2, its antigenic epitopes are dense. AlphaFold v2 established the tertiary structure model of the protein as shown in Figure 2. Figure 4 In summary, the signal peptide of MltA was removed and amino acids 23 to 403 were retained.

[0065] The amino acid sequence of MltA (GenBank: CAJ54742.1) is shown below:

[0066]

[0067] The straight line portion is the signal peptide amino acid sequence, and the wavy line portion is the amino acid sequence shown in SEQ ID NO: 1.

[0068] The gene sequence of MltA (GenBank: AM180252.1) is shown below:

[0069]

[0070] The straight line portion is the gene sequence of the signal peptide, and the wavy line portion is the gene sequence shown in SEQ ID NO: 2.

[0071] 1.2 MltA protein primer design and synthesis

[0072] The gene sequence of MltA protein was downloaded from NCBI (GenBank: AM180252.1), and PCR primers were designed using Primer Premier 5.0 software as shown in Table 1 and sent to Sangon Biotech (Shanghai) Co., Ltd. for synthesis.

[0073] Table 1 PCR primers for MltA protein

[0074]

[0075] Note: The underlined sequence is the BamHI restriction enzyme site

[0076] 1.3 Gene sequence amplification and purification of MltA protein

[0077] by The LI genomic nucleic acid in the live attenuated Ileitis vaccine was used as a template to amplify the MltA gene fragment with the signal peptide removed. The PCR amplification system is shown in Table 2. The PCR amplification conditions were 98°C pre-denaturation for 3 minutes, 98°C denaturation for 10 seconds, 58°C annealing for 30 seconds, 68°C extension for 2 minutes, for a total of 35 cycles, and 68°C extension for 10 minutes. The PCR product was identified by 1% agarose gel electrophoresis and the MltA gene fragment was recovered after gel excision. Figure 5 shown.

[0078] Table 2 PCR amplification system of the gene sequence of MltA protein

[0079]

[0080] 1.4 Construction and enzyme digestion identification of the MAL-C6T-MltA recombinant vector

[0081] The pMAL-C6T expression vector was incubated with BamHI restriction enzyme at 37℃ for 1 hour, identified by agarose gel electrophoresis, and the target band was recovered by gel excision. The enzyme digestion system is as follows: 1μL Fast Digest BamHI, 1μL 10×FastDigest Buffer, 1μg pMAL-C6T, and ddH2O is added to 10μL. The pMAL-C6T expression vector fragment after enzyme digestion was ligated with the MltA gene fragment to obtain the recombinant expression vector pMAL-C6T-MltA. Figure 17 As shown. The Takara seamless cloning system is as follows: 1μL 5×In-FusionSnap Assembly MasterMix; 1μL pMAL-C6T, 3μL target fragment, incubate at 50℃ for 30min, and store at 4℃ for later use. The recombinant expression vector pMAL-C6T-MltA is spread on an ampicillin-resistant LB plate in E. coli DH5α competent cells for overnight culture; a single colony of overnight culture is picked and expanded, and then PCR identification of the bacterial liquid is performed to obtain a positive colony containing the pMAL-C6T-MltA recombinant vector. After the positive colony is expanded, the pMAL-C6T-MltA recombinant vector is extracted and digested and sequenced for identification. The sequencing results are shown as follows Figure 6 As shown; the pMAL-C6T-MltA recombinant vector that was correctly identified by sequencing was transformed into E. coli BL21 competent cells and cultured. The positive colonies containing the recombinant expression vector pMAL-C6T-MltA were expanded, identified and then frozen.

[0082] 1.5 Expression and purification of rMltA protein

[0083] 5 mL of a single colony containing the recombinant expression vector pMAL-C6T-MltA was cultured overnight and added to 100 mL of Amp-resistant LB broth at a ratio of 1:100. The culture was carried out at 37°C and 220 rpm for 3 h (OD 600: 0.6-0.8), then IPTG was added to a final concentration of 0.3 mM and induced at 120 rpm and 16°C for 16 h. The induced bacterial solution was collected and centrifuged at 8000 rpm at 4°C for 3 min to collect the bacterial pellet, which was then resuspended in 20 mL of PBS by vortexing. The bacterial solution was placed on ice and ultrasonically disrupted until clear and translucent, at 60 Hz for 3 seconds, followed by a 7-second pause. The ultrasonicated solution was centrifuged at 10000 rpm at 4°C for 15 min, and the supernatant was collected and filtered through a 0.45 μm filter for later use. Take 40 μL of uninduced bacterial solution, induced bacterial solution, supernatant and superprecipitate respectively for SDS-PAGE electrophoresis. The detailed steps are as follows: add 10 μL of 5×SDS-PAGE protein loading buffer to each sample and mix well, boil at 100℃ for 10 minutes; take out the pre-configured protein gel, assemble it into the electrophoresis tank and load the sample, adjust the electrophoresis voltage and time, 80V, 30min, 120V, 90min; after the end of SDS-PAGE electrophoresis, stain with Coomassie brilliant blue for 30min, decolorize with ultrapure water 3 times, and observe the results with the gel imaging system to identify the expression of rMltA recombinant protein and the expression form. Figure 7 As shown. The rMltA recombinant protein was purified, and the detailed steps are as follows: take 5mL Dextrin Beads and load them into the gravity column, open the lower outlet to drain the protective liquid, add 3 times the column volume of ultrapure water to rinse the Dextrin Beads, and then add 2 times the column volume of PBS for balance. After the liquid is drained, close the outlet, add the filtered ultrapure supernatant, seal the gravity column, and incubate at 4°C for 2h. After the incubation is completed, collect the flow-through liquid, which can be temporarily stored at 4°C for 3d and purified repeatedly 1-2 times, and then add 3 times the column volume of washing solution to wash to remove non-specifically adsorbed impurities, and repeat the washing 3 times. After the washing solution is drained, close the lower outlet, add an appropriate amount of eluent, incubate for 3-5min to fully elute the protein and collect the eluent, collect the second eluent to obtain high-purity rMltA recombinant protein as shown. Figure 8 As shown, the size is 79 kDa.

[0084] 1.6 Removal of the rMltA recombinant protein tag and purification of MltA protein

[0085] The rMltA recombinant protein carries a His-MBP tag and a TEV enzyme cleavage site. It needs to be cleaved and purified using TEV protease. The detailed steps are as follows: add an appropriate amount of TE to the rMltA recombinant protein. V protease and 10×TEVBuffer, incubated on a 4°C rocking shaker for 20 hours, and then the MltA protein was purified. The specific steps are as follows: the proteins were cleaved by enzymes and subjected to SDS-PAGE electrophoresis. After the electrophoresis, the protein gel was placed in a clean dish and stained with an appropriate amount of 0.25M KCl solution for 30 minutes. The silver-white MltA protein band was carefully cut with a clean scalpel and transferred to another clean dish. After washing with PBS three times, the gel was placed in a grinding tube and three 5mm grinding beads were added. The gel was ground in a -20°C pre-cooled tissue grinder at 60Hz for 15 seconds and paused for 15 seconds until the gel was crushed. An appropriate amount of PBS was then added to the grinding tube, the gel was in an 80°C water bath for 10 minutes, and the gel was centrifuged at 4°C and 1200rpm for 20 minutes. The supernatant was the MltA protein. Figure 9 As shown, the size is 37 kDa.

[0086] The amino acid sequence of the rMltA recombinant protein (SEQ ID NO: 3) is shown below:

[0087]

[0088] The “straight line” is the amino acid sequence of the His-MBP protein, the “dashed line” is the TEV protease cleavage site, and the “wavy line” is the amino acid sequence of the MltA protein after removing the signal peptide.

[0089] The gene sequence of rMltA recombinant protein (SEQ ID NO: 4) is as follows:

[0090]

[0091] Example 2 Analysis of immunogenicity of MltA protein

[0092] 2.1 Western blot identification of rMltA recombinant protein

[0093] The purified rMltA recombinant protein was identified by Western blot test with rabbit anti-LI high immune serum. The detailed steps are as follows: After SDS-PAGE electrophoresis, the pre-cut PVDF membrane was immersed in anhydrous ethanol for activation, and the membrane transfer equipment was assembled and electrotransferred at 250mA for 1 hour; after that, it was rinsed with PBST once and placed in 5% skim milk, blocked at 37℃ for 1 hour, washed 3 times with PBST, placed in 1:500 diluted rabbit anti-LI high immune serum, incubated at 37℃ for 1 hour; then washed 3 times with PBST, placed in 1:10000 HRP-labeled goat anti-rabbit IgG antibody, incubated at 37℃ for 1 hour; finally washed 3 times with PBST, and the PVDF membrane was exposed and developed after soaking in developer. The results are as follows Figure 10 As shown, the rMltA recombinant protein can be recognized by rabbit anti-LI high immune serum and has reactogenicity.

[0094] 2.2 Immunization of mice with rMltA recombinant protein

[0095] Fifteen six-week-old SPF female BALB / c mice were randomly divided into three groups (n=5): a blank group, an MBP group, and an rMltA group. After 5 days of feeding to ensure they were free of LI infection, mice were immunized according to the following schedule: 100 μg of His-MBP and rMltA recombinant proteins, respectively, were mixed with an equal volume of aluminum hydroxide adjuvant and injected subcutaneously at multiple sites. Two weeks later, mice received a second immunization and a booster immunization with the same antigen dose. Serum was collected every two weeks after each immunization by tail docking.

[0096] 2.3 Western blot identification of MltA protein

[0097] The purified MltA protein and rMltA recombinant protein positive serum were subjected to Western blot test identification. The detailed steps are as follows: after SDS-PAGE electrophoresis, the pre-cut PVDF membrane was immersed in anhydrous ethanol for activation, and the membrane transfer equipment was assembled and electrotransferred at 250mA for 1 hour; after completion, it was rinsed with PBST and placed in 5% skim milk, blocked at 37℃ for 1 hour, washed 3 times with PBST, and placed in 1:500 diluted rabbit anti-LI high immune serum, incubated at 37℃ for 1 hour; then washed 3 times with PBST, placed in 1:10000 HRP-labeled goat anti-rabbit IgG antibody, incubated at 37℃ for 1 hour; finally washed 3 times with PBST, and the PVDF membrane was exposed to light after soaking in developer solution, as shown in Figure 2. Figure 11As shown, MltA protein can be recognized by rMltA recombinant protein positive serum, and the target band appears near 37kDa.

[0098] 2.4 Detection of MltA protein-specific IgG antibody levels

[0099] Dissolve the MltA protein in coating solution at a final concentration of 2 μg / mL and add 100 μL / well to a 96-well ELISA plate. Coat at 37°C for 2 hours and wash three times with 300 μL PBST. Add 200 μL / well of blocking solution and incubate at 37°C for 1 hour and wash three times with 300 μL PBST. Add 100 μL / well of diluted MltA recombinant protein positive serum and incubate at 37°C for 1 hour and wash three times with 300 μL PBST. Add 1:10000 diluted HRP-labeled goat anti-mouse antibody and incubate at 37°C for 1 hour and wash three times with 300 μL PBST. Pat dry the remaining liquid and repeat three times. Add 100 μL TMB solution to develop the color for 30 minutes in the dark. Add 100 μL stop solution and gently tap the 96-well ELISA plate. Read the OD value on a microplate reader. 650 The result is as follows Figure 12 As shown, when the rMltA recombinant protein positive serum was diluted to 1∶1638400, the OD 650nm Greater than negative serum OD 650nm Therefore, the titers of MltA protein-specific IgG antibodies were 1:1638400. The serum of mice was collected 2 weeks after each immunization and the changes in the levels of MltA protein-specific IgG antibodies at different immunization times were detected at a dilution of 1:100. Figure 13 As shown in the figure, the specific IgG antibodies induced by MltA protein in the first immunization were not significantly different from those in the second and third immunizations (p>0.05), indicating that MltA protein had induced a strong immune response in the first immunization.

[0100] 2.5 Detection of total IgG, IgG1, and IgG2a levels in serum positive for rMltA recombinant protein

[0101] The double antibody sandwich ELISA detection kit was used to detect the total IgG, IgG1, and IgG2a levels in the serum of mice in the blank, MBP, and rMltA groups 2 weeks after the third immunization. The general steps were as follows: 300 μL of 1× washing solution was added to the ELISA plate and allowed to soak for 30 seconds. After discarding the washing solution, the ELISA plate was patted dry on absorbent paper, and the mouse positive serum was diluted appropriately, added to 100 μL / well, shaken at 100-300 r / min, and incubated at room temperature for 2 hours; 300 μL of 1× washing solution was added to each well. Wash the plate with washing solution for 6 times, patting it dry on absorbent paper after each wash; dilute the concentrated detection antibody at 1:100, add 100 μL of detection antibody working solution to each well, shake at 100-300 rpm, and incubate at room temperature for 1 hour; wash 6 times, add 100 μL of color development substrate to each well in the dark, and incubate at room temperature for 20 minutes; add 100 μL of stop solution to each well, gently tap the plate frame to mix thoroughly; measure the OD value at the maximum absorption wavelength of 450 nm and the reference wavelength of 570 nm or 630 nm. The results are shown as follows Figure 14-16 As shown, His-MBP protein and rMltA recombinant protein induced the production of total IgG, IgG1 and IgG2a antibody levels after immunization of mice. The total IgG, IgG1 and IgG2a antibody levels induced by rMlt A recombinant protein were significantly higher than those in the blank group and MBP group, which could synergistically activate Th2 and Th1 responses, with IgG2a / IgG1 <1. rMltA recombinant protein can induce a high level of humoral immunity.

[0102] In summary, the present invention conducted a bioinformatics analysis of the LTs of Lawsonia intracellularis and found that Lawsonia intracellularis can specifically encode the MltA protein and successfully achieve soluble expression of it together with the His-MBP protein; the MltA protein can be recognized by rabbit anti-LI hyperimmune serum and elicited a strong MltA-specific IgG antibody response in mice (the highest titer reached 1:1638400), which can effectively activate humoral immunity. The His-MBP protein also has the function of enhancing the immune response.

[0103] The above embodiments are preferred implementation modes of the present application, but the implementation modes of the present application are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principles of the present application shall be considered as equivalent replacement methods and shall be included in the scope of protection of the present application.

Claims

1. Use of an outer membrane protein of Lawsonia intracellularis for preparing vaccines and diagnostic kits, wherein the outer membrane protein of Lawsonia intracellularis is MltA protein.

2. The use according to claim 1, characterized in that The amino acid sequence of the MItA protein includes the amino acid sequence fragment shown in SEQ ID NO:

1.

3. The use according to claim 1, characterized in that The gene sequence of the Mlta protein includes the gene sequence fragment shown in SEQ ID NO:

2.

4. The use according to claim 1, characterized in that The amino acid sequence of the MItA protein is shown in SEQ ID NO: 1 or SEQ ID NO:

3.

5. The use according to claim 1, characterized in that The gene sequence of the MItA protein is shown in SEQ ID NO: 2 or SEQ ID NO:

4.

6. A recombinant protein, characterized in that The amino acid sequence of the recombinant protein is shown in SEQ ID NO: 1 or SEQ ID NO:

3.

7. A vaccine, characterized in that The recombinant protein according to claim 6 is used as an antigen.

8. A kit, characterized in that The recombinant protein according to claim 6 is used as an antigen.

Images